Induction of cytochrome P-450 isozymes by hexachlorobenzene in rats and aromatic hydrocarbon (Ah)—Responsive mice

Hexachlorobenzene (HCB) differs markedly from other chlorinated benzenes (CBs) as an inducer of cytochrome P-450 (P-450) isozymes as determined by radioimmunoassay and immunoblotting. At > 99% pure, HCB induced both the phenobarbital-inducible forms, cytochromes P-450b + e (70X), and the 3-methylcholanthrene-inducible forms, cytochromes P-450c (58X) and P-450d (8X), in rat liver microsomes. The concentration of P-450d was considerably greater than that of P-450c in HCB-induced rat liver. In contrast to HCB, all lower chlorinated benzenes tested were PB-type inducers. Hexachlorobenzene increased the amounts of translatable messenger RNAs (mRNAs) for P-450b, P-450c, and P-450d in rat liver polysomes, suggesting that it increases the synthesis of these proteins. Evidence that HCB interacted with the putative Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was equivocal. Western blots of liver microsomes from Ahresponsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice demonstrated that HCB produced a large increase in P₃-450 and a very small increase in P₁-450 in the responsive strain. The increase in P₁-450 was not observed after HCB administration to nonresponsive mice, but a small increase in P₃-450 was noted. These findings suggested that HCB may act through the Ah receptor. However, HCB was at best a very weak competitor for specific binding of [³H]-TCDD to the putative receptor in rat or mouse hepatic cytosol in vitro, producing decreases in binding of [³H]-TCDD only at very high concentrations (10^?6 to 10^?5 M).     

Cytochrome P-450 mediated reductive dehalogenation of the perhalogenated aromatic compound hexachlorobenzene

Hexachlorobenzene (HCB) elicits concentration-dependent and saturable type 1 binding spectra when added to oxidized (Fe3+) cytochrome P-450 (CYT P-450) in control, phenobarbital- (PB) induced, and beta-naphthoflavone- (BNF) induced male Sprague-Dawley rat liver microsomes. The spectral binding constants (Ks) for HCB in control and PB-induced microsomes are 180 microM and 83 microM, respectively, and correlate inversely with the specific content of CYT P-450 (0.9 and 2.1 nmol/mg) in the two microsomal preparations. BNF-induced microsomes show type 1 interaction only at low HCB concentration. Overall biotransformation of HCB, monitored by loss of [14C]HCB from the reaction medium, is dependent on NADPH and intact microsomes. Dimethyl sulfoxide (Me2SO), a potent hydroxyl radical scavenger and the solvent used for HCB dissolution, does not affect the biotransformation of HCB in aerobic reactions. Pentachlorobenzene (PCB) appears to be the initial and major isolatable CYT P-450 mediated dechlorination product of HCB with NADPH-fortified rat liver microsomes. Trace levels of pentachlorophenol (PCP) and an unidentified metabolite are also observed. PCB formation is enhanced under anaerobic conditions but is inhibited by metyrapone and carbon monoxide. PCB formation is also inhibited with aerobic reaction conditions, while PCP formation is observed. The data indicate that CYT P-450 in hepatic microsomes supports the reductive dechlorination of HCB to PCB.     

The role of the Ah locus in hexachlorobenzene-induced porphyria. Studies in congenic C57BL/6J mice.

