Edward K. Lai

Specificity of a phenobarbital-induced cytochrome P-450 for metabolism of carbon tetrachloride to the trichloromethyl radical

Evidence is presented which demonstrates that the first polypeptide to disappear in liver microsomes of phenobarbital-induced rats treated with CC14 was the 52,000 dalton p-450 cytochrome. Data are also presented which show that this form of cytochrome P-450 was capable of generating the trichloromethyl radical from CCl4 in a reconstituted system containing the purified cytochrome, NADPH-cytochrome P-450 reductase, NADPH, CCl4, and the spin-trapping agent, phenyl-t-butyl nitrone. Other cytochrome P-450 fractions not containing the 52,000 dalton form did not produce this radical. The formation of this highly reactive radical may have resulted in localized damage to the cytochrome, causing the cytochrome either to be released from the microsomal membrane or to form large aggregates which did not migrate in the gel electrophoretic procedures employed.

Confirmation of assignment of the trichloromethyl radical spin adduct detected by spin trapping during 13C-carbon tetrachloride metabolism in, vitro, and, in, vivo

Abstract Rat liver microsomal incubation systems containing the free radical spin trap, phenyl-t-butyl nitrone, as well as an NADPH generating system and [13C]CCl4 (90 atom % 13C) produce electron spin resonance spectra consistent with that expected for a trichloromethyl-phenyl-t-butyl nitrone adduct. This same spectrum is observed in a lipid extract of the liver from a rat orally administered [13C]CCl4 as well as in a solution of phenyl-t-butyl nitrone and [13C]CCl4 irradiated with ultraviolet light.

In vivo spin trapping of free radicals generated in brain, spleen, and liver during γ radiation of mice

Spin trapping techniques combined with electron spin resonance spectroscopy were used to capture and detect free radicals generated in vivo during exposure to ionizing radiation. Tissue extracts of mice given an intraperitoneal or intragastric dose of the spin trap, alpha-phenyl-t-butyl nitrone prior to exposure to gamma radiation (2 to 5 Gy), contained a radical adduct with hyperfine splitting constants characteristic of spin adducts of carbon-centered lipid radicals. Considerably more radicals were trapped in tissues when the trap was given 3 h before radiation as compared to 30 min before exposure. The radicals observed may either be secondary species resulting from an attack on cellular components by products of water radiolysis, or primary radicals resulting from direct interaction of the radiation with biological molecules. The results indicate that the spin trapping agent is able to penetrate well into animal tissues, and to capture radical species under conditions where the latter would be expected to occur.

Selective early loss of polypeptides in liver microsomes of CCl4-treated rats. Relationship to cytochrome P-450 content.

Treatment of rats with carbon tetrachloride (CCl4) resulted in early reproducible losses of either one or two specific polypeptides (depending on the inducing agent with which the animals had been treated) in the molecular weight range of the multiple forms of cytochrome P-450. The loss was correlated with a decrease in total cytochrome P-450 content in the microsome. The results of this study and those in the accompanying report indicate that CCl4 was metabolized by a specific form of cytochrome P-450 (52,000 daltons), which was rapidly destroyed in the process. The early loss of this peptide occurred simultaneously with the previously demonstrated production of highly reactive trichloromethyl radicals (CCl3). This polypeptide, which was shown to disappear from liver microsomes following treatment of rats with CCl4 was demonstrated in the accompanying report to be the form of cytochrome P-450 specifically required for production of the highly reactive trichloromethyl radical in a reconstituted monooxygenase system.

Evidence That Alpha‐Tocopherol Functions Cyclically to Quench Free Radicals in Hepatic Microsomes Requirement for Glutathione and a Heat‐Labile Factor

In the early part of this decade, Burk and co-workers reported that lipid peroxidation in rat liver microsomes was inhibited by the presence of glutathione.I4 These investigators found that the inhibition of peroxidation by glutathione was heat-labile and that this protective activity involved alpha-to~opherolP.~ It was concluded that the thiol may function in conjunction with a trypsin-sensitive microsomal factor4 to protect the endoplasmic reticulum of the liver against oxidative stress that might trigger peroxidative breakdown, possibly by recycling tocopheroxyl radicals back to tocopherol, as the latter quenches free radicals formed in the initiating phase of lipid peroxidation.’-’ The hepatic endoplasmic reticulum is the site of a number of strongly oxidizing cytochrome P-450 enzyme systems that have the capability of metabolizing some xenobiotic compounds to free radicals? This report describes an investigation in which additional evidence has been obtained to support the hypothesis that a labile glutathione-dependent factor, presumably an enzyme, cycles the tocopheroxyl radical back to tocopherol, as the latter quenches free radical reactions that may be initiated by enzymic process in the hepatic endoplasmic reticulum. The latter appears to be the only organelle in animal tissues containing such a protective mechanism. The data also suggest that the recycling to the tocopheroxyl radical by ascorbic acid may not be efficient in this organelle.

