The atrophic effect of a synthetic steroidal antiandrogen, chlormadinone acetate(CMA), on spontaneous benign prostatic hyperplasia(BPH)in dogs was investigated. Male beagle dogs(5-8 years old)were divided into four experimental groups. Group 1 consisted of untreated controls. Group 2 to 4 received CMA 0.03, 0.1, and 0.3 mg/kg/day, p.o., respectively, for 6 months. In group 1, glandular hyperplasia of the prostate was clearly detected. The glandular epithelial cells showed uniformly intense nuclear staining for androgen receptor(AR). AR was also localized in the nuclei of the fibro-muscular stromal cells. Immunoreactivity of 5 alpha-reductase type I was positive in most glandular epithelial cells. No fibro-muscular stromal cells were stained. Immunolocalization of 5 alpha-reductase type II was clearly detected in the interacinar fibro-muscular stromal cells, but not in the glandular epithelial cells. In groups 2 to 4, CMA produced marked atrophy of the glandular epithelium. The interacinar fibro-muscular stroma was prominent. The nuclear staining for AR in both epithelial and stromal cells was remarkably decreased. Furthermore, the immunoreaction for 5 alpha-reductase type I in most glandular epithelial cells was negative or very weak. The immunoreaction on 5 alpha-reductase type II in the interacinar fibro-muscular stromal cells was negative or very weak. These results indicate that the uptake of testosterone and/or its androgenic effect on the prostate may be suppressed by CMA. The decreased AR-immunostaining may be explained by the decrease in the number of AR and/or antibody binding sites for AR. Therefore, the atrophy after treatment with CMA may be due to shrinkage of both glandular and stromal compartments in the prostate tissue.

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We examined the correlation between histopathological changes and porphyrin levels in the Harderian glands of male rats after treatment with atropine sulfate. After a single administration of atropine sulfate(250 mg/kg/day), the porphyrin levels in the Harderian glands gradually increased, beginning from 2 hr after administration, and at 36 hr reached a maximum level, which was about 7 times higher than that of the control animals. Histopathologically, the Harderian glands showed marked luminal dilatation and a brownish pigment accumulation in the lumina 6 hr after a single dose. Under daily repeated administrations of atropine sulfate(250 mg/kg/day), the highest porphysin levels in the Harderian glands were observed 24 hr after the third dose, and were about 9 times higher than those of the control animals. However, beginning from one week after the initial dose, much lower peak porphyrin levels were observed 6 hr after each dose. The maximum porphyrin levels were only twice as high as those of the control animals, and they returned to the control levels 24 hr after each atropine dose. Histological examinations of the Harderian glands revealed that repeated administrations of atropine sulfate induced the same histopathological changes observed after a single atropine administration, and that no aggravated dilation of the lumina or pigmentation in the lumina appeared after such repeated administrations. The degree and incidence of the histopathological changes observed correlated well with the porphyrin levels. Some animals showed a degeneration of the glandular epithelium after 4 weeks of treatment, and the frequency increased slightly after 13 weeks of treatment. The present study suggests that atropine suppresses the expulsion of secretory materials, including porphyrin, from the glandular lumen of the Harderian glands, and thereafter an excessive accumulation of porphyrin induced luminal dilatation. These changes were gradually reduced by repeated administrations. The degeneration of the glandular epithelium after repeated administration might be a consequence of retention of an excessive accumulation of porphyrin.

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In this study, a new simple method to measure erythrocyte fragility with stirring of diluted blood(stirring method)was introduced and evaluated with anemic rats given β-acetylphenylhydrazine(APHZ)or clofibrate. APHZ at a dose of 40 mg/kg caused significant decreases in hemoglobin and hematocrit 24 hr after administration. However, the marked elevation of erythrocyte fragility was already detectable after 6 hr by our stirring method. At a dose of 10 mg/kg APHZ, although no significant changes in the erythrocytic parameters were observed throughout the experimental period(72 hr), the blood stirring method revealed a marked elevation of erythrocyte fragility 6 hr after administration. Similarly with clofibrate, no changes in erythrocytic parameters were noted following 100 mg/kg or 300 mg/kg administration, but the enhanced fragility was evident with the stirring method. Thus, using our approach, the erythrocyte fragility could be detected at an earlier stage and with greater sensitivity than by decreases in erythrocytic parameters. The results suggest that the stirring method will prove to be useful for detecting erythrocyte fragility in safety studies.

