Michihito Takahashi

NaCl induced ornithine decarboxylase and DNA synthesis in rat stomach mucosa

Inductions of 200-fold increase in ornithine decarboxylase activity within 6 hours and 9-fold increase in DNA synthesis within 3 hours in rat stomach mucosa were observed after a single oral dose of saturated NaCl solution. NaCl caused dose-dependent induction of ornithine decarboxylase activity at doses of 0.25 to 1. 5g /kg body weight, and of DNA synthesis at doses of 0. 5g to 1. 5g /kg body weight. Administration of 1,3-diaminopropane one hour before NaCl inhibited the induction of ornithine decarboxylase activity in the stomach mucosa 3 and 6 hours after NaCl administration, but a single dose of 1,3-diaminopropane itself induced ornithine decarboxylase after 16 hours.

A Collaborative Study in Japan on Optimal Treatment Period and Parameters for Detection of Male Fertility Disorders Induced by Drugs in Rats

The tripartite-harmonized International Conference on Harmonization reproductive guideline (1993) recommends administration of test substances for 4 weeks to male rats before mating. However, scientific or experimental rationale for this recommendation is not firmly based, and the most appropriate parameters have not been established in experimental models. Therefore, a team consisting of 16 Japanese pharmaceutical companies and the National Institute of Health Sciences performed a collaborative study to determine the optimal period and parameters for detection of male fertility disorders in rats. Sixteen compounds, including four anticancer drugs, two psychotropic drugs, two nootropic drugs, two vitamins, two hormones, one antihypertensive agent, one diuretic drug, and two general chemicals were administered to male rats for 4 or 9 weeks before mating. Parameters used to examine effects on the male reproductive system were organ weights, sper-matogenic endpoints, mating behavior, cesarean section findings, and histopathology. From the results, treatment for 4 weeks before mating was concluded to be sufficient to detect adverse effects on male fertility, with the histopathology of the testis being the most sensitive index for the drugs used. Sperm parameters, especially number, and genital organ weight determination provided information confirming toxicity. Tests of reproductive activity were generally found to be insensitive, except where the drugs affected sperm maturation. Based on this study, it is concluded that a 4-week treatment period is appropriate for detection of drug effects on male fertility, and that histopath-ological examination of the testis is the most sensitive approach.

Effect of thyroid stimulating hormone on the development and progression of rat thyroid follicular cell tumors

Time course changes in cell proliferative activity of thyroid focal hyperplastic and tumorous lesions as well as blood thyroid-related hormones in male F344 rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN: 2800 mg/kg body weight, single s.c. injection) were examined following chronic administration of 0.1% sulfadimethoxine (SM) in the drinking water for 1, 4, 8, 12 and 16 weeks and at the end of a subsequent 4-week recovery period. Serum thyroid stimulating hormone (TSH) levels increased rapidly from week 1 of SM treatment, reaching a peak at week 8, and then decreased gradually with prolongation of treatment period, although remaining significantly elevated as compared with the corresponding controls at all time points up to week 16. Follicular cell hyperplasias and adenomas of the thyroid occurred from week 4 and carcinomas from week 8. All of these lesions showed high cell proliferative activities corresponding to high serum TSH levels during the early stage, but the levels in hyperplasias and adenomas decreased rapidly with prolongation of SM treatment. After the recovery period, serum TSH levels had returned to below the normal range and cell proliferation in follicular hyperplasias and adenomas had stopped or was very low. Some carcinomas demonstrating invasive growth also showed remarkable decreases in the cell proliferative activity. The results of our study strongly suggest that a high serum TSH level plays an important role in the early stage of thyroid tumorigenesis and that some tumors exhibiting invasive growth are still dependent on TSH stimulation.

Differentiation of pepsinogen-producing cells in the fundic and pyloric mucosa of developing rats

Abstract In the rat fundic mucosa, increase in the peptic activity of pepsinogen, changes in the molecular species of pepsinogen separated by electrophoresis and changes in the morphology of the chief cells indicated that maturation of the chief cells began around 15 days after birth, and was complete 25–30 days after birth. In the rat pyloric mucosa, no changes in the peptic activity or molecular species of pepsinogen or in the morphology of pyloric gland cells occurred after birth. These results suggest that maturation of pyloric gland cells is complete before birth.

Inhibitory Effects of Antioxidants on N‐Bis(2‐hydroxypropyl)nitrosamine‐induced Lung Carcinogenesis in Rats

Potential second‐stage modifying effects of 8 antioxidants on lung tumorigenesis initiated by N‐bis(2‐hydroxypropyl)nitrosamine (DHPN) were examined in male F344 rats. After an initial 2‐week treatment with DHPN (0.1% in drinking water), rats were administered one of the antioxidants supplemented in the diet for 30 weeks. Although the incidences of lung adenomas were not affected, those of carcinomas were lowered by 2% butylated hydroxyanisole (BHA, 2 rats/20 rats), 1% butylated hydroxytoluene (BHT, 1/20), 0.8% ethoxyquin (EQ, 3/20) and 1%α‐tocopherol (α‐TP, 2/20) treatments as compared to the control level (9/20), while 5% sodium l‐ascorbate (SA), 0.8% catechol (CC), 0.8% resorcinol (RN), and 0.8% hydroquinone (HQ) did not exert any significant effect on incidence. Quantitative analysis of adenomas and carcinomas (numbers and areas of lesions per unit area of lung section) revealed obvious inhibitory effects of SA, CC, and RN as well as BHA, BHT, EQ, and α‐TP. Among the antioxidants, BHT exerted the strongest inhibitory activity. In contrast, DHPN‐induced thyroid tumorigenesis was significantly enhanced by BHT (14/20) and EQ (20/20) treatments (control=5/20). Thus the antioxidants showed opposite effects on lung and thyroid carcinogenesis in the rat.

