Induction Of Dominant Lethal Mutations Research Articles

Reproductive toxicity assessment of Olusosun municipal landfill leachate in Mus musculus using abnormal sperm morphology and dominant lethal mutation assays.

Mixture of xenobiotics in Olusosun landfill leachates (OSL) induced somatic and germ cell genotoxicity in eukaryotic cells. However, whether the DNA-damaged germ cells can increase reproductive failure, embryotoxicity and/or teratogenicity during fertilization remained unclear. The study herein investigated reproductive toxicity and embryotoxic effects of OSL in mice using abnormal sperm morphology and dominant lethal mutation assays. Six mice per group were exposed to 0.5 mL of 5, 10, 25, and 50% OSL (v/v, leachate/distilled water) for 5 consecutive days. Similar treatment was giving to distilled water and cyclophosphamide (20 mg/kg body weight), as negative and positive control groups, respectively. At 35 day post-treatment, the mice were sacrificed and examined for cauda epididymal abnormal sperm morphology, testicular weight change and histopathological lesions. Male mice were mated to untreated nulliparous females (1 treated male per 2 untreated females) until vaginal plugs were observed. Pregnant females were sacrificed on 14th day post-mating and uterine content examined for number of implants per female, resorptions, and live and dead fetuses. OSL significantly increased teratozoospermia by 31.02% with amorphous sperm head having the highest frequency and sperm with two heads, the least. Histology of the testes revealed congestion of the interstitial blood vessels, spermatid retention and disorganization of the germinal epithelium from the basal compartment. OSL insignificantly increased absolute and relative testes weights but reduced body weight of exposed mice. There was insignificant induction of dominant lethal mutation, although low fertility was observed in the treated mice. The analyzed physico-chemical parameters and heavy metals/metalloids in OSL were higher than permissible limits. The metals, physico-chemical parameters and other unanalyzed substances in OSL induced abnormal sperm morphology, altered testes pathology, but did not significantly induce dominant lethal mutation in mice. Constituents of OSL are toxic to the male reproductive organs in mice and suggests harmful impact on public health. Indiscriminate disposal of solid wastes in the environment should be prevented.

English

Dominant lethal mutation assay was carried out on rats after being treated with graded doses of ethanol extract of the seeds of Mucuna urens. Male albino rats (Wistar strain) were caged in three groups labeled, groups II, III and IV and treated with three different dosages of the ethanol extract of the seeds of M. urens; 70, 140 and 210 mg/kg body weight (BW), respectively, for 14 days. The positive control animals (group I), were treated with distilled water for the entire period. At the end of the feeding period of two weeks, they were co-habited with virgin female albino rats at a ratio of 1:1 for 3 days. 14 days after mating, the females were sacrificed for the dominant lethal mutation assay. The results of the dominant lethal mutation assay showed that only female rats in group II had implants on the uterine horn, of all the treated groups. The rats in groups III and IV did not have any implants at all. Biological evaluations (pre-implantation losses) carried out showed 0, 76, 100 and 100% lethal mutations in groups I, II, III and IV, respectively. The statistical evaluations obtained showed 8.6b ± 0.47, 6.6a ± 0.94, 0 ± 0.0 and 0 ± 0.0 in groups I, II, III and IV, respectively. Photographs of the corpora lutea were obtained using a digital camera (DCR-HC48E, KODAK). The results obtained can be attributed to the induction of dominant lethal mutations in spermatocytes and early spermatids in the male Albino rats, showing the mutagenic effect of the seeds of the plant M. urens and its potential as a male contraceptive. Key words: Mucuna urens seeds, dominant lethal mutations assay, mutagenesis, male Albino rats.

