Comparison of germ cell mutagenicity in male CYP2E1-null and wild-type mice treated with acrylamide: evidence supporting a glycidamide-mediated effect.

Acrylamide is an animal carcinogen and probable human carcinogen present in appreciable amounts in heated carbohydrate-rich foodstuffs. It is also a germ cell mutagen, inducing dominant lethal mutations and heritable chromosomal translocations in postmeiotic sperm of treated mice. Acrylamide's affinity for male germ cells has sometimes been overlooked in assessing its toxicity and defining human health risks. Previous investigations of acrylamide's germ cell activity in mice showed stronger effects after repeated administration of low doses compared with a single high dose, suggesting the possible involvement of a stable metabolite. A key oxidative metabolite of acrylamide is the epoxide glycidamide, generated by cytochrome P4502E1 (CYP2E1). To explore the role of CYP2E1 metabolism in the germ cell mutagenicity of acrylamide, CYP2E1-null and wild-type male mice were treated by intraperitoneal injection with 0, 12.5, 25, or 50 mg acrylamide (5 ml saline)(-1) kg(-1) day(-1) for 5 consecutive days. At defined times after exposure, males were mated to untreated B6C3F1 females. Females were killed in late gestation and uterine contents were examined. Dose-related increases in resorption moles (chromosomally aberrant embryos) and decreases in the numbers of pregnant females and the proportion of living fetuses were seen in females mated to acrylamide-treated wild-type mice. No changes in any fertility parameters were seen in females mated to acrylamide-treated CYP2E1-null mice. Our results constitute the first unequivocal demonstration that acrylamide-induced germ cell mutations in male mice require CYP2E1-mediated epoxidation of acrylamide. Thus, CYP2E1 polymorphisms in human populations, resulting in variable enzyme metabolic activities, may produce differential susceptibilities to acrylamide toxicities.

Antiretroviral therapy effects on genetic and morphologic end points in lymphocytes and sperm of men with human immunodeficiency virus infection.

Many human immunodeficiency virus (HIV)-infected persons receive prolonged treatment with DNA-reactive antiretroviral drugs. A prospective study was conducted of 26 HIV-infected men who provided samples before treatment and at multiple times after beginning treatment, to investigate effects of antiretrovirals on lymphocyte and sperm chromosomes and semen quality. Several antiretroviral regimens, all including a nucleoside component, were used. Lymphocyte metaphase analysis and sperm fluorescence in situ hybridization were used for cytogenetic studies. Semen analyses included conventional parameters (volume, concentration, viability, motility, and morphology). No significant effects on cytogenetic parameters, semen volume, or sperm concentration were detected. However, there were significant improvements in sperm motility for men with study entry CD4 cell counts >200 cells/mm(3), sperm morphology for men with entry CD4 cell counts < or =200 cells/mm(3), and the percentage of viable sperm in both groups. These findings suggest that nucleoside-containing antiretrovirals administered via recommended protocols do not induce chromosomal changes in lymphocytes or sperm but may produce improvements in semen quality.

Micronucleated erythrocyte frequency in peripheral blood of B6C3F(1) mice from short-term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program.

The mouse peripheral blood micronucleus (MN) test was performed on samples collected from 20 short-term, 67 subchronic, and 5 chronic toxicity and carcinogenicity studies conducted by the National Toxicology Program (NTP). Data are presented for studies not previously published. Aspects of protocol that distinguish this test from conventional short-term bone marrow MN tests are duration of exposure, and absence of repeat tests and concurrent positive controls. Furthermore, in contrast to short-term bone marrow MN tests where scoring is limited to polychromatic erythrocytes (PCE), longer term studies using peripheral blood may evaluate MN in both, or either, the normochromatic (NCE) or PCE populations. The incidence of MN-PCE provides an index of damage induced within 72 hr of sampling, whereas the incidence of MN in the NCE population at steady state provides an index of average damage during the 30-day period preceding sampling. The mouse peripheral blood MN test has been proposed as a useful adjunct to rodent toxicity tests and has been effectively incorporated as a routine part of overall toxicity testing by the NTP. Data derived from peripheral blood MN analyses of dosed animals provide a useful indication of the in vivo potential for induced genetic damage and supply an important piece of evidence to be considered in the overall assessment of toxicity and health risk of a particular chemical. Although results indicate that the test has low sensitivity for prediction of carcinogenicity, a convincingly positive result in this assay appears to be highly predictive of rodent carcinogenicity.