The role of the Ah locus in hexachlorobenzene (HCB)-induced porphyria and the possible involvement of P-450 cytochromes P(1)450 and P(3)450 in the pathogenesis of this disease were investigated in two congenic strains of C57BL/6J mice that differ only at this locus. Female B6-Ahb mice (Ah receptor: approximately 30-70 fmol/mg of cytosolic protein) and B6-Ahd mice (Ah receptor: undetectable) were pretreated with iron (500 mg/kg) and then fed a diet containing 0 or 200 p.p.m. of HCB for up to 17 weeks. Mice from the two strains consumed similar amounts of HCB. Urinary excretion of porphyrins was increased after 7 weeks of HCB treatment in B6-Ahb mice, and after 15 weeks was over 200 times greater than that of mice given iron only. In B6-Ahd mice, porphyrin excretion did not begin to increase until after 13 weeks, and after 15 weeks was only six times greater than that of controls. Similar differences were seen in the 15-week hepatic porphyrin concentrations (B6-Ahb: 1110 +/- 393; B6-Ahd: 17.6 +/- 14.5; controls: approximately 0.20 nmol/g). Uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was diminished by 70 and 20% in B6-Ahb B6-Ahd mice respectively after 15 weeks of treatment with HCB. Cytochromes P(1)450 and P(3)450 were measured in hepatic microsomes (microsomal fractions) by radioimmunoassay and immunoblotting, using antisera raised against the orthologous rat isoenzymes P450c and P450d. HCB induced small amounts of a protein recognized by anti-P450c (P(1)450) in B6-Ahd mice, but not in B6-Ahd mice. Relatively large amounts of a protein recognized by anti-P450d (P(3)450) were induced in both strains, but to a somewhat greater extent in the B6-Ahb mice. The hepatic accumulation of HCB at 15 weeks was greater in B6-Ahb than in B6-Ahd mice, in association with elevated hepatic lipid levels in the former strain. The results of this experiment indicate that the Ah locus influences the susceptibility of C57BL/6J mice to HCB-induced porphyria and are consistent with the suggestion that the sustained induction of P(3)450 and/or P(1)450 may be a causative factor in the development of this disease.     

Interaction of hexachlorobenzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo. Evidence that hexachlorobenzene is a weak Ah receptor agonist

Hexachlorobenzene (HCB) produces hepatic porphyria and induces the hepatic cytochrome P450 isozymes P450c (P450IA1) and P450d (P450IA2) in rodents. These and other effects of HCB resemble those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which acts via its binding to the aromatic hydrocarbon (Ah) receptor. We therefore examined the ability of HCB to interact with this receptor in vitro and in vivo. HCB, at concentrations of 1 microM or higher, inhibited the specific binding of [3H]TCDD (0.3 nM) to the Ah receptor in vitro, whereas the solubility of [3H]TCDD was affected only at 100 microM HCB. The inhibition was competitive, with a KI of approximately 2.1 microM. In rats fed a diet containing 3000 ppm HCB for varying times (4 h to 7 days), the specific binding of [3H]TCDD in hepatic cytosol was reduced by up to 40%, as observed previously for known Ah receptor agonists. The decrease in [3H]TCDD specific binding in cytosol of HCB-treated rats was due principally to a decrease in the number of binding sites for [3H]TCDD rather than competition from residual HCB. As shown by immunoblotting and radioimmunoassay, HCB induced the cytochrome P450 isozymes P450c and P450d, which are regulated by the Ah receptor, as well as the phenobarbital-inducible isozymes P450b and P450e. Together these results indicate that HCB is a weak agonist for the Ah receptor, and suggest that some of its effects may be mediated by its interaction with this gene-regulatory protein.     

Catalytic activity of isolated cytochromes P-450d and P-450c

Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined.     

Structure-activity relationships of chlorinated benzenes as inducers of different forms of cytochrome P-450 in rat liver

Hexachlorobenzene (HCB) produced increases in ethoxyresorufin (ERR) O-deethylase, aryl hydrocarbon hydroxylase (AHH) and aminopyrine N-demethylase activities in rat liver microsomes which were intermediate between those produced by phenobarbital and 3,4-benzpyrene (BP). α-Naphthoflavone (ANF) selectively inhibited ERR activity in BP and HCB-induced microsomes (94% and 88%). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of liver microsomes indicated that HCB did not produce a detectable increase in a polypeptide with electrophoretic properties similar to those of purified cytochrome P-448 (M_r = 56 000). However, HCB did induce a polypeptide with M_r = 53 000 corresponding to one of two polypeptide bands induced by BP. This polypeptide may represent a second form of cytochrome P-448. Purification of HCB to remove possible dibenzo-p-dioxin impurities did not alter the ‘mixed-type’ induction produced by HCB. In contrast to HCB, all other chlorinated benzenes tested resembled phenobarbital as inducers.     

