ALBERT

Description: The ICH S6(R1) recommendations on safety evaluation of biotherapeutics have led to uncertainty in determining what would constitute a cause for concern that would require genotoxicity testing. A Health and Environmental Sciences Institute’s Genetic Toxicology Technical Committee Workgroup was formed to review the current practice of genotoxicity assessment of peptide/protein-related biotherapeutics. There are a number of properties of peptide/protein-related biotherapeutics that distinguish such products from traditional ‘small molecule’ drugs and need to be taken into consideration when assessing whether genotoxicity testing may be warranted and if so, how to do it appropriately. Case examples were provided by participating companies and decision trees were elaborated to determine whether and when genotoxicity evaluation is needed for peptides containing natural amino acids, non-natural amino acids and other chemical entities and for unconjugated and conjugated proteins. From a scientific point of view, there is no reason for testing peptides containing exclusively natural amino acids irrespective of the manufacturing process. If non-natural amino acids, organic linkers and other non-linker chemical components have already been tested for genotoxicity, there is no need to re-evaluate them when used in different peptide/protein-related biotherapeutics. Unless the peptides have been modified to be able to enter the cells, it is generally more appropriate to evaluate the peptides containing the non-natural amino acids and other non-linker chemical moieties in vivo where the cleavage products can be formed. For linkers, it is important to determine if exposure to reactive forms are likely to occur and from which origin. When the linkers are anticipated to be potential mutagenic impurities they should be evaluated according to ICH M7. If linkers are expected to be catabolic products, it is recommended to test the entire conjugate in vivo , as this would ensure that the relevant ‘free’ linker forms stemming from in vivo catabolism are tested.

Description: Genome sequences that contain tandem repeats of guanine can form stable four-stranded structures known as G-quadruplex, or G4 DNA. While the molecular mechanisms are not fully defined, such guanine-rich loci are prone to mutagenesis and recombination. Various repair pathways function to reduce the potential for genome instability by correcting base damage and replication errors; however, it is not yet fully defined how well these processes function at G4 DNA. One frequent form of base damage occurs from cytidine deamination, resulting in deoxyuracil and UG mismatches. In duplex and single-stranded DNA, uracil bases are recognised and excised by uracil glycosylases. Here, we tested the efficiency of uracil glycosylase activity in vitro on uracil bases located directly adjacent to guanine repeats and G4 DNA. We show that uracil excision by bacterial UDG and human hUNG2 is reduced at uracils positioned directly 5' or 3' of a guanine tetrad. Control reactions using oligonucleotides disrupted for G4 formation or reaction conditions that do not favour G4 formation resulted in full uracil excision activity. Based on these in vitro results, we suggest that folding of guanine-rich DNA into G4 DNA results in a DNA conformation that is resistant to uracil glycosylase-initiated repair and this has the potential to increase the risk of instability at guanine repeats in the genome.

Description: Galectin-4 is a member of the galectin family which consists of 15 galactoside-binding proteins. Previously, galectin-4 has been shown to have a role in cancer progression and metastasis and it is found upregulated in many solid tumours, including colorectal cancer (CRC). Recently, the role in the metastatic process was suggested to be via promoting cancer cells to adhere to blood vascular endothelium. In the present study, the regulatory region of LGALS4 (galectin-4) in seven colon cell lines was investigated with respect to genetic variation that could be linked to expression levels and therefore a tumourigenic effect. Interestingly, qRT-PCR and sequencing results revealed that galectin-4 upregulation is associated with SNPs rs116896264 and rs73933062. By use of luciferase reporter- and pull-down assays, we confirmed the association between the gene upregulation and the two SNPs. Also, using pull-down assay followed by mass spectrometry, we found that the presence rs116896264 and rs73933062 is changing transcription factors binding sites. In order to assess the frequencies of the two SNPs among colon cancer patients and healthy individuals, we genotyped 75 colon cancer patients, 12 patients with adenomatous polyposis and 17 patients with ulcerative colitis and we performed data mining in the 1000 genomes databank. We found the two SNPs co-occuring in 21% of 75 CRC patients, 0 out of 12 patients of adenomatous polyposis, and 6 out of 17 patients (35%) with ulcerative colitis. Both in the patient samples and in the 1000 genomes project, the two SNPs were found to co-occur whenever present (D' = 1).

Description: The dose effect between nucleoplasmic bridges (NPB) and relatively low doses of ionising radiation remains unknown. Accordingly, this study investigated the NPB frequencies in human peripheral blood lymphocytes exposed to low-dose 60 Co -rays. Complex anomalies, including fused nuclei (FUS), horse-shoe nuclei (HS) and circular nuclei (CIR), which possibly originated from multiple NPBs, were also scored. Human peripheral blood samples were collected from three healthy males and irradiated with 0–1 and 0–0.4 Gy 60 Co -rays. A cytokinesis-block micronucleus cytome assay was then conducted to analyse NPB, PFHC (NPB plus three complex nuclear anomalies) and micronucleus (MN) in binucleated cells. All dose–response curves followed the linear model for both NPB frequency and PFHC cell frequency. The dose–response curves between NPB frequency and absorbed dose at 0–1 and 0–0.4 Gy were y = 0.0037 x + 0.0005 ( R 2 = 0.979, P 〈 0.05) and y = 0.0043 x + 0.0004 ( R 2 = 0.941, P 〈 0.05), respectively. The dose–response curves between PFHC cell frequency and absorbed dose at 0–1 and 0–0.4 Gy were y = 0.0044 x + 0.0007 ( R 2 = 0.982, P 〈 0.05) and y = 0.0059 x + 0.0005 ( R 2 = 0.969, P 〈 0.05), respectively. The statistical significance of differences between the irradiated groups (0–0.4 Gy) and background levels of NPB, PFHC and MN were also analysed. The lowest analysable doses of NPB, PFHC and MN were 0.12, 0.08 and 0.08 Gy, respectively. In conclusion, NPBs and PFHC positively correlated with the absorbed radiation at a relatively low dose.

Description: The aim of the study was to investigate how coadministration of resveratrol (RSV) at different time after the start of irradiation influences the frequency of micronuclei (MN) in reticulocytes of bone marrow and peripheral blood, and if the RSV supplementation after termination of irradiation may influence the recovery process of damaged cells. Coadministration of RSV with 1-day delay after 1 Gy irradiation significantly decreased the levels of MN in bone marrow and in peripheral blood, whereas with 1-week delay, only in bone marrow reticulocytes. Above combined treatment did not improve the process of recovery. RSV supplementation with 1-day delay relatively to 0.5 Gy irradiation, significantly decreased the frequencies of MN, especially after coadministration with 28mg/kg bw of RSV. Coadministration of RSV since eighth day did not influence the frequencies of MN compared to irradiated cells. The recovery process in the presence of RSV proceeded faster. Supplementation of RSV following initiation of irradiation is beneficial in case of irradiation with lower doses. RSV should be supplemented as soon as possible. Supplementation of RSV after termination of irradiation significantly speed up the recovery. Current results confirmed the ability of RSV to mitigate the effect of irradiation.

Description: Various naturally occurring stilbene-like compounds that are related to resveratrol (RSV) possess some of the beneficial effects of the parent molecule and provide even further benefits. Therefore, a series of methoxylated analogues of RSV were prepared with the aim of increasing antitumour and proapoptotic activity. In a previous article, we studied two methoxy-derivatives, pterostilbene (PTERO) and trimethoxystilbene (TRIMETHOXY), in which the first was formed by the substitution of two hydroxyl groups with two methoxy groups ( trans -3,5-dimethoxy-4'-hydroxystilbene) and the second was formed by the replacement of all three OH groups with methoxy groups ( trans -3,5,4'-trimethoxystilbene). Both methoxy-derivatives showed stronger antioxidant activity when compared with RSV. In the present article, we focused on the analysis of the ability of RSV and its two methoxylated derivatives to protect proliferating non-tumoural cells from the damage induced by ionising radiation (IR). First we showed that the methoxy derivatives, contrary to their parental compound, are unable to affect topoisomerase enzyme and consequently are not clastogenic per se . Second we showed that both PTERO and TRIMETHOXY more efficiently reduce the chromosome damage induced by IR. Furthermore, TRIMETHOXY, but not PTERO, causes a delay in cell proliferation, particularly in mitosis progression increasing the number of cells in metaphase at the expense of prophases and ana/telophases.

Description: α-, β- and -asarone are naturally occurring phenylpropenes that occur in different plant families, mainly in Aristolochiaceae , Acoraceae and Lauraceae. Plants containing asarones are used as flavouring ingredients in alcoholic beverages (bitters), traditional phytomedicines and the rhizome of e.g. Acorus calamus is used to prepare tea. Although α- and β-asarone show a potential in the treatment of several diseases, previous studies have shown carcinogenicity in rodents (duodenum, liver). However, the mechanism of action remained unclear. Studies on the mutagenicity of propenylic α- and β-asarone are inconsistent and data on carcinogenicity and genotoxicity of allylic -asarone are lacking completely. Thus, the present study determined the mutagenicity of the three asarone isomers using the Ames fluctuation assay with and without exogenous metabolic activation (S9 mix) in the standard Salmonella typhimurium strains TA98 and TA100. A concentration dependent increase in mutagenicity could be verified for α- and β-asarone in strain TA100 in the presence of rat liver homogenate. The side-chain epoxides of α- and β-asarone, major metabolites formed in liver microsomes, caused mutations in TA100, supporting the hypothesis that epoxidation of the side chain plays a key role in mutagenicity of the propenylic alkenylbenzenes. The allylic -asarone, not undergoing detectable side-chain epoxidation in liver microsomes, was supposed to be activated via side-chain hydroxylation and further sulphonation, a typical pathway for other allylic alkenylbenzenes like estragole or methyleugenol. However, neither y-asarone nor 1'-OH--asarone showed any mutagenic effect even in the human SULT-expressing Salmonella strains (TA100-hSULT1A1 and TA100-hSULT1C2), while 1'-OH-methyleugenol used as a positive control was mutagenic under these conditions. These results indicate that the propenylic asarones are genotoxic via metabolic formation of side-chain epoxides while the side-chain hydroxylation/sulphonation pathway is either not operative or does not lead to mutagenicity with the allylic -asarone.

Description: Prior to the downstream development of chemical substances, including pharmaceuticals and cosmetics, their influence on the genetic apparatus has to be tested. Several in vitro and in vivo assays have been developed to test for genotoxicity. In a first tier, a battery of two to three in vitro tests is recommended to cover mutagenicity, clastogenicity and aneugenicity as main endpoints. This regulatory in vitro test battery is known to have a high sensitivity, which is at the expense of the specificity. The high number of false positive in vitro results leads to excessive in vivo follow-up studies. In the case of cosmetics it may even induce the ban of the particular compound since in Europe the use of experimental animals is no longer allowed for cosmetics. In this article, an alternative approach to derisk a misleading positive Ames test is explored. Hereto we first tested the performance of five existing computational tools to predict the potential mutagenicity of a data set of 132 cosmetic compounds with a known genotoxicity profile. Furthermore, we present, as a proof-of-principle, a strategy in which a combination of computational tools and mechanistic information derived from in vitro transcriptomics analyses is used to derisk a misleading positive Ames test result. Our data shows that this strategy may represent a valuable tool in a weight-of-evidence approach to further evaluate a positive outcome in an Ames test.

Description: Pioglitazone (PTZ) is an oral antidiabetic agent whose anti-cancer properties have been described recently. Since PTZ increases the production of reactive oxygen species in mammalian cells, the aim of current study was to evaluate the cytotoxic, mutagenic and recombinogenic effects of PTZ using respectively the in vitro mitotic index assay and the in vitro mammalian cell micronucleus test in human peripheral lymphocytes, and the in vivo homozygotization assay in Aspergillus nidulans , which detects the loss of heterozygosity due to somatic recombination. Although the lowest PTZ concentrations (4–36 μM) did not show any significant rise in the micronucleus production, the higher PTZ concentration (108 μM) produced a statistically higher number of micronuclei than the negative control and significantly altered the cell-proliferation kinetics, demonstrating the mutagenic and antiproliferative effects of PTZ, respectively. The recombinogenic activity of PTZ, demonstrated here for the first time, was observed at the highest tested concentration (400 μM) through the homozygotization index rates significantly different from the negative control. Taken together, our results show that PTZ is genotoxic at a concentration higher than the therapeutic plasma concentration. This PTZ genotoxicity may be a potential benefit to its previously described antitumour activity.

Description: Environmental pollutants are complex mixtures in which metals are ubiquitous. Metal mixtures of arsenic, cadmium and lead are present in the occupational environment and generate health effects such as cardiovascular, renal and cancer diseases. Cell transformation induced by metal mixtures that depend on reactive oxygen species (ROS) generation, cell viability maintenance and avoidance of senescence was previously reported by our group. The aim of the present study was to explore the role of a Obg-like ATPase1 (OLA1) in the cell transformation of BALB/c 3T3 A31-1-1 clonal cells induced by a metal mixture (2 µM NaAsO 2 , 2 µM CdCl 2 and 5 µM Pb(C 2 H 3 O 2 ) 2 . 3H 2 O) through ROS generation. The interest in OLA1 is justified because this protein has been proposed to be a negative regulator of the cellular antioxidant response. Small interfering RNA (siRNA) was used to knockdown OLA1 before the initiation stage of the transformation assay. We evaluated (ROS) and OLA1 protein expression throughout the initiation and promotion stages of transformation. OLA1 knockdown modulated metal mixture-induced cell transformation more strongly when the metal mixture was an initiator stimulus than when it was a promoter. The ability of the metal mixture to initiate cell transformation was diminished by OLA1 knockdown, an effect that depended on intracellular ROS levels. The effect of OLA1 was synergistic with N -Acetyl- l -cysteine (NAC) co-treatment. Oxidative stress-associated transcription factors Egr1 and Smad were also down-regulated by the OLA1 knockdown, contributing to the rescue of metal mixture cell transformation.

Description: G-quadruplexes (G4) are highly stable tetra-stranded DNA secondary structures known to mediate gene regulation and to trigger genomic instability events during replication. G4 structural stability can be affected by DNA methylation and oxidation modifications; thus nutrients such as folate that have the ability to alter these processes could potentially modify the genomic occurrence of G4 elements. Hela cells were cultured in a range of folate concentrations or in the presence or absence of 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor. G4 structures were then quantified by immunofluorescence using an automated quantitative imaging system. G4 frequency in Hela cells and nuclei area mean were increased in 20nM folate medium compared with 2000nM folate, as well as in the presence of 5-aza-2'-deoxycytidine when compared to cells non-exposed to 5-aza-2'-deoxycytidine. These changes were exacerbated when pyridostatin, a G4 stabilising ligand, was added to the culture medium. G4 intensity in Hela cells cultured in deficient folate condition with pyridostatin was highly correlated with DNA damage as measured by H2AX immunofluorescence ( r = 0.71). This study showed for the first time that cellular G4 balance is modifiable by low folate concentrations and that these changes may occur as a consequence of DNA hypomethylation. Although the exact mechanism by which these changes occur is unclear, these findings establish the possibility that nutrients could be utilised as a tool for sustaining genome integrity by modifying G4 frequency at a cellular level.

