Regulation of heme metabolism and cytochrome P-450 levels in primary culture of rat hepatocytes in a defined medium

Liver cells were prepared from adult Sprague-Dawley rats and used for the determination of delta-aminolevulinic acid synthetase (ALAS) activity and cytochrome P-450 concentrations at different time intervals in tissue culture in a serum-free synthetic medium. During the first 24 hr in culture, the level of cytochrome P-450 decreased to 30-40% of the level in isolated liver cells from untreated animals. The disappearance of cytochrome P-450 was especially fast in hepatocytes obtained from female phenobarbital-treated rats where only 40% of the original cytochrome P-450 was present after 2 hr in culture and 80% had disappeared in 2 days. The activity of ALAS increased 3- to 4-fold when measured 2 hr after plating, and it reached the maximum level in 19-24 hr when its activity was about eight times the original activity. In 2-4 days in culture, the activity of ALAS was four to five times above the original level. When the amount of delta-aminolevulinic acid (ALA) in the medium was increased from 1 to 100 microM, a decrease in ALAS was obtained, but no significant increase in cytochrome P-450 level was observed. Addition of heme to the medium gave a dose-dependent decrease in the activity of ALAS. Our data indicate that during the first 24 hr in culture the increase of ALAS activity was prevented by exogenous heme. This effect may be due to inhibition of the catalytic activity, suppression of the synthesis of the enzyme, or accelerated breakdown of the enzyme by heme.     

Induction of cytochrome P-450c and P-450d by metyrapone in the primary culture of rat hepatocytes

The effects of metyrapone on qualitative changes in cytochrome P-450-dependent drug metabolizing activities in primary cultures of rat hepatocytes were investigated. Metyrapone apparently increased benzo(a)pyrene hydroxylation and maintained both ethoxycoumarin-O-deethylation and propoxycoumarin-O-depropylation, whereas it had little effect on methoxycoumarin-O-demethylation. Furthermore, P-450d (high spin type of P-448) as well as P-450c (low spin type of P-448) were induced by metyrapone, while P-450b and P-450e were not. In conclusion, metyrapone act as a 3-methylcholanthrene-like inducer in the primary cultures of rat hepatocytes.     

Effects of cytochrome P-450 inducers on the perazine metabolism in a primary culture of human hepatocytes

The metabolism of perazine in a primary culture of human hepatocytes after treatment of cells with TCDD (a CYP1A1/2 inducer) or rifampicin (mainly a CYP3A4 inducer) were studied in vitro. The concentrations of perazine and its main metabolites (perazine 5-sulfoxide, N-desmethylperazine) formed in hepatocytes were assayed in the extracellular medium using the HPLC method. TCDD and rifampicin induced the formation of perazine 5-sulfoxide, however, such an effect was not observed in the case of N-desmethylperazine. The accumulation of perazine 5-sulfoxide in the extracellular medium was enhanced until up to 4 h by rifampicin, and until up to 8 h byTCDD. After 24 h, perazine and perazine 5-sulfoxide were not detected in the extracellular medium of the inducer-treated cultures, except for perazine 5-sulfoxide in the TCDD-treated cultures The obtained results indicate that CYP1A2 and CYP3A4 are involved in the perazine metabolism via 5-sulfoxidation pathway.     

Dependence of the induction of cytochrome P-450 by phenobarbital in primary cultures of adult rat hepatocytes on the composition of the culture medium

A chemically defined medium developed for the maintenance of differentiated adult rat hepatocytes (T1) was compared with two commercially available media (Waymouth 752/1 and Leibovitz L-15) for maintenance of cytochrome P-450 metabolic activity in cultured hepatocytes. Specific metabolic activities of initially isolated cells and 72-hr control and phenobarbital-treated cultures were determined with 7-ethoxycoumarin, 7-ethoxyresorufin, and 7-pentoxyresorufin as substrates. Control and phenobarbital-treated cultures in T1 medium had a higher metabolic activity towards each of the three substrates than comparable cultures in the other media. These studies indicated that the metabolic activity and the response to phenobarbital of the major isozyme of the phenobarbital-inducible family of cytochrome P-450 were maintained in hepatocytes in T1 medium. However, there was anomalous expression and induction by phenobarbital of the major 3-methylcholanthrene-inducible isozyme, cytochrome P-450c, in cultured hepatocytes in each of the three media tested, but this response was more pronounced in T1 medium. In conclusion, the regulation of cytochrome P-450 metabolic activity in cultured hepatocytes was shown to be dependent on the composition of the culture medium.     

