Evaluation of aneugenic effects of bisphenol A in somatic and germ cells of the mouse.

Bisphenol A (BPA) is a synthetic monomer widely used to polymerize polycarbonate plastics and resins. It is shown in vitro to interfere with microtubules, producing aberations in mitotic and meiotic spindles. An increase of meiotic abnormalities in untreated female mice from an experimental colony was temporally correlated with the accidental release of BPA from polycarbonate cages and bottles damaged by inadvertent treatment with harsh alkaline detergents [P.A. Hunt, K.E. Koehler, M. Susiarjo, C.A. Hodges, A. Ilagan, R.C. Voigt, S. Thomas, B.F. Thomas, T.J. Hassold, Bisphenol A exposure causes meiotic aneuploidy in the female mouse, Curr. Biol. 13 (2003) 546-553]. In the present study, potential aneugenic effects of BPA on mouse male and female germ cells and bone marrow cells have been evaluated after acute, sub-chronic or chronic in vivo exposure. Female mice were orally treated with a single BPA dose, with 7 daily administrations or exposed for 7 weeks to BPA in drinking water. No significant induction of hyperploidy or polyploidy was observed in oocytes and zygotes at any treatment condition. The only detectable effect was a significant increase of metaphase II oocytes with prematurely separated chromatids after chronic exposure; this effect, however, had no irreversible consequence upon the fidelity of chromosome segregation during the second meiotic division, as demonstrated by the normal chromosome constitution of zygotes under the same exposure condition. With male mice, no delay of meiotic divisions was found after six daily oral doses of BPA with the BrdU assay. Similarly, no induction of hyperploidy and polyploidy was shown in epydidimal sperm hybrized with probes for chromosomes 8, X and Y, 22 days after six daily oral BPA doses. Finally, two daily oral BPA doses did not induce any increase of micronucleus frequencies in polychromatic erythrocytes of mouse bone marrow. In conclusion, our results do not add evidence to the suspected aneugenic activity of BPA and suggest that other factors or co-factors should be considered to explain the unexpected burst of meiotic abnormalities previously attributed to accidental BPA exposure.

Orthovanadate increased the frequency of aneuploid mouse sperm without micronucleus induction in mouse bone marrow erythrocytes at the same dose level.

The objective of the current study was to investigate the ability of orthovanadate to induce aneuploidy in mouse sperm and micronuclei in mouse bone marrow cells at the same dose levels. The BrdU-incorporation assay was performed to test if the chemical treatment altered the duration of the meiotic divisions. It was found that orthovanadate (25mg/kg bw) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated by intraperitoneal (i.p.) injection with 5, 15 or 25mg/kg bw orthovanadate and sperm were sampled from the Caudae epididymes 22 days later. Fluorescence in situ hybridization (FISH) was performed with DNA-probes for chromosomes 8, X or Y. Significant increases in the frequencies of total hyperhaploid sperm (p<0.01) were found with 15 and 25mg/kg bw orthovanadate, indicating induced non-disjunction during male meiosis. The dose-response was described best by a linear equation. Orthovanadate did not significantly increase the frequencies of diploid sperm at any of the three doses tested, indicating that no complete meiotic arrest occurred. Orthovanadate was investigated also by the micronucleus test at i.p. doses of 1, 5, 15 or 25mg/kg bw, followed by bone marrow sampling 24h after treatment. None of the orthovanadate doses caused a significant increase in the rates of micronuclei (MN). Since the results show that orthovanadate induced non-disjunction during male meiosis without an accompanying induction of MN in bone marrow erythrocytes under the present experimental conditions and doses, it is concluded that male germ cells (meiosis) are more sensitive to the aneugenic effects of orthovanadate than somatic cells (mitosis). However, induction of micronuclei was reported in the literature with orthovanadate, vanadylsulfate and ammonium metavanadate, which contradicts the notion that vanadium compounds might be unique germ cell aneugens.

Nicotine is not clastogenic at doses of 1 or 2 mg/kg body weight given orally to male mice.

