In vivo and in vitro peroxisome proliferation properties of selected clofibrate analogues in the rat

We have examined, relative to clofibric acid (CPIB), the effects of a chemical series of phenoxyacetic acids and of two asymmetric CPIB analogues, the R (+)- and S (−)-enantiomers of 2-(4-chlorophenoxy) propionic acid (4-CPPA) and 2-(4-chlorophenoxy)butyric acid (4-CPBA), on hepatic peroxisome proliferation both in vivo and in vitro utilizing cholesterol-fed rats and primary cultured rat hepatocytes respectively. Peroxisome proliferation was assessed by measuring changes in peroxisomal fatty acyl-CoA oxidase (FACO) and microsomal laurate hydroxylase (LH) activities as well as by electron microscopic examination of 3,3'-diaminobenzidine-stained liver slices. CPIB and enantiomers of 4-CPPA and 4-CPBA (0.6 mmol/kg/day for 7 days) produced hepatomegaly, lowered serum cholesterol levels, and caused 4.7- to 12.9-fold and 2.9- to 6.1-fold increases in hepatic FACO and LH activities, respectively, in cholesterol-fed rats. Electron micrographs of liver cells showed an increased number of peroxisomes from cholesterol-fed rats given S (−)-4-CPBA and CPIB. Likewise, these compounds (0.03 to 1.0 mM) induced FACO and LH in primary rat hepatocyte cultures after 72 hr. R (+)- and S (−)-Enantiomers of 4-CPPA produced similar concentration-dependent and maximal increases in both FACO and LH activities, whereas enantiomeric selectivity [ s (−) > R (+)] for the induction of these two enzymes was observed with the isomers of 4-CPBA. The increases in the activities of FACO and LH caused by S (−)-4-CPBA were similar to those elicited by 1.0 mM CPIB (58.6- and 9.8-fold respectively). These results show that the enantiomers of 4-CPPA and 4-CPBA induce the peroxisome proliferation-associated enzymes FACO and LH in vivo and in vitro , and that the S (−)-isomer of 4-CPBA causes a greater induction of FACO and LH in vitro than its corresponding R (+)-isomer, indicating that these two enzymes are induced in an enantioselective manner. Optimal induction of the peroxisome proliferation-associated enzymes FACO and LH in rat hepatocyte cultures was produced by phenoxyacetic acids possessing (1) a chlorine atom at the 4-position of the phenyl ring, (2) a dimethyl or mono-ethyl substitution at the α-carbon atom of the carboxylic acid side chain; and (3) an S (−)-orientation for chiral analogues possessing a mono-ethyl group at the α-carbon atom of the carboxylic acid side chain. These results indicate that changes in the chemical and stereoisomeric structures of phenoxyacetic acids alter peroxisome proliferation and are consistent with a hypothesized receptor-mediated mechanism, and by employing the proper enantiomeric form, a greater dissociation of beneficial lipid lowering actions from adverse carcinogenic effects of peroxisome proliferators may be obtained.