Report of the IWGT working group on strategy/interpretation for regulatory in vivo tests II. Identification of in vivo-only positive compounds in the bone marrow micronucleus test.

A survey conducted as part of an International Workshop on Genotoxicity Testing (IWGT) has identified a number of compounds that appear to be more readily detected in vivo than in vitro. The reasons for this property varies from compound to compound and includes metabolic differences; the influence of gut flora; higher exposures in vivo compared to in vitro; effects on pharmacology, in particular folate depletion or receptor kinase inhibition. It is possible that at least some of these compounds are detectable in vitro if a specific in vitro test is chosen as part of the test battery, but the 'correct' choice of test may not always be obvious when testing a compound of unknown genotoxicity. It is noted that many of the compounds identified in this study interfere with cell cycle kinetics and this can result in either aneugenicity or chromosome breakage. A decision tree is outlined as a guide for the evaluation of compounds that appear to be genotoxic agents in vivo but not in vitro. The regulatory implications of these findings are discussed.

Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards.

In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus, a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in increases in micronucleated cells in the bone marrow that are not related to the intrinsic genotoxicity of the compound under test. For rodent bone marrow or peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell division in these cells. This paper reviews relevant data from the literature and also previously unpublished data obtained from a questionnaire devised by the IWGT working group. Regulatory implications of these findings are discussed and flow diagrams have been provided to aid in interpretation and decision-making when such changes in physiology are suspected.

Spindle poisons induce allelic loss in mouse lymphoma cells through mitotic non-disjunction.

Aneuploidy is an important contributor to reproductive failure and tumor development. It arises spontaneously or as a result of exposure to aneugenic agents through non-disjunction. Two spindle poisons, colchicine (COL) and vinblastine (VBL) are mutagenic in the mouse lymphoma assay (MLA), a gene mutation assay that targets the heterozygous thymidine kinase (tk) gene on chromosome 11 in mouse lymphoma L5178Y tk+/- 3.7.2c cells. To investigate the mechanisms of spindle poison mutagenesis, we analyzed the COL- and VBL-induced TK mutants at the molecular and cytogenetic level. Loss of heterozygosity (LOH) analysis employing a microsatellite region within the tk locus revealed that almost all mutants had lost the functional tk allele. To determine the extent of the LOH, we further examined LOH mutants for heterozygosity at nine microsatellite loci spanning the entire chromosome 11. Interestingly, every microsatellite marker showed LOH in all COL- and VBL-induced LOH mutants, suggesting that these mutants were generated by loss of the whole chromosome 11 through mitotic non-disjunction. Chromosome painting analysis supported this hypothesis; there were no mutants showing structural changes such as deletions or translocations involving chromosome 11. In contrast, spontaneous TK mutants followed from point mutations, deletions and recombinational events as well as whole chromosome loss. Our present study indicates that spindle poisons induce mutations through mitotic non-disjunction without structural DNA changes and supports a possible mechanism in which a recessive mutation mediated by aneuploidy may develop tumors.

Hepatocarcinogen quinoline induces G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (MutaMouse).

Quinoline is carcinogenic to the liver in rodents, but it is not clear whether it acts by a genotoxic mechanism. We previously demonstrated that quinoline does induce gene mutation in the liver of lambda/lacZ transgenic mice. In the present report, we reveal the molecular nature of the mutations induced by quinoline in the lambda cII gene, which is also a phenotypically selectable marker in the lambda transgene. (The cII gene has 294bp, which enables much easier sequence analysis than the original lacZ gene (3kb)). The liver cII mutant frequency was nine times higher in quinoline-treated mice than in control mice. Sequence analysis revealed that quinoline induced primarily G:C to C:G transversions (25 of 34). Thus, we have confirmed that quinoline is genotoxic in its target organ, and the G:C to C:G transversion is the molecular signature of quinoline-induced mutations.

Requirement of wild-type p53 protein for maintenance of chromosomal integrity.

