Induction of different species of cytochrome P-450 by coplanar and noncoplanar isomers of hexachlorobiphenyl

The effects of coplanar⁺ 3,4,5,3′,4′,5′-hexachlorobiphenyl (HCB) and noncoplanar 2,4,5,2′,4′,5′-HCB, 2,3,5,2′,3′,5′-HCB, phenobarbitone (PB) and 3-methylcholanthrene (3-MC) on drug metabolizing enzymes have been studied 72 hr after dosing in male rat liver. 3-MC and 3,4,5,3′,4′,5′-HCB induced the activity of ethoxyresorufin deethylase dramatically. NADPH cytochrome P-450 reductase and benzphetamine $\text{N}$-demethylase were induced by PB and noncoplanar isomers and not by 3-MC or 3,4,5,3′,4′,5′-HCB. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the microsomes obtained from various groups showed that 3-MC and 3,4,5,3′,4′,5′-HCB induced the synthesis of a polypeptide of approximate 54,500 daltons which was absent in the microsomes obtained from control, PB or noncoplanar isomer treated animals. Noncoplanar isomers and PB induced the synthesis of a polypeptide of approximate 51,000 daltons. These results, along with the reduced, CO difference spectra, demonstrate that 3,4,5,3′,4′,5′-HCB induces the synthesis of cytochrome P-448 and resembled 3-MC in its mechanism of action, while noncoplanar isomers induced the synthesis of cytochrome P-450 and resembled PB in its mechanism of action. Further administration of various doses of 3,4,5,3′,4′,5′-HCB to genetically responsive mice (C57BL/6J), induced cytochrome P-450, caused one nm shift in the difference spectrum of reduced microsomes and induced the activity of ethoxyresorufin deethylase, whereas it did not induce the activity of ethoxyresorufin deethylase in non-responsive mice (DBA/2J) even at the highest dose studied. These studies indicate the fact that coplanar and noncoplanar isomers have differential interaction with $\text{Ah locus}$.     

Differences in the carbon tetrachloride-induced damage to components of the smooth and rough endoplasmic reticulum from rat liver

There is a higher activity of ethyl morphine N-demethylase (EM-ase) and cytochrome P-450 (P-450) reductase as well as higher P-450 content in the smooth endoplasmic reticulum (SER) than in the rough endoplasmic reticulum (RER). The extent of the irreversible binding of the¹⁴C from¹⁴CCl₄ to lipids and proteins, as well as the CCl₄-induced destruction of P-450 is more intense in SER than in RER while the opposite was found for glucose 6-phosphatase (G6P-ase) destruction. CCl₄-induced lipid peroxidation is as intense in SER as is in RER.¹⁴C from¹⁴CCl₄ gets irreversibly bound to ribosomal proteins.     

Mechanism of in vivo carbon tetrachloride-induced liver microsomal cytochrome P-450 destruction.

Prior administration of aminotriazole (3-amino-1,2,4-triazole) or pyrazole to rats resulted in a significant prevention of the CCl₄-induced decrease in the liver microsomal P-450 content. In A/J mice the CCl₄ activation and P-450 destruction occurred in absolute absence of lipid peroxidation as determined by uv absorption. The data suggest that P-450 destruction is mainly mediated by direct attack of CCl₄ metabolites rather than by CCl₄-induced lipid peroxidation.     

Synthesis of liver cytochrome P-450b in a cell-free protein synthesizing system

Live ppolysomes isolated from rats that had been treated with phenobarbital (PB) are able to incorporate [³H]leucine into total protein $\text{in}\text{vitro}$ at a rate almost five times that of polysomes prepared from control animals. Specific immunoprecipitation of translational products has shown that polysomes from induced animals synthesize cytochrome P-450b at a rate almost seven times greater than polysomes from control animals. The increased protein and cytochrome P-450b synthesis can be detected as early as 6 h following phenobarbital administration and reaches a maximum at 12–18 h. The results suggest that PB administration effects an increase in mRNA for cytochrome P-450b.     

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450.

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{vitro}$ with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O₂ (8:2) and addition of a cytochrome $\text{c}$ reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome $\text{c}$ reductase activity was unchanged, the covalent binding of CCl₄, CHCl₃, and CBrCl₃ was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N₂ enhanced the binding of CCl₄, inhibited the binding of CHCl₃ and did not influence the binding of CBrCl₃. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.     