Nicotine has been tested in the conventional mouse bone marrow assay. Single doses of 1mg/kg bw or 2mg/kg bw were given by oral intubations and bone marrow was sampled at 24h (1mg/kg) or at 6, 12 and 18 h after treatment (2mg/kg). Nicotine treatment did not increase the micronucleus frequencies in polychromatic erythrocytes while the positive control compound mitomycin C yielded the expected result. These data contradict the only published in vivo study of nicotine in which 1.1mg/kg bw was called positive for the induction of chromosomal aberrations in mouse bone marrow cells at all sampling intervals, even as early as 6h after treatment. It is discussed that aberration scoring is a matter of subjectivity and depends on strict discrimination criteria between gaps and true DNA discontinuities, i.e. breaks. International collaboration has shown that micronucleus scoring is less subjective, hence more reliable. Therefore it is concluded that nicotine is not clastogenic at the doses and time intervals tested in the present experiments.

Etoposide and merbarone are clastogenic and aneugenic in the mouse bone marrow micronucleus test complemented by fluorescence in situ hybridization with the mouse minor satellite DNA probe.

The topoisomerase II (topo II) inhibitors etoposide (VP-16) and merbarone (MER) were investigated with the in vivo micronucleus test (MN test) combined with fluorescence in situ hybridization (FISH) using the mouse minor satellite DNA probe to discriminate MN of clastogenic and aneugenic origin. All experiments were performed with male (102/ElxC3H/El) F1 mice bred in the mouse colony of the GSF Research Center. The sample size per experimental group was five animals and 2,000 polychromatic erythrocytes (PCE) were scored per animal from coded slides in the conventional MN test. A separate set of coded slides was used for the FISH analysis. All treatments consisted of single intraperitoneal injections. Colchicine (COL, 3 mg/kg) and mitomycin (MMC, 1 mg/kg) were used as a positive control aneugen and clastogen, respectively, and these compounds produced the expected responses. A dose of 1 mg/kg VP-16 induced 3.44% MNPCE (compared to the concurrent solvent control of 0.37%, P < 0.001) and of these 39.9% (1.4% MNPCE) showed one or more fluorescent signals. MER (7.5-60 mg/kg) increased the MNPCE frequencies in a dose-dependent manner, with 15 mg/kg being the lowest positive dose. At the highest dose of 60 mg/kg of MER, a total of 4.26% MNPCE were found (compared to 0.31% in the concurrent solvent control, P < 0.001) and of these 46.2% (2.0% MNPCE) contained one or more fluorescent signals. The data demonstrate that VP-16 and MER induced both clastogenic and aneugenic events despite their different modes of topo II inhibition.

Molecular cytogenetic analysis in mouse sperm of chemically induced aneuploidy: studies with topoisomerase II inhibitors.

The ability of two topoisomerase II (topo II) inhibitors, etoposide (VP-16) and merbarone (MER), to induce meiotic delay and aneuploidy in mouse spermatocytes was investigated. The progression from meiotic divisions to epididymal sperm was determined by injecting male mice with 5-bromo-2'-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. At 20-24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). It was found that VP-16 (50mg/kg) treatment induced a meiotic delay of about 24h. A significant reduction of BrdU-labelled sperm was observed at 22 days compared to the controls (VP-16 group: 14.20%; controls: 41.10%, P<0.001). At 23 and 24 days, there were no significant differences between the VP-16 and the control groups. MER (80 mg/kg) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated with 12.5, 25 and 50mg/kg VP-16 or 15, 30 and 60 mg/kg MER. Sperm were sampled from the Caudae epididymes 24 days after VP-16 treatment or 22 days after MER treatment. Significant increases above the concurrent controls in the frequencies of total hyperhaploid sperm were found after treatment with 25, 50mg/kg VP-16 (0.074 and 0.122% versus 0.052%) and after treatment with 60 mg/kg MER (0.098% versus 0.044%). Furthermore, significant increases in the frequencies of diploid sperm were found after treatment of mice with all three doses of VP-16 (0.024, 0.032 and 0.056% versus 0.004 and 0.00%, respectively) and with 30 and 60 mg/kg MER (0.022 and 0.05% versus 0.004 and 0.002%, respectively). All dose responses could be expressed by linear equations. The results indicate that cancer patients may stand transient risk for siring chromosomally abnormal offspring after chemotherapy with these topo II inhibitors.

Induction of chromosomal aberrations by dacarbazine in somatic and germinal cells of mice.

