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.

Heritable translocations induced by dermal exposure of male mice to acrylamide.

Acrylamide (AA) is an important industrial chemical used mainly in the production of polymers. It can be absorbed through the skin. AA was shown to be a germ cell clastogen that entails a genetic risk for exposed workers. The genetic risk calculation was based on mouse heritable translocation test data obtained after acute intraperitoneal (ip) exposure (Adler et al., 1994). To obtain a correction factor between ip and dermal exposure, dominant lethal and heritable translocation tests were carried out with dermal exposure of male mice to AA. In the dominant lethal test, male (102/El x C3H/El)F1 mice were exposed by dermal application to the shaved backs of 50 mg/kg AA per day on five consecutive days or to five daily ip injections of 50 mg/kg AA. One day after the end of exposure, the males were mated to untreated females of the same hybrid stock for four days and females were changed every four days for a total of five matings. Dominant lethal effects were found during matings 1-3. For ip exposure, these values were 81.7, 85.7 and 45.4%, respectively; for dermal exposure the corresponding values were 22.1, 30.6 and 16.5%, respectively. In the heritable translocation assay, male C3H/El mice were treated with five dermal exposures of 50 mg/kg AA and mated 1.5-8.5 days after the end of exposure to untreated female 102/El mice. Pregnant females were allowed to come to term and all offspring were raised to maturity. Translocation carriers among the F1 progeny were selected by a sequential fertility testing and cytogenetic analysis including G-band karyotyping and M-FISH. A total of 475 offspring were screened and 41 translocation carriers were identified. The observed translocation frequency after dermal exposure was 8.6% as compared to 21.9% after similar ip exposure (Adler, 1990). The calculated ratio of ip vs. dermal exposure of 0.39 can be applied to obtain a more realistic calculation of genetic risk for dermally exposed workers.

Future research directions to study genetic damage in germ cells and estimate genetic risk.

The late Frits Sobels developed a parallelogram model to estimate genetic risk to humans based on experimental data in somatic cells (peripheral blood) of exposed animals and humans and on data from progeny studies of exposed animals (mice). Recently, an extension to the original parallelogram model was proposed to bridge the gap of extrapolation between rodent and human germ cells by studying sperm samples. The comparison in the parallelogram of rodent/human sperm data with data from rodent progeny tests to derive at an estimate of human progeny at risk is more promising. Therefore, data on all possible end points, DNA adducts, mutations, chromosomal aberrations, and aneuploidy, should be obtained in sperm of exposed rodents and humans. The technology from somatic cell studies is available or adaptable to sperm studies. Sperm samples lend themselves to automated analyses because they are a homogeneous cell population. By flow cytometry or image analysis, large cell samples can be studied per individual. Animal experiments could be conducted in the actual range of chronic human exposure to low doses. The acceptability of extrapolation from the high acute doses so far used in animal experiments to low chronic doses of human exposure could be assessed. Proof could be obtained in human germ cells for the assumption that data from animal experiments can be extrapolated to humans. Data from transgenic rodent systems may play an important role in the extension of the parallelogram approach to genetic risk estimation by providing a link between cancer and genetic risk estimates.

Development of screening tests for aneuploidy induction by environmental pollutants.

When legally required mutagenicity testing of chemicals is undertaken, the important genetic end point of aneuploidy is not included because validated test methods are lacking. Therefore, the Commission of the European Communities (CEC) has funded a research program to develop and validate tests for aneuploidy induction. Ten chemicals, selected on the basis of their ability to interact with cell organelles relevant for aneuploidy induction, were tested in 11 laboratories. The assays ranged from in vitro tubulin assembly studies to in vivo germ-cell tests. The results allow several conclusions: a) Fungal aneuploidy tests are not capable of detecting inhibitors of mammalian tubulin polymerization such as colchicine and vinblastine. Therefore, they will not play a role in screening for aneuploidy but are of value for studying the relationship between induced aneuploidy and recombination. b) Chemicals that induce aneuploidy in mammalian germ cells are readily detected in the in vitro mammalian cell systems. Some chemicals such as thiabendazole and thimerosal induce aneuploidy in vitro but do not appear to be very effective in vivo. c) Cell division aberrations induced in mammalian cells in vitro seem to be predictive for aneuploidy induction in the same cell type. Likewise, c-mitotic effects and cell cycle delay in vivo in mitotic and meiotic cells correlate with aneuploidy induction in the respective tissue. A second CEC Aneuploidy Program has started recently to refine the most promising test protocols, to provide understanding of variety of mechanisms by which chemicals induce aneuploidy, and to establish a data base for aneugens among environmental pollutants.

Inhalation exposure of rats and mice to 1,3-butadiene induces N6-adenine adducts of epoxybutene detected by 32P-postlabeling and HPLC.

