Anders Tunek

Multi-step metabolic activation of benzene. Effect of superoxide dismutase on covalent binding to microsomal macromolecules, and identification of glutathione conjugates using high pressure liquid chromatography and field desorption mass spectrometry

Abstract Incubation of [ 14 C]benzene or [ 14 C]phenol with liver microsomes from untreated rats, in the presence of a NADPH-generating system, gave rise to irreversible binding of metabolites to microsomal macromolecules. For both substrates this binding was inhibited by more than 50% by addition of superoxide dismutase to the incubation mixtures. The decrease in binding was compensated for by accumulation of [ 14 C]hydroquinone, indicating superoxide-mediated oxidation of hydroquinone as one step in the activation of benzene to metabolites binding to microsomal macromolecules. Since our previous work had shown that binding occurred mainly with protein rather than ribonucleic acid and was virtually completely prevented by glutathione, suggesting identity of metabolite(s) responsible for binding to protein and glutathione, a conjugate was chemically prepared from p -benzoquinone and reduced glutathione (GSH) and identified by field desorption mass spectrometry (FDMS) as 2-( S -glutathionyl) hydroquinone. Microsomal incubations, containing an NADPH-generating system, with benzene, phenol, hydroquinone or p -benzoquinone in the presence of [ 3 H]glutathione or, alternatively, with [ 14 C]benzene or [ 14 C]phenol in the presence of unlabeled glutathione, were performed. All of these incubations gave rise to a peak of radioactivity eluting from the high pressure liquid chromatograph (HPLC) at a retention time identical to that of the chemically prepared 2-( S -glutathionyl) hydroquinone, whilst microsomal incubation of catechol in the presence of [ 3 H]glutathione led to a conjugate with a very different retention time which was not observed after incubation of benzene or phenol. The microsomal metabolites of p -benzoquinone, hydroquinone and phenol thus eluting from the HPLC were further identified as the 2-( S -glutathionyl) hydroquinone by field desorption mass spectrometry. The glutathione adduct formed from benzene during microsomal activation eluted from HPLC with the same retention time and its mass spectrum also contained the molecular ion (MH + ) ( m/e 416) of this conjugate as an intense peak, but the fragmentation patterns did not allow definite assignments probably due to the considerably smaller amounts of ultimate reactive metabolites formed from this pre-precursor and thus relatively larger amounts of impurities. The results indicate that rat liver microsomes activate benzene via phenol and hydroquinone to p -benzosemiquinone and/or p -benzoquinone as quantitatively important reactive metabolites.

Mechanism of benzene toxicity. Effects of benzene and benzene metabolites on bone marrow cellularity, number of granulopoietic stem cells and frequency of micronuclei in mice

The effects of benzene and benzene metabolites *hydroquinone and catechol) on bone marrow cellularity, number of granulopoietic stem cells and on the frequency of micronuclei in polychromatic erythrocytes were investigated in mice. The dose-effect curve for benzene revealed that there was a threshold dose (approx. 100 mg benzene/kg body wt./day injected subcutaneously on 6 consecutive days) above which severe toxicity occurred in all three parameters. Also hydroquinone gave rise to adverse effects in the parameters studied, but the sequence of occurrence was different from that observed with benzene. These data are interpreted to indicate that hydroquinone is a hemotoxic metabolite of benzene in mice in vivo, but that other metabolites, or benzene itself, also probably contribute to the toxicity. Catechol gave no effects. However, due to acute effects like tremor and convulsions only rather low doses could be tested. Simultaneous administration of toluene dramatically reduced the toxicity of benzene, but gave only a small reduction of the hydroquinone-induced effects.

A rapid and sensitive method for determination of covalent binding of benzo[a]pyrene to proteins

A method is presented for the quantitative determination of covalent binding of metabolically activated benzo[a]pyrene to microsomal proteins. After incubation of radiolabelled benzo[a]pyrene with microsomes and NADPH, the mixture is applied to filter paper discs. These are immersed in ethanol to precipitate the proteins. Unbound radiolabel is removed by repeated washes of the filters in organic solvents before scintillation counting. The method is simple, rapid, sensitive and accurate, and works both with 14C- and 3H-labelled compounds. The method is suitable for measuring the incorporation of other radiolabelled xenobiotics to proteins of both microsomes and other subcellular fractions and for the analysis of binding to isolated proteins.

