Sidney J. Stohs

Lipid peroxidation as a possible cause of TCDD toxicity

The target tissues of TCDD, the dysfunctions that result in death in experimental animals, and the ultimate biochemical lesion(s) caused by TCDD are not known despite numerous studies. We have shown by the thiobarbituric acid and conjugated diene methods that TCDD induces hepatic lipid peroxidation in rats. The lipid peroxidation produced by TCDD is both dose and time dependent. A 5-6 fold increase in lipid peroxidation occurs within 6 days following the administration of 40 micrograms TCDD/kg body weight/day for 3 days. Thus, the toxicity of TCDD may be caused in part by free radical-mediated lipid peroxidation that leads to general cell membrane damage which can ultimately produce death in experimental animals at acutely toxic doses.

Altered Activity of Hepatic Mixed-Function Monooxygenase Enzymes in Streptozotocin-induced Diabetic Rats

1. In streptozotocin-induced diabetic male rats, hepatic microsomal aminopyrine N-demethylase activity was depressed, whereas aniline hydroxylase activity and cytochrome P-450 content were increased over control values. 2. In diabetic female rats, hepatic microsomal aminopyrine N-demethylase activity, aniline hydroxylase activity, biphenyl 4-hydroxylase activity, and cytochrome P-450 content were increased over control values. 3. Insulin treatment of diabetic male and female rats antagonized all physical and biochemical abnormalities of the diabetic state; 4. Methyl analogues of streptozotocin did not produce a diabetic state when injected into female rats, and resulted in no changes in aminopyrine N-demethylase activity, aniline hydroxylase activity, or cytochrome P-450 content. 5. Insulin treatment of non-diabetic female rats resulted in slight decreases in aminopyrine N-demethylase and aniline hydroxylase activities, but no changes in cytochrome P-450 content. These observations suggest that insulin primarily influences drug metabolism of diabetic animals through correction of the insulin-deficient diabetic state.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced lipid peroxidation in genetically responsive and non-responsive mice

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) administration on lipid peroxidation in various strains of responsive and non-responsive mice was determined. The hepatic content of thiobarbituric acid reactive substances (TBARS) was used as an index of lipid peroxidation, employing malondialdehyde as the standard. Six days after the administration of a single oral dose of 0.5 μg TCDD/kg to congenic male C57BL/6J mice, which were either homozygous (bb) or heterozygous (bd) with respect to the gene for the Ah locus, significant increases in hepatic lipid peroxidation were induced. Hepatic lipid peroxidation was induced in homozygous non-responsive (dd) mice by a dose of 25 μg TCDD /kg but not 0.5 μg/kg. In kidney, heart and testis, lower doses of TCDD produced lipid peroxidation in C57BL/6J bb and bd mice as compared to dd mice. The ability of TCDD to induce lipid peroxidation in B6D2F1/J, DBA2J and Swiss-Webster mice was also examined. A dose of 2,500 μg TCDD/kg produced the same level of lipid peroxidation in DBA/2J mice that was observed with 180 μg TCDD/kg in C57BL/6J bb and B6D2F1/J mice. Lipid peroxidation was not induced in kidney, heart, or testis at any of the doses of TCDD that were administered to DBA/2J mice. Thus, large strain differences exist in the ability of TCDD to induce lipid peroxidation in mice. In the liver, the ability to induce lipid peroxidation by TCDD is controlled in part by the Ah “b” allele, although other loci may play a major role.

Effects of oltipraz, BHA, ADT and cabbage on glutathione metabolism, DNA damage and lipid peroxidation in old mice ☆

Eighteen-month-old female mice were fed defined diets for 2 weeks which contained 0.05% or 0.10% oltipraz, 0.10% anethole dithione (ADT), 0.10% butylated hydroxyanisole (BHA) or 20% lyophilized cabbage. All diets resulted in significant increases in hepatic reduced glutathione (GSH) content. Glutathione reductase and glutathione S-transferase activities were also significantly higher than the control values. All diets produced significant decreases in hepatic DNA damage (single strand breaks) and lipid peroxidation (malondialdehyde content). In general, similar effects were produced by the two dithiolthiones, oltipraz and ADT. More pronounced effects were produced by oltipraz and ADT than by BHA or cabbage in the diet. Diets high in antioxidants may be effective in retarding free radical reaction processes associated with aging and cancer.

Glutathione levels in hepatic and extrahepatic tissues of mice as a function of age

Glutathione is the most abundant thiol-containing component in living cells and is believed to play an important role as an antioxidant. We have examined the levels of reduced, oxidized, and total glutathione in liver, blood, kidneys, and intestinal mucosa of mice as a function of age. Reduced glutathione levels decreased in kidneys, Intestinal mucosa, and blood while total glutathione levels decreased in all tissue with advanced age. Highest concentrations of reduced glutathione per μg protein were present in liver and intestinal mucosa. Our results support the hypothesis that a decrease in reduced glutathione may contribute to changes associated with aging as well as to the increased susceptibility to disease processes which occur with advanced age.

