Bioactivation of hexachlorobutadiene by glutathione conjugation

Glutathione (GSH) conjugation reactions in the metabolism of hexachlorobutadiene (HCBD), in rats and mice, initiate a series of metabolic events resulting in the formation of reactive intermediates in the proximal tubular cells of the kidney. The GSH S-conjugate 1-(glutathion-S-yl)-1,2,3,4,4-pentachlorobutadiene (GPCB), which is formed by conjugation of HCBD with GSH in the liver, is not reactive and is eliminated from the liver in the bile or plasma, or both. GPCB may be translocated intact to the kidney and processed there by gamma-glutamyl transpeptidase and dipeptidases to the corresponding cysteine S-conjugate. Alternatively, gamma-glutamyl transpeptidase and dipeptidases present in epithelial cells of the bile duct and small intestine may catalyse the conversion of GPCB to cysteine S-conjugates. The kidney concentrates both GSH and cysteine S-conjugates and processes GSH conjugates to cysteine S-conjugates. A substantial fraction of HCBD cysteine S-conjugate thus concentrated in the kidney is metabolized by renal cysteine conjugate beta-lyase to reactive intermediates. The selective formation of reactive intermediates in the kidney most likely accounts for the organ-specific effects of HCBD. Alternatively, cysteine S-conjugates may be acetylated to yield excretable mercapturic acids.     

Dual role of phospholipid in the reconstitution of cytochrome P-450 LM2-dependent activities

The effect of dilauroylphosphatidylcholine (DLPC) concentration on cytochrome P-450 LM2 (LM2)-dependent reduction and monooxygenase activities was examined as a function of preincubation time. Purified NADPH-cytochrome P-450 reductase (reductase) and LM2 were reconstituted at different DLPC to LM2 ratios by preincubation of the proteins in the presence of DLPC for either 5 min or 2 hr at room temperature. After preincubation was complete, the samples were assayed for either monooxygenase activity or first-electron transfer activity. When preincubated for 5 min, overall monooxygenase activity was dependent on the [DLPC]:[LM2] ratio, beginning at a low level in the absence of phospholipid and increasing to a maximum at a 160:1 ratio. At [DLPC]:[LM2] ratios above 160:1, the rate was decreased to 80% of the maximum rate. When the samples were preincubated for 2 hr, again low monooxygenase activities were obtained in the absence of DLPC, which increased to a maximum at 160:1 [DLPC]:[LM2] ratio. Above this [DLPC]:[LM2]ratio, the rate was decreased to less than 50% of the maximum value. These changes in overall activities appear to be related to changes in the amount of functional reductase-LM2 complex formed. Similar results were found when LM2 reduction was examined. When preincubated for 5 min, LM2 reduction was shown to be diminished as the DLPC to LM2 ratio decreased below 160:1. The DLPC-dependent effect on reduction was primarily characterized by alterations in the fraction of LM2 reduced in the first phase, with the first-phase rate constant and the slow phase parameters being largely unaffected. Below a 16:1 ratio [( DLPC]:[LM2]), no phospholipid stimulation of LM2 reduction was observed. When the [DLPC]:[LM2] ratio was increased above a 160:1 ratio, only a small effect on the kinetic constants was observed, which was characterized by a 20% decrease in the fraction of LM2 reduced in the first phase. LM2 reduction was more sensitive to DLPC concentration after longer preincubations (2 hr), with a 50% decrease in the fraction of reduction in the first phase being observed at [DLPC]:[LM2] ratios above 160:1. The results are consistent with a dual role for phospholipid in the stimulation of LM2-dependent activities. First, DLPC facilitates the association of reductase and LM2 and, second, DLPC provides a matrix for the incorporation of LM2 and reductase. Facilitation of the protein association appears to be a relatively rapid process, occurring after a 5-min preincubation, whereas a 2-hr preincubation altered the protein interactions in a manner consistent with incorporation of the LM2 and reductase into the phospholipid.     