Dacarbazine (DTIC) is a chemotherapeutic agent that has been successfully applied to treat various types of cancer such as Hodgkin's disease, malignant melanomas, soft tissue sarcomas and advanced neuroblastomas. Many of the patients are of reproductive age and express concern over the genetic risk of the treatment they receive. Therefore, DTIC was tested for its clastogenic effects in somatic and germinal cells of mice. In the bone marrow micronucleus assay DTIC induced micronuclei that increased linearly in the dose range 0-125 mg/kg. In a dominant lethal study DTIC gave a positive response at the dose of 500 mg/kg when conceptions occurred 5-16 days after treatment, corresponding to treated spermatids and early spermatozoa. The induction of heritable translocations was tested in that sensitive period. The observed translocation rate among the F(1) progeny of male mice treated with 500 mg/kg DTIC was 2.13% (P < 00.1 against the historical control of 0.05%). Assuming linearity of the dose-response effect, the point estimate was used to calculate a doubling dose for the induction of heritable translocations of 12 mg/kg. Alternatively, an induced translocation rate of 41.6x10(-6) per unit dose was calculated. Both figures indicate that an increased genetic risk may exist for male patients after chemotherapy with DTIC under the assumption that germ cells of mice and humans are equally sensitive to the clastogenic effects of DTIC. However, the genetic risk is restricted to conceptions within a period of 40 days after the end of chemotherapy, since the sensitive stages of spermatogenesis are spermatids and early spermatozoa.

Effect of chemicals on the duration of male meiosis in mice detected with laser scanning cytometry.

Aneuploidy studies in sperm such as the sperm-FISH assay require a precise knowledge of the duration of spermatogenesis, especially of the meiotic stages. This is important in order to sample sperm from the epididymis at appropriate intervals after animal treatment. However, aneugens may delay the cell cycle. The progression from meiotic divisions to epididymal sperm was determined by labelling the last S-phase before meiosis with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. In a time frame of 20--24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). We studied the effects of the chemicals acrylamide, colchicine, diazepam, griseofulvin, taxol, thiobendazole, trichlorfon and vinblastine on the duration of meiotic divisions in male mice. Colchicine treatment prolonged the duration of meiotic divisions by about 48 h. On days 21 and 22, the frequencies of BrdU-labelled sperm in the colchicine group were 11.7 and 9.4%, respectively, while they were 28.4 and 30.6%, respectively, in the concurrent controls (P > 0.01). On day 24 after treatment, the frequency of labelled sperm in the colchicine group reached the control level. Etoposide treatment resulted in an elevation of BrdU-labelled sperm at 23 rather than 22 days. The other chemicals showed no significant effect of prolonging meiotic cell cycle progression. On the basis of the colchicine and etoposide data, it is suggested that the effect of a chemical on the meiotic cell cycle progression is determined first in order to chose the appropriate sperm sampling time to detect aneuploidy induction.

Recommendations for the categorization of germ cell mutagens.

Germ cell mutagens are currently classified into three categories in the German List of MAK and BAT Values. These categories have been revised and extended by analogy with the new categories for carcinogenic chemicals. Germ cell mutagens produce heritable gene mutations, and heritable structural and numerical chromosome aberrations in germ cells. The original categories 1 and 2 for germ cell mutagens remain unchanged. Two new categories 3A and 3B are proposed for chemicals suspected to be germ cell mutagens. A new category 5 is proposed for germ cell mutagens with low potency that contribute negligibly to human genetic risk provided the MAK value is observed.

Molecular cloning and functional characterization of the mouse organic-anion-transporting polypeptide 1 (Oatp1) and mapping of the gene to chromosome X.

We have cloned a murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane-transport proteins from mouse liver. The cloned cDNA insert of 2783 bp with an open reading frame of 2011 bp codes for a 12-transmembrane 670-amino-acid protein with highest amino acid identity with the rat Oatp1. When expressed in Xenopus laevis oocytes, the mouse Oatp exhibited the same substrate specificity as the rat Oatp1. Besides the common Oatp substrates bromosulphophthalein, taurocholate, oestrone 3-sulphate and ouabain, the new mouse Oatp also mediates transport of the Oatp1-specific magnetic-resonance-imaging agent gadoxetate. The Oatp2-specific cardiac glycoside digoxin, however, is not transported. Kinetic analyses performed for taurocholate and oestrone 3-sulphate revealed apparent K(m) values of 12 microM and 5 microM respectively. Northern-blot analysis demonstrated a predominant expression in the liver with an additional moderate expression in the kidney. Taken together, the amino acid identity, the functional characteristics and the tissue distribution suggest that we have isolated the murine orthologue of the rat Oatp1, and consequently the identified protein will be called Oatp1. Using fluorescence in situ hybridization, the murine Oatp1 gene was mapped to chromosome XA3-A5.

