Purified Rat Liver NADPH-cytochrome Research Articles

Mechanism of bioactivation and covalent binding of 2,4,6-trinitrotoluene

Studies were undertaken to investigate the mechanism of bioactivation and covalent binding of TNT. Incubation of [ 14C]TNT with rat liver microsomes in the presence of an NADPH generating system resulted in metabolism and covalent binding to microsomal proteins. Timedependence studies showed that TNT was rapidly reduced to yield 4-hydroxylamino-2,6-dinitrotoluene (4HA), 4-amino-2,6-dinitrotoluene (4A) and 2-amino-4,6-dinitrotoluene (2A) as intermediates which were further metabolized to form 2,4-diamino-6-nitrotoluene (2,4DA) and 2,6-diamino-4-nitrotoluene (2,6DA). In contrast to the rapid disappearance of TNT, formation of covalent protein adducts increased with time, suggesting that the reactive intermediate was likely to be formed not directly from TNT but from proximal intermediates such as 4HA. The hypothesis that 4HA was more readily converted to the reactive intermediate than TNT was further supported by the increased levels of covalent adduct formation when [ 14C]4HA was incubated directly with liver microsomes. Covalent binding of TNT and 4HA was dependent on oxygen concentration. Higher levels of covalent adducts were formed when TNT was incubated aerobically (up to 50% oxygen concentration) than under anaerobic conditions. Covalent binding of [ 14C]4HA also increased with increasing oxygen concentrations. These results suggest that the reactive intermediate is likely to be an oxidized metabolite of 4HA, e.g. 4-nitroso-2,6-dinitrotoluene. Compounds containing a free sulfhydryl group (cysteine, N-acetylcysteine, GSH or 3,4-dichlorobenzenethiol) decreased the amount of covalent binding to various degrees, suggesting the involvement of the sulfhydryl group in adduct formation with TNT following bioactivation. Metabolic activation of TNT by liver microsomes required NADPH but not NADH as the cofactor. Incubation of [ 14C]TNT with purified rat liver NADPH cytochrome P450 reductase under either aerobic or anaerobic conditions yielded exclusively 4HA. In contrast, 2A and 4A were formed following incubation of TNT with the reconstituted system containing cytochrome P450, NADPH cytochrome P450 reductase and dilauroyl phosphatidylcholine. These observations suggest that the initial reduction of the nitro group can be catalyzed by NADPH cytochrome P450 reductase alone but cytochrome P450 is needed in the reduction of the hydroxylamine to the amine.

Expression and purification of functional human 17 alpha-hydroxylase/17,20-lyase (P450c17) in Escherichia coli. Use of this system for study of a novel form of combined 17 alpha-hydroxylase/17,20-lyase deficiency.

Enzymatically active human 17 alpha-hydroxylase cytochrome P450 (P450c17) has been expressed in and purified from Escherichia coli. The cDNA containing modifications within the amino-terminal eight codons which are favorable for expression in E. coli, as well as codons for 4 histidine residues at the carboxyl terminus, was placed in the pCWori+ expression vector. The modified human P450c17 was detected spectrophotometrically (400 nmol of P450c17/liter culture) and was found to be integrated into E. coli membranes. This previously inaccessible human P450 was purified to electrophoretic homogeneity (10.7 nmol of P450/mg) from solubilized bacterial membranes using two sequential chromatographic steps, nickel nitrilotriacetate followed by hydroxylapatite. The expected enzymatic activities of human P450c17 were reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase, giving turnover numbers of 8.0 nmol/min/nmol P450 for pregnenolone, 6.5 nmol/min/nmol P450 for progesterone, 0.06 nmol/min/nmol P450 for 17 alpha-hydroxypregnenolone, and no detectable activity for 17 alpha-hydroxyprogesterone. This system was utilized to study the molecular basis of a novel form of combined 17 alpha-hydroxylase, 17,20-lyase deficiency resulting from compound heterozygous mutations, a missense point mutation Tyr64(TAT)--> Ser (TCT), and an Ile112 duplication (ATCATC). Upon expression of these mutant proteins in E. coli, the Tyr64 mutant has 15% of the wild type 17 alpha-hydroxylase activity, whereas the Ile112 duplication shows no activity, results consistent with the observed clinical phenotype.