Chromosomal double-strand breaks (DSBs) occurring in mammalian cells can initiate genomic instability, and their misrepairs result in chromosomal deletion, amplification, and translocation, common findings in human tumors. The tumor-suppressor protein p53 is involved in maintaining genomic stability. In this study, we demonstrate that the deficiency of wild-type p53 protein may allow unrepaired DSBs to initiate chromosomal instability. The human lymphoblastoid cell line TK6-E6 was established by transfection with human papilloma virus 16 (HPV16) E6 cDNA into parental TK6 cells via a retroviral vector. Abrogation of p53 function by E6 resulted in an increase in the spontaneous mutation frequencies at the heterozygous thymidine kinase (TK) locus but not at the hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus. Almost all TK-deficient mutants from TK6-E6 cells exhibited loss of heterozygosity (LOH) with the hemizygous TK allele. LOH analysis with microsatellite loci spanning the long arm of chromosome 17, which harbors the TK locus, showed that LOH extended over half of 17q toward the terminal end. Cytogenetic analysis of LOH mutants by chromosome painting indicated a mosaic of chromosomal aberrations involving chromosome 17, in which partial chromosome deletions, amplifications, and multiple translocations appeared heterogeneously in a single mutant. We speculate that spontaneous DSBs trigger the breakage-fusion bridge cycle leading to such multiple chromosome aberrations. In contrast, no chromosomal alterations were observed in TK-deficient mutants from TK6-20C cells expressing wild-type p53. In wild-type p53 cells, spontaneous DSBs appear to be promptly repaired through recombination between homologous chromosomes. These results support a model in which p53 protein contributes to the maintenance of genomic integrity through recombinational repair.

In vivo genotoxicity of 2-amino-3,8-dimethylimidazo[4, 5-f]quinoxaline in lacI transgenic (Big Blue) mice.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a heterocyclic amine found in cooked meat, is a strong mutagen in the Salmonella/microsome assay and was proven to be a hepatocarcinogen in rodents. We used the lacI transgenic (Big Blue(R)) mouse to investigate MeIQx genotoxicity in vivo. lacI mutant frequencies were examined in liver and colon after single intragastric administration of MeIQx (males) or 12 weeks of feeding in the diet (males and females). Micronucleus induction was monitored in the peripheral blood and cell proliferating activity was monitored by proliferating cell nuclear antigen (PCNA) immunostaining, but only after the intragastric administration. Intragastric treatment with MeIQx (100 mg/kg) did not increase mutant frequency (MF) in liver or colon but it did induce a slight but statistically significant increase in the incidence of micronucleated reticulocytes 48 h after the treatment. No apparent increase in PCNA-positive foci was observed in any of tissues analyzed 14 days after the treatment. Administration of MeIQx (300 ppm) in diet for 12 weeks, however, caused MF increases in liver and colon in male and female mice, with greater increases in the females. An increase was also obvious after 4 weeks, but only in females. The sex difference in MF is consistent with the fact that female mice are more susceptible to MeIQx carcinogenesis. These results demonstrated that in the transgenic mouse mutation assay, long-term feeding of MeIQx was more effective than single gastric exposures in revealing the compound's mutagenicity in the target organs of carcinogenicity and that sex differences in susceptibility can also be observed.

Appropriate levels of cytotoxicity for genotoxicity tests using mammalian cells in vitro.

Among standard battery genotoxicity assays, the in vitro chromosome aberration test and the mouse lymphoma tk assay (MLA) yield about fourfold higher incidences of positive test results than the bacterial reverse mutation test or in vivo bone marrow tests. This is a result of experience with submissions of 335 new pharmaceuticals to the German Federal Institute for Drugs and Medical Devices. While all of the standard systems have their value in detecting relevant genotoxins, there is no supportive evidence for DNA reactivity for a considerable number of in vitro clastogens and MLA positives. In particular the clastogenic response of such compounds is often associated with high cytotoxicity. This may invoke the need to change the approach to test for clastogenicity in vitro. A combination of measures such as (1) a change in the upper limits of cytotoxicity that are currently given in International Conference on Harmonisation (ICH) and Organization for Economic Co-Operation & Economic Development (OECD) guidelines, (2) the creation of a common ground of understanding for interpretation of in vitro (positive) test results, and (3) lowering the upper limits of test compound concentration irrespective of cytotoxicity may prove useful to ensure a sufficient reliability of genotoxicity testing with mammalian cells in vitro.

Validation study of the in vitro micronucleus test in a Chinese hamster lung cell line (CHL/IU).

