Forms Of Hepatic Cytochrome P-450 Research Articles

Predictive value of animal models for human cytochrome P450 (CYP)-mediated metabolism: A comparative study in vitro

One major challenge in drug development is defining of the optimal animal species to serve as a model of metabolism in man. The study compared the hepatic drug metabolism characteristics of humans and six widely used experimental animal species. Classical in vitro model enzyme assays with known human cytochrome P450 (CYP) enzyme selectivity were employed and optimized to target human hepatic CYP forms. The profile of CYP activities best resembling the human was seen in mouse followed by monkey, minipig, and dog liver microsomes, with rats displaying the most divergent. The widest interindividual variability was found in CYP3A-mediated midazolam α-hydroxylase, and omeprazole sulphoxidase activities in human and monkey liver microsomes. These data demonstrate that if hepatic xenobiotic-metabolizing characteristics were to be the sole reason for the selection of animal species for toxicity studies, then the rat might not be the most appropriate model to mimic human CYP activity patterns.

Selectivity of 7-alkoxycoumarins as probe substrates for rat hepatic cytochrome P450 forms is influenced by the substitution pattern on the coumarin nucleus

The O-dealkylation of 7-alkoxycoumarins is widely used as an assay to characterize cytochrome P450 (CYP) activity. These substrates can also undergo oxidative attack at additional sites on the coumarin nucleus, which may influence their apparent selectivity for particular CYP forms.Accordingly, the effect of blockade of these additional sites was investigated on the selectivity towards rat hepatic CYP forms, with emphasis on the CYP1A and 2B forms.Blockade of the 3-/4- and 6-positions resulted in substrates for which the CYP1A1/2 selectivity of the unsubstituted 7-alkoxycoumarins was altered to a CYP2B selectivity; this was achieved with little overall change in the molecular dimensions of the substrate. Limited analysis of other inducible CYP forms indicated at most only small effects of structure modification on activity.The findings suggest that the sensitivity of probe substrates for CYP forms may be limited by the occurrence of competing side reactions of the substrate, and that better probes may be derived by blocking the sites of these side reactions.

Urinary 6beta-hydroxycortisol/17-hydroxycorticosteroids ratio as a measure of hepatic CYP3A4 capacity after enzyme induction.

The correlation between the urinary 6beta-hydroxycortisol/17-hydroxycorticosteroids (6beta-OHF/17-OHCS) ratio and the metabolic capacity of the most abundant form of hepatic cytochrome P450 (CYP3A4) after induction remains unclear. Concentrations of 6beta-OHF and 17-OHCS in spot urine specimens obtained from 61 epileptic children receiving continuous carbamazepine therapy were measured by high-performance liquid chromatography. The relationship between the urinary 6beta-OHF/17-OHCS ratio and the serum carbamazepine concentration, corrected for dose and body weight, was examined. Serum carbamazepine was inversely associated with the urinary 6beta-OHF/17-OHCS ratio, and the hyperbolic relationship between the two parameters was statistically significant (P < 0.01). Carbamazepine is well known as a potent inducer and a substrate of hepatic CYP3A4. The present results suggest that measurement of the urinary 6betaOHF/17-OHCS ratio is helpful for assessing individuals' hepatic CYP3A4 capacity after enzyme induction.

Metabolism of a novel phosphodiesterase-IV inhibitor (V11294) by human hepatic cytochrome P450 forms