In this paper we report DNA binding of butadiene monoepoxide, a first metabolite of 1,3-butadiene catalyzed by monooxygenases. We prepared alkylated purines as marker compounds for 32-P-postlabeling and electrochemical analysis and developed methods to measure the corresponding products. The traditional postlabeling assay was modified by incorporating a solid phase extraction column and high-performance liquid chromatography (HPLC) enrichment steps to the assay prior to labeling. The final analysis of adducted N6 adenines is based on two dimensional thin-layer chromatography (TLC) and an on-line HPLC/radioactivity analysis. The qualitative and quantitative results are based on positively identified marker compounds. Alkylated N7 guanines were released from DNA by neutral thermal hydrolysis, prepurified by HPLC, and analyzed by HPLC with a sensitive electrochemical detection procedure. By using these methods, we found alkylation of calf thymus DNA exposed to butadiene monoepoxide in vitro at adenine N6 and guanine N7 sites. Analysis of lung DNA samples from mice and rats exposed to butadiene through inhalation showed that adenine N6 adducts were formed in vivo in a dose responsive manner.

Establishment and characterization of C-type RNA virus-producing cell lines from radiation-induced murine osteosarcomas.

Eight cell lines were established from murine osteosarcomas induced in vivo with the radionuclides 224Ra and 227Th. They have been compared by light and electron microscopy, by karyology, and by their growth properties. The morphology, the growth pattern, and the ability to induce tumors in mice indicate that five of them are tumor cell lines. Chromosome studies demonstrated that the five cell lines have marker chromosomes. The other cell lines only showed some criteria generally used to score for transformation of fibroblasts and they may be derived from stromal cells. All cell lines release virus particles in the culture fluid which have the typical properties of RNA tumor viruses. They possess C-type morphology, a density of 1.16--1.18 g/cm3, a 60--70 S RNA, a RNA dependent DNA polymerase and they induce syncytia in rat XC cells. The possible significance of these virus particles in radiation osteosarcomagenesis is discussed.

Numerical and structural chromosomal abnormalities detected in human sperm with a combination of multicolor FISH assays.

A pair of multicolor FISH assays (X-Y-21 and A-M-16) was developed for human sperm to simultaneously measure sex ratios; aneuploidies involving chromosomes 1, 16, 21, X, and Y; meiotic diploidies; and structural aberrations involving chromosome 1p. Sex ratios in sperm were not significantly different from unity among healthy men. Baseline frequencies of disomic sperm for chromosomes 1, 8, and 21 were similar (6.7 per 10(4) sperm, 95% CI of 5.6-8.1), suggesting that among these three chromosomes, chromosome 21 was not especially prone to nondisjunction. Frequencies of disomy 16 sperm were significantly lower, however (3.5 per 10(4) sperm, 95% CI of 2.0-6.2; P < 0.02). The baseline frequencies of sperm disomy by FISH for chromosomes 16 and 21 were validated against aneuploidy data obtained by the hamster-egg technique for human sperm cytogenetics. The frequencies of X-X, Y-Y, X-Y ("Klinefelter") sperm and sex-null ("Turner") sperm were 5.5, 5.1, 5.5, and 7.8 per 10(4) sperm, respectively. For chromosomes 16 and 21, the frequencies of nullisomic and disomic sperm were similar, suggesting that gain and loss events occurred symmetrically. However, more gain than loss was reported for chromosomes 1, X, and Y. The frequency of MI and MII diploid sperm (with flagella) was approximately 12 per 10(4) (range 8.3-16.7 per 10(4) sperm). Based on flagella data, the frequency of somatic cells in the semen was estimated to be approximately 1.8 per 10(4) sperm. Loss or gain of a portion of chromosome-arm 1p occurred in 5.5 per 10(4) sperm, and the percentage of sperm carrying structural aberrations within the haploid genome as calculated from FISH (1.4%), was similar to that obtained with the hamster-egg technique. These complementary sperm FISH assays have promising applications in studies of chromosomally abnormal sperm after exposure to occupational, medical, and environmental toxicants.

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.

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half-life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application.

Synopsis of the in vivo results obtained with the 10 known or suspected aneugens tested in the CEC collaborative study.

The synopsis of the in vivo test results in the first collaborative CEC Aneuploidy Project with 10 selected chemicals, colchicine (COL), econazole (EZ), chloral hydrate (CH), hydroquinone (HQ), diazepam (DZ), thiabendazole (TB), cadmium chloride (CD), thimerosal (TM), pyrimethamine (PY) and vinblastine (VBL), allowed several conclusions. (1) The spindle poisons, COL and VBL, were positive in all bone marrow and germ cell tests; (2) the clastogen HQ also induced aneuploidy in somatic and germinal cells; (3) the other seven compounds gave contradictory results either between laboratories or between test systems which require further experimental clarification; (4) CREST labeling or in situ hybridization for centromere identification showed about 70% fluorescent signals in micronuclei induced by COL or VBL but only about 15% in HQ induced micronuclei; (5) the tests for induction of a delay in cell division progression can be recommended as a prescreen for possible aneugens; (6) all test methods applied in these experiments require standardization with respect to sample size, sampling times and statistical treatment of the data. A second CEC Aneuploidy Programme has started recently to answer some of the questions raised by the first study regarding tissue and sex specificities.