Description: Metal oxide nanoparticles (NPs), including zinc oxide (ZnO) NPs have shown success for use as vehicles for drug delivery and targeting gene delivery in many diseases like cancer. Current anticancer chemotherapeutics fail to effectively differentiate between cancerous and normal cells. There is an urgent need to develop novel drug delivery system that can better target cancer cells while sparing normal cells and tissues. Particularly, ZnO NPs exhibit a high degree of cancer cell selectivity and induce cell death, oxidative stress, interference with the cell cycle progression and genotoxicity in cancerous cells. In this scenario, effective cellular uptake of NP seems to be crucial, which is shown to be affected by cell cycle progression. In the present study, the cytotoxic potential of ZnO NPs and the effect of different cell cycle phases on the uptake of ZnO NPs were examined in A431 cells. It is shown that the ZnO NPs led to cell death and reactive oxygen species generation and were able to induce cell cycle arrest in S and G 2 /M phase with the higher uptake in G 2 /M phase compared with other phases.

Description: Malondialdehyde (MDA), a biomarker of lipid peroxidation and oxidative stress, is a mutagenic and carcinogenic compound that can react with DNA to form several types of DNA adducts including the deoxyguanosine adduct (M 1 dG). The aim of this cross-sectional study was to evaluate the association between individual dietary and lifestyle habits and M 1 dG levels, measured in peripheral leukocytes in a large representative sample of the general population of Florence City (Italy). Selected anthropometric measurements, detailed information on dietary and lifestyle habits and blood samples were available for 313 adults of the Florence City Sample enrolled in the frame of European Prospective Investigation into Cancer and nutrition (EPIC) study. A multivariate regression analysis adjusted for selected individual characteristics possibly related to M 1 dG levels (sex, age, BMI, smoke, physical activity level, education level, total caloric intake and a Mediterranean dietary score) was performed to estimate the association between these parameters and M 1 dG levels. M 1 dG levels were significantly higher in women ( P = 0.014) and lower in moderately active or active subjects ( P = 0.037).We also found a significant inverse association with the Modified Mediterranean dietary score ( P for trend = 0.049), particularly evident for the highest categories of adherence. Our results indicate that M 1 dG levels can be modulated by selected individual characteristics such as gender, physical activity and a Mediterranean dietary pattern.

Description: Arsenic-induced health effects may be associated with critically shortened telomeres. However, few data are available on the effects of arsenic exposure on telomere length. The aim of this study was to investigate the effects of chronic arsenic exposure on leukocyte telomere length (LTL) as well as the contribution of common polymorphisms in genes implicated in arsenic metabolism (GSTT1 and GSTM1) and DNA repair (hOGG1 and XRCC1). A group of 241 healthy subjects was enrolled from four areas of Italy known to be affected by natural or anthropogenic arsenic pollution. Urine samples were tested for inorganic As (iAs), monomethylarsinic (MMA) and dimethylarsinic acid (DMA). LTL was evaluated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Genotyping was carried out by PCR-RFLP on leukocyte DNA. In multiple linear regression analysis, LTL was significantly and inversely correlated with age (β = –0.231, P = 0.006) and showed a certain trend toward significance with iAs urinary concentration (log 10 iAs, β = –0.106, P = 0.08). The genotype distribution showed significant associations between GSTT1 and the As concentration (log 10 iAs, P = 0.01) and metabolite patterns (log 10 DMA, P = 0.05) in the urine. However, GST genes did not interact with arsenic exposure in the modulation of LTL. Conversely, the combined presence of a higher level of iAs + MMA + DMA ≥ 19.3 μg/l ( F = 6.0, P interaction = 0.01), Asi ≥ 3.86 ( F = 3.9, P interaction = 0.04) μg/l, iAs + MMA + DMA ≥ 15 μg/l ( F = 4.2, P interaction = 0.04) and hOGG1 Cys allele was associated with a significantly lower LTL. An interaction between XRCC1 Arg399Gln and arsenic exposure was also observed (all P interaction = 0.04). These findings suggest that telomere shortening may represent a mechanism that contributes to arsenic-related disease. The interaction of hOGG1 and XRCC1 DNA repair polymorphisms and exposure enhances telomeric DNA damage. Future studies are warranted to understand better the epidemiologic impact of arsenic on telomere function as well as to identify the subgroups of exposed subjects who need better health surveillance.

Description: The comet assay is frequently used in human biomonitoring for the detection of exposure to genotoxic agents. Peripheral blood samples are most frequently used and tested either as whole blood or after isolation of lymphocytes (i.e. peripheral blood mononuclear cells, PBMC). To investigate a potential impact of lymphocyte isolation on induced DNA damage in human blood samples, we exposed blood ex vivo to mutagens with different modes of genotoxic action. The comet assay was performed either directly with whole blood at the end of the exposure period or with lymphocytes isolated directly after exposure. In addition to the recommended standard protocol for lymphocyte isolation, a shortened protocol was established to optimise the isolation procedure. The results indicate that the effects of induced DNA strand breaks and alkali-labile sites induced by ionising radiation and alkylants, respectively, are significantly reduced in isolated lymphocytes. In contrast, oxidative DNA base damage (induced by potassium bromate) and stable bulky adducts (induced by benzo[ a ]pyrene-7,8-dihydrodiol-9,10-epoxide; BPDE) seem to be less affected. Our findings suggest that in vivo -induced DNA damage might also be reduced in isolated lymphocytes in comparison with the whole blood depending of the types of DNA damage induced. Because only small genotoxic effects can generally be expected in human biomonitoring studies with the comet assay after occupational and environmental exposure to genotoxic agents, any loss might be relevant and should be avoided. The possibility of such effects and their potential impact on variability of comet assay results in human biomonitoring should be considered when performing or evaluating such kind of studies.

Description: Numerical and structural chromosomal abnormalities are the hallmarks of cancer. Whereas the structural chromosome aberrations got more substantial attention for cancer risk assessment in a healthy population, the role of aneuploidy is much less understood in this respect. We analysed the frequency of numerical (and structural) aberrations in peripheral blood lymphocytes of 2145 healthy individuals between 1989 and 2010, taking into account different biological- and exposure-conditions. We also studied to what extent chromosome gains or losses may predict the probability of cancer. The average frequency of all aneuploid cells was 1.78±0.06% in the entire study population, which increased linearly with age. Gender and smoking did not influence the values, however, occupational exposures did. The highest frequency of aneuploidy was found in chemical industry-workers (1.89±0.05%) compared with the lowest value of medical radiation workers (1.44±0.10%), respectively. No correlation was found between numerical and structural chromosomal aberrations. Cancer incidence followed for 1–23 years after the chromosome analysis showed a 1.26-fold relative risk (confidence interval: 1.02–1.58; P = 0.04) for those with higher frequency of aneuploid cells (1.82% vs. 1.44% in controls). Hypodiploidy had higher impact on the cancer risk than hyperdiploidy (1.72% vs. 0.10%). Our findings on the frequency of numerical aberrations in a healthy cohort represent the largest cytogenetic database from one laboratory with an unchanged mechanistic scoring method during a 30-year period, and provide basic information not only for genotoxicological studies but also confirm the association between numerical aberrations and cancer risk.

Description: 2,5-Dimethylfuran (DMF) and furfuryl alcohol (FFA) are two substituted furans that are formed during the processing of foods and have also been used as food flavorings. DMF and FFA are proposed to be bioactivated by human sulfotransferases (SULTs) which are not expressed in conventional cell lines used for genotoxicity testing. Therefore, in addition to the standard V79 cell line, we used a transfected V79 derived cell line co-expressing human cytochrome P450 (CYP) 2E1 and human SULT1A1 to assess the genotoxicity of DMF and FFA. The alkaline single cell gel electrophoresis (SCGE) assay was used to detect DNA damage in the form of single strand breaks and alkali-labile sites after exposure to DMF (0.5h; 0.5, 1, 1.5 or 2mM) or FFA (3h; 1, 3, 6 or 15mM). DMF induced DNA damage in V79 cells in a concentration-dependent manner irrespective of the expression of human CYP2E1 and SULT1A1. Almost no increase in the level of DNA damage was detected after exposure to FFA, except for a weak effect at the highest concentration in the transfected cell line. The results suggest that DNA damage in V79 cells from exposure to DMF detected by the alkaline SCGE assay is independent of human CYP2E1 and SULT1A1, and the genotoxic effect of FFA, as assessed by SCGE, is minimal in V79 cells.

Description: Alcohol is a human carcinogen. A causal link has been established between alcohol drinking and cancers of the upper aerodigestive tract, colon, liver and breast. Despite this established association, the underlying mechanisms of alcohol-induced carcinogenesis remain unclear. Various mechanisms may come into play depending on the type of cancer; however, convincing evidence supports the concept that ethanol’s major metabolite acetaldehyde may play a major role. Acetaldehyde can react with DNA forming adducts which can serve as biomarkers of carcinogen exposure and potentially of cancer risk. The major DNA adduct formed from this reaction is N 2 -ethylidenedeoxyguanosine, which can be quantified as its reduced form N 2 -ethyl-dG by LC-ESI-MS/MS. To investigate the potential use of N 2 -ethyl-dG as a biomarker of alcohol-induced DNA damage, we quantified this adduct in DNA from the oral, oesophageal and mammary gland tissues from rhesus monkeys exposed to alcohol drinking over their lifetimes and compared it to controls. N 2 -Ethyl-dG levels were significantly higher in the oral mucosa DNA of the exposed animals. Levels of the DNA adduct measured in the oesophageal mucosa of exposed animals were not significantly different from controls. A correlation between the levels measured in the oral and oesophageal DNA, however, was observed, suggesting a common source of formation of the DNA adducts. N 2 -Ethyl-dG was measured in mammary gland DNA from a small cohort of female animals, but no difference was observed between exposed animals and controls. These results support the hypothesis that acetaldehyde induces DNA damage in the oral mucosa of alcohol-exposed animals and that it may play role in the alcohol-induced carcinogenic process. The decrease of N 2 -ethyl-dG levels in exposed tissues further removed from the mouth also suggests a role of alcohol metabolism in the oral cavity, which may be considered separately from ethanol liver metabolism in the investigation of ethanol-related cancer risk.

Description: Potential ionising radiation exposure scenarios are varied, but all bring risks beyond the simple issues of short-term survival. Whether accidentally exposed to a single, whole-body dose in an act of terrorism or purposefully exposed to fractionated doses as part of a therapeutic regimen, radiation exposure carries the consequence of elevated cancer risk. The long-term impact of both intentional and unintentional exposure could potentially be mitigated by treatments specifically developed to limit the mutations and precancerous replication that ensue in the wake of irradiation The development of such agents would undoubtedly require a substantial degree of in vitro testing, but in order to accurately recapitulate the complex process of radiation-induced carcinogenesis, well-understood animal models are necessary. Inbred strains of the laboratory mouse, Mus musculus , present the most logical choice due to the high number of molecular and physiological similarities they share with humans. Their small size, high rate of breeding and fully sequenced genome further increase its value for use in cancer research. This chapter will review relevant m. musculus inbred and F 1 hybrid animals of radiation-induced myeloid leukemia, thymic lymphoma, breast and lung cancers. Method of cancer induction and associated molecular pathologies will also be described for each model.

Description: Furocoumarin extracts from Psoralea morisiana , the endemic Sardinian legume species, were tested for their mutagenic potential on river buffalo blood cells. The results obtained performing the sister chromatid exchange (SCE) test in blood cultures of five river buffalo calves (exposure to furocoumarins for 72h) and five cows (exposure to furocoumarins for 3h, in the absence and presence of S9 metabolic activator) are reported. Significant differences in mean values of SCEs were observed in cells of calves compared to control cells (unexposed), but no differences in SCE mean values were found between treated and untreated cells of cows in the presence or absence of S9. SCE mean values were much higher in cells of cows (exposed and control) than in cells of calves. Indeed, in calf cells, SCE mean values/cell (±SD) were 6.66±2.45 in the control and 7.63±3.01, 9.03±3.90, 9.53±3.60 and 9.99±3.41 in treated cells at 50, 100, 200 and 400 µg/ml of furocoumarin extracts, respectively. In cow cells, grown in presence of S9, SCE mean values/cell were 11.49±4.78 and 11.65±5.19 in treated cells at 100 and 200 µg/ml of furocoumarins and 11.66±5.45 in the control. In cow cells grown in absence of S9, SCE mean values were 11.81±6.14 in the control and 12.35±7.09 and 12.01±5.43, respectively, in the presence of 100 and 200 µg/ml of furocoumarins. Despite their higher SCE values in the absence of S9, no statistically significant differences were found when these values were compared with those shown in presence of S9, suggesting no mutagenic action of furocoumarins in cows, at the doses used in this study.

Description: The well-defined battery of in vitro systems applied within chemical cancer risk assessment is often characterised by a high false-positive rate, thus repeatedly failing to correctly predict the in vivo genotoxic and carcinogenic properties of test compounds. Toxicogenomics, i.e. mRNA-profiling, has been proven successful in improving the prediction of genotoxicity in vivo and the understanding of underlying mechanisms. Recently, microRNAs have been discovered as post-transcriptional regulators of mRNAs. It is thus hypothesised that using microRNA response-patterns may further improve current prediction methods. This study aimed at predicting genotoxicity and non-genotoxic carcinogenicity in vivo, by comparing microRNA- and mRNA-based profiles, using a frequently applied in vitro liver model and exposing this to a range of well-chosen prototypical carcinogens. Primary mouse hepatocytes (PMH) were treated for 24 and 48h with 21 chemical compounds [genotoxins (GTX) vs. non-genotoxins (NGTX) and non-genotoxic carcinogens (NGTX-C) versus non-carcinogens (NC)]. MicroRNA and mRNA expression changes were analysed by means of Exiqon and Affymetrix microarray-platforms, respectively. Classification was performed by using Prediction Analysis for Microarrays (PAM). Compounds were randomly assigned to training and validation sets (repeated 10 times). Before prediction analysis, pre-selection of microRNAs and mRNAs was performed by using a leave-one-out t -test. No microRNAs could be identified that accurately predicted genotoxicity or non-genotoxic carcinogenicity in vivo. However, mRNAs could be detected which appeared reliable in predicting genotoxicity in vivo after 24h (7 genes) and 48h (2 genes) of exposure (accuracy: 90% and 93%, sensitivity: 65% and 75%, specificity: 100% and 100%). Tributylinoxide and para-Cresidine were misclassified. Also, mRNAs were identified capable of classifying NGTX-C after 24h (5 genes) as well as after 48h (3 genes) of treatment (accuracy: 78% and 88%, sensitivity: 83% and 83%, specificity: 75% and 93%). Wy-14,643, phenobarbital and ampicillin trihydrate were misclassified. We conclude that genotoxicity and non-genotoxic carcinogenicity probably cannot be accurately predicted based on microRNA profiles. Overall, transcript-based prediction analyses appeared to clearly outperform microRNA-based analyses.

Description: Male infertility is a relevant social and medical problem. Male infertility is mostly caused by genetic disorders. The purpose of the study was to analyze the correlation of chromosome aberrations, as well as DNA fragmentation and various manifestations of spermatogenesis disorder. Sperm samples of 58 males with infertility and 23 conditionally healthy males were studied. All patients diagnosed with asthenozoospermia, teratozoospermia, oligoasthenozoospermia and oligoteratozoospermia underwent subsequent analysis of sperm DNA fragmentation. Sperm DNA fragmentation was examined with sperm chromatin dispersion test (sperm chromatin dispersion, Spermprocessor, India) with an Axioscope 40 fluorescent microscope. Fluorescence in situ hybridization with fluorescent probes (Vysis Multi Vysion PGT, Abbot Molecular) was used to study chromosome abnormalities in sperm nuclei with regard to X and Y chromosomes, as well as to chromosomes 18 and 21. It was found that the development of pathospermia was characterized by genetic discontinuity, which manifests as DNA fragmentation and disjunction of chromosomes in meiosis with spermatogenesis. It was also found that the prevailing type of pathospermia in men with infertility was oligozoospermia. In addition, this group also had the highest rate of numerical chromosome abnormalities. This was caused by the degeneration of spermatozoids with aneuploidies in chromosomes.