Butadiene diolepoxide- and diepoxybutane-derived DNA adducts at N7-guanine: a high occurrence of diolepoxide-derived adducts in mouse lung after 1,3-butadiene exposure.

Butadiene (BD) is a high production volume chemical and is known to be tumorigenic in rodents. BD is metabolized to butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). These epoxides are genotoxic and alkylate DNA both in vitro and in vivo, mainly at the N7 position of guanine. In this study, a 32P-post-labeling/thin-layer chromatography (TLC)/high-pressure liquid chromatography (HPLC) assay for BDE and DEB adducts at the N7 of guanine was developed and was used in determining the enantiomeric composition of the adducts and the organ dose of BD exposure in lung. Exposure of 2'-deoxyguanosine (dGuo), 2'-deoxyguanosine-5'-phosphate (5'-dGMP) and 2'-deoxyguanosine-3'-phosphate (3'-dGMP) to racemic BDE followed by neutral thermal hydrolysis gave two products (products 1 and 2) that were identified by MS and UV and NMR spectroscopy as a diastereomeric pair of N7-(2,3,4-trihydroxybutan-1-yl)-guanines. Exposure of dGuo nucleotides to RR/SS DEB (also referred to as dl DEB) followed by thermal depurination resulted in a single product coeluting with the BDE product 1. If the reaction mixture of BDE and 5'-dGMP was analyzed by HPLC before hydrolysis of the glycosidic bond, four major nucleotide alkylation products (A, B, C and D) with identical UV sepectra were detected. The products were isolated and hydrolyzed, after which A and C coeluted with product 1 and B and D coeluted with the product 2. The major adduct of DEB-exposed 5'-dGMP was N7-(2-hydroxy-3,4-epoxy-1-yl)-dGMP (product E). A 32P-post-labeling assay was used to detect BDE- and DEB-derived N7-dGMP adducts in DNA. Levels of adducts increased with a dose of BDE and DEB and exhibited a half life of 30 +/- 3 (r = 0.98) and 31 +/- 4 h (r = 0.95), respectively. Incubation of DEB-modified DNA at 37 degrees C at neutral pH for up to 142 h did not lead to an increase of N7-(2,3,4-trihydroxybutan-1-yl)-dGMP in the DNA. These observations led to the conclusion that the N7-(2,3, 4-trihydroxybutan-1-yl)-dGMP adducts in DNA can be used as a marker of BDE exposure and that N7-(2-hydroxy-3,4-epoxy-1-yl)-dGMP adducts are related to DEB exposure. Dose-related levels of BDE- and DEB-derived adducts were detected in lungs of mice inhaling butadiene. Most of the N7-dGMP adducts (73%; product D) were derived from the 2R-diol-3S-epoxide of 1,3-butadiene. The data presented in this paper indicate that in vivo, 98% of N7-dGMP alkylation after BD exposure is derived from BDE, and approximately 2% of the adducts were derived from DEB and BMO.

Detection of aneuploidy by multicolor FISH in mouse sperm after in vivo treatment with acrylamide, colchicine, diazepam or thiabendazole.

