Rika Ogata

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.

Oestradiol-induced relaxation of rabbit basilar artery by inhibition of voltage-dependent Ca channels through GTP-binding protein

1 Effects of oestradiol on the electrical and mechanical properties of the rabbit basilar artery were investigated by use of microelectrode, patch‐clamp and isometric tension recording methods. 2 Oestradiol (10 μm‐100 μm) relaxed arterial tissue pre‐contracted by excess [K]0 solution (30 mM) in a concentration‐dependent manner. In Ca‐free solution, histamine (10 μm) and caffeine (20 mM) each produced a phasic contraction, but oestradiol (10 μm) did not significantly affect their amplitude. 3 Oestradiol (≥ 100 μm) did not change the resting membrane potential of the artery whether in the presence or absence of TEA (10 mM). Action potentials observed in the presence of 10 mM TEA were abolished by oestradiol (100 μm). 4 Oestradiol (1 μm‐100 μm) inhibited the voltage‐dependent Ba current in a concentration‐dependent manner. Oestradiol (100 μm) inhibited the Ba current observed in the presence of nicardipine (1 μm) more than that in the absence of nicardipine (to 31.0% vs 62.0% of control). 5 GTPγS (30 μm) in the pipette enhanced the inhibitory actions of oestradiol on the Ba current. On the other hand, with GDPβS (1 mM) in the pipette, oestradiol failed to inhibit the Ba current. Pertussis toxin (PTX 3 μg ml−1) in the pipette totally prevented the inhibitory action of oestradiol on the Ba current. 6 Oestradiol (≷ 100 μm) had no significant effect on the outward K currents evoked by a membrane depolarization. 7 These results strongly suggest that oestradiol relaxes arterial tissue by inhibition of voltage‐dependent Ca channels and that it inhibits both nicardipine‐sensitive and‐resistant Ca currents via a PTX‐sensitive GTP‐binding protein. The main target of oestradiol among the arterial Ca channels seems to be the nicardipine‐resistant Ca channel, rather than the nicardipine‐sensitive one.

Inhibitory effects of genistein on ATP‐sensitive K+ channels in rabbit portal vein smooth muscle

1 Effects on the pinacidil‐induced outward current of inhibitors of tyrosine kinases and phosphatases were investigated by use of a patch‐clamp method in smooth muscle cells of the rabbit portal vein. 2 A specific tyrosine kinase inhibitor, genistein, inhibited the pinacidil‐induced current in a concentration‐dependent manner with an IC50 of 5.5 μM. Superfusion of Ca2+‐free solution did not affect this inhibitory effect of genistein. At higher concentrations, genistein inhibited the voltage‐dependent Ba2+ and K+ currents with IC50 values of >100 μM and 75 μM respectively. Tyrphostin B46 (30 μM), a tyrosine kinase inhibitor, also inhibited the pinacidil‐induced current by 70 % of the control. 3 Sodium orthovanadate (100 μM), an inhibitor of tyrosine phosphatase, slightly but significantly enhanced both the pinacidil‐induced and delayed rectifier K+ currents. Daidzein (100 μM), an inactive analogue of genistein, did not inhibit these currents. 4 Neither herbimycin A (1 μM), lavendustin A (30 μM), tyrphostin 23 (10 μM), which are also tyrosine kinase inhibitors, nor wortmannin (10 μM), a phosphatidylinositol 3‐kinase inhibitor, had an effect on either the pinacidil‐induced or delayed rectifier K+ currents. Epidermal growth factor (EGF; 1 μg ml−1) did not induce an outward current or enhance the pinacidil‐induced current. 5 Pinacidil alone, in the cell‐attached configuration, or pinacidil with GDP, in the inside‐out configuration, activated a 42 pS channel in the smooth muscle cells of the rabbit portal vein. Genistein (30 μM) reduced the channels open probability without inducing a change in unitary conductance at any holding potential (−30 to +20 mV). 6 In the inside‐out configuration, genistein at 30 μM did not change the mean channel open time, but reduced the burst duration. At 100 μM genistein abolished channel opening. The inhibitory potencies with which 30 and 100 μM genistein acted on the unitary current of the ATP‐sensitive K+ channel were similar to those seen in the whole‐cell voltage‐clamp configuration. 7 Although direct inhibitory actions of genistein on the ATP‐sensitive K+ channels are not ruled out, our results suggest that a protein tyrosine kinase may play a role in the regulation of ATP‐sensitive K+ channel activity in the rabbit portal vein.

Changes in coagulation factors and fibrinolytic components of postmenopausal women receiving continuous hormone replacement therapy