Use of monoclonal antibody probes against rat hepatic cytochromes P-450c and P-450d to detect immunochemically related isozymes in liver microsomes from different species

Nine distinct monoclonal antibodies raised against purified rat liver cytochrome P-450c react with six different epitopes on the antigen, and one of these epitopes is shared by cytochrome P-450d. None of these monoclonal antibodies recognize seven other purified rat liver isozymes (cytochromes P-450a, b, and e-i) or other proteins in the cytochrome P-450 region of "Western blots" of liver microsomes. Each of the monoclonal antibodies was used to probe "Western blots" of liver microsomes from untreated, or 3-methylcholanthrene-, or isosafrole-treated animals to determine if laboratory animals other than rats possess isozymes immunochemically related to cytochromes P-450c and P-450d. Two protein-staining bands immunorelated to cytochromes P-450c and P-450d were observed in all animals treated with 3-methylcholanthrene (rabbit, hamster, guinea pig, and C57BL/6J mouse) except the DBA/2J mouse, where no polypeptide immunorelated to cytochrome P-450c was detected. The conservation of the number of rat cytochrome P-450c epitopes among these species varied from as few as two (guinea pig) to as many as five epitopes (C57BL/6J mouse and rabbit). The relative mobility in sodium dodecyl sulfate-gels of polypeptides immunorelated to cytochromes P-450c and P-450d was similar in all species examined except the guinea pig, where the polypeptide related to cytochrome P-450c had a smaller Mr than cytochrome P-450d. With the use of both monoclonal and polyclonal antibodies, we were able to establish that purified rabbit cytochromes P-450 LM4 and P-450 LM6 are immunorelated to rat cytochromes P-450d and P-450c, respectively.     

Regulation of three forms of cytochrome P-450 and epoxide hydrolase in rat liver microsomes. Effects of age, sex, and induction

A procedure for the preparation of monospecific antibody directed against rat liver microsomal cytochrome P-45-a is described. This antibody, together with monospecific antibodies to cytochromes P-450b and P-450c, has been used to show that these three forms of cytochrome P-450 are distinct and share no common antigenic determinants. These antibodies (a) give single immunoprecipitin bands with detergent-solubilized microsomes; (b) do not cross-react with the purified heterologous antigens in Ouchterlony double diffusion analyses; (c) have no effect on catalytic activity of the heterologous antigens but completely inhibit the enzymatic activity of the homologous antigens; and (d) remove only the homologous antigen from detergent-solubilized microsomes when covalently bound to a solid support. With radial immunodiffusion assay, we have quantitated these three forms of cytochrome P-450 in liver microsomes after treatment of rats with seven different inducers of cytochrome P-450. The levels of these cytochrome P-450 isozymes vary independently and are also regulated by the age and sex of the animal. The antibodies have also been used to assess the contribution of cytochromes P-450a, P-450b, and P-450c in the metabolism of xenobiotics by rat liver microsomes. A large proportion of benzo(a)pyrene metabolism and testosterone 16 alpha-hydroxylation in microsomes from untreated rats is not catalyzed by cytochromes P-450a, P-450b, and P-450c. Epoxide hydrolase, another microsomal enzyme involved in the metabolism of xenobiotics, was also quantitated by radial immunodiffusion after prior treatment of rats with microsomal enzyme inducers. The inductions of epoxide hydrolase varies independently of the induction of cytochromes P-450a, P-450b, and P-450c.     

Purification of hepatic microsomal cytochromes P-450 from beta-naphthoflavone-treated Atlantic cod (Gadus morhua), a marine teleost fish

Four isozymes of cytochrome P-450 were purified to varying degrees of homogeneity from liver microsomes of cod, a marine teleost fish. The cod were treated with beta-naphthoflavone by intraperitoneal injection, and liver microsomes were prepared by calcium aggregation. After solubilization of cytochromes P-450 with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate, chromatography on Phenyl-Sepharose CL-4B, and subsequently on DEAE-Sepharose, resulted in two cytochrome P-450 fractions. These were further resolved on hydroxyapatite into a total of four fractions containing different isozymes of cytochromes P-450. One fraction, designated cod cytochrome P-450c, was electrophoretically homogeneous, was recovered in the highest yield and constituted the major form of the isozymes. The relative molecular mass of this form (58 000) corresponds well with a protein band appearing in cod liver microsomes after treatment with beta-naphthoflavone. Both cytochrome P-450c and a minor form called cytochrome P-450d (56000) showed activity towards 7-ethoxyresorufin in a reconstituted system containing rat liver NADPH-cytochrome P-450 reductase and phospholipid. Differences between these two forms were observed in the rate and optimal pH for conversion of this substrate, and in optical properties. Rabbit antiserum to cod cytochrome P-450c did not show any cross-reactions with cod cytochrome P-450a (Mr 55000) or cytochrome P-450d in Ouchterlony immunodiffusion, but gave a precipitin line of partial identity with cod cytochrome P-450b (Mr 54000), possibly as a result of contaminating cytochrome P-450c in this fraction.     