1. The metabolism of a novel phosphodiesterase-IV inhibitor (V11294) was studied in human liver microsomal and cytosol preparations and in cDNA-expressed human hepatic CYP forms. 2. Human liver microsomes, but not cytosol, catalysed the NADPH-dependent metabolism of V11294 to V10331 (formed by hydroxylation of the cyclopentyl ring), V10332 (N -desethyl V11294) and V11689 (formed by hydroxylation of the isopropyl side chain). In addition, smaller amounts of a secondary metabolite V11690 (which can be formed from either V10332 or V11689) were also produced. 3. Kinetic analysis of V11294 metabolism to V10331, V10332 and V11689 in two preparations of pooled human liver microsomes revealed average K m = 2.5, 8.1 and 3.9 µM, respectively. 4. The metabolism of V11294 was determined with a characterized bank of 16 individual human liver microsomal preparations employing a V11294 substrate concentration of 8µM (i.e. approximately the K m for V10332 formation and around twice the K m for V10331 and V11689 formation). Good correlations (r 2 = 0.570-0.903) were observed between V10331, V10332 and V11689 formation and markers of CYP3A forms. In contrast, poorer correlations (r 2 = 0.0002-0.428) were observed with markers of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 5. Using human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP forms, V11294 (8 µM) was metabolized by cDNA-expressed CYP3A4 to V10331, V10332 and V11689, with lower amounts of V11690 also being formed. Lower rates of V11294 metabolism to some V11294 metabolites were also observed with cDNA-expressed CYP2C9, CYP2C19 and CYP2D6, whereas only very low or undetectable rates of V11294 metabolism were observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8 and CYP2E1. 6. The metabolism of V11294 (8 µM) to V10331, V10332 and V11689 was markedly inhibited by the CYP3A mechanism-based inhibitor troleandomycin. In contrast, V11294 metabolism was not significantly affected by inhibitors of CYP1A2, CYP2C9, CYP2D6 and CYP2E1 or by the CYP2C19 substrate S -mephenytoin. 7. In summary, by correlation analysis, chemical inhibition studies and the use of cDNA-expressed CYPs, V11294 metabolism in human liver to V10331, V10332 and V11689 appears to be primarily catalysed by CYP3A forms.

Effects of enzyme inducers or inhibitors on the pharmacokinetics of intravenous parathion in rats.

In order to find what form of hepatic cytochrome P450 (CYP) is involved in the metabolism of parathion to form paraoxon, rats were pretreated with the enzyme inhibitors, such as SKF 525-A and ketoconazole or enzyme inducers, such as dexamethasone, isoniazid, phenobarbital, and 3-methylcholanthrene. Parathion, 3 mg/kg, was infused in 1 min via the jugular vein. In rats pretreated with SKF 525-A or ketoconazole, nonspecific CYP inhibitors, the area under the plasma concentration-time curve from time zero to time infinity (AUC) and total body clearance (Cl) of parathion were significantly greater and slower, respectively, than those in respective control rats, suggesting that parathion was metabolized by CYPs. In rats pretreated with dexamethasone (CYP3A23 inducer), the AUC was significantly smaller (41.5 compared with 52.5 microg min/mL), Cl was significantly faster (72.2 compared with 57.1 mL/min/kg), and the amounts and/or tissue-to-plasma ratios of parathion was significantly (or tended to be) smaller than those in control rats. However, the pharmacokinetic parameters of parathion were not significantly different after pretreatment with other enzyme inducers compared with respective control rats. The above data suggested that parathion was metabolized to paraoxon by dexamethasone-inducible CYP3A23, the induction of which was confirmed by Western blot analysis. This was supported by in vitro intrinsic clearance (Cl(int)) of parathion to form paraoxon in hepatic microsomal fraction; the Cl(int) in rats pretreated with dexamethasone was significantly faster (0.0900 compared with 0.0290 mL/min/mg protein) than that in control rats.

Effects of Chlorinated Ethylenes on Expression of Rat CYP Forms. Comparative Study on Correlation between Biological Activities and Chemical Structures.

Chlorinated ethylenes (CEs) such as tetrachloroethylene (PCE), trichloroethylene (TCE), 1, 1-dichloroethylene (1, 1-DCE), cis-1, 2-dichloroethylene (cis-DCE), and trans-1, 2-dichloroethylene (trans-DCE) are members of the class of volatile halogenated hydrocarbons. Each was administered intraperitoneally at 0.5g/kg alone or simultaneously with phenobarbital (PB, 80mg/kg/d) to male Wistar rats weighing about 200g (7-weeks-old). Microsomal fractions of livers and lungs removed from animals sacrificed 24h after treatment were tested for monooxygenase activities and protein content of 4 cytochrome P450 (CYP) forms, i.e., CYP1A1/2, 2B1/2, 2E1 and 3A1/2. In terms of constitutive expression, they were compensatory in liver and lung with CYP1A1/2, 2E1 and 3A1/2 being detected only in liver and CYP2B1 only in lung. All 5 CEs employed in this work suppressed hepatic CYP1A1/2, 2E1 and 3A1/2 in the descending order of 1, 1-DCE > cis-DCE > trans-DCE > TCE > PCE. The magnitude of suppression of pulmonary CYP2B1 by CEs were compared as follows; PCE > trans-DCE > 1, 1-DCE > cis-DCE > TCE. CYP1A1/2, CYP2E1 and CYP3A1/2 in hepatic microsomes from PB-treated animals responded to CEs similarly to those from PB-untreated animals. These 3 enzyme activities could not be detected in pulmonary microsomal fractions, irrespective of the PB-treatment. Hepatic CYP2B1/2 could be detected only when treated with PB in marked contrast to the constitutive expression of pulmonary CYP2B1. The suppression of PB-induced hepatic CYP2B1/2 was observed with all 5 CEs, with special emphasis on complete inhibition with 1, 1-DCE. The adverse effects of TCE and PCE on pulmonary CYP2B1 from PB-treated rats were comparable with those from PB-untreated animals. In contrast, 3 dichloro-isomers were more suppressive to the enzyme in the absence than in the presence of PB. The protein levels of hepatic CYP forms were generally proportional to the enzyme activities in the case of 1, 1-DCE-treatment. The amounts of pulmonary CYP2B1 apoprotein were in good accordance with the enzyme activities when treated with CEs individually.

