ALBERT

Description: Much evidence show that over-expression of epidermal growth factor receptor (EGFR) plays an important role in regulating carcinogenesis. Genetic variations in 3' untranslated region (3'UTR) of gene have been reported to affect gene expression by interfering with microRNAs (miRNAs), which are thought to function as either tumour suppressors or oncogenes by binding to their target mRNA. In this study, we investigated the association between the EGFR 3'UTR 774T〉C polymorphism and bladder cancer risk. We used the TaqMan technology to genotype this genetic variant in a hospital-based case–control study of 908 bladder cancer patients and 1239 controls in a Chinese population. We found that the 774CC genotype was associated with a statistically significantly increased risk of bladder cancer [adjusted odds ratio = 1.29, 95% confidence interval = 1.05–1.58], compared with the 774TT/TC genotype, and this increased risk was more pronounced among subgroups of age 〉 65 years, non-smokers and patients’ tumour invasive stage. Furthermore, luciferase assays in T24 cell showed that EGFR 3'UTR 774 T to C substitution could increase the expression of EGFR, which was consistent with the association study finding. Additionally, we also provide evidence that 774T〉C polymorphism increasing EGFR expression was not regulated by hsa-miR-214 binding. These findings suggested that EGFR 3'UTR 774T〉C polymorphism may contribute to susceptibility to bladder cancer.

Description: Many colorectal cancers (CRCs) develop in genetically susceptible individuals most of whom are not carriers of germ line mismatch repair or APC gene mutations and much of the heritable risk of CRC appears to be attributable to the co-inheritance of multiple low-risk variants. The accumulated experience to date in identifying this class of susceptibility allele has highlighted the need to conduct statistically and methodologically rigorous studies and the need for the multi-centre collaboration. This has been the motivation for establishing the COGENT (COlorectal cancer GENeTics) consortium which now includes over 20 research groups in Europe, Australia, the Americas, China and Japan actively working on CRC genetics. Here, we review the rationale for identifying low-penetrance variants for CRC and the current and future challenges for COGENT.

Description: Colorectal cancer (CRC) is one of the most common cancers worldwide with a peak of incidence in industrialised countries. It is a complex disease related to environmental and genetic risk factors. Low-penetrance genetic variations contribute significantly to sporadic and familial form of CRC. Genome-wide association studies (GWAS) have uncovered numerous robust associations between common variants and CRC risk; only a few of those were protein altering non-synonymous polymorphisms. One of the hypotheses is that non-coding and intergenic variants may change the expression levels of one or several target genes and, thus, account for a fraction of phenotypic differences, including susceptibility to CRC. Such genetic variations have been detected as expression quantitative loci (eQTLs) that show linkage/association to a large number of genes and have been defined as "master regulators of transcription". In the present work, we overview the potentialities to use results from GWAS and eQTL studies in the identification as well as investigation of master regulators in CRC susceptibility.

Description: Colorectal cancer (CRC) is a major cause of mortality throughout the world and risk of CRC is known to be modulated by nutritional factors. Low intake of the micronutrient selenium (Se) has been implicated as a risk factor in CRC, and in this article we describe the biochemical functions of selenium in selenoproteins, review the evidence for an association of selenium status with CRC and adenoma risk and describe the genetic epidemiological data on selenoprotein genes and CRC risk. Epidemiological evidence linking Se intake to CRC risk is limited but there is strong evidence for a link to adenoma risk. Two studies show an association between a genetic variant in the selenoprotein S gene and CRC risk. Selenium intake modulates selenoprotein expression in the colon, especially selenoproteins W, H, M, 15 kDa selenoprotein and glutathione peroxidase 1, and downstream targets such as endoplasmic reticulum stress response, oxidative stress and inflammatory pathways. We hypothesis that Se, through the selenoproteins, plays a key role in the ability of colonic epithelial cells to respond to microbial and oxidative challenges and that a combination of low Se intake and SNP in selenoprotein genes can impair that role and so lead to increased risk of pre-neoplastic lesions. There is a need for both further studies of selenoprotein function in the colon and major genetic epidemiological and intervention studies.

Description: Every year, approximately 1 million new colorectal cancer (CRC) cases are diagnosed and about half a million people worldwide die due to this cancer. Known differences in CRC incidence rates are mainly attributed to differences in diet and other environmental factors represented, among others, by nutrition-related complex diseases (e.g. obesity and diabetes mellitus type II). Within the last years, it has become evident that environmental risk factors can be complemented by a genetic component when considering the risk of CRC. For example, a number of polymorphisms are known to be associated with an increased risk of obesity and obesity is a risk factor for CRC. Several studies have shown that the ‘ancestral-susceptibility model’ can be reasonably applied to nutrition-related complex diseases such as obesity. The work in hand shortly discusses whether the ancestral-susceptibility model can also be applied to CRC as a nutrition-related complex disease.

Description: Alteration of DNA integrity is a potential cause of cancer and it is assumed that reduced DNA repair capacity and accumulation of DNA damage may represent intermediate markers in carcinogenesis. In this case-control study, DNA damage and nucleotide excision repair capacity (NER-DRC) were assessed in association with sporadic colorectal cancer (CRC). Both parameters were quantified by comet assay in blood cells of 70 untreated incident patients and 70 age-matched healthy controls. mRNA expression and polymorphisms in relevant NER genes were concurrently analyzed. The aim of this study was to characterize incident CRC patients for NER-DRC and to clarify possible relations between investigated variables. Comet assay and mRNA expression analysis showed that CRC patients differ in repair capacity as compared to controls. Patients had a lower NER-DRC and simultaneously they exhibited higher endogenous DNA damage (for both P 〈 0.001). Accumulation of DNA damage and decreasing NER-DRC behaved as independent modulating parameters strongly associated with CRC. Expression levels of 6 out of 9 studied genes differed between groups ( P ≤ 0.001), but none of them was related to DRC or to any of the studied NER polymorphisms. However, in patients only, XPC Ala499Val modulated expression levels of XPC , XPB and XPD gene, whereas XPC Lys939Gln was associated with XPA expression level in controls (for all P 〈 0.05). This study provides evidence on altered DRC and DNA damage levels in sporadic CRC and proposes the relevance of the NER pathway in this malignancy. Further, alterations in a complex multigene process like DNA repair may be better characterized by functional quantification of repair capacity than by quantification of individual genes transcripts or gene variants alone.

Description: APC is a key ‘gatekeeper’ gene in colorectal tumorigenesis. The high frequency of APC defects observed in colorectal cancer tissue is the result of selective growth advantage of cells with loss-of-function mutations at that locus. However, mutations may also arise due to inherent sequence instability. Defective DNA mismatch repair (MMR) and base excision repair (BER) also contribute to colorectal carcinogenesis and may compound such instability. To avoid the effect of clonal selective advantage imparted by APC mutation in cancer cells, we assessed in vitro APC mutation frequency in cell lines of lymphoid lineage to investigate the influence of defective MMR and BER. In DNA repair proficient cells, we observed substantially greater inherent sequence instability in APC gene coding sequences compared to reference sequences. Surprisingly, however, this difference was abrogated in MMR defective lines. We also found greater mutation frequency at exonic DNA sequences outwith the APC region in cells defective for either MMR or BER defects. The underlying propensity for mutation at the APC gene is intriguing, while the greater frequency of mutation in cells defective for DNA repair has relevance to understanding events leading to colorectal cancer and other malignancies.

Description: Inherited predisposition plays a role in 10–30% of colorectal cancer (CRC) cases. Of the large families with a clearly positive family history of CRC, ~40% is not affected by known CRC syndromes. The existence of families with unexplained forms of inherited CRC—familial CRC—suggests the presence of still unknown high- or moderate-risk CRC predisposing factors. While the genomic profiles of sporadic CRCs have been studied extensively, few studies have analysed the tumour profiles of hereditary or familial CRC. Here, we review recent advances in genomic tumour profiling in familial CRC in comparison with sporadic CRC. In addition, we discuss the role of known CRC risk factors in familial CRC.

Description: Despite their prime candidate status, polymorphisms near genes involved in DNA repair or in other functions related to genome stability have been conspicuously under-represented in the significant associations reported from genome-wide association studies (GWAS) of cancer susceptibility. In this study, we assessed a set of single-nucleotide polymorphisms (SNPs) near 157 DNA repair genes in three colorectal cancer (CRC) GWAS. Although no individual SNP showed evidence of association, the set of SNPs as a whole was associated with colorectal cancer risk. When candidate SNPs were examined, our data did not support most of the previously reported associations with CRC susceptibility, an exception being an effect of the MLH1 promoter SNP –93G〉A (rs1800734). Rare variants in CHEK2 (I157T and possibly del1100C) also appear to be associated with CRC risk. Overall, the absence to date of disease-associated DNA repair SNPs in cancer GWAS may be explained by a combination of the following: (i) many loci with individually very small effects on risk; (ii) rare alleles of moderate effect and (iii) subgroups of CRC, such as those with microsatellite instability, associated with specific variants. It will be particularly intriguing to determine whether any GWAS across cancer types identify DNA variants that predispose to cancers of more than one site.

Description: Over 2000 microRNA (miRNA) sequences from different species have been submitted to the miRBase, the central online repository for miRNAs, making a total of 5071 miRNA loci, expressing 5922 distinct mature miRNA sequences. In this review, we have addressed the importance of the genetic variations in humans affecting miRNAs, their target genes and the genes involved in miRNA processing for individual risk of cancer, with particular emphasis on colorectal cancer. In fact, the number of studies suggesting that individual predisposition to cancer is modulated by genetic polymorphisms affecting the biogenesis of miRNA and the interaction between miRNAs and targets has risen steeply in the last few years. We also report the first evidence that variant alleles of single-nucleotide polymorphisms (SNPs) within miRNA genes and miRNA targets, previously associated with the risk of cancer, behave differently when tested in functional studies. The SNPs belonging to the miRNA world are certainly contributing to new insights in the field of the genetic predisposition to disease.

Description: The EPICOLON consortium was initiated in 1999 by the Gastrointestinal Oncology Group of the Spanish Gastroenterology Association. It recruited consecutive, unselected, population-based colorectal cancer (CRC) cases and control subjects matched by age and gender without personal or familial history of cancer all over Spain with the main goal of gaining knowledge in Lynch syndrome and familial CRC. This epidemiological, prospective and multicentre study collected extensive clinical data and biological samples from ~2000 CRC cases and 2000 controls in Phases 1 and 2 involving 25 and 14 participating hospitals, respectively. Genetic susceptibility projects in EPICOLON have included candidate-gene approaches evaluating single-nucleotide polymorphisms/genes from the historical category (linked to CRC risk by previous studies), from human syntenic CRC susceptibility regions identified in mouse, from the CRC carcinogenesis-related pathways Wnt and BMP, from regions 9q22 and 3q22 with positive linkage in CRC families, and from the mucin gene family. This consortium has also participated actively in the identification 5 of the 16 common, low-penetrance CRC genetic variants identified so far by genome-wide association studies. Finishing their own pangenomic study and performing whole-exome sequencing in selected CRC samples are among EPICOLON future research prospects.

Description: Two forms of genomic instability can be distinguished in colorectal cancer (CRC) tumourigenesis. One is characterised by pronounced chromosomal instability (CIN), while the other relates to alterations produced at the nucleotide level that preferentially target microsatellite sequences. Tumours developing under the latter form of genomic instability possess a microsatellite instability-high (MSI-H) phenotype due to inactivation of the DNA mismatch repair system. The most recently described CRC syndrome, MUTYH -associated polyposis (MAP), shares characteristics with both MSI-H and CIN cancers. MAP carcinomas develop from the impairment of the base excision repair system, where MUTYH is involved, but also present a peculiar form of CIN. Several clinicopathological characteristics of MSI-H and MAP CRCs overlap such as tumour location, clinical prognosis and histological features. We propose that MSI-H and MAP CRCs are particularly prone to interact with their tumour microenvironment. A great deal of this interaction is probably stimulated by the immunogenic character of those tumours, known to possess a high mutagenic potential. The accumulation of mutations in coding regions of the genome of MSI-H and MAP carcinomas is likely to translate into a surplus of neo-antigens that trigger an anti-tumour immune response. The immune system constitutes thus an important vector of selective pressure that favours the outgrowth of tumour clones with immune-evasive phenotypes. In this review, we summarise the evidence for the influence of the tumour microenvironment in MSI-H and MAP tumourigenesis. Furthermore, we discuss how particular features of MSI-H and MAP CRCs can be exploited for the development of therapeutic strategies for affected patients.

Description: Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Epidemiological risk factors for CRC included dietary fat intake; consequently, the role of genes in the fatty acid biosynthesis and metabolism pathways is of particular interest. Moreover, hyperlipidaemia has been associated with different type of cancer and serum lipid levels could be affected by genetic factors, including polymorphisms in the lipid metabolism pathway. The aim of this study is to assess the association between single-nucleotide polymorphisms (SNPs) in fatty acid metabolism genes, serum lipid levels, body mass index (BMI) and dietary fat intake and CRC risk; 30 SNPs from 8 candidate genes included in fatty acid biosynthesis and metabolism pathways were genotyped in 1780 CRC cases and 1864 matched controls from the Molecular Epidemiology of Colorectal Cancer study. Information on clinicopathological characteristics, lifestyle and dietary habits were also obtained. Logistic regression and association analysis were conducted. Several LIPC (lipase, hepatic) polymorphisms were found to be associated with CRC risk, although no particular haplotype was related to CRC. The SNP rs12299484 showed an association with CRC risk after Bonferroni correction. We replicate the association between the T allele of the LIPC SNP rs1800588 and higher serum high-density lipoprotein levels. Weak associations between selected polymorphism in the LIPC and PPARG genes and BMI were observed. A path analysis based on structural equation modelling showed a direct effect of LIPC gene polymorphisms on colorectal carcinogenesis as well as an indirect effect mediated through serum lipid levels. Genetic polymorphisms in the hepatic lipase gene have a potential role in colorectal carcinogenesis, perhaps though the regulation of serum lipid levels.

Description: Worldwide, colorectal cancer (CRC) is the third most common cancer, with the highest mortality rates occurring in Central Europe. The use of chemotherapy to treat CRC is limited by the inter-individual variability in drug response and the development of cancer cell resistance. ATP-binding cassette (ABC) transporters play a crucial role in the development of resistance by the efflux of anticancer agents outside of cancer cells. The aim of this study was to explore transcript levels of all human ABCs in tumours and non-neoplastic control tissues from CRC patients collected before the first line of treatment by 5-fluorouracil (5-FU)-containing regimen. The prognostic potential of ABCs was evaluated by the correlation of transcript levels with clinical factors. Relations between transcript levels of ABCs in tumours and chemotherapy efficacy were also addressed. The transcript profile of all known human ABCs was assessed using real-time polymerase chain reaction with a relative standard curve. The majority of the studied ABCs were down-regulated or unchanged between tumours and control tissues. ABCA12, ABCA13, ABCB6, ABCC1, ABCC2 and ABCE1 were up-regulated in tumours versus control tissues. Transcript levels of ABCA12, ABCC7 and ABCC8 increased in direction from colon to rectum. Additionally, transcript levels of ABCB9, ABCB11, ABCG5 and ABCG8 followed the reverse significant trend, i.e. a decrease in direction from colon to rectum. The transcript level of ABCC10 in tumours correlated with the grade ( P = 0.01). Transcript levels of ABCC6, ABCC11, ABCF1 and ABCF2 were significantly lower in non-responders to palliative chemotherapy in comparison with responders. The disease-free interval of patients treated by adjuvant chemotherapy was significantly shorter in patients with low transcript levels of ABCA7, ABCA13, ABCB4, ABCC11 and ABCD4. In conclusion, ABCC11 may be a promising candidate marker for a validation study on 5-FU therapy outcome.

Description: A functionally normal TP53 is essential to protect organisms from developing cancer. Somatic mutations in the gene represent one of the highest recurring perturbations in human tumours, including colorectal cancer (CRC). However, the variegated phenotype of wide spectrum of somatic mutations in TP53 and the complexity of the disease prevent a straight interpretation of the mutational analysis in tumours. In addition to the presence of somatic mutations, polymorphic features of the gene may also contribute to alteration of the normal TP53 functioning and variants, mainly in the form of single nucleotide polymorphisms, can be expected to impact susceptibility to sporadic CRC. In the present study, we reviewed the potential role of alterations in the TP53 gene, both somatic mutations and inherited sequence variations, in predisposition to CRC and in the prognosis and response to therapy. The available data from association studies have mostly shown contradictory outcomes. The majority of the studies were based on limited sample sizes and focussed on a limited number of polymorphisms, with main being the rs1042522 (Arg72Pro). Thus far, there is no possible generalisation of the role of TP53 as also a predictor of therapeutic response and prognosis. The effects of TP53 , and its abnormalities, on the response of tumours to cytotoxic drugs, radiation and chemoradiation are complex. However, from studies it is emerging that the inherited genetics of TP53 pathway components could be utilised to further define patient populations in their abilities to induce p53 activity in response to either DNA damaging or p53-targeted therapies.

