An appropriate statistical methodology in toxicity studies has been discussed over the last two decades and many statistical methods have already been proposed. Many practical problems, however, still remain unresolved and most pharmaceutical industries have been using a tree-type algorithm routinely to analyze repeated-dose toxicity study data. In considering routine use of statistical analysis in toxicological studies, standardization of statistical methodology is necessary and the decision tree has an important role. In this article, the problems, relating to tree-type algorithms are summarized. Then we propose a new tree-type algorithm, which targets quantitative data in repeated-dose studies in rodents, usually sample size per group between 10 to 20, based on the following two important principles:"using a parametric method "and "suitable for intuition of toxicologists". An example of its application to actual toxicity study data is demonstrated. The performance of this new method is also evaluated using historical data. However, it should be noted that the intention of this paper is not to make a definite solution of the decision tree. Several other alternatives can be considered. Since there is no single theoretically correct solution of tree-type algorithms, too formal a use of the decision tree is not recommended. We must not forget the exploratory nature of evaluating repeated toxicity data.
To ascertain whether NO-elicited cell death is mediated by decreased intracellular ATP, the effect of sodium nitroprusside(SNP), a NO-generator, on ATP content in PC12 cells was examined. After treatment with SNP, the ATP content in PC12 cells was found to decline in a time-and concentration-dependent manner. The decline of ATP content in PC12 cells caused by SNP was found to occur before the appearance of cytotoxicity estimated by MTT staining. In addition, the ATP content of neuronally differentiated PC12 cells, which has been shown to have a higher resistance to SNP than the undifferentiated cells(Nakamura et al., 1997), was less affected by SNP treatment that the undifferentiated cells. These findings suggest that reduction of intracellular ATP content may be one of the mechanisms responsible for the NO toxicity in PC12 cells.
The usefulness of cellular size measurement for differentiating erythroid and myeloid cells was investigated using rat and canine bone marrow film prepared by the Cytospin method and Wright-Giemsa staining. 1.In the erythroid series, basophilic and polychromatic erythroblasts were distinguishable in terms of cellular diameter;i.e., 99% of rat and 95% of canine polychromatic erythroblasts were distributed in a range≤9.5μm, at which basophilic erythroblasts did not exist. 2.In the myeloid series, myelocytes and metamyelocytes were to some extent distinguishable by their diameters;in rats, myelocytes(75% of the population)were≥13.5μm, and metamyelocytes(61%)≤11μm;and in dogs, myelocytes(45%)were≥16μm, and metamyelocytes(66%)≤12μm. 3.With regard to the metamyelocytes and myelocytes existing in the same range, their nuclear sizes(width)allowed further differentiation;in rats, the nuclear width of myelocytes(87%)was≥5μm, and that of metamyelocytes(84%)<5μm;and in dogs, myelocytes(96%)≥7μm, and metamyelocytes(88%)<7μm. The present results indicate that cellular size, together with nuclear size, contribute to distinguish the active mitotic group from less-or non-mitotic group in erythroids and myeloids, thus being helpful for toxicological evaluation on chemicals.
We have attempted to determine if there is a hazardous effect of acute microwaves at 2.45GHz on the eye of a rabbit under lengthy exposure without anesthesia, the contralateral eye serving as a control. Unilateral eyes of 9adult Japanese white rabbits(10-12 weeks of age)were irradiated by 2.45GHz continuous wave(CW)microwave for 160 min. to 240 min. under restraint without anesthesia. The specific absorption rate(SAR:phantom material)was 26.5 W/kg. The corneal surface temperature increment was 3.0°C for 15 min. on average. Miosis occurred in all rabbits within 15 min. Post-exposure ophthalmological signs, first detected as the effect of CWirradiation, included 1)miosis and pupillary congestion; 2)keratoleucoma and corneal edema; 3)endothelial cell detachment and floating in aqua oculi, 4)fibrinogenesis in the anterior chamber, and 5)conjunctiva edema, which disappeared one week after exposure. There was no cataract formation. The acute microwave irradiation to the rabbit eye, causing the miosis and pupillary congestion in all irradiated eyes, was first to be detected.
Chloroform, an industrial solvent and one of the most common environmental contaminants which produces carcinogenic effects in the liver and kidney of rodents, is not genotoxic in most traditional bacterial and mammalian test systems. Its carcinogenic potential appears attributable to the sustained cell turnover(regenerative hyperplasia)which results from chronic chloroform toxicity. In this present study, cell proliferation(replicative DNA synthesis, RDS)and histopathological changes in hepatocytes and renal tubular epithelial cells were assessed in male F344 rats following a single gavage chloroform exposure(50, 150 or 500 mg/kg). In addition, biochemical parameters(BUN, GOT, LDH and NAG)were examined using plasma and urine samples. Cell proliferation and histopathological changes(e.g. hypertrophy, necrosis, vacuolation)were only seen at the dose of 500 mg/kg in the liver and kidney. At the same dose, all biochemical markers were increased at the 24 to 48 hr time points. These results obtained are thus in line with earlier findings pointing to epigenetic carcinogenicity.