Bio-organic chemistry and cytochrome P-450-dependent catalysis

This review presents current ideas, models and experimental data relating to the precise chemistry that links the transition metal active centre of cytochrome P-450 systems, the unactivated alkane substrate and the triplet atmospheric dioxygen molecule. Aspects considered include the hypervalent transition metal, the reductive activation of the dioxygen molecule by two electrons as an intermediate in the four-equivalent oxidase mechanism, and the details of carbon-hydrogen and carbon-carbon fragmentation. Studies of the microbial camphor 5-exo hydroxylase system are used to exemplify the principles discussed.     

Bio-organic chemistry and cytochrome P-450-dependent catalysis

1. This review presents current ideas, models and experimental data relating to the precise chemistry that links the transition metal active centre of cytochrome P-450 systems, the unactivated alkane substrate and the triplet atmospheric dioxygen molecule.

2. Aspects considered include the hypervalent transition metal, the reductive activation of the dioxygen molecule by two electrons as an intermediate in the four-equivalent oxidase mechanism, and the details of carbon-hydrogen and carbon-carbon fragmentation.

3. Studies of the microbial camphor 5-exo hydroxylase system are used to exemplify the principles discussed.     

Depression of cytochrome P-450-dependent drug biotransformation by adriamycin

The cytochrome P-450-dependent mixed function oxidase system was depressed following the administration of adriamycin. Cytochrome P-450, aminopyrine N-demethylase, and benzo(a)pyrene hydroxylase were significantly decreased in hepatic microsomes prepared from rats treated with a single dose of adriamycin (10 mg/kg subcutaneously) 4 days previously. Total microsomal protein levels and cytochrome b₅ levels remained unchanged. The administration of cysteamine 1 hr prior to adriamycin prevented the loss of the cytochrome P-450 and the decrease in drug biotransformation. The administration of diethylmaleate had no effect on the ability of adriamycin to decrease this enzyme system. The loss of drug biotransformation and the protection offered by cysteamine is correlated with the lipid peroxidation activity in hepatic microsomes. This suggests that the loss of cytochrome P-450 and related drug biotransformation is related to the generation of free radicals and subsequent lipid peroxidation in the liver.     

Increased glutathione in cultured hepatocytes associated with induction of cytochrome P-450. Lack of effect of glutathione depletion on induction of cytochrome P-450 and delta-aminolevulinate synthase

Cellular glutathione concentrations in primary cultures of chick embryo hepatocytes were 15.3 +/- 5.3 nmoles/mg protein (mean +/- S.D.) and remained stable for up to 3 days in culture. The presence of insulin was not essential for the maintenance of glutathione concentrations. Induction of cytochrome P-450 by phenobarbital-like inducers (2-propyl-2-isopropylacetamide, 2-allyl-2-isopropylacetamide, and 2,4,5,2',4',5'-hexabromobiphenyl) was accompanied by 2- to 3-fold increases in glutathione concentrations and by increased glucuronidation of phenol red. The 3-methylcholanthrene-like inducers of cytochrome P-450 (beta-naphthoflavone and 3,4,3',4'-tetrachlorobiphenyl) did not have these effects. Glutathione was rapidly depleted to 15-30% of control levels in hepatocytes treated with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthase. No toxicity was observed with glutathione depletion. Glutathione depletion did not affect the ability of 2-propyl-2-isopropylacetamide to induce cytochrome P-450, glucuronidation of phenol red, or delta-aminolevulinate synthase.     

Genetic variation in cytochrome P-450-dependent demethylation in Drosophila melanogaster

The genetic variation in the basal capacity to N-demethylate aminopyrine, d-benzphetamine and ethylmorphine was studied in microsomes from adult Drosophila of 9 different strains. Ethylmorphine and d-benzphetamine N-demethylase activity varied about fourfold between the strains, with the highest capacity for both reactions in the Aflatoxin B1-sensitive Florida 9 and the lowest in the insecticide-resistant Hikone R. The two activities were closely correlated with each other but not with aminopyrine demethylation or any previously studied cytochrome P-450-dependent reaction, indicating a common determination by a separate cytochrome P-450 form(s). Aminopyrine N-demethylase activity was more than fourfold higher in the DDT-resistant Oregon R than in Berlin K. A genetic analysis of aminopyrine N-demethylation revealed that the high activity in the Oregon R(R) strain was inherited as an apparently semidominant second chromosome trait. The similar mode of inheritance as well as the close correlation between aminopyrine demethylase and the previously analysed biphenyl 4-hydroxylase activity suggests that these activities are under the same genetic control.     