Description: The potential of cytologically reconstructed barley line D-2946 to cope with the major lesions that hamper genome integrity, namely DNA single- and double-strand breaks was investigated. Strand breaks induced by -rays and Li ions were assessed by neutral and alkaline comet assay. Repair capacity after bleomycin treatment was evaluated by agarose gel electrophoresis under neutral and alkaline conditions. Frequencies of radiation-induced chromosome aberrations were also determined. Results indicate that radiation-mediated constitutive rearrangement of the chromosome complement has led to a substantial modulation of the sensitivity of barley genome towards DNA strand breaks, produced by ionising radiation, Li ion implantation and bleomycin in an agent-specific manner, as well as of the clastogenic response to -rays. Based on these findings, reconstructed barley karyotype D-2946 can be considered a candidate radio-sensitive line with reduced ability to maintain genome integrity with respect to both DNA and chromosomal damage.

Description: The trace element selenium is an essential micronutrient that has received considerable attention for its potential use in the prevention of cancer. In spite of this interest, the mechanism(s) by which selenium might function as a chemopreventive remain to be determined. Considerable experimental evidence indicates that one possible mechanism by which selenium supplementation may exert its benefits is by enhancing the DNA damage repair response, and this includes data obtained using cultured cells, animal models as well as in human clinical studies. In these studies, selenium supplementation has been shown to be beneficial in reducing the frequency of DNA adducts and chromosome breaks, consequentially reducing the likelihood of detrimental mutations that ultimately contribute to carcinogenesis. The benefits of selenium can be envisioned as being due, at least in part, to it being a critical constituent of selenoproteins such as glutathione peroxidases and thioredoxin reductases, proteins that play important roles in antioxidant defence and maintaining the cellular reducing environment. Selenium, therefore, may be protective by preventing DNA damage from occurring as well as by increasing the activity of repair enzymes such as DNA glycosylases and DNA damage repair pathways that involve p53, BRCA1 and Gadd45. An improved understanding of the mechanism of selenium’s impact on DNA repair processes may help to resolve the apparently contradicting data obtained from decades of animal work, human epidemiology and more recently, clinical supplementation studies.

Description: The tumour-suppressor gene BRCA2 has been demonstrated to be involved in maintenance of genome integrity by affecting DNA double-strand break repair and homologous recombination. Protein-truncating mutations in BRCA2 predispose women to early onset breast and ovarian cancers and account for 15–30% of familial breast cancer risk. In contrast, the human cancer risk due to missense mutations, intronic variants, and in-frame deletions and insertions in the BRCA2 gene, called unclassified variants, has not been determined. Here, we want to define if the yeast Saccharomyces cerevisiae is a good model to study the role of BRCA2 in DNA recombination and repair and to characterise the unclassified BRCA2 missense variants. Therefore, we expressed the wild-type BRCA2 in yeast and determined the effect of BRCA2 on yeast homologous recombination, methyl methanesulphonate (MMS)-induced Rad51 and Rad52 foci and MMS sensitivity. The expression of BRCA2 induces a high increase in both intra- and inter-recombination events and confers a higher MMS resistance as compared with the negative control. This may suggest that BRCA2 gets involved in DNA repair pathways in yeast. Moreover, the expression of BRCA2 did not affect the number of cells carrying Rad51 or Rad52 nuclear foci. Finally, we aimed to investigate if yeast could be reliable system to set up a functional assay to distinguish a mutated protein from a neutral polymorphism. Therefore, we have expressed two neutral (M1915T and A2951T) and one pathogenic variant (G2748D) in yeast and checked the effect on recombination. The neutral M1915T variant increased intra-chromosomal recombination by almost 2-fold and the other neutral A2975T variant increased intra-chromosomal recombination 2.5-fold as compared with the control. On the other end, the pathogenic variant G2748D did not increase intra- and inter-chromosomal recombination in yeast and, consequently, confers a phenotype very different from the wild-type BRCA2. Moreover, we have also evaluated whether the expression of the selected unclassified variants affects homologous recombination in yeast. Results indicated that the expression of the selected BRCA2 variants differentially affects yeast recombination suggesting that yeast could be a very promising genetic system to characterise BRCA2 missense variants.

Description: Alcohol consumption is frequently associated with various cancers and the enhancement of the metabolic activation of carcinogens has been proposed as a mechanism underlying this relationship. The ethanol-induced enhancement of N -nitrosodiethylamine (DEN)-mediated carcinogenesis can be attributed to an increase in hepatic activity. However, the mechanism of elevation of N -nitrosomethylbenzylamine (NMBA)-induced tumorigenesis remains unclear. To elucidate the mechanism underlying the role of ethanol in the enhancement of NMBA-induced oesophageal carcinogenesis, we evaluated the hepatic and extrahepatic levels of the cytochrome P450 (CYP) and mutagenic activation of environmental carcinogens by immunoblot analyses and Ames preincubation test, respectively, in F344 rats treated with ethanol. Five weeks of treatment with 10% ethanol added to the drinking water or two intragastric treatments with 50% ethanol, both resulted in elevated levels of CYP2E1 (1.5- to 2.3-fold) and mutagenic activities of DEN, N -nitrosodimethylamine and N -nitrosopyrrolidine in the presence of rat liver S9 (1.5- to 2.4-fold). This was not the case with CYP1A1/2, CYP2A1/2, CYP2B1/2 or CYP3A2, nor with the activities of 2-amino-3-methylimidazo[4,5- f ]quinoline, 3-amino-1-methyl-5 H -pyrido[4,3- b ]indole, aflatoxin B 1 or other N -nitroso compounds (NOCs), including NMBA. Ethanol-induced elevations of CYP2A and CYP2E1 were observed in the oesophagus (up to 1.7- and 2.3-fold) and kidney (up to 1.5- and 1.8-fold), but not in the lung or colon. In oesophagus and kidney, the mutagenic activities of NMBA and four NOCs were markedly increased (1.3- to 2.4-fold) in treated rats. The application of several CYP inhibitors revealed that CYP2A were likely to contribute to the enhancing effect of ethanol on NMBA activation in the rat oesophagus and kidney, but that CYP2E1 failed to do so. These results showed that the enhancing effect of ethanol on NMBA-induced oesophageal carcinogenesis could be attributed to an increase in the metabolic activation of NMBA by oesophageal CYP2A during the initiation phase, and that this occurred independently of CYP2E1.

Description: The comet assay is increasingly used to measure the repair of various types of DNA damage. Modifications of the standard protocol have been introduced to determine the repair capacity of specific DNA repair pathways by the removal of pathway-specific DNA lesions. Recently, a cellular phenotype assay for nucleotide excision repair (NER) by quantifying the DNA strand breaks after in vitro challenge of peripheral blood mononucleated cells with benzo[ a ]pyrene diol epoxide (BPDE) in the presence or absence of the DNA polymerase inhibitor aphidicolin (APC) was developed (Vande Loock, K., Decordier, I., Ciardelli, R., Haumont, D. and Kirsch-Volders, M. (2010) An aphidicolin-block nucleotide excision repair assay measuring DNA incision and repair capacity. Mutagenesis , 25, 25–32). Individual repair capacity (RC) was defined as the amount of DNA damage induced by BPDE in the presence of APC minus the damage induced by BPDE and APC alone. This value should mainly reflect the incision capacity of the NER enzymes. Following this approach, we investigated the RC of cultured isolated peripheral blood mononuclear cells of nine donors in repeated experiments. We also performed the same experiments with peripheral whole blood cultures from these donors. Our results indicated considerable intra- and inter-individual variability and substantial differences between the RC of isolated mononuclear cells and whole blood from the same donor. Furthermore, the RC of unstimulated blood did not differ significantly from the repair capacity of stimulated blood but also showed considerable inter-individual variability. Altogether, our results suggest that there is still need for standardisation and validation of this assay before it can be reliably used in human biomonitoring studies.

Description: Genetic polymorphisms can partially explain the large inter-individual variation in DNA adduct levels following exposure to polycyclic aromatic hydrocarbons. Effects of genetic polymorphisms on DNA adduct formation are difficult to assess in human studies because exposure misclassification attenuates underlying relationships. Conversely, ex vivo studies offer the advantage of controlled exposure settings, allowing the possibility to better elucidate genotype–phenotype relationships and gene–gene interactions. Therefore, we exposed lymphocytes of 168 non-smoking volunteers ex vivo to the environmental pollutant benzo(a)pyrene (BaP) and BaP-related DNA adducts were quantified. Thirty-four genetic polymorphisms were assessed in genes involved in carcinogen metabolism, oxidative stress and DNA repair. Polymorphisms in catalase ( CAT , rs1001179) and cytochrome P450 1B1 ( CYP1B1 , rs1800440) were significantly associated with DNA adduct levels, especially when combined. Moreover, reverse transcription–polymerase chain reaction (RT–PCR) analysis in a subset of 30 subjects revealed that expression of catalase correlated strongly with expression of CYP1B1 ( R = 0.92, P 〈 0.001). To further investigate the mechanism by which catalase influences CYP1B1 and how they simultaneously affect BaP-related DNA adduct levels, catalase expression was transiently knocked down in the human lung epithelial cell line A549. Although catalase knockdown did not immediately change CYP1B1 gene expression, recovery of catalase expression 8 h after the knockdown coincided with a 2.2-fold increased expression of CYP1B1 ( P 〈 0.05). We conclude that the genetic polymorphism in the promoter region of CAT may determine the amount and activity of catalase, which may subsequently regulate the expression of CYP1B1. As a result, both genetic polymorphisms modulate DNA adduct levels in lymphocytes by BaP ex vivo .

Description: The aetiology of head and neck cancer (HNC) has been shown to be associated with genetic and certain environmental factors that produce DNA damage. Base excision repair (BER) genes are responsible for repair of DNA damage caused by reactive oxygen species and other electrophiles and therefore are good candidate susceptibility genes for HNC. Apurinic/apyrimidinic endonuclease-1 ( APEX1 ) proteins have important functions in the BER pathway. In this case–control study, all exons of the APEX1 gene and its exon/intron boundaries were amplified in 300 HNC cases and 300 matched healthy controls and then analysed by single-stranded conformational polymorphism. Amplified products showing altered mobility patterns were sequenced and analysed. To confirm our observations, we examined APEX1 expression at mRNA level on 50 head and neck squamous cell carcinoma (HNSCC) and 50 normal control samples by quantitative real-time polymerase chain reaction. At germ line level, three novel mutations (13T 〉 G, Ser129Arg and Val131Gly) of APEX1 were observed. The homozygous and heterozygous genotypes of APEX1 13T 〉 G, Ser129Arg and Val131Gly appear to be significantly involved in the development of HNC. In the case of expressional level, APEX1 mRNA expression was positively correlated with tumour size, clinical stage and positive lymph node metastasis. Statistical analysis showed a significantly higher APEX1 mRNA level in HNC tumour tissue than in control samples. Our study demonstrated that APEX1 mutations and deregulation of APEX1 are associated with increased risk of HNC in the Pakistani population.

Description: Patients with chronic kidney disease (CKD) have signs of genomic instability and, as a consequence, extensive genetic damage, possibly due to accumulation of uraemic toxins, oxidative stress mediators and other endogenous substances with genotoxic properties. We explored factors associated with the presence and background levels of genetic damage in CKD. A cross-sectional study was performed in 91 CKD patients including pre-dialysis (CKD patients; n = 23) and patients undergoing peritoneal dialysis (PD; n = 33) or haemodialysis (HD; n = 35) and with 61 healthy subjects, divided into two subgroups with the older group being in the age range of the patients, serving as controls. Alkaline comet assay and cytokinesis-block micronucleus assay in peripheral blood lymphocytes were used to determine DNA and chromosome damage, respectively, present in CKD. Markers of oxidative stress [malondialdehyde (MDA), advanced glycation end products (AGEs), thiols, advanced oxidation protein products and 8-hydroxy-2'-deoxyguanosine] and markers of inflammation (C-reactive protein, interleukin-6 and tumour necrosis factor alpha) were also measured. Micronucleus (MN) frequency was significantly higher ( P 〈 0.05) in the CKD group (46±4) when compared with the older control (oC) group (27.7±14). A significant increase in MN frequency ( P 〈 0.05) was also seen in PD patients (41.9±14) versus the oC group. There was no statistically significant difference for the HD group (29.7±15.6; P = NS) versus the oC group. Comet assay data showed a significant increase ( P 〈 0.001) of tail DNA intensity in cells of patients with CKD (15.6±7%) with respect to the total control (TC) group (11±1%). PD patients (14.8±7%) also have a significant increase ( P 〈 0.001) versus the TC group. Again, there was no statistically significant difference for the HD group (12.5±3%) compared with the TC group. Patients with MN values in the upper quartile had increased cholesterol, triglycerides, AGEs and MDA levels and lower albumin levels. Multiple logistic regression analysis showed that male gender, diabetes and treatment modality were independently associated with higher levels of DNA damage. Our results suggest that oxidative stress, diabetes, gender and dialysis modality in CKD patients increased DNA and chromosome damage. To confirm these data, prospective clinical trials need to be performed.

Description: Recent studies suggest increased cancer risk in patients with type 2 diabetes mellitus (T2DM) compared with healthy individuals. The present study aims to assess whether T2DM is associated with increased genome instability and whether a healthy diet with natural foods can improve genome stability in peripheral blood lymphocytes (PBLs). Seventy-six diabetic and 21 non-diabetic individuals were randomly assigned to either an ‘intervention’ or an ‘information only’ group. All participants received information about the beneficial effects of a healthy diet, while subjects of the intervention group received additionally 300g of vegetables and 25ml of plant oil rich in polyunsaturated fatty acids per day for 8 weeks. Chromosomal damage was assessed using the cytokinesis-block micronucleus (MN) cytome assay. Levels of chromosomal damage did not differ between diabetic and non-diabetic individuals. However, diabetic individuals with MN frequency above the high 50th percentile had significantly higher levels of fasting plasma glucose, glycosylated haemoglobin and were at higher risk for cardiovascular disease (CVD), assessed by the Framingham general cardiovascular risk score. Non-diabetic individuals with MN frequency above the 50th percentile had significantly lower vitamin B12 levels. The intervention with vegetables and plant oil led to significant increases in folate, -tocopherol, α- and β-carotene while vitamin B12 was significantly reduced. Levels of chromosomal damage were not altered, only apoptosis was slightly increased. The results suggest interactions between glycaemic control, CVD risk and genome stability in individuals with T2DM. However, a healthy diet does not improve genome damage in PBLs.

Description: A novel selective glucocorticoid receptor (GR) agonist, AZD2906, was found to increase the incidence of micronucleated immature erythrocytes (MIE) in the bone marrow of rats given two oral doses at the maximum tolerated level. Because GR agonists as a class are considered not to be genotoxic and AZD2906 showed no activity in the standard in vitro tests or in vivo in a rat liver comet assay, investigative studies were performed to compare AZD2906 with a reference traditional GR agonist, prednisolone. Emphasis was placed on blood and bone marrow parameters in these studies because GR activation has been reported to induce erythropoiesis which, in turn, is known to increase MIE in the bone marrow. Both compounds induced almost identical, small increases in micronucleus frequency at all doses tested. Directly comparable changes in haematological and bone marrow parameters were also seen with significant decreases in lymphoid cells in both compartments and significant increases in numbers of circulating neutrophils. Although no evidence of increased erythropoiesis was seen as increased immature erythrocyte numbers either in the blood or in the bone marrow, histopathological examination showed focal areas in the bone marrow where the erythroid population was enriched in association with an atrophic myeloid lineage. This could have been due to direct stimulation of the erythroid lineage or a secondary effect of myelosuppression inducing a rebound increase in erythropoiesis into the vacant haematopoietic cell compartment. It was concluded that the increased MIE frequencies induced by both AZD2906 and prednisolone are a consequence of their pharmacological effects on the bone marrow, either by directly inducing erythropoiesis or by some other unknown effect on cellular function, and do not indicate potential genotoxicity. This conclusion is supported by the lack of carcinogenic risk in man demonstrated by decades of clinical use of prednisolone and other GR agonists.

