Responses to genotoxicity in mouse testicular germ cells and epididymal spermatozoa are affected by increased age.

The increased number of cell divisions undergone by spermatogonia of older fathers cannot fully account for the observed increase in germline genetic damage. Studies have shown that the mechanisms induced in germ cells in response to oxidative damage varies with age, that DNA repair efficiency declines, and both sperm DNA damage and spontaneous mutations increase. However, it is not known whether the altered response with age is a cause, or consequence, of an age-associated change in cell susceptibility to genetic damage. Following a single 150 mg/kg dose of cyclophosphamide (CP), young (8-weeks old) and aged (17-month old) male mice were examined 24 h later for induced genetic damage in epididymal spermatozoa using the alkaline comet and sperm chromatin stability assays. Apoptosis among testicular cells was examined on tissue cross-sections using the TUNEL assay. Sperm showed no significant increase in DNA strand breaks with age (detected by the comet assay) and no change in sperm chromatin stability (detected by the SCSA assay). Following CP treatment, there was no effect on DNA-strand breakage but sperm chromatin instability was significantly higher. Furthermore, it was also significantly elevated in old treated, compared with young treated, animals suggesting that increased age affects the sensitivity of epididymal sperm to chromatin damage. There was no difference in apoptosis in testicular germ cells from either young or old control animals, while CP administration resulted in a significant increase in apoptosis among young animals but not old animals. Following genotoxin exposure, an increase in chromatin instability in the spermatozoa of old animals and a decrease in the ability of their testicular germ cells undergo apoptosis suggests an age-related decrease in genome protection mechanisms. Since those germ cells are only transiently present in the testis, it is likely that this age-related deterioration originates in the spermatogonial stem cells. The findings are also evidence that the safety evaluation of reproductive genotoxins should consider young and old individuals separately.

Detection of structural and numerical chromosomal abnormalities by ACM-FISH analysis in sperm of oligozoospermic infertility patients.

BACKGROUND: Modern reproductive technologies are enabling the treatment of infertile men with severe disturbances of spermatogenesis. The possibility of elevated frequencies of genetically and chromosomally defective sperm has become an issue of concern with the increased usage of ICSI, which can enable men with severely impaired sperm production to father children. Several papers have been published reporting aneuploidy in oligozoospermic patients, but relatively little is known about chromosome structural aberrations in the sperm of these patients. METHODS: We examined sperm from infertile, oligozoospermic individuals for structural and numerical chromosomal abnormalities using a multicolour ACM fluorescence in situ hybridization (FISH) assay that utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical chromosomal abnormalities plus two categories of structural aberrations: duplications and deletions of 1pter and 1cen, and chromosomal breaks within the 1cen-1q12 region. RESULTS: There was a significant increase in the average frequencies of sperm with duplications and deletions in the infertility patients compared with the healthy concurrent controls. There was also a significantly elevated level of breaks within the 1cen-1q12 region. There was no evidence for an increase in chromosome 1 disomy, or in diploidy. CONCLUSIONS: Our data reveal that oligozoospermia is associated with chromosomal structural abnormalities, suggesting that oligozoospermic men carry a higher burden of transmissible, chromosome damage. The findings raise the possibility of elevated levels of transmissible chromosomal defects following ICSI treatment.

Effect of age on testicular germ cell apoptosis and sperm aneuploidy in MF-1 mice.

The spontaneous mutation rate in the male germ-line increases with age. The reason for this is unknown, but presumably involves an age-related degeneration in the efficacy of cellular processes. To investigate the possibility that rates of apoptosis and genetic damage (represented by aneuploidy) might vary with age in mice, the testes and sperm of 2- and 12-month-old male MF-1 mice were examined by a modified TUNEL technique and 3-colour sperm-FISH assay, respectively. Sperm were labeled with probes to chromosomes 8, X and Y and 20,000 sperm scored from each of 5 animals per group. A significant increase in gonosomal disomy was found in the aged mice, especially X-X-8. This suggests that advanced paternal age is associated primarily with meiosis II rather than meiosis I disjunction errors. Neither diploidy nor autosomal disomy was affected in the older group. The rate of germ cell apoptosis (apoptotic cells per seminiferous tubule cross-section per animal per group) was higher in the old mice than controls, but not significantly. Considerable inter-animal variability was observed in the older group. The finding of an increase in levels of sperm aneuploidy is novel for 1-year-old mice and confirms the genotoxic effect of ageing in mice. Since apoptosis is assumed to eliminate cells with unrepaired damage, it may be that the apoptotic response in older mice is compromised, resulting in the higher levels of aneuploidy in sperm. However, given the inter-animal variability in testicular germ cell apoptosis, this awaits confirmation.

