Genetic toxicology of 1,2-dibromo-3-chloropropane (DBCP)

1,2-Dibromo-3-chloropropane (DBCP) is a nematocide, which has been used extensively as a soil fumigant in agriculture. Since sterility was found among male workers involved in the manufacture of DBCP, great concern has been focused on the genetic hazards of DBCP. DBCP gave positive results in many tests such as microbial, in vitro cytogenetics, and Drosophila studies. In mammalian test systems, DBCP caused chromosomal aberrations in the bone marrow cells and dominant-lethal mutations in germ cells in rats. In mice, there were no signs of DBCP-induced heritable mutation in germ cells, although point mutations were detected in somatic cells. The occurrence of Y-chromosomal non-disjunction was indicated in DBCP-exposed male workers by an increased number of sperm containing 2 Y-chromosomes.     

Studies on the dominant-lethal and fertility effects of the heavy metal compounds methylmercuric hydroxide, mercuric chloride, and cadmium chloride in male and female mice

Dominant-lethal effects of 10 mg/kg methylmercuric hydroxide were studied in male mice from two hybrid stocks and in females from one of these stocks. Two other compounds, mercuric chloride (2 mg/kg) and cadmium chloride (2 mg/kg), were studied only in females for dominant-lethal (in one hybrid stock) and reproductive capacity effects (in two hybrid and one mixed stocks). All compounds were administered in a single intraperitoneal injection. When males of one of the two stocks studied were treated with methylmercuric hydroxide, the females to which they were mated exhibited a slight reduction in the total number of implantations and in the number of living embryos. These reductions were accompanied by a very small increase in the incidence of dead implantations. In females, cadmium chloride had no detectable dominant-lethal or other fertility effects, except superovulation. On the other hand, the two mercury compounds slightly reduced the numbers of implants and living embryos in females subjected to dominant-lethal studies. The two mercury compounds also induced a slight reduction in the long-term reproductive performance of one stock of females. These results and those reported earlier by others, indicate that the mercury compounds studied so far are not potent inducers of dominant-lethal mutations in male and female mice. It is not clear whether the small effects on male or female fertility induced in some cases, particularly the increase in dead implantations and reductions in the number of living embryos, were attributable to dominant-lethal mutations or to nongenetic causes.     

Lack of association between induction of dominant-lethal mutations and induction of heritable translocations with benzo[a]pyrene in postmeiotic germ cells of male mice

Benzo[a]pyrene was tested for induction of dominant-lethal mutations in germ cells of male mice. Clear-cut dominant-lethal effects were induced in middle and early spermatoza. In contrast to the dominant-lethal effects observed the study showed no detectable increase in hertiable translocations for these stages over the spontaneous level. Thus, the results provide another example of a chemical mutagen that is effective in inducing dominant-lethal mutations but relatively ineffective in inducing heritable translocations in male postmeiotic germ cells.     

Female-specific mutagenic response of mice to hycanthone

Male and female gametogeneses differ markedly in all mammals. While male germ cells are continuously being produced from stem cells throughout the reproductive life span, the number of female germ cells is fixed during prenatal development and, soon after birth, all of the oocytes are arrested in a modified diplotene, or dictyate, stage. Following puberty, dictyate oocytes are hormonally triggered to mature either singly or in groups, resulting in ovulation and the completion of the first meiotic division. It has been hypothesized that female mice are more susceptible to dominant lethal effects of intercalating agents than male mice because oocyte chromosomes, which are arrested in a diffuse state, are generally more accessable to intercalation than are the more condensed chromosomes present within most male germ cell stages. This hypothesis was further tested using the intercalating agent hycanthone methane-sulfonate. Effects of hycanthone were studied in maturing and primordial oocytes and in male germ cells throughout spermatogenesis. No induction of dominant lethality was observed for treated males while a significant increase in embryonic death, expressed around the time of implantation, was observed in females that mated within 4.5 days after treatment. These effects were the result of dominant lethal mutations induced in maturing oocytes and not of maternal toxicity as indicated by the presence of chromosomal aberrations observed at first-cleavage metaphase of zygotes obtained from treated females. These results add support to the hypothesis that certain intercalating chemicals, which are not mutagenic to male mice, may be mutagenic to females and point to a need for more in-depth studies of female-specific mutagenesis.     