OBJECTIVE The effect of hormone replacement therapy (HRT) on coagulation factors and fibrinolytic components in postmenopausal women was studied for 6 months to elucidate whether continuous HRT has an influence on thrombosis. METHODS One hundred and thirty-four postmenopausal women were divided into three groups according to treatment: 39 women who had undergone hysterectomy and oophorectomy received 0.625 mg/day of conjugated equine estrogen (CEE) continuously (CEE therapy), 48 postmenopausal women received both 0.625 mg/day of CEE and 2.5 mg/day of medroxyprogesterone acetate (MPA) continuously (CEE/MPA therapy) and 47 postmenopausal women received placebo as control. The following variables were measured before treatment as well as after 1, 3 and 6 months of treatment: factor VII activity, protein C activity, fibrinogen level, antithrombin III activity, plasminogen activator inhibitor-1 (PAI-1) level and the plasma concentration of tissue-type plasminogen activator (t-PA). RESULTS After 1 month of treatment, protein C activity increased by 9.6% and 11.4% of the initial value (p < 0.05), fibrinogen level decreased by 7.8% and 6.1% of the initial value (p < 0.05) and PAI-1 decreased by 19.4% and 14.3% of the initial value (p < 0.05) in the CEE therapy group and the CEE/MPA therapy group, respectively. Factor VII activity increased by 10.1% of the initial value (p < 0.05) in the CEE therapy group only. Antithrombin III and t-PA levels did not change throughout either treatment. CONCLUSION Except for an increase in factor VII activity in the case of continuous CEE therapy, continuous HRT had no unfavorable effects on either coagulation factors or fibrinolytic components.

Treatment of bone loss in oophorectomized women with a combination of ipriflavone and conjugated equine estrogen.

Objective: We previously reported that 0.625 mg/day of conjugated equine estrogen (CEE) could not prevent acute bone loss in the first year after oophorectomy. The effect of additional administration of ipriflavone on bone mineral density (BMD) and biochemical indices of bone remodeling were studied to investigate whether concurrent use of CEE and ipriflavone prevent acute bone loss in the early stages following surgical menopause. Methods: One‐hundred and sixteen oophorectomized women were randomly divided into four groups according to treatment; group 1: placebo, n=30; group 2: CEE (0.625 mg/day), n=29; group 3: ipriflavone (600 mg/day), n=30; group 4: CEE (0.625 mg/day) plus ipriflavone (600 mg/day), n=27. Vertebral BMD was measured using dual energy X‐ray absorptiometry (DEXA) and two biochemical indices of bone metabolism, urinary pyridinoline (Pyr) and serum intact human osteocalcin (hOC), were also measured before, 24 weeks, and 48 weeks after initiation of treatment. Results: BMD was reduced 48 weeks after treatment by 6.1, 3.9 and 5.1% in groups 1–3, respectively, but by only 1.2% in group 4. Pyr decreased by 49.5, 32.0 and 41.5% in groups 2–4, respectively. hOC also decreased by 45.2 and 21.6% in groups 2 and 4, but increased by 40.5% in group 3, suggesting an inhibitory action of CEE and ipriflavone on the turnover of bone metabolism and stimulatory action of ipriflavone on bone formation. Conclusion: Concomitant use of ipriflavone with CEE from an early stage after oophorectomy inhibited bone loss and was considered to be effective in maintaining bone mass after oophorectomy.

Efficacy of selective venous sampling to localize a small ovarian androgen-producing tumor.

Two cases of androgen‐producing tumors, including a Sertoli‐Leydig cell tumor in a woman of reproductive age and a Leydig cell tumor in a postmenopausal woman, are reported herein. In both cases, only selective venous sampling was able to detect the presence of the androgen‐producing ovarian tumors.

Effects of Perinatal Simultaneous Exposure to Tributyltin (TBT) and p, p′-DDE (1,1-Dichloro-2,2-Bis(p-Chlorophenyl) Ethylene) on Male Offspring of Wistar Rats

p,p′-DDE [1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene; DDE] and tributyltin (TBT) are ubiquitous in the environment and in Japan were shown to bioaccumulate in marine products. Thus these chemicals serve as a source of contaminant in the mammalian food chain. Fetuses and neonates through maternal ingestion may be exposed to DDE and TBT. Therefore, the effects of concurrent exposure to DDE and TBT were investigated in male Wistar rat offspring of dams ingesting these two contaminants. In this study, TBT suppressed the growth and delayed eye opening. However, both growth retardation and delayed eye opening produced by TBT failed to occur in the presence of DDE. Unexpectedly, the prostate weight of male rat offspring was significantly reduced with the administration of TBT but restored in the presence of DDE. These results indicate that TBT and DDE affected the development of male rat offspring following maternal exposure, and simultaneous administration of DDE prevented some of the observed effects of TBT, especially of an antagonistic nature, through a mechanism, still to be determined.

Systemic Effects of Orally Administered p, p'-DDE on Immature Male Wistar Rats during Pubertal Period

Systemic Effects of Orally Administered p, p’‐DDE on Immature Male Wistar Rats during Pubertal Period: Yuji Makita, et al. Department of Hygiene, Graduate School of Medical Sciences, Kyushu University—Systemic effects of p, p’‐DDE (1, 1‐dichloro‐2, 2 bis (p‐chlorophenyl) ethylene; DDE) on immature male rats were investigated in pubertal Wistar rats after oral administration of DDE. Special rat chow containing 125 ppm DDE (approximately 10 mg/kg DDE) had been administered daily for 42 d since 6 wk of age and its effects had been observed until 12 wk of age. The administration of DDE did not produce any overt signs of toxicity. Neither physical development nor sexual maturation was affected, and serum biochemistry was not impaired at the dose used in this experiment. Moreover, the male reproductive organs and epididymal sperm count were not affected by the administration of DDE during the pubertal period. Our results showed that even immature male rats were resistant to DDE exposure at the daily dose of ca. 10 mg/kg, but metabolic and immunological changes still remained uncertain. Further investigation should be conducted to reveal all the effects of DDE on immature male rats.