Enzyme-Specific Transport of Rat Liver Cytochrome P450 to the Golgi Apparatus

It has been reported that cytochrome P450 is expressed in the plasma membrane of hepatocytes isolated from human and rat. Cytochrome P450s expressed on the cell surface are potential targets for the immune response of drug-induced and autoimmune hepatitis. However, the mechanisms behind transport of cytochrome P450 to the plasma membrane are obscure. The present investigation aimed at identifying cytochrome P450 expressed in the Golgi apparatus. Golgi membrane fractions from rat liver were prepared and characterized: one enriched withcis-Golgi, one highly enriched withtrans-Golgi, and one intermediate Golgi fraction representingmedial-Golgi. In these three fractions, significant amounts of cytochrome P450 and NADPH cytochrome P450 reductase were present, which could not be accounted for by contamination with endoplasmic reticulum. A marked difference between the relative content of different cytochrome P450 enzymes was found. CYP4A1 was found at the highest concentration, CYP2E1 at an intermediary level, and CYP1A2 at low levels, whereas no Golgi-specific CYP3A1 was detectable. It was also shown that the CYP2E1 present in the Golgi fractions was catalytically active. It is suggested that various forms of hepatic cytochrome P450 are transported to the plasma membrane through the Golgi apparatus in an enzyme-specific manner.

Cytochrome P450 and associated monooxygenase activities in the rat and human spinal cord: induction, immunological characterization and immunocytochemical localization.

We have discovered cytochrome P450 and associated monooxygenase activities in microsomes prepared from spinal cord tissues from rats and a human. Cytochrome P450 levels and nicotinamide adenine dinucleotide phosphate cytochrome c reductase activities in microsomes from rat spinal cord were similar to those observed from the whole brain. However, certain monooxygenase activities were significantly lower in the rat spinal cord microsomes as compared to the corresponding activities observed in the whole brain. Cytochrome P450-mediated monooxygenase activities were also detectable in microsomes prepared from human spinal cord. Immunoblot analyses of rat and human spinal cord microsomes using antisera to various forms of hepatic cytochrome P450 namely (2B1 + 2B2), 1A1, 1A2 and 2E1 revealed the presence of immunologically similar forms. The spinal cord microsomes also cross-reacted with the antiserum to the phenobarbital-inducible form of rat brain cytochrome P450. Immunocytochemical stain was predominant in the gray horns of the rat spinal cord. At the cervical level, lamina 1 and 2 representing the substantia gelatinosa were intensely stained. In the ventral horns, lamina 7, 8 and 9 containing the large motor neurons were strongly labelled, while small neurons revealed variable staining. In the white matter, the glial cells were stained but the axons remained non-reactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Gender differences in drug metabolism regulated by growth hormone