Description: Although amorphous silica is used in food products, cosmetics and paints and as vector for drug delivery, data on its potential health hazard are limited. The aim of this study was to investigate the cytotoxic and genotoxic potential of silica particles of different sizes (250 and 500nm) and structures (dense and mesoporous). Dense silica (DS) spheres were prepared by sol–gel synthesis, mesoporous silica particles (MCM-41) were prepared using hexadecyltrimethyl ammonium bromide as a structure-directing agent and tetraethylorthosilicate as silica source. Particles were accurately characterised by dynamic light scattering, nitrogen adsorption, X-ray diffraction and field emission scanning electron microscopy. Murine macrophages (RAW264.7) and human epithelial lung (A549) cell lines were selected for investigation. Genotoxicity was evaluated by Comet assay and micronucleus test. Cytotoxicity was tested by the trypan blue method. Cells were treated with 0, 5, 10, 20, 40 and 80 µg/cm 2 of different silica powders for 4 and 24 h. The intracellular localisation of silica was investigated by transmission electron microscopy. Amorphous particles penetrated into the cells, being compartmentalised within endocytic vacuoles. DS and MCM-41 particles induced cytotoxic and genotoxic effects in A549 and RAW264.7 although to different extent in the two cell lines. A549 were resistant in terms of cell viability, but showed a generalised induction of DNA strand breaks. RAW264.7 were susceptible to amorphous silica exposure, exhibiting both cytotoxic and genotoxic responses as DNA strand breaks and chromosomal alterations. The cytotoxic response of RAW264.7 was particularly relevant after MCM-41 exposure. The genotoxicity of amorphous silica highlights the need for a proper assessment of its potential hazard for human health.

Description: Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with -rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by -rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.

Description: AZD9708 is a new chemical entity with selective and long-acting β2-agonistic properties currently being evaluated by AstraZeneca for potential use in treatment of respiratory diseases by the inhaled route. As part of the toxicological characterisation of this compound, an increased incidence of micronucleated immature erythrocytes (MIEs) was seen in the bone marrow of rats following single intravenous doses near the maximum tolerated. This effect was seen in the absence of in vitro genotoxicity in bacterial and mammalian cells and no consistent evidence of in vivo DNA damage in the the bone marrow or liver using the comet assay was observed. Because of the lack of signals for mutagenic potential, combined with the observation that MIE frequencies appeared to be increased in only some of the rats and the clearest response was seen at the intermediate dose, it was hypothesised that the effect was secondary to β2-adrenergic receptor overstimulation. Because it appears that this has not been previously described for β2-agonists and because pharmacodynamic/pharmacokinetic factors may influence the response, studies using repeated dosing were performed to investigate whether this would lead to compound-induced tachyphylaxis with tolerance induction and decreased responses indicated by β2-effect biomarkers. A series of experiments confirmed that a sequence of five escalating daily doses leading to systemic exposure corresponding to that after a single dose led to symptomatic tolerance, declining or diminished effects on plasma biomarkers of β2-effects (plasma glucose and potassium) and elimination of the micronucleus response. This suggests that the increased MIE frequencies after single doses of AZD9708 are secondary to physiological overstimulation of β2-adrenergic receptors, not a consequence of genotoxicity.

Description: In human skin, the 3895-bp deletion of mitochondrial DNA (mtDNA 3895 ) is catalysed by ultraviolet (UV) light through the generation of reactive oxygen species. Given its function in vision, the human eye is exposed to oxidising UV and blue light in its anterior (cornea, iris) and posterior (retina) structures. In this study, we employed a highly sensitive quantitative PCR technique to determine mtDNA 3895 occurrence in human eye. Our analysis shows that the mtDNA 3895 is concentrated in both the cornea and the retina. Within the cornea, the highest mtDNA 3895 level is found in the stroma, the cellular layer conferring transparency and rigidity to the human cornea. Moreover, mtDNA 3895 accumulates with age in the stroma, suggesting a role of this deletion in corneal ageing. Within the retina, mtDNA 3895 is concentrated in the macular region of both the neural retina and the retinal pigment epithelium, supporting the hypothesis that this deletion is implicated in retinal pathologies such as age-related macular degenerescence. Taken together, our results imply that UV and blue light catalyse mtDNA 3895 induction in the human eye.

Description: Although there are several in vivo tests for potential genotoxicity, with the possible exception of the transgenic rodent mutation models, none is specifically intended to assess increasing damage with chronic administration. In principle, peripheral blood lymphocytes would be expected to accumulate DNA damage with repeated dosing because the majority are not in active division and appear to have limited DNA repair capability, and they are exposed to plasma levels of test materials and metabolites. However, there appear to be no published reports confirming this principle. Therefore, in the current study, after optimising culture conditions for rat lymphocytes in this laboratory, rats were given oral doses of cyclophosphamide or hexamethylphosphoramide (HMPA) for up to 28 days and peripheral lymphocytes analysed for chromosome aberrations at various time points. The results clearly show that, for both compounds, doses that gave no significant increases in aberration frequency after 2 days induced clear increases after 15 days with further damage detectable after 28 doses. With HMPA, it was shown that DNA damage persisted for at least 10 days after cessation of treatment. These data show that repeat dose studies in the rat measuring chromosome aberration frequency in lymphocytes can give a genuine indication that genotoxicity may increase with chronic administration and, therefore, maybe useful in assessing the risk of potentially genotoxic substances.

Description: The parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness, is a highly neglected fatal condition endemic in sub-Saharan Africa, which is poorly treated with medicines that are toxic, no longer effective or very difficult to administer. New, safe, effective and easy-to-use treatments are urgently needed. Many nitroimidazoles possess antibacterial and antiprotozoal activity and examples such as tinidazole are used to treat trichomoniasis and guardiasis, but concerns about toxicity including genotoxicity limit their usefulness. Fexinidazole, a 2-substituted 5-nitroimidazole rediscovered by the Drugs for Neglected Diseases initiative (DNDi) after extensive compound mining of public and pharmaceutical company databases, has the potential to become a short-course, safe and effective oral treatment, curing both acute and chronic HAT. This paper describes the genotoxicity profile of fexinidazole and its two active metabolites, the sulfoxide and sulfone derivatives. All the three compounds are mutagenic in the Salmonella /Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo ( ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative. Thus, fexinidazole does not pose a genotoxic hazard to patients and represents a promising drug candidate for HAT. Fexinidazole is expected to enter Phase II clinical trials in 2012.

Description: The mouse liver tumorigenic conazole fungicides triadimefon and propiconazole have previously been shown to be in vivo mouse liver mutagens in the Big Blue™ transgenic mutation assay when administered in feed at tumorigenic doses, whereas the nontumorigenic conazole myclobutanil was not mutagenic. DNA sequencing of the mutants recovered from each treatment group as well as from animals receiving control diet revealed that propiconazole- and triadimefon-induced mutations do not represent general clonal expansion of background mutations, and support the hypothesis that they arise from the accumulation of endogenous reactive metabolic intermediates within the liver in vivo . We therefore measured the spectra of endogenous DNA adducts in the livers of mice from these studies to determine if there were quantitative or qualitative differences between mice receiving tumorigenic or nontumorigenic conazoles compared to concurrent control animals. We resolved and quantitated 16 individual adduct spots by 32 P postlabelling and thin layer chromatography using three solvent systems. Qualitatively, we observed the same DNA adducts in control mice as in mice receiving conazoles. However, the 13 adducts with the highest chromatographic mobility were, as a group, present at significantly higher amounts in the livers of mice treated with propiconazole and triadimefon than in their concurrent controls, whereas this same group of DNA adducts in the myclobutanil-treated mice was not different from controls. This same group of endogenous adducts were significantly correlated with mutant frequency across all treatment groups ( P = 0.002), as were total endogenous DNA adduct levels ( P = 0.005). We hypothesise that this treatment-related increase in endogenous DNA adducts, together with concomitant increases in cell proliferation previously reported to be induced by conazoles, explain the observed increased in vivo mutation frequencies previously reported to be induced by treatment with propiconazole and triadimefon.

Description: The comet assay or single cell gel electrophoresis has proven to be a versatile and sensitive method of measuring the induction and repair of DNA damage in individual cells. However, one of the drawbacks of the assay is the bias caused by changes in the ability of cells to repair DNA damage in different cell cycle phases. Whereas the bias seems less important when G0 peripheral blood lymphocytes are studied, it might cause problems when proliferating cells are investigated. In this paper, we validate the assumption that the total comet fluorescence intensity corresponds to the position of the cell in the cell cycle and can be used to assign single cells to specific cell cycle phases. To validate the approach, we used a very homogenous blood mononuclear CD34 + cell population in G0 phase (unstimulated) or stimulated to enter the cell cycle. An analysis of the cell cycle distribution revealed that the 15 comet intensity classes and the 100 comets usually analyzed in a typical comet experiment are sufficient to obtain a reliable cell cycle distribution comparable with the results obtained by the flow cytometry for the same cell population. The effect of the cell cycle position on the results obtained by the comet assay for proliferating and non-proliferating cell populations irradiated with 3 Gy of X-radiation is also discussed.

Description: Disruptions of normal apoptotic pathways, which are mainly mediated by caspases, play an essential role in cancer development. Caspase-8 (CASP8) is encoded by the CASP8 gene and is centrally involved in the apoptosis of T lymphocytes. The association between a six-nucleotide deletion polymorphism (-652 6N del) of the CASP8 gene and the risk of cancer is widely reported; however, study results have been inconsistent and contradictory. To evaluate the association between the CASP8 -652 6N del polymorphism and the risk of cancer and to overcome the limitations of any individual study, a meta-analysis based on a total of 23 700 cases and 26 412 controls from 30 case–control studies was conducted. The results of the overall analysis suggested that the CASP8 -652 6N del polymorphism is associated with decreased risk of cancer for the allele contrast [del versus ins: odd ratio (OR) = 0.86, 95% confidence interval (CI) = 0.80–0.92], the additive genetic model (del/del versus ins/ins: OR = 0.78, 95% CI = 0.69–0.88), the dominant genetic model (del/del+del/ins versus ins/ins: OR = 0.83, 95% CI = 0.78–0.89) and the recessive genetic model (del/del versus ins/ins+del/ins: OR = 0.84, 95% CI = 0.75–0.93). In addition, after stratification for ethnicity and cancer type, significantly reduced risk was found for Asians and Caucasians as well as for individuals in the colorectal cancer group and the ‘other cancers’ group. Accordingly, there is an association between the CASP8 -652 6N del polymorphism and reduced cancer risk, especially among Asians, Caucasians and those with colorectal cancer. However, further research, such as studies focusing on additional ethnic groups and cancer types, is needed to provide a more exact and comprehensive synthesis conclusion.

Description: Increased serum bile salt levels have been associated to a single-nucleotide polymorphism in the bile salt export pump (BSEP; ABCB11 ) in several acquired cholestatic liver diseases but there is little evidence in alcoholic liver disease (ALD). Furthermore, a crosstalk between vitamin D and bile acid synthesis has recently been discovered. Whether this crosstalk has an influence on the course of ALD is unclear to date. Our aim was to analyse the role of genetic polymorphisms in BSEP and the vitamin D receptor gene ( NR1I1 ) on the emergence of cirrhosis in patients with ALD. Therefore, 511 alcoholic patients (131 with cirrhosis and 380 without cirrhosis) underwent ABCB11 genotyping (rs2287622). Of these, 321 (131 with cirrhosis and 190 without cirrhosis) were also tested for NR1I1 polymorphisms (bat-haplotype: BsmI rs1544410, ApaI rs7975232 and TaqI rs731236). Frequencies of ABCB11 and NR1I1 genotypes and haplotypes were compared between alcoholic patients with and without cirrhosis and correlated to serum bile salt, bilirubin and aspartate aminotransferase levels in those with cirrhosis. Frequencies of ABCB11 and NR1I1 genotypes and haplotypes did not differ between the two subgroups and no significant association between genotypes/haplotypes and liver function tests could be determined for neither polymorphism. We conclude that ABCB11 and NR1I1 polymorphisms are obviously not associated with development of cirrhosis in patients with ALD.

Description: Newborns have to cope with hypoxia during delivery and a sudden increase in oxygen at birth. Oxygen will partly be released as reactive oxygen species having the potential to cause damage to DNA and proteins. In utero , increase of most (non)-enzymatic antioxidants occurs during last weeks of gestation, making preterm neonates probably more sensitive to oxidative stress. Moreover, it has been hypothesized that oxidative stress might be the common etiological factor for certain neonatal diseases in preterm infants. The aim of this study was to assess background DNA damage; in vitro H 2 O 2 induced oxidative DNA damage and repair capacity (residual DNA damage) in peripheral blood mononucleated cells from 25 preterm newborns and their mothers. In addition, demographic data were taken into account and repair capacity of preterm was compared with full-term newborns. Multivariate linear regression analysis revealed that preterm infants from smoking fathers have higher background DNA damage levels than those from non-smoking fathers, emphasizing the risk of paternal smoking behaviour for the progeny. Significantly higher residual DNA damage found after 15-min repair in preterm children compared to their mothers and higher residual DNA damage after 2 h compared to full-term newborns suggest a slower DNA repair capacity in preterm children. In comparison with preterm infants born by caesarean delivery, preterm infants born by vaginal delivery do repair more slowly the in vitro induced oxidative DNA damage. Final impact of passive smoking and of the slower DNA repair activity of preterm infants need to be confirmed in a larger study population combining transgenerational genetic and/or epigenetic effects, antioxidant levels, genotypes, repair enzyme efficiency/levels and infant morbidity.

Description: Experimental evidences suggest that most essential oils possess a wide range of biological and pharmacological activities that may protect tissues against oxidative damage. In this study, we investigated DNA-protective effect of borneol, a component of many essential oils, against oxidative DNA damage induced in primary cultures of rat hepatocytes. Borneol was added to drinking water of Sprague–Dawley rats and DNA resistance against oxidative agents was compared in hepatocytes originated from control and borneol-treated rats. Oxidative stress induced by visible light-excited methylene blue (MB/VL) or 2,3-dimethoxy-1,4-naphthoquionone (DMNQ) resulted in increased levels of DNA lesions measured by the modified single cell gel electrophoresis. Borneol (17 or 34 mg/kg body weight) added to drinking water of rats for 7 days reduced the level of oxidative DNA lesions induced in their hepatocytes by MB/VL or DMNQ. To explain the increased resistance of DNA towards oxidative stress, we measured the base-excision repair (BER) capacity in liver cell extracts of control and borneol-supplemented rats on DNA substrate of HepG2 cells containing oxidative damage. Our results showed that administration of borneol in drinking water had no effect on incision activity of hepatocytes isolated from supplemented rats. The spectrophotometric assessment of enzymatic antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and the flow cytometric assessment of total intracellular glutathione (iGSH) in primary hepatocytes of borneol-supplemented rats showed no changes in SOD and GPx activities but higher iGSH content particularly in hepatocytes of higher borneol dose (34 mg/kg) supplemented rats in comparison to control animals. Despite the fact that borneol had no effect either on BER of oxidative DNA damage or on the levels of antioxidant enzymes and manifested no reducing power and radicals scavenging activity, it increased significantly the level of non-enzymatic antioxidant iGSH which could reduce the oxidative DNA lesions induced by MB/VL or DMNQ.

Description: It is important to identify the mechanism by which ionising irradiation induces various genomic alterations in the progeny of surviving cells. Ionising irradiation activates mobile elements like retrotransposons, although the mechanism of its phenomena consisting of transcriptions and insertions of the products into new sites of the genome remains unclear. In this study, we analysed the effects of sparsely ionising X-rays and densely ionising carbon-ion beams on the activities of a family of active retrotransposons, long interspersed nuclear elements 1 (L1). We used the L1/reporter knock-in human glioma cell line, NP-2/L1RP-enhanced GFP (EGFP), that harbours full-length L1 tagged with EGFP retrotransposition detection cassette (L1RP-EGFP) in the chromosomal DNA. X-rays and carbon-ion beams similarly increased frequencies the transcription from L1RP-EGFP and its retrotransposition. Short-sized de novo L1RP-EGFP insertions with 5'-truncation were induced by X-rays, while full-length or long-sized insertions (〉5 kb, containing ORF1 and ORF2) were found only in cell clones irradiated by the carbon-ion beams. These data suggest that X-rays and carbon-ion beams induce different length of de novo L1 insertions, respectively. Our findings thus highlight the necessity to investigate the mechanisms of mutations caused by transposable elements by ionising irradiation.