Bleomycin, unlike other male-mouse mutagens, is most effective in spermatogonia, inducing primarily deletions.

Dominant-lethal tests [P.D. Sudman, J.C. Rutledge, J.B. Bishop, W.M. Generoso, Bleomycin: female-specific dominant lethal effects in mice, Mutat. Res. 296 (1992) 205-217] had suggested that Bleomycin sulfate (Blenoxane), BLM, might be a female-specific mutagen. While confirming that BLM is indeed a powerful inducer of dominant-lethal mutations in females that fails to induce such mutations in postspermatogonial stages of males, we have shown in a specific-locus test that BLM is, in fact, mutagenic in males. This mutagenicity, however, is restricted to spermatogonia (stem-cell and differentiating stages), for which the specific-locus mutation rate differed significantly (P<0.008) from the historical control rate. In treated groups, dominant mutations, also, originated only in spermatogonia. With regard to mutation frequencies, this germ-cell-stage pattern is different from that for radiation and for any other chemical studied to date, except ethylnitrosourea (ENU). However, the nature of the spermatogonial specific-locus mutations differentiates BLM from ENU as well, because BLM induced primarily (or, perhaps, exclusively) multilocus deletions. Heretofore, no chemical that induced specific-locus mutations in spermatogonia did not also induce specific-locus as well as dominant-lethal mutations in postspermatogonial stages, making the dominant lethal test, up till now, predictive of male mutagenicity in general. The BLM results now demonstrate that there are chemicals that can induce specific-locus mutations in spermatogonia without testing positive in postspermatogonial stages. Thus, BLM, while not female-specific, is unique, (a) in its germ-cell-stage specificity in males, and (b) in inducing a type of mutation (deletions) that is atypical for the responding germ-cell stages (spermatogonia).

Strategies for assessing the implications of malformed frogs for environmental health.

The recent increase in the incidence of deformities among natural frog populations has raised concern about the state of the environment and the possible impact of unidentified causative agents on the health of wildlife and human populations. An open workshop on Strategies for Assessing the Implications of Malformed Frogs for Environmental Health was convened on 4-5 December 1997 at the National Institute of Environmental Health Sciences in Research Triangle Park, North Carolina. The purpose of the workshop was to share information among a multidisciplinary group with scientific interest and responsibility for human and environmental health at the federal and state level. Discussions highlighted possible causes and recent findings directly related to frog deformities and provided insight into problems and strategies applicable to continuing investigation in several areas. Possible causes of the deformities were evaluated in terms of diagnostics performed on field amphibians, biologic mechanisms that can lead to the types of malformations observed, and parallel laboratory and field studies. Hydrogeochemistry must be more integrated into environmental toxicology because of the pivotal role of the aquatic environment and the importance of fates and transport relative to any potential exposure. There is no indication of whether there may be a human health factor associated with the deformities. However, the possibility that causal agents may be waterborne indicates a need to identify the relevant factors and establish the relationship between environmental and human health in terms of hazard assessment.

Induction of micronucleated erythrocytes in rodents by diisopropylcarbodiimide and dicyclohexylcarbodiimide: dependence on exposure protocol.

The induction of micronucleated erythrocytes by diisopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC) was investigated as part of a U.S. National Toxicology Program (NTP) evaluation of the subchronic toxicity of these chemicals. Analysis of peripheral blood smears from male and female B6C3F1 mice exposed to 17.5-140.0 mg DIC/kg/day by skin painting for 13 weeks revealed dose-related increases in the frequency of micronucleated normochromatic erythrocytes (MN-NCE) in both sexes. Results of a similar 13-week peripheral blood micronucleus (MN) test with DCC (1.5-12.0 mg/kg/day) were also positive, although the increases in MN-NCE were not as great as those observed with DIC. In contrast to the positive results of the subchronic skin-painting studies in mice, acute bone marrow MN studies with DIC and DCC in male F344 rats, using intraperitoneal (i.p.) injection, yielded negative results. Both the acute and the subchronic exposures included doses that produced clinical signs of toxicity. Acute mouse bone marrow MN tests with DIC administered in single or triple i.p. injection protocols were subsequently conducted to determine if the differing responses between mice and rats were due to species or protocol differences. The results of these acute tests were negative or equivocal. Because the subchronic studies produced positive results, it was hypothesized that these carbodiimides required multiple treatments over an extended period of time to produce an increase in MN-erythrocytes. To confirm the original response, a second dermal subchronic study was conducted with DIC; the protocol was modified to include sequential blood samplings to permit monitoring MN frequencies over time. The data demonstrated a small but consistent induction of micronucleated erythrocytes in mice treated with DIC by skin painting.