Scoparone O-demethylase assay is not useful to differentiate the effects of model inducers of cytochrome P-450 in rabbit and guinea pig liver

Coumarin derivative, scoparone (6,7-dimethoxycoumarin), is regioselectively O-demethylated into isoscopoletin (I) and scopoletin (S). This oxidation is inversely influenced by cytochrome P-450 inducers in the rat such as 3-methylcholantrene (3-MC) and phenobarbital (PB). The I/S ratio is higher than 1.5 with 3-MC treatment whereas it is lower than 0.5 with PB treatment. With regards to this contrasting effect, it gas been suggested that the I/S ratio should be useful to differemtiate between the effects of these types of inducers. We studied the consequences of in vivo PB and 3-MC treatment on scoparone biotransformation in guinea pig and rabbit. In these two species, at the basal state, scoparone biotransformation was enhanced in comparison to the rat. Moreover, in these untreated animals, two other metabolites were formed. After 3-MC or PB treatment, scoparone metabolism is, in contrast to the rat, inappropriate to differentiate between the P-450 profile of other animals.     

Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene

Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.     

Induction and catalytic activities of cytochromes P-450b/e and P-450c in the liver microsomes of neonatal rats

The activities of cytochrome P-450-dependent monooxygenases has been investigated in the liver microsomes of newborn rats (3-16 days after birth) induced with PB or 3-MC. It has been shown that the induction by PB and 3-MC results in the increase of both the total amount of cytochrome P-450 as determined by the CO-reduced spectrum and the amount of induced forms P-450b/e and P-450c respectively. In the course of induction of the specific forms of cytochrome P-450 BP-hydroxylase and 7-ER-O-deethylase activities increased at 3-MC-induction, while BPh-N-demethylase and BP-hydroxylase increased at PB-induction. Analysis of inhibition of monooxygenase reactions with antibodies has showed that only P-450c was involved in metabolism of BP and 7-ER. Participation of P-450b/e in BPh N-demethylation was notably lower in the neonates in comparison to the adult rats. In the one-week-old rats induced with 3-MC a considerable rate of BP hydroxylation and 7-ER O-deethylation (2-4.5 nmol of product min-1 mg-1) has been observed despite a small amount of P-450 (0.02-0.1 nmol/mg of protein). This fact shows the higher catalytic activity of this cytochrome P-450 in the neonates compared to similar characteristics of P-450c in the 3-MC-induced microsomes. Metabolism of BP in the PB-microsomes of the neonatal rats was inhibited neither by anti-P-450b/e nor anti-P-450c in contrast to the adults, where this reaction was inhibited by antibodies against P-450b/e.     

Incorporation of radioactive- -aminolevulinic acid into microsomal cytochrome P 450 : selective breakdown of the hemoprotein by allylisopropylacetamide and carbon tetrachloride

Administration of allylisopropylacetamide (AIA) or CCl₄ to rats previously treated with phenobarbital leads to a rapid decrease in cytochrome P₄₅₀ within 1 hr. The amount of cytochrome b₅ and NADPH cytochrome c reductase in liver microsomes remains unchanged following AIA treatment. In contrast, CCl₄ administration causes a decrease in total microsomal protein thus leading to a net loss in cytochrome b₅ and NADPH cytochrome c reductase. By using ³H-δ-aminolevulinic acid to label microsomal cytochrome P₄₅₀ heme, the effect of AIA and CCl₄ on this cytochrome was shown to be caused by destruction of preexisting CO-binding pigment and not from inhibition of synthesis. In addition, the breakdown products of cytochrome P₄₅₀ heme accumulate in the liver after AIA or CCl₄ treatment.     

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4

Hepatic microsomes isolated from untreated male rats or from rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) were labeled with the hydrophobic, photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of [125I]TID into uninduced liver microsomes. The major hepatic microsomal cytochrome P-450 forms inducible by PB and 3-MC, respectively designated P-450s PB-4 and BNF-B, were shown to be the principal polypeptides labeled by [125I]TID in the correspondingly induced microsomes. Trypsin cleavage of [125I]TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr approximately 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. [125I]TID incorporation into microsomal P-450 PB-4 was inhibited in a dose-dependent manner by the P-450 PB-4 substrate benzphetamine. In the absence of photoactivation, TID inhibited competitively about 80% of the cytochrome P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced microsomes with a Ki of 10 microM; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or beta-naphthoflavone-induced livers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of microsomal electron transport components from control, phenobarbital and 3-methylcholanthrene treated mice: II. Resolution and quantitation of cytochromes P-450 and P1-450 and the so-called "factor X" in SDS-polyacrylamide gels of total microsomes.

By improving the resolution of microsomal proteins in SDS-polyacrylamide gels, we have succeeded in assigning one major protein not only to each of the four known components of microsomal electron transport but also to the proposed components, cytochrome P₁-450 and “Factor X”. The increases in P-450 protein after PB induction and P₁-450 protein after 3-MC induction were comparable to the respective increases in CO binding. They demonstrated molecular weights of 56,000 and 54,000 daltons respectively. The “Factor X” protein, molecular weight = 48,000, appears to be of non-heme iron character, and probably is an intermediated electron carrier between flavo- and hemoproteins.     