Description: The ultraviolet (UV)-B spectrum in solar UV radiation is essential for stimulating the epidermal production of vitamin D but also damages DNA and causes cancer in exposed cells. We examined the role of solar UV in inducing DNA damage in blood lymphocytes and the possible modulation of this damage by serum 25-hydroxy vitamin D (25(OH)D) in 207 male and female participants from South Australia. Personal solar UV exposure was estimated from hours of outdoor exposure recalled at the time of blood collection for analysis of DNA damage in lymphocytes, using the cytokinesis-block micronucleus cytome (CBMN-cyt) assay and of serum 25(OH)D. We examined the association between solar UV exposure, serum 25(OH)D and DNA damage using multiple linear regression, with age, sex, body mass index and alcohol consumption as covariates. The frequency of cells with micronuclei (a biomarker of chromosome breakage or loss) increased with increasing sun exposure [% increase = 5.24; 95% confidence interval (CI): 0.35 to 10.37 P -value = 0.04] but cells with nucleoplasmic bridges (a biomarker of misrepair of DNA strand breaks or telomere end fusions) decreased (% increase = –8.38; 95% CI: –14.32 to –2.03 P -value = 0.01). There was also a fall in the nuclear division index (NDI) (% increase = –1.01; 95% CI: –2.00 to 0.00 P -value = 0.05), suggesting diminished mitogenic response and, possibly, immune suppression. There was no overall relationship between 25(OH)D and DNA damage. There were, however, weak modulating effects of 25(OH)D on the associations of solar UV exposure with micronucleus formation and with NDI ( P -interaction = 0.03 and 0.05, respectively), where the increase in micronuclei and fall in NDI with increasing solar UV were greater at serum 25(OH)D levels 〈50 nmol/l. Thus, the influence of solar UV exposure in causing DNA damage or immune suppression in internal tissues may be stronger when vitamin D levels are low.

Description: We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)–DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a ]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE–DNA SCIA has a limit of detection of 3 adducts per 10 9 nucleotides with 5 μg DNA per well. We have validated the BPDE–DNA SCIA using DNA modified in vitro , DNA from benzo[ a ]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30–60%) than levels of bulky DNA adducts measured in the same samples by 32 P-postlabelling. The BPDE–DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE–DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between 32 P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies.

Description: 1, 3-Butadiene (BD) is a high-efficiency carcinogen in rodents and was classified as a human carcinogen in 2008 by the International Agency for Research on Cancer. However, its ability to induce genetic damage and the influence of metabolic polymorphisms to such damage in humans are both controversial claims. This study was conducted to investigate the relationships between exposure to BD, the polymorphisms of metabolic genes and the chromosomal damage in 45 pairs of occupationally exposed workers in a BD product workshop and matched control workers in an administrative office and circulatory water workshop in China. Exposure to BD was evaluated by personal sampling and stationary sampling. Different chromosomal damage endpoints in peripheral blood lymphocytes were determined using the cytokinesis-blocked micronucleus (CBMN) cytome assay; polymorphisms of metabolic genes [cytochrome P450 2E1 ( CYP2E1 ), glutathione S -transferases ( GST ) and microsomal epoxide hydrolase ( mEH )] in BD-exposed group were detected by polymerase chain reaction (PCR) or PCR–restriction fragment length polymorphism analysis. The results show that the average BD measurements of the exposed group were significantly higher than those for the control group (a personal sampling and stationary sampling, respectively). The BD-exposed workers exhibited increased frequencies of micronuclei (MNi) (8.00 ± 3.78 versus 5.62 ± 2.41) and nucleoplasmic bridges (NPBs) (2.58 ± 2.79 versus 1.13 ± 1.34) and a decreased nuclear division index (2.20 ± 0.14 versus 2.35 ± 0.27) when compared subjects in the control group. Meanwhile, BD-exposed workers carrying CYP2E1 c1c2/c2c2 or mEH intermediate (I)/high (H) group had a significantly higher NPB frequency than those carrying CYP2E1 c1c1 [frequency ratio (FR) = 2.60, 95% confidence interval (CI) 1.72–3.93; P 〈 0.0001) or the mEH low(S) group (FR = 2.06, 95% CI% 1.17–3.62; P 〈 0.05), respectively. Our study suggests that MNi and NPB frequency in CBMN cytome assay could be potential genotoxic biomarkers for BD exposure in humans. The polymorphism of CYP2E1 and mEH could also affect the chromosomal instability of BD workers.

Description: Cultured human cells are invaluable biological models for mechanistic studies of genotoxic chemicals and drugs. Continuing replacement of animals in toxicity testing will further increase the importance of in vitro cell systems, which should accurately reproduce key in vivo characteristics of toxicants such as their profiles of metabolites and DNA lesions. In this work, we examined how a common severe deficiency of cultured cells in ascorbate (Asc) impacts the formation of oxidative DNA damage by hexavalent chromium (chromate). Cr(VI) is reductively activated inside the cells by both Asc and small thiols but with different rates and spectra of intermediates and DNA adducts. We found that Cr(VI) exposure of H460 human lung epithelial cells in standard culture (〈0.01 mM cellular Asc) induced biologically significant amounts of oxidative DNA damage. Inhibition of oxidative damage repair in these cells by stable XRCC1 knockdown strongly enhanced cytotoxic effects of Cr(VI) and led to depletion of cells from G 1 and accumulation in S and G 2 phases. However, restoration of physiological levels of Asc (~1 mM) completely eliminated Cr(VI) hypersensitivity of XRCC1 knockdown. The induction of chromosomal breaks assayed by the micronucleus test in Asc-restored H460, primary human lung fibroblasts, and CHO cells was also unaffected by the XRCC1 status. Centromere-negative (clastogenic) micronuclei accounted for 80–90% of all Cr(VI)-induced micronuclei. Consistent with the micronuclei results, Asc-restored cells also showed no increase in the levels of poly(ADP-ribose), which is a biochemical marker of single-stranded breaks. Asc had no effect on cytotoxicity of O 6 -methylguanine, a lesion produced by direct DNA alkylation. Overall, our results indicate that the presence of physiological levels of Asc strongly suppresses pro-oxidant pathways in Cr(VI) metabolism and that the use of standard cell cultures creates a distorted profile of its genotoxic properties.

Description: Variation in xenobiotic metabolism cannot entirely be explained by genetic diversity in metabolic enzymes. We suggest that maternal diet during gestation can contribute to variation in metabolism by creating an in utero environment that shapes the offspring's defence against chemical carcinogens. Therefore, pregnant mice were supplemented with the natural aryl hydrocarbon receptor (AhR) agonist quercetin (1 mmol quercetin/kg feed) until delivery. Next, it was investigated whether the adult offspring at the age of 12 weeks had altered biotransformation of the environmental pollutant benzo[ a ]pyrene (B[ a ]P). In utero quercetin exposure resulted in significantly enhanced gene expression of Cyp1a1 , Cyp1b1 , Nqo1 and Ugt1a6 in liver of foetuses at Day 14.5 of gestation. Despite cessation of supplementation after delivery, altered gene expression persisted into adulthood, but in a tissue- and gender-dependent manner. Expression of Phase I enzymes ( Cyp1a1 and Cyp1b1 ) was up-regulated in the liver of adult female mice in utero exposed to quercetin, whereas expression of Phase II enzymes ( Gstp1 , Nqo1 and Ugt1a6 ) was predominantly enhanced in the lung tissue of female mice. Epigenetic mechanisms may contribute to this adapted gene expression, as the repetitive elements (SINEB1) were hypomethylated in liver of female mice prenatally exposed to quercetin. Studies on ex vivo metabolism of B[ a ]P by lung and liver microsomes showed that the amount of B[ a ]P-9,10-dehydrodiol, B[ a ]P-7,8-dihydrodiol and 3-hydroxy-B[ a ]P did not change, but the amount of unmetabolised B[ a ]P was significantly lower after incubation with lung microsomes from offspring that received quercetin during gestation. Moreover, ex vivo B[ a ]P-induced DNA adduct formation was significantly lower for liver microsomes of offspring that were exposed to quercetin during gestation. These results suggest that prenatal diet leads to persistent alterations in Phase I and II enzymes of adult mice and may affect cancer risk.

Description: Intra-individual variation in G 2 chromosomal radiosensitivity was examined by repeatedly taking blood samples from two individuals. Two healthy female volunteers provided a total of 44 blood samples, Donor 1 gave 28 samples in four time periods between 2001 and 2006 and Donor 2 gave 16 samples in two of the same time periods. Lymphocytes were cultured for 72 h prior to irradiation with 0.5 Gy, 300 kV X-rays. Colcemid was added 30 min post-irradiation. Cultures were harvested 90 min post-irradiation and analysed for chromatid gaps and breaks. Donor 1 exhibited significant intra-individual variation in G 2 chromosomal radiosensitivity for two of the four time periods. Variation was not significant for Period 1 (13 samples, P = 0.111) and Period 2 (six samples, P = 0.311) but was significant for Period 3 (two samples, P = 0.030) and Period 4 (seven samples, P = 0.005). Significant intra-individual variation was observed for both time periods involving Donor 2, these being Period 2 (nine samples, P = 0.002) and Period 4 (seven samples, P 〈 0.001). The combined data from all time periods exhibited a significant intra-individual variation for Donor 1 ( P 〈 0.001) and Donor 2 ( P 〈 0.001). These findings led to the conclusion that too much reliance should not be placed on the result from a single sample when assessing individual radiosensitivity status.

Description: The exposure of the population to non-ionising electromagnetic radiation is still increasing, mainly due to mobile communication. Whether low-intensity electromagnetic fields can cause other effects apart from heating has been a subject of debate. One of the effects, which were proposed to be caused by mobile phone radiation, is the occurrence of mitotic disturbances. The aim of this study was to investigate possible consequences of these mitotic disturbances as manifest genomic damage, i.e. micronucleus induction. Cells were irradiated at a frequency of 900 MHz, which is located in one of the main frequency bands applied for mobile communication. Two cell types were used, HaCaT cells as human cells and A L cells (human-hamster hybrid cells), in which mitotic disturbances had been reported to occur. After different post-exposure incubation periods, cells were fixed and micronucleus frequencies were evaluated. Both cell types did not show any genomic damage after exposure. To adapt the protocol for the micronucleus test into the direction of the protocol for mitotic disturbances, the post-exposure incubation period was reduced and exposure time was extended to one cell cycle length. This did not result in any increase of the genomic damage. In conclusion, micronucleus induction was not observed as a consequence of exposure to non-ionising radiation, even though this agent was reported to cause mitotic disturbances under similar experimental conditions.

Description: Alcohol consumption is an established risk factor for cancers of the head and neck, colorectum, liver and female breast. Acetaldehyde, the primary metabolite of ethanol, is suspected to play a major role in alcohol-related carcinogenesis. Acetaldehyde binds to DNA resulting in formation of adducts. DNA adducts are involved in mutagenesis and carcinogenesis. N 2 -Ethylidenedeoxyguanosine ( N 2 -ethylidene-dGuo) is the major adduct formed in this reaction. Studies have shown an association between alcohol drinking and levels of this DNA adduct, suggesting its potential use as a biomarker for studying alcohol-related carcinogenesis. However, there are no reports on the kinetics of formation and repair of N 2 -ethylidene-dGuo after alcohol consumption. Therefore, we investigated levels of N 2 -ethylidene-dGuo in DNA from human peripheral blood cells at several time points after consumption of increasing doses of alcohol. Ten healthy non-smokers were recruited and asked to abstain from alcohol consumption except for the study doses. The subjects were given measured doses of alcohol once a week for 3 weeks, targeting increasing blood alcohol levels. Blood was collected at several time points before and after each dose, DNA was isolated from granulocytes and lymphocytes and N 2 -ethylidene-dGuo was quantified as its NaBH 3 CN reduction product N 2 -ethyldeoxyguanosine by liquid chromatography–electrospray ionisation–tandem mass spectrometry. Significant increases in N 2 -ethylidene-dGuo were observed after all doses and in both cell types. However, there was substantial intraindividual variability, indicating that there are other important sources of this adduct in peripheral blood DNA. Further studies are needed to better understand the origins of N 2 -ethylidene-dGuo in blood cells, the exposures it reflects, and thus its potential use as a marker of alcohol’s genotoxic effects.

Description: Here, we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene used in insecticides, solvents and perfumes. Morphological analysis, performed in V79-Cl3 cells exposed for 1 h to increasing concentrations (25 up to 50 μM) of α-pinene, indicated a statistically significant increase in micronucleated and multinucleated cell frequencies; apoptotic cells were seen at 40 and 50 μM. This monoterpene caused genomic instability by interfering with mitotic process; in fact, 50% of cells (versus 19% of control cells) showed irregular mitosis with multipolar or incorrectly localised spindles. Cytogenetic analysis demonstrated high-frequency hypodiploid metaphases as well as endoreduplicated cells and chromosome breaks. Clastogenic damage was prevalent over aneuploidogenic damage as demonstred by the higher proportion of kinetochore-negative micronuclei. Alkaline comet confirmed that monoterpene exposure caused DNA lesions in a concentration-dependent manner. This damage probably arose by increased reactive oxygen species (ROS) production. In order to assess the generation of ROS, the cells were incubated with CM-H 2 DCFDA and then analysed by flow cytometry. Results demonstrated an increase in fluorescence intensity after α-pinene treatment indicating increased oxidative stress. On the whole, these findings strongly suggest that α-pinene is able to compromise genome stability preferentially through mitotic alterations and to damage DNA through ROS production.

Description: Tobacco smoke causes lung cancer in smokers and in never-smokers exposed to second-hand tobacco smoke (SHS). Nonetheless, molecular mechanisms of lung cancer in SHS-exposed never-smokers are still elusive. We studied lung cancers from current smokers ( n = 109), former smokers ( n = 56) and never-smokers ( n = 47) for promoter hypermethylation of five tumour suppressor genes— p16 , RARB , RASSF1 , MGMT and DAPK1 —using methylation-specific polymerase chain reaction. Lung tumours from ever-smokers suggested an increased risk of p16 hypermethylation as compared to never-smokers ( P = 0.073), with former smokers having the highest frequency of p16 hypermethylation ( P = 0.044 versus current smokers and P = 0.009 versus never-smokers). In the never-smoking group, p16 hypermethylation was seen in lung tumours from SHS-exposed individuals (4/33; 12%) but in none of the non-exposed individuals (0/9). The overall occurrence of hypermethylation (measured both as methylation index and as number of genes affected) was similar in those ever exposed to tobacco smoke (smokers, SHS-exposed never-smokers) and differed from non-exposed never-smokers. In multivariate analysis, p16 hypermethylation was more prevalent in lung tumours from male than female patients ( P = 0.018) and in squamous cell carcinomas than in adenocarcinomas ( P = 0.025). Occurrence of TP53 mutation in the tumour was associated with hypermethylation of at least one gene ( P = 0.027). In all, our data suggest that promoter hypermethylation pattern in SHS-exposed never-smokers resembles that observed in smokers. Association between TP53 mutation, a hallmark of smokers’ lung cancer, and methylation of one or more of the lung cancer-related genes studied, provides further evidence for common tobacco smoke-related origin for both types of molecular alterations.

Description: Vitamins with antioxidant properties have the ability to act as pro-oxidants, inducing oxidative damage and oxidative stress as opposed to preventing it. While vitamin supplements are commonly consumed, the scientific evidence for their health beneficial effects is inconclusive. In fact, even harmful effects have been reported. The present study aimed to investigate and compare pro-oxidant properties of different antioxidants and vitamins commonly found in dietary supplements, at concentrations of physiological relevance, alone or in combination with metals also found in supplements. Focus was on damages related to DNA. The vitamins’ chemical oxidation potencies were studied by measuring the amount of the oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formed from the DNA nucleoside deoxyguanosine (dG) after vitamin exposure, using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. To study the vitamins’ ability to cause DNA damage to cultured cells, promyelocytic leukemia cells (HL-60) were exposed to vitamins, and strand breaks, alkali-labile sites and oxidative DNA lesions, i.e. formamido pyrimidine DNA glycosylase-sensitive sites, were detected using the comet assay. Vitamins A and C chemically induced oxidation of dG, alone and in synergism with iron or copper, whereas only vitamin C and copper induced DNA damage in cultured cells. Contrary, vitamins B1, B2, B3, B6 and B12, β-carotene, folic acid, α-tocopherol, -tocopherol or -tocopherol did not induce oxidative damage to dG, while lycopene induced a weak dose–response increase. Taken together, vitamin C and copper stood out with the strongest oxidative potency, which is of potential concern since both substances are commonly found in multivitamins.

