Low dose genotoxicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in gpt delta transgenic mice

Although humans are chronically exposed to most environmental chemicals at low doses, genotoxicity assays with rodents are usually performed at high doses with short treatment period. To investigate the dose-response of genotoxicity at lower doses, gpt delta transgenic mice were fed a diet containing 300, 30 or 3 parts per million (ppm) of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) for 12 weeks and the gpt mutations in the liver were analyzed. In addition, the mice were continuously fed a diet containing MeIQx at a dose of 300 ppm for 78 weeks to examine the effect of a long-term treatment. In the mice treated for 12 weeks, the gpt mutant frequencies (MFs) were 8.6-, 2.3- and 1.2-fold higher than the control level at the doses of 300, 30 and 3 ppm, respectively. G:C to T:A transversion was the most predominant type of mutations and the fold increases in the specific MF of G:C to T:A were 58.2, 4.4 and 1.7 above the control at the three doses, respectively. The increases in the whole gpt and specific MFs at 3 ppm were not statistically significant. In the mice treated with 300 ppm of MeIQx for 78 weeks, the gpt MF was about 20 times higher than that of the untreated mice fed a control diet for 78 weeks, which was about two times higher than that of the untreated mice at 12 weeks. These results suggest that no obvious genotoxic effects can be detectable at the dose of MeIQx at 3 ppm in the liver and a longer treatment substantially enhances the genotoxicity. Factors constituting the practical threshold dose are discussed.     

Combined genotoxic effects of radiation and a tobacco-specific nitrosamine in the lung of gpt delta transgenic mice

It is important to evaluate the health effects of low-dose-rate or low-dose radiation in combination with chemicals as humans are exposed to a variety of chemical agents. Here, we examined combined genotoxic effects of low-dose-rate radiation and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most carcinogenic tobacco-specific nitrosamine, in the lung of gpt delta transgenic mice. In this mouse model, base substitutions and deletions can be separately analyzed by gpt and Spi- selections, respectively. Female gpt delta mice were either treated with gamma-irradiation alone at a dose rate of 0.5, 1.0 or 1.5 mGy/h for 22 h/day for 31 days or combined with NNK treatments at a dose of 2 mg/mouse/day, i.p. for four consecutive days in the middle course of irradiation. In the gpt selection, the NNK treatments enhanced the mutation frequencies (MFs) significantly, but no obvious combined effects of gamma-irradiation were observable at any given radiation dose. In contrast, NNK treatments appeared to suppress the Spi- large deletions. In the Spi- selection, the MFs of deletions more than 1 kb in size increased in a dose-dependent manner. When NNK treatments were combined, the dose-response curve became bell-shaped where the MF at the highest radiation dose decreased substantially. These results suggest that NNK treatments may elicit an adaptive response that eliminates cells bearing radiation-induced double-strand breaks in DNA. Possible mechanisms underlying the combined genotoxicity of radiation and NNK are discussed, and the importance of evaluation of combined genotoxicity of more than one agent is emphasized.     

Reduction of GC --> TA transversion mutation by overexpression of MutS in Escherichia coli K-12

Overexpression of the MutS repair protein significantly decreased the rate of lacZ GC --> TA transversion mutation in stationary-phase and exponentially growing bacteria and in mutY and mutM mutants, which accumulate mismatches between 8-oxoguanine (8-oxoG) and adenine residues in DNA. Conversely, GC --> TA transversion increased in mutL or mutS mutants in stationary phase. In contrast, overexpression of MutS did not appreciably reduce lacZ AT --> CG transversion mutation in a mutT mutant. These results suggest that MutS-dependent repair can correct 8-oxoG:A mismatches in Escherichia coli cells but may not be able to compete with mutation fixation by MutY in mutT mutants.     

