Shin-ichi Katsuda

Subcutaneous treatment of p-tert-octylphenol exerts estrogenic activity on the female reproductive tract in normal cycling rats of two different strains

Effects of p-tert-octylphenol (OP), an endocrine disrupting chemical (EDC), on the female reproductive tract of normal cycling Fischer 344 (F344) and Donryu rats were investigated. OP was subcutaneously injected at concentrations of 12.5, 25, 50 or 100 mg/kg for 28 days. The most notable changes were the disappearance of normal cyclicity in the 50 mg/kg or more OP-treated groups of both the strains, and the appearance of persistent estrus (PE) evident on examination of vaginal smears in the 100 mg/kg groups of both the strains, the effects being time- and dose-dependent. In PE rats of both the strains, the uterine morphology deviated from the normal for each estrous stage of the cycling rats, and proliferation in the endometrium was slightly increased. The data for uterine weights, luminal epithelial cell-heights and/or numbers of epithelial cells in the endometrium demonstrated equivocal alteration. In both the strains, the serum 17beta-estradiol (E2) levels were decreased with 50 mg/kg of OP or more. Serum concentrations of the administrated chemical were dose and duration-dependently increased in all the treated groups of both the strains. The results demonstrate that subcutaneous administration of OP at doses of 50 mg/kg or more exerts time- and dose-dependent estrogenic activity on the reproductive tract of normal cycling female Donryu and F344 rats, indicating similar qualitative sensitivity to the effects in both the strains. Vaginal cytology may be the most sensitive endpoint for the detection of estrogenic activity of potential EDCs using adult cycling rats.

Exposure of Neonatal Female Rats to p-tert-Octylphenol Disrupts Afternoon Surges of Luteinizing Hormone, Follicle-Stimulating Hormone and Prolactin Secretion, and Interferes with Sexual Receptive Behavior in Adulthood

Abstract The present study investigated the effects of exposure of neonatal female rats to p-tert-octylphenol (OP) on estrogen-induced afternoon surges of LH, FSH, and prolactin (PRL) secretion, and on sexual behavior in adulthood. After birth, one group of female Wistar rat pups received s.c. injections of OP (100 mg/kg body weight [BW]; OP group) dissolved in DMSO, while the control group received DMSO only (DMSO group). In order to make a qualitative comparison, a third group was injected with estradiol-17β (500 μg/kg BW; estradiol group) dissolved in DMSO. Injections were given on Days 1, 3, 5, 7, 9, 11, 13, and 15 of age. The rats from the OP and estradiol groups that were used for subsequent experiments were in persistent vaginal estrus. Spontaneous LH surge measured at Postnatal Days (PND) 78–81 was observed only in the DMSO group on the afternoon of the day of proestrus. At PND 115, randomly selected rats from each of three treatment groups were bilaterally ovariectomized (ovx), and 8 days later, Silastic capsules containing estradiol-17β were implanted under the skin. Estrogen implants stimulated afternoon surges of LH, FSH, and PRL for two consecutive days in the DMSO group, but not in the OP and estradiol groups. Rats from the OP and DMSO groups underwent ovx at PND 186, and 6 days later they were treated with a combination of estradiol benzoate s.c. (15 μg/kg BW) and progesterone s.c. (2 mg/kg BW) to test the lordosis reflex. In response to this hormone treatment and mounting stimulus delivered by the stud male rats, the OP-treated rats were less receptive compared with control DMSO-treated rats, and thus the lordosis quotient and lordosis rating were significantly (P < 0.05) reduced in the OP group compared with the DMSO group. Analysis of the area of the sexually dimorphic nucleus of the preoptic area of the brain revealed that the area of this nucleus was larger in the OP group than it was in control DMSO rats. We conclude that the exposure of neonatal female rats to higher doses of OP disrupts the cyclic release of LH, FSH, and PRL, and interferes with the display of sexual receptive behavior in adulthood.

