H. Kappus

Evidence for carbon tetrachloride- and ethanol-induced lipid peroxidation in Vivo demonstrated by ethane production in mice and rats

Mice and rats were dosed with carbon tetrachloride (1 g/kg) and placed in a gas-tight all-glass desiccator connected to a gasometer containing pure oxygen. At different times gas samples from the desiccator were examined for their ethane content. Mice produced about 75 nmol of ethane/kg during 4 hr, whereas rats exhaled 45 nmol/kg during this time period. Control mice and rats produced only small amounts of ethane. Rats which were pretreated chronically with carbon tetrachloride (0.4 g/kg daily) for 6 weeks produced only about 21 nmol of ethane/kg during the 4 hr following an acute dose of carbon tetrachloride (1 g/kg). After treating rats with ethanol (5 g/kg) they exhaled about 17 nmol of ethane/kg during 4 hr. Compared to mice, the experiments with rats show that, qualitatively, rats and mice do not differ in ethane production after carbon tetrachloride. However, quantitatively, there are species variations. The results described here give further evidence for the suggestion that acute doses of ethanol can induce lipid peroxidation in vivo .

Disposition of [1,2-14C] vinyl chloride in the rat

Rats were exposed to [1,2-14C] vinyl chloride in a closed system at initial concentrations below 100 ppm. When the system was occupied by 3 rats, a half-life of vinyl chloride in the systems atmosphere of 1.13 ± 0.12 h was observed. The volume of the system was 10.3 l. Calculation of the clearance of vinyl chloride from the system revealed that about 40% of inspired vinyl chloride is absorbed by lung. Therefore, changes in respiration did not influence uptake of vinyl chloride.Uptake of vinyl chloride by the rats was completely blocked by acute pretreatment with potent inhibitors of cytochrome-P-450-dependent microsomal drug metabolism (i.e., by 35 mg/kg 3-bromophenyl-4(5)-imidazole or 50 mg/kg 6-nitro-1,2,3-benzothiadiazole in 0.6 ml/kg DMSO). A weaker inhibition was observed after dosing SKF 525 A or 5,6-dimethyl-1,2,3-benzothiadiazole (50 mg/kg in 0.6 ml/kg DMSO). Metyrapone did not cause inhibition.Uptake of vinyl chloride was increased by pretreatment with DDT and, to a lesser extent, with clotrimazol. No significant stimulation of uptake was observed after pretreatment with phenobarbital, 3-methylcholanthrene, rifampicin, or chronic ethanol treatment.Immediately after exposure, highest radioactivity levels were observed in liver and kidney. The radioactive metabolites of 14C-vinyl chloride were rapidly excreted, largely by the kidneys. Excretion of radioactivity in the urine was 69.4 ± 2.6% within 24 h.ZusammenfassungIn einem geschlossenen System wurden Ratten initialen Konzentrationen an [1,2-14C] Vinylchlorid von unter 100 ppm ausgesetzt. Bei einer Besetzung des Systems durch 3 Ratten wurde in der Atmosphäre eine Halbwertszeit des gasförmigen Vinylchlorid von 1,13 ± 0,12 Std gemessen. Bei einem Volumen des Systems von 10,3 l ergab die Berechnung der Vinylchlorid Clearance, daß nur ca. 40% des von den Ratten eingeatmeten Vinylchlorid resorbiert wurde. Aus diesem Grunde führten Änderungen der Atemtätigkeit nicht zu Änderungen der Aufnahmegeschwindigkeit von Vinylchlorid.Durch sehr wirksame Inhibitoren von Cytochrom-P-450-abhängigen mikrosomalen Oxidationen (3-Bromphenyl-4(5)-imidazol und 6-Nitro-1,2,3-benzothiadiazol) konnte die Aufnahme von Vinylchlorid vollständig verhindert werden. SKF 525 A und 5,6-Dimethyl-1,2,3-benzothiadiazol waren in dieser Hinsicht weit weniger wirksam.Durch Vorbehandlung der Ratten mit DDT und, zum geringeren Maße, mit Clotrimazol wurde die Aufnahme von Vinylchlorid gesteigert. Keine signifikante Steigerung trat auf nach Vorbehandlung mit Phenobarbital, 3-Methylcholanthren, Rifampicin und nach chronischer Alkoholgabe.Unmittelbar nach Beendigung der Exposition wurden die höchsten Radioaktivitätswerte in Leber und Niere festgestellt. Die Metabolite von 14C-Vinylchlorid wurden sehr schnell ausgeschieden. Bereits nach 24 Std wurden im Urin 69,4±2,6% der inkorporierten Radioaktivität gemessen.

