Estrous cyclicity and ovarian follicles in female rats after prenatal exposure to 3,3',4,4',5-pentachlorobiphenyl

Alterations of estrous cyclicity and ovarian follicles following prenatal exposure to PCB126 were examined. Female SD rats were given (i.g.) 25 pg, 2.5, 250 ng and 7.5 microg of PCB126/kg or the vehicle on days 13-19 postconception. Vaginal opening (VO) in the 250 ng and 7.5 microg offspring was significantly delayed. All groups showed irregular estrous cyclicity following VO, but it became normal after a few days. However, the start of normal estrous cyclicity following VO in the 2.5, 250 ng and 7.5 microg groups was significantly delayed. At 30 and 50 days old, the 2.5, 250 ng and 7.5 microg groups showed significantly fewer antral follicles and a higher number of atretic follicles. The 7.5 microg group at 50 days old revealed significantly fewer corpus luteums. In 50-day-old offspring, the 2.5, 250 ng and 7.5 microg groups showed a significant reduction in serum 17beta-estradiol and progesterone levels and significantly higher levels of PCB126 in the fatty tissue compared with the vehicle group. Thus, while the prenatal dose of PCB126 used in this study did not induce malformation of the external genitalia or persistent ovarian disruption, disruption of ovarian function at puberty was found in the 2.5 ng group of pups born to dams exposed to 17.5 ng/kg PCB126. The present study suggests that PCB126, at least in part, exerted direct effects on the ovary as shown by the disruption of estrous cyclicity.     

Mammary gland differentiation in female rats after prenatal exposure to 3,3',4,4',5-pentachlorobiphenyl

Recently we reported finding that prenatal exposure to a relatively low dose of 3,3',4,4',5-pentachlorobiphenyl (PCB126) increases the rate of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma, while a high dose decreases it. One of the most important factors determining sensitivity of the mammary gland to neoplastic stimuli is its stage of differentiation at the time of exposure to the carcinogenic agent. Hence, to verify a biphasic dose-response relationship (enhancement of carcinogenesis at low dose, and inhibition at high dose), we investigated the effects of prenatal exposure to PCB126 on mammary gland differentiation. Female SD rats were injected (i.g.) with 25 pg, 2.5 ng, 250 ng, 7.5 microg of PCB126/kg, or the vehicle, on days 13-19 postconception. In 50-day-old offspring, regardless of the day of exposure to DMBA, only the 7.5 microg group showed statistically significant high levels of PCB126 in the fatty tissue of their mammary glands. Fifty-day-old female offspring of the 250 ng group showed apparent inhibition of the normal differentiation of terminal end buds (TEB) to alveolar buds and lobules (ABL), while those of the 7.5 microg group showed mammary gland hypoplasia. Expression levels of the estrogen receptor-alpha (ER) in TEBs and the ER mRNA in mammary glands were higher in the 7.5 microg, 250 ng, 2.5 ng groups. Proliferating cell nuclear antigen (PCNA) expression in TEBs of 50-day-old rats was statistically significantly higher in the 250 ng group and lower in the 7.5 microg group. In the developing mammary gland, TEBs are considered the most susceptible to mammary carcinogenesis, while ABLs are relatively protected from mammary carcinogenesis. Thus, prenatal exposure to a relatively low dose of PCB126 induced an alteration of mammary gland differentiation that might potentially increase the risk of DMBA-induced mammary carcinoma.     

Testicular spermiation failure in rats exposed prenatally to 3,3',4,4',5-pentachlorobiphenyl

Testicular spermatogenesis was studied in 7-, 10-, 13- and 17-week-old Sprague-Dawley rats whose dams had been administered intragastrically with 2.5, 25, or 250 ng of 3,3',4,4',5-pentachlorobiphenyl (PCB126) or vehicle on days 13-19 of gestation. The 250 ng groups among the 7-, 10- and 13-week-old offspring showed significant inhibition of mature spermatid release (spermiation), but 17-week-old offspring did not show this. These alterations were not observed in other PCB126 and vehicle groups, and no germ cell or Sertoli cell degeneration were observed in any group. Spermiation failure at puberty appeared in those rats born to dams exposed 250 ng/kg PCB126 on days 13-19 of gestation was reversible change that recovered at adulthood. Because the serum testosterone, luteinizing hormone and follicle-stimulating hormone concentrations were similar in the PCB126 and vehicle groups, a direct endocrine cause for the observed effects was unlikely.     