Effects of neonatal exposure to a high-dose p-tert-octylphenol on the male reproductive tract in rats

Time-course alterations in morphological changes of the reproductive tract including spermatogenesis as well as pituitary and gonadal hormones, reproductive ability, and the size of the sexually dimorphic nucleus of the preoptic area (SDN-POA) were investigated in male rats neonatally exposed to 100 mg/kg p-tert-octylphenol (OP) subcutaneously. OP treatment affected hormone levels of follicle stimulating hormone (FSH) and testosterone, reproductive organ weights and sperm counts. Slightly depressed FSH levels at prepuberty and prolonged suppression of testosterone till 7 weeks of age were observed as two hormonal alterations. The lasting reduction in testosterone appeared to be associated with growth inhibition of male reproductive organs such as the testis, prostate and epididymis, these demonstrating low organ weights compared with those of age-matched controls till 7 weeks of age. The FSH concentrations after puberty showed a rise to values equal to or higher than those of the control group, suggesting recovery of maturation of the reproductive tract. No morphological abnormalities, even with morphometric stage analysis of spermatogenesis, were detected in the male reproductive tract throughout the study. Size of the SDN-POA and reproductive ability was comparable to those in controls. At the termination (18 weeks of age), however, a reduction in the sperm count in the epididymis of OP-treated animals demonstrated a possibility that the male reproductive system might be still affected by neonatal exposure to OP. The results observed demonstrate that neonatal exposure to a high-dose OP exerts estrogenic action directly or indirectly, resulting in slight but prolonged impairment of the male reproductive tract. The suppression of FSH caused by modulation of the hypothalamus-pituitary control system may be the trigger for the impairment, while the possibility of direct estrogenic action of OP is not ruled out. Our results also indicate that more sensitive endpoints should be established to detect the effects of neonatal exposure to estrogens or estrogenic compounds on the male reproductive tract.

Dose- and treatment duration-related effects of p-tert-octylphenol on female rats.

We conducted a study to investigate the relation between estrogenic effects of p-tert-octylphenol (OP) and serum concentration as well as treatment duration. Adult ovariectomized rats were given daily subcutaneous injections of OP for 2 and 14 days. 17beta-Estradiol was also administrated at a dose of 5 microg/kg. OP was detected in serum at doses of 25 mg/kg and above for 2 days and with multiple doses (14 days) of 12.5 mg/kg. Uterine weights and luminal epithelial height were increased although the effects were weak compared to 17beta-estradiol. Estrous conversion of the vaginal smear was detected only in 14-day-treated animals. OP-treatment for 2 days caused a dose-related increase in proliferation of uterine luminal, glandular, and stromal cells and vaginal epithelial cells. From the 14-day experiment, the minimum estrogenic dose level of OP was concluded to be 25 mg/kg/day and the serum level at the dose was determined to be about 80 ng/ml. These findings demonstrated that OP exerts estrogenic activity in the female reproductive tract of ovariectomized rats only at high levels of exposure and that the effects are fundamentally related to serum OP levels.

Neonatal Exposure to p-tert-octylphenol Causes Abnormal Expression of Estrogen Receptor α and Subsequent Alteration of Cell Proliferating Activity in the Developing Donryu Rat Uterus:

In the present study, we investigated immunohistochemicall y the time-course alterations in estrogen receptor α (ER) expression and cell proliferating activity in the developing uteri of Donryu rats exposed neonatally to a high dose p-tert-octylphenol (OP), an endocrine disrupting chemical (EDC). OP-treatment (sc injections of 100 mg/kg, every other day from postnatal days 1 to 15) induced an early and enhanced ER expression in the luminal epithelium compared with age-matched controls from postnatal day (PND) 10, and increased proliferating cell nuclear antigen (PCNA) positive cells up to PND21. At PND28, ER expression in the luminal epithelium of the OP-treated group was decreased, in association with decline in the luminal epithelial areas. PND14, the second week of life, is coincident with the normal time for differentiation when the luminal epithelium invaginates into the stroma to form uterine glands. OP-treatment, however, delayed and inhibited gland-formation, and suppressed ER expression in the invaginated-luminal and glandular epithelium at this time. These results indicate that ER expression in these sites is strongly linked with cell proliferating activity. In stromal cells, ER was expressed from PND6 in both groups without any PCNA positive cells, but significantly lower values were noted in the OP-treated group up to PND10. Our immunohistochemical investigation did not reveal any abnormalities in expression of the proto-oncogen e c-fos, mitotic inhibitor p21, or epidermal growth factor antigen, although the apoptotic index in the luminal epithelium was slightly increased in the OP-treated group. These results demonstrate neonatal effects of a high dose of OP, already detectable at PND10, with early and enhanced ER expression, resulting in increase of cell proliferative activity in the luminal epithelium, though expression in the glandular epithelium was suppressed in relation to inhibited gland-genesis. The present study thus suggests that neonatal exposure to high doses of EDCs with estrogenic activity can induce abnormal differentiation in the developing rat uteri via abnormal ER expression and subsequent alteration of cell proliferating activity.

