Biochemical characterization of glutathione-deficient mutants of Escherichia coli K12 and Salmonella strains TA1535 and TA100.

Glutathione-deficient mutants of Escherichia coli K12/343/408 and Salmonella typhimurium TA1535 and TA100 were characterized biochemically by measuring the rate of formation of (14C)gamma-glutamylcysteine and (14C)glutathione in cell-free extracts of the strains. gamma-Glutamylcysteine synthetase activity was found to be absent in the NGR-2 mutant of E. coli and in the Salmonella mutants TA1535/NG-19, TA100/NG-57 and TA100/NG-11, while only low activities were found in the NGR-9 and NG-54 mutant of E. coli and Salmonella respectively. These results correspond with the decreased levels of glutathione found in these strains. Extracts of the parent strains have normal glutathione levels and show high gamma-glutamylcysteine synthetase activities. It is concluded that the present GSH-deficient strains of E. coli and Salmonella are gshA mutants, analogous to those previously described in E. coli. In addition, the present results show that the fluorometric method used for the determination of glutathione, employing o-phthalaldehyde as a reagent, is not specific for glutathione (at pH 8.0), but also sensitively reacts with gamma-glutamylcysteine.

Mutagenic activity of various chemicals in Salmonella strain TA100 and glutathione-deficient derivatives. On the role of glutathione in the detoxification or activation of mutagens inside bacterial cells.

Several mutants with decreased levels of reduced glutathione (GSH) were isolated from the sensitive mutagen tester strain Salmonella typhimurium TA100 after treatment with u.v. and selection for resistance to N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and its methyl analogue MNNG. Estimation of the GSH concentration and GSH S-transferase activity in extracts of these strains and of TA100 indicates that the GSH- derivatives contain 10-30% of the GSH level found in TA100, and that they exhibit normal GSH S-transferase activity. The mutagenic activities of 7 chemicals, namely, MNNG, ENNG, 1,2-dibromoethane (DBE), 1-chloro-2,4-dinitrobenzene (CDNB), styrene-7,8-oxide (STOX), N-ethyl-N-nitrosourea (ENU) and methyl methane sulphonate (MMS) were compared in TA100 and in one representative GSH- strain, denominated NG-57. MNNG, ENNG, DBE and CDNB are potent to extremely potent mutagens in TA100, but induce very low levels of His+ mutants in NG-57. Pretreatment of NG-57 with 1 mM GSH (partially) restores the mutant yields to the levels usually found in TA100. The mutagenic activities of STOX, ENU and MMS are similar in both strains. These results support some previous findings, namely that ENNG, MNNG and DBE, but not ENU are activated to mutagens inside the test bacteria, and also suggest that CDNB is activated by bacterial GSH. The latter finding is in contrast with the current view that CDNB is detoxified by GSH, as is also presently evidenced by a strong reduction of the compound's mutagenicity in the presence of extracts of rat liver, which contains GSH and GSH S-transferase activity. The results with STOX indicate that GSH plays in bacteria a much less important role in the detoxification of xenobiotics than in mammalian tissue, presumably due to a much lower GSH S-transferase activity in the first organism.

1,2-Dibromo compounds. Their mutagenicity in Salmonella strains differing in glutathione content and their alkylating potential.

The mutagenic activities of several structurally related dibromo compounds were compared in Salmonella strains sensitive to base substitution mutagenesis (TA1535 and/or TA100) and in the glutathione (GSH)-deficient derivative TA100/NG-57, using a preincubation procedure. The compounds tested were 1,2-dibromoethane (DBE), 1,2-dibromopropane (DBP), 1,2-dibromo-1-phenylethane (DBPE) and model compounds for the half-mustards resulting from their conjugation with GSH, i.e. the N-acetyl-S-2-bromoalkyl-L-cysteine methyl esters SBE, SBP, and SBPE, respectively. The alkylating potential of all compounds was assayed with the 4-(p-nitrobenzyl)pyridine (NBP) alkylation test. Five of the compounds showed a good correlation between relative mutagenic activity in TA100 and electrophilic reactivity in the NBP-test, the order of decreasing potency being SBE greater than SBP greater than DBPE greater than DBP. SBPE displayed the highest reactivity in the NBP-test, but was devoid of mutagenic activity. The mutagenic activity of DBE was substantially decreased in the GSH-deficient strain TA100/NG-57 and could be restored by pretreating the cells with GSH. None of the other chemicals showed different mutagenic activities in TA100 and TA100/NG-57. From the results it can be concluded that 2-bromothioethers possess higher alkylating activities than the 1,2-dibromo compounds. Methyl substitution has a deactivating effect on the mutagenic activity. The results with the phenyl-substituted analogue, DBPE, show that a higher alkylating activity does not always lead to a higher mutagenic activity.