Description: The long non-coding RNA (lncRNA) H19 as an imprinted gene transcribed from only the maternal allele has the vital role in carcinogenesis. Aberrant H19 expression is involved in bladder cancer development. In this study, we explored the association between single nucleotide polymorphisms (SNPs) in H19 and bladder cancer risk. Four tagging SNPs (tagSNPs) were selected from the 1000 Genomes Project database. In total, 1049 bladder cancer cases and 1399 controls were recruited in this case–control study. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated by using unconditional univariate and multivariate logistic regression models to evaluate associations between the H19 tagSNPs genotypes and risk of bladder cancer. We found a statistically significant increased risk of bladder cancer in the carriers of the rs217727 AA genotype compared with carriers of GG/GA genotype (OR = 1.31, 95% CI = 1.03–1.67). The subsequently stratified analyses also revealed that the H19 rs217727 AA genotype remarkably elevated the risk of bladder cancer in subgroups of young subjects (OR = 1.80, 95% CI = 1.16–2.81), males (OR = 1.44, 95% CI = 1.10–1.89) and smokers (OR = 1.55, 95% CI = 1.06–2.27), as well as high tumour grade (OR = 1.89, 95% CI = 1.23–2.91) and invasive disease (OR = 1.62, 95% CI = 1.01–2.60). This finding indicates that the rs217727 polymorphism is significantly associated with the risk of bladder cancer.

Description: Current therapy approaches in melanoma targeting have met with the development of resistance and tumour recurrence with a more aggressive phenotype. In a quest for alternative therapy targets, we had previously identified Signal Sequence Receptor 2 (SSR2) as a gene with high expression in a subgroup of human primary melanomas. Now we show that SSR2 exerts a prosurvival functionality in human melanoma cells and that high expression levels of SSR2 are associated with an unfavourable disease outcome in primary melanoma patients. Consistent with SSR’s role in translocation of proteins from the ribosome across the endoplasmic reticulum (ER) membrane, our data supports induction of SSR2 as a part of the ER stress response. This response included SSR2 upregulation upon development of therapy resistance to BRAF inhibitors, as well as the dependency of cell survival of BRAF inhibitor-resistant melanoma cells on SSR2. Complementary gain and loss of function data showed the Unfolded Protein Response (UPR) to ER stress as an inducer of SSR2 via transcriptional regulation through X-Box Binding Protein 1s (XBP1s) and support an ER stress-UPR-Transcription Factor XBP1s-SSR2 response axis in human melanocytic cells. Together with its dispensability for survival in normal human cells, these data propose SSR2 as a potential therapeutic target in (therapy-resistant) human melanoma.

Description: Pulp therapy is the last resort for preserving deciduous teeth. However, the genotoxic and cytotoxic effects of many products used in this therapy are not well established. The aim of this study was to use the micronucleus test on bone marrow from mice to evaluate the genotoxic and cytotoxic effects of four filling pastes: zinc oxide, calcium hydroxide P.A., mineral trioxide aggregate and an iodoform paste (iodoform + camphorated + paramonochlorophenol + rifamycin + prednisolone). Male Swiss mice were divided into 4 groups of 10 animals, each exposed to one of the pastes, and were subdivided according to the dilutions tested: 1/10, 1/50, 1/500 and 1/1000 administered intraperitoneally (0.1ml/10g of weight). Cyclophosphamide was the positive control. The negative controls were dimethylsulfoxide and buffered saline solution. Five animals were killed 24h and five 48h after the treatment. The material was processed in accordance with Schmid (1976) and micronuclei were counted in 1000 polychromatic erythrocytes (PCE), under an optical microscope in a blinded test. Cytotoxicity was evaluated using the PCE/normochromatic erythrocyte (NCE) ratio in 200 erythrocytes. The micronucleus analysis results were evaluated using the conditional test for comparing proportions in situations of rare events. Analysis of variance and Tukey’s test were used to evaluate the PCE/NCE ratio. There was significantly greater occurrence of micronuclei in the animals treated with iodoform paste at all the dilutions tested, at both sacrifice times. Greater occurrence of micronuclei was observed among the animals treated with zinc oxide and sacrificed 48h after the treatment, at the dilutions 1:50; 1:500 and 1:1000. Calcium hydroxide P.A. and mineral trioxide aggregate did not present any genotoxic or cytotoxic effects. The genotoxicity and cytotoxicity of zinc oxide and iodoform paste revealed here constitute an initial step towards their contraindication, but additional studies will be necessary in order to securely establish the risks involved in their use.

Description: In this study, we investigated the applicability of using in vivo mouse micronucleus (MN) data to derive cancer potency information. We also present a new statistical methodology for correlating estimated potencies between in vivo MN tests and cancer studies, which could similarly be used for other systems (e.g. in vitro vs. in vivo genotoxicity tests). The dose–response modelling program PROAST was used to calculate benchmark doses (BMDs) for estimating the genotoxic and carcinogenic potency for 48 compounds in mice; most of the data were retrieved from the National Toxicology Program (NTP) database, while some additional data were retrieved from the Carcinogenic Potency Database and published studies. BMD 05 s (doses with 5% increase in MN frequency) were derived from MN data, and BMD 10 s (doses with 10% extra cancer risk) were derived from carcinogenicity data, along with their respective lower (BMDL) and upper (BMDU) confidence bounds. A clear correlation between the in vivo MN BMD 05 s and the cancer BMD 10 s was observed when the lowest BMD 05 from the in vivo MN was plotted against the lowest BMD 10 from the carcinogenicity data for each individual compound. By making a further selection of BMDs related to more or less equally severe cancer lesions, the correlation was considerably improved. Getting a general scientific consensus on how we can quantitatively compare different tumour lesion types and investigating the impact of MN study duration are needed to refine this correlation analysis. Nevertheless, our results suggest that a BMD derived from genotoxicity data might provide a prediction of the tumour potency (BMD 10 ) with an uncertainty range spanning roughly a factor of 100.

Description: Assessment of genetic toxicity and/or carcinogenic activity is an essential element of chemical screening programs employed to protect human health. Dose–response and gene mutation data are frequently analysed by industry, academia and governmental agencies for regulatory evaluations and decision making. Over the years, a number of efforts at different institutions have led to the creation and curation of databases to house genetic toxicology data, largely, with the aim of providing public access to facilitate research and regulatory assessments. This article provides a brief introduction to a new genetic toxicology portal called Mutation Analysis Informatics Tools (MutAIT) ( www.mutait.org ) that provides easy access to two of the largest genetic toxicology databases, the Mammalian Gene Mutation Database (MGMD) and TransgenicDB. TransgenicDB is a comprehensive collection of transgenic rodent mutation data initially compiled and collated by Health Canada. The updated MGMD contains approximately 50 000 individual mutation spectral records from the published literature. The portal not only gives access to an enormous quantity of genetic toxicology data, but also provides statistical tools for dose–response analysis and calculation of benchmark dose. Two important R packages for dose–response analysis are provided as web-distributed applications with user-friendly graphical interfaces. The ‘drsmooth’ package performs dose–response shape analysis and determines various points of departure (PoD) metrics and the ‘PROAST’ package provides algorithms for dose–response modelling. The MutAIT statistical tools, which are currently being enhanced, provide users with an efficient and comprehensive platform to conduct quantitative dose–response analyses and determine PoD values that can then be used to calculate human exposure limits or margins of exposure.

Description: Genotoxic substances are generally not permitted for deliberate use in food production. However, an appreciable number of known or suspected genotoxic substances occur unavoidably in food, e.g. from natural occurrence, environmental contamination and generation during cooking and processing. Over the past decade a margin of exposure (MOE) approach has increasingly been used in assessing the exposure to substances in food that are genotoxic and carcinogenic. The MOE is defined as a reference point on the dose-response curve (e.g. a benchmark dose lower confidences limit derived from a rodent carcinogenicity study) divided by the estimated human intake. A small MOE indicates a higher concern than a very large MOE. Whilst the MOE cannot be directly equated to risk, it supports prioritisation of substances for further research or for possible regulatory action, and provides a basis for communicating to the public. So far, the MOE approach has been confined to substances for which carcinogenicity data are available. In the absence of carcinogenicity data, evidence of genotoxicity is used only in hazard identification. The challenge to the genetic toxicology community is to develop approaches for characterising risk to human health based on data from genotoxicity studies. In order to achieve wide acceptance, it would be important to further address the issues that have been discussed in the context of dose-response modelling of carcinogenicity data in order to assign levels of concern to particular MOE values, and also whether it is possible to make generic conclusions on how potency in genotoxicity assays relates to carcinogenic potency.

Description: Despite ongoing efforts to better understand the mechanisms underlying safety and toxicity, ~30% of the attrition in drug discovery and development is still due to safety concerns. Changes in current practice regarding the assessment of safety and toxicity are required to reduce late stage attrition and enable effective development of novel medicines. This review focuses on the implications of empirical evidence generation for the evaluation of safety and toxicity during drug development. A shift in paradigm is needed to (i) ensure that pharmacological concepts are incorporated into the evaluation of safety and toxicity; (ii) facilitate the integration of historical evidence and thereby the translation of findings across species as well as between in vitro and in vivo experiments and (iii) promote the use of experimental protocols tailored to address specific safety and toxicity questions. Based on historical examples, we highlight the challenges for the early characterisation of the safety profile of a new molecule and discuss how model-based methodologies can be applied for the design and analysis of experimental protocols. Issues relative to the scientific rationale are categorised and presented as a hierarchical tree describing the decision-making process. Focus is given to four different areas, namely, optimisation, translation, analytical construct and decision criteria. From a methodological perspective, the relevance of quantitative methods for estimation and extrapolation of risk from toxicology and safety pharmacology experimental protocols, such as points of departure and potency, is discussed in light of advancements in population and Bayesian modelling techniques (e.g. non-linear mixed effects modelling). Their use in the evaluation of pharmacokinetics (PK) and pharmacokinetic–pharmacodynamic relationships (PKPD) has enabled great insight into the dose rationale for medicines in humans, both in terms of efficacy and adverse events. Comparable benefits can be anticipated for the assessment of safety and toxicity profile of novel molecules.

Description: Assessing the shape of dose–response curves for DNA-damage in cellular systems and for the consequences of DNA damage in intact animals remains a controversial topic. This overview looks at aspects of the pharmacokinetics (PK) and pharmacodynamics (PD) of cellular DNA-damage/repair and their role in defining the shape of dose–response curves using an in vivo example with formaldehyde and in vitro examples for micronuclei (MN) formation with several test compounds. Formaldehyde is both strongly mutagenic and an endogenous metabolite in cells. With increasing inhaled concentrations, there were transitions in gene changes, from activation of selective stress pathway genes at low concentrations, to activation of pathways for cell-cycle control, p53-DNA damage, and stem cell niche pathways at higher exposures. These gene expression changes were more consistent with dose-dependent transitions in the PD responses to formaldehyde in epithelial cells in the intact rat rather than the low-dose linear extrapolation methods currently used for carcinogens. However, more complete PD explanations of non-linear dose response for creation of fixed damage in cells require detailed examination of cellular responses in vitro using measures of DNA damage and repair that are not easily accessible in the intact animal. In the second section of the article, we illustrate an approach from our laboratory that develops fit-for-purpose, in vitro assays and evaluates the PD of DNA damage and repair through studies using prototypical DNA-damaging agents. Examination of a broad range of responses in these cells showed that transcriptional upregulation of cell cycle control and DNA repair pathways only occurred at doses higher than those causing overt damage fixed damage—measured as MN formation. Lower levels of damage appear to be handled by post-translational repair process using pre-existing proteins. In depth evaluation of the PD properties of one such post-translational process (formation of DNA repair centers; DRCs) has indicated that the formation of DRCs and their ability to complete repair before replication are consistent with threshold behaviours for mutagenesis and, by extension, with chemical carcinogenesis.

Description: Qualitative and quantitative approaches are important issues in field of carcinogenic risk assessment of the genotoxic carcinogens. Herein, we provide quantitative data on low-dose hepatocarcinogenicity studies for three genotoxic hepatocarcinogens: 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ) and N -nitrosodiethylamine (DEN). Hepatocarcinogenicity was examined by quantitative analysis of glutathione S -transferase placental form (GST-P) positive foci, which are the preneoplastic lesions in rat hepatocarcinogenesis and the endpoint carcinogenic marker in the rat liver medium-term carcinogenicity bioassay. We also examined DNA damage and gene mutations which occurred through the initiation stage of carcinogenesis. For the establishment of points of departure (PoD) from which the cancer-related risk can be estimated, we analyzed the above events by quantitative no-observed-effect level and benchmark dose approaches. MeIQx at low doses induced formation of DNA–MeIQx adducts; somewhat higher doses caused elevation of 8-hydroxy-2'-deoxyquanosine levels; at still higher doses gene mutations occurred; and the highest dose induced formation of GST-P positive foci. These data indicate that early genotoxic events in the pathway to carcinogenesis showed the expected trend of lower PoDs for earlier events in the carcinogenic process. Similarly, only the highest dose of IQ caused an increase in the number of GST-P positive foci in the liver, while IQ-DNA adduct formation was observed with low doses. Moreover, treatment with DEN at low doses had no effect on development of GST-P positive foci in the liver. These data on PoDs for the markers contribute to understand whether genotoxic carcinogens have a threshold for their carcinogenicity. The most appropriate approach to use in low dose–response assessment must be approved on the basis of scientific judgment.

Description: Applied genetic toxicology is undergoing a transition from qualitative hazard identification to quantitative dose–response analysis and risk assessment. To facilitate this change, the Health and Environmental Sciences Institute (HESI) Genetic Toxicology Technical Committee (GTTC) sponsored a workshop held in Lancaster, UK on July 10–11, 2014. The event included invited speakers from several institutions and the contents was divided into three themes—1: Point-of-departure Metrics for Quantitative Dose–Response Analysis in Genetic Toxicology; 2: Measurement and Estimation of Exposures for Better Extrapolation to Humans and 3: The Use of Quantitative Approaches in Genetic Toxicology for human health risk assessment (HHRA). A host of pertinent issues were discussed relating to the use of in vitro and in vivo dose–response data, the development of methods for in vitro to in vivo extrapolation and approaches to use in vivo dose–response data to determine human exposure limits for regulatory evaluations and decision-making. This Special Issue, which was inspired by the workshop, contains a series of papers that collectively address topics related to the aforementioned themes. The Issue includes contributions that collectively evaluate, describe and discuss in silico , in vitro , in vivo and statistical approaches that are facilitating the shift from qualitative hazard evaluation to quantitative risk assessment. The use and application of the benchmark dose approach was a central theme in many of the workshop presentations and discussions, and the Special Issue includes several contributions that outline novel applications for the analysis and interpretation of genetic toxicity data. Although the contents of the Special Issue constitutes an important step towards the adoption of quantitative methods for regulatory assessment of genetic toxicity, formal acceptance of quantitative methods for HHRA and regulatory decision-making will require consensus regarding the relationships between genetic damage and disease, and the concomitant ability to use genetic toxicity results per se .