Significant increase in the dominant lethal mutations: benzo(a)pyrene

Background: Use of biomarkers particularly under in vivo condition is of importance in risk assessment. This study is proposed to identify the in vivo antigenotoxic potential using dominant lethal mutation test in B(a)P exposed, turmeric fed animals and evaluate the role of turmeric in counteracting the germ cell mutations. Methods: Twenty four male mice were taken and divided into 4 groups each group containing 6 mice/group. The first and second group received stock diet (control) while the third and fourth groups received 5% turmeric diet for a period of one month. At the end of feeding period, second and fourth group received a single dose of benzo(a)pyrene 1mg/mouse intraperitoneally. After 1 week each male mouse was mated to 3 female mice at 1,4, 8 and 12 week intervals. On 13th day from mid point of mating, the females were sacrificed and live and dead embryos were counted to study the pre and post implantation loss of embryos. The differences in the frequencies of pre and post implant deaths were done by comparing treated with control group at 4-time periods by analysis of variance (ANOVA) and chi-square test which is used to compare treatment group and positive control with negative control. Results: Feeding of 5% turmeric and treatment with a single dose of B(a)P did not show any significant effect on the mutagenic index or the frequency of pregnancy in treated groups compared with control. There was no apparent induction of dominant lethal mutations in B(a)P or B(a)P+Turmeric treated groups. No post implantation dominant lethal effects produced by B(a)P could be detected in this study. The induction of B(a)P and treatment with turmeric did not influence the dominant lethal test. Conclusion: B(a)P did not produce a significant increase in the dominant lethal mutations. Turmeric also did not give any positive response and may be considered as non-mutagenic. The negative result suggests that under the conditions of the test the test substance may not be genotoxic in the germ cell of the treated sex of the test species.

ДОЦІЛЬНІСТЬ ВИКОРИСТАННЯ ВІТАМІННИХ ХІМІОПРЕВЕНТЕРІВ ДЛЯ КОРЕГУВАННЯ ІНДУКЦІЇ ХРОМОСОМНИХ АБЕРАЦІЙ СИНТЕТИЧНИМ ХАРЧОВИМ АРОМАТИЗАТОРОМ «КАРАМЕЛЬ»

Flavors “Caramel” induced in root meristem cells of onion Allium cepa L. single and paired fragments of chromosome and dicentric chromosomes, which are the result of chromosomal aberrations such as deletions and translocations. It was found induction of dominant lethal mutations and unfertilized eggs by different concentration of flavors in Drosophila melanogaster test. As well as under flavors influence we have shown the induction of a wide range of chromosome aberrations in metaphase bone marrow cells of white mice Mus musculus L., which included not only chromosomes but also genomic mutations. This could be evidence of spindle apparatus disruption and chromosomes nondisjunction as an influence of mutagenic agents, which may include a flavoring component themselves as well as their metabolites appearing in the biotransformation in mammal’s organism. We observed a partial decrease of aberrant metaphases and dominant lethal mutations after vitamin A and C application.

Reproductive Toxicity of <em>Orthosiphon stamineus</em> Benth (Java tea) in Swiss Albino Mice

The java tea is significantly used in traditional medicine for its anti-allergic, antihypertensive, anti-inflammatory and diuretic properties. The present study is designed to assess the effect of java tea after its chronic use on male reproduction, cytological and biochemical changes in Swiss albino mice. Java tea was administered orally at the doses 62.50, 125 and 250 mg/kg/day for 90 days. Higher dose of Java tea caused a significantly reduction in sperm count and motility along with an induction of morphological abnormalities in spermatozoa and also induced aberrations in sperm chromosomes compare to normal mice. Higher dose caused a significant increase in plasma levels of estradiol and decrease in testosterone levels. It also reduced the fertility index in male and female mice and that indicates towards the induction of dominant lethal mutations after sub-chronic administration of Java tea particularly in higher dose treated group. Malondialdehyde (MDA) level was found to be increased along with a concomitant reduction in Non-Protein Sulfhydryl (NP-SH) in testes tissue. In conclusion, Java tea administration at higher dose induces reproductive toxicity in mice and the study paves a path to further investigate its clinical effects on reproductive system.

Cardiac effect of piroxicam and DFU in rat fetuses

The present study investigates the effects of Bisphenol A on the induction of dominant lethal mutation and male reproductive functions. The male rats were gavaged with BPA (10 μg, and 5.0 mg/kg/bw) over a period of six days and control group with vehicle. Each male was cohabited with untreated females sequentially over the period of eight weeks. The mated females were sacrificed on 15th day of gestation. The results revealed a significant increase in dominant lethal mutation rate during fourth and sixth week of mating intervals at 5.0 mg/kgbw dose of BPA. These findings demonstrate that mid-spermatids and spermatocytes are more sensitive to BPA exposure. The male rats sacrificed at the end of mating study showed an increase in the sperm DNA damage, and decrease in motility at higher dose. However, significant reductions in sperm production effects were observed at both lower and higher doses of BPA. These preliminary results indicate that BPA may be a weak male germ cell mutagen.