Inducers of cytochrome P-450d: influence on microsomal catalytic activities and differential regulation by enzyme stabilization

The interaction of isosafrole, 3,4,5,3',4',5'-hexabromobiphenyl (HBB) and hexachlorobiphenyl (HCB) with cytochrome P-450d was evaluated by characterization of estradiol 2-hydroxylase activity. Displacement of the isosafrole metabolite from microsomal cytochrome P-450d derived from isosafrole-treated rats resulted in a 160% increase in estradiol 2-hydroxylase. The increase was fully reversed by incubation with 1 microM HBB. Although isosafrole is capable of forming a complex with many different cytochrome P-450 isozymes, it appears to bind largely to cytochrome P-450d in vivo as was demonstrated by measuring the enzymatic activity of microsomal cytochromes P-450b, P-450c, and P-450d from isosafrole-treated rats. When estradiol 2-hydroxylase was measured in rats treated with increasing doses of HCB, there was a gradual decrease in microsomal enzyme activity despite a 20-fold increase in cytochrome P-450d. The ability of cytochrome P-450d ligands to stabilize the enzyme was investigated in two ways. First, cytochromes P-450c and P-450d were quantitated immunochemically in microsomes from rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a dose which maximally induced total cytochrome P-450, followed by a single dose of a second inducer. The specific content of cytochrome P-450d was significantly increased when isosafrole or HCB was the second inducer but not when 3-methylcholanthrene was the second inducer. Second, the relative turnover of cytochrome P-450d was measured by the dual label technique. Following TCDD treatment, microsomal protein was labeled in vivo with [3H]leucine, the second inducer was given and protein was again labeled 3 days later with [14C]leucine. A higher ratio of 3H/14C in the cytochrome P-450d from isosafrole + TCDD- and HCB + TCDD-treated rats relative to TCDD (control)-treated rats suggested that isosafrole and HCB were able to retard the degradation of cytochrome P-450d, presumably by virtue of being tightly bound to the enzyme.     

Immunochemical evidences that hexachlorobenzene induces two forms of cytochrome P-450 in the rat liver microsomes

Induction by hexachlorobenzene (HCB) of the liver microsomal system of metabolism of xenobiotics has been studied in comparison with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that HCB increases the content of cytochrome P-450 in the microsomes. Like PB, HCB induces the activities of aminopyrine- and benzphetamine-N-demethylases. At the same time HCB increases also the activities of benzpyrenehydroxylase and 7-ethoxyresorufin-O-deethylase, which are characteristic of the MC-induction. However, sodium dodecyl sulphate (SDS)-electrophoresis on polyacrylamide gel has revealed that HCB, similar to PB, induces protein with Mr = 52 000 (cytochrome P-450), but not the protein with Mr = 56 000, which is the main isoenzyme of cytochrome P-450 in MC-microsomes (P-448). Using specific antibodies to isolated cytochromes P-450 and P-448 (anti-P-450 and anti-P-448) it has been found by rocket immunoelectrophoresis that in HCB-treated microsomes 20% of the total cytochrome P-450 consist of PB-form and about 10% comprise cytochrome P-488. It has also been found that anti-P-448 totally inhibit 7-ethoxyresorufin-O-deethylase activity of HCB-microsomes while anti-P-450 was inactive. The data presented give direct proof that HCB exemplifies an individual chemical compound which is able to initiate the synthesis of both PB-form and MC-form of the cytochrome P-450.     

