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Dietary intake of methylmercury from fish was estimated via Monte Carlo simulation using data for methylmercury concentrations in 210 fish samples and data regarding fish consumption extracted from the Japanese National Health and Nutrition Survey. The fish analyzed were classified into 5 groups according to categories used in the survey. The distribution of consumption of fish from each group was used without fitting to statistical distributions. A log-normal distribution was fitted to the distribution of methylmercury concentration in each fish group. Two random numbers that followed these distributions were generated, and a trial value was calculated by multiplying these random numbers. The trial value was divided by the body weight (50 kg) to arrive at an estimate of dietary methylmercury intake. A total of 100,000 Monte Carlo simulation iterations were performed. The estimated mean daily intake of methylmercury was 0.093 µg/kg body weight (bw)/day. This value is well below the tolerable daily intake of 0.292 µg/kg bw/day calculated from the tolerable weekly intake (2.0 µg/kg bw/week) established by the Food Safety Commission of Japan. The probability that the daily intake of methylmercury exceeds the tolerable daily intake was 7.6%. As there are no data regarding fish consumption for consecutive days, estimation of the weekly intake of methylmercury is a subject for future studies.

A 13-week repeated oral dose toxicity study of madder color (MC), a natural food colorant extracted from the roots of Rubia tinctorum L., was performed using F344 rats. Five groups of animals, each consisting of 10 males and 10 females, were fed diet containing 0, 0.6, 1.2, 2.5 or 5.0% MC for 13 weeks. During the experiment, lower body weight was evident from the 2.5% dose. Hematologically, fluctuation in red blood cell (RBC) parameters suggestive of weak anemia (females), and slight increases of platelet counts (both sexes) and white blood cell (WBC) counts (males) were observed at higher doses. Serum biochemically, slight fluctuations were observed in many parameters, including increased total protein (TP), conjugated bilirubin, Ca, and inorganic phosphate, and decrease of the albumin/globulin (A/G) ratio in both sexes, with dose-dependence for TP and A/G from 0.6% in females. Histopathological changes were mainly observed in the renal proximal tubules, such as microvesicular vacuolar degeneration in the cortex and karyomegaly in the outer medulla involving both sexes, lesions being evident even with 0.6%. In the outer medulla, elevation of cell proliferation activity as assessed with proliferating cell nuclear antigen was observed in males from 2.5%. Severity of focal necrosis of hepatocytes was increased only in females at 5.0%, while the increased relative liver weight as with the increased conjugated bilirubin was evident in both sexes from 1.2%. The results thus suggest that MC exerts mild toxicity, targeting liver, kidneys, and possibly RBCs and WBCs, some renal changes being evident from 0.6% in diet, that is attributable to be the lowest-observed adverse effect level (305.8–309.2 mg/kg body weight/day).

Two potential endocrine-disrupting chemicals, bisphenol A (BPA) and nonylphenol (NP), were assessed for their long-lasting effects on endocrine/reproductive systems following transplacental and lactational exposure to rat offspring during a time-window that included the critical period for brain sexual differentiation. Each chemical was mixed with diet at concentrations of 60, 600 and 3000 ppm and was provided to maternal Sprague-Dawley rats from gestational day (GD) 15 to postnatal day (PND) 10. Ethinylestradiol (EE) at 0.5 ppm was used as an estrogenic reference drug. During pregnancy and lactation, including the exposure period, a soy-free rodent diet was provided to eliminate possible modification of the study results by plant-derived phytoestrogens. Effects on endocrine/reproductive systems were evaluated by examining the anogenital distance, organ weights before puberty, onset of puberty, estrous cyclicity, and organ weights and histopathology of adult endocrine organs (at 11 weeks of age), as well as the volume of the sexually dimorphic nucleus of preoptic area. Both NP and BPA, at high doses, caused decreases in maternal body weights and retardation of offspring growth, but neither affected any of the endocrine/reproductive endpoints of offspring, whereas EE induced irreversible changes in estrous cyclicity and histopathology of ovaries and uterus of adult females. The results indicated that maternal dietary exposure to NP or BPA at concentrations up to 3000 ppm from GD 15 through PND 10 do not exert any apparent adverse effects on the endocrine/reproductive systems of offspring.

