John A. Schmitz

Mechanism of chemical-induced toxicity: I. Use of a rapid centrifugation technique for the separation of viable and nonviable hepatocytes

A major obstacle in defining the mechanism of chemical-induced toxicity has been the inability to distinguish between events that cause cell death and those that result from cell death. This problem results from measuring biochemical parameters in tissues or cell pellets containing both viable and nonviable cells. In the present study, we described a method for the rapid separation of viable hepatocytes from nonviable cells and medium prior to biochemical analysis. Separation of viable hepatocytes was accomplished in a microcentrifuge tube by layering a sample of isolated hepatocyte suspension over a dibutyl phthalate oil layer and centrifuging for several seconds. As a result, greater than 90% of the hepatocytes centrifuged through dibutyl phthalate were viable while greater than 90% of the cells recovered above the oil layer were nonviable. The separation of viable hepatocytes by the dibutyl phthalate method was not affected by the presence of the hepatotoxins, adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or ethyl methanesulfonate (EMS), though the ratio of viable to nonviable cells in the suspension was drastically reduced. The metabolic and morphological integrity of hepatocytes centrifuged through dibutyl phthalate was altered after cell suspensions were treated with the ADR-BCNU or EMS. These chemically treated viable hepatocytes showed degenerative ultrastructural changes and a greater than 80% reduction in intracellular K+ and glutathione concentrations. Because centrifugation through dibutyl phthalate does not significantly alter the concentration of intracellular constituents nor the ultrastructure of control hepatocytes, the signs of reversible injury observed in hepatocytes centrifuged through oil resulted from the chemical treatment. These data indicate that the dibutyl phthalate separation technique offers the advantage of monitoring only viable hepatocytes for changes in membrane integrity or metabolic performance during a toxic chemical insult.

Immunotoxicity of pentachlorophenol (PCP): Increased susceptibility to tumor growth in adult mice fed technical PCP-contaminated diets☆

Adult male C57B16 mice were fed diets containing 50 or 500 ppm pure (99+%) or technical grade (86%) pentachlorophenol (PCP) for 10–12 weeks prior to assessment of immunocompetence. Assays for immunocompetence included in vivo host susceptibility to virus infection and tumor growth and in vitro quantitation of T-cell cytotoxicity and macrophage phagocytosis. Exposure of mice to technical PCP resulted in profound enhancement of tumor susceptibility. The incidence of progressive methylcholanthrene (MCA)-induced transplanted tumors increased from 35% in controls to 67 and 82% in animals exposed to 50 and 500 ppm technical PCP, respectively. Mortality following primary Moloney sarcoma virus (MSV) inoculation and secondary MSB challenge increased from 19% in controls to 45 and 73% in animals exposed to 50 and 500 ppm technical PCP, respectively. An additional 50% of the MSVMSB challenged mice exposed to 50 ppm technical PCP exhibited tumor dissemination in the spleen. Animals exposed to pure PCP did not exhibit enhancement of primary MCA- or MSV-induced tumor growth. However, splenic tumor development was observed in 22 and 44% of the MSVMSB challenged mice exposed to 50 and 500 ppm pure PCP, respectively. Control mice did not develop splenic tumors. In contrast to the enhancement of tumor susceptibility, mortality associated with encephalomyocarditis virus infection tended to be reduced by technical PCP exposure. In vitro immune functional assessment indicated significant depression in T-cell cytolytic activity and enhancement of macrophage phagocytosis in animals exposed to technical PCP. No significant changes in in vitro immune response were noted with cells from pure PCP-exposed animals.