Comparison of cytochrome P-450 forms induced by phenobarbital and 1,4-bis[3,5-dichloropyridyloxy)]benzene in the mouse and rat liver

A form of cytochrome P-450 (P-450PB) with a molecular weight of 53.5-54.0 kD possessing a high benzphetamine-N-demethylase activity (100-120 nmol formaldehyde/min/nmol cytochrome) was isolated from liver microsomes of phenobarbital-induced C57Bl/6 mice. This cytochrome P-450 form is immunologically identical to its rat liver counterpart-P-450b (Mr = 52 kD) which is also characterized by a high rate of benzphetamine-N-demethylation. It was shown that 1.4-bis[2-(3.5-dichloropyridyloxy])benzene (TCPOBOP) induces in mouse liver the synthesis of the monoxygenase form whose substrate specificity and immunologic properties are identical to those of cytochromes P-450PB and P-450b. The immunochemically quantitated content of this form makes up to 20% of the total P-450 pool in liver microsomes of phenobarbital- or TCPOBOP-induced mice. Immunochemical analysis of microsomes with the use of antibodies to cytochromes P-450PB and P-450b revealed the presence on the electrophoregrams of phenobarbital-induced rat liver microsomes of two immunologically identical forms of cytochrome P-450, i.e., P-450b and P-450e (the latter had a low ability to benzphetamine N-demethylation). Liver microsomes of phenobarbital- or TCPOBP-induced mice gave only one precipitation band corresponding to cytochrome P-450PB.     

Functional characteristics of cytochromes P-4501A1 and P-4501A2 in rodent liver

Antibodies to mouse liver cytochrome P3-450 (anti-P3-450) and antibodies to rat liver cytochrome P-450d (anti-P-450d-c) inhibit the 0-deethylation of 7-ethoxyresorufin (ER) in liver microsomes of benz(a)pyrene-induced (BP) mice but do not inhibit the 0-deethylase activity in liver microsomes of BP-induced rats. Anti-P3-450 and anti-P-450c inhibit BP-hydroxylation in BP-induced mouse liver microsomes by 20%, but they do not inhibit this reaction at all in BP-induced rat liver microsomes. In a reconstituted monooxygenase system isolated cytochrome P3-450 metabolized 7-ER and BP. In contrast, its homologue, cytochrome P-450d, did not metabolize these substrates. The fraction containing cytochrome P1-450 metabolized 7-ER at a low rate and BP at a rate of 3.6 nmol product/min/nmol cytochrome. Western blot analysis with anti-P-450c + d revealed two bands in SDS-PAGE gels containing BP-induced mouse liver microsomes. The interaction of mouse liver BP-microsomes with anti-P3-450 and anti-P-450d-c was accompanied by the appearance of a single band (cytochrome P3-450).     

Identification of the forms of cytochrome P-450 induced in rat liver by 2-acetylaminofluorene using immunoblotting and partial purification

The amounts of 5 different forms of cytochrome P-450 in liver microsomes from rats treated with 2-acetylaminofluorene were determined and compared with the corresponding patterns in microsomes from control, 3-methylcholanthrene- and phenobarbital-treated animals. 2-Acetylaminofluorene was found to increase the amount of cytochromes P-450b + e 10-fold and of cytochrome P-450d 3-fold, while there was a 54% increase in the level of cytochrome P-450 PB/PCN-E. Cytochrome P-450c was increased from a level too low to detect (less than 0.001 pmol/mg protein) to 0.019 pmol/mg protein. These findings were also confirmed by partial purification of cytochromes P-450b + e and c after 2-acetylaminofluorene treatment.     

Pronounced and differential effects of ionic strength and pH on testosterone oxidation by membrane-bound and purified forms of rat liver microsomal cytochrome P-450