Dammar resin, a non-mutagen, inducts oxidative stress and metabolic enzymes in the liver of gpt delta transgenic mouse which is different from a mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline

Dammar resin has long been used in foods as either a clouding or a glazing agent. In a recent study, 2% Dammar resin showed significant hepatocarcinogenicity in a rat 2-year bioassay. Therefore, for an accurate estimate of human risk, it is necessary to understand whether Dammar resin induces liver genotoxicity and the underlying mechanisms of its hepatocarcinogenicity. Modifying effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a typical genotoxic carcinogen produced during cooking of protein-rich foods, was also studied in the present study. Exposure of gpt delta mice to Dammar resin at a dose of 2% for 12 weeks did not induce any obvious mutagenicity in the liver. However, the index of cell proliferation, the level of 8-OHdG, and bax, bcl-2, p53, cyp1a2, cyp2e1, gpx1 and gstm2 gene expression were all significantly increased when compared with the control group. In the IQ treatment group, at a dose of 300ppm, mutagenicity was readily detected, the index of cell proliferation increased, and p53, cyp2e1 and gpx1 gene expression was down-regulated in the liver. Down-regulation of p53, P450s, and gpx1 in the livers of IQ treated mice are consistent with its genotoxic mechanism of carcinogenicity observed in a 675-day study. In contrast, our results using gpt delta mice suggest that Dammar resin is not genotoxic. Instead, the Dammar resin-induced hepatocarcinogenicity seen in our previous 2-year study with rats may have been mediated by non-genotoxic mechanisms, including increased P450 enzyme activity, increased oxidative stress, altered gene expression, and promotion of cell proliferation.     

A newly established GDL1 cell line from gpt delta mice well reflects the in vivo mutation spectra induced by mitomycin C

In order to create a novel in vitro test system for detection of large deletions and point mutations, we developed an immortalized cell line. A SV40 large T antigen expression unit was introduced into fibroblasts derived from gpt delta mouse lung tissue and a selected clone was established as the gpt delta L1 (GDL1) cell line. The novel GDL1 cells were examined for mutant frequencies (MFs) and for molecular characterization of mutations induced by mitomycin C (MMC). The GDL1 cells were treated with MMC at doses of 0.025, 0.05, and 0.1 microg/mL for 24h and mutations were detected by Spi- and 6-thioguanine (6-TG) selections. The MFs of the MMC-treated cells increased up to 3.4-fold with Spi- selection and 3.5-fold with 6-TG selection compared to MFs of untreated cells. In the Spi- mutants, the number of large (up to 76 kilo base pair (kbp)) deletion mutations increased. A majority of the large deletion mutations had 1-4 base pairs (bp) of microhomology in the deletion junctions. A number of the rearranged deletion mutations were accompanied with deletions and insertions of up to 1.1 kbp. In the gpt mutants obtained from 6-TG selection, single base substitutions of G:C to T:A, tandem base substitutions occurring at the 5'-GG-3' or 5'-CG-3' sequence, and deletion mutations larger than 2 bp were increased. We compared the spectrum of MMC-induced mutations observed in vitro to that of in vivo using gpt delta mice, which we reported previously. Although a slight difference was observed in MMC-induced mutation spectra between in vitro and in vivo, the mutations detected in vitro included all of the types of mutations observed in vivo. The present study demonstrates that the newly established GDL1 cell line is a useful tool to detect and analyze various mutations including large deletions in mammalian cells.     

Intermediate frequency magnetic fields do not have mutagenic, co-mutagenic or gene conversion potentials in microbial genotoxicity tests

We used bacterial mutation and yeast genotoxicity tests to evaluate the effects of intermediate frequency (IF; 2 kHz, 20 kHz and 60 kHz) magnetic fields (MFs) on mutagenicity, co-mutagenicity and gene conversion. We constructed a Helmholtz type exposure system that generated vertical and sinusoidal IF MFs, such as 0.91 mT at 2 kHz, 1.1 mT at 20 kHz and 0.11 mT at 60 kHz. Mutagenicity, co-mutagenicity and gene conversion assays were performed for each of the three MF exposure conditions. Mutagenicity testing was performed in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and two strains of Escherichia coli (WP2 uvrA and WP2 uvrA/pKM101) to cover a wide spectrum of point mutations. For co-mutagenicity tests, we used four sensitive test strains (TA98, TA100, WP2 uvrA and WP2 uvrA/pKM101) with five chemical mutagens (t-butyl hydroperoxide (BH, a hydroxyl free radical precursor), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, DNA reactive reagents), benz[a]pyrene (BaP) and 2-aminoanthracene (2AA, DNA reactive promutagens). Gene conversion testing was performed in the yeast test strain, Saccharomyces cerevisiae XD83. We also examined the effects on the repair process of DNA damage by UV irradiation. No statistically significant effects were observed between exposed and control groups in any of the genotoxicity tests, indicating that the IF MFs (0.91 mT at 2 kHz, 1.1 mT at 20 kHz or 0.11 mT at 60 kHz) do not have mutagenic or co-mutagenic potentials for the chemical mutagens tested under these experimental conditions. Our findings also indicate that these IF MFs do not induce gene conversion or affect the repair process of DNA damage in eukaryotic cells.     

