Minoru Omura

Low dose effects of bisphenol A on sexual differentiation of the brain and behavior in rats

There is an endocrinological concern that environmental endocrine disrupters (EEDs) may influence sexual differentiation. Bisphenol A (BPA), one of EEDs, is released from polycarbonate plastics, and has been detected in the human umbilical cord. In this study, we examined the effect of BPA on the sexual differentiation of open-field behavior and the sexually dimorphic nuclei in the brain in the offspring of rats exposed to BPA during the fetal and suckling periods at a dosage below the human tolerable daily intake (TDI) level. In the control group, females were more active in the open field and had a larger locus coeruleus (LC) volume than males. BPA abolished and inverted the sex differences of the open-field behavior and the LC volume, respectively, without affecting the reproductive system. We also compared the effects of estrogenic compounds, diethylstilbestrol (DES) and resveratrol (RVT), to that of BPA because of their structural similarities. DES affected the open-field behavior, LC volume and reproductive system, while RVT affected the LC volume and the reproductive system. These results suggest that the brain is highly sensitive to BPA at a dosage below TDI and that the disrupting effects of BPA on sexual differentiation may vary from those of RVT and DES.

Exposure to bisphenol A during the fetal and suckling periods disrupts sexual differentiation of the locus coeruleus and of behavior in the rat

This study tested the effect of exposure to bisphenol A (BPA) early in life on the sexual differentiation in the brain and behavior in Wistar rats. We administered BPA only to mother rats during pregnancy and lactation at a dosage of approximately 1.5 mg/kg per day far less than the no-observed-adverse-effect level (NOAEL; 50 mg/kg per day). Control female offspring showed a higher activity, a lower avoidance memory, and larger locus coeruleus than the male controls, while the BPA-exposed group did not show any sexual dimorphism. BPA did not affect the reproductive organs or sex hormones. Our results suggest that the current methods to determine the NOAEL of artificial industrial chemicals may not be sufficient to detect a disruption of the sexual differentiation in the brain.

Two-generation reproductive toxicity study of tributyltin chloride in female rats

A two-generation reproductive toxicity study of the effects of tributyltin chloride (TBTCl) was conducted in female rats using dietary concentrations of 5, 25, and 125 ppm TBTCl. Reproductive outcomes of dams (number and body weight of pups and the percentage of live pups) and the growth of female pups (the day of eye opening and body weight gain) were significantly decreased in the 125 ppm TBTCl group. A delay in vaginal opening and impaired estrous cyclicity were also observed in the 125 ppm TBTCl group. However, an increase in anogenital distance was found in all TBTCl groups on postnatal d 1. A dose-effect relationship was observed in TBTCl-induced changes in anogenital distance. These results indicate that the whole-life exposure to TBTCl affects the sexual development and reproductive function of female rats. In addition, the TBTCl-induced increase in anogenital distance seems to suggest it may exert a masculinizing effect on female neonates. However, the concentrations of TBTCl used in this study are not environmentally relevant.

Histopathological evidence that spermatogonia are the target cells of 2-bromopropane.

To confirm the target cell of 2-bromopropane within the testis, 1355 mg/kg of 2-bromopropane was subcutaneously injected to rats for 1-5 days and the numbers of spermatogonia and spermatocytes were examined 6 h after each last injection. The number of stage I spermatogonia decreased after the first 2-bromopropane injection and the number of spermatogonia at the other stages also decreased following repetitive injection. The number of these spermatogonia decreased further by the repetition of 2-bromopropane injection. In addition, the delay in mitotic division of type B spermatogonia was frequently observed after the fifth 2-bromopropane injection. The number of stage I pachytene spermatocytes also decreased slightly after the first 2-bromopropane injection, although it did not decrease further following repetitive injection. Therefore, we concluded that spermatogonia are the target cells of 2-bromopropane in rats.

