M.T.Stephen Hsia

Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations

SummaryA simple yet effective method (iso-density percoll centrifugation) has been developed for consistently preparing isolated rat liver parenchymal cells with over 98% initial viability. The method has been applied to cells isolated by a variety of collagenase digestion techniques. This procedure involves the low-speed centrifugation (50 ×g) of the initial cell suspension through a percoll medium having a density of 1.06 g/ml and results in the separation of single and viable parenchymal cells from cell aggregates, debris, and nonparenchymal cells. The enriched parenchymal cells have been shown to be superior to untreated cells by a number of criteria including: preparation homogeneity, cell morphology, maintenance of cytochrome P-450, hormonal responsiveness (measured by the induction of tyrosine aminotransferase after treatment with glucagon or dexamethasone, or both), plasma membrane integrity (determined by both trypan blue exclusion and leakage of glutamic-oxaloacetic transaminase), and the DNA repair capability after treatment with benzo[a]pyrene or 2-acetylaminofluorene.

Embryotoxicity of 3,3′,4,4′-tetrachloroazobenzene and 3,3′,4,4′-tetrachioroazoxybenzene in the chick embryo

The toxicity of 3,3′,4,4′-tetrachloroazobenzene (TCAB) and 3,3′,4,4′-tetrachloroazoxybenzene (TCAOB) to chick embryos was examined. TCAB or TCAOB was dissolved in corn oil and injected into the air cell of fertile chicken eggs. The time of injection had a major effect on embryo mortality as eggs injected with TCAB or TCAOB on the fourth day of incubation had a higher incidence of embryo mortality than eggs injected on days 11–13. Both TCAB and TCAOB were more toxic than all other chemicals that have been tested in the chick embryo with the exception of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Comparing the potency of the two compounds, TCAOB was more potent than TCAB in the chick embryo with an estimated LD50 of 12 ng and 44 ng respectively. Rump edema was the major abnormality observed in embryos treated with either TCAB or TCAOB. Other malformations included altered feather pattern and lack of down, hemorrhage, external viscera, reduced body size, failure to withdraw the yolk sac, beak malformation, dilation of blood vessels, and monomicropthalmia. The results of this investigation suggest that both TCAB and TCAOB are teratogenic in the chick embryo.

Alteration of precocene II-induced hepatotoxicity by modulation of hepatic glutathione levels

Precocene II (6,7-dimethoxy-2,2-dimethyl-2H-benzo[b]pyran), an insect growth regulator that is structurally related to several naturally occurring carcinogenic and non-carcinogenic alkenylbenzenes, is genotoxic and produces hepatic centrolobular necrosis in rats. This investigation was conducted to evaluate the effects of modulation of hepatic glutathione levels on the toxicity of precocene II. Administration of a toxic dose of precocene II (175 mg/kg) to male Sprague-Dawley rats rapidly depleted hepatic GSH, produced histopathological changes in the liver, and induced increases in serum aminotransferase activity. Concurrent administration of the cysteine pro-drug L-2-oxothiazolidine-4-carboxylic acid (OTC) prevented these toxic effects of precocene II. In contrast, pretreatment of rats with DL-buthionine-SR-sulfoximine (BSO), an inhibitor of glutathione synthesis, potentiated the toxicity of an otherwise non-toxic dose of precocene II (100 mg/kg). These results indicate that glutathione is important for protection from precocene II-induced hepatotoxicity.

Precocene II nephrotoxicity in the rat

The effects of precocene II (6,7-dimethoxy-2,2-dimethylchromene) on the kidney were examined histopathologically. Marked changes were observed in the proximal convoluted tubules, collecting tubules, and glomeruli. These changes included congestion of blood in the capillaries of the glomeruli, tubular cell degeneration, and tubular cell regeneration. In addition, blood urea nitrogen levels were elevated in precocene II-treated animals. The results indicate that precocene II is nephrotoxic in the male Sprague-Dawley rat.