Description: Quantifying health-related biological effects, like genotoxicity, could provide a way of distinguishing between tobacco products. In order to develop tools for using genotoxicty data to quantitatively evaluate the risk of tobacco products, we tested five carcinogens found in cigarette smoke, 4-aminobiphenyl (4-ABP), benzo[ a ]pyrene (BaP), cadmium (in the form of CdCl 2 ), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in the mouse lymphoma assay (MLA). The resulting mutagenicity dose responses were analyzed by various quantitative approaches and their strengths and weaknesses for distinguishing responses in the MLA were evaluated. L5178Y/ Tk +/– 3.7.2C mouse lymphoma cells were treated with four to seven concentrations of each chemical for 4h. Only CdCl 2 produced a positive response without metabolic activation (S9); all five chemicals produced dose-dependent increases in cytotoxicity and mutagenicity with S9. The lowest dose exceeding the global evaluation factor, the benchmark dose producing a 10%, 50%, 100% or 200% increase in the background frequency (BMD 10 , BMD 50 , BMD 100 and BMD 200 ), the no observed genotoxic effect level (NOGEL), the lowest observed genotoxic effect level (LOGEL) and the mutagenic potency expressed as a mutant frequency per micromole of chemical, were calculated for all the positive responses. All the quantitative metrics had similar rank orders for the agents’ ability to induce mutation, from the most to least potent as CdCl 2 (–S9) 〉 BaP(+S9) 〉 CdCl 2 (+S9) 〉 MeIQ(+S9) 〉 4-ABP(+S9) 〉 NNK(+S9). However, the metric values for the different chemical responses (i.e. the ratio of the greatest value to the least value) for the different chemicals ranged from 16-fold (BMD 10 ) to 572-fold (mutagenic potency). These results suggest that data from the MLA are capable of discriminating the mutagenicity of various constituents of cigarette smoke, and that quantitative analyses are available that can be useful in distinguishing between the exposure responses.

Description: Genetic toxicity testing has traditionally been used for hazard identification, with dichotomous classification of test results serving to identify genotoxic agents. However, the utility of genotoxicity data can be augmented by employing dose–response analysis and point of departure determination. Via interpolation from a fitted dose–response model, the benchmark dose (BMD) approach estimates the dose that elicits a specified (small) effect size. BMD metrics and their confidence intervals can be used for compound potency ranking within an endpoint, as well as potency comparisons across other factors such as cell line or exposure duration. A recently developed computational method, the BMD covariate approach, permits combined analysis of multiple dose–response data sets that are differentiated by covariates such as compound, cell type or exposure regime. The approach provides increased BMD precision for effective potency rankings across compounds and other covariates that pertain to a hypothesised mode of action (MOA). To illustrate these applications, the covariate approach was applied to the analysis of published in vitro micronucleus frequency dose–response data for ionising radiations, a set of aneugens, two mutagenic azo compounds and a topoisomerase II inhibitor. The ionising radiation results show that the precision of BMD estimates can be improved by employing the covariate method. The aneugen analysis provided potency groupings based on the BMD confidence intervals, and analyses of azo compound data from cells lines with differing metabolic capacity confirmed the influence of endogenous metabolism on genotoxic potency. This work, which is the first of a two-part series, shows that BMD-derived potency rankings can be employed to support MOA evaluations as well as facilitate read across to expedite chemical evaluations and regulatory decision-making. The follow-up (Part II) employs the combined covariate approach to analyse in vivo genetic toxicity dose–response data focussing on how improvements in BMD precision can impact the reduction and refinement of animal use in toxicological research.

Description: Methyl methanesulfonate, a well-known direct-acting genotoxicant, was assessed in a multi-endpoint study in rats using six closely spaced dose levels. The main goal of the study was to investigate the genotoxic response at very low doses and to analyse this response with dedicated statistical tools in order to find a Point of Departure (PoD) and related metrics. Software packages like PROAST or EPA-BMDS require the toxicologist to define a so-called critical effect size (CES) or benchmark response (BMR) and this choice has a large impact on the result of the PoD calculation. Currently, increases of 5%, 10% or 1 standard deviation over concurrent vehicle controls have been proposed for CES/BMR, values that may or may not be suited for all genotoxicity endpoints. Based on the data obtained in this study, we propose an endpoint specific CES approach that reflects the typical evaluation process of a regulatory acceptable genotoxicology study. However, we are aware that this ratio-based CES strategy will need to be more fully developed with additional experimentation and should be mainly seen as a starting point for scientific discussion.

Description: Genotoxicity tests have traditionally been used only for hazard identification, with qualitative dichotomous groupings being used to identify compounds that have the capacity to induce mutations and/or cytogenetic alterations. However, there is an increasing interest in employing quantitative analysis of in vivo dose–response data to derive point of departure (PoD) metrics that can be used to establish human exposure limits or margins of exposure (MOEs), thereby supporting human health risk assessments and regulatory decisions. This work is an extension of our companion article on in vitro dose–response analyses and outlines how the combined benchmark dose (BMD) approach across included covariates can be used to improve the analyses and interpretation of in vivo genetic toxicity dose–response data. Using the BMD-covariate approach, we show that empirical comparisons of micronucleus frequency dose–response data across multiple studies justifies dataset merging, with subsequent analyses improving the precision of BMD estimates and permitting attendant potency ranking of seven clastogens. Similarly, empirical comparisons of Pig-a mutant phenotype frequency data collected in males and females justified dataset merging across sex. This permitted more effective scrutiny regarding the effect of post-exposure sampling time on the mutagenicity of N -ethyl- N -nitrosourea observed in reticulocytes and erythrocytes in the Pig-a assay. The BMD-covariate approach revealed tissue-specific differences in the induction of lacZ transgene mutations in Muta™Mouse specimens exposed to benzo[ a ]pyrene (BaP), with the results permitting the formulation of mechanistic hypotheses regarding the observed potency ranking. Lastly, we illustrate how historical dose–response data for assessments that examined numerous doses (i.e. induced lacZ mutant frequency (MF) across 10 doses of BaP) can be used to improve the precision of BMDs derived from datasets with far fewer doses (i.e. lacZ MF for 3 doses of dibenz[ a,h ]anthracene). Collectively, the presented examples illustrate how innovative use of the BMD approach can permit refinement of the use of in vivo data; improving the efficacy of experimental animal use in genetic toxicology without sacrificing PoD precision.

Description: The anti-parasitic benzimidazole flubendazole has been used for many years to treat intestinal infections in humans and animals. Previous genotoxicity studies have shown that the compound is not a bacterial mutagen and a bone marrow micronucleus test, using a formulation that limited systemic absorption, was negative. The purpose of this study is to explore the genotoxicity of flubendazole and its main metabolites in in vitro micronucleus studies and to test a new oral formulation that improves systemic absorption in an in vivo micronucleus test. The isolated metabolites were also screened using the Ames test for bacterial mutagenicity. It was found that flubendazole, like other chemically related benzimidazoles used in anti-parasitic therapies, is a potent aneugen in vitro . The hydrolysed metabolite of flubendazole is negative in these tests, but the reduced metabolite (R- and S-forms) shows both aneugenic and clastogenic activity. However, in vitro micronucleus tests of flubendazole in the presence of rat liver S9 gave almost identical signals for aneugenicity as they did in the absence of S9, suggesting that any clastogenicity from the reduced metabolite is not sufficient to change the overall profile. Like flubendazole itself, both metabolites are negative in the Ames test. Analysis of dose–response curves from the in vitro tests, using recently developed point of departure approaches, demonstrate that the aneugenic potency of flubendazole is very similar to related anti-parasitic benzimidazoles, including albendazole, which is used in mass drug administration programmes to combat endemic filarial diseases. The in vivo micronucleus test of the new formulation of flubendazole also showed evidence of induced aneugenicity. Analysis of the in vivo data allowed a reference dose for aneugenicity to be established which can be compared with therapeutic exposures of flubendazole when this has been established. Analysis of the plasma from the animals used in the in vivo micronucleus test showed that there is increased exposure to flubendazole compared with previously tested formulations, as well as significant formation of the non-genotoxic hydrolysed metabolite of flubendazole and small levels of the reduced metabolite. In conclusion, this study shows that flubendazole is a potent aneugen in vitro with similar potency to chemically related benzimidazoles currently used as anti-parasitic therapies. The reduced metabolite also has aneugenic properties as well as clastogenic properties. Treatment with a new formulation of flubendazole that allows increased systemic exposure, compared with previously used formulations, also results in detectable aneugenicity in vivo. Based on the lack of carcinogenicity of this class of benzimidazoles and the intended short-term dosing, it is unlikely that flubendazole treatment will pose a carcinogenic risk to patients.

Description: The nature of the dose–response relationship for various in vivo endpoints of exposure and effect were investigated using the alkylating agents, methyl methanesulfonate (MMS) and methylnitrosourea (MNU). Six male F344 rats/group were dosed orally with 0, 0.5, 1, 5, 25 or 50mg/kg bw/day (mkd) of MMS, or 0, 0.01, 0.1, 1, 5, 10, 25 or 50 mkd of MNU, for 4 consecutive days and sacrificed 24h after the last dose. The dose–responses for multiple biomarkers of exposure and genotoxic effect were investigated. In MMS-treated rats, the hemoglobin adduct level, a systemic exposure biomarker, increased linearly with dose ( r 2 = 0.9990, P 〈 0.05), indicating the systemic availability of MMS; however, the N7MeG DNA adduct, a target exposure biomarker, exhibited a non-linear dose–response in blood and liver tissues. Blood reticulocyte micronuclei (MN), a genotoxic effect biomarker, exhibited a clear no-observed-genotoxic-effect-level (NOGEL) of 5 mkd as a point of departure (PoD) for MMS. Two separate dose–response models, the Lutz and Lutz model and the stepwise approach using PROC REG both supported a bilinear/threshold dose–response for MN induction. Liver gene expression, a mechanistic endpoint, also exhibited a bilinear dose–response. Similarly, in MNU-treated rats, hepatic DNA adducts, gene expression changes and MN all exhibited clear PoDs, with a NOGEL of 1 mkd for MN induction, although dose–response modeling of the MNU-induced MN data showed a better statistical fit for a linear dose–response. In summary, these results provide in vivo data that support the existence of clear non-linear dose–responses for a number of biologically significant events along the pathway for genotoxicity induced by DNA-reactive agents.

Description: The application of flow cytometry as a scoring platform for both in vivo and in vitro micronucleus (MN) studies has enabled the efficient generation of high quality datasets suitable for comprehensive assessment of dose–response. Using this information, it is possible to obtain precise estimates of the clastogenic potency of chemicals. We illustrate this by estimating the in vivo and the in vitro potencies of seven model clastogenic agents (melphalan, chlorambucil, thiotepa, 1,3-propane sultone, hydroxyurea, azathioprine and methyl methanesulfonate) by deriving BMDs using freely available BMD software (PROAST). After exposing male rats for 3 days with up to nine dose levels of each individual chemical, peripheral blood samples were collected on Day 4. These chemicals were also evaluated for in vitro MN induction by treating TK6 cells with up to 20 concentrations in quadruplicate. In vitro MN frequencies were determined via flow cytometry using a 96-well plate autosampler. The estimated in vitro and in vivo BMDs were found to correlate to each other. The correlation showed considerable scatter, as may be expected given the complexity of the whole animal model versus the simplicity of the cell culture system. Even so, the existence of the correlation suggests that information on the clastogenic potency of a compound can be derived from either whole animal studies or cell culture-based models of chromosomal damage. We also show that the choice of the benchmark response, i.e. the effect size associated with the BMD, is not essential in establishing the correlation between both systems. Our results support the concept that datasets derived from comprehensive genotoxicity studies can provide quantitative dose–response metrics. Such investigational studies, when supported by additional data, might then contribute directly to product safety investigations, regulatory decision-making and human risk assessment.

Description: In vitro genotoxicity assessment routinely employs an exogenous metabolic activation mixture to simulate mammalian metabolism. Activation mixtures commonly contain post-mitochondrial liver supernatant (i.e. S9) from chemically induced Sprague Dawley rats. Although Organization for Economic Cooperation and Development (OECD) test guidelines permit the use of other S9 preparations, assessments rarely employ human-derived S9. The objective of this study is to review and evaluate the use of human-derived S9 for in vitro genetic toxicity assessment. All available published genotoxicity assessments employing human S9 were compiled for analysis. To facilitate comparative analyses, additional matched Ames data using induced rat liver S9 were obtained for certain highly cited chemicals. Historical human and induced rat S9 quality control reports from Moltox were obtained and mined for enzyme activity and mutagenic potency data. Additional in vitro micronucleus data were experimentally generated using human and induced rat S9. The metabolic activity of induced rat S9 was found to be higher than human S9, and linked to high mutagenic potency results. This study revealed that human S9 often yields significantly lower Salmonella mutagenic potency values, especially for polycyclic aromatic hydrocarbons, aflatoxin B1 and heterocyclic amines (~3- to 350-fold). Conversely, assessment with human S9 activation yields higher potency for aromatic amines (~2- to 50-fold). Outliers with extremely high mutagenic potency results were observed in the human S9 data. Similar trends were observed in experimentally generated mammalian micronucleus cell assays, however human S9 elicited potent cytotoxicity L5178Y, CHO and TK6 cell lines. Due to the potential for reduced sensitivity and the absence of a link between enzyme activity levels and mutagenic potency, human liver S9 is not recommended for use alone in in vitro genotoxicity screening assays; however, human S9 may be extremely useful in follow-up tests, especially in the case of chemicals with species-specific metabolic differences, such as aromatic amines.

Description: The present study aims to further characterize benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects. Therefore, we measured DNA effects by the comet assay and adduct levels by high-performance liquid chromatography (HPLC) in human lymphocytes and A549 cells exposed to (±)- anti -benzo[a]pyrene-7,8-diol 9,10-epoxide [(±)- anti -BPDE] or (+)- anti -benzo[a]pyrene-7,8-diol 9,10-epoxide [(+)- anti -BPDE]. Both, the racemic form and (+)- anti -BPDE, which is the most relevant metabolite with regard to mutagenicity and carcinogenicity, induced DNA migration in cultured lymphocytes in the same range of concentrations to a similar extent in the alkaline comet assay after exposure for 2h. Nevertheless, (+)- anti -BPDE induced significantly enhanced DNA migration after 16 and 18h post-cultivation which was not seen in response to (±)- anti -BPDE. Combination of the comet assay with the Fpg (formamidopyrimidine-DNA glycosylase) protein did not enhance BPDE-induced effects and thus indicated the absence of Fpg-sensitive sites (oxidized purines, N7-guanine adducts, AP-sites). The aphidicolin (APC)-modified comet assay suggested significant excision repair activity of cultured lymphocytes during the first 18h of culture after a 2 h-exposure to BPDE. In contrast to these repair-related effects measured by the comet assay, HPLC analysis of stable adducts did not reveal any significant removal of (+)- anti -BPDE-induced adducts from lymphocytes during the first 22h of culture. On the other hand, HPLC measurements indicated that A549 cells repaired about 70% of (+)- anti -BPDE-induced DNA-adducts within 22h of release. However, various experiments with the APC-modified comet assay did not indicate significant repair activity during this period in A549 cells. The conflicting results obtained with the comet assay and the HPLC-based adduct analysis question the real cause for BPDE-induced DNA migration in the comet assay and the reliability of the APC-modified comet assay for the determination of DNA excision repair activity in response to BPDE in different cell types.