Multicolor fluorescence in situ hybridization (FISH) was used to investigate the induction of aneuploidy during meiosis in young adult male mice treated with chemicals chosen for the EU sponsored aneuploidy project (acrylamide, colchicine, diazepam and thiabendazole). The aim of the present study was to evaluate the frequency of aneuploid sperm induced by each of these chemicals by sperm FISH. Male (102/ElxC3H/El)F1 mice were treated with acrylamide (120 and 60 mg/kg single dose i.p.), colchicine (1.5 and 3 mg/kg single dose, i.p.), diazepam (300, 150 and 75 mg/kg single dose by oral intubation) or thiabendazole (100 and 300 mg/kg daily for 11 days by oral intubation). At 22 days after the last treatment, sperm were collected from the cauda epididymis. Three chromosome FISH was applied to determine hyperhaploid and diploid sperm with DNA probes specific for the chromosomes X, Y and 8. Five animals were treated per dose group and sperm aneuploidy was evaluated in 10,000 sperm per animal. We found significant increases in the frequency of total hyperhaploidy for the males treated with 3.0 mg/kg colchicine (0.092 versus 0.056%, P < 0.05) and with 1.5 mg/kg colchicine (0.082 versus 0.050%, P < 0.05), as well for the males treated with 300 mg/kg diazepam (0.081 versus 0.050%, P < 0.05), indicating that colchicine and diazepam each induced germ cell aneuploidy. We also found significant increases in the frequency of total diploidy for the males treated with 300 mg/kg diazepam (P < 0.05) and with 300 mg/kg thiabendazole (P < 0.05). No significant effects were found for 120 and 60 mg/kg acrylamide or for the other doses of diazepam and thiabendazole. These first results indicate that the multicolor FISH method is useful to determine aneuploidy induction in sperm of mice.

Isolation and embryonic expression of the novel mouse gene Hic1, the homologue of HIC1, a candidate gene for the Miller-Dieker syndrome.

The human gene HIC1 (hypermethylated in cancer) maps to chromosome 17p13.3 and is deleted in the contiguous gene disorder Miller-Dieker syndrome (MDS) [Makos-Wales et al. (1995) Nature Med., 1, 570-577; Chong et al. (1996) Genome Res., 6, 735-741]. We isolated the murine homologue Hic1, encoding a zinc-finger protein with a poxvirus and zinc-finger (POZ) domain and mapped it to mouse chromosome 11 in a region exhibiting conserved synteny to human chromosome 17. Comparison of genomic and cDNA sequences predicts two exons for the murine Hic1. The second exon exhibits 88% identity to the human HIC1 on DNA level. During embryonic development, Hic1 is expressed in mesenchymes of the sclerotomes, lateral body wall, limb and cranio-facial regions embedding the outgrowing peripheral nerves during their differentiation. During fetal development, Hic1 additionally is expressed in mesenchymes apposed to precartilaginous condensations, at many interfaces to budding epithelia of inner organs, and weakly in muscles. We observed activation of Hic1 expression in the embryonic anlagen of many tissues displaying anomalies in MDS patients. Besides lissencephaly, MDS patients exhibit facial dysmorphism and frequently additional birth defects, e.g. anomalies of the heart, kidney, gastrointestinal tract and the limbs (OMIM 247200). Thus, HIC1 activity may correlate with the defective development of the nose, jaws, extremities, gastrointestinal tract and kidney in MDS patients.

Colcemid and econazole do not show aneugenicity in male mouse germ cells.

Colcemid (COM) and econazole (EZ) were tested for induction of mitotic arrest and C-mitotic effects in mouse bone marrow cells and for meiotic delay and induction of hyperploidy in mouse spermatocytes after single injection. Doses of 1 and 3 mg/kg COM were used and bone marrow and spermatocytes were sampled at 2, 6, 10, 14 and 18 h. For EZ a dose of 120 mg/kg and intervals of 6, 10, 14 and 18 h were chosen. At 2 h after COM treatment of bone marrow cells the mitotic index and C-mitotic effect were highest, then both decreased from 6 to 18 h. At 18 h the mitotic indices decreased to or significantly below the control level at doses of 1 and 3 mg/kg respectively, whereas the corresponding frequencies of C-mitotic cells were still significantly higher than in the controls. EZ also induced mitotic arrest and C-mitotic effects in mouse bone marrow cells. In contrast to COM, the effects of EZ were highest at 18 h after treatment. COM and EZ caused disturbances in progression from the first to second meiotic division, however, after COM treatment the ratios of MMII to MMI were significantly below the control. In contrast, after EZ treatment the ratios were significantly higher than in the controls. Such differences may result from the different mechanisms by which COM and EZ act on cell cycle progression. COM and EZ did not induce non-disjunction under the experimental conditions. However, EZ induced structural chromosomal aberrations.