Identification and evaluation of anti-inflammatory compounds from Kaempferia parviflora

The rhizome of Kaempferia parviflora has been used in traditional Thai medicine. In this study, we identified and compared specific compounds from the hexane extract of K. parviflora with those from other Zingiberaceous plants by using gas chromatography–mass spectrometry. We identified 5,7-dimethoxyflavone (DMF), 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (TMF), estimated 3,5,7-trimethoxyflavone, 5-hydroxy-7,4′-dimethoxyflavone, 3,5,7,4′-tetramethoxyflavone, and investigated their anti-inflammatory effects in rat basophilic leukemia (RBL-2H3) cells stimulated with an IgE antigen or a calcium ionophore. We found that DMF and TMF more potently inhibited antigen-induced degranulation than did nobiletin, a well-known anti-inflammatory agent. In addition, compared to RBL-2H3 cells stimulated with a calcium ionophore, those treated with DMF and TMF showed more marked inhibition of the degranulation and the production and mRNA expression of inflammatory mediators. These results suggest that DMF and TMF inhibit an early step in the high-affinity IgE receptor signaling cascade rather than intracellular calcium release and protein kinase C activation. Graphical Abstract The extracts from Kaempferia parviflora effectively suppress the antigen-mediated degranulation in RBL-2H3 cells.

Uterine Adenocarcinoma in N-Ethyl-N-nitro-N-nitrosoguanidine-treated Rats with High-dose Exposure to p-tert-Octylphenol during Adulthood

Since many risk factors are associated with the development of uterine adenocarcinomas in humans, the etiology is unclear in most cases, although it has been pointed out that estrogen may play essential roles. To clarify the effects of exposure to p‐tert‐octylphenol (OP), an environmental xenoestrogen, on uterine carcinogenesis, adult Donryu rats were initiated with a single intrauterine treatment of N‐ethyl‐N‐nitro‐N‐nitrosoguanidine (ENNG) at 11 weeks of age and exposed thereafter to 100 mg/kg OP by s.c. injection until 15 months of age. Adult ovariectomized (OVX) rats were also treated in a similar way. OP had no effect on occurrence of persistent estrus in middle age, although uterotrophic effects were obvious in OVX rats. At the termination, development of uterine adenocarcinomas was significantly increased in animals exposed to OP during adulthood. No tumors, but a few focal hyperplasias, developed in OVX rats. These findings suggest that OP has tumor‐promoting effects on ENNG‐treated endometrium of rats, possibly due to direct action on the uterus, as indicated by the uterotrophic effect when a high dose of OP was given. The results provide clues to the mechanisms of influence of hormonal disrupters on uterine carcinogenesis.

Lack of effects of oxolinic acid on spermatogenesis in young adult and aged Wistar rats