Difference in the Ratio of Dominant-Lethal Mutations to Heritable Translocations Produced in Mouse Spermatids and Fully Mature Sperm after Treatment with Triethylenemelamine (Tem)

The relative induction of dominant-lethal mutations and heritable translocations in triethylenemelamine-treated male postmeiotic germ cells of mice was determined depending on the stage treated. Males were mated either 11.5–14.5 days after treatment (middle spermatids) or less than 2.5 hours after treatment (fully mature sperm). Results clearly showed that, even though similar levels of dominant-lethal mutations were induced in fully mature sperm and in middle spermatids, the frequency of heritable translocations induced in mature sperm was markedly lower than that induced in middle spermatids. This observation was used, together with earlier ones, to suggest a mechanism by which dominant-lethal mutations and heritable translocations are produced following chemical treatment of male postmeiotic germ cels.     

Late onset of spermatogenesis and gain of fertility in POG-deficient mice indicate that POG is not necessary for the proliferation of spermatogonia

The germ cell-deficient (gcd) mouse mutation is a recessive, transgenic insertional mutation associated with the disruption of two Chr11 genes, Pog (proliferation of germ cells) and Vrk2 (vaccinia virus-related protein kinase 2). We have recently shown that like gcd/gcd mice, targeted Pog-/- males and females show virtually no spermatogenesis or oogenesis at 4-6 wk of age. Because Pog is deleted in gcd/gcd and Pog-/- mice, a comparison of the phenotypes of the two mouse models is appropriate. Here, we report that unlike in POG-deficient females, the germ cells in POG-deficient males eventually populate the seminiferous tubules at 9 wk, and fertility can be achieved by 12 wk. Homozygous gcd/gcd males did not show a similar degree of germ cell population, and most gcd/gcd males remained infertile at 16 and 22 wk of age. A comparison of the degree of germ cell deficiency at 13.5 days postcoitum and 1 day postpartum between Pog-/- and gcd/gcd males revealed that gcd/gcd males had far fewer germ cells than Pog-/- males at both time points. Our data suggest that Pog is essential for proper primordial germ cell proliferation in the embryonic stage but is not needed for spermatogonial proliferation after birth. Thus, the difference in the spermatogenetic potential in adult Pog-/- and gcd/gcd mice may result from the severity of germ cell deficiency rather than from the inability of gcd/gcd spermatogonia to proliferate efficiently. The greater deficiency of germ cells before the onset of spermatogenesis seen in gcd/gcd males compared to Pog-/- mice suggests either that the different background affects the outcome of Pog deletion or that Vrk2 has additional effects on germ cell development.     

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

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

Bleomycin: female-specific dominant lethal effects in mice.

Limited comparative data in mice indicate that chemical mutagens that induce dominant lethal mutations in males are not necessarily effective in females, but those which are effective in females are generally equally or more effective in males. Recently, however, a few chemicals have been identified that are female-specific with respect to induction of dominant lethal mutations. The antitumor antibiotic adriamycin is among them. Another antitumor antibiotic, bleomycin was examined for its ability to induce dominant lethal mutations in the reproductive cells of male and female mice. No dominant lethal or cytotoxic effects were observed in males treated with bleomycin, even at a maximum tolerated dose. In females, on the other hand, a dose nearly 1/4 of that used in males induced not only a high level of dominant lethal mutations but also killed oocytes in certain stages of follicular development. The effectiveness of bleomycin in inducing dominant lethal mutations in mouse oocytes makes it a valuable tool for investigating whether gonadal transport, inherent differences in the configuration of chromatin in the germ cells of the two sexes or other factors are responsible for the differential susceptibility to bleomycin, which implies potential gender-specific genetic risk in cancer chemotherapy.     

