A micromethod for the in vitro micronucleus assay.
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A micromethod for the in vitro micronucleus assay was developed using L5178Y cells to enable the rapid screening of a large number of molecules. The method is quick, simple to perform and needs very small amounts of compound, i.e. <10 mg. In this methodology, three types of treatment were carried out in parallel, enabling an optimal detection of both aneugenic and clastogenic compounds: two treatments without metabolic activation with or without a recovery period after a 24 h continuous treatment and one treatment with metabolic activation by Aroclor 1254-induced rat or hamster liver S9 mix. Seventeen known genotoxins (12 clastogens and five aneugens) and seven known non-genotoxins were tested. The in vitro micronucleus micromethod using L5178Y cells exhibited good sensitivity (16 positive/17 known genotoxins tested) and specificity (7 negative/7 known non-genotoxins tested) for the 24 test compounds studied with or without metabolic activation. Furthermore, this test showed a good correlation with other in vitro micronucleus tests performed using macromethods with various mammalian cell cultures. We conclude that the in vitro micronucleus micromethod with L5178Y cells could be used in the earliest stages of development of new molecules as a preliminary short-term screening assay before starting regulatory tests. (+info)
Development of a new bioluminescent mutagenicity assay based on the Ames test.
(66/3817)
A newly developed rapid mutagenicity assay based on the adenosine triphosphate (ATP)-bioluminescence technique and the Ames test is described. Salmonella typhimurium strains TA98 and TA100 were exposed in an appropriate liquid medium to the direct mutagens 4-nitroquinoline-N-oxide and methyl methanesulphonate, respectively, and to the indirect mutagen 2-aminoanthracene. Both auxotrophic and prototrophic growth were monitored throughout the incubation period as variations in the intracellular ATP levels by means of the luciferin-luciferase assay. After 9-12 h of incubation a dose-response increase in the levels of ATP was readily detected. In order to demonstrate that this increase was due to the growth of revertant bacteria, aliquots from each culture were plated on minimal agar plates. A very good correlation between the changes in ATP levels and the appearance of revertant colonies on the plates was found. Given the rapidity of this method as compared with conventional mutagenicity assays, it has potential for industrial and environmental applications. Other potential applications are also discussed. (+info)
Mice over-expressing human O6 alkylguanine-DNA alkyltransferase selectively reduce O6 methylguanine mediated carcinogenic mutations to threshold levels after N-methyl-N-nitrosourea.
(67/3817)
While it is well known that MNU induces thymic lymphomas in the mouse, it remains unclear which pre-mutagenic lesions are responsible for lymphomagenic transformation. One lesion thought to play a critical role is O6methylguanine[O6mG]which initiates G: C to A:T transition mutations in K-ras and other oncogenes. O6alkylguanine-DNA alkyltransferase (AGT), encoded by the methylguanine methyltransferase gene [MGMT], removes the methyl group thereby preventing the mutation from occurring. When overexpressed in the thymus, MGMT protects mice from MNU-induced thymic lymphomas. To determine whether MGMT overexpression reduced G: C to A: T mutation frequency after MNU, Big Blue lacI and MGMT+/Big Blue mice were treated with MNU and analysed for mutations in the lacI and K-ras genes. The incidence of MNU-induced lymphomas was 84% in Big Blue lacI mice compared to 14% in MGMT+Big Blue lacI mice. Sixty-two per cent of the lymphomas had a GGT to GAT activating mutation in codon 12 of K-ras consistent with O6mG adduct-mediated point mutagenesis. LacI mutation frequency in thymus of MNU treated Big Blue mice was 45-fold above background whereas it was 11-fold above background in MNU treated MGMT+/Big Blue mice. Most lacI mutations were G:C to A:T transitions, implicating O6mG even in the MGMT+mice. No mutations were attributable to chromosomal aberrations or rearrangements. Thus, O6mG adducts account for the carcinogenic effect of MNU and MGMT overexpression is selectively able to reduce O6methylguanine adducts below a carcinogenic threshold. Other adducts are mutagenic but appear to contribute much less to malignant transformation or oncogene activation. (+info)
Inherited susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes.