Liver microsomal cytochrome P-450-dependent O-dealkylation reaction in various animals

Liver microsomal O-dealkylation activity was determined using O-methyl, O-ethyl and O-propyl derivatives of p-nitrophenol, 7-hydroxycoumarin (umbelliferon) and 7-hydroxyphenoxazone (resorufin) as substrates. Microsomal O-dealkylation activities of p-nitrophenol and 7-hydroxycoumarin O-alkyl derivatives were of similar levels, but the activities of 7-hydroxyphenoxazone O-alkyl derivatives were very low compared with those of other substrates. Pretreatment of rats with beta-naphthoflavone resulted in the preferential increase of O-deethylation and O-depropylation activities regardless of the ring structure of the substrates, and the ratio of O-deethylation and O-depropylation activities to that of O-demethylation increased markedly. On the other hand, the O-dealkylase activity of all substrates increased generally upon pretreatment of the rats with phenobarbital, but the ratio of O-deethylase or O-depropylase activity to that of O-demethylase in the pretreated rats was not very different from that of the untreated animals. Hexobarbital inhibited competitively the O-dealkylation activity in control and phenobarbital-pretreated rat microsomes. On the other hand, the O-dealkylase activity in microsomes obtained from beta-naphthoflavone-pretreated rats was inhibited remarkably by alpha-naphthoflavone, but not in microsomes prepared from untreated and phenobarbital-pretreated rats. Based on these results, this report discusses the relationship between the alteration of O-dealkylation activity and the composition change of cytochrome P-450 in microsomal membrane. Species differences in the substrate specificity of the O-dealkylation reaction and in the responsiveness of the animals to typical inducers were also observed using liver microsomes obtained from several animals under various conditions.     

Circadian changes of cytochrome P-450-dependent monooxygenase system in the rat liver.

Circadian changes in cytochrome P-450 and cytochrome b5 content and activity of NADPH-cytochrome P-450 and NADH-cytochrome b5 reductases have been studied in rat liver microsomes in season autumn. The obtained results indicate, that cytochrome P-450 in 6-month-old animals shows 12 h rhythm, but in older ones 24 h rhythm. NADPH-cytochrome P-450 reductase activity shows 24 h rhythm in oldest animals only. Cytochrome b5 and its reductase has 24 h rhythm in all examined groups of rats.     

Cytochrome P-450-dependent mixed-function oxidase and glutathione S-transferase activities in spontaneous obesity-diabetes.

The effect of non-insulin-dependent diabetes on the hepatic microsomal cytochrome P450-dependent mixed-function oxidase system and on cytosolic glutathione S-transferase activity was determined using the spontaneously obese-diabetic (ob/ob) mouse model. The activities of the xenobiotic-metabolizing cytochrome P450 proteins were monitored by the use of chemical probes. Non-insulin-dependent diabetes did not influence the hepatic metabolism of substrates associated with the P450 I, IIB, IIE, III and IV families of cytochromes. In contrast, cytosolic glutathione S-transferase activity was markedly reduced and glutathione levels were significantly lowered. These findings raise the possibility that patients suffering from this disease may be more susceptible to chemicals that rely on glutathione conjugation for their deactivation.     