Effects of β-carotene in cultured mouse embryos exposed to nicotine

The present study investigates the effects of Bisphenol A on the induction of dominant lethal mutation and male reproductive functions. The male rats were gavaged with BPA (10 μg, and 5.0 mg/kg/bw) over a period of six days and control group with vehicle. Each male was cohabited with untreated females sequentially over the period of eight weeks. The mated females were sacrificed on 15th day of gestation. The results revealed a significant increase in dominant lethal mutation rate during fourth and sixth week of mating intervals at 5.0 mg/kgbw dose of BPA. These findings demonstrate that mid-spermatids and spermatocytes are more sensitive to BPA exposure. The male rats sacrificed at the end of mating study showed an increase in the sperm DNA damage, and decrease in motility at higher dose. However, significant reductions in sperm production effects were observed at both lower and higher doses of BPA. These preliminary results indicate that BPA may be a weak male germ cell mutagen.

Mutagenic effect of Bisphenol A on adult rat male germ cells and their fertility

The present study investigates the effects of Bisphenol A on the induction of dominant lethal mutation and male reproductive functions. The male rats were gavaged with BPA (10μg, and 5.0mg/kg/bw) over a period of six days and control group with vehicle. Each male was cohabited with untreated females sequentially over the period of eight weeks. The mated females were sacrificed on 15th day of gestation. The results revealed a significant increase in dominant lethal mutation rate during fourth and sixth week of mating intervals at 5.0mg/kgbw dose of BPA. These findings demonstrate that mid-spermatids and spermatocytes are more sensitive to BPA exposure. The male rats sacrificed at the end of mating study showed an increase in the sperm DNA damage, and decrease in motility at higher dose. However, significant reductions in sperm production effects were observed at both lower and higher doses of BPA. These preliminary results indicate that BPA may be a weak male germ cell mutagen.

Dominant lethal mutations of topoisomerase II inhibitors etoposide and merbarone in male mice: a mechanistic study

Two topoisomerase II inhibitors, etoposide and merbarone, were tested for the induction of dominant lethal mutations in male mice. Etoposide was administered at a dosage of 30 or 60 mg/kg. Merbarone was administered at a dosage of 40 or 80 mg/kg. These males were mated at weekly intervals to virgin females for 6 weeks. In the present experiments, regardless of the agent, spermatids appeared to be the most sensitive germ-cell stage to dominant lethal induction. Etoposide and merbarone clearly induced dominant lethal mutations in the early spermatid stage only with the highest tested doses. The mutagenic effects were also directly correlated with reactive oxygen species accumulation as an obvious increase in 2',7'-dichlorofluorescein fluorescence level was noted in the sperm of animals treated with higher doses of etoposide and merbarone. Treatment of male mice with N-acetylcysteine significantly protected mice from etoposide- and merbarone-induced dominant lethality. Moreover, N-acetylcysteine treatment had no antagonizing effect on etoposide- and merbarone-induced topoisomerase II inhibition. Overall, this study provides for the first time that etoposide and merbarone induce dominant lethal mutations in the early spermatid stage through a mechanism that involves increases in oxidative stress. The demonstrated mutagenicity profile of etoposide and merbarone may support further development of effective chemotherapy with less mutagenicity.

Induction of dominant lethal mutations by alkylating agents in germ-cells of the silkworm, Bombyx mori

Induction of dominant lethal mutations by alkylating agents in germ-cells of the silkworm, Bombyx mori