Changes of TSH-producing cells in the pituitary and thyroid expression of the growth factors, transforming growth factor α(TGFα)and epidermal growth factor receptor(EGFR), as well as cyclin D1, were investigated immunohistochemically in order to clarify their contribution to the enhancing effects of excess vitamin A(VA)on thyroidal carcinogenesis induced by thiourea(TU). Male rats were allocated to 4 groups, control, TU, VA, and TU+VA, respectively, receiving on treatment, water containing 0.2% TU, diet containing 0.1% VA, and both for 10 or 19 weeks after a single s.c. injection of DHPN(2800 mg/kg)for initiation. Immunohistochemistry using antibodies against TSH demonstrated enlargement of TSH-producing cells in the TU-VA group as compared to the TU group, supporting our conclusion that enhanced TSH stimulation is mainly responsible for promoting the effects of excess VA. Since the expression of TGFα, EGFR, and cyclin D1 in thyroid proliferative lesions did not exhibit any differences between the TU and TU+VA groups in the present study, these factors are unlikely to participate in VA enhancement of carcinogenesis.
Mepanipyrim, a new fungicide, was administered orally to rats, mice and dogs for 13 weeks to clarify its toxic profiles. Hepatotoxicities were observed characteristically in these species with high concentrations of mepanipyrim; more than 200 ppm in rats, more than 1, 000 ppm in mice, and more than 50 mg/kg/day in dogs. In rats, obvious fatty vacuolation in the perilobular hepatocytes and changes in the serum-lipid concentrations such as total cholesterol(TC), triglyceride(TG), phospholipid(PL)and non-esterified fatty acid(NEFA)were observed. This fatty liver appeared to be based on the alteration of lipid metabolism. In contrast, no remarkable changes were observed in mice and dogs except for an enhancement of anisonucleosis in mice or a lipofuscin deposition in Kuppffer cells and hepatocytes in dogs. It appeared that there species differences in the hepatotoxicities of mepanipyrim.
Our preceding paper reported that mepanipyrim, a new fungicide, induced fatty liver in the rat. This study was undertaken to examine this phenomenon further on hepatic triglyceride(TG)synthesis, on liver and serum lipid concentrations, and on concentration of serum very-low-density lipoprotein(VLDL)in rats fed 3 weeks on the drug at 4, 000 ppm. Mepanipyrim decreased the incorporation of <14>C-acetate into hepatic TG, total cholesterol(TC)and total lipids. In addition, mepanipyrim treatment induced a drastic increase in hepatic TG accompanying a decrease in serum TG. Esterified cholesterol(CE), phospholipid(PL)and non-esterified fatty acid(NEFA)also increased in the liver with a concomitant decrease in the serum. The decrease of serum VLDL by mepanipyrim was comparable to the decrease in serum TG. Because hepatic TG is secreted into the blood by forming VLDL, which consists of TG, TC, PL, and apoprotein, the decrease in serum TG would be mainly ascribable to that in serum VLDL. Mepanipyrim also decreased serum concentrations of low-density lipoprotein(LDL)and high-density lipoprotein(HDL), and the relative weights of the epididymal adipose tissue, indicating that a reduction in serum VLDL dose not reflect acceleration of serum VLDL dissimulation. These results suggest that the fatty liver induced by mepanipyrim would be due to the inhibition of hepatic VLDL synthesis or its secretion into the blood.
To investigate the accumulation pattern of cadmium(Cd)in the liver and kidney following Cd intake from diet, female SD rats were fed cadmium choride(CdCl2)-contained diets(1.24 and 4.96ppm Cd)for 2 or 4 months. The other rats were fed CdCl2-contained diets(8, 40, 200, and 600ppm Cd)for 2, 4 or 8 months. The control rats were given diet without Cd addition(lower than 0.01ppm Cd). The concentrations of Cd in the liver and kidney derived from all rats were determined. The concentrations of Cd in the liver and kidney increased depending on the dosage of Cd. The concentrations of Cd in the liver did not reach plateau level even in the 200 and 600ppm groups. On the other hand, the concentrations of Cd in the kidney in the 200 and 600ppm groups reached a plateau level, which was approximately 250 μg/g. In the 600ppm group, the concentrations of Cd in the kidney reached 250 μg/g at 2 months, but did not exceed that level at 4 months. In the 200ppm group, the concentrations of Cd in the kidney increased to nearly the level of 250 μg/g at 8 months. The ratio of the concentrations of Cd in the kidney versus liver decreased as the dosage of Cd increased, suggesting that a low dosage of Cd was distributed preferentially to the kidney, but a high dosage of Cd w distributed to the liver. The relation curves between total amounts of Cd intake and Cd levels in the kidney in the 2-, 4-, and 8-month groups showed a parabola. The curves were shifted in parallel in the direction of higher levels of ingested Cd in order of length of Cd exposure period. These results suggested that when Cd is ingested over a long time at low concentrations, the amount of Cd accumulation in the kidney is small even for equal amounts of total ingested Cd.