Genetic toxicology of 1,1,2-trichloroethylene

1,1,2-Trichloroethylene (TCE) is a widely used halogenated solvent, produced in hundreds of millions of kg each year for industrial purposes. Occupational and environmental exposure of human populations to TCE has been reported in industrialized areas. Long-term carcinogenicity studies in rodents demonstrate that exposure to high doses of TCE results in the induction of liver and lung tumors in the mouse, and tumors of the kidney and the testis in the rat. An indirect mechanism, based on the stimulation of liver peroxisome proliferation by TCE metabolites, was proposed to explain species differences in TCE hepatocarcinogenicity. Mutagenicity studies indicate that TCE is weakly active both in vitro, where liver microsomes produce electrophilic TCE metabolites, and also in vivo in mouse bone marrow, where high rates of micronuclei, but no structural chromosome aberrations, are found. Among TCE metabolites, trichloroacetic acid was reported to be carcinogenic to mouse liver. Furthermore, both trichloroacetic acid and chloral hydrate were found to be genotoxic in vivo, inducing structural and numerical chromosome abnormalities, respectively.     

Proposed reference dose for toxaphene carcinogenicity based on constitutive androstane receptor-mediated mode of action

Toxaphene is a liver tumor promoter in B6C3F1 mice but not in F344 rats or hamsters. Recent studies demonstrate that key events leading to the mouse liver tumor response for toxaphene are mediated by activation of the constitutive androstane receptor (CAR). Benchmark dose modeling was conducted on available data for five endpoints in B6C3F1 mouse liver tissue or cultured liver cells (tumor response, cytotoxicity, proliferation, gap junction intercellular communication inhibition, and CAR-mediated CYP2B10 induction) and for CAR activation in human HepG2 cells, all reported in previous studies. The available evidence supports a nonlinear CAR-mediated mode of action (MOA) for toxaphene-induced mouse tumors including demonstration of a J-shaped dose-response pattern for human CAR activation, indicating that linear risk extrapolation at low doses is not supported for this MOA. Based on analysis of benchmark dose lower confidence limits at 10% response (BMDL10) and no observed effect levels (NOELs) for potential key events in the mouse liver tumor MOA for toxaphene, an RfD of 0.13 mg/kg-d is proposed based on a the BMDL10 for human CAR activation in human HepG2 cells. This value is below candidate RfD values based on BMDL10 estimates for both mouse liver tumors and mouse hepatocyte proliferation and therefore can be considered protective for human risk of liver tumor promotion and other CAR-mediated adverse health effects based on available data.     

Use of a cell hybrid test system to demonstrate that benomyl induces aneuploidy and polyploidy

We have monitored the segregation of a single human chromosome in a human-Chinese hamster hybrid cell line, EUBI, following exposure to benomyl. We found a dose-dependent increase in frequency of aneuploidy, but a much more marked induction of polyploidy was noted at the highest benomyl concentration. We confirm the usefulness of this assay for determining genetic risk associated with human exposure to environmental chemicals.     

Discrimination of aneuploidogens from clastogens by C-banding, DNA and area measurements of micronuclei from mouse bone marrow.

Micronuclei (MN) obtained from mouse bone marrow cells, in vivo exposed to 3 typical clastogens (procarbazine, azathioprine, ethyl methanesulfonate) and 3 typical aneuploidogens (vinblastine, tubulazole, colchicine), were examined for C-band, area and DNA content. C-banding allows a clear discrimination between clastogens and aneuploidogens: the clastogens do not exceed 50% C-band-positive MN and the aneuploidogens all 3 produce 65-75% C-band-positive MN. Concerning the DNA content the percentages of MN containing more DNA than an average chromosome (chr) are lower than 12% for the clastogens and 38-60% for the aneuploidogens. As far as the area of the MN is concerned the percentages of MN which have a larger area than chr are lower than 23% for the clastogens and range from 47% to 71% for the aneuploidogens. Additionally 3 other mutagens were studied. Hydroquinone induces 43% C-band-positive MN with DNA content far below the content of chr; considering the area measurements, however, hydroquinone behaves as an aneuploidogen (65% of the MN are larger than chr). Mitomycin C lies between the clastogens and the aneuploidogens for all 3 criteria but 5-azacytidine is comparable to the model aneuploidogens.     