Irreversile binding of ethynyl-estradiol metabolites to protein and nucleic acids as catalyzed by rat liver microsomes and mushroom tyrosinas

Abstract 17α-Ethynyl-estradiol is bound irreversibly to protein by the catalytic action of rat liver microsomes and also by mushroom tyrosinase. Whereas the binding reaction in the tyrosinase system could be inhibited by cysteine, cysteine derivatives, lysine and analogous, the microsomal binding reaction was not affected by lysine and amines, but was markedly inhibited by cysteine and its derivatives. Concordantly, an irreversible binding of ethynyl-estradiol metabolites to poly-lysine could only be achieved with tyrosinase, not with rat liver microsomes. The observations support the concept of different reactive intermediates involved in the binding of estrogens to protein by both enzymic systems examined. Since estrogen o-quinones are known to be formed from estrogen phenoles, the ability of 17β-hydroxy-4, 10(1)-estradiene-2,3-dione to react non-enzymatically with cysteine, lysine and related compounds was examined and found in agreement with the postulate of estrogen o-quinones being the intermediates involved in the tyrosinase-catalyzed protein binding of estrogens. Ethynyl-estradiol was not bound irreversibly to DNA and RNA by rat liver microsomes, while tyrosinase could catalyze this type of reaction. This behaviour is seen as a result of the dissimilarity in the active intermediates formed from ethynyl-estradiol by microsomes and tyrosinase.

Irreversible protein binding of metabolites of ethynylestradiol in vivo and in vitro

Abstract During incubation of 6,7-3H-ethynylestradiol with rat liver microsomes up to 20 % of the radioactivity was bound irreversibly to the microsomal proteins. Incubations in presence of albumin resulted in a further radioactive labelling of the albumin. The irreversible nature of the steroid-protein bond was established by solvent extraction and charcoal treatment. Further evidence was obtained after hydrolyzing the microsomal protein with trypsin and submitting the labelled tryptic peptides to ion exchange chromatography and electrophoresis. The labelled albumin was applied to sephadex gel filtration which showed the association of the ethynylestradiol radioactivity to the albumin peak. The binding reaction required supply of NADPH, could be stimulated by pretreatment of the animals with phenobarbital and was inhibited by CO and SKF 525 A. On these characteristics the concept was based that, in analogy to the well known binding of estradiol and estrone, 2hydroxylation is also an essential prerequisite for the binding of ethynylestradiol. The concept was confirmed by trapping off the 2-hydroxy-ethynylestradiol with glutathione, which led to a decrease of the ethynylestradiol-protein binding. Further evidence resulted from experiments in vivo , dosing rats with 6,7-3H-ethynylestradiol and 6,7-3H-estradiol 48 hrs prior to sacrifice and examining the amount of radioactivity irreversibly bound to the liver endoplasmic reticulum. 3H-ethynylestradiol caused a radioactive labelling of microsomes twice as much as that after 3H-estradiol.

Irreversible binding of DOPA and dopamine metabolites to protein by rat liver microsomes.

Abstract Rat liver microsomes catalyze NADPH-dependent irreversible binding of metabolites of DOPA and DOPAmine to microsomal protein and to BSA. Binding is inhibited by cysteine and the singlet oxygen quencher 1,4-diaza-bicyclo(2.2.2)octane. Irreversible binding to BSA is also catalyzed by mushroom tyrosinase, xanthine oxidase, and NADPH-cytochrome c reductase. The results suggest that in the microsomal system the participation of the hemoprotein, cytochrome P-450, is not an absolute requirement for the irreversible binding of metabolites of DOPA and DOPAmine to proteins.

Studies on the Metabolism of Ethynylestradiol in vitro and in vivo: The Significance of 2-Hydroxylation and the Formation of Polar Products

Abstract1. The metabolic fate of ethynylestradiol in vitro and in vivo has been followed by examining the displacement of tritium from [6,7-3H]ethynylestradiol and [2,4,6,7-3H]ethynylestradiol by other substituents. Incubations with rat liver microsomes and NADPH demonstrated that 2-hydroxylation is the major pathway of ethynylestradiol metabolism. In contrast, hydroxylations of ethynylestradiol at C-6 and C-7 are only of minor importance.2. The easy inducibility of this microsomal 2-hydroxylation by pretreatment with phenobarbital, the requirement for NADPH and inhibition by CO and SKF 525A indicate that a haeme protein is involved.3. The microsomal elimination of 3H from C-2 and C-4 of ethynylestradiol was markedly increased by glutathione, which is known to bind at C-1 and C-4 of estrogens forming ‘polar’ products. This formation of polar products was dependent on NADPH and was inhibited by CO and SKF 525A, supporting the concept that 2-hydroxylation of an estrogen is prerequisite for binding of glutat...

Liver microsomal uptake of (14C)vinyl chloride and transformation to protein alkylating metabolites in vitro.

