Regulation of human liver cytochromes P-450 in family 3A in primary and continuous culture of human hepatocytes.

The cytochrome P-450 3A gene family comprises the dominant forms of cytochrome P-450 found in human liver. We examined as a possible useful system for studying the regulation of cytochrome P-450 3A under controlled conditions in vitro, primary monolayer cultures of human hepatocytes and compared the results with those obtained from the study of cytochrome P-450 3A in the human hepatoblastoma cell line HepG2 or in the human hepatocellular carcinoma cell line TONG/HCC. Using 3A antibodies, 3A cDNAs and 3A3, 3A4, 3A5 and 3A7 isozyme-specific oligonucleotides as probes, we determined that primary human hepatocyte cultures routinely expressed a 3A3/4* immunoreactive protein and 3A mRNA. These gene products were well maintained for many days and were induced by treatment of the cultures with dexamethasone, phenobarbital, macrolide antibiotics, the HMG CoA reductase inhibitor lovastatin or an antifungal agent, clotrimazole. Of six donor livers examined, only two contained mRNA or protein for 3A5, a form found in only a few adult human subjects. In cultures prepared from one of these two livers, 3A5 mRNA was detectable for several days. In cultures of hepatocytes from the remaining four human livers that did not contain 3A5 mRNA or protein, we detected neither spontaneous nor inducible 3A5 proteins or mRNAs. HepG2 cells contained only 3A7 protein, a form found in human fetal liver, even after treatment with inducers. treatment of HepG2 cells with dexamethasone, macrolide antibiotics, phenobarbital and phenobarbital-like inducers or lovastatin produced dose-dependent induction of 3A7 mRNA and 3A7 immunoreactive protein. TONG/HCC cells contained 3A3, 3A4 and 3A5 mRNAs, but only 3A5 immunoreactive protein could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional regulation of the human cholesterol 7 alpha-hydroxylase gene.

As an initial step toward understanding the transcriptional regulation of cholesterol 7 alpha-hydroxylase (CYP7) in man, we isolated and functionally characterized the 5'-flanking region of the human CYP7 gene. The nucleotide sequences of the first exon and 1.6 kb preceding the exon were determined and found to contain a TATA box at position -30, a modified CAAT box at position -92, three potential hepatocyte nuclear factor 3 (HNF-3) recognition sites at nucleotides -316, -288, and -255, respectively, and a modified sterol response element at position -271. DNA sequences containing 1.3 kb of the 5'-flanking region and 29 nucleotides of the first exon were linked to the chloramphenicol acetyltransferase gene and transiently transfected into several cell lines. Promoter activity was very strong in the human hepatoma cell line HepG2 but absent in cells of nonhepatic origin. Mutational analysis of the promoter identified several regions that function in the transcriptional regulation of CYP7. Introduction of a fragment containing the region from -432 to -220 upstream of a heterologous promoter, in either orientation, resulted in a tremendous stimulation of activity in HepG2 cells. DNase I footprint analysis identified three regions within this fragment which were protected from digestion. The overexpression of HNF-3 in HepG2 cells resulted in a 4-fold stimulation of CYP7 transcriptional activity. We suggest that the region between -432 and -220 functions as a cell-specific enhancer whose activity is controlled, in part, by HNF-3.

Regulation of rat hepatic cytochrome P450IIE1 in primary monolayer hepatocyte culture.

1. Rat hepatic cytochrome P450IIE1 is an ethanol-inducible enzyme which catalyses ethanol oxidation and activation of the procarcinogen, N-nitrosodimethylamine (NDMA) to its carcinogenic metabolite. 2. Initial studies in adult rat indicated that the regulation of cytochrome P450IIE1 is complex, therefore we strove to identify a central regulatory mechanism, using primary monolayer hepatocyte culture. These studies examined the effect of a range of agents (i.e. inducers, hormones, sodium butyrate and 5-aminolaevulinic acid) on amounts of cytochrome P450IIE1 protein and mRNA expression in rat hepatocytes maintained in serum-free medium on both Vitrogen and Matrigel, a laminin-rich basement membrane. 3. At time 0, immunoreactive cytochrome P450IIE1 protein was easily detectable in control cultures, yet decreased rapidly with time in culture to nearly undetectable levels at 120 h. Addition of inducers (notably, pyrazole) to the culture medium increased cytochrome P450IIE1 above that of untreated cultures at similar time points, yet did not elevate cytochrome P450IIE1 or NDMA demethylation above their levels at time 0. 4. Cytochrome P450IIE1 hybridizable mRNA also rapidly declined in culture. The decline in mRNA was not significantly altered in cultures exposed to pyrazole or any other agent. Thus, post-transcriptional factors appear to play an important role in the regulation of hepatic cytochrome P450IIE1, with protein stabilization being the most probable mechanism.

Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3).

The human P450III family has been shown to be composed of at least four members, P450IIIA3 (HLp), P450IIIA4 (P450NF), P450IIIA5 (HLp3), and P450IIIA6 (HLp2). Due to the lack of probes that specifically recognize the individual members of this family, little is known about their relative expression. We prepared a form-specific antibody to P450IIIA5 by immunoabsorption of anti-P450IIIA5 IgG against Sepharose 4B upon which microsomes that did not contain P450IIIA5 or purified P450IIIA3 had been bound. Immunoblot analyses demonstrated that P450IIIA5 was expressed at detectable levels in only 19 of 66 (29%) human livers. The expression of P450IIIA5 was not influenced by the gender or medical history of the patients. When the expression of P450IIIA5 in different age groups was examined, it was observed that P450IIIA5 was detected in a statistically significantly higher percentage of children and adolescents (19 years old and under), as compared with the remaining population (8 of 17, 47%, versus 11 of 46, 24%, respectively). Furthermore, P450IIIA5 was detected in 1 of 10 human fetal livers. Of the large number of compounds identified as substrates of P450III family members, P450IIIA5 was found to actively metabolize nifedipine, testosterone, estradiol, dehydroepiandrosterone 3-sulfate, and cortisol, whereas it metabolized poorly or did not metabolize erythromycin, quinidine, 17 alpha-ethynylestradiol, and aflatoxins. The acetylenic steroid gestodene was found to be an effective mechanism-based inhibitor of both P450IIIA4 and P450IIIA5. Immunoblots of microsomes isolated from untreated and dexamethasone-, phenobarbital-, or 3-methylcholanthrene-treated HepG2 cells that were developed with an antibody that recognizes all the P450III family members demonstrated that no proteins in the P450III family were expressed by the HepG2 cells. In conclusion, our studies indicate that P450IIIA5 is polymorphically expressed at all stages of human development and is more limited in its metabolic capabilities than is P450IIIA4.

Isolation and characterization of cloned cDNAs encoding human liver chlordecone reductase.

Chlordecone (Kepone), a toxic organochlorine pesticide, undergoes bioreduction to chlordecone alcohol in human liver. This reaction is controlled by a cytosolic enzyme, chlordecone reductase (CDR), which may be of the aldo-keto reductase family of xenobiotic metabolizing enzymes [Molowa et al. (1986) J. Biol. Chem. 261, 12624-12627]. To further investigate the primary structure and expression of CDR, we screened a library of human liver cDNAs cloned in the expression vector lambda gt11 and isolated an 800 bp cDNA that directed synthesis of a fusion protein recognized by polyclonal anti-CDR antibodies. Using this cDNA as a probe, we screened two human liver cDNA libraries and found several 1.2-kb cDNAs which would code for a polypeptide with 308 residues (35.8 kDa). However, a similar full-length cDNA, possibly the transcript of a pseudogene, contained an in-frame nonsense codon. The deduced protein sequence of CDR showed 65% similarity to the primary structure of human liver aldehyde reductase and 66% similarity to the inferred protein sequence of rat lens aldose reductase. A search of GenBank revealed significant nucleotide similarity to a cDNA coding for bovine lung prostaglandin f synthase and to a partial cDNA coding for frog lens rho-crystallin. Southern blot analysis of human genomic DNA displayed between 45 and 65 kilobases of DNA hybridizable to CDR cDNA and demonstrated several restriction fragment length polymorphisms among 26 individuals. Northern blot analysis of RNA from human, gerbil, rabbit, hamster, mouse, and rat livers disclosed hybridization with CDR cDNA only for the first three species.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a cDNA encoding a new member of the glucocorticoid-responsive cytochromes P450 in human liver.