DNA adducts in mouse testis and lung after inhalation exposure to 1,3-butadiene.

1,3-Butadiene is a widely used industrial chemical and also an environmental contaminant. Recent findings have shown that butadiene can also be a male germ cell mutagen. In this study, DNA adduct formation in testis and lung has been explored by using N7-alkylated guanines as a marker of biological effective dose. The adducts measured were the four structurally different guanine N7-adducts alkylated by butadienemonoepoxide, the main metabolite of 1,3-butadiene. This study demonstrates the dose-dependent adduct formation in lung and testis. At lower exposures (50 and 200 ppm) the adduct levels were about the same in the two organs, but at 500 ppm the adduct level was significantly (p < 0.03) higher in testis than in lung. The enantiomeric composition of the adducts detected was also different. In lung, all 4 possible adducts were present (S-C-1" dominating, 49%), but in testis only two out of four adducts were detected (S-C-2" being the most abundant adduct, 71%). These novel observations indicate that the DNA repair is different in these two organs studied and that heritable genetic effects observed may be mediated through the DNA adducts.

Dose response study for 1,3-butadiene-induced dominant lethal mutations and heritable translocations in germs cells of male mice.

Butadiene (BD) and its metabolites have extensively been studied in the EU sponsored research project "Multi-endpoint analysis of genetic damage induced by 1,3-butadiene and its major metabolites". Within this project a dominant lethal test and a heritable translocation test were performed with male mice to study the dose-response relationships for the respective endpoints. BD concentrations of 130 and 500 ppm were tested in the dominant lethal assay by exposing male mice on 6 h/day for five consecutive days resulting in doses of 3900 and 15,000 ppmh, respectively. Males were mated for four consecutive weeks at a ratio of 1:2 to untreated females. A positive dominant lethal effect was observed in the first mating week in the experiment with 15,000 ppmh but no dominant lethality was found with the lower dose of 3900 ppmh. The present dominant lethal data have to be viewed together with the data already published for a BD dose of 39,000 ppmh (1300 ppm at 6 h/day on 5 consecutive days) [1]. The main difference between results with the highest and the middle dose is that mating weeks one and two (sperm and late spermatids) showed an effect at 39,000 ppmh while only mating week one (sperm) showed an effect at 15,000 ppmh. In the heritable translocation assay, males mice were exposed with a BD dose of 15,000 ppmh and mated for one week to untreated females. Among 434 F1 offspring, we found 5 translocation carriers (1.15% vs. 0.05% in the historical control, p < 0.001). In the previous heritable translocation experiment with a BD dose of 39,000 ppmh of DB exposure, 2.7% of the offspring carried a reciprocal translocation [2]. These data can be used for quantification of genetic risk. The dose response for BD-induced heritable translocations in sperm and late spermatids of mice was linear (Y = 0.05 + 6.9 x 10(-5)X) and a doubling dose of 725 ppmh could be calculated.

Genetic effects of 1,3-butadiene and associated risk for heritable damage.