Genetic damage in oligozoospermic patients detected by fluorescence in-situ hybridization, inverse restriction site mutation assay, sperm chromatin structure assay and the Comet assay.

BACKGROUND: The possibility that oligozoospermic men may have elevated levels of genetic damage in their sperm is of particular concern as they could transmit defects to their offspring. METHODS: Sperm samples were obtained from 12 infertile, oligozoospermic patients and 12 healthy normozoospermic volunteers. Fluorescence in-situ hybridization (FISH) was used to determine aneuploidy rates in sperm and inverse restriction site mutation (iRSM) assay to determine gene mutations; defective chromatin packaging was quantified by sperm chromatin structure assay (SCSA) and DNA strand breaks by the Comet assay. RESULTS: FISH analysis showed a significant increase in gonosomal X,Y,18 (P < 0.01) disomy and diploid sperm with X,Y,18,18 (P < 0.05) in the infertility patients compared with the controls. A significant increase (P < 0.01) in disturbed sperm chromatin was found in the infertility patients compared with the control group using the SCSA assay. In the Comet assay, a significant increase (P < 0.01) in the tail moment was found in the infertility patients compared with the control group, indicating significantly high levels of DNA strand breaks. There was no significant increase in point mutations detected by iRSM assay. CONCLUSIONS: The data indicate that infertile oligozoospermic men have an elevated level of XY aneuploidy and XY diploidy in the germ-line, as well as elevated levels of sperm chromatin disturbances and sperm DNA strand breaks. These data demonstrate that oligozoospermic infertility patients show several different types of genetic damage in their sperm. Thus, such men appear to have defects at a variety of levels of spermatogenesis and their infertility may not just be a result of the oligozoospermia.

Quantitative analysis of spermatogenesis and apoptosis in the common marmoset (Callithrix jacchus) reveals high rates of spermatogonial turnover and high spermatogenic efficiency.

Spermatogenesis is characterized by the succession in time and space of specific germ cell associations (stages). There can be a single stage (e.g., rodents and some macaques) or more than one stage (e.g., chimpanzee and human) per tubular cross section. We analyzed the organization of the seminiferous epithelium and quantified testicular germ cell production and apoptosis in a New World primate, the common marmoset (Callithrix jacchus). Tubule cross sections contained more than one stage, and the human six-stage system could be applied to marmoset spermatogenesis. Stereological (optical disector) analysis (n = 5) revealed high spermatogenic efficiency during meiosis and no loss of spermatids during spermiogenesis. The conversion of type A to type B spermatogonia was several-fold higher than that reported for other primates. Highest apoptotic rates were found for S-phase cells (20%) and 4C cells (15%) by flow cytometric analysis (n = 6 animals); histological analysis confirmed spermatogonial apoptosis. Haploid germ cell apoptosis was <2%. Marmoset spermatogenesis is very efficient and involves substantial spermatogonial proliferation. The prime determinants of germ cell production in primates appear to be proliferation and survival of spermatogonia rather than the efficiency of meiotic divisions. Based on the organizational similarities, common marmosets could provide a new animal model for experimental studies of human spermatogenesis.

Paternal transmission of genetic damage: findings in animals and humans.

The concept that mutations can be induced in the male germ-line and result in adverse effects in the offspring has achieved only limited acceptance despite considerable theoretical appeal. This is partly because fetal malformations are generally perceived to be induced solely as a result of maternally mediated events during gestation and partly because the low incidence of the end-points concerned make experimental approaches costly and time-consuming. Nonetheless, a substantial body of work relating to the hypothesis has accumulated in the last 20 years, which has never been reviewed in its entirety. A consideration of the available evidence indicates that preconceptional paternal exposure to mutagens (particularly radiation, cyclophosphamide and ethylnitrosourea) can indeed, under certain conditions, have adverse effects on offspring. The results suggest two principal mechanisms by which such effects may be induced: the induction of germ-line genomic instability or the suppression of germ cell apoptosis.