Expression of a catalytically active human cytochrome P-4502E1 in Escherichia coli

The cDNA coding for human cytochrome P-4502E1 has been expressed in Escherichia coli by placing it under the control of the isopropylthiogalactoside inducible trc promoter. Production of P4502E1 was demonstrated by immunoblots and by catalytic activity with dimethylnitrosamine as substrate. Modifications which favor expression in E. coli were made within the first seven codons. This resulted in approx. a 2- to 2.5-fold increase in P4502E1 isolated from the bacterial membranes as detected by immunoblots and catalytic activity. CO-reduced difference spectra of the modified P4502E1 revealed a peak at 452 nm, which is characteristic of hepatic P4502E1, and a molecular weight of 54 kDa. A partially purified preparation of recombinant P450 protein was active with dimethylnitrosamine, a substrate specific for this isozyme, when reconstituted with purified rat liver NADPH-cytochrome P-450 oxidoreductase. This activity was enhanced in the presence of cytochrome b 5 and inhibited by the addition of antibody to the P4502E1 purified from pyrazole-treated rats. E. coli were capable of oxidizing p-nitrophenol when transformed with vector containing the human P4502E1 cDNA but not with vector alone. This in vivo metabolism of p-nitrophenol was increased 2-fold when the modified P4502E1 cDNA was used, which corresponds to the increase in P4502E1 content. Expression of human P4502E1 in E. coli appears to be an attractive system for producing large amounts of this isozyme, and for studies on the toxicological properties and structure-function relationship of the human P4502E1.

Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system.

Free radical formation and DNA strand breakage during metabolism of diaziquone by NAD(P)H quinone-acceptor oxidoreductase (DT-diaphorase) and NADPH cytochrome c reductase

One-electron reduction of diaziquone (AZQ) by purified rat liver NADPH cytochrome c reductase was associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as indicated by ESR spin-trapping studies. Reactive oxygen formation correlated with AZQ-dependent production of single and double PM2 plasmid DNA strand breaks mediated by this system as detected by gel electrophoresis. Direct two-electron reduction of AZQ by purified rat liver NAD(P)H (quinone acceptor) oxidoreductase (QAO) was also associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as detected by ESR spin trapping. Furthermore, PM2 plasmid DNA strand breaks were detected in the presence of this system. Plasmid DNA strand breakage was inhibited by dicumarol (49 ± 5%), catalase (57 ± 2.3%), SOD (42.2 ± 3.6%) and ethanol (41.1 ± 3.9%) showing QAO and reactive oxygen formation was involved in the PM2 plasmid DNA strand breaks observed. These results show that both one- and two-electron enzymatic reduction of AZQ give rise to formation of reactive oxygen species and DNA strand breaks. Autoxidation of the AZQ semiquinone and hydroquinone in the presence of molecular oxygen appears to be responsible for these processes. QAO appears to be involved in the metabolic activation of AZQ to free radical species. The cellular levels and distribution of this enzyme may play an important role in the response of tumor and normal cells to this antitumor agent.

Cis-trans isomerization of a nitrofuran AF-2 by rat liver microsomal preparations.

The enzymatic cis-trans isomerization of nitrofurans such as 3-(5-nitro-2-furyl)-2-(2-furyl) acrylamide (AF-2) has been proved to occur via the formation of the corresponding nitro anion radicals. Rat liver microsomes supplemented with NADPH exhibited the cis-trans isomerase activity toward AF-2, which is markedly inhibited by p-chloromercuribenzoic acid, but not by carbon monoxide. Purified rat liver NADPH-cytochrome c reductase supplemented with NADPH also exhibited a significant isomerase activity toward the nitrofuran, whereas purified rat liver NADH-cytochrome b5 reductase exhibited only a little activity in the presence of NADPH or NADH. These results indicate that mainly NADPH-cytochrome c reductase is involved in the cis-trans isomerization of nitrofurans, i.e. the formation of nitro anion radicals catalyzed by rat liver microsomes.

Oxidation-reduction properties of rat liver cytochromes P-450 and NADPH-cytochrome p-450 reductase related to catalysis in reconstituted systems.

