Neonatal exposure to SERMs disrupts neuroendocrine development and postnatal reproductive function through alteration of hypothalamic kisspeptin neurons in female rats.

Selective estrogen receptor modulators (SERMs) are a class of therapeutic chemicals which present tissue-specific estrogen receptor modulating activity. Neonatal exposure to SERMs has been reported to adversely affect central nervous system development, however, mechanism and involvement of hypothalamic kisspeptin neurone in this impairment remains undetermined. To clarify this uncertainty, neonates from female Donryu rats were subcutaneously injected with raloxifene (RLX) at 0.1, 1, and 10mg/kg or tamoxifen (TMX) at 10mg/kg on postnatal day 0, and then hypothalamic KiSS1 mRNA expression and gonadotropin levels were investigated during young adulthood and estrous cycling was monitored until middle age. Treatment with RLX or TMX at 10mg/kg significantly depressed luteinizing hormone surge levels and KiSS1 mRNA expression in the anteroventral periventricular nucleus (AVPV), the control center of estrous cyclicity. The 10mg/kg TMX group also showed decreased levels of follicle-stimulating hormone and KiSS1 mRNA expression in the arcuate nucleus (ARC). Early cessation of normal estrous cycling was observed in the 10mg/kg RLX group, while the estrous cycle in the 10mg/kg TMX group had ceased by the start of the analysis. The same dose of tamoxifen or raloxifene had either weak-estrogenic or anti-estrogenic activity on the uterus, respectively; however, treatment in adulthood with both SERMs did not affect KiSS1 mRNA expression in either the AVPV or ARC in the present study. These results indicate that neonatal exposure to SERMs could disrupt neuroendocrine development and postnatal reproductive function through the alteration of kisspeptin neurons.

Dose-dependent difference of nuclear receptors involved in murine liver hypertrophy by piperonyl butoxide.

Nuclear receptors play important roles in chemically induced liver hypertrophy in rodents. To clarify the involvement of constitutive androstane receptor (CAR) and other nuclear receptors in mouse liver hypertrophy induced by different doses of piperonyl butoxide (PBO), wild-type and CAR-knockout mice were administered PBO (200, 1,000, or 5,000 ppm) in the basal diet for 1 week. Increased liver weight and diffuse hepatocellular hypertrophy were observed at 5,000 ppm for both genotypes, accompanied by increased Cyp3a11 mRNA and CYP3A protein expression, suggesting that CAR-independent pathway, possibly pregnane X receptor (PXR), plays a major role in the induction of hypertrophy. Moreover, wild-type mice at 5,000 ppm showed enhanced hepatocellular hypertrophy and strong positive staining for CYP2B in the centrilobular area, suggesting the localized contribution of CAR. At 1,000 ppm, only wild-type mice showed liver weight increase and centrilobular hepatocellular hypertrophy concurrent with elevated Cyp2b10 mRNA expression and strong CYP2B staining, indicating that CAR was essential at 1,000 ppm. We concluded that high-dose PBO induced hypertrophy via CAR and another pathway, while lower dose of PBO induced a pathway mediated predominantly by CAR. The dose-responsiveness on liver hypertrophy is important for understanding the involvement of nuclear receptors.

The Critical Hormone-Sensitive Window for the Development of Delayed Effects Extends to 10 Days after Birth in Female Rats Postnatally Exposed to 17alpha-Ethynylestradiol.

Neonatal exposure to estrogens is known to cause delayed effects, a late-occurring adverse effect on adult female reproductive functions, such as early onset of age-matched abnormal estrous cycling. However, the critical period in which neonates are sensitive to delayed effects inducible by exogenous estrogen exposure has not been clearly identified. To clarify this window, we examined the intensity and timing of delayed effects using rats exposed to ethynylestradiol (EE) at various postnatal ages. After subcutaneous administration of a single dose of EE (20 µg/kg, which induces delayed effects) on Postnatal Day (PND) 0, 5, 10, or 14 in Wistar rats, hypothalamic and hormonal alterations in young adults and long-term estrous cycling status were investigated as indicators of delayed effects. In young adults, peak luteinizing hormone concentrations at the time of the luteinizing hormone surge showed a decreasing trend, and KiSS1 mRNA expression of the anterior hypothalamus and number of KiSS1-positive cells in the anteroventral periventricular nucleus were significantly decreased in the PND 0, 5, and 10 groups. The reduction in KiSS1 mRNA and KiSS1-postive cells was inversely correlated with age at time of exposure. These groups also exhibited early onset of abnormal estrous cycling, starting from 17 wk of age in the PND0 group and 19 wk of age in the PND5 and 10 groups. These indicators were not apparent in the PND14 group. Our results suggest that PND0-PND10 is the critical window of susceptibility for delayed effects, and PND14 is presumed to be the provisional endpoint of the window.