Gender differences in drug metabolism in rats have been known for more than 60 years when it was first reported that the much shorter duration of drug action in the male was due to the effects of testicular androgens. More recent studies have demonstrated that this sexual dimorphism in rat drug metabolism results from the differential expression of a possible dozen, or so sex-dependent hepatic forms of cytochrome P450. Moreover, it is the sexually dimorphic plasma profiles of growth hormone, and not androgens, that directly regulate the expression of these individual forms of hepatic cytochrome P450. Male rats secrete growth hormone in an “on-off” episodic rhythm in which interpulse periods contain no detectable levels of the hormone. Growth hormone secretion in the female rat is also pulsatile, but can be characterized as “continuous” since hormone levels are always present in the circulation. It would appear that the duration of the interpulse period is at least one “signal” in the growth hormone profile regulating hepatic expression of the sex-dependent forms of cytochrome P450, and thus establishing the gender differences in drug metabolism. The exaggerated gender differences in rat drug metabolism (i.e. 300–500%) have made it the standard, and understandably an ideal model in which to investigate the mechanisms regulating these dimorphisms. However, it is also possible that these studies have limited value when extrapolated to other species, such as humans, in which the magnitude of the sexual differences are much smaller, and the dimorphism may be reversed (F > M). In this regard, the mouse model, in which the sexual differences (F > M) in drug metabolizing enzyme activities vary by only 40–100%, are also regulated by sex-dependent plasma growth hormone profiles, and may be more representative of the vast majority of outbred species in which only subtle gender differences occur in drug metabolism.

Cytochrome P450 mediated metabolism of diazepam in human and rat: involvement of human CYP2C in N-demethylation in the substrate concentration-dependent manner

Metabolism of diazepam (DZP) was studied in vitro to clarify the involvement of different forms of hepatic cytochrome P450 (CYP) in rats, and humans of Japanese and Caucasian origin. Microsomal 3-hydroxylation was the major pathway of DZP metabolism in rats and was inhibited by anti-CYP3A antibodies. Purified CYP3As and CYP2C11 catalysed 3-hydroxylation and N-demethylation, respectively, in the reconstituted systems. The rates of both reactions in human liver microsomes depended on the substrate concentration: the rate of 3-hydroxylation was 3-4 times higher than N-demethylation at 0.2 mM; the two activities were essentially the same at a lower substrate concentration (0.02 mM). Inhibitions of the N-demethylation by anti-CYP2C antibody and S-mephenytoin also depended on the substrate concentration and was detectable only at a low substrate concentration. Kinetic studies revealed the presence of two distinct catalytic activities for the N-demethylation; low Km and low Vmax, and high Km and high Vmax. The former activity seems to be mediated by a CYP2C P450 form. On the other hand, DZP 3-hydroxylation was rather selectively catalysed by a CYP3A P450 at the low and high substrate concentrations. These results were consistent with the observation in vivo that DZP N-demethylation and S-mephenytoin 4'-hydroxylation are closely correlated in humans. These results also suggest that the apparent discrepancy on the role of CYP forms in DZP metabolism in vitro and in vivo may reside in the difference in substrate concentration.

Effects of tributyltin in vivo on hepatic cytochrome P450 forms in marine fish

The interaction of tributyltin chloride (TBT) in vivo with different forms of hepatic microsomal cytochrome P450 and ethoxyresorfin O-deethylase (EROD) activity was studied in scup ( Stenotomus chrysops). Fish were injected with single doses of 3.3, 8.1 and 16.3 mg/kg TBT. Hepatic microsomes were analyzed 24 h later for total cytochrome P450 content, and three P450 forms by immunoblotting: scup P450E, the major polyaromatic hydrocarbon-inducible form (CYPlAl), scup P450A, the major contributor to microsomal testosterone 6β-hydroxylase activity, and scup P450B, which oxidizes testosterone at several different sites including the 15α position. Spectrally determined conversion of P450 to its degraded form cytochrome P420 occurred at all TBT doses, the conversion being considerable only at the highest dose with more P420 (65%) than P450 (35%). EROD activity tended to be decreased by TBT in all doses. with a significant reduction at 16.3 mg/kg. Microsomal protein degradation as revealed by SDS-polyacrylamide gel electrophoresis occurred at the highest dose. Immunoblot analysis with a monoclonal antibody to P450E (CYPlAl) showed a decrease of CYPlA protein content at all TBT doses, with a significant loss at 16.3 mg/kg, similar to the EROD activity pattern. Immunoblot analysis with polyclonal antibodies to P450A and P450B showed decreases in P450A and P450B protein content only at the highest dose, the decrease being significant for P450A only. Cytochrome b 5 content was unaffected. In the liver, metabolites of TBT, dibutyltin and monobutyltin, were identified at all doses, with a decreased content, however, in fish given the highest dose. This study indicates important biochemical effects of TBT in fish liver, and suggests that exposure to TBT may alter both cytochrome P450 dependent metabolism, and induction response to other environmental pollutants.