Comparative testicular toxicity of bis(2-methoxyethyl) ether and 2-methoxyethanol in rats

Male rats were exposed to 0, 110, 370, or 1100 ppm bis(2-methoxyethyl)ether (diglyme) 6 h/day, 5 days/week for 2 weeks. One group of male rats was exposed to 300 ppm 2-methoxyethanol (2-ME) for 2 weeks as a positive control. Exposed rats were killed after 10 days of exposure and 14, 42, or 84 days post-exposure (PE), respectively. At 110 ppm diglyme, spermatocytes in pachytene and meiotic division at spermatogenic stages XII-XIV were mainly affected. At 370 ppm diglyme, affected germ cells were similar to those seen at 110 ppm diglyme, but round spermatids at spermatogenic stages I-VII were also affected. The testes regained normal spermatogenesis by 84 days PE. At 1100 ppm diglyme or 300 ppm 2-ME, marked testicular atrophy was found affecting all spermatogenic stages. Damaged seminiferous tubules were lined with regenerating pachytene spermatocytes at 14 days PE and with spermatocytes and round spermatids after 42 days PE. Most but not all testes in rats exposed to 300 ppm 2-ME or 1100 ppm diglyme had normal morphology after 84 days PE. Based on the observation of germ cell damage, spermatozoa population in the epidymal tubules, reversibility of spermatogenesis after various PE periods, testicular toxicity induced by 300 ppm 2-ME was more severe than that seen at 370 ppm diglyme but was slightly less remarkable than that of 1100 ppm diglyme.     

CYP1 and AhR expression in 7,12-dimethylbenz[a]anthracene-induced mammary carcinoma of rats prenatally exposed to 3,3',4,4',5-pentachlorobiphenyl

We previously reported the finding that prenatal exposure to a relatively low dose of 3,3',4,4',5-pentachlorobiphenyl (PCB126) acted as an enhancing agent for 17-beta-estradiol (E2)-dependent 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma, while a high dose decreased it. E2 is a known risk factor for mammary carcinoma, and CYP1A1 and 1B1 (CYP1) are the major enzymes catalyzing 2- and 4-hydroxylation of E2, respectively. We investigated the induction of CYP1 and aryl hydrocarbon receptor (AhR) in DMBA-induced mammary carcinoma using female Sprague-Dawley rats whose dams had been treated (i.g.) with 2.5 ng, 250 ng, 7.5 microg of PCB126/kg or the vehicle on days 13-19 post-conception. Immunohistochemical analysis revealed that the mammary carcinoma of the 250 ng group showed a significantly higher number of nuclei expressing estrogen receptor alpha (ER) and proliferating cell nuclear antigen (PCNA) compared to those of the other groups. Quantitative real-time RT-PCR analysis revealed that the 7.5 microg group showed a significantly higher level of CYP1A1 mRNA, and that the 250 ng group showed significantly higher levels of CYP1B1 mRNA. The level of AhR mRNA was significantly higher in both the 7.5 microg and 250 ng groups. Western blotting analysis was consistent with mRNA changes. It has been revealed that CYP1B1 catalyzes a step in the formation of 4-hydroxylated E2 metabolites, which show quite high mammary carcinogenicity. This study indicates that the enhancement of DMBA-induced mammary carcinogenicity in a relatively low PCB126 dose group might partially involve the higher expression of CYP1B1 and AhR in these carcinomas.     

Testicular toxicity of rats exposed to hexafluoroacetone (HFA) for 90 days

Rats were exposed to 0, 0.1, 1, and 12 ppm of hexafluoroacetone (HFA) for 6 h/day, 5 days/week for 90 days. The exposed rats were killed after 30 or 90 days exposure, and 28 or 84 days post-exposure (PE). There were no exposure-related pathological lesions in the rats exposed to 0.1 or 1.0 HFA for 90 days. After 30 days exposure to 12 ppm HFA, rats showed lower body weight gain, testicular atrophy, and oligospermia or aspermia in the epididymal tubules. At 30 days exposure, the atrophic testes had marked depletion of round spermatids in spermatogenic stages I-VIII and elongated spermatids in spermatogenic stages IX-XIV, but mature spermatids appeared only slightly decreased. Numerous spermatocytes in meiotic division in spermatogenic stage XIV were necrotic. At 90 days exposure, the testes showed severe atrophy with almost all seminiferous tubules affected and both immature and mature spermatids had disappeared from the seminiferous tubules. The epididymal tubules were devoid of spermatozoa. After 28 days PE, regeneration of atrophic testes was evident but varied markedly among the exposed rats. The number of seminiferous tubules producing elongated and mature spermatids was significantly lower than that of normal testes. Many seminiferous tubules had not regained normal spermatogenesis and the epididymal tubules showed marked oligospermia. After 84 days PE, normal spermatogenesis was still only partially restored to the atrophic testes, with many of the regenerating tubules still devoid of normal spermatogenesis.     