Essential role of constitutive androstane receptor in Ginkgo biloba extract induced liver hypertrophy and hepatocarcinogenesis.

Ginkgo biloba extract (GBE) is commonly used as a herbal supplement. The National Toxicology Program (NTP) study of GBE reported clear evidence of hepatocarcinogenicity in mice. To clarify the mode of action (MOA) for hepatocarcinogenesis by GBE, we investigated the involvement of the constitutive androstane receptor (CAR) in hepatocarcinogenesis induced by GBE using CAR-knockout (CARKO) and wild type (WT) mice. We used the same lot of GBE that was used for the NTP study. In 1-week GBE dietary treatment, hepatocellular DNA replication was increased in WT mice but not in CARKO mice. In 4- or 13-week treatment, greater hepatic Cyp2b10 induction and hepatocellular hypertrophy were observed in WT mice, whereas these effects of GBE were much smaller in CARKO mice. In a two-stage hepatocarcinogenesis model initiated by diethylnitrosamine, 27-week treatment with GBE resulted in an increase of eosinophilic altered foci and adenomas in WT mice. By contrast, foci and adenomas were clearly less evident in CARKO mice. These results indicate that GBE-induced hepatocarcinogenesis is mainly CAR-mediated. Since CAR-mediated MOA for hepatocarcinogenesis in rodents is considered to be qualitatively implausible for humans, our findings would be helpful to evaluate the carcinogenic characterization of GBE to humans.

Early indicators of delayed adverse effects in female reproductive organs in rats receiving neonatal exposure to 17alpha-ethynylestradiol.

We previously reported that neonatal exposure to 17α-ethynylestradiol (EE) led to delayed adverse effects in which age-related anovulation after sexual maturation was accelerated. To identify early indicators of these adverse effects, female Wistar Hannover GALAS rats received a single EE injection (0, 0.02, 0.2, 2, 20, or 200 µg/kg) within 24 hr of birth. Histopathological changes in ovarian and uterine development were investigated from postnatal day (PND) 14 to 10 weeks of age. Immunohistochemical expression of estrogen receptor alpha (ERα) in the uterus, serum levels of sex-related hormones and gene expression in the hypothalamus were examined. Although neonatal exposure to EE did not affect body growth or ovarian development, serum FSH tended to decrease at doses ≥ 2 µg/kg, and Kiss1 mRNA level in the whole hypothalamus was significantly decreased in all EE-treated groups at PND14.The number of uterine glands at PND21 was suppressed at doses ≥ 20 µg/kg, and ERα expression in the uterine epithelium at estrus stage decreased in a dose-dependent manner at 10 weeks of age. These results demonstrated that the various identified changes that occurred before the appearance of delayed adverse effects could be candidate early indicators.

Adaptive parotid gland hypertrophy induced by dietary treatment of GSE in rats.

In a 13-week feeding toxicity study of grape skin extract (GSE) performed previously, 5.0% GSE showed diffuse hypertrophy and basophilia in rat parotid glands. To clarify whether the change in the parotid glands was an adverse effect of GSE, 6-week-old male F344 rats were fed a diet containing 5.0% GSE or were administered a dose corresponding to the dietary concentration via gavage for 4 weeks, and the treatment was stopped for 2 weeks. To ascertain the effect of astringency, other animals were fed a diet containing 5.0% tannic acid (TA) using the same protocol as the GSE feed group. Control groups were fed a basal diet or were administered sterilized distilled water by gavage. In the GSE and TA feed groups, diffuse severe hypertrophy and basophilia in the parotid glandular epithelial cells were observed. Macroscopic, microscopic, and ultrastructural characteristics consistent with cellular hypertrophy was less apparent after the recovery period in both feed groups. In contrast, no changes were observed in the parotid glands of the gavage GSE and control groups at week 4. Based on these findings of parotid hypertrophy without cytotoxicity, the data from this and previous studies suggest that hypertrophy of the parotid glands induced by feeding treatment with GSE is an adaptive non-adverse effect that is reversible upon removal of the sialotrophic agent.

A 13-week subchronic toxicity study of grape skin extract in F344 rats.