Studies on chemically induced dominant lethality. II. Cytogenetic studies of MMS-induced dominant lethality in maturing dictyate mouse oocytes.

Young adult female mice were injected intravenously with either 50- or 100- mg/kg doses of methyl methanesulfonate. The females were superovulated and mated to untreated males at intervals ranging from 0.5 to 14.5 days after treatment. The fertilized ova were collected and cultured to the first cleavage mitosis, at which time the female chromosome complement was analyzed for structural chromosomal damage. Chromatid-type aberrations were observed, but at a much lower frequency than previously reported for treatment of post-meiotic male germ cells. The time after treatment at which chromosomal damage was observed and the frequency of affected cells agree, qualitatively, with existing dominant-lethal data derived from treatment of maturing oocytes. Parallel experiments in which metaphase I oocytes were analyzed indicate a lack of MMS-induced chromosomal damage in the meiotic stages. This observation is consistent with the suggestion that an intervening round of DNA synthesis is necessary for MMS-induced lesions to be translated into chromosomal damage. The low yield of chromosomal damage is consistent with the idea that maturing oocytes, unlike later spermatids and spermatozoa, are capable of performing macromolecular repair of premutational lesions.     

Effects of 1,2-dibromo-3-chloropropane on male reproductive function in the rat

The pesticide 1,2-dibromo-3-chloropropane (DBCP) is a known male reproductive toxin. Previous studies employed production-grade DBCP containing allyl chloride and epichlorohydrin, both capable of producing effects similar to DBCP. The purpose of this study was to determine if purified DBCP caused the same effects as DBCP containing allyl chloride. Rats were injected for 6 mo with varying doses of pure or production-grade DBCP. Very few differences were observed in the parameters measured between pure and production-grade DBCP-treated animals at any one dose level. Treatment with 25 mg/kg DBCP, pure or production grade, reduced body weight as well as the weight of the testes, prostate glands and seminal vesicles, and elevated serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This dose of pure DBCP reduced serum testosterone. Treatment with 5 mg/kg of either grade DBCP caused decreases only in body and testis weights. Animals treated with 1 mg/kg did not show any major differences from controls. These results indicate that DBCP, and probably not a contaminant, is responsible for the effects observed on male reproduction. Furthermore, DBCP appears to affect either androgen action or production since multiple androgenic indices are affected by DBCP administration.     

6-Mercaptopurine,an inducer of cytogenetic and dominant-lethal effects in premeiotic and early meiotic germ cells of male mice

Dominant-lethal effects of 6-mercaptopurine on male mice were studied using eight doses, ranging from 150 to 482 mg/kg. Effects of the 150-mg/kg dose were studied over the entire spermatogenic cycle, and those of the higher doses for matings made between days 28.5 and 41.5 after treatment. It was found that, with low doses, there was only one period in which clearcut increases in induced dominant-lethal mutations were detected, namely in matings that occurred 32.5 to 35.5 days after treatment. With higher doses, effects could be detected beyond that period through day 39.5. Spermatozoa utilized for matings during the period of greatest response were presumably derived from germ cells that were in late differentiating spermatogonial and early meiotic spermatocyte stages at the time of treatment. These results are similar to those of Ray and Hyneck. To date, 6-mercaptopurine is unique in inducing dominant lethality only at these particular stages.A study of chromatid aberration induction in the treated males themselves was carried out for 150 and 250 mg/kg doses of 6-mercaptopurine over the period of 9 to 16 days after treatment. A considerable increase in isochromatid and chromatid deletions was observed in diakinesis-metaphase-I spermatocytes on days 14 and 15 after treatment. For reasons discussed, the cells sampled at this time may be assumed to have been in early meiosis (preleptotene), with some in late differentiating spermatogonial stages, at the time of treatment. The rough agreement in sensitive cell type for dominant lethality and chromatid aberration induction suggests that chromatid deletions are the cause of dominant lethality in this study. Conservative estimates of the frequency of dominant lethality expected from the chromatid aberration frequencies tend to substantiate this suggestion.     