Induction of mortality and malformation in Xenopus laevis embryos by water sources associated with field frog deformities.

Water samples from several ponds in Minnesota were evaluated for their capacity to induce malformations in embryos of Xenopus laevis. The FETAX assay was used to assess the occurrence of malformations following a 96-hr period of exposure to water samples. These studies were conducted following reports of high incidences of malformation in natural populations of frogs in Minnesota wetlands. The purpose of these studies was to determine if a biologically active agent(s) was present in the waters and could be detected using the FETAX assay. Water samples from ponds with high incidences of frog malformations (affected sites), along with water samples from ponds with unaffected frog populations (reference sites), were studied. Initial experiments clearly showed that water from affected sites induced mortality and malformation in Xenopus embryos, while water from reference sites had little or no effect. Induction of malformation was dose dependent and highly reproducible, both with stored samples and with samples taken at different times throughout the summer. The biological activity of the samples was reduced or eliminated when samples were passed through activated carbon. Limited evidence from these samples indicates that the causal factor(s) is not an infectious organism nor are ion concentrations or metals responsible for the effects observed. Results do indicate that the water matrix has a significant effect on the severity of toxicity. Based on the FETAX results and the occurrence of frog malformations observed in the field, these studies suggest that water in the affected sites contains one or more unknown agents that induce developmental abnormalities in Xenopus. These same factors may contribute to the increased incidence of malformation in native species.

Unlike other chemicals, etoposide (a topoisomerase-II inhibitor) produces peak mutagenicity in primary spermatocytes of the mouse.

The cancer chemotherapy agent, and topoisomerase-II inhibitor, etoposide (VP-16) produced both recessive mutations at specific loci and dominants at other loci with peak frequencies in primary spermatocytes, a cell type in which the topo-II gene has been shown to be activated. Etoposide thus differs from all other chemicals whose germ-cell-stage specificity has been analyzed. No effects of etoposide exposure of spermatogonial stem cells ( approximately 15, 000 offspring scored) were detectable by either mutagenicity or productivity endpoints. The significant mutagenic response that followed exposure of poststem-cell stages ( approximately 25,000 offspring scored) showed a clear peak, with three of four specific-locus mutants, and three of four dominant mutants conceived during weeks 4 or 5 (days 22-35) post-injection, a period that also encompassed the dominant-lethal peak. For this period, the induced specific-locus rate (with 95% confidence limits) at a weighted-average exposure of 75.1 mg etop/kg was 59.5 (14.6, 170. 9)x10-6/locus. At least 3 of the 4 specific-locus mutations were deletions, paralleling findings with etoposide or analogs in other test systems where a recombinational origin of the deletions has been suggested. Because, unlike other chemicals that induce deletions in male germ cells, etoposide is effective in stages normally associated with recombinational events, it will be of interest to determine whether this chemical can affect meiotic recombination.

Determination of tamoxifen and metabolites in serum by capillary electrophoresis using a nonaqueous buffer system.

Tamoxifen (TAM), an antiestrogen, is widely used to treat hormone-dependent breast cancer in post-menopausal women. TAM may be used as a chemopreventive agent in women of child-bearing age; however, few data exist describing potential TAM-induced fetal toxicity. In support of the National Toxicology Program's characterization of reproductive and developmental effects of TAM, this work describes an analytical technique utilizing capillary electrophoresis (CE) for the detection of circulating levels of TAM, N-desmethyltamoxifen (DMT), and 4-hydroxytamoxifen (4-HT) in maternal rodent serum. Greater than 90% of 3H-labeled TAM was extractable from serum using 98:2 hexane-isoamyl alcohol. Optimum separation of TAM, DMT, and 4-HT was obtained on a 57 cmx50 microm capillary using a nonaqueous buffer system of 1:1 methanol-acetonitrile containing 50 mM ammonium acetate and 1% acetic acid. 4-Dimethylaminopyridine was used as internal standard. Temperature and voltage were optimized at 40 degrees C and 15 kV, respectively. The limit of detection of TAM by UV detection at 214 nm was approximately 800 amol. TAM and DMT were confirmed in serum of female rats 4 h following a single oral dose of 120 mg/kg. Transplacental exposure of TAM to fetal tissue will be evaluated using this technique.