Mutagenicity of 43 structurally related heterocyclic compounds and its relationship to their carcinogenicity.

43 heteropolycyclic compounds belonging to a homologous series were investigated for mutagenicity. The results are compared with carcinogenicity data obtained with the same batches of compounds under conditions identical for all of them. Mutagenicity was tested in the Ames test with Salmonella typhimurium strains TA1535, TA1537 and TA100 in the presence and absence of liver 10 000 g supernatant from rats treated with Aroclor 1254. Carcinogenicity was tested by injection of the compounds into subcutaneous tissue of XVIInc/Z mice. 18 test compounds showed carcinogenic activity, some strongly, others only weakly. Of these, 17 were detected as mutagens: one weak carcinogen did not revert the Salmonella strains. No quantitative correlation was observed between the extents of the mutagenic and the carcinogenic effects. Of the 25 substances that did not produce tumours, 13 showed mutagenicity (12 in the presence, 2 in the absence, of the liver homogenate). The mutagenic effects of these compounds were quantitatively similar to those of the compounds that produced tumours. The most sensitive strain of Salmonella typhimurium was TA100. It detected all 30 mutagens. TA98 was mutated by 25 compounds, TA1537 by 16 compounds. No mutagenic effects were seen with TA1535. Possible reasons for the high percentage of apparently "false positives" in the Ames test and the lack of a quantitative correlation between the potency of the mutagenic and carcinogenic effects are discussed. It is suggested that the complexity of the metabolism of these heterocyclic compounds may lead to critical differences in metabolism in mouse subcutaneous tissue in vivo and in liver homogenates from rats treated with Aroclor. Therefore the present study will be extended to life-long oral and intrahepatic carcinogenicity tests leading to a higher proportion of metabolism in the liver.     

Carbendazim (MBC) disrupts oocyte spindle function and induces aneuploidy in hamsters exposed during fertilization (meiosis II)

Peri-fertilization exposure to Carbendazim (MBC; a microtubule poison) induces infertility and early pregnancy loss in hamsters. Presently, both in vivo and in vitro techniques were employed to characterize the effects of MBC on cellular aspects of fertilization in hamsters. Exposure to MBC during either in vivo or in vitro fertilization (IVF) induced identical morphological abnormalities in the maternal chromatin of zygotes and embryos. These abnormalities included either multiple second polar bodies (PB₂), and/or multiple small female pronuclei (PN), or meiotic arrest. Multiple PB₂, multiple female PN, multiple PB₂ with multiple female PN, or meiotic arrest were exhibited by approximately 31%, 15%, 12%, and 2% of the in vivo zygotes; and 3%, 16%, 36%, and 20% of IVF zygotes, respectively. The effects of MBC persisted to day 2 of pregnancy as indicated by decreased (P ≤ 0.05) embryo development to the two-cell stage and the presence of micronuclei in 6% of two-cell embryos from MBC-treated females. Immunofluorescence analysis of microtubules (MTs) confirmed that MBC disrupted spindle MTs during IVF. Numerical chromosome analysis revealed that a single dose of MBC administered during in vivo fertilization induced aneuploidy in the resulting pronuclear-stage zygotes. The present data point to two mechanisms by which peri-fertilization MBC exposure may induce early pregnancy loss: 1) arrested meiosis with no zygotic cleavage; or 2) induction of zygotic aneuploidy with subsequent developmental arrest. © 1995 wiley-Liss, Inc.     