A 13-week repeated oral dose toxicity study of grape skin extract (GSE) was performed using F344 rats. Four groups of animals, each consisting of ten males and ten females, were fed a diet containing 0%, 0.2%, 1.0% or 5.0% GSE for 13 weeks. Throughout the experiment, there were no treatment-related changes in clinical signs, body weight or mean food intake in any of the treated groups of either gender. Hematological studies and serum biochemical analyses revealed no treatment-related changes in all groups in both genders. In the glandular epithelial cells of the parotid glands, diffuse hypertrophy and basophilia was observed in all animals in both 5.0% groups. Hypertrophy of the parotid glands was not detected in the 0.2% or the 1.0% dose groups. In female kidneys, slight calcification in the renal proximal tubules of the cortex and medulla was observed in all groups including controls. This is a common spontaneous change in female rats, and the incidence was comparable between controls and treated groups. However, the number of tubules with calcification was higher in the 5.0% group based on a semi-morphometric analysis. Based on the histopathology of the parotid glands and the minor change in the kidneys, the no observed adverse effect level (NOAEL) of GSE in the present study was a 1.0% treatment dose in both genders (males: 0.6 ± 0.2 g/kg body weight/day; females: 0.7 ± 0.1 g/kg body weight/day).

Carcinogenic potential of alizarin and rubiadin, components of madder color, in a rat medium-term multi-organ bioassay.

Madder color (MC), a food coloring extracted from roots of Rubia tinctorum L., has been proven to exert carcinogenicity in the rat kidney and liver. Furthermore, it induces DNA adducts in the kidney, liver, and colon. MC is in fact composed of anthraquinones such as lucidin-3-O-primeveroside and alizarin. To clarify which of these might be responsible for the carcinogenicity, a rat medium-term multi-organ carcinogenesis bioassay was performed focusing on the kidney, liver, and colon. Male 6-week-old F344 rats after receiving five different carcinogens were fed a diet containing either 0.008% or 0.04% of alizarin or rubiadin, a metabolite of lucidin-3-O-primeveroside, for 23 weeks. Treatment with 0.04% rubiadin significantly increased atypical renal tubules/hyperplasias and induced renal cell adenomas and carcinomas. Renal cell tumors were also increased with 0.04% alizarin, although at lower incidence than with rubiadin. In addition, glutathione S-transferase placental form-positive liver cell foci and large intestinal dysplasias were significantly increased with 0.04% rubiadin. These results indicate that both rubiadin and alizarin can increase renal preneoplastic lesions, the potential of the latter being weaker. Rubiadin may also target the liver and large intestine, suggesting a major role in madder color-induced carcinogenicity.

Possible contribution of rubiadin, a metabolite of madder color, to renal carcinogenesis in rats.

Madder color (MC) has been shown to exert carcinogenic potential in the rat kidney in association with degeneration, karyomegaly, increased cell proliferation of renal tubule cells and increased renal 8-OHdG levels. To clarify the causal relationship of components and metabolites of MC to renal carcinogenesis, male F344 rats were fed lucidin-3-O-primeveroside (LuP) or alizarin (Alz), and the genotoxic LuP metabolites lucidin (Luc) or rubiadin (Rub) for up to 26 weeks. After one week and four weeks, Luc did not induce any renal changes. In contrast, after one week, cortical tubule degeneration was apparent in the Alz and LuP groups, and cytoplasmic swelling with basophilic change and karyomegaly in the outer medulla was observed only in the Rub group. LuP and Rub increased the proliferative activity of tubule cells in the outer medulla, and Alz and LuP increased renal 8-OHdG levels. After 26 weeks, Rub but not Alz induced atypical tubules, a putative preneoplastic lesion, and karyomegaly in the outer medulla. These results indicate that Rub may be a potent carcinogenic metabolite of MC, targeting proximal tubule cells in the outer medulla, although oxidative stress increased by Alz or LuP might also be involved in renal carcinogenesis by MC.

Induction of kidney and liver cancers by the natural food additive madder color in a two-year rat carcinogenicity study.

Madder color (MC) extracted from the roots of Rubia tinctorum (madder root) has been used as a food coloring in Japan. Our previous studies revealed MC to have obvious subchronic and chronic toxicity and potent carcinogenicity targeting rat liver and kidney. In the present two-year carcinogenicity study, conducted to further elucidate the long-term effects of MC and its target organs, male and female F344 rats were fed diet containing 0%, 2.5%, and 5.0% MC for 104 weeks. Body weights were significantly decreased in treated groups of both sexes throughout the feeding period. However, survival rates at week 104 were higher in treated groups of both sexes than in controls. Relative weights of the kidneys and liver were significantly increased in treated groups of both sexes. Histopathologically, karyomegaly and atypical tubules/hyperplasias, as well as renal cell adenomas and carcinomas were significantly increased in treated groups of both sexes with dose-dependence. Moreover, the incidence of hepatocellular adenomas and/or carcinomas was increased significantly with a dose-relation in treated groups of both sexes. These data provide clear evidence that MC exerts unequivocal carcinogenicity against renal tubule cells and hepatocytes in rats.

One-year chronic toxicity of madder color in F344 rats--induction of preneoplastic/neoplastic lesions in the kidney and liver.