We conducted a collaborative validation study, under the auspices of the Japanese Ministry of Labour, on the in vitro micronucleus test to see if it could be used as an alternative to the in vitro chromosome aberration test for evaluation of chemical safety. We used the Chinese hamster lung cell line (CHL/IU), which is the most widely used system for the latter test in Japan, and evaluated 66 chemicals, including clastogens and polyploidy inducers. The cytochalasin B cytokinesis blocking method, which is commonly used in human lymphocyte culture, was applied to the established cell line, but did not improve the detection of chemically-induced micronuclei in continuously growing cells. The highest micronucleus frequencies were obtained at 48 or 72 h continuous treatments. In short treatments (6 h), a 42 h recovery time yielded the best responses. Concordance between the results of the micronucleus test and the chromosomal aberration test was satisfactorily high (88.7%), and we concluded that the in vitro micronucleus test could be used in place of the chromosomal aberration test as a simple and rapid method for detecting clastogens and aneugens in vitro. We also propose a protocol for the test.

Procarbazine genotoxicity in the MutaMouse; strong clastogenicity and organ-specific induction of lacZ mutations.

Procarbazine, a drug used for cancer chemotherapy, is carcinogenic in rodent bioassays. We analyzed the mutagenicity of procarbazine in various organs and the clastogenicity of the drug in hematopoietic cells of the lacZ transgenic MutaMouse. This was part of the second collaborative study of the Mammalian Mutagenesis Study Group of the Japanese Environmental Mutagen Society on the transgenic mouse mutation assay. At 50 mg kg(-1), procarbazine induced micronuclei in hematopoietic cells, but it did not increase the lacZ mutant frequency (MF) in bone marrow. It was also negative in liver, testis, spleen, kidney, and lung. Five daily administrations of 150 mg kg(-1) yielded highly positive responses in the drug's target organs for carcinogenesis (lung, bone marrow, and spleen). Lower positive responses were detected in kidney, which is a minor target organ. Liver showed only a slight increase in lacZ MF and brain showed no increase. The testis MF more than doubled which suggest that procarbazine is mutagenic to germ cells. Thus, we demonstrated that procarbazine has a strong clastogenic effect in hematopoietic cells and is mutagenic in a variety organs after high dose treatment. The induced MF was especially high in procarbazine's target organs for carcinogenesis, which supports the relevance of the transgenic mouse mutation assay for the assessment of potential genotoxins in vivo.

Effects of O (6)-alkylguanine-DNA alkyltransferase deficiency in Escherichia coli as the host for the detection of mutations in lacI transgenic mice.

Transgenic mice are widely used to detect gene mutations in vivo induced by a variety of chemicals. It is known, however, that no mutagenicity of methyl methanesulfonate (MMS) is detected in epididymal sperm in various transgenic mice assays, although MMS induces the dominant lethal and specific locus mutations in male mice. To investigate the issue of whether unrepaired lesions in DNA of mature sperm can be transformed into mutations during replication of the lambda phage in Escherichia coli cells, we developed an E. coli strain YG5152, which is a derivative of strain SCS-8 but is deficient in the genes encoding O (6)-alkylguanine-DNA alkyltransferases. When lambda LIZalpha phages were treated with MMS or N-ethyl-N-nitrosourea (ENU) in vitro and infected to the E. coli strains, the mutant frequencies of lacI were markedly higher in strain YG5152 than in strain SCS-8. When Big Blue(trade mark) mice were treated with MMS (160 mg/kg) or ENU (125 or 250 mg/kg) and the phages rescued from mature sperm were infected to the strains, the mutation frequency (MF) of phages from ENU-treated mice at a dose of 250 mg/kg in strain YG5152 was about two times higher than that in strain SCS-8. However, no increase in the MF was observed in the MMS-treated mice even in strain YG5152. These results suggest that, although strain YG5152 efficiently detects ex vivo mutations caused by mutagenic alkyl adducts formed by MMS in lambda phage DNA, no detectable levels of mutagenic methyl adducts are present in mature sperm of MMS-treated mice. Possible reasons for this lack of mutagenicity of MMS in mature sperm using transgenic mice assays are discussed.

Toxicity study of a rubber antioxidant, mixture of 2-mercaptomethylbenzimidazoles, by repeated oral administration to rats.