Description: Gene–environment interactions influence an individual’s risk of disease development. A common human 8-oxoguanine DNA glycosylase 1 (OGG1) variant, Cys326-hOGG1, has been associated with increased cancer risk. Evidence suggests that this is due to reduced repair ability, particularly under oxidising conditions but the underlying mechanism is poorly understood. Oxidising conditions may arise due to internal cellular processes, such as inflammation or external chemical or radiation exposure. To investigate wild-type and variant OGG1 regulation and activity under oxidising conditions, we generated m Ogg1 –/– null mouse embryonic fibroblasts cells stably expressing Ser326- and Cys326-hOGG1 and measured activity, gene expression, protein expression and localisation following treatment with the glutathione-depleting compound L-buthionine-S-sulfoximine (BSO). Assessment of OGG1 activity using a 7,8-dihydro-8-oxodeoxyguanine (8-oxo dG) containing molecular beacon demonstrated that the activity of both Ser326- and Cys326-hOGG1 was increased following oxidative treatment but with different kinetics. Peak activity of Ser326-hOGG1 occurred 12 h prior to that of Cys326-hOGG1. In both variants, the increased activity was not associated with any gene expression or protein increase or change in protein localisation. These findings suggest that up-regulation of OGG1 activity in response to BSO-induced oxidative stress is via post-transcriptional regulation and provide further evidence for impaired Cys326-hOGG1 repair ability under conditions of oxidative stress. This may have important implications for increased mutation frequency resulting from increased oxidative stress in individuals homozygous for the Cys326 h OGG1 allele.

Description: A recent genome-wide association study of renal cell carcinoma (RCC) in European population has identified genetic variants in the regions of 2p21 (rs7579899), 11q13.3 (rs7105934) and 12q24.31 (rs4765623) conferred susceptibility to RCC. In our study, we assessed whether these polymorphisms are also associated with RCC risk in a Chinese population. We genotyped these polymorphisms using TaqMan method and assessed their associations with RCC risk in a case–control study of 710 patients with histologically confirmed RCC and 760 cancer-free controls. Normal renal tissues adjacent to tumors were used to evaluate the functional consequences of these polymorphisms. We found that rs7105934 was significantly associated with reduced RCC risk [adjusted odds ratio (OR) = 0.67, 95% confidence intervals (CIs) = 0.47–0.95, GA+AA versus GG], particularly among subgroups of normal-weight individuals (OR = 0.51, 95%CI = 0.29–0.88), never-smokers (OR = 0.53, 95%CI = 0.33–0.85) and non-drinkers (OR = 0.57, 95%CI = 0.370.87). Furthermore, the rs7105934 GA genotype was associated with lower levels of CCND1 mRNA compared with GG genotype, although this association was only marginally significant ( P = 0.055). No significant association between rs7579899 or rs7105934 and RCC risk was observed. Our results suggest that rs7105934 on 11q13.3 may confer susceptibility to RCC in our population. Large population-based prospective and functional studies are required to validate the associations between these loci and RCC risk.

Description: To use lymphocytes as surrogate cells to investigate their in vitro sensitivities to ultraviolet (UV) treatment in different cancers and precancerous states by comparison with lymphocytes from healthy control individuals was the main aim of this research. UV light induces precise cellular and genomic mutations. In this study, the effect of ultraviolet A (UVA) (320–400 nm) was used as a generic mutagen to evaluate in vitro different sensitivities from lymphocytes of patients with suspected melanoma (SM), malignant melanoma (MM), polyposis coli (PC) and colorectal cancer (CRC). DNA damage was evaluated by two different methods: the micronucleus (MN) assay and the Comet assay. The baseline frequency of MNs was significantly increased in lymphocytes from all patients (SM, MM, PC and CRC) when compared to healthy individuals. After UV irradiation, MN frequencies were significantly increased in lymphocytes of all groups, both patients and healthy individuals. However, the MN frequency in all patient groups was significantly higher than in the healthy individual group. Similar results for the induction of genomic DNA damage were obtained for the Comet assay. Also for the Comet assay, UVA-induced DNA damage for all four patient groups was significantly increased when compared to healthy individuals (SM, MM, PC and CRC groups: P 〈 0.001). Conclusively, peripheral lymphocytes from patients with cancers MM and CRC or precancerous states SM and PC are more sensitive to a generic mutagen such as UVA than lymphocytes from healthy individuals. This feature may be used as an essential biomarker to screen and diagnose precancerous states and cancers in early stages.

Description: Nibrin, product of the NBN gene, together with MRE11 and RAD50 is involved in DNA double-strand breaks (DSBs) sensing and repair, induction of apoptosis and cell cycle control. Biallelic NBN mutations cause the Nijmegen breakage syndrome, a chromosomal instability disorder characterised by, among other things, radiosensitivity, immunodeficiency and an increased cancer risk. Several studies have shown an association of heterozygous c.657-661del, p.I171V and p.R215W mutations in the NBN gene with a variety of malignancies but the data are controversial. Little is known, however, whether and to what extent do these mutations in heterozygous state affect nibrin functions. We examined frequency of chromatid breaks, DSB repair, defects in S-phase checkpoint and radiosensitivity in X-ray-irradiated cells from control individuals, NBS patients and heterozygous carriers of the c.657-661del, p.I171V and p.R215W mutations. While cells homozygous for c.657-661del displayed a significantly increased number of chromatid breaks and residual -H2AX foci, as well as abrogation of the intra-S-phase checkpoint following irradiation, which resulted in increased radiosensitivity, cells with heterozygous c.657-661del, p.I171V and p.R215W mutations behaved similarly to control cells. Significant differences in the frequency of spontaneous and ionising radiation-induced chromatid breaks and the level of persistent -H2AX foci were observed when comparing control and mutant cells heterozygous for c.657-661del. However, it is still possible that heterozygous NBN mutations may contribute to cancer development.

Description: Antibiotics like fluoroquinolones (FQs) that target bacterial type II topoisomerases pose a potential genotoxic risk due to interactions with mammalian topoisomerase II (TOPO II) counterparts. Inhibition of TOPO II can lead to the generation of clastogenic DNA double-strand breaks (DSBs) that can in turn manifest in mutagenesis. Thus, methods that allow early identification of drugs that present the greatest hazard are warranted. A rapid, medium-throughput and predictive genotoxicity screen that can be applied to bacterial type II topoisomerase inhibitors is described herein. Maximal induction of the DSB biomarker serine 139-phosphorylated histone H2AX (H2AX) in L5178Y cells was quantified via flow cytometry and correlated with data derived from the mouse lymphoma screen (MLS), a default assay used to rank genotoxic potential. When applied to a class of novel bacterial type II topoisomerase inhibitors (NBTIs) in lead-optimisation, maximal H2AX induction 〉1.4-fold (relative to controls) identified 22/27 NBTIs that induced 〉6-fold relative mutation frequency (MF) in MLS. Moreover, response signatures comprising of H2AX induction and G 2 M cell cycle arrest elucidated using this approach suggested that these NBTIs, primarily of the H class, operated via a TOPO II poison-like mechanism of action (MoA) similar to FQs. NBTIs that induced ≤6-fold relative MF, which were mainly A class-derived, had less impact on H2AX (≤1.4-fold) and also evoked G 1 arrest, indicating that their cytotoxic effects were likely mediated through a non-poison MoA. Concordance between assays was 86% (54/63) when 1.4- and 6-fold ‘cut offs’ were applied. These findings were corroborated through inspection of human TOPO IIα IC 50 data as NBTIs exhibiting equivalent inhibitory capacities had differing genotoxic potencies. Deployed in an early screening capacity, the H2AX by flow assay coupled with structure–activity relationship evaluation can provide insight into MoA and impact medicinal chemistry efforts, ultimately leading to the production of inherently safer molecules.

Description: Interleukin-10 (IL-10) is a multifunctional cytokine with both immunosuppressive and anti-angiogenic properties and play an important role in the pathogenesis of cancer. IL-10 -1082A〉G polymorphism is the most extensively studied polymorphism in the IL-10 gene in cancer susceptibility. To date, a number of case–control studies were conducted to investigate the association between IL-10 -1082A〉G polymorphism and cancer risk in humans. However, the association between the IL-10 -1082A〉G polymorphism and cancer risk is still ambiguous. In an effort to solve this controversy, we performed a meta-analysis based on 61 case–control studies, including 14 499 cancer cases and 16 967 controls. We used odds ratios (ORs) with 95% confidence intervals (CIs) to assess the strength of the association. In the stratified analyses by specific cancer type, increased risk was found in lung cancer (OR = 3.16, 95% CI = 1.16–8.63 for GA versus AA; OR = 2.07, 95% CI = 1.16–3.70 for GG versus AA; OR = 3.17, 95% CI = 1.31–7.68 for GA/GG versus AA) and non-Hodgkin’s lymphoma (OR = 1.18, 95% CI = 1.02–1.36 for GA versus AA; OR = 1.17, 95% CI = 1.02–1.35 for GA/GG versus AA). The meta-analysis also indicated that the variant genotypes were associated with a moderately increased risk in Asians in all genetic models (OR = 1.80, 95% CI = 1.17–2.76 for GA versus AA; OR = 3.32, 95% CI = 1.62–6.82 for GG versus AA; OR = 1.67, 95% CI = 1.07–2.60 for GA/GG versus AA; OR= 2.93, 95% CI = 1.43–6.03 for GG versus AA/GA). The meta-analysis suggested that the IL-10 -1082A〉G polymorphism was associated with increased risk of cancer in Asians and lung cancer and non-Hodgkin’s lymphoma. To draw comprehensive and true conclusions, more researches with larger numbers of worldwide participants are needed to examine associations between IL-10 -1082A〉G polymorphism and cancer risk.

Description: A micronucleus is a small nucleus-like structure found in the cytoplasm of dividing cells that suffered from genotoxic stress. It is generally hypothesised that micronuclei content is eventually lost from cells, though the mechanism of how this occurs is unknown. If DNA located within the micronucleus is not replicated, it may explain the loss of micronuclei content. Because there had been no compelling evidence for this issue, we have addressed whether DNA located within the micronucleus is replicated this issue. Pulse labelling of bromodeoxyuridine revealed that DNA synthesis takes place in a portion of micronuclei that contain nuclear lamin B protein. By using iodine 3'-deoxyuridine/chlorodeoxyuridine double labelling, we found that all micronuclei containing lamin B are replicated during one cell cycle, whereas micronuclei lacking lamin B are never replicated. This result suggests that the content of lamin B-negative micronuclei is lost during cell division. Furthermore, we simultaneously visualised sites of DNA synthesis, lamin B and the extrachromosomal double minutes chromatin, which contain amplified oncogenes. We found that although the replication timing of double minutes was generally preserved in micronuclei, at times it differed greatly from the timing in the nucleus, which may perturb the expression of the amplified oncogenes. Taken together, these findings uncovered the DNA replication occurring inside micronuclei.

Description: The in vitro micronucleus test is considered as an attractive tool for genotoxicity testing of chemicals because of its simplicity of scoring and wide applicability in different cell types. However, most of the cells currently in use are devoid of the enzyme equipment required for activation of promutagens in the genotoxic metabolites. We postulated that the human HepaRG cell line, which can express xenobiotic metabolising enzymes at levels close to those found in primary human hepatocytes and has retained the indefinite growth capacity of transformed cells, could represent a more suitable model for genotoxicity testing of chemicals requiring metabolic activation. Based on the recommendations of the Organisation for Economic Co-operation and Development test guideline TG 487 for testing of chemicals, HepaRG cell cultures containing 〉80% mature hepatocytes were treated in situ with various chemicals for 24 h followed by a 3-day mitogenic stimulation with epidermal growth factor without cytokinesis block. In such culture conditions, HepaRG cells underwent 〉1.5 cell cycle per cell during the mitogenic stimulation. While non-genotoxic compounds (mannitol and staurosporine) did not increase the rate of micronucleated mononucleated cells, all aneugens (colchicine, nocodazole and dichlorodiphenyldichloroethylene) as well as the direct acting clastogen methyl methanesulfonate and clastogens requiring metabolic activation (aflatoxin B1, benzo(a)pyrene and 2-nitrofluorene) induced a statistically significant concentration-related increase in the number of mono-micronucleated cells. The micronucleus test was also performed after 7-day repeat exposure of HepaRG cells to the chemicals. Noticeably, a time-dependent effect was obtained with the three clastogens requiring metabolic activation. In conclusion, our results obtained with HepaRG hepatocytes exposed to various genotoxic compounds requiring or not bioactivation, compared favorably with those reported in various other cell types. They support the view that metabolically competent HepaRG cells have unique potential benefits for testing genotoxic compounds using the in vitro micronucleus assay.

Description: The Ser326Cys polymorphism in the human 8-oxogunaine DNA glycosylase ( hOGG1 ) gene had been implicated in cancer susceptibility. Studies investigating the associations between the Ser326Cys polymorphism and cancer susceptibility showed conflicting results. To derive a more precise estimation of the relationship, a meta-analysis was performed. This meta-analysis was performed from 83 case–control studies, including 27 918 cases and 33 399 controls. The fixed and random effect models were used to estimate the odds ratios (ORs) and their 95% confidence interval (CI) for various contrasts of this polymorphism. The combined results based on all studies showed that the hOGG1 Ser326Cys polymorphism was associated with an increased cancer susceptibility in different genetic models. In the stratified analyses, the association was significantly in head and neck cancer (homozygote comparison: OR = 2.19, 95% CI: 1.20–4.01, P heterogeneity = 0.002; heterozygote comparison: OR = 1.48, 95% CI: 1.11–1.99, P heterogeneity = 0.004; dominant model comparison: OR = 1.58, 95% CI: 1.14–2.19, P heterogeneity 〈 0.001; recessive model comparison: OR = 1.73, 95% CI: 1.02–2.94, P heterogeneity = 0.002; and additive model comparison: OR = 1.43, 95% CI: 1.09–1.88, P heterogeneity 〈 0.001) which remained for studies of the Asian populations and hospital-based of control sources. But it was not observed in other cancer types of the European population and population based of control sources. This meta-analysis suggested that the hOGG1 Ser326Cys polymorphism might contribute to an increased risk on cancer susceptibility. More studies based on larger sample size should be performed to confirm the findings.

Description: The aim of this study was to investigate the association of receptor interacting protein 2 (RIP2) single-nucleotide polymorphisms (SNPs) with susceptibility to systemic lupus erythematosus (SLE) in a Chinese population. A case–control study was performed on the SNPs rs16900617 and rs16900627 in 590 Chinese SLE patients and 660 healthy controls. These SNPs were typed by TaqMan allele discrimination assays. We found a significant association of rs16900617 G allele [odds ratio (OR) = 0.54, 95% confidence interval (CI) 0.41–0.72] and rs16900627 G allele (OR = 1.28, 95% CI 1.04–1.58) with SLE. Significant differences in genotype frequency distribution were also found in SLE and control individuals (rs16900617: AG versus AA, OR = 0.59, 95% CI 0.44–0.81; GG versus AA, OR = 0.08, 95% CI 0.01–0.65; AG + GG versus AA, OR = 0.55, 95% CI 0.41–0.75; rs16900627: AG versus AA, OR = 1.51, 95% CI 1.17–1.93; AG + GG versus AA, OR = 1.43, 95% CI 1.13–1.82). Analysis of the haplotypes revealed that two haplotypes of AG and GA were also significantly associated with SLE (OR = 1.37, 95% CI 1.11–1.70; OR = 0.60, 95% CI 0.45–0.79). Our findings suggest that the RIP2 gene polymorphisms may be associated with susceptibility to SLE in the Chinese population.