To evaluate the impact of dietary exposure to endocrine disrupting chemicals (EDCs) during the sensitive period of brain sexual differentiation, maternal Sprague–Dawley rats were fed three representative chemicals, methoxychlor (MXC; 24, 240, and 1200 ppm), genistein (GEN; 20, 200, and 1000 ppm), or diisononyl phthalate (DINP; 400, 4000, and 20,000 ppm), from gestational day 15 to postnatal day 10. Soy-free diet was used as a basal diet to eliminate possible estrogenic effects from the standard diet. Offspring were examined in terms of anogenital distances, prepubertal organ weights, onset of puberty, estrous cyclicity, and organ weights and histopathology of endocrine organs at adult stage (week 11) as well as the volumes of sexually dimorphic nucleus of preoptic area (SDN-POA). All chemicals caused signs of maternal toxicity at high doses. MXC, at 1200 ppm, facilitated and delayed the onset of puberty in females and males, respectively, females also showing endocrine disrupting effects thereafter, such as irregular estrous cyclicity and histopathological alterations in the reproductive tract and anterior pituitary. GEN, at all doses, reduced body weight (BW) at week 11, but did not affect endocrine parameters. Treatment with DINP at 20,000 ppm resulted in degeneration of meiotic spermatocytes and Sertoli cells in the testis and decrease of corpora lutea in the ovary at week 11, although changes remained minimal or slight. The SDN-POA volume remained unchanged with all three chemicals. The results demonstrated that perinatal dietary exposure to EDCs for a limited period causes endocrine disruption in offspring only at high doses.

In search of genes that are steadily up-regulated during the promotion stage in carcinogenesis, suppression PCR subtractive hybridization and following northern blot screening were performed using a phenobarbital (PB)-promotion model based on a medium-term liver bioassay. Two weeks after a single injection of diethylnitrosamine (DEN; 200 mg/kg body wt, i.p.), rats were given 600 p.p.m. PB in the drinking water for up to 64 weeks. For comparison, animals fed 1 p.p.m. ethinylestradiol (EE) or 3000 p.p.m. butylated hydroxytoluene (BHT) in the diet at promotion stage were also included. Rats were subjected to partial hepatectomy (PH) at week 3. In addition, dose-dependence of PB at week 8 of promotion and responsiveness to representative non-genotoxic carcinogens without DEN initiation were examined. Fragments of a total of 67 different genes were isolated from the up-regulated gene population in the liver at day 10 of PB treatment by subtracting from basal expression of DEN + PH alone. Using northern blot screening for signal-detectable 48 genes, 16 genes showed up-regulation in the livers at week 8 of promotion, common to the PB and EE treatments with the levels being three times or more than the basal expression of unpromoted liver. The majority of these genes were also up-regulated at week 8 by BHT treatment, and were also constitutively expressed in the DEN(-), PH(-) untreated rat livers. Among the up-regulated genes common to the PB and EE promotion, and not responding to the non-genotoxic carcinogens in uninitiated liver, the following six genes showed overexpression in PB-promoted hepatocellular carcinomas at week 64, with the levels three times or more than untreated rat liver: ubiquitously expressed mammalian ABC half transporter, apolipoprotein A4, nuclear receptor binding factor-2, CD81, hypothetical protein (HSPC014) and one unidentified gene. These genes might be candidates for biomarkers in screening of non-genotoxic hepatocarcinogens by analysis in two-stage carcinogenesis models.

In the present study, we reviewed food poisoning data published by the Ministry of Health, Labour and Welfare of Japan from 1961 to 2010 in Japan to analyze the trends of plant toxin food poisonings (PTFPs). The annual number of incidents of PTFPs has gradually increased since 1995. PTFPs were predominantly caused by Datura spp., Veratrum spp. and Aconitum spp. Although PTFPs frequently occurred in April and May, poisonings caused by some plants occurred in all seasons, e.g. Datura spp. The major location of PTFPs was "at home", and most of the patients had accidentally harvested poisonous plants. During the past decade, the numbers of incidents of PTFPs caused by Veratrum spp., Narcissus spp., Solanum tuberosum and Alocasia odora were especially increased. Consumer advice to prevent PTFPs associated with certain plants that are cultivated in gardens and contain toxic substances is needed, because PTFPs caused by such plants are increasing. In addition, education of elementary school teachers and children about the potential risks of natural toxins in plants, particularly Solanum tuberosum, is desirable.

Interrelationships among induction of cytochrome P-450 (CYP) 1A1/2, decrease in connexin 32 (Cx32), and liver tumor-promoting activity by β-naphthoflavone (BNF) in the promotion stage were examined in a 2-stage liver carcinogenesis model. A total of 20 male Fischer 344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone. Starting 2 weeks later, they were fed a diet containing 2%, 1%, or 0% BNF for 6 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Absolute and relative liver weights were significantly increased in the DEN+BNF groups as compared to the DEN-alone group. Diffuse hepatocellular hypertrophy with cytoplasmic eosinophilia, sometimes accompanied by development of adenoma-like hepatic foci, was observed in the BNF-treated rats. Remarkable induction of cytochrome CYP 1A1/2 and significant increase in CYP 2E1 were noted in the DEN+BNF groups, and positive immunohistochemical staining for both was observed diffusely. The areas of Cx32-positive spots per hepatocyte in the centrilobular areas of livers of the BNF-treated rats were significantly decreased, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form were increased in the BNF-treated groups. These results suggest that BNF is a liver tumor promoter that, unlike phenobarbital, does not induce CYP 2B1/2 isozymes, and there seems to be no direct relationship between CYP 1A1/2 induction and Cx32 reduction in BNF hepatocarcinogenesis.