2-Mercaptobenzimidazole (2-MBI), a rubber antioxidant, is known to exhibit potent antithyroid toxicity in rats and is a candidate as an environmental endocrine disrupter. 2-Mercaptomethylbenzimidazoles (a 1:1 mixture of 4-methyl and 5-methyl isomers, MMBIs), are also employed industrially as rubber antioxidants and are suspected to exert antithyroid toxicity such as 2-MBI. In this investigation, acute and subacute oral toxicity studies of MMBIs in Wistar rats were conducted. The clinical signs of acute oral toxicity were observed including decreased spontaneous movement, a paralytic gait, salivation and lacrimation, and adoption of prone and lateral positions. The LD50 was estimated to be 330 mg/kg. In the subacute oral toxicity study, male and female rats were treated with MMBIs by gavage at doses of 0 (corn oil), 4, 20 and 100 mg/kg for 28 consecutive days followed by a 2-week recovery period for the control and highest dose groups. Body weight and food consumption, clinical signs, organ weights, clinical biochemistry and haematological parameters including clotting times and micronuclei induction in bone marrow erythropoeitic cells, and histopathology were examined. Relative organ weights of lung, liver and kidney, and serum cholesterol and phospholipid significantly increased in male rats treated with MMBIs at doses of 20 and 100 mg/kg. Male rats administered 100 mg/kg MMBIs exhibited a 1.8-fold increase in thyroid weight associated with histopathological changes but not altered serum thyroid hormone levels. Female rats administered 100 mg MMBIs/kg exhibited significant increases of liver and kidney but not thyroid weights, and serum cholesterol level. The antithyroid toxicity of MMBIs in rats was estimated to be one-tenth that of 2-MBI. No-observed-effect levels for male and female rats were found to be 4 and 20 mg/kg, respectively, in this subacute oral toxicity study.

Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test.

In order to evaluate the utility of the mouse lymphoma assay (MLA) for detecting in vitro clastogens and spindle poisons and to compare it with the in vitro chromosomal aberration test (CA), we conducted an international collaborative study of the MLA that included 45 Japanese laboratories and seven overseas laboratories under the cooperation of the Ministry of Health and Welfare of Japan and the Japanese Pharmaceutical Manufacturer's Association. We examined 40 chemicals; 33 were reportedly positive in the CA but negative in the bacterial reverse mutation assay, six were negative in both assays and one was positive in both. We assayed mutations of the thymidine kinase (TK) locus (tk) of L5178Y tk +/- mouse lymphoma cells using the microwell method. According to our standard protocol, cells were exposed to the chemical for 3 h, cultured for 2 days and TK-deficient mutants were expressed in 96-well plates under trifluorothymidine. Each chemical was coded and tested by two or three laboratories. Among the 34 CA-positive chemicals, positive MLA results were obtained for 20 and negative results were obtained for nine. The remaining five chemicals were inconclusive or equivocal because of discrepant inter-laboratory results or reproduced discrepant results, respectively. Among the six CA-negative chemicals, one was negative in the MLA, two were positive and three were inconclusive. Thus, the MLA could detect only 59% (20/34) of CA-positive chemicals. We concluded that the MLA was not as sensitive as the CA. Some MLA-negative chemicals evoked positive responses in the CA only after long continuous treatment. These might also be genotoxic in the MLA with long continuous treatment. Improvement of the MLA protocol, including alteration of the duration of the treatment, might render the MLA as sensitive as the CA.

The need for long-term treatment in the mouse lymphoma assay.

The L5178Y tk +/- mouse lymphoma assay (MLA) has been widely used as a genotoxicity test for the detection of mutagens and clastogens. The standard MLA, as well as other mammalian cell gene mutation assays, usually employs a short treatment period (3-6 h). Our previous report, however, suggested that such short treatments may be insufficient for detecting some clastogens and spindle poisons. For the present study, we introduced and evaluated a longer treatment (24 h) in the MLA. We examined 15 chemicals which were evaluated as negative or inconclusive in the short-term study. Cells were exposed to the chemical for 24 h without S9 mix, cultured for 2 days and then thymidine kinase-deficient mutants were selected in 96-well microtiter plates under trifluorothymidine. Eleven chemicals yielded positive responses in the 24 h treatment MLA. They included nucleoside analogs (2'-deoxycoformycin and dideoxycytidine), a base analog (1,3-dimethylxanthine) and spindle poisons (colchicine and vinblastine sulfate), all of which do not directly affect DNA, but bring about mutations and chromosome alterations through nucleoside metabolism and chromosome segregation. Because the mutagenicities of these non-DNA targeting chemicals appear to be cell cycle dependent, treatment extending over more than one cell cycle may be required for their effect. Combining results from the present and previous studies, 31 of 34 (91%) chromosome aberration-positive chemicals exhibited positive responses in the MLA, suggesting that the sensitivity of the MLA with 24 h treatment periods approaches that of the chromosome aberration test.