Metabolic fate of the hepatotoxic anti-juvenile hormone precocene II in the rat☆

The metabolic fate of precocene II (6,7-dimethoxy-2,2-dimethyl-2H-benzo[b]pyran), a potent anti-juvenile hormone, was examined in male Sprague-Dawley rats. When a single intraperitoneal dose of [3H]precocene II was administered, approximately 34% of the administered dose was excreted within the first 24 h. Examination of the various urinary metabolites indicated that precocene II is metabolized to a stereoisomeric cis/trans mixture of the 3,4-dihydroxy-3,4-dihydroprecocene II. In addition, a mercapturic acid derivative of precocene II was tentatively identified. Thus, it is postulated that a highly reactive 3,4-epoxide intermediate is generated in vivo by the rat during oxidative metabolism.

Reproductive, immunologic, and cytogenetic effects of dietary 3,3',4,4'-tetrachloroazoxybenzene exposure to mice.

The effects of chronic dietary exposure to 3,3′,4,4′-tetrachloroazoxybenzene (TCAOB) on the reproductive efficiency of female Swiss-Webster mice were measured. The immunocompetence of their offspring was assayed at weaning. No indications of toxicosis were seen in the adult females with the exception of a reduction in the thymus weight of the animals consuming 10 ppm TCAOB. Pup mortality to weaning and the percentage of females whelping were not affected by 0.1 ppm, 1 ppm, or 10 ppm of TCAOB in the diet. There was, however, a significant reduction in the number of pups (at birth and at weaning) per female whelping at 10 ppm TCAOB in the diet. The thymus weight and plaque-forming-cell response of the pups were also significantly reduced below control levels. The lymphocyte blastogenic response to Concanavalin-A and lipopolysaccharide, and total peripheral blood leukocyte count, were not affected by the concentrations of TCAOB tested. Dietary treatment of female mice with relatively high concentrations of this chemical resulted in reduced reproductivity capacity, but only moderate immuno-suppression of their offspring exposed duringin utero and early postnatal development.Spleen cells of mice exposedin vivo to TCAOB for 28 days via the diet were examined for chromosome damage and sister chromatid exchange. TCAOB was investigated because of its similarity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); TCAOB caused significantly increased chromatid breakage, suggesting that it may be affecting protein synthesis or may be a mutagen. However, TCAOB did not result in a significant increase in sister chromatid exchanges or isochromatid breaks even at a dietary concentration of 40 ppm.

Effects of 3,3′,4,4′-tetrachloroazoxybenzene on selected immune parameters of the mouse

The effects of 3,3′,4,4′-tetrachloroazoxy-benzene (TCAOB) on the immune system of mice was examined and compared with cyclophosphamide (CY). This chemical can be produced as a result of microbial degradation of commonly used chloroaniline herbicides. Herbicides of the acylan-iline class generally have a low mammalian toxicity while remaining relatively inexpensive. Therefore, TCAOB could be formed anywhere in the environment where choroaniline pesticides are used. TCAOB treatment caused thymic atrophy and a decrease in white blood cell (WBC) count after sheep red blood cell (SRBC) immunization. A decline in the number of Lyt-1+ cells (T-helper lymphocytes) was seen in all TCAOB animals, with unimmunized mice also showing a decreased number of Lyt-2.2+ cells (T-suppressor lymphocytes). No change was found in the ratio of these two cell types. TCAOB did, however, result in a severe reduction in the number of plaque-forming-cells (PFCs) and in serum antibody concentration. CY caused a decrease in thymus weight, WBC count, the number of cells recovered per spleen, and the relative percentages of Lyt-l+ and Lyt-2.2+ cells recovered. The mice exhibited a lower lymphocyte blastogenic response to lipopolysaccharide (LPS) than control animals, but no change in Concanavalin A (Con-A) responsiveness. CY also resulted in a severe drop in the number of PFCs and the quantity of antibody produced following SRBC immunization.