Absence of noxious effects of selected neuroleptics in dominant-lethal mutagenesis assay.

In a dominant-lethal assay in mice the following tricyclic neuroleptics were tested: prothiaden, imipramine, oxyprothepin decanoate and docloxythepin. No dominant-lethal effect was induced by these neuroleptics, even when administered at doses many times as great as clinical doses. The reduced percentages of pregnancies, in females who had copulated with males receiving docloxythepin, observed during and immediately after its administration, were directly connected with marked sedation induced in the males by this neuroleptic.     

Exposure to a novel male during late pregnancy influences subsequent growth of offspring during lactation

In mammals, allocation to reproduction can either be primed or suppressed in relation to cues from other individuals. Some conspecifics (e.g. potential mates) may enhance an individual's ability to reproduce but others may have a detrimental effect on reproductive success. One widely studied response to conspecific cues, the ‘Bruce effect’, occurs when pregnant females abort their pregnancies after exposure to a novel male. It has been suggested that this response has evolved as a counter‐tactic to the threat of infanticide posed by novel males. In some species, like mice, pregnancy termination will only occur if females are exposed to the unfamiliar male during a brief critical period early in pregnancy, which is surprising considering that an unfamiliar male threatens infanticide whenever present, and in particular near to birth. We demonstrate that female mice experiencing novel males during late pregnancy also alter their investment in progeny, but in a more subtle manner than previously observed. Females exposed to an unfamiliar male during late pregnancy give birth to offspring of a comparable weight to those produced by females exposed to the paternal male, but these offspring grow more slowly over lactation. As a consequence, offspring from these females weigh less at weaning. Modification of their growth trajectory, however, allows these offspring to catch up to normal weights by adulthood. Thus, cues of unfamiliar males, and possibly their associated threat of infanticide, can produce more wide‐ranging effects on maternal investment than previously recognized.     

The proto-oncogene Ret is required for male foetal germ cell survival

The spermatogenic and oogenic lineages originate from bipotential primordial germ cells in response to signalling in the foetal testis or ovary, respectively. The signals required for male germ cell commitment and their entry into mitotic arrest remain largely unknown. Recent data show that the ligand GDNF is up regulated in the foetal testis indicating that it may be involved in male germ cell development. In this study genetic analysis of GDNF-RET signalling shows that RET is required for germ cell survival. Affected germ cells in Ret-/- mice lose expression of key germ cell markers, abnormally express cell cycle markers and undergo apoptosis. Surprisingly, a similar phenotype was not detected in Gdnf-/- mice indicating that either redundancy with a Gdnf related gene might compensate for its loss, or that RET operates in a GDNF independent manner in mouse foetal germ cells. Either way, this study identifies the proto-oncogene RET as a novel component of the foetal male germ cell development pathway.     

Induction of specific-locus and dominant-lethal mutations in male mice by diethyl sulfate (DES)

Diethyl sulfate (DES), a monofunctional alkylating agent, induces mutations and chromosomal aberrations in many different organisms and cell systems, including dominant-lethal mutations in male mice. However, until now it could not be demonstrated that DES induces specific-locus mutations in mice. This observation would contradict the close correlation observed between the induction of dominant-lethal mutations and specific-locus mutations in mice with other chemicals. DES induces dominant-lethal and specific-locus mutations in spermatozoa and late spermatids of mice. The mutation frequency for dominant-lethal mutations is dose-dependent, while for specific-locus mutations it is independent of the dose. In the mating interval 5-8 days post-treatment the mutation frequency for 200 mg/kg DES is 17.0 X 10(-5) and for 300 mg/kg 7.5 X 10(-5) mutations per locus. The dose-dependent increase of dominant-lethal mutations probably reduced the chance of recovering specific-locus mutations. The importance of these findings for mutagenicity testing is discussed.     

