Mutagenesis - Advance Access

Genotoxic effects of neutrophils and hypochlorous acid

Thu, 05 Nov 2009 04:48:00 PST

Chronic inflammation has been recognized as a contributing factor in the pathogenesis of lung cancer. In this process, reactive oxygen species released by neutrophils may play an important role. The aim of the present study was to investigate the capacity of the major neutrophilic oxidant hypochlorous acid (HOCl), which is formed by myeloperoxidase (MPO), to induce DNA damage and mutagenicity in lung cells. HOCl was mutagenic in lung epithelial A549 cells in vitro, showing at physiological concentrations a significant induction of mutations in the HPRT gene. We studied three major types of DNA lesions that could be relevant for this HOCl-induced mutagenicity. Single strand DNA breakage and 8-oxo-7,8-dihydro-2'-deoxyguanosine were not found to be increased following HOCl treatment. On the other hand, HOCl caused a significant increase in the formation of 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-]purin-10(3H)-one (M₁dG), which can be formed by either malondialdehyde (MDA) or base propenals. We observed an increased MDA formation upon exposure of A549 cells to HOCl, but a role of base propenals cannot be excluded. In line with this, we observed 4-fold increased M₁dG adduct levels in mice that were intratracheally instilled with lipopolysaccharide to induce a pulmonary inflammation with neutrophil influx. Depletion of circulating neutrophils significantly reduced pulmonary MPO activity as well as M₁dG adducts levels, thereby providing a causal link between neutrophils/HOCl and pulmonary genotoxicity in vivo. Taken together, these data indicate that MPO catalysed formation of HOCl during lung inflammation should be considered as a significant source of neutrophil-induced genotoxicity.

Lethal and mutagenic properties of MMS-generated DNA lesions in Escherichia coli cells deficient in BER and AlkB-directed DNA repair

Thu, 05 Nov 2009 04:47:59 PST

Methylmethane sulphonate (MMS), an S_N2-type alkylating agent, generates DNA methylated bases exhibiting cytotoxic and mutagenic properties. Such damaged bases can be removed by a system of base excision repair (BER) and by oxidative DNA demethylation catalysed by AlkB protein. Here, we have shown that the lack of the BER system and functional AlkB dioxygenase results in (i) increased sensitivity to MMS, (ii) elevated level of spontaneous and MMS-induced mutations (measured by argE3 -> Arg⁺ reversion) and (iii) induction of the SOS response shown by visualization of filamentous growth of bacteria. In the xth nth nfo strain additionally mutated in alkB gene, all these effects were extreme and led to ‘error catastrophe’, resulting from the presence of unrepaired apurinic/apyrimidinic (AP) sites and 1-methyladenine (1meA)/3-methylcytosine (3meC) lesions caused by deficiency in, respectively, BER and AlkB dioxygenase. The decreased level of MMS-induced Arg⁺ revertants in the strains deficient in polymerase V (PolV) (bearing the deletion of the umuDC operon), and the increased frequency of these revertants in bacteria overproducing PolV (harbouring the pRW134 plasmid) indicate the involvement of PolV in the error-prone repair of 1meA/3meC and AP sites. Comparison of the sensitivity to MMS and the induction of Arg⁺ revertants in the double nfo alkB and xth alkB, and the quadruple xth nth nfo alkB mutants showed that the more AP sites there are in DNA, the stronger the effect of the lack of AlkB protein. Since the sum of MMS-induced Arg⁺ revertants in xth, nfo and nth xth nfo and alkB mutants is smaller than the frequency of these revertants in the BER^– alkB^– strain, we consider two possibilities: (i) the presence of AP sites in DNA results in relaxation of its structure that facilitates methylation and (ii) additional AP sites are formed in the BER^– alkB^– mutants.