Adult human liver contains a form of cytochrome P450, termed HLp, that resembles the glucocorticoid-inducible cytochrome P450p in rat liver in its structure, function, and regulation and catalyzes the oxidation of such clinically important substrates as cyclosporin, nifedipine, erythromycin, and midazolam. Recent evidence, however, suggests that HLp may represent two or more closely related forms of cytochromes P450, one of which is termed P450nf. To search for additional members of the Class III human subfamily of HLp related genes, we screened a human liver cDNA library cloned in phage vector lambda gt11 with oligonucleotides and with a cDNA fragment related to HLp. We isolated a full-length cDNA (1709 nucleotides) encoding a new form of human cytochrome P450 termed HLp2. Analysis of HLp2 cDNA predicted a protein of 502 amino acids, weighing 57,294 Da 83% similar to HLp. HLp2 appears to represent a distinct gene as judged by partial sequence analysis of a cloned human gene and by hybridizations of Southern blots, under conditions of varying stringency, with a 3'-portion of HLp cDNA and with an oligonucleotide specific for HLp2. Northern blot analysis revealed that HLp/P450nf was present in all samples of liver mRNA from adult patients not treated with inducers of HLp, whereas HLp2 mRNA was undetectable in more than two-thirds. Human fetal liver RNA contained mRNA species 2.1 and 1.9 kb which hybridized with an HLp2 oligonucleotide. We conclude that HLp2 represents a third member of the Class III glucocorticoid-responsive gene family that is expressed in both fetal and adult human liver and may account for polymorphism in metabolism of clinically important drugs.

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells.

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.

Co-ordinate regulation of low-density-lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase and synthase gene expression in HepG2 cells.

Cellular processes responsible for maintaining cholesterol homoeostasis are highly regulated. To determine whether two of these processes, cholesterol biosynthesis and receptor-mediated uptake of low-density lipoprotein (LDL), are co-ordinately regulated in human liver, we employed a human hepatoma cell line (HepG2) and measured the accumulation of mRNA for LDL receptor, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and HMG-CoA synthase under a variety of conditions. Genomic Southern-blot analysis demonstrated that the integrity of these genes is maintained in the transformed cell. Treatment of HepG2 cells with mevalonate, 25-hydroxycholesterol, LDL, lovastatin or miconazole resulted in a similar effect on the accumulation of all three mRNAs at the concentrations tested. The onset of the response to drug, whether repression or induction of mRNA accumulation, occurred after approximately the same period of exposure for each mRNA. We conclude that the expression of the LDL receptor, HMG-CoA reductase and HMG-CoA synthase is co-ordinately regulated in HepG2 cells.

Coinduction of multiple hepatic cytochrome P-450 proteins and their mRNAs in rats treated with imidazole antimycotic agents.

To characterize the molecular basis by which imidazole antimycotic drugs increase cytochrome P-450, we examined the effects of treating female rats with clotrimazole, miconazole, or ketoconazole on expression of the major inducible forms of hepatic cytochromes P-450 (P-450p, P-450b/e, P-450c/d, and P-450j). From measurements of the content of immunoreactive cytochromes P-450 in liver microsomes and of the amounts of liver RNA hybridizing to cloned P-450 cDNAs, we established that the glucocorticoid-responsive P-450p is the form predominantly induced by clotrimazole, miconazole, and ketoconazole, to as much as 382 times above control values. The phenobarbital-responsive cytochromes P-450b/e were also induced strongly by clotrimazole and miconazole, but not by ketoconazole. Aromatic hydrocarbon-inducible cytochromes P-450c/d were modestly elevated by each of these three antifungal drugs whereas ethanol-responsive P-450j was marginally induced by ketoconazole, but not by clotrimazole or miconazole. In some, but not all cases, treatment of rats with antifungal drugs resulted in accumulation of P-450 protein that significantly exceeded the increase in the corresponding P-450 mRNA. In conclusion, imidazole antifungal drugs differentially modulate the expression of at least four distinct gene subfamilies of rat hepatic cytochrome P-450 by separate mechanisms involving accumulation of P-450 mRNA and protein.