Description: End-stage renal failure patients exhibit a high incidence of genetic damage and genomic instability. Part of this genetic damage is assumed to be caused by the hemodialysis (HD) procedure. To reduce these effects, different alternative HD procedures have been proposed, such as the use of high efficiency convective therapies to improve the reactive oxygen species/antioxidant ratio. To determine the efficiency of online hemodiafiltration (HDF) technique on the levels of DNA damage, we have measured the frequency of micronucleus in peripheral blood lymphocytes of 33 individuals moving from low-flux HD to post-dilution online HDF. In addition to basal levels of genetic damage, potential changes in radiosensitivity were measured as indicators of genomic instability. Plasma antioxidant capacity was also determined. Second samples were obtained after 6 months on the HDF protocol. Results indicate that moving to online HDF therapy produce a significant reduction of the basal levels of genetic damage, but does not affect the genomic instability status. In addition, a greater increase in plasma antioxidant capacity was observed. In spite of the lack of correlation between these parameters, our results confirm the usefulness of the online HDF technique as a way to reduce DNA damage in HD patients.

Description: Machaerium hirtum (Vell.) Stellfeld ( M.hirtum ) is a plant known as ‘jacarandá-bico-de-pato’ whose bark is commonly used against diarrhea, cough and cancer. The aim of this study was to phytochemically characterise the hydroethanolic extract of this plant, investigate its antimutagenic activities using the Ames test and evaluate its effects on cell viability, genomic instability, gene expression and cell protection in human hepatocellular carcinoma cells (HepG2). Antimutagenic activity was assessed by simultaneous pre- and post-treatment with direct and indirect mutagens, such as 4-nitro- o -phenylenediamine (NPD), mitomycin C (MMC), benzo[ a ]pyrene (B[ a ]P) and aflatoxin B 1 (AFB 1 ), using the Ames test, cytokinesis blocking micronucleus and apoptosis assays. Only 3 of the 10 concentrations evaluated in the MTT assay were cytotoxic in HepG2 cells. Micronucleated or apoptotic cells were not observed with any of the tested concentrations, and there were no mutagenic effects in the bacterial system. However, the Nuclear Division Index and flow cytometry data showed a decrease in cell proliferation. The extract showed an inhibitory effect against direct (NPD) and indirect mutagens (B[a]P and AFB 1 ). Furthermore, pre- and post-treated cells showed significant reduction in the number of apoptotic and micronucleated cells. This effect is not likely to be associated with the modulation of antioxidant genes, as shown by the RT-qPCR results. Six known flavonoids were identified in the hydroethanolic extract of Machaerium hirtum leaves, and their structures were elucidated by spectroscopic and spectrophotometric methods. The presence of the antioxidants apigenin and luteolin may explain these protective effects, because these components can inhibit the formation of reactive species and prevent apoptosis and DNA damage. In conclusion, the M.hirtum extract showed chemopreventive potential and was not hazardous at the tested concentrations in the experiments presented here. Moreover, this extract should be investigated further as a chemopreventive agent.

Description: Various types of polycyclic aromatic compounds (PACs) in diesel exhaust particles are thought to contribute to carcinogenesis in mammals. Although the carcinogenicity, mutagenicity and tumour-initiating activity of these compounds have been evaluated, their tumour-promoting activity is unclear. In the present study, to determine the tumour-inducing activity of PACs, including previously known mutagenic compounds in atmospheric environments, a transformation assay for promoting activity mediated by the release of contact inhibition was conducted for six polycyclic aromatic hydrocarbons (PAHs), seven oxygenated PAHs (oxy-PAHs) and seven nitrated PAHs (nitro-PAHs) using mouse embryonic fibroblast cells transfected with the v-Ha- ras gene (Bhas 42 cells). Of these, two PAHs [benzo[ k ]fluoranthene (B[ k ]FA) and benzo[ b ]fluoranthene (B[ b ]FA)], one oxy-PAH [6 H -benzo[ cd ]pyren-6-one (BPO)] and two nitro-PAHs (3-nitro-7 H -benz[ de ]anthracen-7-one and 6-nitrochrysene) were found to exhibit particularly powerful tumour-promoting activity (≥10 foci following exposure to 〈100nM). In addition, clear mRNA expression of CYP1A1, which is associated with aryl hydrocarbon receptor (AhR)-mediated activation, was observed following the exposure of cells to two PAHs (B[ k ]FA and B[ b ]FA) and three oxy-PAHs (1,2-naphthoquinone, 11 H -benzo[ b ]fluoren-11-one and BPO). Further, an HO-1 antioxidant response activation was observed following exposure to B[ k ]FA, B[ b ]FA and BPO, suggesting that the induction of tumour-promoting activity in these compounds is correlated with the dysfunction of signal transduction via AhR-mediated responses and/or oxidative stress responses.

Description: The increasing use of mobile phones and wireless networks raised a great debate about the real carcinogenic potential of radiofrequency–electromagnetic field (RF–EMF) exposure associated with these devices. Conflicting results are reported by the great majority of in vivo and in vitro studies on the capability of RF–EMF exposure to induce DNA damage and mutations in mammalian systems. Aimed at understanding whether less ambiguous responses to RF–EMF exposure might be evidenced in plant systems with respect to mammalian ones, in the present work the mutagenic effect of RF–EMF has been studied through the micronucleus (MN) test in secondary roots of Vicia faba seedlings exposed to mobile phone transmission in controlled conditions, inside a transverse electro magnetic (TEM) cell. Exposure of roots was carried out for 72h using a continuous wave (CW) of 915 MHz radiation at three values of equivalent plane wave power densities (23, 35 and 46W/m 2 ). The specific absorption rate (SAR) was measured with a calorimetric method and the corresponding values were found to fall in the range of 0.4–1.5W/kg. Results of three independent experiments show the induction of a significant increase of MN frequency after exposure, ranging from a 2.3-fold increase above the sham value, at the lowest SAR level, up to a 7-fold increase at the highest SAR. These findings are in agreement with the limited number of data on cytogenetic effects detected in other plant systems exposed to mobile phone RF–EMF frequencies and clearly show the capability of radiofrequency exposure to induce DNA damage in this eukaryotic cell system.

Description: The micronucleus assay (MN assay) is a well-established assay in genetic toxicology, biomonitoring of mutagen-exposed populations and chromosomal radiosensitivity testing. To evaluate the effect of storage time on the chromosomal radiosensitivity assessment in lymphocytes, micronuclei (MN) yields in blood samples received and processed on the same day were compared with MN yields obtained when blood cultures were set up 24 and 48h after blood sampling. Furthermore, the influence of general anaesthesia on MN and binucleated cells (BN) yields in the MN assay was considered. Blood samples of 10 healthy donors were irradiated and blood cultures were set up during the same day of blood sampling or with a delay of 24 or 48h. The MN assay was also performed on two blood samples from 60 women undergoing breast surgery. The first blood sample was taken before general anaesthesia and the second sample, 2h after anaesthesia induction. Fifty percent of the blood samples were transported to the cytogenetics lab within 2h while the other 50% reached the lab after 24h. The results of this study show a decrease in BN and an increase in MN yields with increasing storage time before irradiation and setting up of the MN assay for both healthy controls and patients. The administration of general anaesthesia in patients resulted in lower BN yields, higher spontaneous MN yields but no differences in radiation-induced MN yields. In conclusion, this study indicates that the time between blood sampling and the in vitro irradiation of the samples for the MN assay influences the MN yields. Delays of more than 24h should be avoided. To assess chromosomal radiosensitivity in patients, blood samples should be taken before induction of general anaesthesia as anaesthesia can have an impact on the reliability of the MN results.

Description: 4-Nitroquinoline 1-oxide (4NQO) is used as a positive control in various genotoxicity assays because of its known mutagenic and carcinogenic properties. The chemical is converted into 4-hydroxyaminoquinoline 1-oxide and gives rise to three main DNA adducts, N -(deoxyguanosin-8-yl)-4AQO, 3-(desoxyguanosin- N 2 -yl)-4AQO and 3-(deoxyadenosin- N 6 -yl)-4AQO. This study was designed to assess the shape of the dose–response curve at low concentrations of 4NQO in three human lymphoblastoid cell lines, MCL-5, AHH-1 and TK6 as well as the mouse lymphoma L5178Y cell line in vitro . Chromosomal damage was investigated using the in vitro micronucleus assay, while further gene mutation and DNA damage studies were carried out using the hypoxanthine–guanine phosphoribosyltransferase forward mutation and comet assays. 4NQO showed little to no significant increases in micronucleus induction in the human lymphoblastoid cell lines, even up to 55±5% toxicity. A dose–response relationship could only be observed in the mouse lymphoma cell line L5178Y after 4NQO treatment, even at concentrations with no reduction in cell viability. Further significant increases in gene mutation and DNA damage induction were observed. Hence, 4NQO is a more effective point mutagen than clastogen, and its suitability as a positive control for genotoxicity testing has to be evaluated for every individual assay.

Description: Chromatin remodelling is critical for repairing DNA damage and maintaining genomic integrity. Previous studies have reported that histone acetyltransferase p300 and ATP-dependent chromatin remodeler chromodomain helicase DNA-binding protein 4 (CHD4) functions, respectively, in DNA double-strand breaks (DSBs) repair. But the physiological significance of their interaction remains elusive. Here, we showed that p300 and CHD4 were both recruited to the sites of DSBs. Their ablation led to impaired DSBs repair and sensitised cells to laser and the anti-cancer drug, etoposide. Using DR-GFP and EJ5-GFP reporter systems, we found that knockdown of p300 or CHD4 impaired the homologous recombination (HR) repair but no the non-homologous end joining (NHEJ) repair. Furthermore, p300 or CHD4 knockdown respectively suppressed the recruitment of replication protein A (RPA), a key protein for HR, to the DSB sites. In addition, immunofluorescence results showed that knockdown of p300 reduced the recruitment of CHD4 at DSB sites. In turn, CHD4 knockdown also decreased p300 assembly. Moreover, immunoprecipitation and purified protein pull down assay revealed that p300 physically interacted with CHD4 at DNA damage sites, and this interaction was dependent on the chromodomain and ATPase/helicase domain of CHD4 and the CH2, Bd and HAT domains of p300. These results indicate that p300 and CHD4 could function cooperatively at DSB sites and provide a new insight into the detailed crosstalk among the chromatin remodelling proteins.

Description: Coal miners are exposed to a wide range of genotoxic agents that can induce genome damage. In addition, miners are characterised by a high risk of the initiation of different occupational inflammatory as well as non-inflammatory diseases. The aim of this investigation is to analyse the modifying influence of occupational pulmonary inflammatory diseases on the level of chromosome aberrations (CAs) in miners working in underground coal mines in Kemerovo Region (Russian Federation). The study group included 90 coal miners with the following pulmonary diseases: chronic dust-induced bronchitis (CDB) and coal-workers’ pneumoconiosis (CWP) (mean age = 53.52±2.95 years; mean work experience in coal-mining conditions = 27.70±3.61 years). As a population control (control 1), we have used venous blood extracted from 124 healthy unexposed men. The mean age in this group was 50.92±4.56 years. Control 2 was the venous blood extracted from 42 healthy coal miners (mean age = 51.56±6.38 years; mean work experience in coal-mining conditions = 25.43±8.14 years). We have discovered that coal miners are characterised by an increased general level of CAs as well as an increased frequency of several types of CAs. The significant increase in the frequency of aberration per 100 cells and aberration of chromosome type was discovered in the group of pulmonary disease patients (study group). No correlations of the level of chromosome damage with age, smoking status and work experience in coal-mining conditions were discovered.

Description: We previously reported that a portion of blueberries reversed endothelial dysfunction induced by acute cigarette smoking. Since smoking-induced endothelial dysfunction is associated with a condition of oxidative stress, we evaluated whether the observed effect was mediated by modulation of markers of oxidative stress and antioxidant defence. Fourteen out of 16 male healthy smokers previously enrolled, participated in a three-armed randomized controlled study with the following experimental conditions: smoking treatment (one cigarette); blueberry treatment (300g of blueberries) + smoking (one cigarette); control treatment (300ml of water with sugar) + smoking (one cigarette). The cigarette was smoked 100min after blueberry/control/water consumption. Each treatment was separated by 1 week of washout period. Plasma vitamin (C, B 12 and folate) and aminothiol concentrations, endogenous [formamidopyrimidine-DNA glycosylase (FPG)-sensitive sites] and oxidatively induced DNA damage (resistance to H 2 O 2 -induced DNA damage) in peripheral blood mononuclear cells (PBMCs) were measured at baseline and 20, 60, 90, 120min and 24h after smoking. On the whole, analysis of variance did not show a significant effect of treatment on the modulation of markers of oxidative stress and antioxidant defence but revealed an effect of time for plasma concentrations of vitamin C ( P = 0.003), B 12 ( P 〈 0.001), folate ( P 〈 0.001), total cysteine ( P = 0.007) and cysteine–glycine ( P = 0.010) that increased following the three treatments after smoking. No significant effect of treatment was observed for the levels of FPG-sensitive sites ( P 〉 0.05) and H 2 O 2 -induced DNA damage ( P 〉 0.05) in PBMCs. In conclusion, the consumption of a single blueberry portion failed to modulate markers of oxidative stress and antioxidant defence investigated in our experimental conditions. Further studies are necessary to elucidate this finding and help clarifying the mechanisms of protection of blueberries against smoking-induced endothelial dysfunction.

Description: Dexrazoxane is the only clinically approved cardioprotectant against anthracyclines-induced cardiotoxicity. Thus, detailed evaluation of the genotoxic potential of dexrazoxane and anthracyclines combination is essential to provide more insights into genotoxic and anti-genotoxic alterations that may play a role in the development of the secondary malignancies after treatment with anthracyclines. Thus, our aim was to determine whether non-genotoxic doses of dexrazoxane in combination with the anthracycline, epirubicin can modulate epirubicin-induced genotoxicity and apoptosis in somatic cells. Bone marrow micronucleus test complemented with fluorescence in situ hybridization assay and comet assay were performed to assess the genotoxicity of dexrazoxane and/or epirubicin. Apoptosis was analysed by using the annexin V assay and the occurrence of the hypodiploid DNA content. Generation of reactive oxygen species was also assessed in bone marrow by using the oxidant-sensing fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate. Dexrazoxane was neither genotoxic nor apoptogenic in mice at a single dose of 75 or 150mg/kg. Moreover, it has been shown that dexrazoxane affords significant protection against epirubicin-induced genotoxicity and apoptosis in the bone marrow cells in a dose-dependent manner. Epirubicin induced marked generation of intracellular reactive oxygen species and prior administration of dexrazoxane ahead of epirubicin challenge ameliorated accumulation of these free radicals. It is thus concluded that dexrazoxane can be safely combined with epirubicin and that pre-treatment with dexrazoxane attenuates epirubicin-induced generation of reactive oxygen species and subsequent genotoxicity and apoptosis. Thus, epirubicin-induced genotoxicity can be effectively mitigated by using dexrazoxane.

Description: The FP7 Sanowork project was aimed to minimise occupational hazard and exposure to engineered nanomaterials (ENM) through the surface modification in order to prevent possible health effects. In this frame, a number of nanoparticles (NP) have been selected, among which zirconium (ZrO 2 ) and titanium (TiO 2 ) dioxide. In this study, we tested ZrO 2 NP and TiO 2 NP either in their pristine (uncoated) form, or modified with citrate and/or silica on their surface. As benchmark material, Aeroxide® P25 was used. We assessed cytotoxicity, genotoxicity and induction of morphological neoplastic transformation of NP by using a panel of in vitro assays in an established mammalian cell line of murine origin (Balb/3T3). Cell viability was evaluated by means of colony-forming efficiency assay (CFE). Genotoxicity was investigated by cytokinesis-block micronucleus cytome assay (CBMN cyt) and comet assay, and by the use of the restriction enzymes EndoIII and Fpg, oxidatively damaged DNA was detected; finally, the morphological neoplastic transformation of NP was assayed in vitro by cell transformation assay (CTA). Our results show that the surface remediation has not been effective in modifying cyto- and genotoxic properties of the nanomaterials tested; indeed, in the case of remediation of zirconia and titania with citrate, there is a tendency to emphasise the toxic effects. The use of a panel of assays, such as those we have employed, allowing the evaluation of multiple endpoints, including cell transformation, seems particularly advisable especially in the case of long-term exposure effects in the same cell type.

