Abstract
1. Species differences in elements of the peroxisome proliferator domain (PPD), including the cytochrome P450 4A subfamily of fatty acid hydroxylases and peroxisomal fatty acid β-oxidation have been examined. 2. Microsomal preparations from a panel of human livers showed cross-reactivity with a sheep anti-sera to rat cytochrome P450 4A1 and gave a single band on Western blot analysis with a relative Mr of approximately 51kDa (compared to approximately 51.5kDa for the rat). Spectral analysis of ferrous-CO complexes revealed peak maxima between 450-452nm with the total cytochrome P450 specific content and NADPH-cytochrome P450 reductase activity displaying about three and two-fold variations, respectively. Immunoquantification of the cytochrome P450 4A1 human orthologue by an ELISA protocol gave between 4-12% of total cytochrome P450 content. NADPH-fortified human liver microsomal preparations from all the samples used both laurate and arachidonate as substrate and formed the ω- and (ω-1)-metabolites at varying rates. Moreover, anti-rat P450 4A1-enriched IgG fraction inhibited the human ω-laurate hydroxylase activity to greater than 55% (20% in the rat) at a concentration of 5mg/nmol total cytochrome P450. Taken collectively, the information thus presented is strongly suggestive of the expression of a member(s) of the cytochrome P450 4A subfamily in the panel of human liver tissues examined. 3. To further elucidate the mechanism of cytochrome P450 4A1 induction and peroxisome proliferation by a diversity of peroxisome proliferators, I also examined the stereochemical specificity in the induction of members of the peroxisome proliferation domain by optically active enantiomers of a clofibrate-analogue. In all the parameters examined, the R(-)- enantiomer was identified as the eutomer and its corresponding S(+)-antipode as the distomer, with the racemic mixture intermediate in inducing these enzyme activities. 4. The effect of a relatively newly described peroxisome proliferator, perfluoro-n-decanoic acid (PFDA), on the peroxisome proliferation domain in a responsive (rat) and a non-responsive (guinea pig) species was also investigated. At the dose level investigated (20 mg/kg), PFDA administration did not appear to impair food intake nor weight gain but resulted in distinct differences in the hepatic and renal response between the two species examined. In the rat, hepatomegaly was observed with the coordinate induction of total cytochrome P450 levels (1.5 fold), NADPH-cytochrome P450 reductase (1.5-fold), palmitoyl CoA oxidase (6-fold), carnitine acetyl transferase (peroxisomal and mitochondrial, 15-fold) and preferential cytochrome P450 4A1-mediated ω-laurate hydroxylase activity (5-fold). In addition, a substantial inhibition in the activity of cytochrome P450 1A1-associated enzyme activity as measured by ethoxyresorufin-O-deethylase (EROD) activity was observed in the rat liver (74%) and kidney (39%). Western blotting analysis of hepatic and renal microsomes for cytochromes P450 4A1 and 1A1 isozymes reflected the above changes. A similar analysis showed significant induction of the trifunctional protein of the rat hepatic peroxisomal B-oxidation spiral. Generally, the guinea pig appeared to be non-responsive at this dose level. However, a significant inhibition of the EROD activity was detected in both the liver (35%) and kidney (31%). A similar pattern of results was obtained for the PROD activity in both tissues. Generally, the rat kidney was less responsive than the liver. 5. The ligand-binding site for peroxisome proliferators in the peroxisome proliferator-activated receptor (PPAR) has been computer-modelled and used as a basis to discuss the structural diversity of peroxisome proliferators. 6. Species differences in response to peroxisome proliferators have been discussed in terms of lipid homoeostasis and/or regulatory elements of the relevant genes.