The carcinogenicity of 5-fluorouracil (5-FU), a compound employed as an antineoplastic drug, was investigated in F344 rats of both sexes. 5-FU was administered to groups of 50 male and 50 female rats ad lib. for 104 weeks, added to drinking water at concentrations of 0 (control), 62 and 125 ppm, these dose levels being selected on the basis of results of a 13-week subchronic toxicity study. Body weight gains were slightly depressed in the 125 ppm group of both sexes. While not statistically significant in females, final survival rates at week 111 in the 125 ppm group of both sexes were higher than those in the control group, suggesting an ability of 5-FU to prolong the lifespan. Histopathologically, a decreased incidence of islet cell adenomas in males and increased incidences of pituitary gland adenomas and pheochromocytomas in females were observed in the 62 ppm group without dose dependence. There was no significant induction of any other neoplastic or non-neoplastic lesions. These results indicate a lack of carcinogenicity of 5-FU under the present experimental conditions using rats.

To examine the relationship between the decrease in connexin 32 (Cx32) and induction of P450 isozymes in the early phase of clofibrate hepatocarcinogenesis, a total of 20 male F344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or given the saline vehicle alone and starting 2 weeks later given diet containing 0.18, 0.09, and 0% clofibrate for 6 weeks. All animals were subjected to two-thirds partial hepatectomy at week 3 and killed at week 8. Absolute and relative (ratios to body weight) liver weights were significantly increased in the DEN + clofibrate groups compared with the DEN-alone group. Diffuse hepatocellular hypertrophy with granular cytoplasmic eosinophilia characterized by a marked increase in peroxisomes and smooth endoplasmic reticulum, was observed in the clofibrate treated rats. Induction of cytochrome P450 (CYP) 4A1 and 2B1/2 was noted in the DEN + clofibrate groups, this being most marked in the CYP 2B1 case. Immunohistochemically, positive immunostaining for anti-CYP 4A1 and CYP 2B1 were observed diffusely and centrilobularly, respectively. The numbers and areas of Cx32-positive spots per hepatocyte in the centrilobular areas in the treated rats were significantly decreased in an essentially dose-dependent manner, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form (GST-P) were decreased in a dose dependent manner in the clofibrate treated groups. These results suggest that the CYP 2B1/2 induction and Cx32 decrease in centrilobular hepatocytes, similarly to those thought to be involved in the hepatic promotion mechanism of phenobarbital, may also play important roles in clofibrate actions in the liver, in addition to its causation of oxidative DNA injury.

To clarify whether apoptosis is involved in doxorubicin (DXR)-induced testicular toxicity and to identify the target germ cell type, adult Sprague-Dawley rats were treated with a single intravenous dose of DXR (8 or 12 mg/kg) and euthanized at 3, 6, 12, 24, and 48 h subsequently. Histologically, germ cell degeneration was first found 6 h after dosing in meiotically dividing spermatocytes and early round spermatids of seminiferous tubules at stage 1, and subsequently observed in spermatogonia at stages I-VI showing ultrastructural characteristics of apoptosis. Coincident with the appearance of morphological changes, degenerating germ cells were shown to be undergoing apoptosis as revealed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The frequency of TUNEL-labeled germ cells increased in a stage- and cell type-specific manner, the peak of frequency gradually progressing from stage I of seminiferous tubules to later stages with time after dosing, suggesting that the damaged germ cells, especially spermatogonia, gradually underwent the processes leading to apoptosis. DNA laddering on gel electrophoresis was apparent 24 and 48 h after dosing. The results demonstrate that apoptosis plays an important role in the induction of testicular toxicity caused by DXR with meiotically dividing spermatocytes and type A and intermediate spermatogonia as highly vulnerable target cells.

Nitrobenezene (NB) produces germ cell degeneration, especially of spermatocytes in rats. To examine the possible involvement of apoptosis in this process, the extent and nature of nuclear DNA fragmentation after NB dosing were assessed using both terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and DNA gel electrophoresis, in addition to conventional histological and electron microscopic procedures. Adult Sprague Dawley rats were treated with a single oral dose of NB (250 mg/kg) and euthanized subsequently at 6, 12, and 24 h and 2, 3, 5, and 7 days. The earliest morphological signs of germ cell degeneration in testes were found in pachytene spermatocytes 24 h after dosing. Electron micrographs of degenerating spermatocytes showed marked nuclear chromatin condensation at the nuclear periphery and crowding of cytoplasmic constituents, which are characteristic of apoptosis. Coincident with the appearance of such morphological changes, degenerating spermatocytes contained fragmented DNA as revealed by TUNEL. The presence of DNA laddering, a hallmark of apoptosis on gel electrophoresis, was first apparent and most prominent at 24 h, gradually becoming less detectable. No such changes were observed up to 12 h after dosing or in control animals. These results demonstrated unequivocal involvement of apoptosis in the induction of germ cell degeneration caused by NB.

