The cytochrome P450 IV family: constitutive and inducible haemoproteins.

Mammalian cytochrome 1'-450 exists as a multigene family expressing a spectrum of structurally and functionally distinct isoenzymes involved in the biotransformation of both exogenous and endogenous substrates [ l , 21. Although a generally accepted isoenzyme nomenclature based on divergent evolution and nucleotide/amino acid homologies is now widely used [I], i t is still useful, under certain circumstances, to consider the cytochromes 1'-450 under the more broad subdivisions of constitutive and inducible enzymes. However, the problem with the use of this broader nomenclature lies in the difficulty in defining what a constitutive enzyme actually is. According to a recent definition [ 31, constitutive enzymes are 'enzymes which, in contrast to the inducible enzymes, are consfanfly (sic) produced by the cell, irrespective of the growth conditions'. This definition infers that constitutive cytochromes 1'-450 are regulated by endogenous or dietary inducers in vivo or by the existence of a control mechanism governing constitutive expression, and that the constitutive cytochromes P 4 5 0 are probably associated with relatively ancient genes. Based on the amino acid sequences of the cytochromes P450, an evolutionary tree has been constructed [4]. There are two major divisions in this phylogenetic tree, the first occurring at the separation of eukaryotic and prokaryotic cytochromes f -450 (approximately 1400 million years ago), and the second separating the enzymes that catalyse the metabolism of endogenous versus exogenous substrates (approximately 100 million years ago). The first gene duplication event occurred approximately 1360 million years ago and gave rise to the cytochromes 1'-450 in two different cellular organelles, namely the mitochondrion and the endoplasmic reticulum. According to this evolutionary analysis, the early history of mammalian cytochrome P -450 appears to be related to the evolution of cholesterol, as the earliest eukaryotic cytochrome 1'-450 sequences reported are the cholesterol side-chain cleavage enzymes of the mitochondrion. Accordingly, the above description of constitutive and inducible cytochromes P-450 serves as a useful operational definition to classify the factors that control and regulate the expression of cytochrome "-450 isoenzymes. In this context, the cytochrome P450 IV family of genes is an excellent example of the expression of both constitutive and inducible isoenzymes in that a constitutive high-level enzyme expression is attained [ 5 ] , and this regulation of a 'constitutive' enzyme is additionally regulated by xenobiotics such as the oxyisobutyrate hypolipidaemic drugs [6], the phthalate ester plasticizers [7] and the chlorinated phenoxy acid herbicides, including 2,4,5-trichlorophenoxy acetic acid [ 81. The cytochrome P450 IV gene family was identified unequivocally in 1 Y X 7 when cytochrome P450 IVAl was cloned, isolated and sequenced and found to contain 2062 base pairs with an open reading frame of 504, amino acids 191, a result independently verified in this laboratory [ 101. Because the isolated cytochrome P4S0 IVAl gene showed less than 35% cDNA nucleotide and amino acid similarity with cytochromes 1'-450 from the I, I1 and 111 families, it was therefore designated the first member of a new gene family [Y, 101. Although the precise number of genes in the P450 IV family are not known with any degree of certainty at present, it is clear that there are two subfamilies, containing a minimum of eight genes 11, 21. Cytochromes P450 IVAl, IVA2 and IVA3 have been identified in the rat; IVA4, 1VA5 and 1VA6 in the rabbit and lVBl in the human, rat and rabbit, and the interested reader is referred to [ 11 for a more complete description of this gene family. The most widely studied mcmber of the cytochrome P450 1V gene family is P450 IVAI, the enzyme product of which was first isolated and characterized from the livers of clofibrate-pretreated rats [ 1 1, 121. This isoenzyme shows a marked substrate specificity for the terminal hydroxylation of fatty acids, including lauric acid and arachidonic acid I 131. and using both antibody and cDNA probes, is constitutively expressed in rat liver and kidney, but not in lung 16, 7, Y, 101. For example, cytochrome P450 IVA 1 comprises approximately 5% of the total carbon monoxide-disccrniblc cytochrome 1'-450 population in 'control' male Wistar rat liver [S, 61, and is readily identified in the kidney of Wistar and Sprague-Dawley rats [9, 14). If thc cytochromc P450 IVAl gene is constitutively exprcsscd in rat liver and kidney, what factors control this expression? It would appear that cndogenous lipids may play an as yut mechanistically poorly understood role in this regulatory proccss. in that mcdiumchain fatty acids (C,,-C,) induce cytochromc P450 IVA 1 in cultured hepatocytes [ 151. In addition, thc administration of high fat diets in vivo results in an induction of cytochromc P450 IVAl-dependent 12-hydroxylation o f lauric acid 1 161, further implicating a possiblc role o f lipids in regulating cytochrome P450 IVA 1 genc expression. As initially reported by Parker & Orton 1 171, cytochrome P-450-dependent fatty acid hydroxylation is substantially induced by the oxyisobutyrate hypolipidaemic drugs, such as clofibrate, in rat liver and kidney, and has been recently rationalized by the significant induction (approximately 5-fold) of catalytically active cytochromc P450 IVA I protein [5, 61. Recent evidence, using cDNA probes for cytochrome P450 IVAl, has demonstrated that clofibrate pretreatment results in an accumulation of cytochrome P450 IVAl mRNA [Y, 101, due to transcriptional activation of the cytochrome P450 IVAl gene as early as 1 h after the administration of the inducer in vivo 191. Although it is absolutely clear that clofibrate is responsible for the transcriptional activation of the cytochrome P450 lVAl gene, this laboratory has recently reported that differential splicing occurs in the 3'-non-coding region of the cytochrome P450 lVAl gene, which may be relevant to mRNA stability or translation repression of the gene [ 101, and hence additionally contributes to the overall control of cytochrome P450 IVAl gene expression. Interestingly, induction of cytochrome P450 IVA 1 in rat liver is not solely confined to oxyisobutyrates, such as clofibrate and its analogues, but also includes structurally diverse inducers such as WY-14,643, aspirin and the phthalate ester plasticizers including di-(2-ethylhexyl)phthalate [6, 71. This, and other observations based on the lack of inducer binding to subcellular liver homogenates [ 181, would tend to cast some doubt on the existence of a 'classical' nuclear/cytosolic receptor for the above inducers of cytochrome P.450 IVA1, as has been described for the 2,3,7,8-tetraochlorodibenzo-p-dioxin which regulates cytochrome P450 I receptor [ 191. In addition, based on cycloheximide-inhibition experiments, it would appear that clofibrate induction of the cytochrome P450 lVAl gene does not involve the intermediation of a clofibrate-inducible protein factor, as has been postulated for the cyclic AMPmediated production of labile proteins in the regulation of steroid hydroxylase cytochrome P450 gene expression in the adrenal cortex [20]. How then is the cytochrome P450 IVAl gene regulated by clofibrate? Does the induction mechanism involve the same control and regulatory elements that are responsible for maintenance of the constitutive level of