Molecular cloning of a family of xenobiotic-inducible drosophilid cytochrome P450s: Evidence for involvement in host-plant allelochemical resistance

Cytochrome P450s constitute a superfamily of genes encoding mostly microsomal hemoproteins that play a dominant role in the metabolism of a wide variety of both endogenous and foreign compounds. In insects, xenobiotic metabolism (i.e., metabolism of insecticides and toxic natural plant compounds) is known to involve members of the CYP6 family of cytochrome P450s. Use of a 3′ RACE (rapid amplification of cDNA ends) strategy with a degenerate primer based on the conserved cytochrome P450 heme-binding decapeptide loop resulted in the amplification of four cDNA sequences representing another family of cytochrome P450 genes (CYP28) from two species of isoquinoline alkaloid-resistant Drosophila and the cosmopolitan species Drosophila hydei. The CYP28 family forms a monophyletic clade with strong regional homologies to the vertebrate CYP3 family and the insect CYP6 family (both of which are involved in xenobiotic metabolism) and to the insect CYP9 family (of unknown function). Induction of mRNA levels for three of the CYP28 cytochrome P450s by toxic host-plant allelochemicals (up to 11.5-fold) and phenobarbital (up to 49-fold) corroborates previous in vitro metabolism studies and suggests a potentially important role for the CYP28 family in determining patterns of insect–host-plant relationships through xenobiotic detoxification.

CXR, a chicken xenobiotic-sensing orphan nuclear receptor, is related to both mammalian pregnane X receptor (PXR) and constitutive androstane receptor (CAR)

Nuclear receptors constitute a large family of ligand-modulated transcription factors that mediate cellular responses to small lipophilic molecules, including steroids, retinoids, fatty acids, and exogenous ligands. Orphan nuclear receptors with no known endogenous ligands have been discovered to regulate drug-mediated induction of cytochromes P450 (CYP), the major drug-metabolizing enzymes. Here, we report the cloning of an orphan nuclear receptor from chicken, termed chicken xenobiotic receptor (CXR), that is closely related to two mammalian xenobiotic-activated receptors, the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Expression of CXR is restricted to tissues where drug induction of CYPs predominantly occurs, namely liver, kidney, small intestine, and colon. Furthermore, CXR binds to a previously identified phenobarbital-responsive enhancer unit (PBRU) in the 5′-flanking region of the chicken CYP2H1 gene. A variety of drugs, steroids, and chemicals activate CXR in CV-1 monkey cell transactivation assays. The same agents induce PBRU-dependent reporter gene expression and CYP2H1 transcription in a chicken hepatoma cell line. These results provide convincing evidence for a major role of CXR in the regulation of CYP2H1 and add a member to the family of xenobiotic-activated orphan nuclear receptors.

Cytochrome P450 Monooxygenases for Fatty Acids and Xenobiotics in Marine Macroalgae1

The metabolism of xenobiotics has mainly been investigated in higher plant species. We studied them in various marine macroalgae of the phyla Chlorophyta, Chromophyta, and Rhodophyta. Microsomes contained high oxidative activities for known cytochrome (Cyt) P450 substrates (fatty acids, cinnamic acid, 3- and 4-chlorobiphenyl, 2,3-dichlorobiphenyl, and isoproturon; up to 54 pkat/mg protein). The presence of Cyt P450 (approximately 50 pmol/mg protein) in microsomes of the three algal families was demonstrated by CO-difference absorption spectra. Intact algal tissue converted 3-chlorobiphenyl to the same monohydroxy-metabolite formed in vitro. This conversion was 5-fold stimulated upon addition of phenobarbital, and was abolished by the known P450 inhibitor, 1-aminobenzotriazole. It is concluded that marine macroalgae contain active species of Cyt P450 and could act as a metabolic sink for marine pollutants.

Inhibition of neoplastic development in the liver by hepatocyte growth factor in a transgenic mouse model.

Overexpression of the c-myc oncogene is associated with a variety of both human and experimental tumors, and cooperation of other oncogenes and growth factors with the myc family are critical in the evolution of the malignant phenotype. The interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in a transgenic mouse model has been analyzed. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Furthermore, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice, whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine.

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

Inactivation of ethanol-inducible cytochrome P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal, a major product of membrane lipid peroxidation.

Of the microsomal P450 cytochromes, the ethanol-inducible isoform, P450 2E1, is believed to be predominant in leading to oxidative damage, including the generation of radical species that contribute to lipid peroxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensitivity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After incubation of a purified cytochrome with HNE, the other components of the reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylcholine, and NADPH) were added, and the rate of oxygenation of 1-phenylethanol to yield acetophenone was assayed. Inactivation occurs in a time-dependent and HNE concentration-dependent manner, with P450s 2E1 and 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4. At an HNE concentration of 0.24 microM, which was close to the micromolar concentration of the enzyme, four of the isoforms were significantly inhibited, but not P450 2B4. In other experiments, the reductase was shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phenobarbital, respectively, are as readily inhibited as the purified forms. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects against HNE, and that the inactivation is reversed upon dialysis. Higher levels of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed inhibition of the various P450s is of sufficient magnitude to cause significant changes in the metabolism of foreign compounds such as drugs and chemical carcinogens by the P450 oxygenase system at HNE concentrations that occur in biological membranes. In view of the known activities of P450 2E1 in generating lipid hydroperoxides and in their beta-scission, its inhibition by this product of membrane peroxidation may provide a negative regulatory function.