Association of cyclophosphamide-induced male-mediated, foetal abnormalities with reduced paternal germ-cell apoptosis.

To investigate the mechanism by which malformed offspring can result from the exposure of males to mutagens, we treated adult male rats with 0, 1.4, 3.4 or 5.1 mg/kg cyclophosphamide, 6 days per week for 9 weeks, a treatment regimen known to induce heritable abnormalities. Testis samples from some of the animals were then collected for fixation in Carnoy's fluid and subsequent analysis of germ-cell apoptosis and proliferation. The remainder were mated, resulting in a greater than 11-fold increase in the proportion of abnormal offspring produced in the 5.1 mg/kg group. The number of apoptotic cells per stage XII/XIII tubular cross-section decreased with increasing dose, significantly so at 5.1 mg/kg (P<0.05). No statistically significant effect was found on spermatocyte numbers at this dose, indicating that a reduction in the amount of cells available to undergo apoptosis cannot explain the decrease. The inappropriate survival of damaged germ-cells caused by a lowering of the incidence of apoptosis may, therefore, account for the rise in the proportion of foetal malformations.

Examination of ras oncoproteins in human plasma from healthy controls and workers exposed to petroleum emissions, including benzene-related compounds.

Ras oncoproteins in blood plasma from workers exposed to petroleum emissions and unexposed controls were examined from Polish and Estonian samples. Twenty-four workers and 35 unexposed controls were examined from Poland and 97 exposed and 40 unexposed controls from Estonia. Of the Estonian workers, 50 were exposed to benzene in a benzene production plant and 47 to polyaromatic hydrocarbons and benzene in a cokery. Blood plasma proteins were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence using a monoclonal antibody as the primary antibody. There were no statistically significant differences between the exposed and the control groups in either the Polish or the Estonian samples.

Direct analysis by small-pool PCR of MS205 minisatellite mutation rates in sperm after mutagenic therapies.

We have used small-pool PCR to analyse mutation in samples of sperm taken from men after mutagenic therapy. Small-pool PCR uses direct analysis of germline DNA at a highly unstable tandem-repeated "minisatellite" locus to measure rates of length-change mutation in individual sperm samples. The advantages of this approach are that the normal mutation rate is extremely high (about 0.4% per gamete at the locus analysed here), so that relatively small increases in mutation rate can be detectable in individual samples. It is known from work on sperm from untreated individuals that different alleles at this locus have different mutation rates. For this reason, we have analysed the germline mutation rates in sperm samples from two men, in each case comparing a post-treatment sample with a pre-treatment sample from the same individual. We find no evidence for altered mutation in the post-treatment sample, suggesting that the repopulation of the germ-cell compartment after treatment may be subject to stringent mechanisms for the detection and elimination of germ-cell damage.

An investigation of male-mediated F1 effects in mice treated acutely and sub-chronically with urethane.

In order to investigate the alleged potential of paternally administered urethane to cause foetal abnormalities and heritable tumours, male CD-1 mice were treated with urethane, either acutely by intraperitoneal injection at doses of 1.25 and 1.75 g/kg bodyweight (bwt) or sub-chronically in the drinking water at 1.25 for 10 weeks, and 3.75 mg/ml for 9 weeks or vehicle for the control groups. They were mated to untreated females 1 week later. Uterine contents of half the pregnant females were examined just before full term, while the remaining females were allowed to deliver their litters. The resulting F1 offspring were observed for approximately 18 months and 12 months for acute and sub-chronic exposures respectively and subjected to necropsy examination. Some of the mice treated acutely with 1.75 g/kg bwt exhibited partial infertility but none of those treated with 1.25 g/kg bwt had an adverse effect on their reproductive ability. There was no genetic effect of acute urethane treatment on male germ cells as indicated by dominant lethality. After birth, there was an increase (P < 0.05) in post-implantation deaths possibly due to perinatal mortality. There was an increased incidence and earlier onset of liver tumours induced in F1 male offspring from F0 males treated with 1.75 g/kg bwt, (20.7% vs. 10.1%, P = 0.026) but not in the female offspring. F1 males from both treatment groups had mean bodyweights significantly higher than controls (P < 0.01). Some males from each dose group of the acute study were examined using the restriction site mutation assay involving analysis of exon sequences. No mutations were identified in testes, liver or spleen of DNA isolated from the urethane-treated animals. No reproductive or genetic effects were seen with sub-chronic treatment at either 1.25 or 3.75 mg/ml urethane nor was there any predisposition of F1 animals to tumours although observation times were shorter.