The influence of phenethyl isothiocyanate (PEITC) on cell kinetics in the target organs of N‐nitroso‐bis(2‐oxopropyl)amine (BOP) tumorigenicity and on xenobiotic‐metabolizing enzymes was investigated in hamsters. Female 5‐week‐old Syrian hamsters were given a single s.c. dose of 0, 20 or 50 mg/ kg of BOP 2 h after receiving PEITC by gavage at a dose of 0, 100 or 250 μmol/animal (0, 16.3 or 40.8 mg/animal). Six and 22 h after the BOP administration, hamsters were killed and tissues were sampled. Proliferating cell nuclear antigen immunohistochemistry demonstrated significant reduction (P< 0.05–0.001) by PEITC of the labeling indices in the pancreatic acini and ducts, bronchioles, and renal tubules of the BOP‐treated animals in a dose‐dependent manner. In the lungs, the PEITC pre treat merit significantly (P< 0.001) reduced the 06‐methyldeoxyguanosine levels as compared to the BOP‐alone value. Immunoblot analysis of liver cytochrome P450 isoenzymes showed CYP 2B1 to be mainly involved in the metabolic activation of BOP. PEITC significantly (P

To examine whether oxfendazole has tumour-promoting activity, a total of 100 male Fisher 344 rats were initiated with a single ip injection of 100 mg/kg of diethylnitrosamine (DEN) or given saline vehicle alone and starting 1 wk later given diet containing 500, 250, 100, 10 or 0 ppm of oxfendazole for 8 wk. Sub-groups of five rats each from the DEN plus 250 and 0 ppm groups were killed after wk 1 of oxfendazole treatment and the remaining animals at wk 8. At the termination relative liver weights were significantly increased in the DEN-initiated and non-initiated groups treated with 250 ppm and 100 ppm or more, respectively, compared with the corresponding controls values. Light microscopical examination showed centrilobular hepatocellular hypertrophy in all animals receiving 100 ppm or more. Electron microscopy also revealed marked increases in smooth endoplasmic reticulum in hepatocytes of the DEN plus 500 ppm group. Furthermore, induction of cytochrome P-450 (CYP) 1A1/2, 2B1/2 or 4A1 was observed in the DEN plus 100 ppm group, that of CYP 1A1/2 being most marked. A similar change in CYP 1A1/2 was seen in the DEN plus 10 ppm group. The numbers and areas of connexin 32 (Cx32)-positive spots per hepatocyte were also significantly decreased in a dose-dependent manner. Similar changes in liver weights, P-450 isozymes and Cx32 immunohistochemistry were already evident in the DEN plus 250 ppm group at wk 1. The number of placental form glutathione S-transferase positive single cells was significantly increased in the DEN-initiated groups treated with 250 ppm or more. The results therefore strongly suggest that oxfendazole exerts liver tumour promotion potential.

The carcinogenic potential of stevioside, a compound that is used as a sweetener for food and drink, was examined in F344 rats of both sexes. Stevioside was added to powdered diet at concentrations of 0 (control), 2.5 and 5%. The doses were selected on the basis of results from a 13-wk subchronic toxicity study and administered to groups of 50 male and 50 female rats ad lib. for 104 wk. All surviving rats were killed at wk 108. Body weight gains were slightly depressed in line with the dose of stevioside, in both sexes, and a significant decrease in the final survival rate was observed for the 5% treated males. Histopathologically, however, there was no significantly altered development of neoplastic or non-neoplastic lesions attributable to the stevioside treatment in any organ or tissue, except for a decreased incidence of mammary adenomas in females and a reduced severity of chronic nephropathy in males. It is concluded that stevioside is not carcinogenic in F344 rats under the experimental conditions described.

The effects of dietary antioxidants on bleomycin (BLM)-induced pulmonary fibrosis were investigated in Syrian golden hamsters. In addition, the influence on cell proliferative activity in bronchioloalveolar hyperplastic lesions during the lung fibrosing process was evaluated in terms of argyrophil nucleolar organizer regions (AgNORs) and proliferating cell nuclear antigen (PCNA). Male 6-wk-old hamsters were divided into six groups. Groups 1-3 were intratracheally instilled with BLM at a dose of 2.5 U/kg body weight on days 0 and 14, and then given a diet supplemented with 1% butylated hydroxyanisole (BHA), or 1% butylated hydroxytoluene (BHT), or basal diet alone for the following 41 days. Groups 4-6 were given 1% BHA, 1% BHT or basal diet without BLM treatment for the same time period as that in those of groups 1-3. The mortality rate of animals in group 1 (BLM/BHA) (one in 20; 5%) was lower than in those of groups 2 (BLM/BHT) (three in 20; 15%) and 3 (BLM alone) (four in 20; 20%). BHA and BHT treatments significantly inhibited lung weight gains by BLM (P0.05). Histopathologically, both BHA and BHT reduced BLM-induced pulmonary histopathological changes such as fibrosis, macrophage aggregation and epithelial proliferation, with a tendency for correlation with accumulation of type III collagen. In addition, antioxidant treatment significantly lowered the mean numbers of AgNORs (P0.01) and PCNA-labelling indices (P0.05) in the hyperplastic bronchioloalveolar lesions. The results thus indicate that these antioxidants exert inhibitory effects on proliferation of hyperplastic lesions associated with lung fibrosis.