Mammalian germ cell mutagenicity of ENU, IPMS and MMS, chemicals selected for a transgenic mouse collaborative study.

A collaborative study to systematically assess transgenic mouse mutation assays as screens for germ cell mutagens has been conducted. Three male mouse germ cell mutagens (ENU, iPMS and MMS) were selected for testing. This paper provides a brief review of the effects reported for those 3 chemicals in the most commonly used non-transgenic germ cell mutagenicity assays, namely the dominant lethal, heritable translocation, and specific locus tests. Additionally, information on the DNA reactivity and the molecular nature of mutations induced by these chemicals is summarized.

Mutation assays in male germ cells from transgenic mice: overview of study and conclusions.

Three confirmed mouse germ cell mutagens, ethyl nitrosourea (ENU), isopropyl methanesulphonate (iPMS) and methyl methanesulphonate (MMS), have been evaluated for their activity as mutagens to the germ cell DNA of two strains of transgenic mice (lac I, Big Blue and LacZ, Muta Mouse). Both testicular DNA and epididymal sperm DNA were evaluated. A range of sampling times was studied, from 3 days post-dosing to 100 days post-dosing. ENU and iPMS were mutagenic to both testicular DNA and epididymal sperm DNA. Mutant frequencies were higher for both chemicals in DNA recovered from testicular tissue than in epididymal sperm DNA. Likewise, mutant frequencies were higher for both DNA samples at the later sampling times. MMS was not mutagenic under any condition of test. A good level of qualitative agreement in test results was seen for the two assays and for the same assays conducted in different laboratories. The level of quantitative agreement was not as high, but was, nonetheless, generally good. Recommendations for the future conduct of transgenic rodent germ cell mutation assays are made. The test data are discussed within the context of the larger question of how such assays should be integrated into the chemical hazard assessment process.

Multilaboratory comparison of in vitro tests for chromosome aberrations in CHO and CHL cells tested under the same protocols.

Different test results have been reported for the same chemicals in two in vitro chromosome aberration test systems, CHL cells tested by a Japanese protocol and CHO cells tested by the US National Toxicology Program [Sofuni et al., Mutat Res 241:173-213,1990]. Here, laboratories in Japan, the US and the UK tested 9 such chemicals in CHL and CHO cells using the same protocols and found all 9 positive in both cell types; differences in earlier conclusions with these chemicals were due mainly to test protocol, not to different sensitivities of the cells. The most important protocol difference is sampling time. Chemicals that were negative in the NTP series using a sampling time of 10 to 13 hours often produced positive results when retested here with a 20- to 24-hour sampling time. While positive results were obtained in both cell types, CHL cells sometimes had higher aberration levels and survived at higher doses than CHO cells would tolerate. This may reflect some intrinsic difference in sensitivity but may also be affected by factors such as cell cycle length and culture media (e.g., oxygen scavenging capacity). The collaboration reported here also contributed to a better understanding of scoring aberrations, especially "gaps"; there was good agreement on what types of aberrations should be included in the totals when scoring criteria were clearly defined, for example, many changes classified as "gaps" by the Japanese system were classified as "breaks" in the scoring systems used in the United States and the United Kingdom, and were appropriately included in total aberration counts.

Dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis induced in male mouse germ cells by glycidamide, a metabolite of acrylamide.

The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.

Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays.

Because of rampant concern that estrogenic chemicals in the environment may be adversely affecting the health of humans and wildlife, reliable methods for detecting and characterizing estrogenic chemicals are needed. It is important that general agreement be reached on which tests to use and that these tests then be applied to the testing of both man-made and naturally occurring chemicals. As a step toward developing a comprehensive approach to screening chemicals for estrogenic activity, three assays for detecting estrogenicity were conducted on 10 chemicals with known or suspected estrogenic activity. The assays were 1) competitive binding with the mouse uterine estrogen receptor, 2) transcriptional activation in HeLa cells transfected with plasmids containing an estrogen receptor and a response element, and 3) the uterotropic assay in mice. The chemicals studied were 17 beta-estradiol, diethylstilbestrol, tamoxifen, 4-hydroxytamoxifen, methoxychlor, the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), endosulfan, nonylphenol, o,p'-DDT, and kepone. These studies were conducted to assess the utility of this three-assay combination in the routine screening of chemicals, or combinations of chemicals, for estrogenic activity. Results were consistent among the three assays with respect to what is known about the estrogenic activities of the chemicals tested and their requirements for metabolic activation. By providing information on three levels of hormonal activity (receptor binding, transcriptional activation, and an in vivo effect in an estrogen-responsive tissue), an informative profile of estrogenic activity is obtained with a reasonable investment of resources.