Identification of glucocorticoid-inducible cytochromes P-450 in the intestinal mucosa of rats and man.

We used monoclonal antibodies and complementary DNAs (cDNAs) to glucocorticoid-inducible liver cytochromes P-450 in rats (P-450p) and in man (HLp) to search for related cytochromes in intestinal mucosa. In rat enterocytes, we found two dexamethasone-inducible proteins related to the steroid-inducible liver cytochromes P-450. Induction of these proteins in enterocytes was associated with increases in the amount of a P-450p-related messenger RNA and of erythromycin demethylase, an activity highly characteristic of P-450p and HLp. Similar studies on human jejunal enterocytes revealed a microsomal protein indistinguishable from HLp on immunoblots and an abundance of RNA hybridizing with HLp cDNA. In human enterocytes the specific concentration of the HLp-related cytochrome (measured immunochemically or as erythromycin demethylase activity) was similar to that found in human liver and could account for all of the CO-binding hemo-protein detected. We conclude that the intestinal mucosa contains prominent form(s) of cytochromes P-450 similar to liver cytochrome P-450p in their structure, function, and some regulatory characteristics.

Purification and characterization of aldo-keto reductases from gerbil liver: immunochemical evidence for related proteins in other mammalian species.

We purified a hepatic aldehyde reductase (AR1) and two carbonyl reductases (CR1, CR2) from the Mongolian gerbil, an animal recently shown to closely resemble man in its metabolism of a carbonyl containing organochlorine pesticide. The apparent molecular weights of AR1, CR1, and CR2 were 40,700, 33,000, and 34,700, respectively. Typical of similar enzymes in other species, gerbil AR1 reduced aliphatic and aromatic aldehydes and was inhibited by phenobarbital or valproate, whereas CR1 and CR2 catalyzed the reduction of aromatic aldehydes and ketones as well as quinones and were inhibited by p-chloromercuribenzoate, mercuric chloride, or pyrazole. All three enzymes were insensitive to metal chelating agents and utilized NADPH as their cofactor. CR1 was unique in being equally active with NADH as its cofactor. Antibodies raised against CR1 reacted with purified CR1 and CR2, but not with AR1, as judged by immunoblot analyses. There were three immunochemically related proteins in gerbil liver cytosol (30 to 35 kDa range) recognized by the anti-CR1 IgG. Similar immunoblot analyses of hepatic cytosolic proteins from other mammalian species revealed immunoreactive proteins only in the hamster, the rabbit, and man, and not in the rat, the mouse, or the guinea pig. Quantitative immunoblot analyses of human liver cytosol from seven patients revealed three immunoreactive proteins. These were present in unequal and varying concentrations, although there were only small interindividual differences in the total concentration of the immunoreactive proteins. We conclude that there are multiple molecular forms of immunochemically related hepatic carbonyl reductases in the gerbil and in some other mammalian species, including man.

Characterization of ethanol-inducible human liver N-nitrosodimethylamine demethylase.

Through the use of monospecific antibodies directed against hepatic cytochrome P-450j, an enzyme induced in rats treated with ethanol or isoniazid, we have purified from human liver the related cytochrome P-450 termed HLj. HLj resembles rat P-450j and P-450 LM3a, the homologous cytochrome in rabbit liver, in its NH2-terminal amino acid sequence, in being in highest concentration in liver microsome samples prepared from two patients intoxicated by ethanol and one patient given isoniazid, and in catalyzing the metabolic activation of the procarcinogen N-nitrosodimethylamine. Furthermore, each of nine human liver RNA samples contained a species of mRNA hybridizable to a cloned HLj cDNA. We conclude that HLj is related by structure, function, and some regulatory characteristics to rat P-450j and rabbit P-450 LM3a, cytochromes critical for metabolism of several clinically relevant cytotoxic and carcinogenic agents.

Purification and characterization of chlordecone reductase from human liver.