Cytosolic Ca2+ transients induced by hepatocyte growth factor (HGF) were imaged in primary cultured rat hepatocytes using newly developed rapid scanning confocal microscopes and Indo-1. HGF (40 ng/ml) increased cytosolic free Ca2+ concentration ([Ca2+]i) in about 60% of hepatocytes, in 45% of which the increases were oscillatory. In each of the oscillatory hepatocytes, the repetitive increases in [Ca2+]i originated from a specific same region adjacent to the cell membrane and propagated across the cell like waves. Phenylephrine (10 μM) also induced Ca2+ waves. The locus where HGF-induced Ca2+ waves and phenylephrine-induced Ca2+ waves were originated was the same, and there was a correlation in the peak height between HGF-induced Ca2+ waves and phenylephrine-induced Ca2+ waves in each cell, although the mechanisms of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation induced by HGF should be different from those by phenylephrine. On the other hand, there was no correlation between sensitivity of each cell to HGF and that to phenylephrine which were measured as latent periods prior to Ca2+ rises after an addition of the agonists. These results suggested the following: the spatial patterns of Ca2+ waves were decided by a common mechanism, probably not the propagation of Ins(1,4,5)P3 but the distribution of Ins(1,4,5)P3-sensitive Ca2+ pools; sensitivities of each cell to the agonists did not mainly depend on the common mechanism.

Pesinogens are acid protease enzymes of pepsin usually found in gastric mucosa. In the present study, we demonstrated the presence of pepsinogen isozymes in male Syrian golden hamster lung tissues by a combined immunohistochemical and biochemical approach. Immunohistochemically, using rat pepsinogen 1 antibody, pepsinogen positive cells were observed mainly in the epithelia of the terminal bronchioles. They demonstrated morphological features of Clara cells. The pepsinogen isozyme pattern of lung tissue determined by polyacrylamide gel electrophoresis was similar to that of stomach mucosa. Treatment of hamsters with polychlorinated biphenyls at a dose of 500 mg/kg body weight ip caused a 2.8-fold increase in pepsinogen content ( p < 0.01) as well as increase in numbers of pepsinogen positive cells in the lung.

Steroid hormones are powerful regulators of gene transcription in the brain and have the potential to permanently alter the structure and function of the developing brain. Steroid-mediated altered gene expression may thus be responsible for the molecular cascade for sexual differentiation. In this study, to assess effects of maternal exposure to ethinylestradiol (EE) on brain sexual differentiation of offspring, region-specific mRNA expression of two estrogen-responsive genes, gamma-aminobutyric acid transporter type 1 (GAT-1) and anti-apoptotic bcl-xL was measured in the medial preoptic area (MPOA), including sexually dimorphic nucleus (SDN), at the late stage of brain sexual differentiation in rats. Pregnant Sprague-Dawley animals were fed diets containing EE at concentrations of 0, 0.02, 0.1, and 0.5 ppm from day 15 of pregnancy to day 9 after delivery. In another group, neonates were directly injected with estradiol benzoate (EB: 10 microg/pup, sc) on postnatal day (PND) 2. The MPOA on PND 9 was microdissected from methacarn-fixed paraffin-embedded brain sections to measure mRNA levels by competitive RT-PCR, followed by plate hybridization. EE-exposure decreased GAT-1 expression dose-dependently from 0.02 ppm in females and at 0.5 ppm in males, while EB-treatment caused reduction only in females. EE-exposure did not alter Bcl-xL levels. At week 11, EE-exposed females exhibited a similar spectrum of histopathological changes in endocrine-linked organs as with EB, evident from 0.1 ppm, while in males EE-exposure did not cause histopathological alteration despite clear change with EB-treatment. Measurement of SDN-POA dimensions at week 11 revealed volume reduction in males exposed to 0.5 ppm EE or EB. The results suggest that GAT-1 expression in the developing MPOA is a sensitive measure for the level of disruption of brain sexual differentiation due to maternal dietary exposure to estrogens, despite definite reproductive abnormalities may not be detectable in males with this exposure protocol.