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BACKGROUND: Susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes may reflect the way a person deals with carcinogenic challenges. This susceptibility (also referred to as mutagen sensitivity) has been found to be increased in patients with environmentally related cancers, including cancers of the head and neck, lung, and colon, and, in combination with carcinogenic exposure, this susceptibility can greatly influence cancer risk. The purpose of this study was to assess the heritability of mutagen sensitivity. METHODS: Heritability was determined by use of a maximum likelihood method that employed the FISHER package of pedigree analysis. Bleomycin-induced breaks per cell values for 135 healthy volunteers without cancer were determined. These individuals were from 53 different pedigrees and included 25 monozygotic twin pairs (n = 50), 14 pairs of dizygotes (twin pairs and siblings, n = 28), and 14 families selected on the basis of a first-degree relative who was successfully treated for head and neck cancer and who had no sign of recurrence for at least 1 year. All data were analyzed simultaneously, and different models of familial resemblance were fitted to the data. All P values are two-sided. RESULTS: Our results showed no evidence for the influence of a shared family environment on bleomycin-induced chromatid breaks. Genetic influences, however, were statistically significant (P =. 036) and accounted for 75% of the total variance. CONCLUSIONS: The high heritability estimate of the susceptibility to bleomycin-induced chromatid breaks indicates a clear genetic basis. The findings of this study support the notion that a common genetic susceptibility to DNA damage--and thereby a susceptibility to cancer--may exist in the general population. (+info)
Should we be frightened of bracken? A review of the evidence.
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OBJECTIVE: To assess the risk to human health of the plant bracken (Pteridium sp). DESIGN: An evaluation of studies of human and animal populations exposed to bracken, together with a review of expert reports and advice to the public. MAIN RESULTS: Bracken induced disease has been demonstrated in animals in both laboratory and field studies. Depending on the species, diseases in animals associated with the plant have included; cancers of the alimentary and urogenital tract, lung and breast; haematuria; retinal degeneration; and, thiamine deficiency. Potential exposure of human populations is through: food either directly (people in some parts of the world eat bracken as a traditional dish) or indirectly by consuming animals fed on bracken; milk; water; inhalation and ingestion of spores; and insect vectors. Four studies of human populations (two analytical and two observational) failed to assess adequately confounding factors and other sources of bias, so that conclusions about a risk to human health from bracken cannot firmly be drawn. Establishing exposure is also extremely difficult in populations (such as the United Kingdom) where direct consumption of bracken is rare. CONCLUSION: Bracken is a common plant worldwide. It is toxic to many animal species and to several organ systems. There is no tumour (or other disease) that is pathognomic of exposure in animals, though cancers of the alimentary and urogenital tract seem to be the most commonly associated. It is not possible to extrapolate from animal models to humans. Studies of human populations, do not establish a clear risk of bracken to human health, largely because of methodological problems. Testing the evidence against traditional criteria of causality only fulfils the criterion of biological plausibility. Despite this, current public information implies a serious risk to human health from bracken, and increasing media coverage of the subject is likely to lead to greater public concern. Further epidemiological studies are required. (+info)
Role of O6-alkylguanine-DNA alkyltransferase in protecting against cyclophosphamide-induced toxicity and mutagenicity.