Description: Acrylamide is used in many industrial processes and is present in a variety of fried and baked foods. In rodent carcinogenicity assays, acrylamide exposure leads to tumour formation at doses lower than those demonstrated to induce genotoxic damage. We evaluated the potential of acrylamide to induce structural DNA damage and gene mutations in rodents using highly sensitive flow cytometric analysis of micronucleus and Pig-a mutant frequencies, respectively. Male F344 rats and B6C3F1 mice were administered acrylamide in drinking water for 30 days at doses spanning and exceeding the range of acrylamide exposure tested in cancer bioassays—top dose of 12.0 and 24.0mg/kg/day in mice and in rats, respectively. A positive control, N -ethyl- N -nitrosourea, was administered at the beginning and end of the study to meet the expression time for the two DNA damage phenotypes. The results of the micronucleus and Pig-a assays were negative and equivocal, respectively, for male rats exposed to acrylamide at the concentrations tested. In contrast, acrylamide induced a dose-dependent increase in micronucleus formation but tested negative in the Pig-a assay in mice. Higher plasma concentrations of glycidamide in mice than rats are hypothesized to explain, at least in part, the differences in the response. Benchmark dose modelling indicates that structural DNA damage as opposed to point mutations is most relevant to the genotoxic mode of action of acrylamide-induced carcinogenicity. Moreover, the lack of genotoxicity detected at 〈6.0mg/kg/day is consistent with the notion that non-genotoxic mechanisms contribute to acrylamide-induced carcinogenicity in rodents.

Description: Nanofibrillated cellulose (NFC) is a sustainable and renewable nanomaterial, with diverse potential applications in the paper and medical industries. As NFC consists of long fibres of high aspect ratio, we examined here whether TEMPO-(2,2,6,6-tetramethyl-piperidin-1-oxyl) oxidised NFC (length 300–1000nm, thickness 10–25nm), administrated by a single pharyngeal aspiration, could be genotoxic to mice, locally in the lungs or systemically in the bone marrow. Female C57Bl/6 mice were treated with four different doses of NFC (10, 40, 80 and 200 µg/mouse), and samples were collected 24h later. DNA damage was assessed by the comet assay in bronchoalveolar lavage (BAL) and lung cells, and chromosome damage by the bone marrow erythrocyte micronucleus assay. Inflammation was evaluated by BAL cell counts and analysis of cytokines and histopathological alterations in the lungs. A significant induction of DNA damage was observed at the two lower doses of NFC in lung cells, whereas no increase was seen in BAL cells. No effect was detected in the bone marrow micronucleus assay, either. NFC increased the recruitment of inflammatory cells to the lungs, together with a dose-dependent increase in mRNA expression of tumour necrosis factor α, interleukins 1β and 6, and chemokine (C-X-C motif) ligand 5, although there was no effect on the levels of the respective proteins. The histological analysis showed a dose-related accumulation of NFC in the bronchi, the alveoli and some in the cytoplasm of macrophages. In addition, neutrophilic accumulation in the alveolar lung space was observed with increasing dose. Our findings showed that NFC administered by pharyngeal aspiration caused an acute inflammatory response and DNA damage in the lungs, but no systemic genotoxic effect in the bone marrow. The present experimental design did not, however, allow us to determine whether the responses were transient or could persist for a longer time.

Description: The influence of surface charge of nanomaterials on toxicological effects is not yet fully understood. We investigated the inflammatory response, the acute phase response and the genotoxic effect of two different titanium dioxide nanoparticles (TiO 2 NPs) following a single intratracheal instillation. NRCWE-001 was unmodified rutile TiO 2 with endogenous negative surface charge, whereas NRCWE-002 was surface modified to be positively charged. C57BL/6J BomTac mice received 18, 54 and 162 µg/mouse and were humanely killed 1, 3 and 28 days post-exposure. Vehicle controls were tested alongside for comparison. The cellular composition and protein concentration were determined in bronchoalveolar lavage (BAL) fluid as markers for an inflammatory response. Pulmonary and systemic genotoxicity was analysed by the alkaline comet assay as DNA strand breaks in BAL cells, lung and liver tissue. The pulmonary and hepatic acute phase response was analysed by Saa3 mRNA levels in lung tissue or Saa1 mRNA levels in liver tissue by real-time quantitative polymerase chain reaction. Instillation of NRCWE-001 and -002 both induced a dose-dependent neutrophil influx into the lung lining fluid and Saa3 mRNA levels in lung tissue at all assessed time points. There was no statistically significant difference between NRCWE-001 and NRCWE-002. Exposure to both TiO 2 NPs induced increased levels of DNA strand breaks in lung tissue at all doses 1 and 28 days post-exposure and NRCWE-002 at the low and middle dose 3 days post-exposure. The DNA strand break levels were statistically significantly different for NRCWE-001 and -002 for liver and for BAL cells, but no consistent pattern was observed. In conclusion, functionalisation of reactive negatively charged rutile TiO 2 to positively charged did not consistently influence pulmonary toxicity of the studied TiO 2 NPs.

Description: In a previous study, 15-nm silica nanoparticles (NPs) caused small increases in DNA damage in liver as measured in the in vivo comet and micronucleus assays after intravenous administration to rats at their maximum tolerated dose, a worst-case exposure scenario. Histopathological examination supported a particle-induced, tissue damage-mediated inflammatory response. This study used a targeted approach to provide insight into the mode of action (MoA) by examining transcriptional regulation of genes in liver in a time and dose-dependent manner at 1, 2, 4, 8 and 24 h after intravenous administration of 15-nm silica NPs. DNA damage was assessed using the standard comet assay and hOGG1 glycosylase-modified comet assay that also measures oxidative DNA damage. Potassium bromate, an IARC Class 2B carcinogen that specifically operates via an oxidative stress MoA, was used as a positive control for the hOGG1 comet assay and gave a strong signal in its main target organ, the kidney, while showing less activity in liver. Treatment of rats with silica NPs at 50 mg/kg body weight (bw) caused small, statistically insignificant increases in DNA damage in liver measured by the standard comet assay, while a statistically significant increase was observed at 4 h with the hOGG1 comet assay, consistent with a MoA involving reactive oxygen species. Histopathology showed liver damage and neutrophil involvement while genomic analysis and response pattern of key genes involved in inflammation and oxidative stress supported a tissue damage-mediated inflammatory response involving the complement system for removing/phagocytising damaged cells. No changes were observed for histopathology or gene array for the low-dose (5 mg/kg bw) silica NPs. The results of this study confirm our hypothesis that the weak DNA damage observed by silica NPs occurs secondary to inflammation/immune response, indicating that a threshold can be applied in the risk assessment of these materials.

Description: Titanium dioxide nanoparticles (TiO 2 NPs) are used in paints, plastics, papers, inks, foods, toothpaste, pharmaceuticals and cosmetics. However, TiO 2 NPs cause inflammation, pulmonary damage, fibrosis and lung tumours in animals and are possibly carcinogenic to humans. Although there are a large number of studies on the toxicities of TiO 2 NPs, the data are inconclusive and the mechanisms underlying the toxicity are not clear. In this study, we used the Comet assay to evaluate genotoxicity and whole-genome microarray technology to analyse gene expression pattern in vivo to explore the possible mechanisms for toxicity and genotoxicity of TiO 2 NPs. Mice were treated with three daily i.p. injections of 50 mg/kg 10 nm anatase TiO 2 NPs and sacrificed 4 h after the last treatment. The livers and lungs were then isolated for the Comet assay and whole genome microarray analysis of gene expression. The NPs were heavily accumulated in liver and lung tissues. However, the treatment was positive for DNA strand breaks only in liver measured with the standard Comet assay, but positive for oxidative DNA adducts in both liver and lung as determined with the enzyme-modified Comet assay. The genotoxicity results suggest that DNA damage mainly resulted from oxidised nucleotides. Gene expression profiles and functional analyses revealed that exposure to TiO 2 NPs triggered distinct gene expression patterns in both liver and lung tissues. The gene expression results suggest that TiO 2 NPs impair DNA and cells by interrupting metabolic homeostasis in liver and by inducing oxidative stress, inflammatory responses and apoptosis in lung. These findings have broad implications when evaluating the safety of TiO 2 NPs used in numerous consumer products.

Description: With rising environmental levels of carbon-based nanoparticles (CBNs), there is an urgent need to develop an understanding of their biological effects in order to generate appropriate risk assessment strategies. Herein, we exposed zebrafish via their diet to one of four different CBNs: C 60 fullerene (C 60 ), single-walled carbon nanotubes (SWCNT), short multi-walled carbon nanotubes (MWCNTs) or long MWCNTs. Lipid alterations in male and female zebrafish were explored post-exposure in three target tissues (brain, gonads and gastrointestinal tract) using ‘omic’ procedures based in liquid chromatography coupled with mass spectrometry (LC-MS) data files. These tissues were chosen as they are often target tissues following environmental exposure. Marked alterations in lipid species are noted in all three tissues. To further explore CBN-induced brain alterations, Raman microspectroscopy analysis of lipid extracts was conducted. Marked lipid alterations are observed with males responding differently to females; in addition, there also appears to be consistent elevations in global genomic methylation. This latter observation is most profound in female zebrafish brain tissues post-exposure to short MWCNTs or SWCNTs ( P 〈 0.05). This study demonstrates that even at low levels, CBNs are capable of inducing significant cellular and genomic modifications in a range of tissues. Such alterations could result in modified susceptibility to other influences such as environmental exposures, pathology and, in the case of brain, developmental alterations.

Description: Due to the steeply increased use of nanomaterials (NMs) for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. In this study, we compared the DNA-damaging properties and concurrent cytotoxicity of a panel of 10 engineered NMs in three different cell lines in relation to their intrinsic oxidant generating properties. The human epithelial cell lines A549, HK-2 and HepG2 were chosen to represent relevant target organs for NMs in the lung, kidney and liver. Cytotoxicity, evaluated by WST-1 assay in the treatment concentration range of 0.3–80 µg/cm 2 , was shown for Ag and ZnO NM in all three cell lines. Cytotoxicity was absent for all other NMs, i.e. five types of TiO 2 and two types of multiwalled carbon nanotubes. DNA damage, evaluated by the alkaline comet assay, was observed with Ag and ZnO, albeit only at cytotoxic concentrations. DNA damage varied considerably with the cell line. The oxidant generating properties of the NMs, evaluated by electron spin resonance spectroscopy in cell free conditions, did not correlate with their cytotoxic or DNA-damaging properties. DNA damage by the nanosilver could be partly attributed to its surfactant-containing dispersant. The coating of a TiO 2 sample with the commercial surfactant Curosurf augmented its DNA-damaging properties in A549 cells, while surface modification with serum tended to reduce damage. Our findings indicate that measurement of the intrinsic oxidant-generating capacity of NMs is a poor predictor of DNA damage and that the cytotoxic and DNA-damaging properties of NMs can vary substantially with experimental conditions. Our study also underlines the critical importance of selecting appropriate cell systems and aligned testing protocols. Selection of a cell line on the mere basis of its origin may provide only poor insight on organ-specific hazards of NMs.

Description: In the course of a 2-year combined chronic toxicity—carcinogenicity study performed according to Organisation for Economic Co-operation and Development (OECD) Test Guideline 453, systemic (blood cell) genotoxicity of two OECD representative nanomaterials, CeO 2 NM-212 and BaSO 4 upon 3- or 6-month inhalation exposure to rats was assessed. DNA effects were analysed in leukocytes using the alkaline Comet assay, gene mutations and chromosome aberrations were measured in erythrocytes using the flow cytometric Pig-a gene mutation assay and the micronucleus test (applying both microscopic and flow cytometric evaluation), respectively. Since nano-sized CeO 2 elicited lung effects at concentrations of 5mg/m 3 (burdens of 0.5mg/lung) in the preceding range-finding study, whereas nano-sized BaSO 4 did not induce any effect, female rats were exposed to aerosol concentrations of 0.1 up to 3mg/m 3 CeO 2 or 50mg/m 3 BaSO 4 nanomaterials (6h/day; 5 days/week; whole-body exposure). The blood of animals treated with clean air served as negative control, whereas blood samples from rats treated orally with three doses of 20mg/kg body weight ethylnitrosourea at 24h intervals were used as positive controls. As expected, ethylnitrosourea elicited significant genotoxicity in the alkaline Comet and Pig-a gene mutation assays and in the micronucleus test. By contrast, 3- and 6-month CeO 2 or BaSO 4 nanomaterial inhalation exposure did not elicit significant findings in any of the genotoxicity tests. The results demonstrate that subchronic inhalation exposure to different low doses of CeO 2 or to a high dose of BaSO 4 nanomaterials does not induce genotoxicity on the rat hematopoietic system at the DNA, gene or chromosome levels.

Description: The widespread production and use of nanoparticles calls for faster and more reliable methods to assess their safety. The main aim of this study was to investigate the genotoxicity of three reference TiO 2 nanomaterials (NM) within the frame of the FP7-NANoREG project, with a particular focus on testing the applicability of mini-gel comet assay and micronucleus (MN) scoring by flow cytometry. BEAS-2B cells cultured under serum-free conditions were exposed to NM100 (anatase, 50–150nm), NM101 (anatase, 5–8nm) and NM103 (rutile, 20–28nm) for 3, 24 or 48h mainly at concentrations 1–30 μg/ml. In the mini-gel comet assay (eight gels per slide), we included analysis of (i) DNA strand breaks, (ii) oxidised bases (Fpg-sensitive sites) and (iii) light-induced DNA damage due to photocatalytic activity. Furthermore, MN assays were used and we compared the results of more high-throughput MN scoring with flow cytometry to that of cytokinesis-block MN cytome assay scored manually using a microscope. Various methods were used to assess cytotoxic effects and the results showed in general no or low effects at the doses tested. A weak genotoxic effect of the tested TiO 2 materials was observed with an induction of oxidised bases for all three materials of which NM100 was the most potent. When the comet slides were briefly exposed to lab light, a clear induction of DNA strand breaks was observed for the anatase materials, but not for the rutile. This highlights the risk of false positives when testing photocatalytically active materials if light is not properly avoided. A slight increase in MN formation for NM103 was observed in the different MN assays at the lower doses tested (1 and 5 μg/ml). We conclude that mini-gel comet assay and MN scoring using flow cytometry successfully can be used to efficiently study cytotoxic and genotoxic properties of nanoparticles.

Description: A number of in vitro methodologies have been used to assess the genotoxicity of different nanomaterials, including titanium dioxide nanoparticles (TiO 2 NPs) and silver nanoparticles (AgNPs). The in vitro micronucleus assay is one of the most commonly used test methods for genotoxicity evaluation of nanomaterials. However, due to the novel features of nanomaterials, such as high adsorption capacity and fluorescence properties, there are unexpected interactions with experimental components and detection systems. In this study, we evaluate the interference by two nanoparticles, AgNPs and TiO 2 NPs, with the in vitro micronucleus assay system and possible confounding factors affecting cytotoxicity and genotoxicity assessment of the nanomaterials including cell lines with different p 53 status, nanoparticle coatings and fluorescence, cytochalasin B, fetal bovine serum in cell treatment medium and different measurement methodologies for detecting micronuclei. Our results showed that micronucleus induction by AgNPs was similar when evaluated using flow cytometry or microscope, whereas the induction by TiO 2 NPs was different using the two methods due to TiO 2 ’s fluorescence interference with the cytometry equipment. Cells with the mutated p 53 gene were more sensitive to micronucleus induction by AgNPs than the p 53 wild-type cells. The presence of serum during treatment increased the toxicity of AgNPs. The coatings of nanoparticles played an important role in the genotoxicity of AgNPs. These collective data highlight the importance of considering the unique properties of nanoparticles in assessing their genotoxicity using the in vitro micronucleus assay.