Presence of hexobarbital in primary cultures of rat hepatocytes maintains cytochrome P-450 levels and drug metabolizing enzyme activities

Addition of hexobarbital (1 mM) to the culture medium of rat hepatocytes protected against the rapid decline in the level of cytochrome P-450 and the activities of various drug metabolizing enzymes. While the hepatocytes cultured for 72 hr without hexobarbital had only 30% of their original level of cytochrome P-450, the cells maintained with hexobarbital had 75% of the initial level of the hemoprotein. After 72 hr in culture, the activities of aminopyrine N-demethylase and biphenyl 4-hydroxylase were 22-24% of the original rate for the nontreated cells and 73-78% for the hexobarbital treated cells. The activities of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase in the cultures of treated cells were even higher than those of the freshly isolated hepatocytes. Additions of other substrates of hepatic mixed function oxidase to the culture medium did not protect against the loss of cytochrome P-450 and enzyme activities.     

The content and activity of cytochrome P-450 in long-term culture of hepatocytes from male and female rats

The content of cytochrome P-450 and the capacity for O-demethylation have been measured in cultures of hepatocytes from male and female rats for a period of 21 days. The effect of dexamethasone, insulin, glucagon, phenobarbital and hemin was investigated. In hepatocytes from female rats the content of cytochrome P-450 was unchanged after one day of culture. From day 1 to day 3 the content of cytochrome P-450 decreased by 65% and only the combined addition of dexamethasone, phenobarbital and hemin diminished the fall. After the initial fall, addition of 0.1 microM dexamethasone resulted in a stable value. Addition of 1 microM dexamethasone or 1 mM phenobarbital gave rise to an induction of cytochrome P-450 (285%). The high level of cytochrome P-450 was maintained for 3 weeks. In hepatocytes from male rats the content of cytochrome P-450 decreased by 40% after one day of culture. From day 1 to day 3 the content decreased by 45% and the decrease continued irrespective of the presence of hormones and/or phenobarbital. The O-demethylase activity in cultures of hepatocytes from female rats correlated to the cytochrome P-450 content independent of medium composition and age of the cultures, whereas no correlation was found in cultures from male rats. The present study demonstrates that hepatocytes from female rats in cultures retain O-demethylase activity for at least 3 weeks and that, with the experimental conditions used, the response to the hormones and inducers is different for hepatocytes from male and female rats.     

Induction of cytochrome P-450 by dimethyl sulfoxide in primary cultures of adult rat hepatocytes.

Treatment of hepatocyte cultures with dimethyl sulfoxide (DMSO) induced P-450IIE1-specific aniline 4-hydroxylase activity and P-450IA1-specific ethoxyresorufin O-deethylase activity at a concentration of 0.1% (v/v). The P-450IIB-specific pentoxyresorufin O-deethylase activity was induced only at the 2% (v/v) level. Dot blot analysis of the total cellular RNA and cycloheximide treatment of the culture suggested that induction of ethoxyresorufin O-deethylase activity by DMSO may be due to the increase of de novo synthesis of the P-450IA1 protein, not to accumulation of mRNA in the hepatocyte culture.     

The effect of galactosamine on rat liver cytochrome P-450 activities

The effect of galactosamine on hepatic drug metabolizing activities was examined in rats. In the microsomal fraction, the contents of cytochrome P-450 (P-450) and cytochrome b5 (b5) and the activity of NADPH-cytochrome c reductase (reductase) were examined for 7 days after galactosamine administration. In addition, substrate metabolizing activities in damaged microsomes were examined using four substrates: aminopyrine, aniline, benzo(a)pyrene (B(a)P) and 7-ethoxycoumarine (7-EC). The contents of P-450 and b5 and the activity of reductase showed a minimal value after 3 days of galactosamine administration and then gradually increased, reaching to the control level after 7 days. All four substrate metabolizing activities showed a similar response as the content of P-450, but the decrement among the four activities was not uniform. The activities of B(a)P hydroxylation and 7-EC deethylation were more impared than those of aminopyrine demethylation and aniline hydroxylation. This nonuniformity was clear on the activity based on P-450. This result suggested that galactosamine disturbed the population of multiple P-450 subtypes, and each P-450 subtype was damaged to the various extent by galactosamine administration.     