Prevalence of genotoxic chemicals among animal and human carcinogens evaluated in the IARC Monograph series.

To determine whether genotoxic and non-genotoxic carcinogens contribute similarly to the cancer burden in humans, an analysis was performed on agents that were evaluated in Supplements 6 and 7 to the IARC Monographs for their carcinogenic effects in humans and animals and for the activity in short-term genotoxicity tests. The prevalence of genotoxic carcinogens on four groups of agents, consisting of established human carcinogens (group 1, n = 30), probable human carcinogens (group 2A, n = 37), possible human carcinogens (group 2B, n = 113) and on agents with limited evidence of carcinogenicity in animals (a subset of group 3, n = 149) was determined. A high prevalence in the order of 80 to 90% of genotoxic carcinogens was found in each of the groups 1, 2A and 2B, which were also shown to be multi-species/multi-tissues carcinogens. The distribution of carcinogenic potency in rodents did not reveal any specific characteristic of the human carcinogens in group 1 that would differentiate them from agents in groups 2A, 2B and 3. The results of this analysis indicate that (a) an agent with unknown carcinogenic potential showing sufficient evidence of activity in in vitro/in vivo genotoxicity assays (involving as endpoints DNA damage and chromosomal/mutational damage) may represent a hazard to humans; and b) an agent showing lack of activity in this spectrum of genotoxicity assays should undergo evaluation for carcinogenicity by rodent bioassay, in view of the present lack of validated short-term tests for non-genotoxic carcinogens. Overall, this analysis implies that genotoxic carcinogens add more to the cancer burden in man than non-genotoxic carcinogens. Thus, identification of such genotoxic carcinogens and subsequent lowering of exposure will remain the main goal for primary cancer prevention in man.     

The alkaline single cell gel electrophoresis assay with mouse multiple organs: results with 30 aromatic amines evaluated by the IARC and U.S. NTP

The genotoxicity of 30 aromatic amines selected from IARC (International Agency for Research on Cancer) groups 1, 2A, 2B and 3 and from the U.S. NTP (National Toxicology Program) carcinogenicity database were evaluated using the alkaline single cell gel electrophoresis (SCG) (Comet) assay in mouse organs. We treated groups of four mice once orally at the maximum tolerated dose (MTD) and sampled stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow 3, 8 and 24 h after treatment. For the 20 aromatic amines that are rodent carcinogens, the assay was positive in at least one organ, suggesting a high predictive ability for the assay. For most of the SCG-positive aromatic amines, the organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Organ-specific genotoxicity, therefore, is necessary but not sufficient for the prediction of organ-specific carcinogenicity. For the 10 non-carcinogenic aromatic amines (eight were Ames test-positive and two were Ames test-negative), the assay was negative in all organs studied. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative non-genotoxic (Ames test-negative) carcinogens. The alkaline SCG assay, which detects DNA lesions, is not suitable for identifying non-genotoxic carcinogens. The present SCG study revealed a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic non-carcinogens. These results suggest that the alkaline SCG assay can be usefully used to evaluate the in vivo genotoxicity of chemicals in multiple organs, providing for a good assessment of potential carcinogenicity.     

Genotoxicity and carcinogenicity studies of antihypertensive agents

This survey is a compendium of genotoxicity and carcinogenicity information of antihypertensive drugs. Data from 164 marketed drugs were collected. Of the 164 drugs, 65 (39.6%) had no retrievable genotoxicity or carcinogenicity data; this group was comprised largely of drugs marketed in a limited number of countries. The remaining 99 (60.4%) had at least one genotoxicity or carcinogenicity test result. Of these 99, 48 (48.5%) had at least one positive finding: 32 tested positive in at least one genotoxicity assay, 26 in at least one carcinogenicity assay, and 10 gave a positive result in both at least one genotoxicity assay and at least one carcinogenicity assay. In terms of correlation between results of the various genotoxicity assays and absence of carcinogenic activity in both mice and rats 2 of 44 non-carcinogenic drugs tested positive in the in vitro bacterial mutagenesis assay, 2 of 9 tested positive in the mouse lymphoma assay, none of 14 tested positive for gene mutation at the hprt locus, 5 of 25 tested positive in in vitro cytogenetic assays, none of 31 in in vivo cytogenetic assays, and none of 14 in inducing DNA damage and/or repair in in vitro and/or in vivo assays. Concerning the predictivity of genetic toxicology findings for long-term carcinogenesis assays, 75 drugs had both genotoxicity and carcinogenicity data; of these 37 (49.3%) were neither genotoxic nor carcinogenic, 14 (18.7%) were non-carcinogens which tested positive in at least one genotoxicity assay, 14 (18.7%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, and 10 (13.3%) were both genotoxic and carcinogenic. Only 42 of the 164 marketed antihypertensives (25.6%) had all data required by the guidelines for testing of pharmaceuticals.     