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Cyclophosphamide is used to treat a wide range of human malignancies. However, it is also a known carcinogen associated with induction of therapy-related leukemia and bladder cancer. The DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), protects cells from the toxic and mutagenic effects of O6-alkylating agents. We report here the contribution of AGT in protecting against the toxic and mutagenic effects of cyclophosphamide. CHO cells transduced with wild-type human AGT (CHO(AGT)) and pcDNA3 (CHOpcDNA3) were treated with activated cyclophosphamide derivatives, 4-hydroperoxycyclophosphamide (4-HC), 4-hydroperoxydidechlorocyclophosphamide (4-HDC), a progenitor of acrolein, and phosphoramide mustard (PM). The results show that CHO(AGT) is 7- or 20-fold less sensitive to the toxic effects of 30 microM 4-HC or 300 microM 4-HDC, respectively, than CHOpcDNA3 cells as measured by cell survival using a colony-forming assay. CHO(AGT) cells treated with 20 microM 4-HC or 200 microM 4-HDC produced 4- or 7-fold lower mutation frequency as measured at the HPRT locus than CHOpcDNA3 cells treated with the same dose of drugs. At 30 microM acrolein, the cell survival for CHO(AGT) was 30% compared with 18.7% for CHOpcDNA3. The mutation frequency of acrolein at the same dose was 57 mutants/10(6) cells in CHOpcDNA3 compared with no mutants in CHO(AGT). In contrast, CHO(AGT) and CHOpcDNA3 cells treated with PM had similar survival curves and exhibited no difference in mutation frequency. The present study demonstrates that AGT plays an important role in protecting against the toxic and mutagenic effect of cyclophosphamide and suggests that acrolein, not PM, is responsible for generating the toxic and mutagenic lesion(s) protected by the AGT protein. (+info)
A novel role in DNA metabolism for the binding of Fen1/Rad27 to PCNA and implications for genetic risk.
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Fen1/Rad27 nuclease activity, which is important in DNA metabolism, is stimulated by proliferating cell nuclear antigen (PCNA) in vitro. The in vivo role of the PCNA interaction was investigated in the yeast Rad27. A nuclease-defective rad27 mutation had a dominant-negative effect that was suppressed by a mutation in the PCNA binding site, thereby demonstrating the importance of the Rad27-PCNA interaction. The PCNA-binding defect alone had little effect on mutation, recombination, and the methyl methanesulfonate (MMS) response in repair-competent cells, but it greatly amplified the MMS sensitivity of a rad51 mutant. Furthermore, the PCNA binding mutation resulted in lethality when combined with a homozygous or even a heterozygous pol3-01 mutation in the 3'-->5' exonuclease domain of DNA polymerase delta. These results suggest that phenotypically mild polymorphisms in DNA metabolic proteins can have dramatic consequences when combined. (+info)
Evidence for site-specific bioactivation of alachlor in the olfactory mucosa of the Long-Evans rat.
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Alachlor (2-chloro-2',6'-diethyl-N-[methoxymethyl]-acetanilide) is a restricted-use chloracetanilide herbicide which has been shown previously to produce a dose-dependent incidence of olfactory mucosal tumors in rats following chronic dietary exposure. However, the mechanism of alachlor carcinogenicity is poorly understood. Alachlor was administered i.p. to male Long-Evans rats for up to 28 days at doses that are carcinogenic in chronic studies in order to study olfactory lesion development and alterations in cell proliferation. Neither treatment-related olfactory mucosal lesions nor regenerative cell proliferation, as assessed with BrdU labeling, was detected. In vitro genotoxicity studies using Salmonella typhimurium strain TA100 showed that alachlor was non-mutagenic in the absence of metabolic activation. When pre-incubated with an olfactory mucosal S9 activation system, alachlor induced a weak, dose-dependent mutagenic response at 500-1250 micrograms/plate, with toxicity at higher doses. In contrast, an S9 activation system derived from nasal respiratory mucosa, the tissue physically juxtaposed with the olfactory mucosa but reportedly not susceptible to alachlor-induced tumors, did not produce a mutagenic response for alachlor or the positive control. Thus, this result suggested site-specificity of alachlor activation consistent with the target site of carcinogenicity. The mutagenicity of alachlor to Salmonella, in the presence of an olfactory mucosal-activating system, was confirmed by a limited positive response in the mouse lymphoma assay. Here there were increases in small colony mutants (indicative of chromosomal effects) as well as large colony mutants (which reflect gene mutations). This study suggests that target tissue bioactivation of alachlor results in the formation of one or more mutagenic metabolite(s), which may be critical in alachlor-induced nasal tumorigenesis. (+info)