Induction of dominant lethal mutations by Ascaris trypsin inhibitor in male mice

Trypsin inhibitor (ATI) isolated from Ascaris suum, a gastrointestinal nematode parasite, was tested for the induction of dominant lethal mutations in male mice. Dominant lethal effects of ATI for the main stages of germ cell development were analyzed by mating at specific time points after dosing. Three groups of adult BALB/c males received 50, 100 or 250 mg/kg body weight (bw) single intraperitoneal (ip) injection of ATI in sterile phosphate-buffered saline (PBS). The control group received concurrent injection of PBS. After the administration of ATI or PBS, each male was mated with two untreated females. For fractionated examination with regard to successive germ cell stages (spermatozoa, spermatids, spermatocytes, and spermatogonia), every second week two other untreated virgin females were placed with each male for mating. The uteri of the females were inspected on the 15th day of gestation, and preimplantation loss and postimplantation loss determined from dominant lethal parameters. Exposure of mice germ cells to ATI did not impair mating activity of males. Pregnancy rates were reduced (∼5–10%) by treatment of males with higher doses of ATI, but differences between treatment and control groups were not statistically significant ( P > 0.05). In the females bred to ATI-treated males, significant increase in preimplantation loss was observed at post-injection week 1 (reflecting exposure to spermatozoa) and 3 (reflecting exposure to mid and early spermatids) for higher doses of the inhibitor ( P < 0.05 or P < 0.01). During mating days 15–21 a statistically significant increase in postimplantation loss and dominant lethal effects were observed for all doses of ATI. At higher doses, dominant lethal effects were restricted to spermatozoa ( P < 0.01). These data suggest that ATI induces dominant lethal mutations at postmeiotic stages of spermatogenesis, but spermatids are the most sensitive cell stage to the effect of ATI. These preliminary findings show that ATI may be one of the factors causing disturbances in spermatogenesis leading to a reduction of host reproductive success.

Mutagenicity of Ascaris chymotrypsin inhibitor in germ cells of mice

Chymotrypsin inhibitor isolated from Ascaris suum (ACHI) was tested for the induction of dominant lethal mutations in male mice. Dominant lethal effects of ACHI for the main stages of germ cell development were analyzed by mating at specific time points after dosing. Two groups of adult BALB/c males received 24 or 40 mg per kilogram body weight (BW) per day intraperitoneal (IP) injection of ACHI in sterile phosphate-buffered saline (PBS) for five consecutive days (subacute exposure). Males from a third group were administered single IP injections of ACHI-60 mg/kg BW (acute exposure). The control group received concurrent injections of PBS for five successive days. After the last dose, each male was mated with two untreated females. For fractionated examination with regard to successive germ cell stages (spermatozoa, spermatids, spermatocytes, spermatogonia), every second week, two other untreated virgin females were placed with each male for mating. The uteri of the females were inspected on the 15th day of gestation, and preimplantation loss and postimplantation loss determined from dominant lethal parameters. Exposure of mice germ cells to ACHI did not impair mating activity of males. Fertility index was reduced (P < 0.05) only for females mated at the third week with males exposed to the highest dose of ACHI. In the females bred to ACHI-treated males, significant (P < 0.05) increase in preimplantation loss was observed at postinjection weeks 1 (reflecting exposure to spermatozoa after single treatment and to spermatozoa or late spermatids after subacute dosing) and 3 (reflecting exposure to mid and early spermatids for acute dosing and to mid and early spermatids or late spermatocytes following acute treatment), regardless of dose and length of exposure to the inhibitor. At the 60-mg/kg-BW group, a significant increase of this parameter was also noted at week 5 (reflecting exposure to early spermatocytes). During mating days 15-21, a significant (P < 0.05) increase in postimplantation loss and dominant lethal effects were observed for all doses of ACHI. Acute ACHI exposure 5 weeks prior to mating resulted in dominant lethal effects in early spermatocytes. These preliminary data suggest that ACHI induces dominant lethal mutations at postmeiotic and meiotic stages of spermatogenesis, but spermatids are the most sensitive cell stage to the effect of ACHI. These results show that ACHI may be one of the factors causing disturbances in spermatogenesis leading to a reduction of host reproductive success.