DNA damage detected by the alkaline comet assay in the liver of mice after oral administration of tetrachloroethylene

Cederberg, H., Henriksson, J., Binderup, M.-L. — Thu, 05 Nov 2009 04:47:58 PST

Induction of DNA damage in the liver and kidney of male CD1 mice was studied by means of the alkaline Comet assay after oral administration of tetrachloroethylene at the doses of 1000 and 2000 mg/kg/day. A statistically significant dose-related increase in tail intensity was established in hepatocytes, indicating that tetrachloroethylene induced DNA damage in the liver. No effect on DNA damage was observed in the kidney. The results are in agreement with carcinogenicity data in mice, in which tetrachloroethylene induced tumours in the liver but not in the kidney, and support that a genotoxic mode of action might be involved in liver carcinogenicity in mice. An alternative interpretation of the results conveyed by the Study director at the test facility, involving that tetrachloroethylene did not induce DNA damage in the liver and kidney of mice, is also presented and discussed.

Effects of O6-methylguanine-DNA methyltransferase (MGMT) polymorphisms on cancer: a meta-analysis

Thu, 05 Nov 2009 04:47:57 PST

O⁶-methylguanine-DNA methyltransferase is one of the rare proteins to directly remove alkylating agents in the human DNA direct reversal repair pathway. Its two common single-nucleotide polymorphisms, Leu84Phe and Ile143Val, had previously been identified to contribute to susceptibility of cancer. However, there are conflicting results in studies on the association of the two polymorphisms with cancer. Therefore, we conducted a meta-analysis to clarify the paradox with a large collected sample (13 069 cancer patients and 20 290 controls). We found significant association between the T allele (84Phe) and cancer risk, under the recessive genetic model [P = 0.023, odds ratio (OR) = 1.251, 95% confidence interval (CI) 1.031–1.517, P_{heterogeneity} = 0.270], TT versus CC comparison (P = 0.035, OR = 1.239, 95% CI 1.015–1.511, P_{heterogeneity} = 0.225) and TT versus CT comparison (P = 0.007, OR = 1.292, 95% CI 1.071–1.559, P_{heterogeneity} = 0.374), using the random-effect model. In the ethnicity subgroup analysis, a significant association with cancer among Caucasians was found under the recessive genetic model, homozygote comparison and TT versus TC comparison. In the tumour sites subgroup analysis, only the protective effects of Leu84Phe polymorphism were found in colorectal cancer, under CT versus CC comparison. No significant association between the G allele of Ile143Val and cancer risk was found. The G allele showed an increased lung cancer risk under the dominant genetic model and AG versus AA comparison in all Hardy–Weinberg equilibrium subjects, only when the fixed-effect model was used. However, it was insignificant in the random-effect model.

A review of biomonitoring studies measuring genotoxicity in humans exposed to hair dyes

Preston, R. J., Skare, J. A., Aardema, M. J. — Thu, 05 Nov 2009 04:47:55 PST

Hair dye ingredients frequently produce positive results in short-term in vitro genotoxicity tests, although results from in vivo assays are typically negative, especially for ingredients in use today. The use of hair dyes is quite widespread resulting in the exposure both for persons working in hairdressing salons and for individuals who have their hair dyed. This provides the opportunity to add to the data from standard in vitro and in vivo genotoxicity tests by investigating whether or not genotoxic responses are detected in such exposed individuals. A number of biomonitoring studies of humans exposed to hair dyes have been conducted using either cytogenetic alterations or DNA damage as measures of genotoxicity, or urine mutagenicity as a measure of exposure to genotoxic compounds. In this paper, each study is critically reviewed and interpreted. Overall, there is no consistent evidence of genotoxicity in humans exposed to hair dyes occupationally or through individual use.