One-electron reductive bioactivation of 2,3,5,6-tetramethylbenzoquinone by cytochrome P450.

Bioreductive activation of quinones in mammalian liver has generally been attributed to NADPH-cytochrome P450 reductase. However, in view of the 20-30-fold molar excess of cytochrome P450 over NADPH-cytochrome P450 reductase on the endoplasmic reticulum of the rat liver cell and the capability of cytochrome P450 to bind and reduce xenobiotics, it was considered of interest to investigate the possible role of cytochrome P450 in the bioreduction of quinones. In the present study, 2,3,5,6-tetramethyl-1,4-benzoquinone (TMQ) was chosen as a model quinone. First, TMQ was found to bind at the metabolic active site of phenobarbital (PB)-inducible cytochrome P450s of rat liver microsomes, indicating that TMQ is a potential substrate for cytochrome P450-mediated biotransformation. Second, with electron spin resonance, one-electron reduction of TMQ to a semiquinone free radical (TMSQ) was found to occur in these microsomal fractions. SK&F 525-A, a well-known inhibitor of cytochrome P450, strongly inhibited TMSQ formation in these subcellular fractions without affecting NADPH-cytochrome P450 reductase activity. One-electron reductive bioactivation of TMQ was further investigated with purified NADPH-cytochrome P450 reductase alone and in reconstituted systems of purified cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase. As measured by ESR, purified cytochrome P450-IIB1 in the presence of NADPH-cytochrome P450 reductase was able to reduce TMQ to TMSQ at a much greater rate than in the presence of NADPH-cytochrome P450 reductase alone. Reduction of TMQ was also investigated by measuring the initial rate of NADPH oxidation by TMQ under anaerobic conditions. Inhibitors of cytochrome P450, namely SK&F 525-A and antibodies against PB-inducible cytochrome P450s, caused a substantial decrease in reductive metabolism in PB-treated microsomes. These antibodies were also effective in the inhibition of TMQ-induced NADPH oxidation in a complete reconstituted system of equimolar concentrations of cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase, indicating that the reaction was specific for cytochrome P450-IIB1. Finally, initial rates of NADPH oxidation were determined in reconstituted systems containing varying amounts of NADPH-cytochrome P450 reductase and cytochrome P450-IIB1 to determine the contribution of either enzyme in the reduction of TMQ. As expected, NADPH-cytochrome P450 reductase was able to reduce TMQ to a small extent. However, reconstitution in the presence of increasing amounts of cytochrome P450-IIB1 (relative to NADPH-cytochrome P450 reductase) resulted in increasing rates of TMQ-induced NADPH oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)

Messenger ribonucleoprotein complexes in human KB cells infected with adenovirus type 5 contain tightly bound viral-coded '100K' proteins.

Late after infection of KB cells with adenovirus 5 an extra protein becomes associated with messenger ribonucleoprotein particles present in the polysomes. This protein has a molecular weight of 100000 and is identical to the virus coded '100K' protein found previously. The extra protein is firmly attached to the messenger ribonucleoprotein complexes. Its binding resists exposure to high salt concentrations as used in puromycin/high-salt dissociation and equilibrium centrifugation in Cs2SO4 gradients. In this respect it resembles the binding of two other proteins of Mr 74000 and 48000 which are commonly found in messenger ribonucleoprotein particles of various eukaryotic cells. The identity between the messenger ribonucleoprotein protein of Mr 100000 and the "100K' protein present in the soluble part of the cytoplasm was established by sodium dodecylsulphate/polyacrylamide gel electrophoresis, isoelectric focusing and peptide mapping after limited proteolysis with Staphylococcus aureus protease.