Inhibition of Sudan I genotoxicity in human liver-derived HepG2 cells by the antioxidant hydroxytyrosol

The chemoprotective effect of hydroxytyrosol (HT) against Sudan I-induced genotoxicity was investigated in a human hepatoma cell line, HepG2. The comet assay and micronucleus (MN) assay were used to monitor genotoxicity. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe, 2,7-dichlorofluorescein diacetate (DCFH-DA). The levels of oxidative DNA damage and lipid peroxidation were estimated by immunocytochemistry analysis of 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS), respectively. Intracellular glutathione (GSH) level was estimated by fluorometric methods. The results showed that HT significantly reduced the genotoxicity caused by Sudan I. Furthermore, HT ameliorated lipid pexidation as demonstrated by a reduction in TBARS formation and attenuated GSH depletion in a concentration-dependent manner. It was also found that HT reduced intracellular ROS formation and 8-OHdG level caused by Sudan I. These results strongly suggest that HT has significant protective ability against Sudan I-induced genotoxicity.     

Mutational specificities of 1′-acetoxysafrole,N-benzoyloxy-N-methyl-4-aminoazobenzene,and ethyl methanesulfonate in human cells

We have used an oriP-tk shuttle vector t determine the types of mutations induced in human cells by ethyl methanesulfonate (EMS), 1'-acetoxysafrole (AcOS), and N-benzoyloxy-N-methyl-4-aminoazobenzene (BzOMAB). Plasmid DNA was treated in vitro with mutagen and electroporated into human lymphoblastoid cells. After replication of the vector in human cells, plasmids were analyzed for mutations in the herpes simplex virus type 1 thymidine kinase gene. Ethyl methanesulfonate induced predominantly GC → AT transition mutations. Treatment of the shuttle vector with AcOS induced 5 of the 6 possible base substitution mutations, including GC → AT (32%) and AT → GC (14%) transition mutations, GC → TA (%), GC → CG (18%), and AT → TA (14%) transversion mutations, as well as a low frequency (9%) of −1 frameshift mutations at GC base pairs. Replication in human cells of DNA modified with BzOMAB yielded a significant increase (17-fold) in the frequency of deletion mutations relative to solvent-treated DNA. A majority (94%) of the point mutations induced by BzOMAB occurred at GC base pairs and were predomianntly GC → AT transitions (33%) and −1 frameshift (22%) mutations, with the remainder consisting mainly of transversions at GC base pairs (28%). The broad spectrum of base substitution mutations observed for AcOS and BzOMAB may indicate the frequent insertion of a variety of bases during replicative bypass of aralkylated bases in human cells.     

Evaluation of the GreenScreen GADD45alpha-GFP indicator assay with non-proprietary and proprietary compounds

The GreenScreen GADD45alpha indicator assay has been assessed for its concordance with in vitro genotoxicity and rodent carcinogenicity bioassay data. To test robustness, sensitivity, and specificity of the assay, 91 compounds with known genotoxicity results were screened in a blinded manner. Fifty seven of the compounds were classified as in vitro genotoxic whereas 34 were non-genotoxic. Out of the 91 compounds, 50 had been tested in 2-year carcinogenicity assays, with 33 identified to be rodent carcinogens and 17 non-carcinogens. Gadd45alpha assay sensitivity and specificity for genotoxicity was 30% and 97%, respectively (17/57 and 33/34), whereas its sensitivity and specificity for rodent carcinogenicity was 30% and 88%, respectively (10/33 and 15/17). Gadd45alpha assay genotoxicity results from this validation study exhibited a high concordance with previously published results as well as for compound test results generated at two different sites (91%, 19/21), indicating that the assay is both robust and reproducible. In conclusion, results from this blinded and independent validation study indicate that the GreenScreen GADD45 indicator assay is reproducible and reliable with low sensitivity and high specificity for identifying genotoxic and carcinogenic compounds.     