Glutathione Peroxidase and Reactive Oxygen Species in TCDD-Induced Lipid Peroxidation

Previous studies have shown that high doses of TCDD induce hepatic lipid peroxidation and inhibit selenium dependent glutathione peroxidase (GSH-Px) activity. The dose dependent effects of TCDD on hepatic lipid peroxidation (malondialdehyde content) and GSH-Px activity were determined. A dose as low as 1 microgram/kg induced hepatic lipid peroxidation and inhibited GSH-Px. Based on the use of scavengers of reactive oxygen species, lipid peroxidation (malondialdehyde formation) by hepatic microsomes from both control and TCDD-treated rats appears to be due primarily to H2O2. The results indicate that superoxide, hydroxyl radical and singlet oxygen are also involved. The differences in the reactive oxygen species involved in microsomal lipid peroxidation between control and TCDD treated animals appear to be quantitative rather than qualitative. A 5.9-fold greater rate of malondialdehyde (MDA) formation by microsomes from TCDD treated animals occurred as compared to controls, while livers of TCDD rats had an MDA content that was 5.0-fold greater than the controls. These differences may be due in part to an enhanced production of H2O2 as well as a decrease in the activity of selenium dependent glutathione peroxidase which metabolizes H2O2.

Effects of BHA, d-α-tocopherol and retinol acetate on TCDD-mediated changes in lipid peroxidation, glutathione peroxidase activity and survival

Daily treatment of female rats with butylated hydroxyanisole (BHA) protected against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. This protective effect was associated with reduced microsomal lipid peroxidation, increased glutathione peroxidase (GSH-PX) activity and decreased aryl hydrocarbon hydroxylase (AHH) activity. Retinol acetate (vitamin A) inhibited lipid peroxidation, elevated GSH-PX activity, and enhanced AHH activity. Thirty per cent of vitamin A-treated animals were alive 25 d after a lethal dose of TCDD. d-alpha-Tocopherol (vitamin E) inhibited markedly microsomal lipid peroxidation, enhanced AHH activity, and had no effect on GSH-PX activity. Only 10% of the vitamin E-treated animals were alive 25 d after a lethal dose of TCDD. The mechanism of TCDD toxicity may involve in part inhibition of GSH-PX activity with resultant lipid peroxidation by hydrogen peroxide.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced lipid peroxidation in hepatic and extrahepatic tissues of male and female rats.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)2 administration to rats on lipid peroxidation (microsomal malondialdehyde formation and hepatic malondialdehyde content) was determined by the thiobarbituric acid method. The results demonstrate that dose and time-dependent lipid peroxidation occurs in hepatic as well as extrahepatic tissues. A 3–4 fold increase in hepatic lipid peroxidation was observed following the oral administration of TCDD. Lipid peroxidation also increases in other tissues as kidney and thymus, which are target tissues of TCDD. Sex differences exist with respect to the induction of lipid peroxidation by TCDD. A greater and more rapid induction of hepatic malondialdehyde (MDA) formation in response to TCDD is observed in female rats. When using arachidonic acid as an oxidizable substrate, more MDA was formed with hepatic, kidney and testes microsomes from TCDD-treated rats as compared to microsomes from control animals. No difference was found in the iron content of whole liver following TCDD administration. However, a 47% increase occurred in hepatic mitochondrial iron content, while a 52% decrease was observed in microsomal iron content per mg protein. The addition of ferrous ion to microsomes from control and treated animals resulted in a proportionally greater increase in MDA formation by control microsomes. Desferrioxamine addition to microsomes from control and TCDD-treated rats resulted in a decrease in MDA formation to the same basal level. The increase in MDA production by microsomes from TCDD-treated rats may be due, in part, to an increase in free ferrous ion.

Induction of lipid peroxidation by hexachlorocyclohexane, dieldrin, TCDD, carbon tetrachloride, and hexachlorobenzene in rats

Hexachlorobenzene (HCB), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hexachlorocyclohexane (HCCH) and dieldrin are all halogenated lipophilic environmental contaminants. A common biologic property of these compounds is their ability to induce hepatic microsomal drug metabolizing enzymes. Furthermore, exposure of laboratory animals to these xenobiotics elicits a number of similar effects including porphyria, hypothyroidism, a wasting syndrome and lethality. Perturbation of membrane lipids and lipid peroxidation may be responsible for at least part of the toxic effects of HCCH. TCDD has been shown to induce lipid peroxidation in hepatic and extrahepatic tissues. Based on the similar toxic manifestations of HCB, HCCH, TCDD and dieldrin, the authors have examined the effects of these xenobiotics on hepatic lipid peroxidation following an acutely toxic dose. Lipid peroxidation was assessed by determining the content of thiobarbituric acid reactive substance (TBARS) in the liver, employing malondialdehyde as the standard. Animals were also treated with carbon tetrachloride, a well know inducer of lipid peroxidation, as a positive control. Furthermore, the ability of these xenobiotics to inhibit selenium dependent glutathione peroxidase (GSHPX) activity was determined.

Cyclosporin A — a chemoprotectant against microcystin-LR toxicity

Microcystin-LR (MCLR) is a potent hepatotoxin that rapidly produces death in experimental animals. We have shown that cyclosporin A (CsA) can prevent the toxic and lethal effects of MCLR in mice. The LD50 of MCLR in mice is approximately 61 micrograms/kg, and 100 micrograms/kg produces death in 100% of treated mice. The minimum dose of CsA which prevented lethality in mice given 100 micrograms MCLR/kg i.p. was 10 mg/kg. This dose of CsA did not protect mice against higher doses of MCLR. Survival of all mice given 100 micrograms MCLR/kg was achieved if 10 mg CsA/kg was given 0.5-3 h prior to the MCLR but not if the CsA was co-administered or given after the MCLR. The results indicate that CsA effectively protects mice against a lethal dose of MCLR, but that the time of administration and the dose of CsA are critical determinants of the chemoprotective effects.