The suppression of hepatic cytochrome P4504A mRNA mediated by the interferon inducer polyinosinic acid- poly cytidylic acid

Interferon and interferon inducers are well known to depress the cytochrome P450-dependent hepatic mixed-function oxidase system and cause a decrease in the capacity of the liver to metabolize drugs and xenobiotics. In this study we have shown that the interferon-mediated changes in an induced form of hepatic cytochrome P450 (CYP4A) are mediated via a depression in the levels of mRNA as assessed by Northern blot and slot blot analyses using a 20-base synthetic oligodeoxyribonucleotide hybridization probe. Rats were pretreated with clofibrate to maximize CYP4A mRNA levels prior to the administration of polyinosinic acid-polycytidylic acid (poly 1C), an α/β interferon inducer. Hepatic CYP4A mRNA levels were decreased by 49 and 30% at 6 and 24 hr, respectively, following poly IC administration. In hepatic microsomes cytochome P450 and functional CYP4A as measured by lauric acid hydroxylation, were not affected at 6hr, but were depressed by 39 and 27%, respectively, 24 hr following poly IC administration. These results suggest that interferon depresses induced levels of hepatic drug metabolism by lowering the level of cytochrome P450 mRNAs and subsequent synthesis of cytochrome P450 apoproteins.

Induction of DNA damage by N-nitrosodiethylamine in rat hepatoma cells: correlation with cytochrome P450-mediated aldrin epoxidase activity

The genotoxic potency of the hepatocarcinogen N-nitrosodiethylamine (NDEA) was tested in various Reuber hepatoma cell lines, which differ in their expression of differentiated liver specific functions including hepatic cytochrome P450 forms, and in rat hepatocytes, with the aim of characterizing the enzymes involved in activation. DNA single-strand breaks assessed by alkaline elution served as an indicator of genetic damage. Aldrin epoxidase activity was used as a marker for various hepatic cytochrome P450 forms. The poorly differentiated cell lines RH35 and H4IIEC3/T were apparently not affected by NDEA; moderate effects were observed in the well-differentiated lines H4IIEC3/G- and 2sFou, and major effects in two other well-differentiated lines, Fao and C2Rev7, and in hepatocytes. The degree of DNA damage in the cell lines correlated positively with the expression of aldrin epoxidase. Furthermore, DNA damage induced by N-nitrosodimethylamine (NDMA) was determined in C2Rev7 cells and in rat hepatocytes in order to assess a possible involvement of the NDMA-metabolizing cytochrome P45oIIE1 in the activation of NDEA by comparing the genotoxic potencies of the two compounds. NDMA was distinctly less effective than NDEA in C2Rev7 cells at all concentrations tested. In hepatocytes, NDMA induced more DNA damage than NDEA at low concentrations, but was slightly less active at high concentrations. The results suggest that NDEA is preferentially metabolized to genotoxic products by one or several cytochrome P450 forms different from P450IIE1.

Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells.

We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.

Signalling Elements in the Ultradian Rhythm of Circulating Growth Hormone Regulating Expression of Sex-Dependent Forms of Hepatic Cytochrome P450*

Neonatal male rats were treated with monosodium glutamate (MSG) at either 2 or 4 mg/g BW on alternate days during the first 10 days of life. As adults, the 4 mg MSG-treated rats displayed the obesity, growth retardation, and reduced pituitary weights that typify this syndrome. These animals had no detectable plasma GH as determined from serial blood samples taken every 20 min for 8 consecutive h. Associated with this loss of circulating GH was an induction of the female-specific hepatic cytochrome P450 2d (gene IIC12) and the disappearance of the male-specific form of cytochrome P450 2c (gene IIC11). The catalytic activities of cytochrome P450 2c (i.e. androgen 16 alpha- and 2 alpha-hydroxylase), sex-dependent hexobarbital hydroxylase and total cytochrome P450 were similarly feminized. Rats exposed to the 2-mg dose of MSG were also obese, but their growth rates and pituitary sizes were not as severely affected as in the 4 mg MSG-treated rats. Circulating GH in these lower dosed males was secreted in a pulsatile pattern similar to that in normal males, except that the pulse amplitude was reduced as much as 90%. In spite of this profound decline in GH peak heights in the 2 mg MSG-treated males, liver metabolism was characteristically masculine. That is, female-specific cytochrome P450 2d remained undetectable, while the male forms of cytochrome P450 2c, 2a (gene IIIA2), and RLM2 (gene IIA2), their respective catalytic steroid hydroxylase activities, and associated sex-dependent drug-metabolizing enzymes were expressed at the level of or greater than that in normal males. Thus, while an ultradian pulse of circulating GH is necessary for the characteristically masculine profile of sex-specific forms of hepatic cytochrome P450, neither the amplitude of the secretory peaks nor their total GH content is critical, and these can be greatly reduced from the normal male levels.