Chlorambucil and bleomycin induce mutations in the specific-locus test in female mice.

Specific-locus studies have shown chlorambucil (CHL) and bleomycin (BLE) to be mutagenic in mouse oocytes, almost doubling the number of chemicals previously known to induce mutations in females. The overall CHL-induced mutation rate in oocytes is, however, one order of magnitude below that for male meiotic and postmeiotic stages, and only 1/50 that for early spermatids. For BLE, no specific-locus data for males are available for comparison, but the chemical had earlier been found negative for dominant-lethal induction in males. Both BLE and CHL were significantly mutagenic only in mature and maturing oocytes. In keeping with an earlier report, BLE produced a high incidence of dominant lethals in these stages. CHL failed to induce dominant lethals, indicating that for mature and maturing oocytes, in contrast with results for males, sensitivity to dominant-lethal mutations is not a prerequisite for induction of specific-locus mutations. Exposure of immature oocytes to either BLE or CHL produced neither dominant lethals nor significant induction of specific-locus mutations; however, CHL gave evidence of killing immature oocytes. By contrast, BLE, which has been considered a radiomimetic chemical, does not appear to kill immature oocytes and thus differs markedly from radiation exposures equivalent for dominant-lethal induction. Therefore, the failure to recover specific-locus mutations cannot be ascribed to cell selection resulting from oocyte killing, as has sometimes been done for radiation. Adding results on the nature of the CHL- and BLE-induced mutations to prior information, the estimated minimum proportion of large DNA lesions induced in oocytes by chemicals becomes 35.3%, significantly different from the corresponding figure (approximately 70%) for radiations. For chemical treatments, the oocyte proportion is highly significantly above the 3.6% induced in spermatogonia, but only on the borderline of statistically significant difference from that induced in postspermatogonial stages.

IPCS harmonization of methods for the prediction and quantification of human carcinogenic/mutagenic hazard, and for indicating the probable mechanism of action of carcinogens.

A flow chart is presented as a recommended sequence of tests to predict the carcinogenic hazard, and to predict and quantify the mutagenic hazard to germ cells of chemicals to humans. Ten associated principles of testing for these endpoints are also suggested. These recommendations are the result of a meeting convened under the auspices of the International Programme on Chemical Safety (IPCS), as part of their project on 'Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals'. The meeting was held at Carshalton, Surrey, from 13-17 February 1995.

Dominant lethal and heritable translocation tests with chlorambucil and melphalan in male mice.

Chemicals used in the treatment of cancer include several that are potent mutagens in a range of in vitro and in vivo assays. For some, genetic effects have also been demonstrated in humans, detected as chromosomal aberrations in peripheral lymphocytes. Because (1) many of these agents are confirmed mutagens, (2) humans are exposed to them in relatively high doses, and (3) an increasing number of early cancer victims are surviving to reproductive age, it is important that information be available on the genetic and reproductive hazards associated with exposure to these agents. Chlorambucil and melphalan are structurally related chemicals that are included in our efforts to identify and assess such hazards among cancer chemotherapy agents. To date, both have been reported to induce specific locus mutations in germ cells of male mice (Russell et al., 1989; Russel et al., 1992b) and melphalan is one of very few chemicals shown to induce such mutations in spermatogonial stem cells. More recently, both chemicals were found to have strong reproductive effects in female mice (Bishop and Generoso, 1995, in preparation). In the present studies, these chemicals were tested for the induction of dominant lethal mutations and heritable translocations in male mice. Both chemicals were found to have reproductive effects attributable to cytotoxicity in specific male germ cell stages and to induce dominant lethal mutations and heritable translocations in postmeiotic germ cells, particularly in mid to early stage spermatids. Thus, relatively extensive data are now available for assessing the genetic and reproductive hazards that may result from therapeutic exposures to these chemicals.