Mutagenicity of cadmium in mammalian cells: implication of oxidative DNA damage

Cadmium and cadmium compounds are well established human carcinogens and are ubiquitously present in the environment. The carcinogenic mechanism(s) of cadmium remains largely unknown since direct mutagenic effect is weak in bacterial and in standard mammalian cell mutation assays. In this study, we show that when evaluated using the human-hamster hybrid A(L) cell mutation assay in which both intragenic and multilocus deletions can readily be detected, CdCl(2) is a strong mutagen that induces predominantly large deletion mutations. Concurrent treatment of A(L) cells with the oxyradical scavenger dimethyl sulfoxide significantly reduced the number of cadmium-induced mutations. In contrast, pre-treatment of cells with buthionine sulfoximine that depletes intracellular glutathione, increased cytotoxicity and mutagenicity of cadmium. These results demonstrate that reactive oxygen species mediate cadmium induced mutations in A(L) cells. With laser scanning confocal microscopy and the fluorescent probe 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, we demonstrated that cadmium induced a dose and time dependent formation of intracellular oxyradicals. Using immunoperoxidase staining coupled with a monoclonal antibody-specific for 8-OHdG adducts in DNA, we demonstrated that cadmium induced a dose dependent increase of 8-OHdG adducts, which accumulated with prolonged exposure. Furthermore, we showed that at low concentration, cadmium, attenuated removal of hydrogen peroxide induced 8-OHdG adducts. Thus, the carcinogenicity of cadmium can, in part, be explained by its mutagenic activity, which is mediated by reactive oxygen species induced DNA damage and by its interference with the repair of oxidative DNA damage.     

Potassium bromate treatment predominantly causes large deletions, but not GC>TA transversion in human cells

Potassium bromate (KBrO(3)) is strongly carcinogenic in rodents and mutagenic in bacteria and mammalian cells in vitro. The proposed genotoxic mechanism for KBrO(3) is oxidative DNA damage. KBrO(3) can generate high yields of 8-hydroxydeoxyguanosine (8OHdG) DNA adducts, which cause GC>TA transversions in cell-free systems. In this study, we investigated the in vitro genotoxicity of KBrO(3) in human lymphoblastoid TK6 cells using the comet (COM) assay, the micronucleus (MN) test, and the thymidine kinase (TK) gene mutation assay. After a 4h treatment, the alkaline and neutral COM assay demonstrated that KBrO(3) directly yielded DNA damages including DNA double strand breaks (DSBs). KBrO(3) also induced MN and TK mutations concentration-dependently. At the highest concentration (5mM), KBrO(3) induced MN and TK mutation frequencies that were over 30 times the background level. Molecular analysis revealed that 90% of the induced mutations were large deletions that involved loss of heterozygosity (LOH) at the TK locus. Ionizing-irradiation exhibited similar mutational spectrum in our system. These results indicate that the major genotoxicity of KBrO(3) may be due to DSBs that lead to large deletions rather than to 8OHdG adducts that lead to GC>TA transversions, as is commonly believed. To better understand the genotoxic mechanism of KBrO(3), we analyzed gene expression profiles of TK6 cells using Affymetrix Genechip. Some genes involved in stress, apoptosis, and DNA repair were up-regulated by the treatment of KBrO(3). However, we could not observe the similarity of gene expression profile in the treatment of KBrO(3) to ionizing-irradiation as well as oxidative damage inducers.     