Protective role of dietary butylated hydroxyanisole against chemical-induced acute liver damage in mice

Prior consumption of a diet containing the food antioxidant, butylated hydroxyanisole (BHA), by female mice prevented the development of or minimized the acute liver damage caused by monocrotaline, acetaminophen, or bromobenzene. In contrast, neither the incidence nor the severity of carbon tetrachloride-induced hepatotoxicity was affected by dietary BHA. Hepatotoxicity was judged by plasma alanine aminotransferase and aspartate aminotransferase levels, hepatic cytochrome P-450 content, and liver histology. The protective effect of BHA against acetaminophen-induced hepatotoxicity was not demonstrated in male mice. The observed protection by dietary BHA against acetaminophen- and bromobenzene-induced hepatotoxicity was associated with the increase of liver glutathione. It is concluded that the protective action of BHA is dependent upon the nature of the toxic agent.

Protective action of zinc against pyrrolizidine alkaloid-induced hepatotoxicity in rats.

The influence of Zn on the acute hepatotoxicity of pyrrolizidine alkaloids (PAs) was determined in male rats. Zinc, 72 mumol/kg as ZnCl2, was administered ip for 3 consecutive days, followed 16 h after the last dose by a single ip injection of purified mixed PAs (80, 120, or 160 mg/kg) obtained from tansy ragwort (Senecio jacobaea). Hepatotoxicity of the PAs was assessed by measuring the activities of plasma glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) and by histological examination of the liver. There was a dose-dependent increase in plasma GOT and GTP 24 h after PA administration, whereas no significant increase of these enzymes was seen after administering Zn alone. The 7-fold increase in plasma GOT and 12-fold increase in GPT after PA (120 mg/kg) were reduced to 2.4- and 2.1-fold, respectively, by Zn pretreatment. The PA-induced liver necrosis was either reduced in severity or abolished by Zn when the PA dose was 80 or 120 mg/kg. These results suggest a protective effect of Zn against PA hepatotoxicity. The protective effect was associated with a marked increase in liver metallothionein and a significant decrease in hepatic cytochrome P-450 content, aminopyrine N-demethylase activity, and in vitro microsomal conversion of the PAs to pyrroles. Liver nonprotein sulfhydryls were unchanged. The possible role of metallothionein in the sequestration of pyrrole metabolites merits further investigation.

Influence of diet on the effects of selenium in the genesis of mammary tumors.

Two experiments were conducted to study the interaction of various diets with selenium in the genesis of mammary tumors. In the first experiment, virgin C3H mice were fed either an Oregon State University (O.S.U.) chow or this chow plus 0.5 or 2.0 ppm selenium in the drinking water, or Wayne Lab-Blox Chow with or without 2.0 ppm selenium in the water. Selenium significantly reduced the tumor incidence in mice fed the O.S.U. chow, but not in those fed the Wayne chow. In the second experiment, virgin female C3H mice were fed a purified casein-based diet with either lard, butter, corn oil, or rapeseed oil at a 10% level without or with selenium (2 ppm) in the drinking water. Selenium had no significant effect upon tumor incidence in this experiment. Thus, these results indicate that the type of diet has a marked influence on the beneficial effects of selenium in reducing mammary tumors in C3H mice.

Modifications of chronic hepatotoxicity of pyrrolizidine (Senecio) alkaloids by butylated hydroxyanisole and cysteine

The chronic hepatotoxic effects of mixed pyrrolizidine alkaloids (PAs) from the poisonous plant tansy ragwort (Senecio jacobaea) and the ability of butylated hydroxyanisole (BHA) and cysteine to alter these hepatic effects were studied in male rats. In control animals, the i.p. administration of a single dose of mixed PAs, 160 mg/kg, produced marked fibrosis, biliary hyperplasia, megalocytosis, necrosis and calcification in liver 8 weeks post injection. In contrast, consumption of 0.75% BHA diet 10 days before and 14 days after PA administration reduced the incidence and/or completely prevented the occurrence of these pathological changes. Similar treatment with 1% cysteine, however, only reduced the severity of the hepatic lesions.