Abstract [1,2- 14 C]Vinyl chloride gas was incubated with rat liver microsomes in an all-glass vacuum system. Microsomal uptake and irreversible protein binding of vinyl chloride radioactivity was determined. Both uptake of vinyl chloride by microsomes and alkylation of proteins by vinyl chloride metabolites were dependent on incubation time, enzymatically active microsomes, NADPH, oxygen, and the partial pressure of vinyl chloride in the atmosphere, and could be inhibited by carbon monoxide. During incubation in presence of NADPH, 10 times more vinyl chloride was taken up by microsomes than in absence of NADPH. Uptake of vinyl chloride by albumin solutions and liposomal suspensions was in a similar range compared to the microsomal uptake without NADPH. Addition of glutathione and cytoplasmic fractions to microsomal incubations with NADPH led to an increase in microsomal uptake of vinyl chloride and to a decrease in protein alkylation by vinyl chloride metabolites. If trichloropropene oxide was present in the microsomal incubation, the protein alkylation reaction by vinyl chloride metabolites was increased twofold, while the microsomal uptake of vinyl chloride was not influenced. Our results are consistent with the view that the microsomal uptake of vinyl chloride radioactivity is due to transformation of vinyl chloride gas to nonvolatile metabolites by microsomal enzymes and that chloroethylene oxide might be the primary microsomal metabolite of vinyl chloride capable of reacting with proteins.

Irreversible protein binding of [14C]imipramine with rat and human liver microsomes☆

Abstract After incubation of [ 14 C]imipramine with rat liver microsomcs up to 0–7 mole/mg was irreversibly bound per mg of microsomal protein. If albumin was added to the microsomal incubations [ 14 C]imipramine was also irreversibly bound to this protein. The irreversible binding of imipramine to protein was determined by exhaustive solvent extraction and charcoal adsorption, and measurement of the remaining 14 C-radioactivity in the protein. The binding reaction was dependent on oxygen, NADPH, microsomal protein content and substrate concentration. It was inhibited by CO and SKF 525-A. Pretreatment of rats with phenobarbital did not increase the amount of imipramine irreversibly bound to protein. Glutathione and other cysteine derivatives diminished the binding, whereas incubation with the epoxide hydrase inhibitor trichloropropene oxide resulted in an increase of imipramine irreversibly bound to protein. The results favour the concept that irreversible protein binding of imipramine is catalyzed by a cytochrome P-450-dependent hydroxylation via an epoxidation step. Irreversible protein binding of imipramine was also detectable with three samples of human liver microsomes.

Molecular aspects of catechol and pyrogallol inhibition of liver microsomal lipid peroxidation stimulated by ferrous ion-ADP-complexes or by carbon tetrachloride

SummaryLipid peroxidation was induced in rat liver microsomes either by iron-ADP-complexes or by carbon tetrachloride in the presence of NADPH. Different compounds containing catechol or pyrogallol structures were examined for their activities to inhibit lipid peroxidation in both systems. In general, all compounds tested showed similar inhibitory activities on lipid peroxidation, if induced by ferrous ion-ADP-complexes or by carbon tetrachloride. This inhibition is explained by the suggestion that catechols and pyrogallos inhibit at the lipid site of the membrane, rather than at the enzymic site. Compounds not containing catechol or pyrogallol groups inhibited lipid peroxidation only weakly. O-Methylation resulted in a decrease of the inhibitory effect. Catecholor pyrogallol-derivatives which contained polar functional side chains, like carboxyl- or amino groups showed minor inhibitory effects compared to the esterified or N-alkylated compounds.Dihydroxychlorpromazine, 2-hydroxy-estradiol and 2-hydroxyethinylestradiol were the most effective inhbitors of microsomal lipid peroxidation (I50-values of 1×10−6 to 2×10−7 M). The inhibitory activity of α-tocopherol, glutathione and ascorbic acid, naturally occurring antioxidants, was about three orders of magnitude lower.Inhibition of lipid peroxidation induced by NADPH-cytochrome c reductase and iron-ADP-complexes in the presence of NADPH and liposomes was also observed with catechols.From our results we assume that the molecular structure of a catechol or pyrogallol functional group is a prequisite for an effective inhibition of lipid peroxidation by these chemicals. Furthermore, the results are discussed in relation to the requisite membrane affinity of catechols, pyrogallols and other antioxidants which might be used for inhibition studies on lipid peroxidation in vivo.

Oxygen dependence of CCl4-induced lipid peroxidation in vitro and in vivo

Rat liver microsomes showed an atypical oxygen dependence of carbon tetrachloride (CCL4)-induced malondialdehyde formation with a maximum at ca. 7% O2 and a minimum at ca. 15% O2. Rats treated with CCl4 expired less ethane under high oxygen concentrations and more ethane under low oxygen concentrations. The initiation of CCl4-induced lipid peroxidation in the liver would appear to be influenced by the oxygen concentrations present in the hepatocytes.