Inhibition of azoreductase activity by antibodies against cytochromes P-450 and P-448

Hepatic microsomal azoreductase activity in mice was induced with phenobarbital (PB) and 3-methylcholanthrene (3-MC). Antibodies against cytochrome P-450 inhibited azoreductase activity of PB-treated animals while antibodies against cytochrome P-448 inhibited liver azoreductase activity of 3-MC-treated animals, each by about 90%. These antibodies also inhibited microsomal 7-ethoxycoumarin-O-deethylase activity to the same extent. It is concluded that hepatic microsomal azoreductase activity is almost totally dependent on cytochromes P-450 and P-448 and the contribution, if any, of other microsomal components is negligible.     

Suppression of levels of phenobarbital-inducible rat liver cytochrome P-450 by pituitary hormone

The effect of pituitary factor on the constitutive and inducible levels of hepatic phenobarbital (PB)-inducible major cytochrome P-450, P-450b and P-450e, in male and female rat livers was studied by immunoblot analyses. Although only trace amounts (approximately 4 pmol/mg protein) of P-450b and P-450e were detected in untreated adult rats, hypophysectomy increased the contents of P-450b and P-450e 58- and 14-fold, respectively, in male rats and 118- and 30-fold, respectively, in female rats. The increases were also observed in treatment with dexamethasone, which suppressed the pituitary function. Treatment with PB increased more effectively the hepatic contents of P-450b and P-450e, but their contents were still 4-fold higher in the male than the female. Treatment of hypophysectomized female rats with PB increased the contents of P-450b and P-450e 4-fold higher than the contents in PB-treated nonhypophysectomized female rats. Consequently, the sex-related difference in their contents was reduced less than 1.4-fold in the hypophysectomized rats treated with PB. Similar results were also obtained from the quantitation of microsomal O-pentylresorufin O-depentylation and testosterone 16 beta-hydroxylation. Either intermittent injection or continuous infusion of human growth hormone, but not of ovine prolactin, into hypophysectomized male and female rats decreased the contents of both cytochromes. These results indicate that growth hormone acts as a repressive factor for the constitutive and inducible levels of P-450b and P-450e in a manner different from the regulation of P-450-male and P-450-female.     

Michaelis--Menten kinetic analysis of the hepatic microsomal benzpyrene hydroxylase from control, phenobarbital- and methyl-3-cholanthrene-treated rats.

The sensitive fluorimetric assay for hydroxy-3-benzpyrene (3-OH-BP) described by Dehnen et al., was used to study the effect of microsomal membrane concentration of the benzpyrene hydroxylase activity. Microsomes from phenobarbital (PB) and methyl-3-cholanthrene (3-MC)-treated rats were used in comparison with the microsomal fraction from control animals. At very low protein concentration, benzpyrene hydroxylase follows as Michaelis--Menten type kinetics. When the concentration of microsomal membrane is higher than a minimal value (+/- 6 mug protein/ml) the Km increases with increasing concentration of protein due to competitive inhibition by reversible and non-specific binding of the substrate. The Ki's for such a binding have been calculated. Pretreatment of rats with 3-MC selectively shortens the time linearity, decreases the Ks value, and has no effect on Vmax, while the administration of PB prolongs the time linearity, decreases Vmax and does not modify the Ks. 3-MC and PB specifically act on cytochrome P-450 and do not modify the physico-chemical properties of the microsomal membrane as measured by the non-specific binding of benzpyrene (BP).     

Effect of chronic treatment with phenobarbital or 3-methylcholanthrene on the male reproductive system in rats

Treatment of rats for 4 weeks with phenobarbital (PB) did not inhibit the growth of the seminal vesicles, nor did it affect the biosynthesis of testosterone by testis microsomes. Moreover, neither the concentration of cytochrome p-450 or the 17 α-hydroxylase activity in testis microsomes were affected. In contrast, treatment with 3-methylcholanthrene (3-MC) for 4 weeks markedly decreased the weights of the seminal vesicles. The decrease was probably related to an impairmant of testosterone formation in the gonads, since testosterone biosynthesis as well as the concentration of cytochrome p-450 and the activity of 17 α-hydroxylase in testis microsomes were significantly decreased in the 3-MC treated rats. No histopathological changes were seen in testes from any of the PB or 3-MC treated rats.     

Hepatic mixed function oxidase system and effect of phenobarbital, 3-methylcholanthrene and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane in developing chickens.