Genetic effects of 1,3-butadiene on the mouse testis.

1,3-Butadiene is a known male mouse germ-cell mutagen, to which humans may either be occupationally or environmentally exposed. Prolonged exposure to moderate or high doses in male mice can cause dominant lethal mutations and one report has indicated that 10 week inhalation administration of low doses can result in the production of malformed foetuses. The present study had dual purposes: (a) to attempt to clarify the suspected ability of sub-chronic (6 h/day, 5 days/wk, 10 weeks) low-dose exposure to 1,3-butadiene to induce heritable mutations in mouse male germ cells: (b) investigation of the relationships between testicular DNA damage, testicular DNA repair and foetal outcome. Adult male mice were exposed to low or moderate doses of 1,3-butadiene by inhalation sub-chronically or for a single 6 h period and either used for mating (sub-chronic exposure only) or for studies of DNA damage and repair. Litter size, dominant lethality and numbers of abnormal foetuses were determined the day preceding the normal day of parturition. Testicular DNA damage and repair were assessed by the Comet assay (for DNA damage) and the unscheduled DNA synthesis assay (for DNA repair). 1,3-Butadiene caused a statistically significant increase in dominant lethality at 125 ppm but not 12.5 ppm. No significant increase in DNA repair was found with either dose level or exposure period while only 6 h exposure to 125 ppm caused a small but significant increase in DNA damage as detected by the Comet assay. These effects demonstrate the reproductive genotoxicity of (125 ppm) 1,3-butadiene but do not confirm its ability to cause abnormalities in the offspring via the sperm. It is suggested that the relationship between 1,3-butadiene-induced DNA damage, DNA repair and heritable defects in the offspring may depend on the pattern of metabolites produced.

A comparison of male-mediated effects in rats and mice exposed to 1,3-butadiene.

There is current concern that exposure of men to certain agents such as radiation and smoking can adversely affect their offspring in terms of cancer outcome. Studies in laboratory animals after radiation have supported such an association, and other studies after male exposure to radiation and various chemicals have also resulted in congenital malformations. The present study was undertaken to examine congenital malformations in offspring from males exposed to 1,3-butadiene over a lower dose range than that in an earlier mouse study and to determine if there was a species difference in sensitivity between rats and mice. An earlier extended dominant lethal study of male CD-1 mice exposed by inhalation to 12.5 ppm and 1250 ppm of 1,3-butadiene for 6 h/day, 5 days/wk, for 10 weeks produced an increase in F1 abnormalities and late deaths at 12.5 ppm and in early deaths at 1250 ppm. The present study examined the same reproductive effects after exposure of male CD-1 mice for 6 h/day, 5 days/wk, for 4 weeks to 12.5, 65 and 130 ppm of 1,3-butadiene. There was no increase in early deaths at 12.5 ppm as in the earlier study but there were statistically significant increases in early deaths at 65 and 130 ppm study and these were not dose-related. There was a non-significant increase in F1 gross abnormalities at 130 ppm and no increase in late deaths. The present study also examined male Sprague-Dawley rats after exposure to 65,400 and 1250 ppm for 6 h/day, 5 days/wk, for 10 weeks. There were no effects on early deaths, late deaths, or congenital malformations in the rat study. There was a reduction in implants at 65 ppm but this was not considered to be biologically/genetically significant as there was no corresponding increase in early deaths and the response was not dose-related. The differences observed between the rat and mouse studies would confirm the greater sensitivity to 1,3-butadiene of the mouse by comparison with the rat as reported by other workers for other parameters.

Modulation of ras p21 oncoprotein levels and DNA strand breakage in human cells with chemotherapeutic agents and/or deferoxamine.