Bioactivation and detoxication of the pyrrolizidine alkaloid senecionine by cytochrome P-450 enzymes in rat liver

Rats display a marked sex difference in the oxidation of the pyrrolizidine alkaloid senecionine, especially with respect to N-oxidation. This sex difference was largely eliminated following treatment with dexamethasone. These observations suggested the potential involvement of the male-specific cytochrome P-450 UT-A and the P-450 PCN-E in the metabolism of this pyrrolizidine alkaloid. Reconstituted rat P-450 UT-A exhibited a high rate of N-oxidation (15 nmol min-1 nmol P-450-1) which is almost 3-fold higher than the turnover number observed with male rat liver microsomes. In contrast, rat P-450 UT-A displayed a much lower activity toward necine pyrrole [+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, DHP) formation (1.0 nmol min-1 nmol P-450-1). The N-oxygenation and pyrrole formation activities displayed by rat cytochromes P-450 PB-B and P-450 BNF-B toward senecionine were low, with rates less than 1 nmol min-1 nmol P-450-1. Rabbit antibody to rat P-450 UT-A inhibited the senecionine-N-oxidation activity of untreated male rat liver microsomes by 60%, with lesser inhibition of DHP production. Rabbit antibody to human P-450NF (the human homologue to rat P-450 PCN-E) was a potent inhibitor of DHP production by untreated male rat liver microsomes. With microsomes from dexamethasone-pretreated rats, anti-P-450NF inhibited DHP and N-oxide production in parallel. We conclude that the large sex difference in senecionine N-oxidation probably is the result of the specificity of P-450 isozymes UT-A and PCN-E.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome P-450-dependent nicotine oxidation by liver microsomes of guinea pigs. Immunochemical evidence with antibody against phenobarbital-inducible cytochrome P-450

When guinea pigs were treated with phenobarbital (PB), the specific activity of liver microsomal nicotine oxidase increased by 42%. PB-inducible cytochrome P-450 (PB-P-450) was purified to homogeneity from liver microsomes of PB-treated guinea pigs. Purified PB-P-450 catalyzed nicotine oxidation when reconstituted with NADPH-P-450 reductase and phospholipid system. Antibody prepared against the purified PB-P-450 formed single precipitation lines with both purified PB-P-450 and microsomal components in livers of PB-treated guinea pigs, and both precipitation lines fused. The antibody against PB-P-450 strongly inhibited nicotine oxidation in the reconstituted system. The antibody also inhibited liver microsomal nicotine oxidase activities in PB-treated and untreated guinea pigs by about 30% and less than 5% respectively. About 45% of total P-450 in liver microsomes of PB-treated guinea pigs was precipitated by the antibody. These results show that PB-P-450 participates in liver microsomal nicotine oxidation in PB-treated guinea pigs but not in untreated control animals.     

Role of cytochrome P-450 in the bioactivation of nicotine

Nicotine (100μM) was incubated with microsomes (1mg/ml) prepared from New Zealand White rabbits. On the basis of microsomal weight, the rate of nicotine oxidation was slightly slower in lung compared to liver. However, when the rates of nicotine oxidation were calculated on the basis of cytochrome P-450 concentration, the specific activity of the metabolic oxidation catalyzed by lung was approximately 4 times greater than liver (6.4vs 1.65 nmoles nicotine oxidized. nmole cytochrome P-450^?1 min^?1). These studies employed several methods of altering activities of specific isozymes present in pulmonary microsomes, including the use of the isozyme 2 and 6 specific inhibitor α-methylbenzyl ABT, metabolite inhibitors, norbenzphetamine and N-hydroxyamphetamine, TCDD induction and Arochlor 1260 pretreatment. These results support the conclusion that nicotine metabolism by rabbit lung microsomes is mediated primarily by cytochrome P-450 isozyme 2.     

Monoclonal antibodies inhibitory to rat hepatic cytochromes P-450: P-450 form specificities and use as probes for cytochrome P-450-dependent steroid hydroxylations