To evaluate developmental toxicity of di- n -butyl phthalate (DBP) with exposure during the period from late gestation to following lactation, maternal rats were given DBP at dietary concentrations of 0, 20, 200, 2000 and 10,000 ppm from gestational day 15 to postnatal day (PND) 21. At 10,000 ppm, male offspring showed a decreased neonatal anogenital distance and retention of nipples (PND 14), while females showed a slight non-significant delay in the onset of puberty. At PND 21, reduction of testicular spermatocyte development was evident from 20 ppm, as well as mammary gland changes at low incidence in both sexes. At this time point, population changes of pituitary hormone-immunoreactive cells were observed at 10,000 ppm with a similar pattern of increase in the percentages of luteinizing hormone (LH)-positive and decrease in follicle-stimulating hormone (FSH) and prolactin producing cells in both sexes, effects also being evident on FSH from 200 ppm and LH from 2000 ppm in females. During postnatal week (PNW) 8–11, marginal increase of the number of cases with extended diestrus was found at 10,000 ppm. At adult stage necropsy, testicular lesions appeared to be very faint in most cases, but degeneration and atrophy of mammary gland alveoli were observed in males from 20 ppm. Although without clear monotonic dose-dependence, relative pituitary weights were increased with the intermediate doses in males at PNW 11. In females, relative pituitary weights were decreased after 10,000 ppm at PNW 11, and from 200 ppm at PNW 20. The proportion of FSH-positive cells in the pituitaries at PNW 11 was increased in both sexes at 10,000 ppm. Thus, developmental exposure to DBP affected female sexual development involving pituitary function, while in males testicular toxicity was mostly reversible but mammary gland toxicity was persistent at a dose level as low as 20 ppm.

We previously found that effects of perinatal dietary exposure to ethinylestradiol (EE) on the rat reproductive system differ depending on the diet used, with a more pronounced estrogenic impact with a regular diet that includes soy-derived proteins than with a soy-free (SF) diet. The present study was performed to examine whether genistein (GEN), a soy-derived major phytoestrogen, acts synergistically with EE. Maternal rats were fed SF diet without chemical (control) or containing 0.5-ppm EE, 0.5-ppm EE + 100-ppm GEN, 0.5-ppm EE + 1250-ppm GEN, or 1250-ppm GEN, from gestational day 15 to postnatal day (PND) 11. EE reduced serum testosterone in males at PND 3, and affected the onset of puberty of both sexes and estrous cyclicity and reproductive system in females, irrespective of co-administration of GEN. GEN alone also affected estrous cyclicity and the reproductive system in females. However, no combination effects of GEN with EE were evident, suggesting no synergism between the two.

We previously performed dose-response studies of genistein, diisononyl phthalate, 4-nonylphenol, methoxychlor (MXC), and bisphenol A to examine the impact of maternal dietary exposure from gestational day 15 to postnatal day 10 on the development of rat reproductive system in later life. Among the chemicals MXC alone showed typical estrogenic effects only at the maternally toxic 1200 ppm. The present study was performed to examine the sensitivity of immunohistochemical analysis of pituitary cells of offspring similarly exposed to each chemical for detection of endocrine-disrupting effects. For this purpose, ratios of pituitary cells expressing luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL), were measured at 3 and 11 weeks of age. Ethinylestradiol (EE) at 0.5 ppm was used as a reference chemical. At week 3, decrease in the relative proportions of LH, FSH, and PRL cells in males and LH cells in females was evident with MXC at 1200 ppm. At week 11, increase was found for PRL cells from 240 ppm MXC, and FSH cells at 1200 ppm in females. On the other hand, EE increased the PRL cell percentage in females at week 3 but no effects were apparent at week 11. The other chemicals were without influence at either time point. The results suggest that the assessment of the pituitary cell populations might be a more sensitive approach to detect perinatal endocrine-disrupting effects than other methods. The difference in the pituitary effect between MXC and EE is discussed.

We investigated the effects of two diets, differing in phytoestrogen contents, on the phenotypic changes induced in the endocrine/reproductive system by perinatal exposure to an estrogen agonist during a critical period for brain sexual differentiation in rats. Ethinylestradiol (EE) was mixed at a concentration of 0.5 ppm into two diets: CRF-1, a standard rodent diet containing soybean-derived phytoestrogens; and a soy-free (SF) diet. These diets were provided to maternal Sprague-Dawley rats during gestational day 15 to postnatal day 10. Growth suppression of offspring was evident with EE especially during the exposure period and was slightly enhanced with the SF diet. On the other hand, most of the female offspring exposed to EE with CRF-1 showed early onset of vaginal opening, strong irregularity in estrous cycle (persistent estrus) and profound histopathological alterations, such as multifollicular ovaries, endometrial hypertrophy, and diffuse hyperplasia of the anterior pituitary. These EE-induced changes were much less pronounced with the SF diet. The results thus demonstrated differential effects of perinatal EE depending on the basal diet used, with enhancement of typical estrogenic responses in females by potential soybean-derived factor(s).

Recent advances in microdissection have succeeded the molecular biological approaches on specific cells of interest within tissue specimens by overcoming the obstacle of tissue complexity. Recently, we found that methacam is suitable for analysis of RNA, protein, and genomic DNA in small tissue samples using paraffin-embedded tissue (PET) sections in conjunction with microdissection technique. By application of sensitive quantitation methods, such as those utilizing fluorescent dyes specific for RNA or protein, molecules of small quantity can be normalized between samples, and thus quantitative expression analysis for RNA or protein can be applied in microdissected small tissue specimens. In addition, methacarn-fixation extends its availability for genomic DNA analysis in terms of target fragment size and number of microdissected cells required. Paraffin embedding permits ease of handling tissues that extend the availability of methacarn fixation for genetic analysis in large-scale experiments. In addition, considering its advantages for immunohistochemistry, tissue embedding after methacarn-fixation should be recommended as a valuable approach for routine application possibly in combination with targeted genetic analysis of immunophenotypically defined cell populations. In combination with techniques such as expression library construction, microarray and subtractive hybridization or differential display, microdissection will permit the establishment of “genetic fingerprints” of specific cellular areas.