Carbon tetrachloride and trichloroethylene toxicities to rat hepatocytes in primary monolayer culture: its relationship to the level of cytochrome P-450

The effects of carbon tetrachloride (CCl4) and trichloroethylene (TCE) on the synthesis and the secretion of triacylglycerols (TGs) in primary cultured rat hepatocytes were investigated in relation to the level of cellular cytochrome P-450 (P-450). Pretreatment of rats with phenobarbital (PB) and plating the hepatocytes in the presence of metyrapone attained a marked preservation of P-450 during the preparation of monolayer. CCl4 was able to cause the accumulation of cellular TG in the hepatocytes when the content of P-450 was retained at the level equivalent to that in the liver in vivo.     

The measurement of FAD-containing mono-oxygenase activity in microsomes containing cytochrome P-450

1. Antibodies to NADPH-cytochrome P-450 reductase have been used to essentially abolish the contribution of cytochrome P-450 to xenobiotic metabolism by mammalian microsomes. This permits the determination of the activity of the FAD-containing monooxygenase and the stoichiometry between substrate, O₂ and NADPH, in the microsomal membrane, and in the absence of cytochrome P-450-dependent activity.

2. FAD-containing mono-oxygenase oxidation rates were determined for sulphur- and nitrogen-containing substrates, including: thiols; sulphides; thioamides; primary, secondary and tertiary amines; hydrazines.

3. Although the enzyme in mouse, rabbit, rat and pig microsomes displays similar substrate specificity, some catalytic characteristics are different between species and tissues.     

Influence of extracellular matrix overlay and medium formulation on the induction of cytochrome P-450 2B enzymes in primary cultures of rat hepatocytes.

The effect of medium formulation, composition of extracellular matrix overlay, and culture dish material on liver microsomal cytochrome P-450 (CYP) 2B induction by phenobarbital (PB) was investigated in primary cultures of rat hepatocytes. When hepatocytes were maintained on Permanox dishes with an overlay of either collagen (type I) or Matrigel, Williams' E medium was superior to other medium formulations in terms of the magnitude of induction of CYP2B on a per milligram microsomal protein basis. Modified Chee's medium (MCM) and hepatocyte culture medium were intermediate in their capacity to sustain induction of CYP2B by PB, and Dulbecco's modified Eagle's medium was slightly less effective. The overall induction of CYP2B activity by PB was, on average, 50% lower in hepatocytes cultured on polystyrene dishes (LUX). Little or no difference was observed between hepatocytes overlaid with collagen and those overlaid with Matrigel. MCM was superior to Williams' E medium in terms of the yield of microsomal protein and the ultrastructural features of the hepatocyte monolayers. CYP2B induction by PB was optimal after 3 days of treatment in either medium. CYP1A, CYP3A, and CYP4A activities could be induced in vitro by prototypical inducing agents in hepatocytes cultured on Permanox dishes with MCM and a Matrigel overlay to comparable levels observed in vivo. The results of these studies show that medium formulation and culture vessel material, but not the type of extracellular matrix overlay, have significant effects on the induction of CYP enzymes in cultured rat hepatocytes maintained in a sandwich configuration.     

Effect of cryopreservation on cytochrome P-450 enzyme induction in cultured rat hepatocytes.