Aneuploidy in Drosophila, IV. Inhalation studies on the induction of aneuploidy by nitriles

The Drosophila ZESTE system was used to monitor the induction of sex chromosome aneuploidy following inhalation exposure of adult females to four nitriles: acetonitrile, propionitrile, acrylonitrile and fumaronitrile. Acetonitrile and propionitrile were highly effective aneuploidogens, inducing both chromosome loss and chromosome gain following brief exposures to low concentrations of these chemicals, and these nitriles also induced rapid paralysis. Acrylonitrile-induced chromosome loss only but did not induce paralysis. Fumaronitrile, in contrast with the results reported in yeast, was ineffective in inducing chromosome loss or gain. Virtually all exceptional offspring induced by acetonitrile and propionitrile were recovered in the first sampled eggs, corresponding to treated mature oocytes. Additionally, the time interval between treatment and sampling was shown to be important, suggesting rapid loss or detoxification of the nitriles. Genetic analysis demonstrated that most aneuploids resulted from induced segregation errors during the first division of meiosis. Cold treatments were found to be ineffective in enhancing the effects of acetonitrile, suggesting important differences between the Drosophila and yeast aneuploidy detection systems. Possible mechanisms by which nitriles may disrupt chromosome segregation in Drosophila oocytes are considered.     

Simultaneous measurement of the frequencies of intrachromosomal recombination and chromosome gain using the yeast DEL assay

The yeast DEL assay measures the frequency of intrachromosomal recombination between two partially-deleted his3 alleles on chromosome XV. The his3Delta alleles share approximately 400bp of overlapping homology, and are separated by an intervening LEU2 sequence. Homologous recombination between the his3Delta alleles results in deletion of the intervening LEU2 sequence (DEL), and reversion to histidine prototrophy. In this study we have attempted to further extend the use of the yeast DEL assay to measure the frequency of chromosome XV gain events. Reversion to His(+)Leu(+) in the haploid yeast DEL tester strain RSY6 occurs upon non-disjunction of chromosome XV sister chromatids, coupled with a subsequent DEL event. Here we have tested the ability of the yeast DEL assay to accurately predict the aneugenic potential of the diversely-acting, known or suspected aneugens actinomycin D, benomyl, chloral hydrate, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and methotrexate. Actinomycin D and benomyl strongly induced aneuploidy. EMS and methotrexate modestly induced aneuploidy, while chloral hydrate and MMS failed to illicit any significant induction. In addition, by FACS-analysis of DNA content it was shown that the majority of both spontaneous- and chemically-induced His(+)Leu(+) revertants were heterodiploid. Thus, our results indicate endoreduplication of almost entire chromosome sets as a major mechanism of aneuploidy induction in haploid Saccharomyces cerevisiae.     

Mutagenicity testing of benomyl, methyl-2-benzimidazole carbamate, streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium in vitro and in rodent host-mediated assays.

The fungicide benomyl and its commercial preparations Fundazol 50WP and Benlate 50WP and the benomyl metabolite methyl-2-benzimidazole carbamate and its commercial preparation MBC 50WP were tested for mutagenicity in in vitro spot tests, in microsomal plate assay, in liquid-culture treatments, or in rodent host-mediated assay. The base-pair substitution Salmonella typhimurium mutant hisG46 and the hisG46-bearing uvrB excision-repair-deficient mutants TA100, TA1530, TA1535 or TA1950 were used as test organisms. Complete genotypic information of these mutants is given in Ames et al. [2]. Captain 50WP, streptozotocin (SZN), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-aminopurine and N-acetylaminofluorene were used as positive control compounds. In nonoverlay spot tests Benlate 50WP was not mutagenic over a dose range of 50-5000 microgram/spot in hisG46 and TA1535. In overlay spot tests 50 or 100 microgram/spot Benomyl, MBC, Fundazol 50WP, Benlate 50WP and MBC 50WP were tested in hisG46, TA1530 or TA1950. Only a non-commercial MBC sample at 100 microgram/spot showed weak mutagenic activity in hisG46. In microsomal activation plate assay MBC, benomyl, Fundazol 50WP and Benlate 50WP were tested in TA100 over a dose range of 50-2000 microgram/plate. None of the compounds showed mutagenicity. In a 20-h liquid-culture treatment 10, 100, 1000 and 10 000 microgram/ml Fundazol 50WP were not mutagenic in TA 30. In 1-h liquid-culture treatments benomyl, Benlate 50WP or Fundazol 50WP failed to induce mutations in hisG46, TA100 or TA1950 over a dose range of 0.25-1000 microgram/ml. Appropriate positive controls were mutagenic in each experiment. The consistently negative results in this study with commercial MBC and benomyl preparations are contrary to positive results reported earlier with similar methods and similar commercial preparations. Possible reasons to explain the different results are presented. The alkylating agents SZN and MNNG induced fewer mutations in TA1530 and TA1950 uvrB excision-repair-deficient strains than in the hisG46 excision-proficient strain, indicating that with these mutagens excision-repair is also a mutation-prone process. In rodent host-mediated assays with Fundazol 50WP in mice 3 consecutive subcutaneous hourly doses of 500 mg/kg in hisG46 and TA1950 and in rats or mice an oral dose of 4000 mg/kg in TA1950 were not mutagenic. The positive control SZN was mutagenic.     

