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.     

Carbon tetrachloride activation in liver microsomes from rats induced with 3-methylcholantrene

The irreversible binding of¹⁴C from¹⁴CCl₄ to microsomal lipids is decreased in animals treated with 3-methylcholantrene (3-MC), while it is increased in animals induced with phenobarbital (PB). CCl₄-induced lipid peroxidation in 3-MC treated rats is as intense as in controls. Destruction of glucose 6-phosphatase (G6P-ase) by CCl₄ is smaller in 3-MC treated rats than in controls. Destruction of total cytochrome P-450 (P-450 + P₁-450) by CCl₄ is smaller in 3-MC treated than in PB treated rats but similar to that obtained in controls. Results would indicate that P-450 would participate in CCl₄ activation much more effectively than P₁-450.     

Binding of nitrosamines to cytochrome P-450 of liver microsomes.

The interactions of 5 carcinogenic and 1 non-carcinogenic nitrosamines with hepatic microsomal cytochrome (cyt.) P-450 were investigated, using both optical difference and electron paramagnetic resonance (EPR) spectroscopic methods. Liver microsomes from phenobarbital (PB)-pretreated mice and 3-methylcholanthrene (3-MC)-pretreated rats were used, in order to have an increased specific content of cyt. P-450 and cyt. P-448 respectively. The optical and EPR spectral data obtained in the oxidised state suggest that nitrosamines are able to bind both as substrates and as ligands to the hemoprotein cyt. P-450, depending on the concentration of nitrosamine, its chemical identity and the cytochrome species present. After reduction with dithionite or NADPH in the optical difference spectrum a Soret band developed between 444 and 453 nm to an extent, which is dependent on the particular nitrosamine present. This initial nitrosamine-induced spectrum might represent a ferrous nitric oxide (NO)-cyt. P-450 complex. It appears unstable and is converted kinetically into a spectrum lacking a Soret band, but with a predominant absorbance minimum at about 425 nm. A visible band is located at 585 nm. In the EPR spectrum a sharp 3-line signal around g = 2.01 appears concomitantly. Both spectral parameters are typical of a NO-cyt. P-420 complex. These results, in conjunction with metabolic studies, indicate that nitrosamines are denitrosated by a reductive process in which cyt. P-450 appears to be involved. The resulting NO-cyt. P-450 complex denatures to a NO-cyt. P-420 complex when the dioxygen level is not sufficiently high to complete successfully.     

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.     

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 $\text{Ah locus}$.     

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.     

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.     

Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.

The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove valuable for more detailed investigations of the molecular and mechanistic basis of the response to PB and its modulation by endogenous hormones.     

Genetic differences in response to pulmonary cytochrome P-450 inducers and oxygen toxicity

The effects of cytochrome P-450 inducers on O2 toxicity were studied in mice. We first examined three cytochrome P-450 inducers, which differ by their specific tissue affinity: phenobarbital sodium (PB), essentially active in the liver, and 3-methylcholanthrene (3-MC) and beta-naphthoflavone (BNF), which are also active in the lung. Both BNF and 3-MC increased the survival rate and significantly decreased pulmonary edema (pulmonary water and wet-to-dry weight ratio) in C57BL/6J mice exposed to hyperoxia (O2 greater than or equal to 95%), whereas PB had no protective effect. In the second part of this study, we compared the action of BNF in two strains of mice. In one (C57BL/6J), cytochrome P-450 can be induced by aromatic hydrocarbons, whereas in the other (DBA/2J) cytochrome P-450 is not inducible by these compounds. Protection against O2 toxicity was assessed in terms of lethality and pulmonary edema and of lung lipid peroxidation (assessed by measuring malondialdehyde). BNF only protected against O2 toxicity in the inducible strain. This protective effect of BNF on O2 toxicity in C57BL/6J mice was associated mainly with a large increase in the components of the cytochrome P-450 system (cytochrome P-450 and cytochrome b5) in the lung. The activity of pulmonary superoxide dismutase was also slightly increased, but the enhancement was not statistically significant. In contrast, in DBA/2J mice neither the components of the cytochrome P-450 system nor the activity of superoxide dismutase showed any increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo and in vitro destruction of rat liver cytochrome P-450 by a monoterpene ketone, pulegone