Immunogenicity of a soluble antigen against Pasteurella haemolytica-associated pneumonia in calves

Three experiments were performed to evaluate the immunogenic potency of a soluble fraction ofPasteurellahaemolytica against pneumonic pasteurellosis in calves. A soluble antigen was extracted by a 2.5% saline solution fromP.haemolytica. Weaned Holstein bull calves, seronegative for infectious bovine rhinotracheitis virus (IBRV) and the pasteurella antigen, were vaccinated either by repeated subcutaneous (SC) vaccination, or by exposure 3 times to the aerosol ofP.haemolytica antigen. Challenge exposure to aerosol ofP.haemolytica was preceded by infection with IBRV, or in experiments 2 and 3, the virus exposures were combined with a stress treatment. The lung lesions were examined at necropsy 3 to 8 days post infection. In the first experiment, all the vaccinated calves produced specific antibody response to the pasteurella antigen, and none of the calves including controls showed significant lesions in the lung. In the second experiment 2 aerogenically vaccinated calves had no lesions. One of the two SC-vaccinated calves had mild consolidated lesions. Two control calves, one of which died 3 days following the challenge, developed severe fibrinous pneumonia with consolidation of 50% or more of the lung surfaces.P.haemolytica was isolated only from the 2 control animals. In the third experiment, 2 of the 3 control calves developed moderate to severe consolidation, butP.haemolytica was isolated only from one of them. Two of the three aerosol-vaccinated calves also developed significant lesions and one of them yielded the bacteria from the lung. Three SC-vaccinated calves had slight lesions and the organism was not isolated from their lungs. The results did not consistently indicate an immunogenic potential of the soluble antigen againstP.haemolytica-related pneumonia. The effect of stress on the pathogenesis of bovine viral penumonia and correlation between pneumonic lesions and antibacterial resistanceinsitu are discussed.

Toxicity of Senecio jacobaea (tansy ragwort) in rats

Abstract To investigate the toxicity of the pyrrolizidine alkaloid containing plant Senecio jacobaea (tansy ragwort), rats were fed diets containing 0 or 5% tansy ragwort and then sacrificed at 1, 2, 4, 6, and 8 weeks. Tansy ragwort produced a depression of body weight gain, food intake, and food efficiency as early as 1 week after beginning treatment. In tansy-fed rats, the relative weights of lungs, heart, spleen, adrenals, and testes were increased at 4 weeks while that of kidney was increased after 6 weeks. Leukocyte counts were markedly elevated at 2 weeks whereas the hematocrit and erythrocyte counts were decreased, respectively, at 4 and 6 weeks of treatment. At 4 weeks, total serum protein, serum albumin, and albumin:globulin ratios were decreased in the tansy-fed rats. The principal pathological changes observed in treated rats were enlarged spleen with lymphoid hyperplasia and increased hematopoietic activity, atrophied thymus, ascites, hydrothorax, and diffuse necrotizing hepatitis. The majority of the gross and histopathological changes were evident following 6 and 8 weeks of treatment.

Comparative effects of antioxidants on the toxicity of mixed pyrrolizidine alkaloids from Senecio jacobaea in mice

The comparative effects of the antioxidants, butylated hydroxyanisole (BHA), ethoxyquin, and cysteine on pyrrolizidine-alkaloid-induced (PA-induced) lethality and acute hepatotoxicity were assessed in female mice. Diets containing 0.75% BHA, 0.25% ethoxyquin, or 1% cysteine were fed to mice for 10 d before the ip administration of mixed PAs from Senecio jacobaea (tansy ragwort), 280 mg/kg. Without the dietary antioxidants, the PAs produced 100% mortality in 24 h. The BHA and ethoxyquin diets were completely and partially protective, respectively, against the PA-induced lethality. The deaths were associated with severe hemorrhagic lesions in liver with or without hepatocytic necrosis. Both BHA and ethoxyquin significantly reduced the incidence of the hemorrhagic lesions but not the necrotic lesions in liver. Cysteine had no significant effect on either mortality or the liver lesions induced by the mixed PAs. These results suggest that dietary antioxidants have differential protective effects against lethality and acute hepatotoxicity induced by mixed PAs from tansy ragwort.