Description: For some decades production of titanium dioxide nanoparticle (TiO 2 -NP) has been increasing at a considerable rate; concerns as to the toxicity of these particles upon inhalation have been raised. Indeed, TiO 2 -NPs have been shown to induce significant genotoxicity and to adversely affect both major DNA repair mechanisms: base excision repair (BER) and nucleotide excision repair (NER). The aims of the present study were to (i) compare the genotoxicity of TiO 2 -NPs and their impact on DNA repair processes on A549 alveolar carcinoma and BEAS-2B normal bronchial lung cell lines and (ii) delve deeper into the mechanisms leading to these effects. To achieve these goals, TiO 2 -NPs effects on cytotoxicity, genotoxicity, DNA repair activity and DNA repair gene expression were investigated in both cell lines upon exposure to 1–100 µg/mL of anatase/rutile, 21 nm TiO 2 -NPs. Our results show that TiO 2 -NPs induce comparable cytotoxic and genotoxic responses in BEAS-2B and A549 cells. Functional response to DNA damage is observed in both cell lines and consists of an overall downregulation in DNA repair processes. When evaluating the relative importance of the two DNA repair pathways, we observed a lower impact on BER compared with NER activities, suggesting that repair of oxidatively generated DNA damage is still triggered in these cells. This response becomes measureable at 4 h of exposure in BEAS-2B but only after 48 h of exposure in A549 cells. The delayed response in A549 cells is due to an initial overall and intense downregulation of the genes encoding DNA repair proteins. This overall downregulation correlates with increased methylation of DNA repair gene promoters and downregulation of NRF2 and BRCA1, which may thus be considered as upstream regulators. These results strengthen the evidence that TiO 2 -NP induces indirect genotoxicity in lung cells, via modulation of DNA repair processes, and shed some light on the mechanisms behind this effect.

Description: One type of carbon nanotubes (CNTs) (MWCNT-7, from Mitsui) has been classified as probably carcinogenic to humans, however insufficient data does not warrant the same classification for other types of CNTs. Experimental data indicate that CNT exposure can result in oxidative stress and DNA damage in cultured cells, whereas these materials appear to induce low or no mutagenicity. Therefore, the present study aimed to investigate whether in vitro exposure of cultured airway epithelial cells (A549) to multi-walled CNTs (MWCNTs) could increase the DNA repair activity of oxidatively damaged DNA and drive the cells toward replicative senescence, assessed by attrition of telomeres. To investigate this, H 2 O 2 and KBrO 3 were used to induce DNA damage in the cells and the effect of pre-exposure to MWCNT tested for a change in repair activity inside the cells or in the extract of treated cells. The effect of MWCNT exposure on telomere length was investigated for concentration and time response. We report a significantly increased repair activity in A549 cells exposed to MWCNTs compared to non-exposed cells, suggesting that DNA repair activity may be influenced by exposure to MWCNTs. The telomere length was decreased at times longer than 24h, but this decrease was not concentration dependent. The results suggest that the seemingly low mutagenicity of CNTs in cultured cells may be associated with an increased DNA repair activity and a replicative senescence, which may counteract the manifestation of DNA lesions to mutations.

Description: We used the marine bivalve ( Mytilus galloprovincialis ) to assess a range of biological or biomarker responses following exposure to a model-engineered nanoparticle, C 60 fullerene, either alone or in combination with a model polycyclic aromatic hydrocarbon, benzo(α)pyrene [B(α)P]. An integrated biomarker approach was used that included: (i) determination of ‘clearance rates’ (a physiological indicator at individual level), (ii) histopathological alterations (at tissue level), (iii) DNA strand breaks using the comet assay (at cellular level) and (iv) transcriptional alterations of p53 (anti-oncogene) and ras (oncogene) determined by real-time quantitative polymerase chain reaction (at the molecular/genetic level). In addition, total glutathione in the digestive gland was measured as a proxy for oxidative stress. Here, we report that mussels showed no significant changes in ‘clearance rates’ after 1 day exposure, however significant increases in ‘clearance rates’ were found following exposure for 3 days. Histopathology on selected organs (i.e. gills, digestive glands, adductor muscles and mantles) showed increased occurrence of abnormalities in all tissues types, although not all the exposed organisms showed these abnormalities. Significantly, increased levels of DNA strand breaks were found after exposure for 3-days in most individuals tested. In addition, a significant induction for p53 and ras expression was observed in a tissue and chemical-specific pattern, although large amounts of inter-individual variability, compared with other biomarkers, were clearly apparent. Overall, biological responses at different levels showed variable sensitivity, with DNA strand breaks and gene expression alterations exhibiting higher sensitivities. Furthermore, the observed genotoxic responses were reversible after a recovery period, suggesting the ability of mussels to cope with the toxicants C 60 and/or B(α)P under our experimental conditions. Overall, in this comprehensive study, we have demonstrated mussels as a suitable model marine invertebrate species to study the potential detrimental effects induced by possible genotoxicants and toxicants, either alone or in combinations at different levels of biological organisation (i.e. molecular to individual levels).

Description: Since 1969, the European Union approves food-grade titanium dioxide (TiO 2 ), also known as E171 colouring food additive. E171 is a mixture of micro-sized particles (MPs) and nano-sized particles (NPs). Previous studies have indicated adverse effects of oral exposure to E171, i.e. facilitation of colon tumour growth. This could potentially be partially mediated by the capacity to induce reactive oxygen species (ROS). The aim of the present study is to determine whether E171 exposure induces ROS formation and DNA damage in an in vitro model using human Caco-2 and HCT116 cells and to investigate the contribution of the separate MPs and NPs TiO 2 fractions to these effects. After suspension of the particles in Hanks' balanced salt solution buffer and cell culture medium with either bovine serum albumin (BSA) or foetal bovine serum, characterization of the particles was performed by dynamic light scattering, ROS formation was determined by electron spin/paramagnetic resonance spectroscopy and DNA damage was determined by the comet and micronucleus assays. The results showed that E171, MPs and NPs are stable in cell culture medium with 0.05% BSA. The capacity for ROS generation in a cell-free environment was highest for E171, followed by NPs and MPs. Only MPs were capable to induce ROS formation in exposed Caco-2 cells. E171, MPs and NPs all induced single-strand DNA breaks. Chromosome damage was shown to be induced by E171, as tested with the micronucleus assay in HCT116 cells. In conclusion, E171 has the capability to induce ROS formation in a cell-free environment and E171, MPs and NPs have genotoxic potential. The capacity of E171 to induce ROS formation and DNA damage raises concerns about potential adverse effects associated with E171 (TiO 2 ) in food.

Description: The rapid development of the engineered nanomaterial (ENM) manufacturing industry has accelerated the incorporation of ENMs into a wide variety of consumer products across the globe. Unintentionally or not, some of these ENMs may be introduced into the environment or come into contact with humans or other organisms resulting in unexpected biological effects. It is thus prudent to have rapid and robust analytical metrology in place that can be used to critically assess and/or predict the cytotoxicity, as well as the potential genotoxicity of these ENMs. Many of the traditional genotoxicity test methods [e.g. unscheduled DNA synthesis assay, bacterial reverse mutation (Ames) test, etc.,] for determining the DNA damaging potential of chemical and biological compounds are not suitable for the evaluation of ENMs, due to a variety of methodological issues ranging from potential assay interferences to problems centered on low sample throughput. Recently, a number of sensitive, high-throughput genotoxicity assays/platforms (CometChip assay, flow cytometry/micronucleus assay, flow cytometry/-H2AX assay, automated ‘Fluorimetric Detection of Alkaline DNA Unwinding’ (FADU) assay, ToxTracker reporter assay) have been developed, based on substantial modifications and enhancements of traditional genotoxicity assays. These new assays have been used for the rapid measurement of DNA damage (strand breaks), chromosomal damage (micronuclei) and for detecting upregulated DNA damage signalling pathways resulting from ENM exposures. In this critical review, we describe and discuss the fundamental measurement principles and measurement endpoints of these new assays, as well as the modes of operation, analytical metrics and potential interferences, as applicable to ENM exposures. An unbiased discussion of the major technical advantages and limitations of each assay for evaluating and predicting the genotoxic potential of ENMs is also provided.

Description: With the need to understand the potential biological impact of the plethora of nanoparticles (NPs) being manufactured for a wide range of potential human applications, due to their inevitable human exposure, research activities in the field of NP toxicology has grown exponentially over the last decade. Whilst such increased research efforts have elucidated an increasingly significant knowledge base pertaining to the potential human health hazard posed by NPs, understanding regarding the possibility for NPs to elicit genotoxicity is limited. In vivo models are unable to adequately discriminate between the specific modes of action associated with the onset of genotoxicity. Additionally, in line with the recent European directives, there is an inherent need to move away from invasive animal testing strategies. Thus, in vitro systems are an important tool for expanding our mechanistic insight into NP genotoxicity. Yet uncertainty remains concerning their validity and specificity for this purpose due to the unique challenges presented when correlating NP behaviour in vitro and in vivo . This review therefore highlights the current state of the art in advanced in vitro systems and their specific advantages and disadvantages from a NP genotoxicity testing perspective. Key indicators will be given related to how these systems might be used or improved to enhance understanding of NP genotoxicity.

Description: This Mutagenesis special issue is on the topic of nanogenotoxicology. It unites a collection of reports that provide insight into: (i) the properties of engineered nanomaterials (ENMs) that contribute to genotoxicity, (ii) the genotoxic mechanisms associated with DNA damage observed in both in vitro and in vivo tests and (iii) the future test systems that will provide more accurate prediction of ENM genotoxicity to support regulatory hazard assessment frameworks. The contributions within therefore provide collective oversight of our current understanding, coupled to future perspectives aimed at overcoming technical hurdles and describing novel analytical methods to further advance the field.

Description: Titanium dioxide nanoparticles (TiO 2 NPs) induce lung inflammation in experimental animals. In this study, we conducted a comprehensive toxicogenomic analysis of lung responses in mice exposed to six individual TiO 2 NPs exhibiting different sizes (8, 20 and 300nm), crystalline structure (anatase, rutile or anatase/rutile) and surface modifications (hydrophobic or hydrophilic) to investigate whether the mechanisms leading to TiO 2 NP-induced lung inflammation are property specific. A detailed histopathological analysis was conducted to investigate the long-term disease implications of acute exposure to TiO 2 NPs. C57BL/6 mice were exposed to 18, 54, 162 or 486 µg of TiO 2 NPs/mouse via single intratracheal instillation. Controls were exposed to dispersion medium only. Bronchoalveolar lavage fluid (BALF) and lung tissue were sampled on 1, 28 and 90 days post-exposure. Although all TiO 2 NPs induced lung inflammation as measured by the neutrophil influx in BALF, rutile-type TiO 2 NPs induced higher inflammation with the hydrophilic rutile TiO 2 NP showing the maximum increase. Accordingly, the rutile TiO 2 NPs induced higher number of differentially expressed genes. Histopathological analysis of lung sections on Day 90 post-exposure showed increased collagen staining and fibrosis-like changes following exposure to the rutile TiO 2 NPs at the highest dose tested. Among the anatase, the smallest TiO 2 NP of 8nm showed the maximum response. The anatase TiO 2 NP of 300nm was the least responsive of all. The results suggest that the severity of lung inflammation is property specific; however, the underlying mechanisms (genes and pathways perturbed) leading to inflammation were the same for all particle types. While the particle size clearly influenced the overall acute lung responses, a combination of small size, crystalline structure and hydrophilic surface contributed to the long-term pathological effects observed at the highest dose (486 µg/mouse). Although the dose at which the pathological changes were observed is considered physiologically high, the study highlights the disease potential of certain TiO 2 NPs of specific properties.

Description: There is serious concern about the potential harmful effects of certain nanomaterials (NMs), on account of their ability to penetrate cell membranes and the increased reactivity that results from their increased surface area compared with bulk chemicals. To assess the safety of NMs, reliable tests are needed. We have investigated the possible genotoxicity of four representative NMs, derived from titanium dioxide, zinc oxide, cerium oxide and silver, in two human cell lines, A549 alveolar epithelial cells and lymphoblastoid TK6 cells. A high-throughput version of the comet assay was used to measure DNA strand beaks (SBs) as well as oxidised purines (converted to breaks with the enzyme formamidopyrimidine DNA glycosylase). In parallel, cytotoxicity was measured with the alamarBlue® assay, and the ability of NM-treated cells to survive was assessed by their colony-forming efficiency. TiO 2 and CeO 2 NMs were only slightly cytotoxic by the alamarBlue® test, and had no long-term effect on colony-forming efficiency. However, both induced DNA damage at non-cytotoxic concentrations; the damage decreased from 3 to 24-h exposure, except in the case of CeO 2 -treated A549 cells. ZnO and Ag NMs affected cell survival, and induced high levels of DNA damage at cytotoxic concentrations. At lower concentrations, there was significant damage, which tended to persist over 24 h. The implication is that all four reference metal NMs tested—whether cytotoxic or not—are genotoxic. A full assessment of NM toxicity should include tests on different cell types, different times of incubation and a wide range of (especially non-cytotoxic) concentrations; a test for cell viability should be performed in parallel. Inclusion of Fpg in the comet assay allows detection of indirect genotoxic effects via oxidative stress.

Description: Carbon nanotubes (CNTs) are fibrous carbon-based nanomaterials with a potential to cause carcinogenesis in humans. Alterations in DNA methylation on cytosine–phosphate–guanidine (CpG) sites are potential markers of exposure-induced carcinogenesis. This study examined cytotoxicity, genotoxicity and DNA methylation alterations on human monocytic cells (THP-1) after incubation with single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs). Higher cytotoxicity and genotoxicity were observed after incubation with SWCNTs than incubation with MWCNTs. At the selected concentrations (25 and 100 µg/ml), DNA methylation alterations were studied. Liquid chromatography–mass spectrometry (LC-MS/MS) was used to assess global DNA methylation, and Illumina 450K microarrays were used to assess methylation of single CpG sites. Next, we assessed gene promoter-specific methylation levels. We observed no global methylation or hydroxymethylation alterations, but on gene-specific level, distinct clustering of CNT-treated samples were noted. Collectively, CNTs induced gene promoter-specific altered methylation and those 1127 different genes were identified to be hypomethylated. Differentially methylated genes were involved in several signalling cascade pathways, vascular endothelial growth factor and platelet activation pathways. Moreover, possible contribution of the epigenetic alterations to monocyte differentiation and mixed M1/M2 macrophage polarisation were discussed.