Use of a human X mouse hybrid cell line to detect aneuploidy induced by environmental chemicals

A short-term assay utilizing a human/mouse monochromosomal hybrid cell line R3-5, to detect chemically induced aneuploidy in mammalian cells is described. A single human chromosome transferred into mouse cells was used as a cytogenetic marker to quantitate abnormal chromosome segregation following chemical treatment. The human chromosome present in the mouse cells can be readily identified by differential staining procedures. The frequency of cells containing 0 or 2 human chromosomes in the progeny of chemically treated monochromosomal hybrid cells provided a direct measure of aneuploidy. We tested the sensitivity of the proposed system with 3 model chemicals (colcemid, cyclophosphamide and benomyl) known to induce numerical or structural changes in chromosomes. The frequency of an abnormal segregation of the human chromosome was found to be dose dependent and consistently higher than controls. This system has the capability to detect gain as well as loss of a chromosome resulting from nondisjunction or other mechanisms leading to aneuploidy.     

1-Methyl-2-pyrrolidinone (NMP) does not induce structural and numerical chromosomal aberrations in vivo

1-Methyl-2-pyrrolidinone induces aneuploidy in yeast, but only under special treatment conditions. Other genotoxic effects have not been found in vitro, and in vivo no data are available in the literature. Therefore, NMP was investigated in the mouse micronucleus test and the Chinese hamster bone marrow test for structural and numerical chromosomal aberrations. These tests can detect both types of alterations as demonstrated by appropriate positive control substances (cyclophosphamide, vincristine sulfate and benomyl). NMP at single oral doses up to 3800 mg/kg body weight (∼ 80% of the LD₅₀) did not lead to an increase either in micronucleated erythrocytes or in structural or numerical chromosomal aberrations when bone marrow was sampled 16, 24 and 48 h after treatment in the micronucleus test or after 24 and 48 h for karyotype analysis.     

Griseofulvin-induced aneuploidy and meiotic delay in male mouse germ cells: detected by using conventional cytogenetics and three-color FISH.

Griseofulvin (GF) was tested in male mouse germ cells for the induction of meiotic delay and aneuploidy. Starved mice were orally treated with 500, 1000 and 2000 mg/kg of GF in corn oil and testes were sampled 22 h later for meiotic delay analysis and chromosome counting in spermatocytes at the second meiotic metaphase (MMII). A dose-related increase in meiotic delay by dose-dependently arresting spermatocytes in first meiotic metaphase (MMI) or/and prolonging interkinesis was observed. Hyperhaploid MMII cells were not significantly increased. Sperm were sampled from the Caudae epididymes 22 days after GF-treatment of the males for three-color fluorescence in situ hybridization (FISH). The frequencies of diploidies were 0.01-0.02% in sperm of the solvent control animals and increased dose-dependently to 0.03%, 0.068% and 0.091%, respectively, for 500, 1000 and 2000 mg/kg of GF. The frequencies of disomic sperm were increased significantly above the controls in all GF-treated groups but showed no dose response. The data for individual classes of disomic sperm indicated that MII was more sensitive than MI to GF-induced non-disjunction in male mice. A comparison of the present data from male mice and literature data from female mice suggests that mouse oocytes are more sensitive than mouse spermatocytes to GF-induced meiotic delay and aneuploidy.     