Role of growth hormone in modulating the constitutive and phenobarbital-induced levels of two P-450(6)beta (testosterone 6 beta-hydroxylase) mRNAs in rat livers.

The role of growth hormone in the expression of two forms of hepatic cytochrome P-450(P-450), P-450(6)beta-1(6 beta-3), and P-450(6)beta-4, was investigated using RNA blots. The level of P-450(6)beta-1(6 beta-3) mRNA was twenty times higher than that of P-450(6) beta-4 mRNAs in untreated male rat livers. The levels of P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs were increased two fold and three fold, respectively, by hypophysectomy of adult male rats. By intermittent injection of human growth hormone (hGH) into hypophysectomized male rats, both mRNAs were decreased to the level of normal rats, and almost disappeared after continuous infusion of hGH. In female rats, these two mRNAs were not detected, but were increased remarkably by hypophysectomy. The increases in these mRNAs were almost abolished after continuous infusion of hGH in hypophysectomized female rats. The effect of hGH on PB-mediated induction of P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs was also examined. The PB-mediated increases in P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs were higher in hypophysectomized male rats (2.5-fold and 10.9-fold, respectively) than in normal male rats (1.5-fold and 5.2-fold, respectively). Thus, the levels of P-450(6)beta-1(6-beta-3) and P-450(6)beta-4 mRNAs were 4.1-fold and 7.3-fold, respectively, higher in PB-induced hypophysectomized rats than in normal male rats. Concerning the postnatal developmental profiles, P-450(6)beta-1(6 beta-3) mRNA was detectable at neonate and reached a maximal level at around 17 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of liver regeneration on hepatic cytochrome P450 isozymes and serum sex steroids in the male rat

Partial hepatectomy in male adult rats results in raised serum estrogen levels and demasculinization of certain aspects of hepatic metabolism. Some constitutive forms of hepatic cytochrome P450 are sex-dependent and we have previously demonstrated demasculinization of cytochrome P450 isozyme distribution in a rat model of cirrhosis. As liver regeneration is an integral component of cirrhosis, the present study was performed to ascertain the effects of regeneration on hepatic cytochrome P450 isozyme composition and serum sex steroid concentrations. Adult male rats were subjected to 65% partial hepatectomy or sham-operation. The position-specific hydroxylation of androstenedione was used as a probe for isozyme activity. Serum sex steroids, hepatic enzymes, and hepatic deoxyribonucleic acid synthesis were measured in groups of animals at 0, 6, 24, 48, and 72 h. By 72 h total microsomal cytochrome P450 in partially hepatectomized animals had fallen to 66% of that in non-operated animals. In both partially hepatectomized and sham-operated animals, androstenedione 7α-and 16β-hydroxylase activity returned to preoperative levels by 48 h. However, the male-specific androstenedione 16α- and 6β-hydroxylase activities and aromatase activity remained suppressed in partially hepatectomized liver. Serum estradiol increased eightfold in partially hepatectomized rats and peaked at 6 h followed by a gradual fall to control values. No change in serum estradiol was observed in sham-operated animals. We conclude that demasculinization of hepatic oxidative metabolism occurs in regenerating rat liver. The early rise in serum estradiol is consistent with a role for this hormone in the changes in cytochrome P450 observed, and possibly the process of liver regeneration.