Mutagenicity of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in the new gpt delta transgenic mouse.

Gender differences and organ specificity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis were examined with the new gptdelta transgenic mouse (T. Nohmi, M. Katoh, H. Suzuki, M. Matsui, M. Yamada, M. Watanabe, M. Suzuki, N. Horiya, O. Ueda, T. Shibuya, H. Ikeda, T. Sofuni, A new transgenic mouse mutagenesis test system using Spi-and 6-thioguanine selections (Environ. Mol. Mutagen. 28 (1996) 465-470). In this mouse model, two distinct selections are employed to efficiently detect different types of mutations, i.e 6-thioguanine (6-TG) selection for point mutations and Spi-selection for deletions, respectively. In both selections, the highest mutant frequencies were observed in colon, followed by in spleen and liver. No increases in mutations were observed in testis, brain and bone marrow in PhIP-treated male mice. No significant differences in 6-TG and Spi- mutant frequencies were observed in colon and liver between male and female treated mice. The correlation between PhIP-induced mutagenesis and carcinogenesis in colon is discussed.

Spectra of gpt mutations in ethylnitrosourea-treated and untreated transgenic mice.

We have established a new transgenic mouse mutagenicity assay for the efficient detection of point mutations and deletions in vivo (Nohmi et al. [1996] Env. Mol. Mutagen. 28:465-470). In this assay, the gpt gene of Escherichia coli is used as a reporter for the detection of point mutations. Treatment of mice with ethylnitrosourea (ENU, 150 mg/kg) enhances by several-fold the mutant frequency of gpt in bone marrow. Here, we report the mutation spectra of the gpt gene recovered from bone marrow of ENU-treated and untreated transgenic mice. In the gpt mutants rescued from ENU-treated mice, more than 90% of the mutations were base change mutations; the predominant types were A:T to T:A transversions and G:C to A:T transitions. On the contrary, in the mutants rescued from untreated mice, 54% were base substitutions and the remainders were short deletions and insertions. Among untreated mice, the most frequently observed base substitution was G:C to A:T transitions (7/14 mutants). Three of these occurred at 5'-CpG-3' sites. Interestingly, the mutation spectra of the gpt gene were different from those of the gpt gene in ENU-treated and untreated E.coli, whereas they were similar to those of the lacZ and lacI genes in ENU-treated and untreated other transgenic mice or cultured mammalian cells. We also report the establishment of homozygous transgenic mice that have transgene lambdaEG10 DNA in both chromosome 17 of C57BL/6J mouse.

Spi(-) selection: An efficient method to detect gamma-ray-induced deletions in transgenic mice.

Despite the importance of genome rearrangement in the etiology of cancer and human genetic disease, deletion mutations are poorly detectable by transgenic rodent mutagenicity tests. To facilitate the detection and molecular analysis of deletion mutations in vivo, we established a transgenic mouse model harboring a lambdaEG10 shuttle vector that includes the red and gam genes for Spi(-) (sensitive to P2 interference) selection [Nohmi et al. (1996] Environ. Mol. Mutagen. 28:465-470]. This selection has a great advantage over other genetic systems, because phage deletion mutants can be preferentially selected as Spi(-) plaques, which can then be subjected to molecular analysis. Here, we show nucleotide sequences of 41 junctions of deletion mutations induced by gamma-irradiation. Unlike spontaneous deletion mutants, more than half of the large deletions occurred between short homologous sequences from one to eight bp. The remaining junctions had no such homologous sequences. Intriguingly, two Spi(-) mutants had P (palindrome)-like nucleotide additions at the breakpoints, which are frequently observed in the coding junctions of V(D)J recombination, suggesting that broken DNA molecules with hairpin structures can be intermediates in the repair of radiation-induced double-strand breaks. We conclude that Spi(-) selection is useful for the efficient detection of deletion mutations in vivo and that most rearrangements induced by gamma-rays in mice are mediated by illegitimate recombination through DNA end-joining.

ICH-harmonised guidances on genotoxicity testing of pharmaceuticals: evolution, reasoning and impact.

