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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.

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 Agtil.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 ofimmunoreactive 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.The liver cytochromes P-450 are a supergene family of microsomal hemoproteins that catalyze the oxidative biotransformation of numerous endogenous substrates, such as steroids, as well as xenobiotics, such as drugs. Extensive studies in many species of laboratory animals indicate that there are multiple polypeptide forms of these cytochromes that can be distinguished by differences in their structure, substrate specificity, or responses to various types of inducers (1-3). These hemoproteins appear to have been conserved in evolution. For example, in rats treated with pregnenolone 16a-carbonitrile, a "catatoxic" steroid that protects animals from stress and from the lethal (4) or carcinogenic (5) effects of many toxic substances, P-450p is the major form of liver cytochrome P-450 (6). We have found that there are forms of hepatic cytochrome P-450 in the rabbit (LM3c), the hamster, the gerbil, and the mouse that (i) cross-react with anti-P-450pantibodies; (ii) catalyze oxidation of the same model substtates; (iii) are inducible by glucocorticoids, phenobarbital, and macrolide antibiotics; (iv) are encoded by mRNA species inducible by a potent glucocorticoid, dexa...

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)

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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)

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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.