467 Induction of dominant lethal mutations in male mice by perezone

L5178Y/TK+/− cells were treated with either N-acetyl-2-aminofluorene (AAF), 2-aminoanthracene (2A), benzanthracene (BA), benzo[a]pyrene (BP), 3-methylcholanthrene (3MCA), or dimethylnitrosamine (DMN) in the presence of varied concentrations (2.5–10% v/v) of liver S9 (9000 × g) postmitochondrial fraction from Aroclor 1254-dosed male CD rats. Consistent S9 concentration-dependent decreases in trifluorothymidine-resistant (TFTr) mutant induction were noted following 3 h exposure of L5178Y/TK+/− cells to either 2.5 μg/ml BP, 50 or 67 μg/ml AAF, 0.96 μg/ml 2A, 7.35 μg/ml BA, or 5.4 μg/ml 3MCA. The exception was DMN which yielded a moderate S9 concentration-dependent increase in TFTr mutant induction when L5178Y/TK+/− cells were treated for 3 h with 74.1 μg/ml DMN. Depending upon the promutagen being tested, these results suggest two different metabolic events: (1) activation via non-induced enzymes or other factors whose activities are totally S9 volume-dependent (DMN), and (2) deactivation via induced detoxification pathways, a sequence favored by increasing S9 concentration (all others). Utilization of a single “standard” (10%) concentration of liver S9 from Aroclor 1254-treated rats did not appear to provide optimal metabolic activation for 5 of these 6 promutagens.

Chlornaphazine and chlorambucil induce dominant lethal mutations in male mice.

Two antineoplastic agents, chlornaphazine (CN) and chlorambucil (CHL), were tested for the induction of dominant lethal mutations in male mice. Both compounds are nitrogen mustard derivatives and have been shown to be genotoxic in a variety of organisms. CN was administered intraperitoneally to DBA/2J male mice at a dosage of 0, 500, 1000, or 1500 mg/kg body weight (bw). Immediately following treatment, each male was mated at 4-day intervals to two virgin C57BL/6J females. CHL was administered intraperitoneally to C3H/HeJ and DBA/2J males at a dosage of 0, 2.5, or 5.0 mg/kg bw. These males were mated at weekly intervals to two virgin T-stock females. CN and CHL clearly induced dominant lethal mutations. CN induced dominant lethal effects in all post-meiotic germ-cell stages of treated DBA males, with a clear dose-response relationship. The results with CHL-treated DBA males indicated that all post-meiotic germ-cell stages, except late-spermatids, were affected by CHL treatment, while in C3H males, CHL induced dominant lethal effects in all post-meiotic germ-cell stages. A dose-response relationship was also observed with CHL in C3H male mice. In the present experiments, regardless of the agent or the mouse strain used, spermatids appeared to be the germ-cell stage most sensitive to dominant lethal induction.

Mutations and their use in insect control.

Traditional chemically based methods for insect control have been shown to have serious limitations, and many alternative approaches have been developed and evaluated, including those based on the use of different types of mutation. The mutagenic action of ionizing radiation was well known in the field of genetics long before it was realized by entomologists that it might be used to induce dominant lethal mutations in insects, which, when released, could sterilize wild female insects. The use of radiation to induce dominant lethal mutations in the sterile insect technique (SIT) is now a major component of many large and successful programs for pest suppression and eradication. Adult insects, and their different developmental stages, differ in their sensitivity to the induction of dominant lethal mutations, and care has to be taken to identify the appropriate dose of radiation that produces the required level of sterility without impairing the overall fitness of the released insect. Sterility can also be introduced into populations through genetic mechanisms, including translocations, hybrid incompatibility, and inherited sterility in Lepidoptera. The latter phenomenon is due to the fact that this group of insects has holokinetic chromosomes. Specific types of mutations can also be used to make improvements to the SIT, especially for the development of strains for the production of only male insects for sterilization and release. These strains utilize male translocations and a variety of selectable mutations, either conditional or visible, so that at some stage of development, the males can be separated from the females. In one major insect pest, Ceratitis capitata, these strains are used routinely in large operational programs. This review summarizes these developments, including the possible future use of transgenic technology in pest control.

Mutagenic profiles of carbazole in the male germ cells of Swiss albino mice

Mutagenic effect of carbazole was evaluated by employing dominant lethal mutation and sperm head abnormality assays in male Swiss albino mice. For the dominant lethal mutation assay, adult male mice were treated for five consecutive days either with 30 or 60 mg/kg body weight (b.w.) of carbazole by single intraperitoneal (i.p.) injection. For the sperm head abnormality assay mice were treated with 50, 100, 150, 200 and 300 mg/kg b.w as a single i.p. injection. Treatment of adult male mice with carbazole resulted in induction of dominant lethal mutation and abnormal sperm heads. The results show that carbazole is mutagenic in male germ cells of mice.