The toxic organochlorine pesticide, chlordecone (Kepone), is excreted in human bile primarily as a stable, reduced monoalcohol metabolite. This bioreduction is catalyzed by a hepatic cytosolic enzyme activity termed chlordecone reductase. We purified this enzyme from human liver and found that chlordecone reductase resembles the family of xenobiotic metabolizing enzymes referred to as the aldo-keto reductases based on its biochemical characteristics, including its ability to catalyze the reduction of a carbonyl-containing substrate. However, analyses of liver cytosolic samples on immunoblots developed with anti-chlordecone reductase antibodies revealed that immunoreactive proteins were present only in those mammalian species that convert chlordecone to chlordecone alcohol in vivo (man, gerbil, and rabbit) and not in those species unable to reduce chlordecone (rat, mouse, and hamster). Hence, chlordecone reductase is unique among aldo-keto reductases in being species-specific. Quantitative immunoblot analyses of seven human liver specimens disclosed two immunoreactive proteins whose total concentration varied over a 6-fold range. Moreover, the amount of immunoreactive protein was directly proportional to chlordecone reductase activity in each sample. We conclude that chlordecone reductase is a unique aldo-keto reductase of potential clinical importance whose expression varies markedly among individuals.

Complete cDNA sequence of a cytochrome P-450 inducible by glucocorticoids in human liver.

HLp is a human liver cytochrome P-450 that is immunochemically related to the glucocorticoid-inducible liver cytochrome P-450p in the rat and its homologue in the rabbit, P-450 LM3c. To investigate the structure and regulation of HLp, we used a monoclonal antibody that recognizes purified HLp to screen a human liver cDNA library in lambda gt11. We isolated and sequenced two overlapping cDNA clones that span the entire 2011 bases of an mRNA that codes for a protein of 504 amino acids. The predicted amino-terminal amino acid sequence of this protein is identical to the first 20 residues determined from purified HLp. HLp mRNA shares more than 70% sequence homology with related proteins from the rat and rabbit but less than 40% homology with other published cytochrome P-450 genes. Moreover, Southern blot analysis of human and rat genomic DNA revealed 50 and 60 kilobases of DNA, respectively, hybridizable to the HLp cDNAs. Blot analysis of human liver RNA from five patients revealed major (2.2 kilobase) and minor (3.0 kilobase) bands that hybridized to HLp cDNAs. The apparent concentration of these hybridizable mRNAs as well as the amounts of immunoreactive HLp protein in microsomes from the same liver were increased in a dose-dependent relationship in three patients who received dexamethasone, a potent glucocorticoid. Furthermore, in samples of RNA and of microsomes isolated from cultures of a human hepatoma cell line (Hep G2) incubated for 120 hr in medium containing dexamethasone, there was a 6-fold induction of the two mRNA species hybridizable to HLp cDNAs and a 3-fold induction of immunoreactive HLp protein as compared to the values for cultures incubated in steroid-free medium. We conclude that HLp is a human representative of a conserved glucocorticoid-inducible cytochrome P-450 gene family whose mechanism of induction involves accumulation of HLp mRNA.

Characterization of a unique aldo-keto reductase responsible for the reduction of chlordecone in the liver of the gerbil and man.

It has been established that the major metabolic pathway for chlordecone (CD) (Kepone) both in humans and in the Mongolian gerbil is bioreduction of this organochlorine pesticide to chlordecone alcohol (CDOH) in the liver. In the present study we developed a gas-liquid chromatography assay to measure the enzymatic reduction of CD to CDOH in vitro and characterized "CD reductase" activity in gerbil liver cytosol. CD reductase is a cytosolic enzyme readily detectable in liver samples prepared from humans, rabbits, and gerbils, the only species of many tested that convert CD to CDOH in vivo. Gerbil CD reductase exhibited a Km of 2.6 microM, a Vmax of 0.14 nmol/min, and a pH optimum of 6.5. The enzyme activity required NADPH, was sensitive to thiol reagents, and was distributed in all tissues with the highest activities found in the liver, intestine, and kidneys. These results are consistent with CD reductase belonging to the family of enzymes referred to as the "aldo-keto reductases." However, unlike previously described reductases, CD reductase was undetectable in rats, mice, hamsters, or guinea pigs and was insensitive to the model aldehyde and ketone reductase inhibitors, phenobarbital and quercetin, respectively. In addition, CD reductase activity in liver was increased by 38% (p less than 0.01) following treatment of gerbils with CD. We conclude that CD reductase is a novel aldo-keto reductase that is uniquely inducible by its substrate.