Induction of Cytochrome P450b and Its Relationship to Liver Tumor Promotion

A wide variety of compounds was examined for the ability to induce a specific form of hepatic cytochrome P450 and to promote the development of DEN-initiated liver tumors (adenomas and carcinomas) in rats over a 72 week period. The induction of cytochrome P450b was determined indirectly by measuring the hepatic induction of pentoxy-or benzyloxyresorufin O-dealkylase activities, which are highly specific substrates for the major phenobarbital-inducible forms of cytochrome P450 in the rat.(10) Results in the rat showed: (1) potent inducers (&gt; 40 ×) of P450b (i.e., phenobarbital, barbital, ethylphenyl-hydantoin, and DDT) are all potent liver tumor promoters; (2) structural analogs that are not inducers of P450b (i.e., hexobarbital, monoethylbarbituric acid, monophenyl-barbituric acid, and diethylhydantoin) all fail to display significant liver tumor promoting activity; and (3) the concomitant induction of liver hypertrophy, microsomal epoxide hydrolase, and cytochrome P450b appears to be proportional and argues for some coordinated “pleiotropic” response of liver parenchyma to these inducers. Additional studies showed that phenobarbital induced cytochrome P450b and was a liver tumor promoter not only in rats, but also in mice and patas monkeys, but was inactive as an enzyme inducer and was a nonpromoter in the hamster.

Effect of Liver Regeneration on Hepatic Cytochrome P450 Isozymes and Serum Sex Steroids in the Male Rat

Partial hepatectomy in male adult rats results in raised serum estrogen levels and demasculinization of certain aspects of hepatic metabolism. Some constitutive forms of hepatic cytochrome P450 are sex-dependent and we have previously demonstrated demasculinization of cytochrome P450 isozyme distribution in a rat model of cirrhosis. As liver regeneration is an integral component of cirrhosis, the present study was performed to ascertain the effects of regeneration on hepatic cytochrome P450 isozyme composition and serum sex steroid concentrations. Adult male rats were subjected to 65% partial hepatectomy or sham-operation. The position-specific hydroxylation of androstenedione was used as a probe for isozyme activity. Serum sex steroids, hepatic enzymes, and hepatic deoxyribonucleic acid synthesis were measured in groups of animals at 0, 6, 24, 48, and 72 h. By 72 h total microsomal cytochrome P450 in partially hepatectomized animals had fallen to 66% of that in nonoperated animals. In both partially hepatectomized and sham-operated animals, androstenedione 7 alpha- and 16 beta-hydroxylase activity returned to preoperative levels by 48 h. However, the male-specific androstenedione 16 alpha- and 6 beta-hydroxylase activities and aromatase activity remained suppressed in partially hepatectomized liver. Serum estradiol increased eightfold in partially hepatectomized rats and peaked at 6 h followed by a gradual fall to control values. No change in serum estradiol was observed in sham-operated animals. We conclude that demasculinization of hepatic oxidative metabolism occurs in regenerating rat liver. The early rise in serum estradiol is consistent with a role for this hormone in the changes in cytochrome P450 observed, and possibly the process of liver regeneration.

Antibodies to liver/kidney microsome1 in chronic active hepatitis recognize specific forms of hepatic cytochrome P-450

Anti-liver/kidney microsome1-positive sera from children with chronic active hepatitis were studied in an effort to identify the microsomal antigens selected during induction and progression of this autoimmune disease. Immunoblot analysis of sodium dodecyl sulfate gel-resolved microsomal proteins from human and rat liver using anti-liver/ kidney microsome1-positive sera revealed a single polypeptide of 48 kilodaltons (human microsomes) or 50 kilodaltons (rat microsomes). Levels of the 50-kilodalton rat microsomal polypeptide were suppressed in vivo by several drugs known to modulate expression of individual forms (enzymes) of hepatic cytochrome P-450, with the largest decrease effected by phenobarbital. Dot blot analysis using a panel of 10 electrophoretically homogeneous rat liver cytochrome P-450 forms under nondenaturing conditions established that the two methylcholanthrene-inducible forms, P-450 BNF-B and P-450 ISF-G (P-450 gene subfamily IA), are selectively recognized by the anti-liver/kidney microsome1 antibodies. These findings demonstrate that sera associated with autoimmune (anti-liver/kidney microsome1) chronic active hepatitis are specifically reactive with select rat hepatic P-450 forms and suggest that these autoantibodies may be principally directed against one or more constitutive forms of the corresponding human liver cytochromes.