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To elucidate the effects of kojic acid on thyroid function, the compound was given orally to male rats for 4 weeks at 0, 4, 15, 62.5, 250 and 1, 000 mg/kg. In 1, 000 mg/kg treatment of kojic acid, the rats showed a slight decrease in motility, inhibition of body weight gain, and a decrease in food consumption. An increase in thyroid weight and a morphological change, i.e., hypertrophy of epithelial cells of the thyroid gland follicles, were observed after 1 week of administration. In addition, the uptake of radioactive iodine from blood into the thyroid gland was enhanced and the TCA-precipitable radioactive iodine in the thyroid gland increased in those rats. However, the rates of the iodination in the thyroid gland did not change during the experiment period. Although serum T4 concentration was low in the rats treated with 1, 000 mg/kg kojic acid, it was not observed in any changes in TSH concentration. None of these changes were found in the other groups. These observations suggest that massive administration of kojic acid may decrease blood T4 concentration and that thyroid function may be enhanced compensatorily. On the other hand, the absorption of kojic acid was rapid as manifested by the T_max of blood concentrations of radioactivity, which was as short as 1.0±0.0 hr, and the t_1/2 was 4.8±0.3 hr. Blood concentrations of radioactivity disappeared nearly completely at 24 hr after administration. This result indicates that the toxic effect observed on the thyroid gland treated with only the largest dosage of kojic acid may depend on a fast decrease following a transient increase of concentration of the compound in the blood.

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Young weaning Swiss albino mice were orally administered leaf extract of Putranjiva roxburghii at 0.5, 1.0 or 2.0 g/kg body weight/day for seven consecutive days. The results showed that the leaf extract significantly induced mitosis-disruptive chromosomal changes in bone marrow cells. No change in the incidence of structural abnormalities was, however, noticed among the metaphase chromosomes. It is proposed that the extract might have interfered with the spindle and other proteins causing polyploidy, aneuploidy, c-mitosis, etc.

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Age-related induction of unscheduled DNA synthesis(UDS)by ultraviolet B(UV-B)irradiation was investigated in the epidermis of female hairless mice by means of an in vivo-in vitro assay using a liquid scintillation counting method. Skin samples were taken and cultured in a medium containing[³H]thymidine with or without hydroxyurea(HU)for 2 hr. DNA of the epidermis was extracted, and the incorporation of[³H]thymidine into DNA and the DNA content were determined with a liquid scintillation counter and a fluorescence spectrophotometer, respectively. Induction of UDS by UV-B was judged in terms of the value of the UDS index calculated as a percentage of the respective unexposed control value taken as 100%[the UDS index is given by(the ratio of DNA synthesis in the presence of HU to that in its absence)×100]. DNA synthesis both in the presence and absence of HU decreased with age[12 months old(M)<8M<4M<2M<1M)], concomitantly with a small but significant increase of UDS index. The decrease was high in the younger age groups and moderate in the older age groups. UV-B increased the UDS index approximately 14-, 12- and 9-fold at 1 hr after 1, 000 J/m² irradiation in 1M, 2M and 12M mice, respectively, and these increases were partly reversed at 4 hr after irradiation. UV-B also increased the UDS index approximately 25-, 24- and 21-fold at 1 hr after 4, 000 J/m² irradiation for each age group. However, there was no statistically significant age-related difference in the magnitude of the UDS index after irradiation of UV-B. These results show that replicative DNA synthesis decreases with age, whereas DNA repair capacity after UV-B irradiation does not change with age under these conditions.

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The aromatic retinoid(arotinoid)Ro 40-8757(4-[2-[p-[(E)-2-(5, 6, 7, 8-Tetrahydro-5, 5, 8, 8-tetramethyl-2-naphthyl)propenyl]phenoxy]ethyl]-morpholine), a compound with antitumor activities, has been studied in a combination therapy with the cytostatic antitumor drug cyclophosphamide(CPA), and was found to protect bone marrow from the toxic effects of CPA. To evaluate its protective effects against CPA toxicities on lymphoid systems, we treated BDF1 mice with Ro 40-8757 orally for 1 to 5 weeks in combination with CPA intraperitoneally. After the combination treatment, mice were subjected to immunohistochemical analysis with antibodies against cell surface markers(Thy1.2, Lyt-2, L3T4, Kappa chain, and Ia)and, in addition, general pathologic examinations were done. The protective effects of Ro 40-8757 on CPA toxicities were observed. The lymphocyte reductions(both in T cells and B cells)in lymphoid organs by CPA were apparently less severe. In particular, recovery of immature T cells in the thymic cortex was greater in combination treatment with Ro 40-8757 and CPA than in treatment with CPA alone. From these results, it can be concluded that Ro 40-8757 protects the lymphoid organs(thymus, spleen, and lymphnode)from the immunotoxicity of CPA, and the protective effect is evident, especially in the thymic cortical lymphocytes.

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The protective effects of green tea polyphenols on paraquat-induced genotoxicity in cultured cells were studied. (-)-Epigallocatechin gallate(EGCG), the major constituent of green tea, and (+)-catechin(CT), a minor constituent, equivalently decreased the frequencies of sister-chromatid exchanges(SCE)induced by paraquat(PQ), which is a generator of reactive oxygen species. These polyphenols were effective at concentrations of 1.0 μM and above. A reduction of the effect on the cell cycle rate caused by PQ was found when EGCG and CT were added at concentations of more than 10.0 μM. These concentrations of EGCG and CT alone had no effect on cell cycle rate, which is used as index of cell proliferation. Decreases in the cell cycle rate were found at 200 μM EGCG and CT in the 24 hr exposure period. The equivalent effectiveness of EGCG and CT suggested the possibility of other mechanisms, apart from acting as reactive oxygen species scavengers, because it has been reported that EGCG is the most potent scavenger among tea catechins. From the present study, it was suggested that green tea and foods containing these polyphenols may be beneficial to human health by protecting against reactive oxygen species-induced genotoxicity.