Prolonged treatment with oxolinic acid is known to elevate serum luteinizing hormone (LH) levels, resulting in induction of Leydig cell tumors in rats. In a carcinogenicity study of the compound, tubular atrophy of the testis was also increased, suggesting that oxolinic acid might affect spermatogenesis. The present study was therefore performed using rats of different ages with a particular focus on seminiferous tubule alteration and its relation to Leydig cell proliferation. Young adult (7 weeks of age) and aged (52 weeks of age) males of the Wistar strain were administered oxolinic acid at dietary concentrations of 0 (basal diet), 300, 1000 or 3000 ppm for 4 (all groups), 13 (0 and 3000 ppm groups), 26 (0 and 3000 ppm groups), or 52 weeks (0 and 3000 ppm groups of aged rats). Serum LH levels were elevated in both young adult and aged animals treated with 3000 ppm at most examined time points. While testosterone levels were also increased at the early time points in young adult, this was not the case in older animals. Elevation of the incidences of foci and/or focal hyperplasia of Leydig cells was noted but was only slight limited to aged rats treated with 3000 ppm after 26 weeks. Furthermore, it did not appear to be related to seminiferous tubular alteration. No treatment-related histopathological abnormalities could be detected in any treatment group, and morphometrical stage analysis of spermatogenesis conducted for the control and 3000 ppm-treated groups demonstrated no lesions. These results provide strong evidence that prolonged oxolinic treatment does not directly induce testicular toxicity or altered spermatogenesis in either young adult or aged rats, except for slight increase of Leydig cell proliferative lesions caused by elevated serum LH levels. Aged rats might have higher sensitivity than young adults to the effects of oxolinic acid on proliferative lesions of Leydig cells.

Involvements of Estrogen Receptor, Proliferating Cell Nuclear Antigen and p53 in Endometrial Adenocarcinoma Development in Donryu Rats.

Involvements of estrogen receptor (ER)α, proliferating cell nuclear antigen (PCNA) and p53 in the uterine carcinogenesis process in Donryu rats, a high yield strain of the uterine cancer were investigated immunohistochemically. ERα was expressed in atypical endometrial hyperplasia, accepted as a precancerous lesion of the uterine tumors, as well as well- and in moderately-differentiated endometrial adenocarcinomas, and the intensities of expression were similar to those in the luminal epithelial cells of the atrophic uterus at 15 months of age. The expression, however, was negative in the tumor cells of poorly differentiated type. Good growth of implanted grafts of the poorly-differentiated adenocarcinomas in both sexes with or without gonadectomy supported the estrogen independency of tumor progression to malignancy. PCNA labeling indices were increased with tumor development from atypical hyperplasia to adenocarcinoma. The tumor cells in poorly-differentiated adenocarcinomas were positive for p53 positive but negative for p21 expression, suggesting accumulation of mutated p53. These results indicate that the consistent ERα expression is involved in initiation and promotion steps of uterine carcinogenesis, but not progression. In addition, PCNA is related to tumor development and the expression of mutated p53 might be a late event during endometrial carcinogenesis.

Inhibitory effects of Kaempferia parviflora extract on monocyte adhesion and cellular reactive oxygen species production in human umbilical vein endothelial cells

PurposeThe rhizome of Kaempferia parviflora (KP) is used in traditional Thai medicine. In this study, we investigated the effects of an ethanol KP extract and two of its components [5,7-dimethoxyflavone (DMF) and 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (TMF)] on monocyte adhesion and cellular reactive oxygen species (ROS) production in human umbilical vein endothelial cells (HUVECs), which provide an in vitro model of events relevant to the development and progression of atherosclerosis.MethodsRAW264.7 mouse macrophage-like cells were incubated with various concentrations of KP extract or polymethoxyflavonoids and stimulated with lipopolysaccharide prior to measuring nitrite levels in the culture media. Monocyte adhesion was evaluated by measuring the fluorescently labeled human monocytic leukemia THP-1 cells that is attached to tumor necrosis factor-α (TNF-α)-stimulated HUVECs. Cellular ROS production was assessed by measuring cellular antioxidant activity using pyocyanin-stimulated HUVECs.ResultsKP extract and DMF reduced nitrite levels (as indicator of nitric oxide production) in LPS-stimulated RAW264.7 cells and also inhibited THP-1 cell adhesion to HUVECs. These treatments induced mRNA expression of endothelial nitric oxide synthase in TNF-α-stimulated HUVECs and downregulated that of various cell adhesion molecules, inflammatory mediators, and endothelial function-related genes. Angiotensin-converting enzyme activity was inhibited by KP extract in vitro. Furthermore, KP extract, DMF, and TMF inhibited the production of cellular ROS in pyocyanin-stimulated HUVECs.ConclusionKP extract, DMF, and TMF showed potential anti-inflammatory and antioxidant effects in these in vitro models, properties that would inhibit the development and progression of atherosclerosis.