Testicular toxicity evaluation of arsenic-containing binary compound semiconductors, gallium arsenide and indium arsenide, in hamsters

The testicular toxicities of gallium arsenide (GaAs), indium arsenide (InAs) and arsenic trioxide (As2O3) were examined by repetitive intratracheal instillation using hamsters. GaAs (7.7 mg/kg) and As2O3 (1.3 mg/kg) were instilled twice a week a total of 16 times and InAs (7.7 mg/kg) was instilled a total of 14 times. GaAs caused testicular spermatid retention and epididymal sperm reduction, though the degrees were less severe than those in rats shown in our previous experiment. InAs and As2O3 did not show any testicular toxicities. Serum arsenic concentration in GaAs-treated hamsters was less than half of that in As2O3-treated hamsters in which no testicular toxicities were found. Serum molar concentration of gallium was 32-times higher than that of arsenic in GaAs-treated hamsters. Therefore gallium may play a main role in the testicular toxicity of GaAs in hamsters.

Long Term Pulmonary Toxicity of Indium Arsenide and Indium Phosphide Instilled Intratracheally in Hamsters

Long Term Pulmonary Toxicity of Indium Arsenide and Indium Phosphide Instilled Intratracheally in Hamsters: Koji Yamazaki, et al. Department of Hygiene, Graduate School of Medical Sciences, Kyushu University—We examined the long‐term toxicological effects of III‐V semiconductor particles on laboratory animals. Eight‐week‐old male Syrian golden hamsters were given 4 mg/kg indium arsenide (InAs) or 3 mg/kg indium phosphide (InP) particles, both containing 2.4 mg/kg as indium, intratracheally twice a week for 8 weeks. Control hamsters were given only a vehicle, phosphate buffer solution. Over a 2‐yr period, these animals were euthanized serially and the biological effects were determined. Weight gain was significantly suppressed in both InAs and InP groups, compared to the control group, with greater suppression in the InAs group. The serum indium concentration in the InAs group was about twice as high as that in the InP group, in each period. Histopathologically, severe pulmonary inflammation and localized lesions with bronchiolo‐alveolar cell hyperplasia were present in both InAs and InP groups from just after the last administration. The localized lesions gradually transformed to proteinosis‐like lesions with periodic acid Schiff reagent positive exudation after 16 wk. By means of immunostaining of proliferating cell nuclear antigen and argyrophilic proteins associated with nucleolar organizer regions staining, proliferative activities were evidenced in the localized lesions at each time and were noticeable in their early stage. K‐ras, a known oncogene, was not mutated in association with these lesions. In conclusion, InAs and InP particles caused severe systemic toxicity and pulmonary localized hyperplastic lesions with proliferative activity were derived via the respiratory route. Neoplastic change was nil even in a 2‐yr observation period.

Changes in the testicular damage caused by indium arsenide and indium phosphide in hamsters during two years after intratracheal instillations

Changes in the Testicular Damage Caused by Indium Arsenide and Indium Phosphide in Hamsters during Two Years after Intratracheal Instillations: Minoru Omura, et al. Department of Hygiene, Graduate School of Medical Sciences, Kyushu University—Change in the testicular damage caused by indium arsenide (InAs) and indium phosphide (InP) was examined during two yr after repetitive intratracheal instillations in hamsters. In this study, 4.0 mg/kg body weight/day of InAs or 3.0 mg/kg body weight/day of InP was instilled intratracheally twice weekly for eight wk. A single instillation dose of indium was 2.4 mg/kg body weight in both groups. Testicular damage was evaluated 0, 8, 16, 40, 64 and 88 wk after the last instillation. Both InAs and InP were proved to be definite testicular toxicants. Both materials decreased reproductive organ weight and caudal sperm count, and caused severe histopathologic changes in the testes. InAs‐induced testicular damage was always more serious than InP‐induced testicular damage. The serum indium concentration in the InAs group was always higher than that in the InP group, and indium was probably a toxic element in both materials. In the histopathologic examination, vacuolization of seminiferous epithelium was frequently observed as an early histopathologic change and spermatogonia remained in general even in the seminiferous tubules with severe histopathologic changes in both groups. It is therefore estimated that Sertoli cells, not stem cell spermatogonia, were the target cells of these indium‐containing compound semiconductor materials. The threat of InAs and InP to male reproduction was proved in this study. We concluded that male reproductive disorders should not be overlooked when severe exposure to indium‐containing compound semiconductor materials is apparent in human subjects.