Testicular toxicity of nitrofurazone causing germ cell apoptosis in rats

In order to clarify the mechanism underlying testicular toxicity of nitrofurazone (NF), two experiments were performed. In experiment 1, sequential histopathological examination of testes after a single oral administration of 100 or 300 mg/kg NF to male rats demonstrated that degeneration of pachytene spermatocytes with an eosinophilic, shrunken appearance in stages VII-VIII and vacuolation of Sertoli cells were first observed 12 h after treatment. By 24 h, degeneration of pachytene spermatocytes in stages VII-XII and diplotene spermatocytes were observed. On post-treatment day 4, neither spermatocytes nor spermatids located inside the pachytene spermatocytes in stage VII were seen anywhere. Generation of seminiferous epithelium progressed with recovery to almost normal morphology after 12 weeks, although some morphological changes were still present. No lesions were apparent in spermatogonia, preleptotene spermatocytes, leptotene spermatocytes, zygotene spermatocytes or Leydig cells. Degenerate pachytene spermatocytes and some round spermatids seen after 24 h showed positive TdT-mediated dUTP-biotin nick end labeling (TUNEL). In addition, DNA laddering patterns were detected with agarose gel electrophoresis, and increased electron density of nuclei and cytoplasm of degenerating spermatocytes with nuclear chromatin focal aggregations were observed by electron microscopy, indicating that cell death was attributable to apoptosis. In experiment 2, sequential serum sex-related hormone levels were assayed after a single oral administration of 300 mg/kg NF to male rats and revealed a significant increase of testosterone and a decrease of progesterone at 6 h, and decreases of luteinizing hormone at 12 h and testosterone at 24 h. Prolactin tended to decrease from 12 h after treatment and the decrease was significant at 48 h. No significant changes were observed in levels of follicle-stimulating hormone or estradiol. The probability that NF damages germ cells by causing a hormonal imbalance is extremely low, since no pattern of hormonal imbalance that could be regarded as the cause of the testicular degeneration was observed until 12 h after NF treatment when pachytene spermatocytes began to degenerate. The present experiments suggest that NF damages Sertoli cells and pachytene spermatocytes in stages VII-XII directly.     

Effect of textile waste water on the spermatogenesis of male albino rats

Textile waste water released from dyeing and printing industries situated in Sanganer, Jaipur (India), brought about inhibition of spermatogenesis in male rats. Water analysis showed the presence of heavy metals at more than permissible limits. Oral administration of waste water to the rats at the dose level of 26.6 ml kg(-1) body wt. significantly reduced the weights of testes, epididymides and seminal vesicle. Treated animals showed a notable depression of various stages of spermatogenesis. The production of spermatids was inhibited by 70.8% in waste-water-treated rats. The populations of spermatogonia, preleptotene spermatocytes and secondary spermatocytes were decreased by 67.2, 71.1 and 73.2%, respectively. The total number of Sertoli cells was affected after waste water treatment. Reduced sperm count and motility resulted in treated groups. A significant fall in the content of various biochemical parameters of reproductive tissues was observed after water treatment.     

Reproductive toxicity of 1-bromopropane, a newly introduced alternative to ozone layer depleting solvents, in male rats.

1-Bromopropane has been newly introduced as an alternative to ozone-depleting solvents. We aimed to clarify its dose-dependent reproductive toxicity in male rats. Thirty-six Wistar male rats were randomly divided into 4 groups of 9. The groups were exposed to 200, 400, or 800 ppm 1-bromopropane or only fresh air, 8 h per day for 12 weeks. Epididymal sperm indices were evaluated after a 12-week exposure. The testes, epididymides, seminal vesicle, prostate, and other organs were weighed and examined histopathologically. Spermatogenic cells, in stage VII seminiferous tubules, and retained spermatids, at the basal region of stages IX-XI seminiferous epithelium, were counted. Plasma testosterone levels were measured by radioimmunoassay. The testicular weight did not significantly change, but the weight of epididymides, seminal vesicle, and prostate dose-dependently decreased. The weight of seminal vesicle decreased significantly at the lowest concentration of 200-ppm and over. 1-Bromopropane induced a dose-dependent decrease in the epididymal sperm count and in motility, as well as an increase in tailless sperm and sperm with an immature head shape. The spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, and round spermatids did not decrease significantly at stage VII. Retained, elongated spermatids near the basement membrane at the postspermiation stages IX-XI increased dose-dependently. Plasma testosterone levels significantly decreased at the 800-ppm dosage. 1-Bromopropane caused failure of spermiation. Its reproductive toxicity is different from that of 2-bromopropane, which specifically impairs spermatogonia. Thus, this solvent may have serious reproductive toxic effects in men, and should be used very cautiously in the workplace.     