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Arterial blood pH and blood gas tensions were measured before and after feeding in seven young(1-year-old)and 6 aged(10 to 12 year-old)conscious beagle dogs with a portable "i-STAT"blood gas analyzer to obtain the fundamental physiological data on the laboratory dogs. Before feeding, arterial blood pH, HCO₃^- and base excess(BE)were 7.395±0.022, 20±1.0 mmol/L and -4±1.1 mmol/L in young dogs, and 7.408±0.031, 23±1.5 mmol/L and -2±1.8 mmol/L in aged ones. After feeding, the above values changed to 7.449±0.021, 25±1.1 and 1±1.3 in young dogs, and 7.456±0.022, 28±1.7 and 4±1.9 in aged ones. After feeding, each of the values for pH, HCO₃^- and BE was increased in comparison with those before feeding in both young and aged dogs, and alkalinization of blood, the alkaline tide, was observed in all healthy dogs. The values for PCO₂ also rose from 33.6±1.91 to 36.7±1.95(mmHg)in young dogs, and 36.4±2.97 to 40.3±3.06(mmHg)in aged ones, and in 3 of 6 aged dogs slight hypoxia(PO₂<80 mmHg)was noted. These changes in PCO₂ and PO₂ were considered to be due to compensatory hypoventilation for the alkalinization of blood. To confirm the cause of the alkaline tide phenomenon, we investigated the association of the alkaline tide with gastiric acid secretion by cimetidine, an H₂-blocker ; and found that it was effective to inhibit postprandial alkaline tide in dogs. After feeding, ionized Ca concentration(iCa)was decreased inversely with an arterial blood pH increase in both young and aged dogs. The mean values of iCa in aged dogs after feeding was lower than those in young dogs. In the present experiments, it was demonstrated that in beagle dogs the acid-base balance and blood gas values were largely affected by feeding, especially in aged dogs. In case blood gases would be evaluated in toxicity studies, we should consider the influence of feeding and aging.

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We examined the effect of hemin on rat hepatic microsomal cytochrome P450(P450)and its molecular species(P450 2E1, 3A2, 2C11 and 2C12)under the induction of heme oxygenase activity in male and female rats. Hemin produced an inverse relationship between the induction of heme oxygenase activity and the decrease of P450 content in a dose-dependent manner. A time course study revealed that hemin produced a significant decrease in total P450 content in male rats to about 37% that of the controls at 24 hr after its administration. Western and Northern blot analyses revealed that the increase in both heme oxygenase-1(HO-1)protein and HO-1 mRNA reached a maximum at 24 hr and returned to control levels within 120 hr in both sexes. With respect to P450-dependent monooxygenase activities, we found that there was a significant decrease of aniline p-hydroxylase activity to about 30% of the control animals, but not in erythromycin N-demethylase activity at various time intervals. Immunoblot analysis revealed that P450 2E1 in male rat liver was dramatically decreased at 24 hr to about 20% of the controls, but not P450 3A2. Parallel to the decrease of androstenedione 16α-hydroxylase activity, there was a marked decrease of P450 2C11 or 2C12 in male or female rats, respectively, at 72 hr after the treatment ; however, hemin did not decrease androstenedione 16α-hydroxylase activity in phenobarbital-pretreated rat liver. These findings suggest that hemin predominantly affects constitutively expressed isozymes rather than inducible P450 species in rat liver.

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Toluene, a commonly used industrial solvent, is known to be toxic to both neuronal and glial cells, and has been shown to increase the immunoreactivity of glial fibrillary acidic protein(GFAP)in the brain. However, the mechanism of toluene-induced GFAP expression is poorly understood. Recently, GFAP mRNA expression in cultured astrocytes has been shown to be modulated by various steroid hormones, such as progesterone, testosterone, and their 5α-reduced metabolites. Therefore, it seems possible that steroid hormones may play a potential role in the enhancement of GFAP expression observed following toluene exposure. To address this possibility, the effect of toluene inhalation on the expression of mRNAs encoding GFAP and steroidogenic enzymes in rat brain was examined. Toluene exposure increased GFAP protein contents without any significant alteration in GFAP mRNA levels in the hippocampus. In contrast, the elevation of both GFAP protein contents and its mRNA levels was observed in the cerebellum following toluene exposure. Further studies indicated that toluene exposure increased steroid 5α-reductase(5α-R)mRNA levels prior to the elevation of GFAP mRNA in the cerebellum, whereas neither 5α-R nor GFAP mRNA levels in the hippocampus were significantly affected by toluene exposure. These results suggest that toluene inhalation may enhance GFAP gene expression in the rat cerebellum, and propose the possibility that the elevation of 5α-R expression, and hence 5α-reduced metabolites of steroid hormones, is presumably related to toluene-induced GFAP mRNA expression.

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