We recently found methacarn to be a versatile fixative for analysis of RNA and protein applicable for microdissected specimens from paraffin-embedded tissue (PET). In this study we investigated the performance of methacarn for genomic DNA analysis using microdissected rat tissues. We found that extensive portions of DNA up to 2.8 kb could be amplified by nested PCR using DNA templates extracted by a simple and rapid extraction procedure from a 1 × 1-mm area of cerebral cortex of a 10-μm-thick section. By nested PCR, a 522-bp fragment from a single cell could be amplified in 20% of cresyl violet-stained Purkinje cells, and the minimal number of cells required, as estimated using hippocampal neurons, was on the order of 10-20. Although tissue staining with hematoxylin and eosin affected the PCR, amplification of a 522-bp fragment was successful, with 150-270 cells by 35 cycles of single-step PCR. Immunostaining resulted in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, a 184-bp DNA fragment could be amplified with 150-270 cells by 35 cycles of PCR. The results thus demonstrate the superior performance of methacarn to that reported with formalin in genomic DNA analysis using microdissected PET specimens.

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.

Fluoroacetate (FA), an inhibitor of aconitase, is known to lower the intracellular level of adenosine triphosphate (ATP), which recently has been suggested to be a possible determinant of the form of cell death, apoptosis or necrosis. To investigate which form of germ cell death occurs in FA-induced testicular toxicity, adult Sprague Dawley rats were given a single oral dose of FA (0.5 or 1.0 mg/kg) and euthanized at 3, 6, 12, 24, 48, and 72 h thereafter. Germ cell degeneration was histologically first found in early round spermatids at stage I and in spermatogonia at stages II-IV of seminiferous tubules 6 and 12 h, respectively, after dosing. Degenerating spermatogonia exhibited characteristic features of apoptosis as demonstrated by both electron microscopy and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), whereas spermatids did not. At the 24 and 48 h time points, degenerating spermatids were continually present and subsequently formed multinucleated giant cells, while the number of degenerating spermatogonia and TUNEL-labeled spermatogonia was drastically and/or significantly decreased compared to those from the control group, indicating that spontaneous male germ cell apoptosis is inhibited. Coincident with these morphological changes, DNA laddering on gel electrophoresis was apparent only 12 h after dosing. The results demonstrate that FA induces either apoptosis or necrosis of male germ cells in the early stage after dosing and subsequently inhibits spontaneous apoptosis.

To establish a quantitative method for analysis of gene expressions in small areas of tissue after paraffin embedding, preliminary validation experiments with RT-PCR and Western blotting were performed using methacarn-fixed rodent tissues and a cultured PC12 cell line. A total RNA yield of 52 +/- 15 ng/mm2, sufficient for a quantitative RT-PCR of many genes, could be extracted from a deparaffinized 10-microm-thick rat-liver section by a simple, single-step extraction method. The low concentration of contaminating genomic DNA and the resolution of ribosomal RNAs in RNA gel proved the purity and integrity of the extracted RNA samples, allowing PCR amplification of a long mRNA sequence and mRNA species expressing low copy numbers. PCR amplification of mRNA-derived target gene fragments could be achieved by optimizing the amount of total RNA for reverse transcription and the number of subsequent PCR cycles for each gene. By this validation, organ- and sex-specific mRNA expression could be detected in methacarn-fixed paraffin-embedded tissues without additional DNase treatment of RNA samples. RT-PCR analysis could also be performed with total RNA extracted from deparaffinized tissue dissected with a laser capture microdissection system. In addition, extraction of protein yielded 4.9 +/- 2.1 microg/mm2 from a 10-microm-thick rat-liver section, allowing a quantitative expression analysis of protein by Western blotting. Thus, in addition to its advantages for immunohistochemistry, methacarn-fixed paraffin-embedded tissue has benefits for analysis of both RNAs and proteins in the cells of histologically defined areas.