In vivo administration of pulegone once daily decreased the levels of liver microsomal cyt. P-450 to the extent of 32 and 76% at the end of 24 and 96 hrs respectively. However, cyt. b5 and NAD(P)H-cyt. c reductase activities remained unchanged. In vitro incubation (15 min) of liver microsomes from phenobarbitol (PB)-treated rats with pulegone (10 mM), aerobically or anaerobically resulted in the loss (approximately 60%) of cyt. P-450 in the presence or absence of NADPH. Destruction of cyt. P-450 was more in PB-treated microsomes as compared to 3-methylcholanthrene (MC)-treated and control microsomes. The loss of cyt. P-450 was accompanied by a concomitant loss of microsomal heme. In contrast, menthone or carvone upon incubation with PB-induced microsomes resulted in the conversion (25-40%) of cyt. P-450 to cyt. P-420 without any loss of microsomal heme. The destructive process is irreversible, time dependent, linear upto a substrate concentration of 10 mM and follows first order kinetics.     

Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes

A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to divide into isoenzymes phosphorylated by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor substrates for the kinases employed. On the other hand, glutathione transferases 1-2 and 4-4, but not 3-3, were relatively good substrates for the calcium-phospholipid-dependent kinase.     

Induction of cytochrome P-450 isozymes by mirex and chlordecone

The effect of the insecticides, mirex and chordecone (Kepone), on the cytochrome P-450 monooxygenase system in C57BL/6N mouse liver microsomes was studied. Mice were treated intraperitoneally with low (6 mg/kg) and high (30 mg/kg) doses of mirex and chlordecone in corn oil for 2 days. For comparison, mice were also treated with either phenobarbital (PB) or 3-methylcholanthrene (3-MC). All treatments significantly increased the hepatic microsomal P-450 content over that of controls. Benzphetamine N-demethylase, ethoxyresorufin O-deethylase, benzo[a]pyrene hydroxylase, and acetanilide hydroxylase activities were also determined. Mirex and chlordecone resembled phenobarbital with respect to the induction of monooxygenase activities. Immunoquantitation with antibodies to purified P-450 IIB1 (Pb-induced P-450) and P-450 IA1 (3-MC-induced P-450) indicated that mirex and chlordecone induced P-450 IIB1 in a dose-dependent manner. The high dose of mirex also induced a small amount of a protein cross reacting with the antibody to IA1. The induction of this isozyme did not, however, contribute significantly to the monooxygenase activities measured.     

Induction of specific cytochrome P-450 isozymes by methylenedioxyphenyl compounds and antagonism by 3-methylcholanthrene

Two methylenedioxyphenyl compounds, isosafrole (5-propenyl-1,3-benzodioxole) and an analog, 5-t-butyl-1,3-benzodioxole (BD), differ markedly as inducers of cytochrome P-450 isozymes in rat liver microsomes. Isosafrole is a mixed-type inducer, inducing P-450b, P-450c, and P-450d. In contrast, BD is a phenobarbital-type inducer, increasing P-450b, but producing little or no increase in P-450c or P-450d. Similarly, isosafrole increases the amount of translatable mRNA for P-450b, c and d, while BD induces only the mRNA for P-450b. Dimethylation of the methylene bridge carbon of BD to give 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBD) blocks the formation of NADPH-reduced Type III metabolite-P-450 complexes in vitro, and diminishes but does not abolish the ability of the compound to induce P-450b. Western blots of microsomes from isosafrole and BD-treated rat livers confirm that in contrast to isosafrole, BD does not induce P-450d or P-450c. However, the antibody to P-450d recognizes two new polypeptides (approximately 50K Mr) from sodium dodecyl sulfate-polyacrylamide gels of liver microsomes from BD-treated rats. These polypeptides are not observed in control, isosafrole, 3-methylcholanthrene (3-MC), or DBD-treated rats. They are intensified by coadministration of 3-MC with BD and may represent either modified isozyme-metabolite adducts or degradation products of P-450d. However, the polypeptides could not be generated in vitro by addition of BD to 3-MC-induced microsomes with NADPH under conditions which produced spectral metabolite complexes, or in a reconstituted system with P-450d. The two methylenedioxyphenyl compounds do not form stable metabolite complexes with the same P-450 isozymes. BD formed distinct spectral metabolite complexes in vitro with both P-450b and P-450c but not with P-450d in a reconstituted system. In contrast, isosafrole forms metabolite complexes with all three isozymes. Coadministration of 3-MC with BD blocked induction of P-450b by 80% and produced a similar repression of its translatable mRNA. This finding indicates that 3-MC type inducers not only induce certain cytochrome P-450 isozymes, but also repress synthesis of other isozymes.     