Description: The most important events during the regulation of tooth development were inductive interactions between the epithelial and mesenchymal tissues. The expression of Pax9 had been shown to specifically mark the mesenchymal regions at the prospective sites of all teeth prior to any morphological manifestations. Here, we investigated the PAX9 gene as a candidate gene for hypodontia in five unrelated Chinese patients with tooth agenesis. Direct sequencing and restriction enzyme analysis revealed a novel heterozygous mutation c.480C〉G (p.160Tyr〉X, Y160X) in a patient who was missing 20 permanent teeth (the third molars excluded) and 6 primary teeth. The mutation was a nonsense mutation, leading to a premature stop codon in exon 2 of PAX9 gene. PCR analysis of complementary DNA from cultured lymphocytes of the affected individual could not indicate the complete degradation of the mutated transcript. Promoter reporter assays revealed reduced transcriptional activity of the mutated PAX9 protein suggesting that the severe phenotype may result from haploinsufficiency of PAX9. In another patient with 15 missing permanent teeth (the third molars excluded), we found the c.219insG mutation previously reported by Stockton.

Description: Integrins are transmembrane adhesion molecules that mediate cell–cell and cell–extracellular matrix attachment. Integrins regulate cell growth, proliferation, migration and apoptosis and as a consequence, can have a potential role in tumour progression and metastasis. In this study, we investigated 19 non-synonymous variants in the coding region of the human integrin genes representing 3 beta subunits and 13 alpha subunits, for their potential role in melanoma susceptibility and survival. The variants were selected on the basis of probable functional relevance and theoretical predictions. Our data showed that no genetic variant was significantly associated with survival. However, the variants in ITGA10 and ITGA6 genes showed association with decreased risk, and variants in ITGA2 , ITGAE and ITGAM were associated with increased risk of melanoma. The haplotype analysis revealed association of CA haplotype of ITGAE and TAC haplotype of ITGAX with the risk modulation. A prediction analysis of functional effect, homology modelling and multiple sequence alignments of integrin sequences from different species supported our data for linkage of variants in the ITGA2 and ITGAE genes with susceptibility. The amino acid changes in each of these integrin proteins could affect intramolecularly and/or the interaction of the heterodimers. Our experimental data indicated a possible role for some of the variant alleles and/or haplotypes of the integrin genes in melanoma susceptibility, which is augmented by the theoretical analysis performed.

Description: Using morphological transformation as an endpoint, the Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) is an in vitro system with a high sensitivity and specificity for testing the carcinogenic potential of test agents. Advantages of the assay are that SHE cells are metabolically competent, genetically stable and acquire spontaneous transformation with a low frequency; additionally, it detects both genotoxic and non-genotoxic carcinogens. However, in comparison with other short-term mammalian cell assays, it is time consuming, laborious and, most importantly, the visual scoring of morphological transformation might be subjective. In this review, we examine the background to the test and why it has the potential for use in safety risk assessment. Additionally, we propose a novel approach to objectively interrogate and classify SHE colonies using vibrational spectroscopy coupled to a mathematical framework for high-throughput screening. It is our view that this alternative approach has the potential to improve the sensitivity and specificity of the in vitro SHE assay.

Description: The p 73 gene (1p36–33) is involved in cancer development through cell growth inhibition by inducing apoptosis in a p 53-like manner. The p 73 G4C14-to-A4T14 dinucleotide polymorphism, consisting of two single-nucleotide polymorphisms in the non-coding region of exon 2 that are in complete linkage disequilibrium, has been extensively studied in association with cancer risk. We performed a meta-analysis of published studies that examined the association between this p 73 G4C14-to-A4T14 polymorphism and cancer by searching for relevant studies on Medline and Embase up to February 28, 2010. Pooling data from 19 case–control studies that included 6510 cancer cases and 5711 controls, we found that carriers of the p 73 G4C14-to-A4T14 homozygous variant genotype (AT/AT) had an increased global risk of cancer [odds ratio (OR) = 1.30, 95% confidence interval (CI), 1.03–1.65]. There was no evidence of an effect modification of p 73 AT/AT by age, gender, ethnicity or smoking status in subgroup analyses; however, a 1.35-fold statistically significant increased risk was found among individuals 〈55 years old. In case-only analysis, the homozygous p 73 G4C14-to-A4T14 variant of p 73 genotype was associated with the presence of the p 53 exon 4 Arg72Pro allele (OR = 1.30, 95% CI, 1.02–1.64), which is suggestive of a biological interaction between the two genes in carcinogenesis. In conclusion, the p 73 G4C14-to-A4T14 homozygous variant genotype might be a risk factor for cancer, especially in combination with the p 53 exon 4 Arg72Pro polymorphism. Further studies looking at p 73 G4C14-to-A4T14 and p 53 exon 4 Arg72Pro interaction are required to support our findings.

Description: Alcohol drinking is a major risk factor for head and neck cancer (HNC). This risk may be modified by alcohol dehydrogenase ( ADH ) genes, particularly ADH1B and ADH1C , that oxidise ethanol to its carcinogenic metabolite, acetaldehyde. A meta-analysis was conducted to assess the association between ADH1B and ADH1C and HNC risk. Twenty-nine studies from 28 articles identified from a literature search were included. Summary odds ratios (meta-ORs) were generated using random effect models. A reduced risk for HNC was associated with carrying the ADH1B*2 and ADH1C*1 alleles that confer faster metabolism of ethanol to acetaldehyde [meta-OR ADH1B , 0.50; 95% confidence interval (CI): 0.37–0.68, 13 studies; meta-OR ADH1C , 0.87; 95% CI: 0.76–0.99, 22 studies]. ADH1B*2 and ADH1C*1 alleles appear to be protective for HNC, possibly due to: (i) decreasing the opportunity for oral microflora to produce acetaldehyde locally from a prolonged systemic circulation of ethanol, (ii) preventing ethanol from acting as a solvent for other carcinogens, and (iii) decreasing the amount of ethanol a person consumes since a consequent peak in systemic acetaldehyde could cause discomfort. These results underscore the importance of ADH1B and ADH1C in the association between alcohol consumption and the risk for HNC.

Description: We previously reported that the proportion of large-size micronuclei (MN) can be a reliable parameter to discriminate aneugens from clastogens in the in vitro MN assay using Chinese hamster lung cells. The frequencies of polynuclear (PN) and mitotic (M) cells are also supposed to be useful parameters for the same purpose since they are known to be increased by aneugens. In the present study, we investigated whether morphological observations of the cell nucleus can be applied for the in vitro MN assay using the p53-competent human lymphoblastoid cell line, TK6 cells. Our present MN assay with six clastogens and six aneugens revealed that the frequencies of large-size MN or PN cells cannot distinguish aneugens from clastogens, while the frequencies of M cells can distinguish them, suggesting that the M-cell frequency is a recommended parameter to determine a mode of action for MN induction in the in vitro MN assay using TK6 cells. Our further investigation using p53-null mutant NH32 cells showed that the frequencies of large-size MN or PN cells induced by aneugen treatments were higher than those in TK6 cells but not by clastogen treatments. These findings suggest that p53 abrogation promotes the susceptibility for morphological changes in the nucleus to aneugens and that morphological observation of the cell nucleus including size-classifying MN counting could distinguish aneugens from clastogens in the MN assay using NH32 cells.

Description: The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has utility in the assessment of potential chemical carcinogenicity (both genotoxic and non-genotoxic mechanisms of action). The assay uses morphological transformation as an end point and has a reported sensitivity of 87%, specificity of 83% and overall concordance of 85% with in vivo rodent bioassay data. However, the scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with benzo[ a ]pyrene, 3-methylcholanthrene, anthracene, N -nitroso- N -methylnitroguanidine, ortho-toluidine HCl, 2,4-diaminotoluene or D -mannitol for 7 days before fixation with methanol. Identified colonies were interrogated by acquiring a minimum of five infrared (IR) spectra per colony using attenuated total reflection Fourier-transform IR spectroscopy. Individual IR spectra were acquired over a spatial area of approximately 250 x 250 μm. Resultant data were analysed using Fisher’s linear discriminant analysis and feature histogram algorithms to extract classifying biomarkers of test agent-specific effects or transformation in SHE cells. Clustering of spectral points suggested co-segregation or discrimination of test agent categories based on mechanism of action. Towards transformation, unifying alterations were associated with alterations in the Amide I and Amide II peaks; these were consistently major classifying biomarkers for transformed versus non-transformed SHE cells. Our approach highlights a novel method towards objectively screening and classifying SHE cells, be it to ascertain test agent treatment based on mechanism of action or transformation.

Description: The comet assay can be applied to virtually any tissue and it has been noted that it can be particularly useful in evaluating directly acting genotoxins at their initial site of action. Consequently, it has become relatively common practice to use the stomach comet assay after oral administration to test chemicals that have given positive in vitro genotoxicity results in the absence of metabolic activation. However, to test nontoxic substances up to the limit doses of 1000/2000mg/kg formulations approaching molar concentrations must be used resulting in the stomach mucosa being exposed to excessively high levels. Evidence is beginning to accumulate which shows positive results that do not indicate that potential carcinogenicity may be associated with such high levels of exposure. For pharmaceutical agents, toxicokinetic data are usually available to demonstrate systemic exposure after oral administration. In such cases, it is proposed that exposure of any tissue to levels of the drug substance greater than those that have given positive in vitro results in the absence of metabolic activation is sufficient. However, it is recognised that toxicokinetic data are not available for all chemicals and there are also agents designed not to leave the gastrointestinal tract (GIT). Where it is necessary to examine the GIT, the dose levels selected for examination should cover the likely or intended exposure levels, not necessarily to achieve the maximum tolerated or limit doses, even if the higher doses are required for genotoxicity endpoints in other tissues to be valid. There are usually two or three dose levels in in vivo genotoxicity studies, so when both systemically exposed tissues and the stomach are being examined, it would be possible to use one of the lower doses for the latter without increasing the numbers of animals required. It is important to consider the local concentrations achieved in the stomach or other parts of the GIT in order to avoid the comet assay generating artefactual positive results and it is hoped this will be addressed in the imminent Organisation for Economic Co-operation and Development guideline.

Description: The comet analysis of DNA strand break levels in tissues and cells has become a common method of screening for genotoxicity. The large majority of published studies have used fresh tissues and cells processed immediately after collection. However, we have used frozen tissues and cells for more than 10 years, and we believe that freezing samples improve efficiency of the method. We compared DNA strand break levels measured in fresh and frozen bronchoalveolar cells, and lung and liver tissues from mice exposed to the known mutagen methyl methanesulphonate (0, 25, 75, 112.5mg/kg). We used a high-throughput comet protocol with fully automated scoring of DNA strand break levels. The overall results from fresh and frozen samples were in agreement [ R 2 = 0.93 for %DNA in tail (%TDNA) and R 2 = 0.78 for tail length (TL)]. A slightly increased %TDNA was observed in lung and liver tissue from vehicle controls; and TL was slightly reduced in bronchoalveolar lavage cells from the high-dose group. In our comet protocol, a small block of tissue designated for comet analysis is frozen immediately at tissue collection and kept deep frozen until rapidly homogenised and embedded in agarose. To demonstrate the feasibility of long-term freezing of samples, we analysed the day-to-day variation of our internal historical negative and positive comet assay controls collected over a 10-year period (1128 observations, 11 batches of frozen untreated and H 2 O 2 -treated A549 lung epithelial cells). The H 2 O 2 treatment explained most of the variation 57–77% and the day-to-day variation was only 2–12%. The presented protocol allows analysis of samples collected over longer time span, at different locations, with reduced variation by reducing number of electrophoreses and is suitable for both toxicological and epidemiological studies. The use of frozen tissues; however, requires great care during preparation before analysis, with handling as a major risk factor.

Description: Selenium is an essential trace element for humans, playing an important role in various major metabolic pathways. Selenium helps to protect the body from the poisonous effects of heavy metals and other harmful substances. Medical studies have provided evidence of selenium supplementation in preventing certain cancers. Low and too high selenium (Se) status correlates with increased risk of e.g. lung, larynx, colorectal and prostate cancers. A higher level of selenium and supplementation with selenium has been shown to be associated with substantially reduced cancer mortality. Selenium exerts its biological roles through selenoproteins, which are involved in oxidoreductions, redox signalling, antioxidant defence, thyroid hormone metabolism and immune responses. Checkpoint kinase 2 ( CHEK2 ) is an important signal transducer of cellular responses to DNA damage and acts as a tumour suppressor gene. Mutations in the CHEK2 gene have been shown to be associated with increased risks of several cancers. Four common mutations in CHEK2 gene (1100delC, IVS2+1G〉A, del5395 and I157T) have been identified in the Polish population. Studies have provided evidence that CHEK2 -truncating and/or missense mutations are associated with increased risk of breast, prostate, thyroid, colon and kidney cancers. The variability in penetrance and cancer expression in CHEK2 mutation carriers can probably be explained by the influence of other genetic or environmental factors. One of the possible candidates is Se, which together with genetic variations in selenoprotein genes may influence susceptibility to cancer risk.

Description: Several potentially functional variants of Nijmegen breakage syndrome 1 ( NBS1 ) have been implicated in cancer risk, but individually studies showed inconclusive results. In this study, a meta-analysis based on 60 publications with a total of 39 731 cancer cases and 64 957 controls was performed. The multivariate method and the model-free method were adopted to determine the best genetic model. It was found that rs2735383 variant genotypes were associated with significantly increased overall risk of cancer under the recessive genetic model [odds ratio (OR) =1.12, 95% confidence interval (CI): 1.02–1.22, P = 0.013]. Similar results were found for rs1063054 under the dominant model effect (OR = 1.12, 95% CI: 1.01–1.23, P = 0.024). The I171V mutation, 657del5 mutation and R215W mutation also contribute to the development of cancer (for I171V, OR = 3.93, 95% CI: 1.68–9.20, P = 0.002; for 657del5, OR = 2.79, 95% CI: 2.17–3.68, P 〈 0.001; for R215W, OR = 1.77, 95% CI: 1.07–2.91, P = 0.025). From stratification analyses, an effect modification of cancer risks was found in the subgroups of tumour site and ethnicity for rs2735383, whereas the I171V, 657del5 and R215W showed a deleterious effect of cancer susceptibility in the subgroups of tumour site. However, rs1805794, D95N and P266L did not appear to have an effect on cancer risk. These results suggest that rs2735383, rs1063054, I171V, 657del5 and R215W are low-penetrance risk factors for cancer development.

Description: We examined the clastogenic and cell-transforming potential of ultrafine particulate matter fraction PM 0.056 of urban ambient aerosol using mammalian cells. PM 1.0 , PM 0.56 and PM 0.056 fractions were sampled from roadside atmosphere of an urban area using the cascade impactor MOUDI-NR-110. The potential to induce cytotoxicity, DNA damage and micronuclei formation was examined at the test concentrations of 3, 6, 12.5, 25, 50 and 100 μg/ml using the 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the plasmid relaxation assay and the C3H10T1/2 (10T1/2) cells. The cell-transforming potential was investigated in vitro using 10T1/2 cell transformation assay and the soft agar assay. PM 1 , PM 0.56 and PM 0.056 fractions were found to be toxic in dose-dependent manner. These induced cytotoxicity at five test concentrations, the ultrafine particle fraction PM 0.056 showed greater cytotoxic potential. PM 0.056 induced micronucleus formation in 10T1/2 cells. The effect was statistically significant. The DNA-damaging potential was measured in a plasmid relaxation assay. Both fine and ultrafine particle fraction PM 0.56 and PM 0.056 displayed greater effect as compared to larger PM 1 fraction. DNA damage was found to be dependent on particulate matter intrinsic pro-oxidant chemicals. The ability of the ultrafine particle fraction PM 0.056 to induce morphological cell transformation was demonstrated by significant and dose-dependent increases in type III focus formation by morphologically transformed cells in culture flasks and their clonal expansion in soft agar. It is concluded that the traffic-linked ultrafine particle fraction PM 0.056 in the atmosphere by the roadside of an urban area is clastogenic and able to induce morphological transformation of mammalian cells.

Description: Demand for new technologies that deliver fast, inexpensive and accurate genome information has never been greater. This challenge has catalysed the rapid development of advances in next-generation sequencing (NGS). The generation of large volumes of sequence data and the speed of data acquisition are the primary advantages over previous, more standard methods. In 2013, the Food and Drug Administration granted marketing authorisation for the first high-throughput NG sequencer, Illumina’s MiSeqDx, which allowed the development and use of a large number of new genome-based tests. Here, we present a review of template preparation, nucleic acid sequencing and imaging, genome assembly and alignment approaches as well as recent advances in current and near-term commercially available NGS instruments. We also outline the broad range of applications for NGS technologies and provide guidelines for platform selection to best address biological questions of interest. DNA sequencing has revolutionised biological and medical research, and is poised to have a similar impact on the practice of medicine. This tool is but one of an increasing arsenal of developing tools that enhance our capabilities to identify, quantify and functionally characterise the components of biological networks that keep us healthy or make us sick. Despite advances in other ‘omic’ technologies, DNA sequencing and analysis, in many respects, have played the leading role to date. The new technologies provide a bridge between genotype and phenotype, both in man and model organisms, and have revolutionised how risk of developing a complex human disease may be assessed. The generation of large DNA sequence data sets is producing a wealth of medically relevant information on a large number of individuals and populations that will potentially form the basis of truly individualised medical care in the future.