DNA strand breakage and lipid peroxidation after exposure to welding fumes in vivo

Chuang, C.-H., Huang, C.-E., Chen, H.-L. — Sun, 01 Nov 2009 22:50:32 PST

A remarkable number of complex aerosols are generated from welding processes. The objective of this study was to compare DNA damage and lipid peroxidation in plasma and in lung and in liver tissue of rats exposed to welding fumes in an exposure chamber with those of control animals. Three air samples from the chamber were also collected to assess the exposure dose for each exposure (total samplings = 18). Eight male Sprague–Dawley rats were exposed to welding fumes at a concentration of 1540.76 mg/m³ for 10 min/day six times on day 1, day 3, day 7, day 15, day 30 and day 40. Lung, liver and kidney injury was measured following exposure, as well as in unexposed control rats (n = 4 at the beginning of the study). DNA strand breakage [tail moment (TMOM)] in exposed animals showed significant differences at day 1, day 4, day 7 and day 15 relative to the levels in control animals. Malondialdehyde (MDA, a lipid peroxidation product) levels increased gradually post-welding to 0.4 µM at 7 days. MDA and TMOM both reached maximum levels 7 days after the first exposure. At the start, an increasing trend in DNA strand breakage was more obvious than increases in MDA levels; MDA seemed to reflect long-term effects of exposure to welding fumes since it increased after 7 days and was sustained to 40 days in vivo. Significant differences in both MDA levels and DNA strand breakage were seen in lung, liver and kidney 40 days after the first fume inhalation. We conclude that acute exposure of rats to welding fumes causes noticeable oxidative damage and lipid peroxidation effects and that DNA damage may recover after long and repeat exposure. More chronic inhalation and low-dose studies are needed in order to further assess the effects of inhalation of welding fumes on DNA and to elucidate the possible causal mechanisms associated with the biologically damaging effects of welding fumes.

Reciprocal translocations in somatic and germ cells of mice chronically exposed by inhalation to ethylene oxide: implications for risk assessment

Donner, E. M., Wong, B. A., James, R. A., Preston, R. J. — Sun, 01 Nov 2009 22:50:30 PST

Groups of male B6C3F1 mice were exposed by inhalation to 0, 25, 50, 100 or 200 p.p.m. ethylene oxide (EO) for up to 48 weeks (6 hours/day, 5 days/week). Animals were sacrificed at 6, 12, 24 and 48 weeks after the start of the exposure for analyses of reciprocal translocations in peripheral blood lymphocytes and germ cells. The frequency of the total chromosomal aberrations in the peripheral blood lymphocytes was significantly increased at the 100 and 200 p.p.m. exposure concentrations at the 12-week time point, at 50, 100 and 200 p.p.m. at the 24-week time point and at all EO concentrations at the 48-week time point. The frequency of stable reciprocal translocations, which can be used as biomarkers, was increased (P < 0.05) at 100 and 200 p.p.m. at the 12-week time point, at 100 and 200 p.p.m. at the 24-week time point and at 50, 100 and 200 p.p.m. at the 48-week time point. No statistically significant increase could be observed in translocation frequencies at the 6-week time point in the peripheral blood lymphocytes. The exposure–response curves were non-linear when the frequencies of translocations were plotted against EO exposure durations or against EO exposure concentrations. There was no effect of exposure concentration rate on reciprocal translocation frequency. Reciprocal translocations induced in spermatogonial stem cells (observed at the sprematocyte stage) showed significant increases in translocation frequencies over controls at all EO concentrations at 48 weeks. However, increases were small and they did not occur in a dose-responsive manner. The statistically significant increase observed at 12 weeks in the spermatocytes was equivocal. This study provides low-level chronic exposure somatic cytogenetic data generated in mice that can be used to support the shape of the tumour dose–response in rodents and humans The germ cell cytogenetic data are discussed in terms of its relevance for a threshold response for genetic effects at low exposures.

