Wallace J. Murray

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.

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.

Spectrophotometric study of the photodecomposition kinetics of nifedipine

Nifedipine is a photosensitive compound. Irradiation for 4 h under a fluorescent lamp placed 30 cm from a solution of nifedipine in 95% ethanol leads to complete photo‐oxidation as determined spectrophotometrically. The disappearance of the reduced form and appearance of the oxidized form is best described by zero‐order kinetics at concentrations higher than 4 times 10−4 M. At lower concentrations pseudo‐first order kinetics are followed. Monochromatic irradiation of nifedipine at wavelengths 400 to 700 nm in 25 nm increments showed no change in the absorbance at 280 nm, and, except for a hyperchromic effect at 237 nm, no other spectral changes were observed. Its photo‐oxidation was dependent on the intensity of light and increased exponentially as solutions were irradiated progressively closer to a fluorescent light source. The pH studies showed that aqueous solutions of nifedipine photo‐oxidized fastest at pH 2.

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 (TCDD)-induced decrease in the fluidity of rat liver membranes

1. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCCD)-induced lipid peroxidation has previously been demonstrated by assessing the hepatic content of thiobarbituric acid reactive substances (TBARS) as well as the NADPH-dependent microsomal formation of TBARS as well as the NADPH-dependent microsomal formation of TBARS using malondialdehyde as the standard. 2. Changes in membrane fluidity as a result of lipid peroxidation may occur. Therefore the dose- and time-dependent effects of TCDD on lipid peroxidation in mitochondrial, microsomal, and plasma membranes, and changes in membrane fluidity in these subcellular fractions, were examined. Animals were treated with either 50 or 100 micrograms TCDD/kg orally, and killed 3, 6, or 9 days post-treatment. 3. Time-dependent increases occurred in TBARS content and formation following TCDD administration for all three membranes. Similar results were observed after 50 and 100 micrograms TCDD/kg. 4. Following TCDD administration, fluorescence polarization measurements as determined by the fluorescence polarization (r) and anisotropy parameter (a.p.) values demonstrated significant decreases in membrane fluidity in all membrane fractions, indicative of membrane structural alterations. 5. Excellent inverse correlations between lipid peroxidation and membrane fluidity were observed. Thus, decreased membrane fluidity and increased membrane damage may contribute to the toxic manifestations of TCDD as a consequence of an oxidative stress.

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.

Biochemical and functional effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the heart of female rats

Biochemical, functional and morphologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the hearts of female rats were examined. Six days after the treatment of rats with TCDD, the blood pressures and resting heart rates were significantly less than in control animals. Treated animals were also less responsive to the effects of the beta-1 agonist, (-)isoproterenol. No histopathologic changes were observed in the heart although extensive centrilobular necrosis occurred in the liver after TCDD administration. Serum levels of thyroxine were 66% less than in control animals. Marked lipid peroxidation was produced in the liver with small but significant increases occurring in the heart. TCDD administration had no effect on catalase activity in the heart, but produced a 20% decrease in superoxide dismutase activity relative to control animals. The effects of TCDD on cardiac function do not appear to be due to a direct action of the xenobiotic on the heart but possibly to a down-regulation of beta-receptors in the heart as a result of the hypothyroid state.

Hepatic lipid peroxidation, sulfhydryl status, and toxicity of the blue‐green algal toxin microcystin‐LR in mice

Microcystin-LR (MCLR), a cyclic heptapeptide produced by the blue-green algae Microcystis aeruginosa, produces death in female mice treated with 100 micrograms MCLR/kg. Kupffer-cell hyperplasia was observed histologically after treatment with 50 or 100 micrograms MCLR/kg. No other changes or lethality were observed with the 50 micrograms MCLR/kg, while 100% lethality occurred in less than 2 h in mice treated with 100 micrograms/kg. In these animals liver weights increased by 45% and hepatic hemoglobin content increased 106% at 60 min posttreatment. Liver histology showed loss of hepatic architecture and necrosis 30 min after treatment, and congestion with blood became evident at 45 min after treatment. Serum enzymes were significantly increased 45 min posttreatment. Hepatic nonprotein sulfhydryl content decreased 19% when calculated on the basis of cytosolic protein and 39% when based upon the total protein content, respectively. The sulfhydryl content of the liver cytoskeletal fraction decreased 26% by 30 min after treatment. Decreased enzyme-mediated and increased non-enzyme-mediated lipid peroxidation were observed in hepatic microsomes following both in vivo and in vitro exposure of hepatic microsomes to MCLR. The toxicity of MCLR may be related to alterations in the sulfhydryl content of the cytoskeletal protein. Furthermore, MCLR may either directly or indirectly affect microsomes, suggesting alterations in structure and function of smooth endoplasmic reticulum.

Some factors affecting the photodecomposition of nifedipine

Abstract Nifedipine, like most nitrophenyldihydropyridine derivatives, undergoes rapid photochemical oxidation to the corresponding nitrophenylpyridine when exposed to light. This is accompanied by a remarkable diminution of the pharmacological activity. In this report, the stability of the drug in different organic solvents and in the presence of an antioxidant was investigated. The UV-spectral properties of the oxidized and reduced forms of the drug were similar in acetonitrile, ethanol and chloroform. In cyclohexane the oxidized form showed absorption characteristics different from those in other solvents. Kinetic parameters for the disappearance of the reduced form or the appearance of the oxidized form as a function of bisulfite concentration were determined and the half-lives calculated. The degradation of two structural analogues of nifedipine was also studied. The results have shown that an electron donating substituent in the position of the nitro group imparts high light resistance to this group of substances.