Genotoxicity testing of extracts of a Swedish moist oral snuff

The present study was designed to investigate the potential genotoxicity of aqueous and methylene chloride extracts of Swedish moist oral snuff. The test systems were selected to provide optimal data for the prediction of carcinogenicity in rodents and included assays for the induction of mutation in bacteria, sister-chromatid exchanges (SCE) in human lymphocytes, of chromosome aberrations and gene mutations in V79 Chinese hamster cells and of micronuclei in mouse bone marrow cells. In addition, the methylene chloride extract was tested for the induction of sex-linked recessive lethal mutations in Drosophila melanogaster. The aqueous extract of 'Snus' induced SCE in human lymphocytes and chromosome aberrations in V79 cells, the latter effect being observed both with and without metabolic activation. No induction of point mutations was detected with the Ames test or in V79 cells and the micronucleus test in mice was negative. It was demonstrated that the induction of chromosome aberrations without metabolic activation may be due to a high salt concentration, indicating that the clastogenic agent(s) in this extract required metabolic activation. The methylene chloride extract showed genotoxicity in the Ames test, the SCE test and the chromosome aberration test, whereas no induction of gene mutations in V79 cells was observed. Once again, the results suggested that metabolism is required for genotoxicity. The methylene chloride extract did not cause induction of micronuclei in mice or of sex-linked recessive lethal mutations in Drosophila melanogaster. These combined data on genotoxicity were analyzed using various models for the prediction of carcinogenicity. In a sequential testing model, the probabilities that the aqueous and methylene chloride extracts of 'Snus' are carcinogenic due to a genotoxic mechanism were both predicted to be low. Using carcinogenicity prediction by battery selection (CPBS), the probabilities of the methylene chloride and aqueous extracts being correctly identified as non-carcinogens are 71 and 77%, respectively. Up to date, the CPBS approach has been validated primarily for individual compounds, so some caution should at present be exercised in interpreting the results using this method. Based on these results, the carcinogenic potential of Swedish 'Snus' should be considered to be low, a conclusion in agreement with the low incidence of oral cancer in Sweden compared to other countries.     

Sudan I induces genotoxic effects and oxidative DNA damage in HepG2 cells

Sudan I, a synthetic lipid soluble azo pigment, is widely used in various industrial fields. However, Sudan I has not been approved at any level of food production, since there are many inconclusive reports relating to its genotoxicity and carcinogenicity in humans. The aim of this study was to assess the genotoxic effects of Sudan I and to identify and clarify the reaction mechanisms by use of human hepatoma HepG2 cells. To study the genotoxic effects of Sudan I, the comet assay and micronucleus test (MNT) were used. In the comet assay and MNT, we found increase of DNA migration and of the micronuclei frequencies at all tested concentrations (25-100 microM) of Sudan I in a dose-dependent manner. The data suggest that Sudan I caused DNA strand breaks and chromosome breaks. To elucidate the underlying mechanism of this difference, we monitored the level of reactive oxygen species (ROS) production with the 2,7-dichlorofluorescein diacetate assay. The level of the oxidative DNA damage and lipid peroxidation was evaluated using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS). Significantly increased levels of ROS, 8-OHdG and TBARS were observed in HepG2 cells at higher concentrations, the doses being 100, 50-100 and 50-100 microM, respectively. We conclude that Sudan I causes genotoxic effects, probably via ROS-induced oxidative DNA damage at the higher doses.     

Age-dependent sensitivity of Big Blue transgenic mice to the mutagenicity of N-ethyl-N-nitrosourea (ENU) in liver

The incidence of childhood cancer is increasing and recent evidence suggests an association between childhood cancer and environmental exposure to genotoxins. In the present study, the Big Blue transgenic mouse model was used to determine whether specific periods in early life represent windows of vulnerability to mutation induction by genotoxins in mouse liver. Groups of mice were treated with single doses of 120 mg N-ethyl-N-nitrosourea (ENU)/kg body weight or the vehicle either transplacentally to the 18-day-old fetus or at postnatal days (PNDs) 1, 8, 15, 42 or 126; the animals were sacrificed 6 weeks after their treatment. The cII mutation assay was performed to determine the mutant frequencies (MFs) in the livers of the mice. Liver cII MFs for both sexes were dependent on the age at which the animals were treated. Perinatal treatment with ENU (either transplacental treatment to the 18-day-old fetus or i.p. injection at PND 1) induced relatively high MFs. However, ENU treatment at PNDs 8 and 15 resulted in the highest mutation induction. The lowest mutation induction occurred in those animals treated as adults (PND 126). For instance, the cII MF for the PND 8 female group was 646 x 10(-6) while the MF for female adults was only 145 x 10(-6), a more than 4-fold difference. Molecular analysis of the mutants found that A:T-->T:A transversions and A:T-->G:C transitions characterized the pattern of mutations induced by ENU in both the neonate and adult mice, while the predominate type of mutation in the controls was G:C-->A:T. The results indicate that mouse liver is most sensitive to ENU-induced mutation during infancy. This period correlates well with the age-dependent sensitivity to carcinogenicity in mouse liver, suggesting that mutation is an important rate-limiting factor for age-related carcinogenesis.     