Viral multiplicity of attachment and its implications for human immunodeficiency virus therapies.

The multiplicity of attachment (MOA) of a virion in any particular time interval is the average number of cellular attachment opportunities that must be blocked to keep the virion in suspension. MOA is usually proportional to incubation time and cell concentration. Low MOA (like low multiplicity of infection) is required for reproducible assay of adsorptive blockers, and high MOA by itself can produce spurious synergies between adsorptive blockers, e.g., soluble CD4 (sCD4) and some antibodies. Poliovirus and human immunodeficiency virus (HIV) data show that viral neutralization conforms quantitatively to MOA and kinetic theory over large ranges of incubation times and target cell concentrations. Extrapolating sCD4 data beyond conditions achievable in vitro to those in vivo predicts that sCD4 concentrations above the strain-specific sCD4-gp120 dissociation constant are required to block lymphoid HIV significantly, in at least semiquantitative agreement with clinical results. The extrapolation is applicable to humoral neutralization data as well. MOA analysis also indicates that although completely stopping the attachment of individual virions to cells may still be an effective therapeutic strategy against established HIV infection, merely retarding attachment probably is not. The concept of MOA holds great promise for improving the therapeutic relevance of in vitro data and can be applied to any infectious agent, to many processes that impair or enhance infection steps, and to many assay end points, not just infection.     

A review and analysis of the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase assay to determine the mutagenicity of chemical agents. A report of phase III of the U.S. Environmental Protection Agency Gene-Tox Program

Published literature on the Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay from mid-1979 through June 1986 was reviewed and evaluated. Data from the papers considered acceptable include test results on 121 chemicals belonging to 25 chemical classes. A total of 87 chemicals were evaluated positive, 3 negative, and 31 inconclusive. Mutagenicity data on 49 of the 121 chemicals evaluated could also be compared with in vivo animal carcinogenicity data. 40 of the 43 reported animal carcinogens were considered mutagenic. Caprolactam, the only definitive noncarcinogen in the group of 49, was not mutagenic. The CHO/HGPRT assay was concluded to be an appropriate assay system for use in the screening of chemicals for genotoxicity.     

Importance of detecting numerical versus structural chromosome aberrations

The aim is to review briefly the key questions related to aneuploidy/polyploidy and to compare the advantages and disadvantages of the in vitro micronucleus test to assess aneuploidy/polyploidy in vitro. The key questions that will be addressed, concern the importance of polyploidy for health, and cancer in particular, the mechanisms leading to aneuploidy and polyploidy, and the survival of aneuploid/polyploid cells.The recently recognised contribution of numerical chromosome changes to carcinogenesis triggered the development and the implementation of tests specifically aiming at the detection of aneugens in the test battery for mutagenicity and carcinogenicity. The validation of the in vitro micronucleus test in combination with the identification of in vitro divided cells with the cytokinesis-block methodology and of centromeres with pancentromeric or chromosome specific centromeric probes fluorescence in situ hybridisation (FISH) provides a sensitive, easy to score and powerful test which allows assessment of cell proliferation, the discrimination between chromosome breaks, chromosome loss and chromosome non-disjunction and polyploidy. Moreover, classic histology permits the estimation of necrosis and apoptosis on the same slide. The cytokinesis-blocked micronucleus assay could be considered as a multi-endpoint test for genotoxic responses to clastogens/aneugens. This methodology has also shown to be capable of identifying threshold values for the induction of chromosome loss and/or non-disjunction by microtubule inhibitors, data which are particularly important for risk calculations. Similar approaches were conducted in vivo on bone marrow in mice and rats (except for identification of chromosome non-disjunction), and are in development for gut in mice.     