Aberration induction by mitomycin C in early primary spermatocytes of mice.

MC is well known to induce dominant lethal mutations in mouse spermatocytes. Tests were done to determine whether chromosomal aberrations could be identified in spermatocytes as being responsible for the dominant lethal effects. Male mice were treated with single doses of MC during DNA synthesis preceding meiosis and during early prophase of meiosis. Simultaneous labeling was performed to identify cells that were in S-phase during the time of treatment. Diakineses-metaphases I were analyzed for the occurrence of univalents, gaps, fragments and rearrangements. The frequencies of cells with aberrations increased with dose and time after treatment. Maximal values were obtained after 12 days, indicating that MC was most effective in cells undergoing DNA replication. 95% of these cells were labeled. The majority of aberrant cells contained one or more fragments. These cells will lead to dominant lethality of the zygotes after fertilization. Cells with rearrangements occurred 11 and 12 days after treatment. These cells can develop into sperm carrying a reciprocal translocation which would then give rise to semi-sterile progeny after fertilization. Further investigations are needed to study the transmission of rearrangements observed in primary spermatocytes.

A review of the coordinated research effort on the comparison of test systems for the detection of mutagenic effects, sponsored by the E.E.C.

Under the Environmental Research Programme of the Commission of the European Communities a coordinated comparative test programme was started to assess the mutagenic effects of chemicals in as many different assay systems as possible. A preliminary report is given on the results for 5 compounds selected by the Contact Group "Genetic Effects of Chemicals". 11 laboratories contributed data from tests in their expertise. The compounds were methyl methanesulfonate (MMS), N-nitrosodiethylamine (DEN), mitomycin C (MC), procarbazine (Natulan) and atrazine. The results of testing these 5 compounds in more than 20 test systems are still incomplete but some observations can be made at this time. MMS has been shown to be mutagenic in every system used in this collaborative effort which is not surprising in view of the fact that MMS is a widely used reference mutagen that does not require metabolic activation. Among the indirect mutagens which were tested, MC has been shown to be mutagenic with almost every system which has been applied. DEN which requires microsomal activation gave positive results in most submammalian systems as well as in mammalian cells in culture when the S9 fraction of liver homogenate was added. However, in whole mammals most results were negative. One study suggests that the difference in the results with DEN may be due to the short biological half-life of the mutagenically active metabolite. Procarbazine was negative in the microbial tests but gave a positive response in mammalian cells in culture, in Drosophila and in mammals. Atrazine has only been subjected to a limited amount of testing but the results indicate that plant metabolic activation is required in vitro, and that activation to a mutagenic metabolite also occurs in mammals independent from liver enzymes.

Comparison of types of chemically induced genetic changes in mammals.

The present paper reviews the currently available in vivo systems for detection of chemically induced mutations and chromosome aberrations and summarizes the data of the relevant tests for mammalian germ-cell mutations (specific-locus test and heritable translocation test). The value of in vivo screening tests (somatic mutations and sperm abnormalities) for predicting specific-locus mutations is illustrated by comparing doubling doses. The results from the mammalian germ-cell mutation tests (specific-locus test and heritable translocation test) constitute the base-line for an assessment of predictability. Radiation and chemically induced specific-locus mutations differ in a number of respects, suggesting a need for caution in making risk estimates for chemical mutagen exposures in terms of radiation-equivalent doses. In vivo nondisjunction tests are discussed. Finally, unsolved problems and difficulties in generalizing qualitative and quantitative correlations between test systems are outlined. It is concluded that even qualitative predictions from data on somatic cells to germ cells are at best insecure because germ-cell specificity cannot be foretold, not to mention the fact that quantitative extrapolations from the results of in vivo screening tests, in general, are fraught with even more uncertainties. There is an acute need for collection of more data from studies involving germ cells.

Radiation-induced translocations in spermatogonial stem cells of Macaca fascicularis and Macaca mulatta.