Assessment of in vivo genotoxicity of the rodent carcinogen furan: evaluation of DNA damage and induction of micronuclei in mouse splenocytes

Thu, 22 Oct 2009 04:58:04 PDT

In recent years, several surveys have highlighted the presence of the rodent carcinogen furan in a variety of food items. Even though the evidence of carcinogenicity of furan is unequivocal, the underlying mechanism has not been fully elucidated. In particular, the role of genotoxicity in furan carcinogenicity is still not clear, even though this information is considered pivotal for the assessment of the risk posed by the presence of low doses of furan in food. In this work, the genotoxic potential of furan in vivo has been investigated in mice, under exposure conditions similar to those associated with cancer onset in the National Toxicology Program long-term bioassay. To this aim, male B6C3F1 mice were treated by gavage for 4 weeks with 2, 4, 8 and 15 mg furan/kg b.w./day. Spleen was selected as the target organ for genotoxicity assessment, in view of the capability of quiescent splenocytes to accumulate DNA damage induced by repeat dose exposure. The induction of primary DNA damage in splenocytes was evaluated by alkaline single-cell gel electrophoresis (comet assay) and by the immunofluorescence detection of foci of phosphorylated histone H2AX (-H2AX). The presence of cross-links was probed in a modified comet assay, in which cells were irradiated in vitro with -rays before electrophoresis. Chromosome damage was quantitated through the detection of micronuclei in mitogen-stimulated splenocytes using the cytokinesis-block method. Micronucleus induction was also assessed with a modified protocol, using the repair inhibitor 1-β-arabinofuranosyl-cytosine to convert single-strand breaks in micronuclei. The results obtained show a significant (P < 0.01) increase of -H2AX foci in mitogen-stimulated splenocytes of mice treated with 8 and 15 mg furan/kg b.w. and a statistically significant (P < 0.001) increases of micronuclei in binucleated splenocytes cultured in vitro. Conversely, no effect of in vivo exposure to furan was observed when freshly isolated quiescent splenocytes were analysed by immunofluorescence and in comet assays, both with standard and radiation-modified protocols. These results indicate that the in vivo exposure to furan gives rise to pre-mutagenic DNA damage in resting splenocytes, which remains undetectable until it is converted in frank lesions during the S-phase upon mitogen stimulation. The resulting DNA strand breaks are visualized by the increase in -H2AX foci and may originate micronuclei at the subsequent mitosis.

Polymorphisms in the promoter regions of matrix metalloproteinases 1 and 3 and cancer risk: a meta-analysis of 50 case-control studies

Tue, 20 Oct 2009 06:08:36 PDT

Matrix metalloproteinase (MMP) 1 and MMP3 are enzymes that degrade the extracellular matrix and have been implicated to play an important role in cancer development. Many studies have been carried out on the association between polymorphisms of MMP1 –1607 1G>2G and MMP3 –1171 5A>6A and cancer risk. However, results from these studies remain inconclusive. Here, we performed a meta-analysis of >38 000 subjects to better assess the purported associations. For MMP1, –1607 2G/2G genotype carriers were found to have an increased risk of colorectal cancer [2G/2G versus 2G/1G + 1G/1G, odds ratio (OR) = 1.48, 95% confidence interval (CI) (1.26–1.74), P_{heterogeneity} = 0.066, I² = 49.3%], head and neck cancer [2G/2G versus 2G/1G + 1G/1G, OR = 1.61, 95% CI (1.26–2.07), P_{heterogeneity} = 0.002, I² = 64.7%] and renal cancer [2G/2G versus 2G/1G + 1G/1G, OR = 1.82, 95% CI (1.38–2.39), P_{heterogeneity} = 0.589, I² = 0.0%] risk. For MMP3, no association was found between –1171 5A>6A polymorphism and cancer risk in the overall group [6A versus 5A, OR = 1.00, 95% CI (0.95–1.05), P_{heterogeneity} = 0.124, I² = 24.9%] and individual cancer subgroups, but stratified analysis by smoking status showed that this polymorphism had different effects on smokers and non-smokers under recessive genetic model. In summary, our study suggests that MMP1 –1607 2G may be associated with an increased cancer risk for certain types of cancers, MMP3 –1171 5A>6A may not be a major risk factor for cancer, but it may be modified by certain environmental factors. Future studies with larger sample sizes are warranted to further evaluate these associations in more detail.