Ethanol feeding stimulates trichloromethyl radical formation from carbon tetrachloride in liver

1. Female, Sprague-Dawley rats were fed liquid ethanol or control diets, both of which contained fat either at 35% (high fat, HF) or 12% (low fat, LF) of total calories. The rats were given an oral dose of 13CCl4 along with the spin trapping agent, phenyl tert.-butyl-nitrone (PBN). 2. Analysis of the hepatic lipid extracts revealed a signal due to the trichloromethyl radical (CCl3) adduct of PBN. Ethanol feeding in the HF diet increased the signal intensity two-fold over controls, whereas ethanol feeding in the LF diet caused only a 35% increase. 3. In isolated microsomes, ethanol feeding in HF or LF diets increased CCl3 formation by approx. 8-fold and 4-fold, respectively, over control values. These data support the hypothesis that ethanol induces a cytochrome P-450 isozyme that is highly active in the metabolism of CCl4 to the CCl3 radical. 4. Ethanol feeding markedly enhanced the hepatotoxicity of CCl4; however, there were no differences in the loss of hepatic enzymes into blood between the ethanol plus HF or ethanol plus LF groups. Thus, ethanol is likely to increase CCl4 toxicity by some mechanism in addition to increased trichloromethyl radical formation.

Spin Trapping of Free Radicals Produced in vivo in Heart and Liver During Endotoxemia

Studies using free radical scavengers and measurements of lipid peroxidation have suggested that free radicals are generated during endotoxemia. Conclusions from these studies have implied that free radicals may participate in the sequence of pathologic events following endotoxin challenge in the experimental animal. Current inferences of free radical generation and involvement have been derived from indirect evidence and are therefore inconclusive. To quantitate the generation of free radicals in vivo during endotoxemia this study employed the use of electron paramagnetic resonance spectroscopy (EPR) combined with spin trapping techniques. Five minutes before intraperitoneal endotoxin administration, trimethoxy-a-phenyl-t-butyl-nitrone [(MeO)3 PBN] was administered intraperitoneally. Experimental animals were always matched with control animals receiving no endotoxin. At either five minutes or twenty-five minutes following endotoxin administration animals were decapitated and hearts and livers were rapidly taken for lipid extraction and EPR evaluation. Analysis of the EPR spectra revealed hyperfine splitting constants that indicated the presence of carbon-centered radical spin adducts in both organ tissues from animals exposed to endotoxin for twenty-five minutes. No signals were present in hearts and livers taken five minutes after endotoxin administration. EPR evaluation did not indicate spin adduct formation in control tissue. These data directly demonstrate that activation of processes in vivo involving free radical generation occur early during endotoxemia, but are not detectable immediately after the endotoxin challenge.

Mass Spectroscopy and Chromatography of the Trichloromethyl Radical Adduct of Phenyl Ter T-Butyl Nitrone

Positive structural identification of the PBN-trichloromethyl spin adduct in vitro was accomplished with the use of high pressure liquid chromatography and/or gas chromatography coupled with mass spectrometry. Both thin layer and liquid chromatography were used to separate a complex mixture of compounds from rat liver extracts treated with CCl4 in vitro and in vivo. Deuterated PBNs (PBN-d9; tert-butyl deuteration, or PBN-d14; both phenyl and tert-butyl deuteration) were also used to aid in the mass spectral analysis of spin adducts from liver extracts of CCl4 exposed rat livers, since the tert-butyl group fragment ion. C4D9+ (m/z = 66) is always present for PBN and PBN spin adducts. In addition, the masses of the ion peaks increase by the amount of deuteration, i.e. an increase of 9 for PBN-d9 or PBN-d14 in comparison to normally synthesized PBN.

Studies on the properties of the singlet oxygen-like factor produced during lipid peroxidation.

The singlet oxygen reaction product of various trapping agents is observed during enzymic and nonenzymic peroxidation of microsomes as well as during the peroxidation of pure lipids extracted from microsomes. We now wish to report that purified fatty acid hydroperoxide alone, as well as peroxidized microsomal lipid and cumene hydroperoxide also form the singlet oxygen reaction product with 2,5-diphenylfuran. The reaction product (cis-1,2-dibenzoylethylene) was observed to be formed in an anaerobic system, with or without EDTA. The data indicate that a reaction of hydroxyl radicals with 2,5-diphenylfuran cannot account for the formation of dibenzoylethylene in these systems. These results are consistent with a hypothesis that the singlet oxygen-like factor was formed from the lipid peroxides per se and, in addition, supports the possibility that either the peroxides can react directly with diphenylfuran to produce dibenzoylethylene or that the self-reaction of organic peroxides may form an intermediate product which can react directly with singlet oxygen-trapping agents to produce substances which are identical to a reaction of the trapping agents with singlets oxygen.

Biological Systems Which Suppress Lipid Peroxidation

Cell metabolism and the environmental factors in general place animal tissues at chronic risk of oxidative alteration of membrane lipids and other components. Several oxidation-reduction enzymes in subcellular organelles are capable of initiating lipid peroxidation in those organelles in vitro. For example, the synthesis of ascorbic acid from gulonolactone by gulonolactone oxidase causes a peroxidative degradation of membrane phospholipids in liver microsomes (1). Oxidation of NADPH by both liver microsomes (2) and liver mitochondria (3) results in lipid peroxidation also. The metabolism of some xenobiotic compounds by the drug metabolizing system is also capable of promoting oxidative degradation of both membrane lipids and proteins (4–6). In addition, radiation, airborne chemicals, ozone, and water pollutants may also produce oxidative damage to tissue.