A series of equilibrium and kinetic measurements involving the oxidation-reduction properties of purified rat liver NADPH-cytochrome P-450 reductase and eight different purified rat liver cytochromes P-450 (P-450s) were carried out. Apparent spin states of P-450 iron were determined in the absence and presence of a number of known substrates by using second-derivative and conventional near-UV absorbance spectroscopy. Many of the substrates examined did not produce significant changes in the apparent iron spin state, even when binding could be demonstrated with equilibrium dialysis. Further, the spin state was not correlated to catalytic activity of the P-450s in reconstituted enzyme systems. The oxidation-reduction potentials were determined for the ferric/ferrous couples of each of the eight P-450s in the presence and absence of known substrates, as well as other proteins suspected of altering the potentials. The midpoint potential (Em,7) ranged from -350 to -289 mV for the P-450s under these conditions. In some cases Em,7 was raised with the addition of substrates, but the extent of the increase was no greater than +33 mV. The Em,7 of one P-450 (P-450 beta NF/ISF-G) was not changed significantly when the fraction of high-spin iron varied between 11 and 67%. Steady-state spectral studies provided evidence for the accumulation of an oxygenated ferrous intermediate (or a derived product) of one P-450 (P-450PB-B) in the presence of a substrate, cyclohexane. Studies on the donation of electrons from cytochrome b5 and a series of dyes to this complex suggest that it has an effective Em,7 (for reduction) of approximately +50 mV. In studies with one of the P-450s, steady-state spectral studies indicated that the three-electron-reduced form of NADPH-P-450 reductase accumulates, consistent with the view that this form of the reductase is involved in the reduction of P-450 from the ferric to the ferrous state.

Immunological comparison of rat, rabbit, and human liver NADPH-cytochrome P-450 reductases.

NADPH-cytochrome P-450 reductase (EC 1.6.2.4) preparations were purified to electrophoretic homogeneity from rat, rabbit, and human liver microsomes. These preparations had apparent monomer molecular weights (Mr's) of 72 000-74 000 and were catalytically active in reducing rat and rabbit liver cytochromes P-450 as well as cytochrome c. A form of the human liver reductase devoid of a peptide of about Mr 6000 was isolated in the absence of protease inhibitors; this enzyme catalyzed the reduction of cytochrome c but not cytochromes P-450. Rabbits were immunized with purified rat liver NADPH-cytochrome P-450 reductase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Immunological differences among the three species were detected by using double-diffusion analysis, quantitative microcomplement fixation, and inhibition of enzyme activity. Microcomplement fixation techniques indicated immunological differences in both rat and human reductase preparations due to removal of a peptide of Mr 6000-8000; these differences were not detected by using double-diffusion analysis. The antibody inhibited rat liver microsomal d-benzphetamine N-demethylase activity to the same extent as NADPh-cytochrome c reductase activity, suggesting that the level of reductase controls the rate of this cytochrome P-450-mediated activity. On the other hand, the antibody was much less effective in inhibiting rat liver benzo[a]pyrene hydroxylase activity. The antibody exerted different effects in inhibiting d-benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities as compared to NADPH-cytochrome c reductase activity in human liver microsomes.

Effects of anti-NADPH-cytochrome [formula omitted] reductase and anti-cytochrome b 5 antibodies on the hepatic and pulmonary microsomal metabolism and covalent binding of the pulmonary toxin 4-ipomeanol

An antibody prepared against purified rat liver NADPH-cytochrome c reductase inhibited both the pulmonary and hepatic microsomal covalent binding of 4-ipomeanol as well as the respective NADPH-cytochrome c reductase activities, findings which are consistent with previous studies which indicated the participation of cytochrome P450 in the metabolic activation of the toxin. An antibody prepared against purified rat liver cytochrome b 5, which strongly inhibited both the rat hepatic and pulmonary NADH-dependent cytochrome c reductases, and was inactive against the respective NADPH-dependent cytochrome c reductases, had little effect on metabolic activation of 4-ipomeanol by hepatic microsomes, but strongly inhibited both the NADH-supported and the NADPH-supported pulmonary microsomal metabolism and covalent binding of the compound. These results suggest that metabolic activation of 4-ipomeanol involves a two-electron transfer in which transfer of the second electron via cytochrome b 5 is rate-limiting in lung microsomes.

Participation of NADPH-Cytochrome C Reductase in Thyroid Hormone Biosynthesis

Purified rat liver NADPH-cytochrome c reductase supports iodination of tyrosine in a system including NADPH, cytochrome c and thyroid perioxidase. Catalase inhibits the iodination of tyrosine, while superoxide dismutase has no effect. Antibody developed in the rabbit against purified rat liver NADPH-cytochrome c reductase inhibits both reduction of cytochrome c and tyrosine iodination supported by the enzyme. The antibody forms a single precipitation line with thyroid extract, and inhibits NADPH cytochrome c reductase activity of the thyroid. The antibody partially inhibits iodination in a thyroid mitochondrial-microsomal fraction, but does not inhibit NADH-dependent iodination. The immunochemical studies indicate the participation of NADPH-cytochrome c reductase in thyroidal H2O generation, and the independent existence of NADPH-dependent and NADH-dependent H2O2 generation mechanisms in the thyroid.