Further evaluation of a flow cytometric in vitro micronucleus assay in CHO-K1 cells: a reliable platform that detects micronuclei and discriminates apoptotic bodies

Shi, J., Bezabhie, R., Szkudlinska, A. — Tue, 20 Oct 2009 06:08:35 PDT

The in vitro micronucleus (MN) assay is widely used to assess genotoxic potential of the test substances by measuring frequency of MN in cultured mammalian cells. Traditionally, MN frequency has been determined by microscopy. In recent years, a flow cytometric method for enumeration of MN has been developed, which significantly shortens analysis time and enhances assay throughput. However, a major concern has been raised that the MN results obtained from flow cytometry can be impacted by chromatin bodies produced during apoptosis or necrosis. In this work, we further evaluated this flow cytometry-based in vitro MN assay with CHO-K₁ cells in a 24-well platform. Our results showed that the MN frequency determined using the flow cytometric method was highly correlated with the microscopy results. Importantly, several non-genotoxic apoptosis inducers or cytotoxins that have been previously reported to produce ‘artificial positives’ in various in vitro genotoxicity tests were evaluated in this system. As a result, these non-genotoxic cytotoxins did not produce false-positive MN response in the flow cytometric system in CHO-K₁ cells when cytotoxicity was <50 ± 10%. Moreover, a total of 21 compounds were evaluated in this work, including direct or indirect clastogens, aneugens and non-genotoxic chemicals. A sensitivity of 83.3% and a specificity of 100% were obtained from the compounds we tested. Finally, significant increase of incidents in the hypodiploid region, an aneugenic signature, was confirmed in our evaluation. In conclusion, the flow cytometric in vitro MN assay is a reliable method that can be used to detect clastogenic or aneugenic potential of the test substances in CHO-K₁ cells.

An aphidicolin-block nucleotide excision repair assay measuring DNA incision and repair capacity

Vande Loock, K., Decordier, I., Ciardelli, R., Haumont, D., Kirsch-Volders, M. — Tue, 20 Oct 2009 06:08:33 PDT

The objective of the present study was to develop a cellular phenotype assay for nucleotide excision repair (NER), using benzo[a]pyrene diol epoxide (BPDE) as model mutagen. Since in vitro exposure to BPDE may lead to DNA strand breaks resulting from both direct interaction with DNA and incisions introduced by the repair enzymes, we aimed to discriminate between both types of breaks using the comet assay and quantified the DNA strand breaks after in vitro challenge of peripheral blood mononucleated cells (PBMCs) with BPDE in the presence or absence of the DNA polymerase inhibitor aphidicolin (APC). The assay was performed with a low (0.5 µM) and a high (2.5 µM) BPDE concentration. The individual NER capacity was defined as the amount of DNA damage induced by BPDE in presence of APC, diminished with the damage induced by BPDE and APC alone. First, the assay was applied to a NER-deficient human fibroblast cell line (XPA–/–) to validate the methodology. Lower repair capacity and a higher amount of BPDE-induced DNA adducts were observed for the XPA–/– fibroblasts as compared to the wild-type fibroblasts. Repeated experiments on PBMCs from four donors showed low intra-individual, intra-experimental and inter-assay variation for both concentrations, indicating the reliability of the method. To assess the inter-individual variation, the assay was applied to PBMCs from 22 donors, comparing the repair capacity after exposure to 0.5 µM (N = 10) and 2.5 µM (N = 12) BPDE. The repair capacity showed a higher inter-individual variation as compared to the intra-individual variation. Moreover, this difference was more pronounced using the low concentration. All these results indicate the adequacy of the method using this low concentration. Further improvement, however, should be recommended by applying the study with low BPDE concentration in a larger population and taking into account the relevant genotypes for NER.