Review of the genotoxicity and carcinogenicity of 4,4'-methylene-dianiline and 4,4'-methylene-bis-2-chloroaniline

4,4'-Methylene-dianiline (MDA) and 4,4'-methylene-bis-2-chloroaniline (MOCA) are polycyclic aromatic amines that are currently used in industry. Both compounds have been found to be bacterial mutagens and to be positive in a number of assays for genotoxicity. In animal studies, MDA has induced thyroid and liver neoplasms while exposure to MOCA resulted in a variety of tumors including those of the liver, mammary gland and bladder. Epidemiologic proof of human carcinogenicity of both compounds is lacking; however, there is evidence that MOCA can be metabolized to mutagenic products by human tissue. In this paper, the major finding concerning the biotransformation, genotoxicity and carcinogenicity of MDA and MOCA are reviewed.     

Evidence for and possible mechanisms of non-genotoxic carcinogenesis in rodent liver

Doses of chemicals which induce hepatocellular necrosis usually induce hepatic tumours if the dosing is frequent and is maintained for long periods. Such necrosis is usually evident within 48 h of the first administration. Similarly, chemicals that lead to marked proliferation of peroxisomes in the liver also usually induce hepatic tumours on pretracked regular dosing. For both of these phenomena failure to produce a certain level of effect, or to maintain it for sufficiently long periods, can result in the observation of a non-carcinogenic response. The exact dose/time requirements for carcinogenicity have not been defined and may be species/strain/sex-specific. Some chemicals induce liver enlargement and mitogenesis in the absence of overt hepatotoxic effects. The early phases of hepatomegaly are associated with mitogenic effects that can be measured as cells in S-phase within the first few days of administration. The later stages of hepatomegaly appear to be associated more with cellular hypertrophy. Both effects appear to be threshold-related. Further, sustained hepatomegaly is associated with proliferation of SER and the induction of a range of liver enzymes. These changes (mitogenesis, hepatomegaly, enzyme induction), in isolation, are less definitive indicators of carcinogenicity, but they occur for a sufficient number of liver-specific carcinogens that their role as early indicators is worthy of confirmed study. The major area of study required for all possible early markers of hepatocarcinogenicity is to establish the dose and time dependence of these changes in relation to the eventual appearance of tumours. Finally, the specificity of all these markers require evaluation by the study of appropriate non-carcinogens.     

Transgenic assays for mutations and cancer: current status and future perspectives

Transgenic mouse modelling has proved to be a powerful approach to explore the various steps involved in spontaneous and induced carcinogenesis. Some of the multitude of models currently available have the potential to become a substitute for the expensive, long-term rodent bioassay to predict carcinogenicity of environmental compounds. Here, we review the progress in the development and use of transgenic mouse models specifically for the purpose of carcinogenicity and mutagenicity testing.     

The induction by X-rays of chromosome aberrations in germ cells of male guinea-pigs, golden hamsters and rabbits. I. Dose response in post-meiotic stages.

The induction by X-rays of translocations in post-meiotic germ cells of the guinea-pig, golden hamster and rabbit was studied by cytological analysis of male offspring of the irradiated animals. As reported previously for the mouse, the pattern of sensitivity to dominant lethal induction, as indicated by litter-size, was similar to that for translocation induction in both the guinea-pig and golden hamster. In both speciesspermatids were more sensitive than spermatozoa, and in the golden hamster spermatocytes gave a lower yield than spermatids. The translocation frequency among post-meiotic germ cells treated with 600 rad was higher in the rabbit than the guinea-pig, and both were above that for the golden hamster. However, for spermatozoa, species differences with respect to the recovered translocation yield appeared to depend on dose. In the hamster, the translocation frequency after 600 rad, as measured in the female offspring, was similar to that obtained in the male offspring. A small amount of data on the induction of sex-chromosome aneuploidy by 200 rad in golden hamsters suggested that the hamster might be as sensitive as the mouse.