A summary of the results of the studies conducted in the EU Project "Multi-endpoint analysis of genetic damage induced by 1,3-butadiene and its major metabolites in somatic and germ cells of mice, rats and man" is presented. Results of the project are summarized on the detection of DNA and hemoglobin adducts, on the cytotoxic and clastogenic effects in somatic and germinal cells of mice and rats, on the induction of somatic mutations at the hprt locus of experimental rodents and occupationally exposed workers, on the induction of dominant lethal mutations in mice and rats, and on heritable translocations induced in mice, after exposure to butadiene (BD) or its major metabolites, butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). The primary goal of this project was to collect experimental data on the genetic effects of BD in order to estimate the germ cell genetic risk to humans of exposure to BD. To achieve this, the butadiene exposure are based on data for heritable translocations and bone marrow micronuclei induced in mice and chromosome aberrations observed in lymphocytes of exposed workers. A doubling dose for heritable translocations in human germ cells of 4900 ppm/h is estimated, which, assuming cumulative BD exposure over the sensitive period of spermatogenesis, corresponds to 5-6 weeks of continuous exposure at the workplace to 20-25 ppm. Alternatively, the rate of heritable translocation induction per ppm/h of BD exposure is estimated to be approximately 0.8 per million live born, compared to a spontaneous incidence of balanced translocations in humans of approximately 800 per million live born. These estimates have large confidence intervals and are only intended to indicate orders of magnitude of human genetic risk. These risk estimates are based on data from germ cells of BD-exposed male mice. The demonstration that clastogenic damage was induced by DEB in preovulatory oocytes at doses which were not ovotoxic implies that additional studies on the response of mammalian female germ cells to BD and its metabolites are needed. The basic assumption of the above genetic risk estimates is that experimental mouse data obtained after BD exposure can be extrapolated to humans. Several points exist in the present report and in the literature which contradict this assumption: (1) the level of BMO-hemoglobin adducts was significantly elevated in BD-exposed workers; however, it was considerably lower than would have been predicted from comparable rat and mouse exposures; (2) the concentrations of the metabolites DEB and BMO were significantly higher in mouse than in rat blood after BD exposure. Thus, while metabolism of BD is qualitatively similar in the two species, it is quantitatively different; (3) no increase of HPRT mutations was shown in 19 workers exposed on average to 1.8 ppm of BD, while in a different population of workers from a US plant exposed on average to 3.5 ppm of BD, a significant increase of HPRT variants was detected; and (4) data from cancer bioassays and cancer epidemiology suggest that rat is a more appropriate model than mouse for human cancer risk from BD exposure. However, the dominant lethal study in rats gave a negative result. At present, we do not know which BD metabolite(s) may be responsible for the genetic effects even though the bifunctional alkylating agent DEB is the most likely candidate for the induction of clastogenic events. Unfortunately, methods to measure DEB adducts in hemoglobin or DNA are only presently being developed. Despite these several uncertainties the use of the mouse genetic data is regarded as a justifiable and conservative approach to human genetic risk estimation given the considerable heterogeneity observed in the biotransformation of BD in humans.

Evaluation of the mutagenicity of acetochlor to male rat germ cells.

Male rat dominant lethal (DL) assays conducted on the herbicide acetochlor are described. Single dose studies conducted at the maximum tolerated dose (MTD, < or = 1000 mg/kg) produced no effects on any of the DL assay parameters at any of the ten weekly sampling periods. It is concluded that acetochlor is non-mutagenic to rat germ cells. Due to initial limited knowledge of the MTD of acetochlor it was also evaluated in the DL assay at a dose level of 2000 mg/kg. At this high dose level severe bodyweight loss and some deaths occurred among the treated animals. In addition, reduced implantations and reduced pregnancy rates were observed at the third sampling period (18-25 days post dosing) in the absence of an increase in early post-implantation deaths. These results indicated that the use of supra-MTD doses of acetochlor had reduced the fertility of the treated males leading to the production of a pseudo-DL assay response, as alerted to and defined by Ehling. Although several such pseudo-DL assay responses have been described, none have been explained mechanistically. It was therefore decided to pursue the effects seen in the DL assay when using supra-MTD doses of acetochlor. Ova analysis of female rats mated with male rats exposed to 2000 mg/kg acetochlor revealed unfertilized ova at the critical third sampling time. Normal fertilization of ova was observed at the first and fifth sampling period and, for a dose of 200 mg/kg acetochlor, at the third sampling period. The magnitude and temporal nature of these effects confirmed the induction of a pseudo-DL assay response, and studies were then undertaken to probe its genesis. Rats treated with 2000 mg/kg acetochlor had normal testicular and epididymal pathology and normal sperm numbers and sperm motility at the critical third sampling period. Despite a small reduction in testicular and epididymal glutathione levels 12 h after exposure to 2000 mg/kg acetochlor, testicular LDH and LDH-X enzyme levels were unaffected. Further, no reduction in the level of free sulphydryl groups (-SH) were observed in epididymal caput sperm heads isolated 0.5, 7 or 14 days after treatment of male rats with 2000 mg/kg acetochlor. The only sperm parameter affected by treatment with 2000 mg/kg acetochlor was an increase in epididymal cauda sperm with head abnormalities. The non-specific nature of this effect was considered inadequate to explain fully the high dose fertility effects seen in the DL assays, which therefore remain unexplained. The present data establish that acetochlor is non-mutagenic to rat germ cells. They also confirm the importance of segregating mutagenic and fertility effects in the DL assay, and emphasize the need for appropriate dose-setting studies prior to the conduct of rodent genetic toxicity assays.