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has convened an expert working group which consisted of the authors of this paper and their respective committees, consulting groups and task forces. Two ICH guidances regarding genotoxicity testing have been issued: S2A, 'Guidance on Specific Aspects of Regulatory Genotoxicity Tests' and S2B, 'Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.' Together, these guidance documents now form the regulatory backbone for genotoxicity testing and assessment of pharmaceuticals in the European Union, Japan, and the USA. These guidances do not constitute a revolutionary new approach to genotoxicity testing and assessment, instead they are an evolution from preexisting regional guidelines, guidances and technical approaches. Both guidances describe a number of specific criteria as well as a general test philosophy in genotoxicity testing. Although these guidances were previously released within the participating regions in their respective regulatory communiqués, to ensure their wider distribution and better understanding, the texts of the guidances are reproduced here in their entirety (see Appendix A) and the background for the recommendations are described. The establishment of a standard battery for genotoxicity testing of pharmaceuticals was one of the most important issues of the harmonisation effort. This battery currently consists of: (i) a test for gene mutation in bacteria, (ii) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma tk assay, (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells. A major change in testing philosophy is the acceptance of the interchangeability of testing for chromosomal aberrations in mammalian cells and the mouse lymphoma tk assay. This agreement was reached on the basis of the extensive review of databases and newly generated experimental data which are in part described in this publication. The authors are fully aware of the fact that some of the recommendations given in these ICH guidances are transient in nature and that the dynamic qualities and ongoing evolution of genetic toxicology makes necessary a continuous maintenance process that would serve to update the guidance as necessary.

Specific mutational spectrum of dimethylnitrosamine in the lacI transgene of Big Blue C57BL/6 mice.

Dimethylnitrosamine (DMN) produces tumors in mice predominantly in the liver, but also in the kidney and lung. It forms O6-methylguanine adducts in DNA, which induce G:C-->A:T transitions. We have analyzed the spectra of spontaneous and DMN-induced mutations in the lacI transgene of the Big Blue mouse (C57BL/6). In both cases, mutations in the liver, kidney and lung were predominantly base substitutions, among which G:C-->A:T transitions were the most frequent. In contrast, a high incidence of short deletions (2-23 bp) was only found in the liver of treated mice. The deletions often occurred at direct repeat sequences. Single-base deletion incidence was also higher in the liver than in the kidney and lung. These results imply that accumulation of DNA lesions or their repair in liver is different from other organs. Spontaneous and induced base substitutions and deletions appeared to be randomly distributed in the lacI gene and an apparent hotspot was not observed, except for a 4 bp deletion of a (TGGC)3 sequence at positions 621-632. The present data demonstrate, for the first time, that DMN induces short deletions especially in the liver, although the mechanism involved needs further investigation.

Spindle disturbances induced by benzo[a]pyrene and 7, 12-dimethylbenz[a]anthracene in a Chinese hamster cell line (V79-MZ) and the stability of the numerical chromosome aberrations that follow.

We previously reported that benzo[a]pyrene (BP) and 7, 12-dimethylbenz[a]anthracene (DMBA) induce aneuploidy and polyploidy, respectively, in the Chinese hamster cell line V79-MZ in the absence of S9 mix. In the present study we investigated the effect of BP and DMBA on the mitotic spindle. BP caused incomplete spindle formation and DMBA inhibited spindle formation completely. The combined results indicate that incomplete spindles caused by BP resulted in aneuploidy, and the absence of spindle formation caused by DMBA resulted in polyploidy. The induced polyploidy was stable for several serial passages in fresh medium. BP and DMBA induced different chromosome number distributions. After BP treatment, the normal distribution of chromosome number was restored in 4 days. After DMBA treatment, on the other hand, a tetraploid peak was maintained for 2 months following an initial transient broad distribution of chromosome number after 1 day. The results suggest that different mechanisms were involved in the induction of numerical aberrations by BP and DMBA. Furthermore the induction of numerical aberrations by BP and DMBA was reproducible over 5 months of passages. In four clones tested, the frequency of cells with the modal chromosome number in control cultures gradually decreased from 82% on the average just after cloning to 62% 5 months later. BP and DMBA induced characteristic ploidy changes following succeeding cell passages for up to 5 months, indicating that the ability to respond to BP and DMBA was stable for that period of time. Because these findings were specific to V79-MZ cells, this cell line might be a good tool for studying chemicals that induce numerical chromosome aberrations.