3 adult monkeys, one Macaca fascicularis and two Macaca mulatta, were whole-body irradiated with 1 Gy gamma-rays (60 Co). Reciprocal translocations induced in spermatogonial stem cells were scored as translocation multivalents in primary spermatocytes from 7.5 to 27.5 months after exposure. The translocation yields ranged from 4.1% at the earliest to 1.8% at the latest sampling interval. No significant differences were observed in the responses of the individual animals. A decline in the translocation frequencies with time after treatment was found in all 3 animals. The present data are different from those reported for testicular X-irradiation of the rhesus monkey Macaca mulatta (van Buul, 1980; Lyon et al., 1976) in that the translocation yields are higher. They are consistent with the results reported for testicular gamma-irradiation of the crab-eating monkey Macaca fascicularis (Matsuda et al., 1984, 1985). In view of the present results it appears unlikely that a species difference exists within the genus Macaca in the sensitivity of spermatogonial stem cells to the induction of translocations by ionizing radiation.

The present lack of evidence for unique rodent germ-cell mutagens.

Six chemicals, diethylhexyl phthalate (DEHP), ethanol, cyclohexylamine (CHA), sodium saccharin (NaS), cadmium chloride (CdCl2) and triflupromazine (TFP), were suggested to be unique germ-cell mutagens (Auletta and Ashby, 1988) by the GeneTox Workgroups of the U.S. Environmental Protection Agency (EPA). If this is a correct classification it would have major consequences when screening for mutagenicity and when labelling genotoxic substances. However, our re-evaluation of the GeneTox literature, including some more recent publications, has failed to find substantive evidence that any of these chemicals have been unequivocally established as having unique mutagenic activity in germ cells. For DEHP, NaS and TFP the evidence for genotoxic/mutagenic effects is questionable, in both germinal and somatic cells. Ethanol and CdCl2 showed clastogenic activity, but it was not restricted to germ cells. Both, ethanol and cadmium salts, appear to induce aneuploidy. The unconfirmed clastogenic effect of CHA was restricted to rats, but it occurred in both bone marrow and spermatogonia. Therefore, the general observation that rodent germ-cell mutagens are also genotoxic in somatic cells in vivo (Brusick, 1980; Holden, 1982) remains valid.

Micronucleus test in bone marrow of mice treated with 1-nitropropane, 2-nitropropane and cisplatin.

Micronucleus tests were carried out in bone marrow of mice treated with 1-nitropropane, 2-nitropropane and cisplatin. For 1-nitropropane and 2-nitropropane the results were negative. With cisplatin a dose-dependent increase in the number of polychromatic erythrocytes with micronuclei was observed. The lowest positive dose was 0.1 mg/kg (P less than 0.001, Mann-Whitney-Wilcoxon test). The hepatocarcinogen 2-nitropropane showed clastogenic activity in human lymphocytes in vitro in the presence of S9 (Bauchinger et al., 1987). The negative results in bone marrow suggest that short-lived genotoxic metabolites may be formed in the liver but do not reach the bone marrow.

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.

Induction of hypoploidy and cell cycle delay by acrylamide in somatic and germinal cells of male mice.

Monomeric acrylamide was tested for its potential to induce aneuploidy in spermatocytes and bone marrow cells of mice. For this purpose, chromosomes from metaphase spreads were counted semi-automatically. In both test systems, cell proliferation was monitored, determining the meiotic index of spermatocytes and the average generation time of bone marrow cells after BrdU incorporation, respectively. No indications could be seen for different sensitivity of somatic and germinal cells towards acrylamide. With a dose of 120 mg/kg, the chemical caused cell cycle delay in both germ line and somatic cells. There was diverging response with respect to the balance of hypo- and hyperploidy. While the percentage of chromosome loss was significantly elevated in both test systems, acrylamide treatment did not increase the frequency of hyperploid cells. Interpreting these results on the basis of conventional test protocols, acrylamide should not be considered as an aneugen. The conservative approach, however, may be inadequate for the detection of aneugenic mechanisms different from non-disjunction.

Detection of aneuploidy in human and rodent sperm using FISH and applications of sperm assays of genetic damage in heritable risk evaluation.

Efficient molecular methods are being developed for detecting various types of cytogenetic genetic damage in sperm, especially numerical aneuploidy for chromosomes involved in trisomies that survive at birth. These methods provide new approaches for identifying potentially detrimental environmental exposures, genetic predisposition, chromosomal rearrangements, and physiologic factors which may increase a man's risk of fathering a genetically defective offspring. Corollary methods are also being developed for detecting sperm aneuploidy in laboratory rodents and these will be used to make inter-species comparisons of mutagen sensitivities and for investigating mechanisms of induction and persistence of aneuploidy. Validated assays for detecting genetic alterations in human and rodent sperm (of which sperm aneuploidy is a first example) permit comparisons of somatic and germinal response to mutagens within individuals, comparisons of human and rodent germinal sensitivity to mutagens, and can be applied in an extended parallelogram model to sperm for assessing heritable risk resulting from paternal mutagen exposures.