Mechanistic Insights into Chemopreventive Effects of Phenethyl Isothiocyanate in N‐Nitrosobis(2‐oxopropyl)amine‐treated Hamsters

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<0.05) inhibited the induction of several isoenzymes, including CYP 2B1, while lowering the hepatic glutathione S‐transferase activity as well as glutathione levels, regardless of BOP administration. Our results thus suggest that PEITC exerts its chemopreventive activity against BOP initiation of carcinogenesis in hamsters by decreasing cell turnover and DNA methylation in the target organs, and by influencing hepatic xenobiotic‐metabolizing phase I enzymes, although the relationship, if any, of the latter with the former events remains to be investigated.

Morphometric and immunohistochemical studies on atrophic changes in lympho-hematopoietic organs of rats treated with piperonyl butoxide or subjected to dietary restriction

Abstract Changes observed in lympho-hematopoietic organs in rats given piperonyl butoxide may be attributable either to direct toxic effects or to undernutrition. Male F344 rats were therefore fed diet containing 2.5% piperonyl butoxide or subjected to a 64% restriction of food intake for 2 weeks. Marked inhibition of body weight gain, decreased white blood cell count, depletion of T/B lymphocytes in lymphoid tissues, hypoplasia of the bone marrow, and decreased proliferating cell nuclear antigen (PCNA) labeling indices in these tissues were seen in both dietary restriction and 2.5% piperonyl butoxide groups. The depletion of T lymphocytes in the thymus and spleen was stronger in the 2.5% piperonyl butoxide group, as indicated by PCNA labeling indices and image analysis of T lymphocyte areas of the spleen, however, the toxicological profile observed for the chemically treated group was essentially the same as for animals on the restricted diet. These results suggest that the lympho-hematopoietic findings in rats receiving 2.5% piperonyl butoxide are probably due to undernutrition resulting from a reduced food intake.

Enhancing effects of captafol on the development of GST-P-positive liver cell foci in a medium-term bioassay, and protection by l-cysteine of the enhancement in rats

The modifying effects of captafol and protective effects of L-cysteine on the development of glutathione S-transferase placental form-positive (GST-P +) foci of the liver and expression of proliferating cell nuclear antigen (PCNA) in the kidney were investigated in a medium-term bioassay using D-galactosamine (DGA) in rats. Male 6-week-old F344 rats were initially given a single i.p. injection (200 mg/kg) of diethylnitrosamine (DEN) and after 2 weeks on basal diet, received two i.p. injections of DGA (300 mg/kg) at the ends of weeks 2 and 5, and were fed a diet supplemented with test chemicals for weeks 3-8. Animals in group 1 were given 1500 ppm captafol in the diet, while group 2 received 1500 ppm captafol in diet as well as 1500 ppm L-cysteine in drinking water, animals in control group being given basal diet alone. Positive results regarding increased numbers and areas of GST-P + liver cell foci were obtained in rats treated with captafol alone. On the other hand, significant reduction by L-cysteine in the areas of GST-P + liver cell foci initiated by DEN and promoted by captafol was observed. In addition, the PCNA-labelling indices of renal tubule cells were elevated in rats treated with captafol alone and significantly reduced in rats treated simultaneously with L-cysteine. The protocol used in the present study therefore allowed the in vivo determination of promoting effects of captafol and inhibitory influence of L-cysteine by analyzing GST-P + foci in the livers as marker lesions, within a relatively short period of 8 weeks. Thus, this bioassay protocol could have applicability as a new in vivo assay system for the screening of hepatic carcinogenic or anti-carcinogenic agents.

Familial bilateral papillary cystadenoma of the epididymis. Report of three cases in siblings

This paper describes an unusual, familial bilateral papillary cystadenoma of the epididymis found in three siblings. The histological characteristics of the tumor were: 1) papillary proliferation of clear cells, consisting of both tall columnar and round ovoid cells, in the efferent duct epithelium; and 2) cystic dilatation of the duct with colloidal material. The present cases were unique in two respects: 1) familial bilateral papillary cystadenomas probably associated with Lindaus disease have not been reported previously: and 2) electron microscopic observations suggested that the tumors originated from the efferent duct.

Precocious differentiation of immature chief cells in fundic mucosa of infant rats induced by hydrocortisone

Injection of hydrocortisone into developing rats induced precocious increase in the potential peptic activity of pepsinogen in the fundic mucosa, change from the immature to the mature electrophoretic pattern of pepsinogen isozymes and morphological change from the immature to the mature chief cells. The continual presence of hydrocortisone was required for maintenance of mature chief cells.