The Effects of Subacute Inhalation of Di (2-ethylhexyl) Phthalate (DEHP) on the Testes of Prepubertal Wistar Rats

The Effects of Subacute Inhalation of Di (2‐ethylhexyl) Phthalate (DEHP) on the Testes of Prepubertal Wistar Rats: Norie Kurahashi, et al. Department of Public Health, Hokkaido University Graduate School of Medicine—In animal studies using oral dosing for short periods, di (2‐ethylhexyl) phthalate (DEHP) is well known for its reproductive toxicity, especially for its testicular toxicity. However, extending the period of DEHP exposure in prepubertal rats resulted in significant increases in testosterone. This suggests that the reproductive effect of DEHP might be associated with the timing and the term of exposure. Moreover, the route of exposure may induce differences in its effect because tissue levels of metabolites of DEHP after inhalation are thought to be different from those after oral administration. We researched the effects of inhalation of DEHP on testes of prepubertal rats. Our results showed that inhalation of DEHP by 4‐wk‐old male Wistar rats at doses of 5 or 25 mg/m3, 6 h per day, for 4 and 8 wk significantly increased the concentration of plasma testosterone and weight of seminal vesicles. However, the concentration of luteinizing hormone (LH), follicular stimulating hormone (FSH) and the expression of mRNAs of androgen biosynthesis enzyme, cytochrome P450 cholesterol side‐chain‐cleavage enzyme (P450scc), 3βhydroxysteroid dehydrogenase (3β‐HSD), cytochrome P450 17α‐hydroxylase/17, 20 lyase (CYP17) and aromatase (CYP19) did not change. Rats with precocious testes did not increase in any of the DEHP groups. We also found that the estimated effective dose in this study was less than those reported in previous studies which used oral dosing. Our study showed that inhaled DEHP increased plasma testosterone concentrations in prepubertal rats and suggested that their effects were more sensitive to inhalation of DEHP than oral dosing.

Mutagenic activity of the leachate of municipal solid waste landfill

Organic concentrates were recovered using XAD-2/8 resin adsorption from the leachates of municipal solid waste landfills and their mutagenic activities were tested for 8 months using the Ames Salmonella/microsome assay. Highly polluted leachates (COD and BOD > or = 40 mg/l) generally had equal or higher mutagenic activities than lightly polluted leachates (COD and BOD < 40 mg/l). But there was no clear difference in mutagenicity per amount of concentrate between the two leachates. These results suggest that the mutagenic activity of landfill leachate is decided to some degree by the organic concentration in the leachate. The mutagenic activities detected even in lightly polluted leachates were not so low as those of various kind of surface waters ever reported. It is suggested that it is important to investigate the mutagenic activity of the leachate for evaluation of the impact of landfill leachate on the environment.

Histopathological Changes of the Testis in Rats Caused by Subcutaneous Injection of 2-Bromopropane.

Histopathological Changes of the Testis in Rats Caused by Subcutaneous Injection of 2‐Bromopropane: Minoru Omura, et al. Department of Hygiene, Kyushu University—Mature male rats were injected subcutaneously with 1,355 mg/kg of 2‐bromopropane five days a week for two weeks. In addition to the routine examinations concerning the effect on male reproductive system, we carried out an evaluation of change in the numbers of various types of germ cells in the seminiferous tubule at stages I, V, VII, X and XII to clarify germ cells which were affected by 2‐bromopropane. 2‐Bromopropane caused mild atrophy of the seminal vesicle, but did not show any adverse effects on spermatid/sperm count, sperm motility or sperm morphology. In the histopathological examination of the testis, the numbers of spermatogonia, preleptotene spermatocyte, leptotene spermatocyte, zygotene spermatocyte and pachytene spermatocyte at stages I and V decreased in the rats treated with 2‐bromopropane. However, the numbers of pachytene spermatocyte at stages VII, X and XII and round spermatids in these rats were comparable with those in control rats. In two weeks, spermatocytes develop to pachytene spermatocyte at stage VII or more developed germ cells in rats. Therefore, it seemed likely that 2‐bromopropane had no adverse effects on spermatocytes but affected spermatogonia, assuming that 2‐bromopropane affected germ cells immediately after the injection. In conclusion, we estimated that spermatogonia were the target cells of 2‐bromopropane in the testis in rats.