A novel quantitative morphometry of germ cells for the histopathological evaluation of rat testicular toxicity

A view that 14 stages of rat spermatogenic cycle could be arranged into 4 groups, viz., conventional stages I-VI, VII-VIII, IX-XI and XII-XIV, according to the features of elongated spermatids was previously presented. A novel morphometry of seminiferous epithelia based on these 4 groups was also proposed. In the present study, utility of the proposed morphometry in the histopathological evaluations of testicular toxicities was monitored in comparison with the conventional one. After administrating adriamycin, ethylene glycol monomethyl ether or 1,3-dinitrobenzene to rats, the viability of their germ cells was estimated by the proposed morphometry and the conventional one employed stages II-III, V, VII, X and XII. In every case, the evaluating results of the proposed morphometry were similar to those of the conventional one. Thus, it was verified that the proposed morphometry was identical with the conventional one in respect of reliable detection of the testicular toxicities. In addition, in situ terminal dUTP nick end labeling indicated that death of spermatogonia, pachytene spermatocytes or round spermatids induced by the above 3 toxic compounds was exclusively apoptotic death. In conclusion, the proposed morphometry would be useful as a practical tool in the evaluation of testicular toxicities.     

Effects of 3,3',4,4',5-pentachlorobiphenyl, a coplanar polychlorinated biphenyl congener, on cultured neonatal mouse testis.

3,3',4,4',5-Pentachlorobiphenyl (PCB126), a congener with a planar configuration, has been established to have relatively strong toxicities similar to those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via aryl hydrocarbon receptors. We investigated the effects of this coplanar PCB on mammalian early spermatogenesis and steroidogenesis in a mouse neonatal testicular organ culture system. Testes collected from newborn mice were subjected to organ culture in medium containing 0, 10, 100 or 1000 nM PCB126. Histochemical analysis revealed that the BrdU-labeling indices of both spermatogenic cells and Sertoli cells were unchanged in all testis specimens exposed to the coplanar PCB. CYP1A1 and steroidogenic enzymes (P450scc, P450c17, 3beta-HSD and 17beta-HSD) mRNA levels were determined by semiquantitative RT-PCR. The CYP1A1 mRNA level in cultured testis was significantly increased by PCB126 in a dose-dependent manner. Although mRNA levels of 3beta-HSD and 17beta-HSD were unchanged, the P450scc mRNA level was significantly down-regulated by PCB126 in a dose-dependent manner. In contrast, the P450c17 mRNA level was significantly higher in 1000 nM PCB126-exposed testis than in control testis. These results suggest that the coplanar PCB does not alter the proliferative activity of spermatogenic cells and Sertoli cells in neonatal testis, but that it directly affects the expression of steroidogenic enzyme genes.     

Stage-related increase in the proportion of apoptotic germ cells and altered frequencies of stages in the spermatogenic cycle following gestational, lactational, and direct exposure of male rats to p-nonylphenol.

The cumulative effects of environmental toxicants, for example, the alkylphenol, para-nonylphenol (p-NP) are of concern. Our previous study showed that p-NP reduced several testicular morphometric parameters, including sperm counts. The present study reexamined material collected in that study to determine the mechanistic basis of p-NP action on spermatogenic development in the offspring. Seven-day pregnant Sprague-Dawley rats were treated with vehicle or 100 or 250 mg/kg p-NP through gestation, lactation and afterward directly to all male offspring until 10 weeks of age. Both doses of p-NP significantly (P < 0.02) increased the number of germ cells with in situ end-labeled fragmented DNA (TUNEL positive) by 1.9-fold and 1.7-fold, respectively, and specifically in stages XII-XIV and I-III. TUNEL-labeling was, however, selective, and excluded labeling of basal cells with apoptotic morphology. Cleaved caspase-3 immunohistochemistry strongly labeled basal cells (spermatogonia and early spermatocytes) with condensed marginated chromatin but not degenerate germ cells lacking definitive nuclear material found throughout the epithelium. Only the caspase index (ratio of number of caspase positive to number of degenerate cells) of the 100-mg/kg p-NP group was significantly (p < 0.05) threefold greater than controls. Whereas both doses and either 250 or 100 mg/kg treatment alone significantly (p < 0.002) reduced the frequencies (duration) of stages I-III, VII-VIII, and late VIII-IX (spermiating and recently spermiated tubules), respectively, both doses significantly (p < 0.002) increased the frequencies of stages IV-VI and all stages containing late-stage spermatocytes (XII-XIII) and meiotic cell divisions (XIV). Thus, p-NP, an environmentally persistent xenoestrogen, insidiously alters the spermatogenic cycle and spermatogenic process in male offspring.     