Description: In Central America, the traditional temazcales or wood-fired steam baths, commonly used by many Native American populations, are often heated by wood fires with little ventilation, and this use results in high wood smoke exposure. Urinary mutagenicity has been previously employed as a non-invasive biomarker of human exposure to combustion emissions. This study examined the urinary mutagenicity in 19 indigenous Mayan families from the highlands of Guatemala who regularly use temazcales ( N = 32), as well as control (unexposed) individuals from the same population ( N = 9). Urine samples collected before and after temazcal exposure were enzymatically deconjugated and extracted using solid-phase extraction. The creatinine-adjusted mutagenic potency of urine extracts was assessed using the plate-incorporation version of the Salmonella mutagenicity assay with strain YG1041 in the presence of exogenous metabolic activation. The post-exposure mutagenic potency of urine extracts were, on average, 1.7-fold higher than pre-exposure samples ( P 〈 0.005) and also significantly more mutagenic than the control samples ( P 〈 0.05). Exhaled carbon monoxide (CO) was ~10 times higher following temazcal use ( P 〈 0.0001), and both CO level and time spent in temazcal were positively associated with urinary mutagenic potency (i.e. P 〈 0.0001 and P = 0.01, respectively). Thus, the wood smoke exposure associated with temazcal use contributes to increased excretion of conjugated mutagenic metabolites. Moreover, urinary mutagenic potency is correlated with other metrics of exposure (i.e. exhaled CO, duration of exposure). Since urinary mutagenicity is a biomarker associated with genetic damage, temazcal use may therefore be expected to contribute to an increased risk of DNA damage and mutation, effects associated with the initiation of cancer.

Description: Benzo[ a ]pyrene (BaP), a potent polycyclic aromatic hydrocarbon carcinogen, is widely distributed in the human environment. All humans are exposed to BaP through the diet and contact with the general environment; cigarette smokers have higher exposure. An important pathway of BaP metabolism proceeds through formation of diol epoxides including the ‘bay region diol epoxide’ 7 R ,8 S -dihydroxy-9 S ,10 R -epoxy-7,8,9,10-tetrahydrobenzo[ a ]pyrene [BaP-(7 R ,8 S )-diol-(9 S ,10 R )-epoxide] and the ‘reverse diol epoxide’ 9 S ,10 R -dihydroxy-7 R ,8 S -epoxy-7,8,9,10-tetrahydrobenzo [ a ]pyrene [BaP-(9 S ,10 R )-diol-(7 R ,8 S )-epoxide]. The bay region diol epoxide is considered a major ultimate carcinogen of BaP based on studies in cell culture and laboratory animals, but the available data in humans are less convincing. The bay region diol epoxide and the reverse diol epoxide react with H 2 O to produce enantiomeric BaP-tetraols that are excreted in the urine. We used chiral stationary-phase high-performance liquid chromatography and gas chromatography–negative ion chemical ionisation–tandem mass spectrometry to quantify these enantiomeric BaP-tetraols in the urine of 25 smokers and 25 non-smokers. The results demonstrated that the BaP-tetraol enantiomer representing the carcinogenic bay region diol epoxide pathway accounted for 68±6% (range 56–81%) of total BaP-tetraol in smokers and 64±6% (range 46–78%) in non-smokers. Levels of the major BaP-tetraol enantiomer decreased by 75% in smokers who quit smoking. These data provide convincing evidence in support of the bay region diol epoxide mechanism of BaP carcinogenesis in humans.

Description: Aristolochic acid (AA) is an active component in herbal drugs derived from the Aristolochia species. Although these drugs have been used since antiquity, AA is both genotoxic and carcinogenic in animals and humans, resulting in kidney tumours in rats and upper urinary tract tumours in humans. In the present study, we conducted microarray analysis of microRNA (miRNA) expression in tissues from transgenic Big Blue rats that were treated for 12 weeks with 0.1–10mg/kg AA, using a protocol that previous studies indicate eventually results in kidney tumours and mutations in kidney and liver. Global analysis of miRNA expression of rats treated with 10mg/kg AA indicated that 19 miRNAs were significantly dysregulated in the kidney, with most of the miRNAs related to carcinogenesis. Only one miRNA, miR-34a (a tumour suppressor), was differentially expressed in the liver. The expression of the two most responsive kidney miRNAs (miR-21, an oncomiR and miR-34a) was further examined in the kidney, liver and testis of rats exposed to 0, 0.1, 1.0 and 10mg/kg AA. Expression of miR-21 was up-regulated in the kidney only, while miR-34a was dose-dependently up-regulated in both the kidney and liver; the expression of miR-21 and miR-34a was unaltered by the AA treatment in the testis. Analysis of cII mutations in the testis of treated rats also was negative. Our results indicate that AA treatment of rats produced dysregulation of a large number of miRNAs in the tumour target tissue and that the up-regulation of miR-21 correlated with the carcinogenicity of AA while the up-regulation of miR-34a correlated with its mutagenicity.

Description: Next generation sequencing has become a powerful tool in dissecting and identifying mutations and genomic structural variants that accompany tumourigenesis. Sequence analysis of glioblastoma multiforme (GBM) illustrates the ability to rapidly identify mutations that may affect phenotype. Approximately 50% of human GBMs overexpress epidermal growth factor receptor (EGFR) which renders the EGFR protein a compelling therapeutic target. In brain tumours, attempts to target EGFR as a cancer therapeutic, however, have achieved little or no benefit. The mechanisms that drive therapeutic resistance to EGFR inhibitors in brain tumours are not well defined, and drug resistance contributes to the deadly and aggressive nature of the disease. Whole genome sequencing of four primary GBMs revealed multiple pathways by which EGFR protein abundance becomes deregulated in these tumours and will guide the development of new strategies for treating EGFR overexpressing tumours. Each of the four tumours displayed a different mechanism leading to increased EGFR protein levels. One mechanism is mediated by gene amplification and tandem duplication of the kinase domain. A second involves an intragenic deletion that generates a constitutively active form of the protein. A third combines the loss of a gene which encodes a protein that regulates EGFR abundance as well as an miRNA that modulates EGFR expression. A fourth mechanism entails loss of an ubiquitin ligase docking site in the C-terminal part of the protein whose absence inhibits turnover of the receptor.

Description: The present study aimed to examine if multiple exposure to Aroclor 1254, a commercial mixture of polychlorinated biphenyls, had any genotoxic potential on gonads of male mice; moreover, the molecular mechanism(s) underlying this deleterious effects were elucidated. In the standard comet assay, there were significant increases in the incidence of DNA strand breaks in sperm of mice killed after 3 and 24h of last treatment with 4mg/kg/day Aroclor 1254 for 5 weeks, while no significant difference in the DNA strand breaks was found in mice treatment with 1 and 2 mg/kg/day Aroclor 1254. The same results were also observed with spermatocyte chromosomal analysis as obvious aberrant primary spermatocytes were noted with the highest dose of Aroclor 1254 when testes were examined at 24h after the last exposure. Moreover, digestion with EndoIII resulted in significantly increased levels of DNA damage at 3 and 24h after the last exposure to 2mg/kg/day Aroclor 1254; digestion with Fpg resulted in a significant increase in DNA damage at the 3-h sampling time only as detected by oxidative comet assays. The expression of DNA repair genes p53 , PARP1 and BAX were up-regulated in testes of mice killed after 3 and 24h of last administration of 4mg/kg/day Aroclor 1254. On the other hand, no significant alteration in the expression of XRCC1 gene was observed at both sampling times. It is noteworthy that the expression of OGG1 and APEX1 was significantly decreased at 3h after the last exposure to 4mg/kg/day Aroclor 1254. On the other hand, only the expression level of APEX1 was recovered at the 24-h sampling times. The unrecovered OGG1 may suggest that inhibition of DNA repair can be considered as a potential mode of action of Aroclor 1254 gonadal toxicity and carcinogenesis.

Description: MicroRNAs (miRNAs), a class of small non-coding RNAs, are fundamental for the post-transcriptional regulation of gene expression. Altered expression of miRNAs has been detected in cancers, not only in primary tissue but also in easily obtainable specimens like plasma and stools. miRNA expression is known to be modulated by diet (micro and macronutrients, phytochemicals) and possibly by other lifestyle factors; however, such influence has not yet been exhaustively explored in humans. In the present study, we analysed the expression levels of a panel of seven human miRNAs in plasma and stool samples of a group of 24 healthy individuals characterised by different dietary habits (eight vegans, eight vegetarians and eight subjects with omnivorous diet, all groups with similar age and sex distribution). The dual aim of the study was to identify possible differences in miRNA expression due to diet (or other lifestyle factors recorded from questionnaires) and to compare results in both types of specimens. miR-92a was differentially expressed in both plasma and stool samples and with the same trend, among the three groups with different diets ( P = 0.0002 and P = 0.02, respectively, with expression levels of vegans〉vegetarians〉omnivores). miR-92a was also associated with low body mass index ( P = 0.04 and P = 0.05, respectively) in both types of specimens, and with several dietary factors. Other analysed miRNAs (miR-16, miR-21, mir-34a and miR-222) were associated with dietary and lifestyle factors, but not consistently in both stool and plasma. Our pilot study provides the first evidence of miRNA modulation by diet and other factors, that can be detected consistently in both plasma and stools samples.

Description: Lung inflammation and epithelial to mesenchymal transition (EMT) are two pathogenic features for the two contextual diseases: chronic obstructive pulmonary disease (COPD) and lung cancer. VEGFR1 (or FLT1 ) plays a certain role in promoting tumour growth, inflammation and EMT. To simultaneously test the association between the single nucleotide polymorphisms (SNPs) in VEGFR1 and risk of COPD and lung cancer would reveal genetic mechanisms shared by these two diseases and joint aetiology. We conducted a two-population hospital-based case–control study. Three potential functional SNPs (rs664393, rs7326277 and rs9554314) were genotyped in southern Chinese and validated in eastern Chinese to explore their associations with COPD risk in 1511 COPD patients and 1677 normal lung function controls, and with lung cancer risk in 1559 lung cancer cases and 1679 cancer-free controls. We also detected the function of the promising SNP. Individuals carrying the rs7326277C (CT+CC) variant genotypes of VEGFR1 had a significant decrease in risk of both COPD (OR = 0.78; 95% CI = 0.68–0.90) and lung cancer (OR = 0.79; 95% CI = 0.64–0.98), compared with those carrying the rs7326277TT genotype. Functional assays further showed that the rs7326277C genotypes had lower transcriptional activity and caused decreased VEGFR expression, compared with the rs7326277TT genotype. However, no significant association was observed for the other two SNPs (rs664393 and rs9554314) and either COPD or lung cancer risk. Our data suggested that the rs7326277C variant of VEGFR1 could reduce both COPD and lung cancer risk by lowering VEGFR1 mRNA expression; the SNP might be a common susceptible locus for both COPD and lung cancer.

Description: Early detection and quantification of DNA damage in oral premalignancy or malignancy may help in management of the disease and improve survival rates. The comet assay has been successfully utilised to detect DNA damage in oral premalignant or malignancy. However, due to the invasive nature of collecting blood, it may be painful for many unwilling patients. This study compares the micronucleus (MN) assay in oral buccal mucosa cells with the comet assay in peripheral blood cells in a subset of oral habit-induced precancer and cancer patients. For this, MN assay of exfoliated epithelial cells was compared with comet assay of peripheral blood leucocytes among 260 participants, including those with oral lichen planus (OLP; n = 52), leukoplakia (LPK; n = 51), oral submucous fibrosis (OSF; n = 51), oral squamous cell carcinoma (OSCC; n = 54) and normal volunteers ( n = 52). Among the precancer groups, LPK patients showed significantly higher levels of DNA damage as reflected by both comet tail length ( P 〈 0.0001) and micronuclei (MNi) frequency ( P = 0.0009). The DNA damage pattern in precancer and cancer patients was OLP 〈 OSF 〈 LPK 〈 OSCC, and with respective oral habits, it was multiple habits 〉 cigarette + khaini 〉 cigarette smokers 〉 areca + khaini 〉 areca. There was no significant difference in the comet length and MNi frequency between males and females who had oral chewing habits. An overall significant correlation was observed between MNi frequency and comet tail length with r = 0.844 and P 〈 0.0001. Thus, the extent of DNA damage evaluation by the comet assay in peripheral blood cells is perfectly reflected by the MN assay on oral exfoliated epithelial cells, and MNi frequency can be used with the same effectiveness and greater efficiency in early detection of oral premalignant conditions.

Description: The applications of cerium oxide nanoparticles (CeO 2 NPs; nanoceria) extend to polishing agents, diesel fuel additives and as a putative antioxidant in therapeutics. Therefore, understanding the long-term toxic effects of CeO 2 NPs is of particular importance. This study investigated the 28 days of repeated toxicity of 30, 300 and 600mg/kg body weight (bw)/day of nanoceria and CeO 2 microparticles (MPs) in Wistar rats after oral exposure. Genotoxicity was analysed using comet, micronucleus (MN) and chromosomal aberration (CA) assays. The results demonstrated a significant increase in DNA damage in peripheral blood leukocytes and liver, MN and CA in bone marrow as well as MN in peripheral blood after exposure to CeO 2 NPs at 300 and 600mg/kg bw/day. Significant alterations were observed in alkaline phosphatase and lactate dehydrogenase activity in serum and reduced glutathione content in the liver, kidneys and brain at 300 and 600mg/kg bw/day in a dose-dependent manner. Conversely, CeO 2 MPs did not induce any significant toxicological changes. A much higher absorptivity and significant tissue distribution of CeO 2 NPs was perceived in comparison to CeO 2 MPs in a dose-dependent manner. A substantial fraction of CeO 2 NPs was cleared by urine and faeces. Histopathological analysis revealed that CeO 2 NPs caused alterations in liver, spleen and brain. Further, distinct difference in the data among genders was not obvious. In general, the results suggested that prolonged oral exposure to nanoceria has the potential to cause genetic damage, biochemical alterations and histological changes after retention in vital organs of rats at high concentrations.

Description: Zearalenone (ZEA) is a mycotoxin produced by various Fusarium fungi, which has been shown to cause several cases of mycotoxicosis in farm animals and humans. However, there is no evidence regarding the effect of ZEA on mouse egg developmental competence. In this study, we found that the activation rate of maturated oocytes was affected in mice by ZEA treatment, indicating that ZEA affects egg developmental competence. And we explored possible mechanisms of low mouse maturated oocyte developmental competence after ZEA treatment from an epigenetic modification perspective. The fluorescence intensity analysis showed that 5-methyl cytosine level increased after ZEA treatment, indicating that the general DNA methylation level increased in the treated eggs. Moreover, histone methylations were also altered: H3K4me2 as well as H3K9me3 and H4K20me1, me2, me3 levels decreased in eggs that were cultured in high-dose ZEA medium. Thus, our results indicated that ZEA decreased egg developmental competence by affecting the epigenetic modifications.

Description: The comet assay combined with fluorescence in-situ hybridisation (FISH) is a powerful technique for comparative analyses of damage induction and repair in genomes and in specific DNA sequences within single cells. Recent advances in the methodology of comet-FISH will be considered here, with particular attention to the design and generation of fluorescent probes. In general, all the approaches must fulfil a few basic requirements: the probes should be no longer than ~300 nucleotides in length (single or double stranded) to be able to penetrate the gel in which the target genomic DNA is embedded, they should be sequence-specific, and their signal should be detectable and distinct from the background fluorescence and the dye used to stain the DNA.

Description: The contributions to this special issue of Mutagenesis have been selected to cover the main research areas served by the comet assay, namely genotoxicology, environmental toxicology, human biomonitoring and fundamental investigations into mechanisms of DNA damage and repair. Innovative methods are described, technical issues are explored, and guidelines are given for venturing into relatively new or unexploited areas of research. The popularity of the comet assay in a historical context is illustrated by a bibliometric survey.