Cytochrome P-450 (P-450) form specificities were established for a total of nine monoclonal antibodies (MAbs) raised to four distinct rat hepatic P-450 enzymes (P-450s 2c, PB-2a, PB-4, and BNF-B), using a combination of enzyme-linked immunosorbent analysis, dot immunoblotting, Western blotting, Ouchterlony immunodiffusion, and immunoinhibition analyses. Four of the MAbs were fully (greater than or equal to 85%) inhibitory toward the corresponding immunoreactive P-450s when assayed in purified, reconstituted enzyme systems, while two of the MAbs were partially inhibitory, with a maximum of 50 or 80% inhibition achieved in the presence of saturating MAb. Inhibitory MAbs reactive with P-450s 2c, 3, and PB-4, respectively, were used to demonstrate that the formation of multiple hydroxytestosterone metabolites by each of the respective purified P-450 enzymes is reflective of their inherent catalytic specificities and not due to the presence of immunochemical distinguishable P-450 enzyme contaminants. P-450 form-specific contributions to rat hepatic microsomal steroid hormone hydroxylase activities were then assessed using the inhibitory MAbs as probes. MAb-reactive P-450 2c was shown to be the major (greater than or equal to 85%) catalyst of microsomal testosterone and androstenedione 16 alpha-hydroxylation in both untreated and beta-naphthoflavone-induced rats. However, this P-450 form catalyzed only approximately 30% of hepatic microsomal steroid 16 alpha-hydroxylase activity in phenobarbital-induced adult males, and less than or equal to 10% of steroid 16 alpha-hydroxylase activity in (phenobarbital-induced immature males or adult females, where the balance of 16 alpha-hydroxylase activity is catalyzed by MAb-reactive P-450 PB-4. Although MAb-reactive P-450 PB-4 catalyzed the majority (greater than or equal to 90%) of microsomal androstenedione 16 beta-hydroxylation in phenobarbital-induced rats, this P-450 enzyme did not contribute to the low level 16 beta-hydroxylase activity of uninduced liver samples. Finally, MAb-reactive P-450 3 catalyzed at least 85% of microsomal androstenedione 7 alpha-hydroxylation, independent of the age, sex, or induction status of the animals used as source of liver microsomes. These findings demonstrate the usefulness of MAbs as probes for the contributions of individual P-450 enzymes to the metabolism of steroid hormones susceptible to hydroxylation at multiple sites.     

On the significance of the cytochrome P-450-dependent hydroxyl radical-mediated oxygenation mechanism

1. Reconstituted membrane vesicles containing purified preparations of cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase effectively destroyed 2-deoxy-D-ribose in an NADPH-dependent process.

2. The destruction was mediated by hydroxyl radicals formed in an iron-catalysed Haber-Weiss reaction between superoxide anions and hydrogen peroxide liberated from the haemoprotein.

3. Administration of ethanol or benzene to rabbits, compounds known to be oxygenated by the hydroxyl radical-dependent mechanism, resulted in induction of a species of cytochrome P-450 effective in the radical-dependent metabolism of both chemicals.

4. Benzene treatment of rabbits also resulted in an enhanced hydroxyl radicaldependent metabolism of ethanol and benzene in liver microsomes.

5. It is suggested that, for certain substrates, hydroxyl radical-mediated cytochrome P-450-dependent oxygenation reactions are of importance for the microsomal metabolism of these compounds.

6. It is speculated that radical-producing species of cytochrome P-450 may contribute to hydroxyl radical-mediated cell damage.     

Quinolone inhibition of cytochrome P-450-dependent caffeine metabolism in human liver microsomes

Inhibitory effects of the quinolone antibiotics ofloxacin, lomefloxacin, pipemidic acid, ciprofloxacin, and enoxacin on caffeine metabolism were examined in vitro with human liver microsomes of four donors. All drugs competitively inhibited the activity of 3-demethylation, the major pathway of caffeine metabolism. Enoxacin, ciprofloxacin, and pipemidic acid were strong inhibitors exhibiting Ki values between 0.1 and 0.2 mM. Lomefloxacin and ofloxacin had moderate effects with Ki values of 1.2 and 3.6 mM, respectively. The rate of caffeine 7-demethylation (which amounted to about 25% of that for 3-demethylation) was only slightly affected by the quinolones. Minor, but inconsistent, effects were found on 8-oxidation to 1,3,7-trimethyluric acid. The results indicate that the reduction of caffeine clearance by concomitant quinolone application observed in vivo is primarily due to a competitive interaction of the inhibiting quinolones with the cytochrome P-450 isoenzyme(s) mediating caffeine demethylation.