The dioxin-like pollutant PCB 126 (3,3',4,4',5-pentachlorobiphenyl) affects risk factors for cardiovascular disease in female rats

Epidemiological studies suggest that exposure to persistent organic pollutants such as organochlorines might induce cardiovascular disorders and diabetes. Some of these organochlorines, such as dioxins and some dioxin-like PCBs, have been characterised as anti-estrogenic due to their inhibition of estrogenic-induced responses. In the present pilot study, 40 female rats were subjected to either exposure to the dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) or vehicle, as well as ovariectomy (OVX) or sham operation in a 2×2 factorial design over 12 weeks to explore potential interactions between estrogen status and PCB 126 exposure on cardiovascular risk factors.

PCB 126 increased heart weight and serum cholesterol levels in both groups. PCB 126 increased blood pressure in the sham-operated animals only.

In conclusion, PCB 126 exposure in female rats resulted in effects on cardiovascular risk factors, such as serum cholesterol, blood pressure, and heart weight. Of these effects of PCB 126, the increase in blood pressure was dependent on estrogen status.     

Estrogen supplementation modulates effects of the endocrine disrupting pollutant PCB126 in rat bone and uterus: diverging effects in ovariectomized and intact animals

The aims of the present study are to compare effects of estrogen depletion (OVX) and estradiol (E2) supplementation on the tissue effects of exposure to the endocrine disrupting organochlorine 3,3',4,4',5-pentachlorobiphenyl (PCB126). For this purpose two highly estrogen-dependent tissues, bone and uterus, were studied. Forty rats exposed to PCB126 (ip) for 3 months (total dose 384 microg/kg body weight (bw)) were randomized in to OVX/sham operation or E2 supplementation (ip, 23 microg/kg, 3 days weekly) per vehicle (corn oil) groups in a 2 x 2 factorial design. Sham operated rats were treated with vehicle, PCB or PCB plus E2 (sham, sham + PCB and sham + PCB + E2, n=10 per group) whereas ovariectomized were treated with vehicle, PCB or PCB plus E2(OVX, OVX + PCB and OVX + PCB + E2, n=10 per group). As control groups served OVX or sham, and OVX + E2 (n=10 in each group). In OVX rats PCB126 + E2 treatment increased trabecular bone volume (TBV) (P<0.01), whilst the opposite was found in sham-operated rats (P<0.01). In OVX animals exposed to PCB126, E2 supplementation decreased the uterine weight and increased the uterine ERbeta mRNA level, whilst no difference was found between the PCB126 and PCB126 + E2 exposed groups in the sham-operated animals. In conclusion, estrogen modulates PCB126 induced effects on trabecular bone, as well as several uterine parameters. These results further support an important role of estrogen on the toxic effects of PCB126 on bone and uterus.     

Testicular toxicity of methylmercury: analysis of cellular distribution pattern at different stages of the seminiferous epithelium.

Stage-specific distribution of methylmercury (MM) and spermatogenic changes were analyzed in rats administered 5 or 10 micrograms MM/kg, ip, daily for 15, 30, 60, and 90 days. MM deposition, as grain number/cm2 was noted in basal portions at later stages on day 15, which increased gradually by day 90. MM deposition was in the order of stages IV, VII, XIV, IX, being higher in adluminal portions on days 30 and 60. MM-enriched cytoplasmic masses leaked out through disintegrated tubular membrane on days 60 and 90. Epithelial damage, at stages late XIV through IV, V through VI, VII through VIII, XIII through mid-XIV, and IX through XII, accorded with the gradual deposition of MM. As profound cell death occurred between zygotenes to pachytenes and dividing spermatocytes to step 1 spermatids, the spermatids were conspicuously decreased at later times. It is possible that MM distorts the barrier system at stages IX through XII, gets distributed within the tubule, and hence may pose a direct or Sertoli cell mediated effect at stages XII through early XIV in a dose-duration-MM burden related manner.