Relation between cytochrome P-450 increase and endoplasmic reticulum proliferation in hepatocytes of mice treated with phenobarbital: a microphotometric and morphometric study.

To obtain detailed information on phenobarbital (PB)-induced cytochrome P-450 (P-450) increase and endoplasmic reticulum (ER) proliferation in hepatocytes, we estimated microphotometrically the amount of P-450 per unit cytoplasmic volume and morphometrically the area of ER per unit cytoplasmic volume in hepatocytes adjacent to the portal area or central venule (1 periportal or 1 perivenular cells) and in the second and third layers from the portal area or central venule (2, 3 periportal or 2, 3 perivenular cells) from mice injected with 35, 50, 100, or 150 mg/kg PB once a day for 3 days. By dividing the P-450 amount by the ER area, the number of P-450 molecules per unit ER area was also calculated. In 1 and 2, 3 perivenular cells, except for 2, 3 perivenular cells after injection of 150 mg/kg PB, the amount of P-450 increased with ER proliferation and the number of P-450 molecules in ER remained unchanged after injection of 50, 100, or 150 mg/kg PB. In 2, 3 periportal cells, however, the P-450 amount and the number of P-450 molecules in ER increased markedly without or with some ER proliferation after injection of 50, 100, or 150 mg/kg PB; the P-450 increase appears to be generally independent of ER proliferation. The 1 periportal cells are probably exceptional hepatocytes that usually did not respond to PB stimulation.     

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)     

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.     

Isolated liver perfusion--a tool in mutagenicity testing for the evaluation of carcinogens.

An isolated liver perfusion system suitable for the combination with Chinese hamster V79 cells is described. With this system, it is possible to study, with the V79 cells as genetic targets, the mutagenic effect of a chemical after metabolic activation in the intact organ. Those substances commonly used in mutagenicity testing as inducers of drug metabolising enzymes, i.e. Arochlor 1254. Phenobarbital(PB) and 3-Methylcholantrene(3-MC), were studied for their effect in the isolated perfused liver. PB increased the bile flow, which was not significantly affected by the other inducers. Only Arochlor caused a significant increase in the amino acid incorporation into plasma proteins and total liver proteins (expressed per mg liver protein). None of the inducers had an effect on gluconeogenesis from lactate or urea synthesis. All three inducers caused an increase in the level of microsomal P-450 enzymes, the biggest increase being seen after Arochlor-induction (170%), followed by PB(90%) and 3-MC(50%). Arochlor- and PB-induction had a dramatic effect on N- and C-oxygenation of N, N-dimethylaniline: N-oxygenation was decreased by 35% and 40% respectively and C-oxygenation increased by 130% and 140% respectively. The advantages of the isolated perfused liver as an intact metabolising unit is discussed in relation to other mutagenicity assays, in which subcellular fractions are used as the metabolising system.     

Glucuronidation and sulfation of p-nitrophenol in isolated rat hepatocyte subpopulations. Effects of phenobarbital and 3-methylcholanthrene pretreatment

Parenchymal cells, isolated from untreated (control), phenobarbital(PB)-or 3-methylcholanthrene(3-MC)-treated rats, were separated into four subpopulations according to cell density, and glucuronidation and sulfation of p-nitrophenol (PNP) in the hepatocyte subpopulations were investigated. PB enhanced the glucuronidation almost 2-fold but not the sulfation, while 3-MC enhanced both glucuronidation (3-fold) and sulfation (2-fold) in the original cell suspensions. Some gradation trends were found in the conjugation activities among the hepatocyte subpopulations: In the control experiment, the extent of glucuronidation in four subpopulations was virtually the same but sulfation in high-density hepatocytes was slightly higher than in low-density ones. Both glucuronidation and sulfation were higher in low-density hepatocytes from PB-treated rats, though the gradation was very modest. Glucuronidation and sulfation tended to be slightly higher in middle-density hepatocytes in the 3-MC experiment. However, no definite correlation in conjugation activities vs. cell density, like those seen in cytochrome P-450s vs. cell density in the hepatocytes isolated from PB-treated rats, were found in the subpopulations from control or inducer-treated rats. Simultaneous studies on acetylation of p-aminobenzoic acid (PABA) revealed that the activities in the subpopulations were virtually the same and the inducers had little influence on the activity.     

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.     

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).