Characterization of ciprofibrate and clofibric acid as peroxisomal proliferators in primary cultures of rat hepatocytes

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Abstract
We have determined the comparative activities of peroxisomal proliferators, ciprofibrate and clofibric acid on various hepatic parameters associated with endoplasmic reticulum, mitochondria and peroxisomes in primary cultures of rat hepatocytes. We have measured the activities of carnitine acetyltransferase and fatty acylCoA oxidase, and the amount of 60 and 80 kD polypeptides as biochemical markers of the peroxisomal function; laurate hydroxylase and cytochrome P-450 as markers of the endoplasmic reticulum; and carnitine palmitoyltransferase as a marker of mitochondria in primary cultures of hepatocytes. Ciprofibrate (0.01 to 0.3 mM) and clofibric acid (0.1 to 3 mM) produced similar changes in several components of cultured hepatocytes within 72 hr. Increases of protein (18 and 11%), carnitine palmitoyltransferase (23 and 97%), cytochrome P-450 (37 and 49%), carnitine acetyltransferase (484 and 614%), fatty acylCoA oxidase (529 and 931%) and laurate hydroxylase (624 and 671%) were obtained in hepatocytes after a 72-hr exposure to 0.1 mM ciprofibrate and 1.0 mM clofibric acid, respectively. In cultured hepatocytes, ciprofibrate was about 30-fold more active than clofibric acid for the stimulation of carnitine acetyltransferase, laurate hydroxylase and fatty acylCoA oxidase activities. Ciprofibrate was also more potent than clofibric acid as an inducer of the 60 and 80 kD proteins in hepatocytes. The maximal drug-induced increases in carnitine acetyltransferase activity were not additive, and the induction of carnitine acetyltransferase by ciprofibrate was blocked by addition (1 μg per ml) of cycloheximide or actinomycin D. Changes in protein and RNA synthesis preceded the drug-induced increases of carnitine acetyltransferase activity. Druginduced increases in peroxisomes of hepatocytes were also confirmed by 3,3′-diaminobenzidine staining and electron microscopic examination. These results show that: (i) measurements of carnitine acetyltransferase, fatty acylCoA oxidase, laurate hydroxylase activities and 80 kD polypeptide in primary cultures of rat hepatocytes are useful criteria for characterizing the effects of peroxisome proliferating agents; (ii) ciprofibrate is more active as a peroxisomal proliferating agent than clofibric acid in hepatocyte cultures; and (iii) the isolated, cultured rat hepatocyte system is valuable as an adjunct to the in vivo model for studying biochemical mechanisms of peroxisome proliferation and hepatotoxicity for this class of agents.