Association between mutation spectra and stable and unstable DNA adduct profiles in Salmonella for benzo[a]pyrene and dibenzo[a,l]pyrene

Benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) are two polycyclic aromatic hydrocarbons (PAHs) that exhibit distinctly different mutagenicity and carcinogenicity profiles. Although some studies show that these PAHs produce unstable DNA adducts, conflicting data and arguments have been presented regarding the relative roles of these unstable adducts versus stable adducts, as well as oxidative damage, in the mutagenesis and tumor-mutation spectra of these PAHs. However, no study has determined the mutation spectra along with the stable and unstable DNA adducts in the same system with both PAHs. Thus, we determined the mutagenic potencies and mutation spectra of BP and DBP in strains TA98, TA100 and TA104 of Salmonella, and we also measured the levels of abasic sites (aldehydic-site assay) and characterized the stable DNA adducts ((32)P-postlabeling/HPLC) induced by these PAHs in TA104. Our results for the mutation spectra and site specificity of stable adducts were consistent with those from other systems, showing that DBP was more mutagenic than BP in TA98 and TA100. The mutation spectra of DBP and BP were significantly different in TA98 and TA104, with 24% of the mutations induced by BP in TA98 being complex frameshifts, whereas DBP produced hardly any of these mutations. In TA104, BP produced primarily GC to TA transversions, whereas DBP produced primarily AT to TA transversions. The majority (96%) of stable adducts induced by BP were at guanine, whereas the majority (80%) induced by DBP were at adenine. Although BP induced abasic sites, DBP did not. Most importantly, the proportion of mutations induced by DBP at adenine and guanine paralleled the proportion of stable DNA adducts induced by DBP at adenine and guanine; however, this was not the case for BP. Our results leave open a possible role for unstable DNA adducts in the mutational specificity of BP but not for DBP.     

Mutagenic fingerprint of ozone in human cells

Ozone is an important factor in urban pollution and represents a major concern for human health. The chemical reactivity of ozone toward biological targets and particularly its genotoxicity supports a possible link between exposure and cancer risk, but no molecular data exist on its mutagenic potential in human cells. Using a shuttle vector, we showed that ozone is indeed a potent mutagen and we characterized the mutation spectrum it produced in human cells. Almost all mutations are base substitutions, essentially located at G:Cs (75%), typical of reactive oxygen species (ROS), but occurring in a specific pattern, i.e. a similar extent of GC:TA (28%), GC:CG (23%) and GC:AT (23%). The targeted distribution of mutations and identification of hotspot sequences define the first molecular fingerprint of mutations induced by ozone in human cells. Possible applications derived from our results with respect to ozone genotoxicity should help determining quantifiable biomarkers of ozone exposure in human health, especially for carcinogenesis.     

Update on genotoxicity and carcinogenicity testing of 472 marketed pharmaceuticals

This survey is a compendium of genotoxicity and carcinogenicity information of 838 marketed drugs, whose expected clinical use is continuous for at least 6 months or intermittent over an extended period of time. Of these 838 drugs, 366 (43.7%) do not have retrievable genotoxicity or carcinogenicity data. The remaining 472 (56.3%) have at least one genotoxicity or carcinogenicity test result. Of the 449 drugs with at least one genotoxicity test result, 183 (40.8%) have at least one positive finding. Of the 338 drugs with at least one carcinogenicity test result, 160 (47.3%) have at least one positive result. Concerning the predictivity of genetic toxicology findings for long-term carcinogenesis assays, of the 315 drugs which have both genotoxicity and carcinogenicity data 116 (36.8%) are neither genotoxic nor carcinogenic, 50 (15.9%) are non-carcinogens which test positive in at least one genotoxicity assay, 75 (23.8%) are carcinogenic in at least one sex of mice or rats but test negative in genotoxicity assays, and 74 (23.5%) are both genotoxic and carcinogenic. Only 208 (24.8%) of the 838 drugs considered have all data required by current guidelines for testing of pharmaceuticals. However, it should be noted that a large fraction of the drugs considered were developed and marketed prior to the present regulatory climate. Although the laws do not require re-testing based on revised standards, in the absence of epidemiological studies excluding a carcinogenic risk to humans, a re-evalutation would be appropriate.