Contents of hepatic microsomal protein, aminopyrine N-demethylase, acetanilide hydroxylase, aniline hydroxylase, hydrogen peroxide formation, cytochrome-c-reductase, cytochrome b5 and cytochrome P-450 were examined in control, phenobarbital (PB), 3-methylcholanthrene (3-MC) and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane (DDT) treated group of 1-28 days old chickens. Increase in aminopyrine N-demethylase, acetanilide hydroxylase, aniline hydroxylase, cytochrome-c-reductase, cytochrome b5 and cytochrome P-450 was noticed at all stages of development during administration of PB and 3-MC. But these enzyme activities were not always paralleled by increase in age. Aminopyrine N-demethylase was increased in early stages only during DDT administration, which indicates that the form of cytochrome P-450, responsible for aminopyrine N-demethylation is present in early stages only. However, acetanilide hydroxylase was decreased in all stages of development, in postnatal development the basal activities of the enzymes for various substrates do not exhibit identical pattern, the degree of inducibility by inducers varied in relation to age of animal. Hydrogen peroxide formation increased in all stages of developing chickens due to the administration of PB and DDT. It however decreased due to 3-MC administration which may be due to induction of high spin cytochrome P-450.     

Slaving the cytochrome P-450 dependent monooxygenase system by periodically applied light pulses

The light-induced enhancement of 7-ethoxycoumarin-O-deethylase activity was measured in a reconstituted system consisting of the enzyme P-450 II B1 (P-450_PB-B) and the NADPH-cytochrome P-450 reductase. The phases of the catalytic cycle of 2 · 10¹² protein complexes were locked by periodic application of light pulses (0.1 s duration, 1.2–2.5 s repetition time, and 390–470 nm 0.27 Joule/nmol P-450). More than 80% of the active reconstituted enzyme complexes worked in phase if the repetition time (1.32 s) was slightly smaller than the catalytic cycle time of the free running enzyme (1.54 s). The percentage of synchronized enzyme complexes as a function of the repetition time is shown. It is shown that the lifetime of the product-enzyme complex is shortened by the light.Abbreviations P-450 liver microsomal cytochrome P-450 - PB phenobarbital Offprint requests to: H. Gruler     

Nonadditive effects of combined in vivo hepatic microsomal enzyme inducers in the Wistar rat

Compounds that are known to increase the hepatic microsomal cytochrome P-450 dependent monooxygenases were administered to adult female rats, alone or in combination, to determine whether their effects on certain substrate oxidations were additive. 3-Methylcholanthrene (3-MC) and pregnenolone-16 alpha-carbonitrile (PCN), known to induce different forms of cytochrome P-450, when administered together increased benzo[a]pyrene oxidation to the same level as observed following 3-MC treatment alone. Phenobarbital (Pb) and PCN when administered concomitantly increased benzo[a]pyrene, amino-pyrine, and ethylmorphine metabolism to the same extent as seen following PCN administration alone. Both compounds are known to induce different forms of cytochrome P-450. Nonadditive effects were also observed with Pb and spironolactone, as well as with Pb and trans-stilbene oxide. Treatment of adult male rats with either PCN or 3-MC resulted in significantly smaller increases in benzo[a]pyrene oxidation than observed in adult female rats. These results suggest that oxidative metabolism in hepatic microsomes is not the sum of activities of a number of cytochrome P-450s, but may represent the activity of a single predominant hemeprotein. In addition, it appears that the oxidation of substrate by a particular cytochrome P-450, in intact microsomes, is greatly influenced by the presence of another form.     

Cloning and sequence analysis of a rat liver cDNA coding for a phenobarbital-inducible microheterogenous cytochrome P-450 variant: regulation of its messenger level by xenobiotics

A rat liver cDNA library was prepared from total polyribosomal poly(A)+ RNA extracted from phenobarbital-treated animals. A cDNA clone coding for a phenobarbital-inducible cytochrome P-450 (PB P-450) was identified by differential colony hybridization to cDNAs synthesized from liver poly(A)+RNAs isolated from phenobarbital-treated rats for positive selection and cDNAs from either untreated rats or beta-naphthoflavone-treated rats as negative controls, followed by hybrid-selected translation and analysis of the translation products by immunoprecipitation. As the cloning and screening strategies involve no prior enrichment for specific mRNAs, they also permit the identification of sequences coding for phenobarbital-induced proteins other than cytochromes P-450. This relatively straightforward approach is generally applicable to the molecular cloning of sequences coding for other inducible cytochromes P-450. Nucleic acid sequencing data indicated that the cloned PB P-450 cDNA codes for a cytochrome P-450 variant [designated P-450e(U.C.)] that is very similar, but not identical, to P-450e. Sequence analysis of the section of cDNA specifying the 3'-non-coding region of the mRNA revealed that it lacked the usual poly(A) addition site signal sequence but contained three inverted repeat structures. Solution hybridization analysis demonstrated that PB P-450 mRNA is increased 20-fold by phenobarbital treatment and decreased 3-fold by beta-naphthoflavone treatment.