In the present study, we evaluated the inducibility of cytochrome P-450 (CYP) CYP1A, CYP2B, CYP3A, and CYP4A by beta-naphthoflavone, phenobarbital, dexamethasone, and clofibric acid, respectively, in primary hepatocyte cultures prepared from both fresh and cryopreserved rat hepatocytes. Rat hepatocytes were successfully thawed and cultured after cryopreservation in liquid nitrogen for up to 1 month. Percentage of total recovery, viable cell recovery, and final viability of the cells were 68%, 72%, and 85%, respectively. Regardless of whether they were cryopreserved or not, cultured hepatocytes exhibited near-normal morphology. Treatment of cryopreserved hepatocytes with beta-naphthoflavone caused an 8-fold increase in 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, with an EC50 of 1.5 microM; treatment with phenobarbital caused a 26-fold increase in 7-pentoxyresorufin O-dealkylase (CYP2B1/2) activity, with an EC50 of 10 microM; treatment with dexamethasone caused a 10-fold increase in testosterone 6beta-hydroxylase (CYP3A1/2) activity, with an EC50 of 1.3 microM, whereas treatment with clofibric acid caused a 3-fold increase in lauric acid 12-hydroxylase (CYP4A1-3) activity, with an EC50 of 170 microM. The induction of CYP1A, CYP2B, CYP3A, and CYP4A enzymes by these inducers was confirmed by Western immunoblotting. The patterns of P-450 induction in cryopreserved rat hepatocytes, in terms of concentration response, reproducibility, magnitude, and specificity of response, were similar to those observed in freshly isolated hepatocytes. Additionally, the magnitude and specificity of induction was similar to that observed in vivo in rats. In conclusion, under the conditions examined, cryopreserved rat hepatocytes appear to be a suitable in vitro system for evaluating xenobiotics as inducers of P-450 enzymes.     

Changes in the concentration of seven forms of cytochrome P-450 in primary cultures of adult rat hepatocytes

We prepared primary monolayer cultures of adult rat hepatocytes and measured the losses of cytochromes P-450 with the use of specific antibodies directed against purified forms of hepatic cytochrome P-450 which predominate in untreated rats (P-450UT-A, P-450UT-F) or in rats treated with phenobarbital (P-450PB-B/D, P-450PB-C, P-450PB/PCN-E) or with 3-methylcholanthrene (P-450 beta NF-B, P-450 beta NF/ISF-G). In hepatocytes prepared from an untreated rat and incubated in control medium, total cytochrome P-450, measured spectrally as CO-binding hemoprotein, declined 68% during the first 72 hr in culture. However, the sum of the immunoreactive cytochromes P-450 declined only 24%, indicating that loss of heme rather than of protein accounts for much of the well-known loss of cytochromes P-450 in hepatocyte cultures. In cultures prepared from untreated rats or from rats treated with phenobarbital or with 3-methylcholanthrene, individual forms of cytochrome P-450 declined at markedly differing rates. Incubation of cultures in three different media previously reported to maintain levels of total cytochrome P-450 failed to prevent the decline in total cytochrome P-450 during the first 24 to 72 hr in culture. However, in cultures incubated in medium containing metyrapone, the level of holocytochrome P-450 was maintained at the initial value during the first 72 hr, apparently by preventing the net loss of cytochrome P-450 heme and by increasing the concentrations of immunoreactive P-450PB/PCN-E and P-450 beta NF-B. Medium containing nicotinamide increased the proportion of P-450 beta NF-B relative to the other forms of cytochrome P-450, whereas cysteine-free medium increased P-450UT-F. We conclude that loss of cytochrome P-450 in cultured hepatocytes involves loss of its heme moiety coupled with changes in the concentrations of the individual forms. Recognition of these changes as influenced by specific components of the culture medium is important when using primary hepatocyte cultures for study of xenobiotic metabolism and toxicity in the liver.     

Effect of diabetes on rat liver cytochrome P-450: Evidence for a unique diabetes-dependent rat liver cytochrome P-450

Detergent-solubilized hepatic microsomal fractions from alloxan diabetic rats exhibited a 52,000 molecular weight hemeprotein band that was not present in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles of identically solubilized hepatic microsomal fractions from normal, 3-methylcholanthrene- or phenobarbital-treated rats. This 52,000 mol. wt hemeprotein band disappeared from the protein profile of insulin-treated diabetic rat liver to yield the SDS-PAGE profile of normal rat liver. When P-450 hemeproteins were purified by lauric acid affinity and hydroxylapatite chromatography from solubilized microsomes, only the diabetic rat had a 52,000 mol. wt P-450. This distinct 52,000 mol. wt diabetes-induced P-450 interacted with type II compounds to yield a 2-fold greater absorbance change than was observed with the purified P-450s from either the normal or the chemically induced rats. The properties of this unique 52,000 mol. wt P-450 suggest that it may be the catalytic component responsible for the increased rate of type II substrate (aniline) metabolism observed in the diabetic rat.     