Genetic and reproductive toxicity of butadiene and isoprene

Butadiene (BD) and its 2-methyl analogue, isoprene, have been extensively studied in animals and BD in population studies. Both chemicals are metabolised by liver cytochrome P450 dependent monogenases to monoepoxide and diepoxide intermediates. The diepoxide intermediates of both compounds were mutagenic in Salmonella typhimurium. However, unlike the monoepoxide of BD, the monoepoxides of isoprene were not mutagenic. It appears that they have no alkylating capacity. BD did not induce somatic cell mutation and recombination or sex-linked recessive lethal mutation in Drosophila melanogaster and isoprene produced no increase in chromosomal aberrations in CHO cells in vitro. Comparative concentrations of haemoglobin adducts in the blood of mice and rats after exposure to BD indicated that reaction with blood may decrease the levels of reactive intermediates available to tissues in rats, but not in mice contributing to greater potency of BD in the mouse. For isoprene, the adducts reach approximately the same concentrations in both species. DNA adducts have also been detected in testicular and lung cells of mice after BD exposure. The level of epoxybutene haemoglobin adducts was significantly elevated in BD-exposed workers, but lower than in rats and mice. In conjunction with the toxicology and carcinogenesis studies for BD and isoprene, additional mice were included for the evaluation of cytogenetic effects. Both chemicals produced increases in sister chromatid exchanges in bone marrow cells and in the frequency of micronuclei in normochromatic and polychromatic erythrocytes, but only BD produced an increase in the percent of bone marrow cells with chromosomal aberrations. At similar doses, the effects with BD were 2–3 times larger than with isoprene. There were also increased hprt mutation frequencies in rats and mice after BD exposure. Biomonitoring studies with hprt mutations in lymphocytes showed conflicting results, with both positive and negative findings. BD has been shown to be positive in one human cytogenetic biomonitoring study and not in three others, but chromosomal aberrations were increased in BD-exposed workers after challenge with gamma rays. Re-analysis of GSTTI null individuals showed positive results. There was an increase in spermatid micronuclei in mice by BD and its metabolites and in rats only by its metabolites. The cytotoxic response of germ cells in mice is greater than in rats. Dominant lethal mutations have been induced by BD and diepoxybutane, but not by epoxybutene. There was some evidence of congenital malformations in mice after BD exposure and there was a linear concentration-related induction of heritable translocations in mice. There was no induction of dominant lethal mutations or congenital malformations in rats. Using the heritable translocation data in mice, it has been determined that if a worker is continually exposed over 5 or 6 weeks to 20–25 ppm of BD, the risk of producing a child with a balanced reciprocal translocation is twice as high as the background risk. Since genetic damage cannot be measured directly in human germ cells, risk to such cells can also be estimated from germ cells and somatic cells of the mouse and human somatic cells using the parallelogram approach. Using doubling doses, the fourth corner of the parallelogram was calculated as a doubling dose for human germ cells of 4390 ppm/h. However, it is still questioned if man is more like rat than mouse in terms of sensitivity to exposure. Similar germ cell data do not exist for isoprene. In conventional developmental studies, where rats and mice were exposed to BD, maternal toxicity was shown in rats but there was no evidence of developmental toxicity or teratogenic effects and there was a small effect on sperm morphology. After exposure to isoprene, there was no adverse effect on rat dams or other reproductive indices. In mice, there was reduced foetal body weight and decreased maternal weight gain and isoprene also affected ovarian follicles. There was a reduction in testicular function parameters such as testicular weight and sperm motility.

Germ cell mutagenicity of phthalic acid in mice

Mutagenicity of phthalic acid was evaluated by employing dominant lethal mutation and sperm head abnormality assays in male Swiss albino mice. For the dominant lethal mutation assay, adult male mice received a single intraperitoneal (i.p.) injection of either 40 mg or 80 mg/kg b.w. of phthalic acid for 5 consecutive days. For the sperm head abnormality assay, the mice were treated with 50, 100, 150, 200 and 300 mg/kg b.w. as a single i.p. injection. Treatment of adult male mice with phthalic acid resulted in induction of dominant lethal mutations and abnormal sperm heads. The results obtained indicate that phthalic acid is a germ cell mutagen.

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 parallelogram approach of Sobels was employed. The presented estimates of heritable damage in man as a consequence of 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.

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.