Description: In plants, an increasing interest for the comet assay was shown in the last decade. This versatile technique appears to be promising to detect the genotoxic effect of pollutants and to monitor the environment. However, the lack of a standardised protocol and the low throughput of the assay limit its use in plants. The aims of this paper are to identify key factors affecting comet assay performance and to improve its reliability and reproducibility. We examined the effect of varying several parameters on four different plant species: broad bean ( Vicia faba ), white clover ( Trifolium repens ), English ryegrass ( Lolium perenne ) and miscanthus ( Miscanthus x giganteus ). The influence of both internal (different nucleus isolation methods, presence or absence of filtration and lysis steps) and external (room temperature, light intensity) parameters were evaluated. Results clearly indicate that short chopping is more efficient to isolate nuclei than the standard slicing method. Filtration and lysis steps were shown to be unnecessary and thus should be skipped. Data also demonstrate that high room temperatures and light could induce DNA damage in isolated nuclei. Calibration tests with H 2 O 2 or ethyl methanesulfonate revealed that a special attention should be paid to plant growing stage, leaf position and exposure duration.

Description: Endogenous and oxidatively induced DNA damage, as evaluated by the comet assay, are widely used as biomarkers of oxidative stress in numerous dietary intervention studies. This analysis can be performed on fresh peripheral blood mononuclear cells (PBMCs) or on cryopreserved cells. However, information pertaining to the effects of cryopreservation on DNA damage is often missing, and this may be crucial in studies in which samples are analysed before and after intervention. The purpose of this study was to compare DNA damage in fresh versus cryopreserved PBMCs obtained from subjects following a 6-week intervention with wild blueberry drink or placebo drink. Fresh and 12-month-stored PBMCs were analysed for formamidopyrimidine-DNA glycosylase (FPG)-sensitive sites and H 2 O 2 -induced DNA damage. The levels of FPG-sensitive sites were significantly higher in the cryopreserved compared with the fresh cells ( P 〈 0.001), while H 2 O 2 -induced DNA damage was significantly lower after storage ( P 〈 0.001). Both the fresh and cryopreserved samples showed reductions in FPG-sensitive sites following the wild blueberry treatment (fresh PBMCs: from 12.50±5.61% to 9.62±3.52%, P = 0.039; cryopreserved PBMCs: from 22.7±6.1% to 19.1±7.0%, P = 0.012). In contrast, the decrease in H 2 O 2 -induced DNA damage observed in the cryopreserved cells masked the protective effect of the wild blueberry drink documented in the fresh samples (fresh PBMCs: from 44.73±7.46% to 36.34±9.27%, P 〈 0.001; cryopreserved PBMCs: from 25.8±4.6% to 23.9±4.6%, P = 0.414). In conclusion, our results suggest that FPG-sensitive sites, and more importantly, H 2 O 2 -induced DNA damage could be significantly modified following the long-term storage of samples obtained from individuals participating in a dietary intervention study. Because storage may affect the assessment of the protective role of diet against DNA damage as a marker of oxidative stress, further research is needed.

Description: Green tea has many reported health benefits, including genoprotective and antioxidant effects, but green tea has pro-oxidant activity in vitro . A tea-induced pro-oxidant shift that triggers cytoprotective adaptations has been postulated, but human data are lacking. We investigated effects on oxidation-induced DNA damage and redox-linked cytoprotective factors, including 8-oxoguanine glycosylase (hOGG1) and heme oxygenase 1 (HMOX-1) in lymphocytes in a randomised, placebo-controlled, cross-over supplementation trial. hOGG1 catalyses the first step in base excision repair; increased HMOX-1 is a sign of cytoprotective response to pro-oxidant change. The influence of microsatellite polymorphisms in the HMOX-1 promoter region was also explored. Higher numbers of GT repeats [GT( n )] in this region reportedly diminish response to pro-oxidant change. Green tea [2 x 150ml of 1% w/v tea/day (or water as control)] was taken for 12 weeks by 43 Type 2 diabetes subjects {20 with short [S/S; GT( n ) 〈 25] and 23 with long [L/L; GT( n ) ≥ 25]}. Fasting venous blood was collected before and after each treatment. The formamidopyrimidine DNA glycosylase-assisted comet assay was used to measure DNA damage in lymphocytes. For measuring hOGG1 activity, we used photo-damaged HeLa cells incubated with lymphocyte extracts from test subjects, in combination with the comet assay. Lymphocyte HMOX-1 and hOGG1 protein concentrations and expression (mRNA) of redox-sensitive genes, including HMOX-1 and hOGG1 , were also investigated. Results showed significantly ( P 〈 0.01) lower (~15%) DNA damage, higher (~50%) hOGG1 activity and higher (~40%) HMOX-1 protein concentration after tea. No changes in mRNA expression were seen. Baseline HMOX-1 protein and hOGG1 activity were higher ( P 〈 0.05) in the S/S group, but tea-associated responses were similar in both GT( n ) groups. Green tea is clearly associated with lowered DNA damage, increased hOGG1 activity and higher HMOX-1 protein levels. Further study is needed to confirm a cause and effect relationship and to establish if these effects are mediated by post-translational changes in proteins or by increased gene expression.

Description: Micronucleation of chromosomal DNA is an effective indicator of DNA damage and micronucleus (MN) analysis is a valuable tool for radiation biodosimetry studies. To gain a comprehensive knowledge of micronucleation process after ionising radiation (IR) exposure, whole genome-wide chromosome analysis is desirable. With this objective, multicolour fluorescence in situ hybridization (M-FISH) technique was utilised in the present study to characterise the chromosome content of spontaneous and IR-induced micronuclei in three human donors. M-FISH analysis revealed a radiation dose-dependant increase in the number of micronuclei with multi-chromosome material above 2 Gy and as many as 3–6 multicolour signals were detected in micronuclei after high -rays radiation doses (5–10 Gy). Involvement of each human chromosome material was more frequently detected in multicoloured micronuclei than in single-coloured micronuclei at high radiation doses (〉2 Gy). Observation of dose-dependant increase in the MN frequency with multi-chromosome material may be due to misrepair of DNA double-strand breaks involving multiple chromosomes leading to asymmetric dicentric or ring chromosomes and acentric fragments. Chromosomes belonging to groups A (1, 2 and 3) and B (4 and 5) were frequently detected in 35–45% of the total micronuclei either as single entities or in combination with other chromosomes. Among the A and B groups, chromosome 1 material was consistently detected at high MN frequencies after radiation exposure in all the donors. Additionally, chromosomes 13 and 19 were more frequently observed in micronuclei than the expected frequency based on DNA content. Our whole genome approach utilising the M-FISH technique revealed that MN formation at high radiation doses might be complex involving multiple chromosome fragments. Understanding the fate and biological consequences of these multi-chromosome-containing micronuclei may provide key molecular insights for some aspects of IR-induced genomic instability and cancer development processes.

Description: Hypoxia promotes genetic instability and is therefore an important factor in carcinogenesis. We have previously shown that activation of the hypoxia responsive transcription factor HIFα can enhance the mutagenic phenotype induced by the environmental mutagen benzo[a]pyrene (BaP). To further elucidate the mechanism behind the ability of hypoxia to increase mutagenicity of carcinogens, we examined the activation and detoxification of BaP under hypoxic conditions. To this end, the human lung carcinoma cell line A549 was treated with BaP under 20%, 5% or 0.2% oxygen for 18h and alterations in BaP metabolism were assayed. First, BaP-induced expression of key metabolic enzymes was analysed; expression levels of the activating CYP1A1 and CYP1B1 were increased, while the detoxifying enzymes UGT1A6 and UGT2B7 were significantly reduced by hypoxia. To evaluate whether these changes had an effect on metabolism, levels of BaP and several of its metabolites were determined. Cells under hypoxia have a reduced capacity to metabolise BaP leaving more of the parent molecule intact. Additionally, BaP-7,8-dihydrodiol, the pre-cursor metabolite of the reactive metabolite BaP-7,8-dihydroxy-9,10-epoxide (BPDE), was formed in higher concentrations. Finally, under hypoxia, DNA adducts accumulated over a period of 168h, whereas adducts were efficiently removed in 20% oxygen conditions. The delayed detoxification kinetics resulted in a 1.5-fold increase in DNA adducts. These data indicate that the metabolism under hypoxic conditions has shifted towards increased activation of BaP instead of detoxification and support the idea that modulation of carcinogen metabolism is an important additional mechanism for the observed HIF1 mediated genetic instability.

Description: Derived polycyclic aromatic hydrocarbons (PAHs) such as nitro-PAHs are present in the environment and are known to be much more toxic than PAHs compounds. However, very few studies have analysed their effects on the aquatic environment and none have investigated the freshwater environment. In the present study, we determined whether 1-nitropyrene (1-NP), a model of nitro-PAHs, can induce DNA adducts in gills and digestive glands of the freshwater mussel Dreissena polymorpha . Two concentrations of 1-NP (50 and 500 μM) were tested. In addition, in order to understand the metabolic pathways involved in 1-NP genotoxicity, mRNA expression of genes implicated in biotransformation mechanisms was assessed by quantitative reverse transcription–PCR. Results showed the presence of DNA adducts in both gills and digestive glands, with highest levels obtained after 5 days of exposure to 500 μM. Metallothionein mRNA levels were enhanced in digestive glands exposed to 50 μM. Surprisingly, at the higher concentration (500 μM), aryl hydrocarbon receptor and HSP70 genes were only up-regulated in digestive glands while PgP mRNA levels were increased in both tissues. Results suggested a cytotoxic and genotoxic effect of 1-NP. Mussels seemed to be able to partially detoxify this compound, in view of the low amount of DNA adducts observed after 5 days exposure to 50 μM. For the first time, 1-NP biotransformation and detoxification systems have been characterised in D. polymorpha .

Description: The use of biomarkers of early genetic effects, predictive for cancer, such as micronuclei (MN) in lymphocytes, may help to investigate the association between diet and cancer. We hypothesised that the presence of mutagens in the diet may increase MN formation. A ‘pooled’ standardised analysis was performed by applying the same experimental protocol for the cytokinesis block micronucleus assay in 625 young healthy women after delivery from five European study populations (Greece, Denmark, UK, Spain and Norway). We assessed MN frequencies in mono- and binucleated T-lymphocytes (MNMONO and MNBN) and the cytokinesis blocked proliferation index using a semi-automated image analysis system. Food frequency questionnaires (FFQs) were used to estimate intake of fatty acids and a broad range of immunotoxic and genotoxic/carcinogenic compounds through the diet. Pooled difference based on delivery type revealed higher MNMONO frequencies in caesarean than in vaginal delivery ( P = 0.002). Statistical analysis showed a decrease in MNMONO frequencies with increasing calculated omega-6 PUFA concentrations and a decrease in MNBN frequencies with increasing calculated omega-3 PUFA concentrations. The expected toxic compounds estimated by FFQs were not associated with MN formation in mothers after delivery. In pregnant women, an omega-3 and -6 rich diet estimated by FFQ is associated with lower MN formation during pregnancy and delivery.

Description: Our previous studies showed that the expressions of miR-148a, miR-152 and miR-148b are altered in gastric cancer (GC). The present study aimed to find relationship between individual single nucleotide polymorphisms (SNPs) or haplotypes of these miRNAs and susceptibility, clinicopathological parameters and prognosis of GC in a large sample of the Han population of Northern China. Twelve SNPs were genotyped using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry in a case–control study of 571 Chinese GC patients and 571 cancer-free controls. The rs11170877 G allele ( P = 0.027) and the rs12231393 C allele ( P = 0.034) were associated with a decreased risk of GC. However, these associations were lost after Bonferroni correction. The rs4719839 G allele was associated with Borrmann type III–IV GC ( P = 0.034), increased tumour size ( P = 0.020), an increased rate of lymph node metastasis ( P = 0.047) and advanced TNM stage ( P = 0.009). These associations were also lost after Bonferroni correction. The haplotype of miR-148b was significantly correlated with GC risk. The haplotypes in miRNA-148a were different in Borrmann types. The haplotype of miR-152 distributed various in the positive lymphovascular invasion group compared to negative group. Polymorphisms of miR-148b rs11170877 and 12231393 and their haplotypes were predictive factors of susceptibility to GC. A functional genetic variant of miRNA rs4719839 and the corresponding haplotype were associated with clinicopathological features and prognosis of advanced GCs.

Description: The polycyclic aromatic hydrocarbon (PAH) benzo(a)pyrene (BP) is thought to bind covalently to DNA, through metabolism by cytochrome P450 1A1 (CYP1A1) and CYP1B1, and other enzymes, to form r7, t8, t9-trihydroxy-c-10-( N 2 -deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[ a ]-pyrene (BPdG). Evaluation of RNA expression data, to understand the contribution of different metabolic enzymes to BPdG formation, is typically presented as fold-change observed upon BP exposure, leaving the actual number of RNA transcripts unknown. Here, we have quantified RNA copies/ng cDNA (RNA cpn) for CYP1A1 and CYP1B1 , as well as NAD(P)H:quinone oxidoreductase 1 ( NQO1 ), which may reduce formation of BPdG adducts, using primary normal human mammary epithelial cell (NHMEC) strains, and the MCF-7 breast cancer cell line. In unexposed NHMECs, basal RNA cpn values were 58–836 for CYP1A1 , 336–5587 for CYP1B1 and 5943–40112 for NQO1. In cells exposed to 4.0 µM BP for 12h, RNA cpn values were 251–13234 for CYP1A1 , 4133–57078 for CYP1B1 and 4456–55887 for NQO1. There were 3.5 (mean, range 0.2–15.8) BPdG adducts/10 8 nucleotides in the NHMECs ( n = 16), and 790 in the MCF-7s. In the NHMECs, BP-induced CYP1A1 RNA cpn was highly associated with BPdG ( P = 0.002), but CYP1B1 and NQO1 were not. Western blots of four NHMEC strains, chosen for different levels of BPdG adducts, showed a linear correlation between BPdG and CYP1A1, but not CYP1B1 or NQO1. Ethoxyresorufin-O-deethylase (EROD) activity, which measures CYP1A1 and CYP1B1 together, correlated with BPdG, but NQO1 activity did not. Despite more numerous levels of CYP1B1 and NQO1 RNA cpn in unexposed and BP-exposed NHMECs and MCF-7cells, BPdG formation was only correlated with induction of CYP1A1 RNA cpn. The higher level of BPdG in MCF-7 cells, compared to NHMECs, may have been due to a much increased induction of CYP1A1 and EROD. Overall, BPdG correlation was observed with CYP1A1 protein and CYP1A1/1B1 enzyme activity, but not with CYP1B1 or NQO1 protein, or NQO1 enzyme activity.

Description: Angiogenesis is fundamentally important to the pathogenesis of clear cell renal cell carcinoma (ccRCC). We investigated the association between variations of genes related to angiogenesis and the risk of ccRCC. In a case–control study of 859 ccRCC patients and 1004 cancer-free subjects, we genotyped 24 potentially functional single nucleotide polymorphisms (SNPs) in seven angiogenesis-related genes ( HIF1A , EPAS1 , VEGFA , VEGFR1 , VEGFR2 , VEGFR3 and PDGFRB ) using the TaqMan or Snapshot method. Unconditional logistic regression, adjusted for potential confounding factors, was used to assess the risk associations. The functionality of selected SNPs was assessed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and luciferase reporter gene assays. We found two SNPs ( VEGFA rs2010963 and VEGFR3 rs448012) that were significantly associated with increased risk of ccRCC, after adjusting for multiple comparisons [rs2010963 CC/GC cf. GG: false discovery rate (FDR) = 0.048, odds ratio (OR) = 1.36, 95% confidence interval (95% CI) = 1.12–1.66; rs448012 CC/GC cf. GG: FDR = 0.048, OR = 1.38, 95% CI =1.13–1.69]. Real-time quantitative PCR revealed that the variant genotypes of rs2010963, but not rs448012, were associated with increased gene expression in normal tissues of ccRCC patients (CC/GC cf. GG: P = 0.036). The luciferase reporter assay showed that the rs2010963 C allele significantly increased luciferase activity over that of the rs2010963 G allele. Our results indicate that VEGFA rs2010963 and VEGFR3 rs448012 are associated with risk of ccRCC. Furthermore, rs2010963 is a functional SNP that may affect ccRCC susceptibility by modulating endogenous VEGFA expression.