To clarify the suitability of a newborn-mouse carcinogenesis assay to detect tumor-promoting activities of carcinogens, the non-genotoxic hydroquinone (HQ) and genotoxic 1,1-dimethylhydrazine (UDMH) were administered to mice during the promotion stage after treatment with 1-methyl-1-nitrosourea (MNU) (20 mg/kg body wt, single intraperitoneal injection) at day 9 after birth. Initiated males and females thus received either HQ at 0.8% in basal diet, or UDMH, at 20 mg/kg body wt once weekly by subcutaneous injection, from day 14 until the end of the experiment at 30 weeks of age. Uninitiated newborn mice, given an injection of the vehicle (0.01 M citrate buffer (pH 5.5), 20 mg/kg body wt), also received HQ or UDMH in the same way. Histopathologically, focal proliferative lesions were found in the livers of male mice and in the lungs of both male and female mice in the MNU-treated groups. HQ significantly increased the incidence and multiplicity of altered hepatocellular foci, the combined incidence of hepatocellular adenomas and carcinomas in males and the incidence and multiplicity of lung adenomas and the combined incidence of lung adenomas and carcinomas in female mice. In addition, four out of eleven MNU + HQ-treated male mice developed lung carcinomas, showing a significant elevation in multiplicity. UDMH also exhibited a tendency to increase the incidence and multiplicity of lung adenomas in female mice. Thus tumor-promoting effects of HQ or UDMH were apparently exerted in the target organs and the MNU-initiated two-stage newborn-mouse carcinogenesis assay may be useful for detection of genotoxic or non-genotoxic carcinogenicity.

The carcinogenicity of β-cyclodextrin, a cyclic, water-soluble carbohydrate comprising seven glucose units, was examined in Fischer 344 (F344) rats. Groups of 50 males and 50 females were given the compound in their diet at concentrations of 0 (control), 2.5 or 5% for 104 wk. Surviving rats were then given a basal diet for a further 5 wk and killed at 109 wk. The dose levels were selected from the results of a 13-wk subchronic toxicity study. Dose-dependent inhibitory effects of β-cyclodextrin on growth were observed in both sexes of the treated groups. The survival rates, mean survival times and range, however, demonstrated no significant differences between the control and treated groups. A variety of tumours developed in all groups, including the control group, but all the neoplastic lesions were histologically similar to those known to occur spontaneously in this strain of rat, and no statistically significant increase in the incidence of any tumour was found for either sex of the treated groups. Thus, it is concluded that under the present experimental conditions, the high dose, about 340–400 times higher than the current daily human intake from ingestion as a food additive and from pharmaceutical use, does not have any carcinogenic potential in F344 rats.

Extracellular application of ATP evoked the oscillatory K + currents ( I KCa ) reflecting oscillation in cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ) of megakaryocyte isolated from rat bone marrow. We have reported that the [Ca 2+ ] i oscillation was regulated by intracellular Ca 2+ -pumping activity (Uneyama, H., C. Uneyama and N. Akaike, 1993, J. Biol. Chem. 268, 168). Here we found that the Ca 2+ pump of the megakaryocyte could be divided into at least two classes according to the sensitivity to phosphorylation-modulating drugs. The effects of protein kinase C and cyclic AMP-dependent protein kinase are complementary, and the effect of Ca 2+ /calmodulin is independent of the above two kinases. In addition, this is the first report concerning the physiological regulation of Ca 2+ -ATPase in living cells.

The effects of a mitochondrial uncoupler, FCCP, (carbonyl cyanide-p-trifluromethoxyphenyl-hydrazone) on the regulation of cytoplasmic Ca2+ were investigated in ATP-induced Ca2+ oscillation system of rat megakaryocyte. Application of FCCP did not induce any detectable Ca(2+)-activated K+ current (IKCa) but pretreatment with FCCP modulated the ATP-induced repetitive IKCa. FCCP abolished the IKCa induced by low concentrations of ATP. However, when the concentration of ATP was high, the uncoupler also changed the periodic current to a sustained one. Similar biphasic regulation by the uncoupler was observed in the case of IP3-evoked repetitive IKCa. These results indicate that FCCP inhibits both Ca2+ mobilization and elimination processes after IP3 liberation induced by agonist stimulation.

Dose-dependent modifying effects of quinacrine on induction of preneoplastic liver cell foci were investigated in male F344 rats. Six week old animals were injected i.p. with N-nitrosodiethylamine (DEN) at a dose of 200 mg/kg, and starting 2 weeks later, rats were given quinacrine at dietary levels of 20, 100 and 500 p.p.m. for 6 weeks. Groups without either DEN or quinacrine treatment were used as controls. At week 3 following DEN administration, all animals were subjected to two-thirds partial hepatectomy, and after killing the animals at week 8, development of preneoplastic liver cell foci was investigated using the glutathione S-transferase placental form (GST-P) as a marker. The numbers and unit areas of GST-P-positive foci per cm2 were significantly increased in the DEN/quinacrine (500 p.p.m.) group as compared to DEN-alone group values. An increase in number was also evident in the 100 p.p.m. but not the 20 p.p.m. treated group, no lesions being induced by quinacrine alone (500 p.p.m.). Electron microscopic study confirmed that quinacrine dose-dependently induces lipidosis in hepatocytes, i.e. markedly myeloid lamellar cytoplasmic inclusion bodies were observed. The results thus demonstrated that quinacrine treatment enhances GST-P-positive liver cell foci development in a dose-dependent way, this effect presumably being related to the induction of lipidosis.