Description: Nowadays engineered nanomaterials (ENMs) are increasingly used in a wide range of commercial products and biomedical applications. Despite this, the knowledge of human potential health risk as well as comprehensive biological and toxicological information is still limited. We have investigated the capacity of two frequently used metallic ENMs, nanosilver and magnetite nanoparticles (MNPs), to induce thymidine kinase ( Tk +/– ) mutations in L5178Y mouse lymphoma cells and transformed foci in Bhas 42 cells. Two types of nanosilver, spherical nanoparticles (AgNM300) and fibrous (AgNM302) nanorods/wires, and MNPs differing in surface modifications [MNPs coated with sodium oleate (SO-MNPs), MNPs coated with SO + polyethylene glycol (SO-PEG-MNPs) and MNPs coated with SO + PEG + poly(lactide- co -glycolic acid) SO-PEG-PLGA-MNPs] were included in this study. Spherical AgNM300 showed neither mutagenic nor carcinogenic potential. In contrast, silver nanorods/wires (AgNM302) increased significantly the number of both gene mutations and transformed foci compared with the control (untreated) cells. Under the same treatment conditions, neither SO-MNPs nor SO-PEG-PLGA-MNPs increased the mutant frequency compared with control cells though an equivocal mutagenic effect was estimated for SO-PEG-MNPs. Although SO-MNPs and SO-PEG-MNPs did not show any carcinogenic potential, SO-PEG-PLGA-MNPs increased concentration dependently the number of transformed foci in Bhas 42 cells compared with the control cells. Our results revealed that fibrous shape underlies the mutagenic and carcinogenic potential of nanosilver while surface chemistry affects the biosafety of MNPs. Considering that both nanosilver and MNPs are prospective ENMs for biomedical applications, further toxicological evaluations are warranted to assess comprehensively the biosafety of these nanomaterials.

Description: The potential health effects of exposure to nanomaterials (NMs) is currently heavily studied. Among the most often reported impact is DNA damage, also termed genotoxicity. While several reviews relate the DNA damage induced by NMs and the techniques that can be used to prove such effects, the question of impact of NMs on DNA repair processes has never been specifically reviewed. The present review article proposes to fill this gap of knowledge by critically describing the DNA repair processes that could be affected by nanoparticle (NP) exposure, then by reporting the current state of the art on effects of NPs on DNA repair, at the level of protein function, gene induction and post-transcriptional modifications, and taking into account the advantages and limitations of the different experimental approaches. Since little is known about this impact, working hypothesis for the future are then proposed.

Description: Micronucleus (MN) assay is extensively used to biomonitor aneuploidy and clastogenicity of genotoxic compounds. However, the suitability of this assay for early life stages of model fish species is still poorly documented. In this study, the determination of MN using flow cytometry was successfully applied for the first time to zebrafish ( Danio rerio ) larvae. Mitomycin C (MMC), etoposide (ETO), cyclophosphamide, demecolcine (COL), benzo[ a ]pyrene (BP) and dibenzo[ def,p ]chrysene (DBC) were selected as model genotoxicants. The method was first confirmed in human HepG2 liver cells and then applied in vivo on isolated cells from exposed 4 days post fertilisation zebrafish larvae. All tested compounds induced MN formation. The flow cytometry results were validated by a strong correlation with results from a standard MN microscopy analysis ( P = 0.002). Moreover, flow cytometry analysis enabled the detection of an up to 3.7-fold increase of hypodiploidy in zebrafish exposed to MMC, COL, BP or DBC. MMC, COL and DBC induced more than a 2-fold MN increase by flow cytometry and were therefore considered as the most suitable positive controls for in vivo zebrafish MN determination. These findings make important contribution by proposing a new reliable and sensitive method for using zebrafish as a model for genotoxicity monitoring.

Description: Swiss albino mice were exposed to formulated cypermethrin (CMR) and/or or chlorpyrifos (CPF) through oral gavages for 60 days. Test doses of CMR (0.69, 1.38 or 2.76mg/kg/day) or CPF (0.5, 1.0 or 2.0mg/kg/day) or CMR + CPF (0.69 + 0.5, 1.38 + 1.0 or 2.76 + 2.0mg/kg/day) were based on the acute oral median lethal doses of CMR or CPF. Chromosome aberrations (CA), micronucleus (MN) induction, cell cycle perturbations, apoptosis and reactive oxygen species (ROS) generation were analysed in bone marrow cells. To explore the involvement of ROS induction, HaCat cells were exposed in vitro to arbitrary concentrations of CMR and/or CPF. Exposure of CMR (2.76mg/kg/day) induced significant inhibition of mitotic index. Significant ( P 〈 0.01) frequencies of CA and MN were observed with the CMR at 1.38mg/kg/day, whereas CPF or its mixture CMR + CPF showed at highest doses. Chromosome/chromatid breaks and fragments were found to be major aberrations in all the treatment groups. Highest doses of CMR or CMR + CPF revealed significant ( P 〈 0.01 or 0.001) elevation of G 0 /G 1 peak, while CPF-exposed cells revealed significant ( P 〈 0.01) declined in G 1 phase. Decline in S phase was observed with highest dose of CMR only. Apoptosis induction measured by gating cell population beside G 1 peak showed 3- to 4-fold increase in apoptotic cells in CPF-exposed mice as compared to control or CMR or CMR + CPF-treated mice. Further, all the treatment groups in vivo as well as in vitro revealed significant generation of ROS in comparison with the control group. Present results, together with the earlier reports, which substantiate ROS generation may be major cause of genotoxicity, cell cycle perturbations and apoptosis, nonetheless co-exposure of low doses of CMR and CPF mixture does not potentiate genotoxicity.

Description: Coal miners are exposed to coal dust, containing mineral particles, inorganic compounds and polycyclic aromatic hydrocarbons, and to ionizing radiation. These factors can induce oxidative stress and promote inflammation that leads to DNA damage. The aim of this investigation is to analyse the degree of DNA damage in miners working in underground coal mines in Kemerovo Region (Russian Federation) using the cytokinesis-block micronucleus assay (CBMN) in peripheral blood lymphocytes. The exposed group included 143 coal miners (mean age = 50.11±7.36 years; mean length of service in coal mining conditions = 23.26±9.66 years). As a control group, we have used venous blood extracted from 127 healthy non-exposed men. The mean age in this group was 47.67±8.45 years. We have discovered that coal miners are characterized by a significant increase in the frequency of binucleated lymphocytes with micronuclei (MN), nucleoplasmic bridges (NPBs) and protrusions (NBUDs) compared to non-exposed donors. In addition, we report, for the first time, a reduction of cell proliferation in a cohort of coal miners. These data are evidence of the genotoxic and cytostatic effects of occupational harmful factors of the coal mining industry. No correlation between the level of chromosome damage and age, smoking status or length of service in coal mining conditions were discovered. We suggest that the CBMN assay would be useful in biomonitoring studies to monitor hygiene and prevention strategies in occupational settings in coal mining countries.

Description: We have previously reported that flocculation of a yeast co-transformed with the human DNA methyltransferase 1 ( DNMT1 ) and DNMT3B genes was inhibited by DNMT inhibitors. It is well known that epigenetic mutagens can disturb nucleosome positioning via DNA methylation and/or histone modification. In this study we first examined the effects of trichostatin A (TSA), a histone deacetylase inhibitor, on the flocculation level of yeast. TSA dose-dependently promoted the flocculation exhibited by the yeast transformed with the DNMT genes or empty vectors. Furthermore, TSA induced the expression of the flocculin-encoding gene FLO1 . The anthracene-derived alizarin, a natural madder root dye, has a potential for carcinogenesis promotion; however, the mode of action has not been elucidated. It is considered that epigenetic changes can promote cancer. Alizarin but not anthracene enhanced the flocculation level of the yeast. Similar to TSA, alizarin also upregulated FLO1 mRNA. Surprisingly, western blotting indicated that alizarin, but not anthracene, reduced the level of histone H3 in yeast, and alizarin-treated cells frequently displayed abnormally shaped nuclei. These findings suggest that alizarin uniquely influences nucleosome structure. Taken together with our previous findings, this study suggests that the DNMT gene-transformed yeast strains are a useful tool for screening various classes of epigenetic mutagens.

Description: The increasing incidence of type 2 diabetes mellitus globally has increased the incidence of diabetes-associated complications such as nephropathy. DNA damage induced by oxidative stress might be one of the important mechanisms in the pathogenesis of diabetic complications. Two hundred Iranian individuals with the conditions of type 2 diabetes, diabetic nephropathy and nephropathy patients with no sign of diabetes and normal unaffected sex- and age-matched controls (50 in each group) were enrolled in the study. The background and the net levels of micronucleus (MN) formation as well as other cellular damages induced after in vitro treatment with 25 µg/ml of bleomycin (BLM) were evaluated using cytokinesis block MNs cytome assay (CBMN cyt) in peripheral blood lymphocytes. The background and net BLM-induced levels of MNs were significantly higher in all patient groups compared with the control ( P 〈 0.01, P 〈 0.001, respectively). The frequency of MNs was significantly higher in those patients with prior incidence of nephropathy than those without. A positive association was observed between basal and net MN frequency among study groups and also between net genetic damages and serum creatinine value and duration of diabetes. The rate of basal and net apoptosis was significantly higher in patients with hyperglycemia. Our results indicate that increased genomic instability expressed as MNs is associated with nephropathy in all pathological stages. Therefore, implementation of MN assay in clinical level may potentially enhance the quality of management of patients with diabetes and its complications such as nephropathy.

Description: RUNX2 is an important osteo-specific factor with crucial functions in bone formation and remodelling as well as resorption of teeth. Heterozygous mutation of RUNX2 can cause cleidocranial dysplasia (CCD), a systemic disease with extensive skeletal dysplasia and abnormality of tooth growth. In our study, dental follicle cells (DFCs) and periodontal ligament cells (PDLCs) were isolated, cultured and identified from one patient with CCD and compared with normal controls. This CCD patient was confirmed to have a heterozygous frameshift mutation of RUNX2 (c.514delT, p.Ser172fs) in the previous study. The results showed that the proliferation abilities of DFCs and PDLCs were both disturbed by the RUNX2 mutation in the CCD patient compared with the normal control. A co-culture system of these cells with human peripheral blood mononuclear cells was then used to investigate the effect of RUNX2 mutation on osteoclastogenesis. We found that the RUNX2 mutation in CCD reduced the expression of osteoclast-related genes, such as RUNX2 , CTR , CTSK , RANKL and OPG . The ability of osteoclastogenesis in DFCs and PDLCs detected by tartrate-resistant acid phosphatase staining in the co-culture system was also reduced by the RUNX2 mutation compared with the normal control. These outcomes indicate that the RUNX2 mutation disturbs the modulatory effects of DFCs and PDLCs on the differentiation of osteoclasts and osteoblasts, thereby interfering with bone remodelling. These effects may contribute in part to the pathological manifestations of retention of primary teeth and delayed eruption of permanent teeth in patients with CCD.

Description: Arsenic (AS) is a reactive oxygen species (ROS)-inducer carcinogen, whose mode of action is still unclear. To defend against ROS, cells use enzymatic and non-enzymatic antioxidants, such as superoxide dismutase (SOD) and catalase. Failure of antioxidant systems (AXS) can result in dicentric chromosomes formation as well as telomere associations for the reduced activity of telomerase. In order to clarify the long-term effects of a past AS exposure, we evaluated the efficiency of the AXS and the telomerase activity in the progeny of arsenite-treated cells named ASO (arsenic shake-off) cells, previously obtained from arsenite-treated V79 cells and selected by shake-off. Despite SOD 1 expression level correlated to the level of ROS observed over time, no changes of the relative amount of antioxidant activities were observed in ASO cells. Moreover, we found that clones characterised by low levels of SOD 1 and high levels of ROS acquired a transformed phenotype. Treatment with 5-azacytidine determined an increase of SOD 1 expression in a clone and decrease in one other, suggesting that aberrant DNA methylation may be responsible for the abnormal expression of SOD 1 or SOD 1 inhibitor genes in different clones. TRAP assay results showed that the progeny of arsenite-treated cells were characterised by a time-dependent decrease of telomerase activity. Integrated results suggest that the increases of ROS levels are accompanied by defective telomerase activity. Finally, we propose that cells escaping the arsenite-induced death perpetuated the memory of past exposure via ROS likely because antioxidant and telomerase activity impairment and ultimately acquire a transformed phenotype.

Description: Oxidation-induced damage to DNA can cause mutations, phenotypic changes and apoptosis. Agents that oppose such damage offer potential therapies for disease prevention. Vitamin D administration reportedly lowered DNA damage in type 2 diabetic mice, and higher DNA damage was reported in mononuclear cells of severely asthmatic patients who were vitamin D deficient. We hypothesised that lower vitamin D status associates with higher oxidation-induced DNA damage. Vitamin D deficiency (plasma 25(OH)D 〈 50 nmol/l) is highly prevalent worldwide, and association with DNA damage has high potential importance and impact in regard to the future health of vitamin D deficient young adults. In this study, oxidation-induced DNA damage in peripheral lymphocytes of 121 young (18–26 years) adults was measured using the formamidopyrimidine DNA glycosylase (FPG)-assisted comet assay. Plasma 25(OH)D was measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Correlational analysis was performed between 25(OH)D and DNA damage. Differences in DNA damage across tertiles of 25(OH)D were explored using analysis of variance. DNA damage in those with 25(OH)D 〈50 nmol and ≥50 nmol/l was compared using the unpaired t-test. Mean (SD) DNA damage (as %DNA in comet tail) and plasma 25(OH)D were, respectively, 18.58 (3.39)% and 44.7 (13.03) nmol/l. Most (82/121; 68%) of the subjects were deficient in vitamin D (25(OH)D 〈50nmol/l). No significant correlation was seen between 25(OH)D and DNA damage ( r = –0.0824; P 〉 0.05). No significant difference was seen across 25(OH)D tertiles: mean (SD) %DNA in comet tail/25(OH)D nmol/l values in lowest, middle and highest tertiles were, respectively, 18.64 (3.30)/31.6 (4.4), 18.90 (3.98)/42.9 (3.5), 18.19 (2.84)/59.9 (8.5), nor across the binary divide: 18.73 (3.63)% in 〈50nmol/l group vs. 18.27 (2.84)% in the ≥50 nmol/l group. No association between vitamin D and oxidation-induced DNA damage was observed, but vitamin D deficiency was highly prevalent in the young adults studied, and we cannot rule out an ameliorative effect of correction of vitamin D deficiency on DNA damage.

Description: Human endogenous retrovirus (HERV) sequences make up ~8% of the human genome and increased expression of some HERV proteins has been observed in various pathologies including leukaemia and multiple sclerosis. However, little is known about the function of these HERV proteins or environmental factors which regulate their expression. Silver nanoparticles (AgNPs) are used very extensively as antimicrobials and antivirals in numerous consumer products although their effect on the expression of HERV gene products is unknown. Cell proliferation and cell toxicity assays were carried out on human acute T lymphoblastic leukaemia (MOLT-4) and Fanconi anaemia associated acute myeloid leukaemia (FA-AML1) cells treated with two different sizes of AgNPs (7nm and 50nm diameter). Reverse-transcriptase polymerase chain reaction and western blotting were then used to the assess expression of HERV-W syncytin-1 mRNA and protein in these cells. FA-AML1 cells were more sensitive overall than MOLT-4 to treatment with the smaller 7nm sized AgNp’s being the most toxic in these cells. MOLT-4 cell were more resistant and showed no evidence of differential toxicity to the different sized particles. Syncytin-1 mRNA and protein were induced by both 7 and 50nm AgNPs in both cell types yet with different kinetics. In summary, the observation that AgNPs induce expression of syncytin-1 in FA-AML1 and MOLT-4 cells at doses as little as 5 µg/ml is grounds for concern since this protein is up-regulated in both malignant and neurodegenerative diseases. Considering the widespread use of AgNPs in the environment it is clear that their ability to induce syncytin-1 should be investigated further in other cell types.