The aim of this study was to determine the effects of ionic strength and pH on the different pathways of testosterone oxidation catalyzed by rat liver microsomes. The catalytic activity of cytochromes P-450a (IIA1), P-450b (IIB1), P-450h (IIC11) and P-450p (IIIA1) was measured in liver microsomes from mature male rats and phenobarbital-treated rats as testosterone 7 alpha-, 16 beta-, 2 alpha- and 6 beta-hydroxylase activity, respectively. An increase in the concentration of potassium phosphate (from 25 to 250 mM) caused a marked decrease in the catalytic activity of cytochromes P-450a (to 8%), P-450b (to 22%) and P-450h (to 23%), but caused a pronounced increase in the catalytic activity of cytochrome P-450p (up to 4.2-fold). These effects were attributed to changes in ionic strength, because similar but less pronounced effects were observed with Tris-HCl (which has approximately 1/3 the ionic strength of phosphate buffer at pH 7.4). Testosterone oxidation by microsomal cytochromes P-450a, P-450b, P-450h and P-450p was also differentially affected by pH (over the range 6.8-8.0). The pH optima ranged from 7.1 (for P-450a and P-450h) to 8.0 (for P-450p), with an intermediate value of 7.4 for cytochrome P-450b. Increasing the pH from 6.8 to 8.0 unexpectedly altered the relative amounts of the 3 major metabolites produced by cytochrome P-450h. The decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h that accompanied an increase in ionic strength or pH could be duplicated in reconstitution systems containing purified P-450a, P-450b or P-450h, equimolar amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. This result indicated that the decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h was a direct effect of ionic strength and pH on these enzymes, rather than a secondary effect related to the increase in testosterone oxidation by cytochrome P-450p. Similar studies with purified cytochrome P-450p were complicated by the atypical conditions needed to reconstitute this enzyme. However, studies on the conversion of digitoxin to digitoxigenin bisdigitoxoside by liver microsomes, which is catalyzed specifically by cytochrome P-450p, provided indirect evidence that the increase in catalytic activity of cytochrome P-450p was also a direct effect of ionic strength and pH on this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of the inducing effect of triphenyldioxan, bis-(dichloropyridyloxy)benzene and phenobarbital on the liver monooxygenase

The induction by triphenyldioxane (TPD) of cytochrome P-450 in rat liver microsomes was studied. It was demonstrated that TPD injection in a single dose (10 mg/kg of body mass) is associated with a marked induction of cytochromes P-450 b/e (cytochrome PB-forms) in rat liver microsomes and a significant increase in the benzphetamine-N-demethylase activity typical of cytochrome P-450b. In other words, TPD is a potent inducer of PB-type, the inducing effect being attained by an injection of a single dose of TPD which is by one order of magnitude less than that of phenobarbital. It can be assumed that this compound shows a high affinity for the hypothetical receptor responsible for cytochrome P-450b synthesis. It was shown also that TPD does not induce the monooxygenase system of mouse liver, whereas 1,4-bis[2-(dichloropyridyloxy)]benzene (DPB) is a potent inducer of PB-type in mice, being fairly ineffective in rats. Hence, the species-specific effect of TPD and DPB appears to be opposite.     

Interaction of 9-hydroxyellipticine with two forms of partially purified rabbit lung cytochrome P-450. Inhibition of lung microsomal benzo[a]pyrene hydroxylase.

9-Hydroxyellipticine (9-OHE), a potent inhibitor of rat liver monooxygenase activities, binds to the various forms of partially purified lung cytochromes P-450 from untreated and 3-methylcholanthrene (3-MC)-treated rabbits. The spectral data (lambda max: 428 nm (ox.), 447 nm (red.), Ks: 10 microM and 5 muM for cytochrome I and cytochrome II from 3-MC-treated rabbits respectively) resemble those obtained with cytochrome P-450 purified from liver of Aroclor 1254-pretreated rats (lambda max: 428 nm (ox.), 445 nm (red.), Ks: 8 microM). 9-OHE has been shown to inhibit the benzo[a]pyrene hydroxylase activity of rat and rabbit lung microsomes. The inhibitory effect was higher towards the 3-MC-induced lung microsomes than with the control microsomes. However, the lung microsomes, as well as the liver microsomes of rabbits were less sensitive to inhibition by 9-OHE than the corresponding microsomes from rats. These results suggest that rabbit and rat cytochromes P-450 have subtle structural differences.     

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.     

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.     

The effect of tetrahydrofuran on the microsomal monooxygenase system in vitro and in vivo]

The effects of tetrahydrofuran (THF) on rat liver microsomes in vitro and in vivo were opposite. In vitro THF inhibited the p-nitrophenol (PNP) hydroxylase activity of microsomes from control rats and from rats treated with PB, acetone, and isoniazide--by 50, 20, 60, and 80%, respectively. THF inhibited dimethylnitrosamine (NDMA) demethylation in control and induced microsomes in a lesser degree. THF increased the total cytochrome P-450 content as well as the contents of cytochromes P-450IIE1 and P-450IIB1/B2. The activities of PNP-hydroxylation and NDMA-demethylation increased also, whereas the PR-dealkylation activity was unchanged. An increase in the THF dose caused inhibition of the rat liver microsomal monooxygenase system.