Differential effects of human recombinant interleukin-1 beta on cytochrome P-450-dependent activities in cultured fetal rat hepatocytes.

The immunomodulator interleukin-1 beta (IL-1) is one of the major inflammatory mediators. In vivo, it has been reported to depress some rat liver cytochromes P-450 (cytochrome P-450). Our aim was to study those effects in vitro, using cultured fetal rat hepatocytes as a model. Testosterone 6 beta-hydroxylase (cytochrome P-450 IIIA family activity) was not depressed by IL-1 treatments, but its induction by dexamethasone was prevented. The effect was time- and dose-dependent. Ethoxyresorufine-O-deethylase (cytochrome P-450 IA1 activity) decreased after IL-1 treatment, and dexamethasone partially prevented this inhibition. Acute phase effects of IL-1 were assayed by albumin and transferrin secretions. The cell's sensitivity to glucocorticoids was determined by tyrosine-aminotransferase activity. Our data demonstrate that IL-1 was able to prevent the glucocorticoid induction of cytochrome P-450 IIIA involving at least two different mechanisms. This is in agreement with the theory suggesting that the induction of CYPIIIA family by glucocorticoids requires the presence of the glucocorticoid receptor and some other regulatory elements. Other cytochrome P-450-dependent activities (IIA1, IIB1/2, and IIC11) were inhibited by IL-1 treatments, depending on dose and time, but some were also protected by dexamethasone.     

Influence of medium composition on 7-alkoxycoumarin O-dealkylase activities of rat hepatocytes in primary maintenance culture

1. The activities of 7-methoxycoumarin (7-MCOD), 7-ethoxycoumarin (7-ECOD) and 7-propoxycoumarin (7-PCOD) O-dealkylases decreased by 75-90% during culture of rat hepatocytes for 72 h. 2. The addition of dexamethasone (D) produced a stabilization or modest enhancement of 7-ECOD and 7-PCOD activities depending on the medium used; D was without effect on 7-MCOD activity. 3. The addition of nicotinamide (N) produced some stabilization of 7-ECOD and 7-PCOD activities but not of 7-MCOD activity. 4. The addition of D + N was associated with large increases in 7-ECOD and 7-PCOD activities, again with little effect on 7-MCOD activity. 5. Ethoxyresorufin O-dealkylase activity correlated with 7-ECOD/7-PCOD activities, whereas pentoxyresorufin O-dealkylase activity correlated with 7-MCOD activity. 6. It is concluded that D exerts a selective effect on certain forms of cytochrome P-450, but that more than one mechanism is probably implicated in this effect. 7. The magnitudes of induction of 7-ECOD activity by phenobarbitone and beta-naphthoflavone were blunted when cells were treated in culture when compared to treatment in vivo; the presence of D in the culture medium did not modify this phenomenon.     

Effects of polychlorinated terphenyls and paraffins on rat liver microsomal cytochrome P-450 and in vitro metabolic activities

The polychlorinated terphenyl Aroclor 5460 and the polychlorinated paraffins Witaclor 171 P and Witaclor 149 increased to different degrees the total microsomal concentration of cytochrome P-450 in the rat liver after intraperitoneal injection of 0.3, 1.0, and 1.0 g · kg^–1 body weight, respectively, each day for four days. The multiple forms of cytochrome P-450 were affected differently with an induction of RLvMc P-450₅₀ and RLvMc P-450₅₄ by all chemicals, and an additional induction of RLvMc P-450₅₅ by the polychlorinated terphenyl. The rat liver weights were extensively increased after treatment with the polychlorinated paraffins. Alterations were found in the in vitro metabolism of biphenyl, benzo(a)pyrene and the steroid hormones, 4-androstene-3,17-dione and 5-androstane-3,17-diol, after exposure to all chemicals. Changes in the in vitro formation of benzo(a)pyrene metabolites were found to correlate with changes in the multiple forms of cytochrome P-450. The present study demonstrate that only limited information can be obtained from alterations in the total concentration of cytochrome P-450 and show the importance of studying changes in the multiple forms and the metabolism of different substrates. Our results further indicate that exposure to any of the investigated polychlorinated chemicals may alter the biological effects of other environmental contaminants, drugs and endogenous substances which are metabolized by the cytochrome P-450 enzyme system.