To evaluate chronic toxicity of madder color (MC), a natural food colorant extracted from the roots of Rubia tinctorum L., F344 rats were fed diet containing 0%, 0.2%, 1.0% or 5.0% MC for 53 weeks. Hematological changes including anemia and serum biochemical alterations indicating hepatotoxicity were demonstrated at 5.0% in both sexes. Relative weights of the liver were significantly increased from 1.0% in both sexes, and those of the kidney were significantly increased from 1.0% in males and from 0.2% in females. Histopathologically, atypical renal tubule hyperplasias were increased at 1.0% or higher in both sexes in association with increase of cell proliferative activity in the tubules. A renal cell adenoma was observed in a male rat receiving 5.0% MC. In addition, glutathione S-transferase placental form-positive liver cell foci were significantly increased at 5.0% in both sexes. These results indicate that MC has chronic toxicity targeting kidney, liver and blood cells. Moreover, the results strongly suggest that MC may have the carcinogenic potential in the kidney and the liver.

Lack of preventive effects of dietary fibers or chlorophyllin against acrylamide toxicity in rats.

Dietary fibers and chlorophyllin have shown to exert anti-carcinogenic effects against co-administered carcinogens. To test the possibility of chemoprevention by such dietary supplements on subacutely induced acrylamide (ACR) toxicity, Sprague-Dawley male rats were administered 2.5% sodium alginate, 5% glucomannan, 5% digestion resistant maltodextrin, 2.5% chitin or 1% chlorophyllin in the diet, and starting one week later, co-administered 0.02% ACR in the drinking water for 4 weeks. For comparison, untreated control animals given basal diet and tap water were also included. Neurotoxicity was examined with reference to gait abnormalities and by quantitative assessment of histopathological changes in the sciatic and trigeminal nerves, as well as aberrant dot-like immunoreactivity for synaptophysin in the cerebellar molecular layer. Testicular toxicity was assessed by quantitation of seminiferous tubules with exfoliation of germ cells into the lumen and cell debris in the ducts of the epididymides. Development of testicular toxicity as well as neurotoxicity was evident with ACR-treatment, but was not suppressed by dietary addition of fibers or chlorophyllin, suggesting no apparent beneficial influence of these dietary supplements on experimentally induced subacute ACR toxicity.

Promoting potential of a Jamaica quassia extract in a rat medium-term hepatocarcinogenesis bioassay.

Jamaica quassia extract (JQE), a natural bittering agent, was investigated for hepatocarcinogenesis-promoting potential using a medium-term liver bioassay system. F344 male rats were given a single intraperitoneal injection of diethylnitrosamine (200mg/kg body weight) and then starting 2 weeks later, received JQE in the diet at concentrations of 500, 5000 or 30,000 ppm for 6 weeks. Animals for tumor promotion (+) and (-) controls were fed 500 ppm sodium phenobarbital (PB) and basal diet, respectively during the promotion phase in this model. All animals were subjected to two-thirds partial hepatectomy at week 3 and killed at week 8. As with the PB-promoted case, both numbers and areas of glutathione S-transferase placental form-positive liver cell foci were significantly increased by JQE at 30,000 ppm, with non-significant increases evident at 5000 ppm. The results thus indicate that JQE at high dose has promoting potential for rat hepatocarcinogenesis.

Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatocarcinogenesis in mice.

Piperonyl butoxide (PBO), alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylene-dioxy-2-propyltoluene, is widely used as a synergist for pyrethrins. In order to clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by PBO, molecular pathological analyses consisting of low-density microarray analysis and real-time reverse transcriptase (RT)-PCR were performed in male ICR mice fed a basal powdered diet containing 6000 or 0 ppm PBO for 1, 4, or 8 weeks. The animals were sacrificed at weeks 1, 4, and 8, and the livers were histopathologically examined and analyzed for gene expression using the microarray at weeks 1 and 4 followed by real-time RT-PCR at each time point. Reactive oxygen species (ROS) products were also measured using liver microsomes. At each time point, the hepatocytes of PBO-treated mice showed centrilobular hypertrophy and increased lipofuscin deposition in Schmorl staining. The ROS products were significantly increased in the liver microsomes of PBO-treated mice. In the microarray analysis, the expression of oxidative and metabolic stress-related genes--cytochrome P450 (Cyp) 1A1, Cyp2A5 (week 1 only), Cyp2B9, Cyp2B10, and NADPH-cytochrome P450 oxidoreductase (Por) was over-expressed in mice given PBO at weeks 1 and 4. Fluctuations of these genes were confirmed by real-time RT-PCR in PBO-treated mice at each time point. In additional real-time RT-PCR, the expression of Cyclin D1 gene, key regulator of cell-cycle progression, and Xrcc5 gene, DNA damage repair-related gene, was significantly increased at each time point and at week 8, respectively. These results suggest the possibility that PBO has the potential to generate ROS via the metabolic pathway and to induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in mice.