Oncogenes are involved with the regulation of cellular proliferation. Ras oncogenes can be activated by chemical treatment and any increased activity could be modulated by further chemical treatment. In the present study, therefore, ras p21 protein expression was examined in in vitro cultures of human lymphocytes treated with mitomycin C and in the human colon adenocarcinoma Caco-2 cell line treated with doxorubicin with and without deferoxamine. Both chemotherapeutic agents act partially through oxygen radical mechanisms. Increases in p21 protein levels were seen with mitomycin C but no clear response was seen with doxorubicin. However, deferoxamine, with and without doxorubicin, altered p21 expression. Deferoxamine is an iron chelator so these results support the hypothesis that oxygen radicals were responsible for the altered p21 protein levels. Modulating responses were confirmed by measuring DNA strand-breakage in the Comet assay after treatment with doxorubicin and deferoxamine. Alterations of ras p21 protein expression in vitro might prove a suitable system for examining modulating effects on chemical carcinogens.

Apoptosis as a mechanism of germ cell loss in elderly men.

In comparison with other mammals, human spermatogenesis is known to be an inefficient process, of which germ cell degeneration is a normal part. This study was performed to determine the mechanism of cell death in the testes of elderly men. Testes from 20 patients undergoing orchidectomy for prostate cancer were fixed, sectioned and processed for the detection of apoptosis using the in situ end-labelling technique. In addition, the formation of DNA ladders, a hallmark of apoptosis, was also investigated. Occurrence of apoptosis was not confined to a particular germ cell population but comprised all types of germ cells. Sertoli cell apoptosis was not encountered. The numbers of degenerating germ cells were determined per standard reference area, but no significant relationship was found between the mean values and age or testis weight. Analysis of the median values for germ cell death per reference area suggested that apoptosis occurs in clusters within the testis but is a rare occurrence outside these areas. It is concluded that spontaneous apoptosis can mediate germ cell death in a variety of cell types in the aged human testis.

Somatic and germ cell effects in rats and mice after treatment with 1,3-butadiene and its metabolites, 1,2-epoxybutene and 1,2,3,4-diepoxybutane.

1,3-Butadiene is produced in large quantities for use in the manufacture of synthetic rubber. It is also an environmental pollutant. There is concern about exposure to 1,3-butadiene as it has been shown to produce tumours in rats, mice and an increased risk of leukaemia in humans. It has also been shown to produce germ cell effects in mice. Differences in responses to 1,3-butadiene have been reported in rats and mice, possibly due to different metabolic capabilities. The present study thus investigated somatic and germ cell effects of 1,3-butadiene in mice and its metabolites in both rats and mice to help determine species differences using different endpoints for genotoxic effects. These included DNA strand breakage as measured in the single cell gel electrophoresis (Comet assay) in bone marrow and testicular cells, and micronuclei in bone marrow cells using both the acridine orange and Giemsa staining methods. Unscheduled DNA synthesis (UDS) was also measured in the testes of mice. CD-1 mice were exposed to 1,3-butadiene by inhalation for 6 h/day for 4 weeks, and CD-1 mice and Sprague-Dawley rats to the metabolites after i.p. injection. 1,3-Butadiene did not affect liver, bone marrow and testicular cells in mice as measured in the Comet assay. After treatment with 1,2-epoxybutene in the Comet assay, there was a response in the testes in mice but not in rats and there was little or no effect in the bone marrow assay in mice but there was in rats. After treatment with 1,2,3,4-diepoxybutane in the Comet assay in mice, there was a response in the bone marrow cells but not in the testicular cells, and in rats there was also a response only in bone marrow cells. There was an increase in micronuclei in both rats and mice with both metabolites, but clastogenicity was stronger with 1,2,3,4-diepoxybutane, occurring at lower doses, than with 1,2-epoxybutene. In the UDS assay in the testes of mice, there was an increase in response with 1,2,3,4-diepoxybutane treatment but not with 1,2-epoxybutene. These studies would appear to confirm a species difference of CD-1 mice and Sprague-Dawley rats, where mice were sensitive at lower doses than rats.

cynDAZLA: a cynomolgus monkey homologue of the human autosomal DAZ gene.