Biotransformation of 1,2-dibromopropane in rats into four mercapturic acid derivatives.

1,2-Dibromopropane was administered orally in doses of 50-350 mg/kg to male Wistar rats. Four mercapturic acids were identified in urine by GC/MS, viz. N-acetyl-S-(2-oxopropyl)-L-cysteine (I), N-acetyl-S-(2-hydroxypropyl)-L-cysteine (II), N-acetyl-S-(1-carboxyethyl)-L-cysteine (III), and N-acetyl-S-(2-bromo-2-propenyl)-L-cysteine (IV). Mercapturic acid IV was a minor metabolite which could only be measured at doses of 200 mg/kg or higher. In 24 hr, urinary excretion of mercapturic acids amounted to about 36% of the dose (11% I, 21% II, 4% III, 0.2% IV). No dose dependency was found up to the highest dose. A unified scheme is proposed for the metabolism of 1,2-dibromopropane in the rat, which accounts for the identified mercapturic acids. The role of direct glutathione conjugation in the route leading to the major metabolite II, presumably involving thiiranium ion formation, is discussed. This route probably is biologically not very important because of the absence of detectable activity of 1,2-dibromopropane toward glutathione S-transferases in vitro, the very low mutagenicity of 1,2-dibromopropane, and the high mutagenic activity of N-acetyl-S-(2-bromopropyl)-L-cysteine methyl ester which was studied as a model compound for direct conjugation.

Glutathione conjugation of 1,2-dibromo-1-phenylethane in rats in vivo.

The metabolism of 1,2-dibromo-1-phenylethane (DBPE) was studied in rats. Administration of DBPE orally, in doses of 0.25-1.25 mmol/kg (66-330 mg/kg), to male Wistar rats resulted in the excretion of a single mercapturic acid in urine. The methyl esters of three potential mercapturic acid metabolites were synthesized: N-acetyl-S-(2-oxo-2-phenylethyl)-L-cysteine methyl ester (O),N-acetyl-S-(2-hydroxy-1-phenylethyl)-L-cysteine methyl ester (I), and N-acetyl-S-(2-hydroxy-2-phenylethyl)-L-cysteine methyl ester (II). GC/MS analysis showed that the methyl ester of the excreted mercapturic acid was identical with II. Quantitative measurement of II in urine by GLC showed that, after 24 hr, excretion of the mercapturic acid was almost complete and amounted to 41% of the administered dose. At doses higher than 1.00 mmol/kg, the excretion no longer increased. Inhibition of the oxidative pathways by ip injection of 1-phenylimidazole resulted in an excretion decrease of about 40%. (Pre)treatment with diethyl maleate lowered the excretion of mercapturic acid by 30-60%. Glutathione conjugates synthesized from DBPE and styrene oxide were separated by HPLC. Both compounds can produce the same two pairs of diastereomers, viz. (R)- and (S)-(2-hydroxy-1-phenyl-ethyl)glutathione ((R)-1 and (S)-1), and (R)- and (S)-(2-hydroxy-2-phenylethyl)glutathione ((R)-2 and (S)-2). These could be separated in the order (R)-2, (R)-1, (S)-1, and (S)-2 within 20 min. This method was also applied to examine glutathione conjugates excreted in bile after DBPE administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione S-transferase activity in human fetal and adult tissues.

Quantitative estimations of glutathione S-transferase activities with 1-chloro-2,4-dinitrobenzene as the electrophilic second substrate, in 142 postmortem human tissue specimens derived from 34 different organs of one or more of 13 individuals belonging to various age groups, are presented. Collectively the data indicate: (1) all tissues examined have appreciable levels of enzyme activity; (2) liver, kidney, lung, muscle, heart, adrenal glands, pancreas, and stomach of fetal origin possess higher enzyme activities than those of the adults, and (3) there are wide interindividual variations in the tissue enzyme activities.