Differential induction of peroxisomal and microsomal fatty-acid-oxidising enzymes by peroxisome proliferators in rat liver and kidney. Characterisation of a renal cytochrome P-450 and implications for peroxisome proliferation.

The induction of renal fatty-acid-oxidising enzymes has been investigated following short-term exposure to a group of structurally diverse peroxisome proliferators and compared to the more extensively documented hepatic responses in the rat. There was a marked compound dependence on induction of both cytochrome P-450-IVA1-dependent omega-hydroxylation of lauric acid and enzymes of the peroxisomal fatty acid beta-oxidation pathway (measured as cyanide-insensitive palmitoyl-CoA oxidation and enoyl-CoA hydratase). Cytochrome P-450 IVA1 (or a very closely related isoenzyme in the same gene family) was a major constitutive haemoprotein in rat kidney microsomes and actively supported the omega-hydroxylation of lauric acid. This activity was induced 2-3-fold by peroxisome proliferators such as clofibrate, di-(2-ethylhexyl)phthalate, bezafibrate and nafenopin. By using a cDNA probe to the cytochrome P-450 IVA1 gene in Northern blot analysis, we have shown that increased renal and hepatic omega-hydroxylation of lauric acid, after treatment with peroxisome proliferators is a consequences of a substantial increase in the mRNA coding for this haemoprotein. In addition, programming of an in vitro rabbit reticulocyte translation system with both renal and hepatic RNA resulted in the synthesis of similar (if not identical) cytochrome-P-450-IVA1-related polypeptides. Furthermore, we have provided Western blot evidence that both rat liver and kidney microsomes contain two closely related cytochrome P-450 IVA1 polypeptides, the major one characterised by a monomeric molecular mass of 51.5 kDa (identical to authentic, purified hepatic cytochrome P-450 IVA1) and a minor one of 52 kDa. The kidney-supported fatty acid omega-hydroxylase activity was refractory to inhibition by a polyclonal antibody to liver cytochrome P-450 IVA1, which may be related to the existence of two closely related (but immunochemically distinct) fatty acid hydroxylases in this tissue. Our studies have also demonstrated that certain of the compounds tested (including clofibrate, bezafibrate and nafenopin) induced renal fatty acid beta-oxidation, mirroring the increased omega-hydroxylase activity in the endoplasmic reticulum. Our studies have also indicated that the kidney was more refractory to induction of the endoplasmic reticulum and peroxisomal fatty-acid-oxidising enzymes than the liver. Taken collectively, our data is strongly suggestive of a possible linkage of the renal fatty acid oxidative enzymes in these two organelles, a situation that also occurs in the liver. In addition, our studies have provided a possible conceptual framework that may rationalise the decreased susceptibility of the k

Cytochrome P-450 dependent metabolic activation of 1-naphthol to naphthoquinones and covalent binding species.

1-Naphthol was metabolised by a fully reconstituted cytochrome P-450 system in the presence of NADPH to methanol-soluble and covalently bound products. The formation of 1,4-naphthoquinone, the major methanol-soluble product at early time points, showed an almost total dependence on cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH, and to a lesser extent on dilauroylphosphatidylcholine. The metabolism was rapid and detectable levels of 1,4-naphthoquinone were formed within 30 sec. 1,4-Naphthoquinone formation was dependent on the concentration of both cytochrome P-450 (0.05-0.04 microM) and 1-naphthol (5-50 microM). Whereas 1,4-naphthoquinone was the major product observed at early time points, additional products were observed after prolonged incubation. In the absence of NADPH and NADPH-cytochrome P-450 reductase, 1-naphthol was metabolised, in a cumene hydroperoxide- and cytochrome P-450-dependent reaction, to 1,2- and 1,4-naphthoquinone and covalently bound products. Glutathione and ethylenediamine inhibited both the NADPH- and cumene hydroperoxide-dependent formation of covalently bound products. These data show that cytochrome P-450 catalyses the activation of 1-naphthol to naphthoquinone metabolites and covalently bound species, the latter most likely being derived from naphthoquinones.

Multiple forms of hepatic cytochrome P-450. Purification, characterisation and comparison of a novel clofibrate-induced isozyme with other major forms of cytochrome P-450.

In the present studies, a novel form of highly purified cytochrome P-450 (cytochrome P-452) isolated from the hepatic microsomes of clofibrate-pretreated rats has been compared to the major isozymes isolated from the hepatic microsomes of rats pretreated with phenobarbital (cytochrome P-450) and 2-naphthoflavone (cytochrome P-447) using a number of biochemical criteria. The results show that these three isozymes exhibit marked structural differences from each other as judged by a complete lack of immunochemical cross-reactivity between the isozymes and the heterologous rabbit serum antibodies using Ouchterlony double diffusion, and non-identity between the limited proteolytic digestion maps of the three isozymes obtained in the presence of chymotrypsin, papain and Staphylococcus aureus V8 proteases. Furthermore, the three isozymes exhibited clear differences in their monomeric molecular weights determined on calibrated sodium dodecyl sulphate/polyacrylamide gel electrophoresis in gels of varying acrylamide concentration. Substantial differences were also observed in the substrate specificities of the isozymes, which were reflected in differences in the turnover rates and positional selectivities of the hemoproteins for some model substrates. In addition, the isozymes differed in their substrate binding affinities and their ability to interact with purified hepatic microsomal cytochrome b5, as judged using difference spectrophotometry. Finally, subtle differences were detected in the ultraviolet visible absorbance spectra of the hemoproteins in the ferric, ferrous, and carbonmonoxyferrous states. Taken collectively, the above data provides compelling evidence that fundamental differences exist between these cytochrome P-450 isozymes, further establishing the uniqueness of the major form of cytochrome P-450 induced by clofibrate pretreatment.