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.     

Congenital defects in the offspring of male mice treated with ethylnitrosourea

Daily doses of ENU (25-100 mg/kg) were injected intraperitoneally into ICR strain male mice for 5 days. The males were mated to untreated virgin females of the same strain on days 1-16 and 64-80 after the last dose. Copulations during these periods involve, respectively, treated postmeiotic cells and spermatogonial stem cells. The uterine contents were examined on day 18 of pregnancy for evidence of dominant lethal effects. The fetuses were examined for external and skeletal abnormalities. ENU treatment of either postmeiotic cells or spermatogonial stem cells caused dose-dependent significant increases in the incidence of abnormal fetuses over the control level. The induction rate per live fetus per unit dose in mg/kg by treating spermatogonial stem cells was estimated to be 1.0 X 10(-4), which is 3-fold lower than the rate previously estimated for the same endpoint at the same germ cell stage with MNU. Cleft palate was the most frequent external abnormality in the ENU-treated and the control series. Malformed vertebrae was the most frequent skeletal abnormality in the treated series. Rib fusion was the only skeletal malformation seen in the control series. Dominant lethals were clearly induced when germ cells were treated as postmeiotic cells.     

Biological effect of low doses of alkylating agents in drosophila studies

The low dose (0.05-0.1 mM) influence of alkylating agents on germ cell survival and male fertility, the level of embryonic and postembryonic lethality as well as the sex-linked recessive lethal (SLRL) frequency induced by high alkylating agent doses was studied in Drosophila melanogaster. The pretreatment of adult males with low doses of methyl and ethyl methanesulfonate (MMS and EMS) did not change or even enhanced EMS cytotoxicity and mutagenicity in both mature sperm and premeiotic cells. On the contrary, the low EMS dose pretreatment of larvae protected them against higher mutagen doses increasing male fertility, decreasing embryonic and postembryonic lethality in F1, and leading to three-fold reduction in the SLRL frequency in F2. The adaptive response was dependent on the Drosophila developmental stage exposed to challenge mutagen doses, since the protection was maximal in larvae and practically absent when the high dose was administered to adult males. The adaptive response observed does not seem to be associated with DNA repair, but it is rather due to other protective mechanisms.     

幼小鼠对种内几种化学信息的识别和反应

50年代以来,国际上相继开展了一些关于化学信息在小家鼠繁殖中作用的研究,并在实验室中揭示了一些反应:如集群雌鼠气味使雌鼠性周期延长的Lee-Boot效应,雌鼠气味能解除集群雌鼠对性周期的抑制、并促使它们同期发情的Whitten效应,以及陌生雄鼠气味中断妊娠的Bruce效应等等。但是,在成鼠间起作用的几种化学信息,是否能被幼鼠感受,对幼鼠的发育和性成熟是否产生影响,还是一个未知的问题。 影响小鼠发育的因素很多,除环境因子外,近来还出现了有关社群因素中外激素对小鼠性成熟影响的报道,发现雄鼠或给予雄激素的雌鼠有促进幼雌鼠性成熟的作用,范志勤发现父本气味和陌生雄鼠气味对幼雌鼠性成熟均有促进作用。但是,目前关于种内雌鼠气味、集群雌鼠气味、幼鼠气味影响性成熟的研究尚不多见。有关各种气味对性成熟效应的比较的报道亦少。本文的目的在于通过实验方法,阐明种内的几种化学信息,诸如父本、陌生雄性、雌性、集群雌性、幼雌等几种气味,对幼雌小鼠性成熟的影响,并据此了解幼鼠识别这几种信息的能力。