Induction of specific-locus and dominant lethal mutations in male mice by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU).

1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) induced dominant lethal and specific-locus mutations in male mice. For both compounds the germ cell stage sensitive to the induction of dominant lethal mutations was dose dependent. A dose of 5 mg BCNU per kg b.wt. induced dominant lethal mutations primarily in spermatocytes, whereas higher doses of BCNU induced dominant lethals in spermatids and spermatocytes. Following doses of 5 and 10 mg CCNU per kg b.wt. dominant lethals were induced in spermatids and spermatocytes similar to the results for higher doses of BCNU. Higher dose exposure to BCNU and CCNU was associated with dominant lethals expressed as pre-implantation loss (reduction in total number of implants). In addition, higher doses of CCNU showed a cytotoxic effect in differentiating spermatogonia. Both compounds induced specific-locus mutations in post-spermatogonial germ cell stages of mice. However, CCNU increased also the specific-locus mutation frequency in spermatogonia in two out of three experiments. We conclude in analogy with criteria developed by IARC, that BCNU and CCNU are potential human mutagens.

In vitro and in vivo mutagenicity of the butadiene metabolites butadiene diolepoxide, butadiene monoepoxide and diepoxybutane.

Three metabolites of 1,3-butadiene, namely butadiene diolepoxide, butadiene monoepoxide and diepoxybutane, were tested in the bacterial mutation assay using Salmonella typhimurium strain TA100 with and without metabolic activation (S9 mix). All three compounds showed a mutagenic response. The bifunctional epoxide was more effective than the diolepoxide which was more effective than the monoepoxide. Toxicity appeared to follow the ranking of the chemicals for their mutagenic potency. The monoepoxide and the diolepoxide were also tested for induction of micronuclei in mouse bone marrow erythrocytes and for dominant lethal mutation induction in postmeiotic male mouse germ cells. The effects of the diepoxide in both in vivo tests have been published earlier. In the micronucleus assay, the three metabolites gave a positive response whereby the diepoxide was more effective than the monoepoxide which was more effective than the diolepoxide. In contrast to the diepoxide which was positive at a dose as low as 36 mg/kg, the monoepoxide and the diol did not show an induction of dominant lethal effects up to doses of 120 and 240 mg/kg, respectively. It is concluded that the metabolites were mutagenic in bacteria without metabolic activation and clastogenic in mouse bone marrow; only the bifunctional diepoxide, however, was active in postmeiotic male mouse germ cells.

Quantitative and qualitative studies of micronucleus induction in mouse erythrocytes using flow cytometry. II. Analysis of micronuclei of aneugenic and clastogenic origin by dual-colour FISH on populations of bone marrow PCEs flow sorted on the basis of their relative DNA content.

Simultaneous dual-colour fluorescent in situ hybridization (FISH) with the major and minor mouse satellite probes was performed on flow-sorted fractions of micronuclei with defined Hoeschst 33342 fluorescence intensities, reflecting their relative DNA content. The purpose of the study was to investigate the relationship between probe binding and the size of micronuclei induced by a set of chemicals with different mechanisms of micronucleus induction. The agents studied were vinblastine, cyclophosphamide, hydroquinone and 5-bromo-2-deoxyuridine. The Hoechst 33342 fluorescence distributions of induced micronuclei differ markedly between cyclophosphamide, vinblastine and hydroquinone. While the distributions for cyclophosphamide and vinblastine agree well with those obtained for other model aneugens and clastogens, hydroquinone shows a distribution with characteristics similar to both clastogens and aneugens. A comparison between the Hoechst 33342 fluorescence distributions of hydroquinone-induced micronuclei positive for both major and minor probes and those positive for the major probe only indicates that the former consist of whole chromosomes, while the latter originate from chromosome fragments. The absolute frequency and Hoechst 33342 fluorescence distribution of major-only-positive micronuclei induced by vinblastine indicates that this model aneugen also shows a certain in vivo clastogenic potential. Micronuclei induced by 5-bromo-2-deoxyuridine indicate that this agent is a clastogen, preferentially inducing pericentric breaks, but may also to some extent be aneugenic.