A gene on the human Y chromosome, specifically deleted in azoospermic patients (DAZ: deleted in azoospermia), and a DAZ homologue (DAZH) on human chromosome 3, have been recently described. In the present work we report the isolation and characterization of the corresponding DAZH gene of the cynomolgus monkey (Macaca fascicularis), which we have named cynDAZLA (cynomolgus DAZ-like autosomal). Reverse transcription-polymerase chain reaction was used to amplify the monkey DAZ homologue, and sequence analysis revealed an open reading frame of 888 bp encoding 295 amino acids. Northern blot hybridization of different tissues to a probe derived from the cynDAZLA cDNA detected a transcript of 3.5 kb that, in the male, was expressed only in the testis. Comparison of the cynDAZLA sequence to autosomal DAZ homologues from human, mouse and Drosophila showed two RNA-recognition motifs (RRM) and the presence of only one DAZ consensus repeat compared with the seven repeats found in the human DAZ gene on the Y chromosome. The homology of the cynDAZLA cDNA compared with the human DAZH and mouse dazla cDNAs is 97.97 and 87.46% respectively. The identification of the monkey cynDAZLA enables further studies regarding the putative functions of DAZH, such as onset of expression and hormonal dependence of this gene.

Expression of mitotic cyclin B1 is not confined to proliferating cells in the rat testis.

Spermatogenesis is a precisely controlled and timed process comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, and the maturation and differentiation of haploid spermatids. Cell proliferation is controlled by genes involved in the regulation of the cell cycle. Among the principal regulatory proteins are cyclins, which are categorized according to their appearance during the cell cycle. B-type cyclins are mitotic cyclins and function at the G2/M transition of the cell cycle. We have investigated the expression and regulation of cyclin B1 during rat spermatogenesis. Rat cyclin B1 was isolated from a testis cDNA library and further used as a probe to detect mRNA expression. Northern blot hybridization of testis mRNA revealed the presence of a single 1.7-kilobase transcript. In situ hybridization showed stage-specific expression during spermatogenesis with highest expression found in late pachytene spermatocytes and early round spermatids. This pattern was confirmed in fractions of isolated germ cells. Immunocytochemistry displayed highest protein levels in round spermatids. Depletion of gonadotropins did not change the quantitative and qualitative expression pattern of cyclin B1. Therefore, the signals triggering the onset of cyclin B1 expression seem not to originate from the pituitary-gonadal endocrine axis and might therefore be paracrine factors originating within the germinal epithelium. Our observations suggest that cyclin B1 plays a hitherto unknown role in spermatid maturation in addition to its known function in dividing cells.

Male-mediated F1 effects in mice exposed to 1,3-butadiene.

We examined the effects on dominant lethality, the incidence of fetal abnormalities and tumour incidence in surviving offspring of acute and subchronic exposure of male mice by inhalation to the industrial monomer, 1,3-butadiene. In the acute study, CD-1 mice were exposed to atmospheres containing 0 (n = 25), 1250 (n = 25) or 6250 ppm (n = 50) for 6 h, and each male was caged 5 days later for 1 week with two untreated virgin females. One of the females was killed humanely on day 17 of gestation. The other was allowed to deliver and rear her litter and the litters were monitored throughout adulthood. The killed female was examined for the number of live foetuses, the number of post implantation deaths (early and late) and the number and type of any gross malformations. In the subchronic study, males were exposed to 0 (n = 25), 12.5 (n = 25) or 1250 (n = 50) for 6 h per day on 5 days per week for 10 weeks and then mated the next morning. Mating and observation details were as for the acute study. Acute exposure to butadiene resulted in only a small decrease in implantations; after 10 weeks' subchronic exposure with either the high or low concentration, however, a wide variety of statistically significant effects was seen. At 1250' ppm, the number of implantations was reduced, dominant lethal mutations were induced, and the incidences of early and late deaths were increased; some of the live foetuses were malformed. The low dose also increased the frequency of malformations and late deaths but it did not affect the number of early deaths. Skeletal examination of malformed foetuses, randomly selected normal litter mates and controls confirmed the abnormalities seen at necropsy in malformed foetuses. However, karyotypic analysis of foetal liver from malformed foetuses, randomly selected normal litter mates and controls showed no karyotypic abnormalities. The number of gross suspected tumours in the F1 adults did not appear to reveal an increase over control values. Thus, butadiene is mutagenic in the germ cells of male mice, as shown by the induction of dominant lethality at 1250 ppm, and the frequencies of late deaths and congenital malformations appear to be increased at the subchronic level of 12.5 ppm and skeletal examination of malformed foetuses confirmed the macroscopic abnormalities.