Toxic effects on mouse bone marrow caused by inhalation of benzene

Male NMRI mice were exposed to benzene in air, concentrations ranging between 1–200 ppm. The following parameters in the bone marrows were examined:1. number of nucleated cells/tibia, 2. number of colony forming granulopoietic stem cells (CFU-C)/tibia, and 3. frequency of micronuclei in polychromatic erythrocytes.At continuous exposure as low benzene concentrations as 21 ppm during 4–10 days significantly affected the three parameters. Intermittent exposure (8 h/day, 5 days/week, 2 weeks) also resulted in measurable toxicity, particularly on number of CFU-C/tibia and frequency of micronuclei, at 21 ppm and higher doses. Short peak exposures had very limited effects but did increase the proliferation rate of the bone marrow, i.e., the number of CFU-C/105 cells became elevated.

Microsomal target proteins of metabolically activated aromatic hydrocarbons

The specificity of binding to microsomal proteins of metabolically activated hydrocarbons has been studied. Radioactively labelled benzene, phenol, chlorobenzene, BP and MC were incubated with liver microsomes from control, phenobarbital- and MC-treated rats in the presence of an NADPH-generating system. The patterns of metabolite binding to microsomal proteins were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. Benzene, phenol and chlorobenzene metabolites showed one type of binding pattern dominated by a band at 72 000 Mr. This band was strong both in control and induced microsomes. Additional radioactive bands were seen in the 50 000--60 000 Mr region particularly in MC-induced microsomes. BP and MC metabolites showed a different type of binding pattern with incorporation of radioactivity into several fractions in the 50 000--60 000 Mr region of MC-induced microsomes. Two other strongly labelled fractions occurred at 68 000 and 72 000 Mr. The incorporation was low into control and phenobarbital-induced microsomes. Two labelled bands (Mr 56 000 and 72 000) were common for all hydrocarbons in MC-induced microsomes. The 56 000 Mr band had the same mobility in the gel as an MC-induced protein likely to be cytochrome P-448. The NADPH-generating system was essential for metabolite binding and GSH and UDPGA greatly reduced binding. We suggest that differences in metabolite binding patterns reflect differences in the routes of metabolite formation and that activated hydrocarbons are likely to bind to proteins close to their site of formation.

Glutathione in human melanoma cells: Effects of cysteine, cysteine esters and glutathione isopropyl ester

Thiols are of great importance for the regulation of many cellular functions including metabolism, transport and cell protection. In this study the usefulness of L-cysteine methyl and octyl esters, of N,S-diacetyl-L-cysteine methyl ester and glutathione isopropyl ester as cellular cysteine and GSH delivery systems was investigated in the human IGR 1 melanoma cell line. The L-cysteine methyl and octyl esters proved to be highly toxic to the cells. Treatment of the cultures with 1 mM N,S-diacetyl-L-cysteine methyl ester or 3 mM glutathione isopropyl ester for 24 h resulted in marked elevation of the cellular glutathione level without apparent or with slight cell loss, respectively. Thus the administration of the latter two compounds seems to be suitable for inducing GSH elevation in the cultured melanoma cells.

Multi-Step Metabolic Activation of Benzene in Rat Liver Microsomes

Benzene is a ubiquitous component of our chemical age. Due to its very special chemical and physical properties, benzene is widely used in the chemical industry and in the manufacture of rubber and plastics. In addition, benzene is used in motor fuel as an anti-knocking agent (Berlin et al., 1974).

Biological Threshold Limits for Benzene Based on Pharmacokinetics of Inhaled Benzene in Man

Volunteers were exposed to benzene, 2--10 ppm, under controlled conditions up to 6 h a day during five consecutive days. The accumulation and elimination of benzene was measured by determination of benzene concentration -- down to 0.001 ppm -- in exhaled breath. From these observations, a multicompartment model, which approximately describes the kinetics of benzene elimination and accumulation has been designed. On the basis of this model, benzene concentrations in breath, corresponding to exposure levels of benzene, have been estimated. Thus, at a daily exposure to 10 ppm the exhaled benzene concentration in the morning after a day of exposure will not exceed 0.1 ppm.

Covalent Binding of Metabolically Activated Hydrocarbons to Specific Microsomal Proteins

Many toxic and/or carcinogenic compounds become covalently bound to DNA, RNA, and proteins of target cells (Jollow et al., 1977). These compounds often have to be metabolised to chemically reactive forms before they can bind to macromolecules (Miller and Miller, 1972; Gillette et al., 1974). Usually this metabolic activation is catalysed by the mixed-function oxidase system located principally in the endoplasmic reticulum.