Patricia S. Foxworthy

Effect of ciprofibrate, bezafibrate, and LY171883 on peroxisomal β-oxidation in cultured rat, dog, and rhesus monkey hepatocytes

Cultured rat hepatocytes have been used extensively to study the mechanisms of chemically induced peroxisome proliferation. Hepatocytes from nonrodent species have been used on a limited scale to study interspecies differences in the response. Because of their importance in pharmaceutical safety assessment, we have developed a model to study the response of beagle dog and rhesus monkey hepatocytes to peroxisome proliferators. Treatment of the hepatocytes with peroxisome proliferators was begun after 20 hr in culture and continued for 72 hr. Untreated rat, dog, and monkey hepatocytes retained 62, 42, and 43% of their initial (20 hr) peroxisomal beta-oxidation activity throughout 92 hr of culture. Ciprofibrate, bezafibrate, and LY171883 caused a dose-related increase in beta-oxidation in rat hepatocytes to a maximum of 10-, 8-, and 5-fold, respectively. In dog and monkey hepatocytes the increases in beta-oxidation were less than 2-fold. Peroxisome morphology in dog and monkey hepatocytes appeared to be unchanged by the drugs. Morphometric analysis in monkey hepatocytes showed no increase in peroxisome volume fraction in response to the chemicals. Treatment of dog and monkey hepatocytes with dexamethasone and glucagon during the final 24 hr in culture caused a 4- to 6-fold increase in tyrosine aminotransferase activity. This induction is characteristic of the in vivo response. The small increase in beta-oxidation reflects the relative insensitivity of the dog and monkey liver to peroxisome proliferators in vivo rather than a loss of sensitivity during culture. Cultured hepatocytes from beagle dog and rhesus monkey may provide a model for studying the mechanisms underlying the interspecies differences. Such information would help clarify the relevance of rodent data in human risk assessment.

Induction of peroxisomal β-oxidation in the rat liver in vivo and in vitro by tetrazole-substituted acetophenones: Structure-activity relationships

LY171883, a leukotriene D4 antagonist in the tetrazole-substituted acetophenone structural class, previously was demonstrated to cause peroxisome proliferation in rodents. In the present studies, several analogs were tested to determine if there are structural requirements for the induction of peroxisomal beta-oxidation in the rat liver in vivo and in cultured rat hepatocytes. Liver weight and serum triglycerides also were measured in vivo. The increases in peroxisomal beta-oxidation caused by the tetrazole-substituted acetophenones in vivo ranged from negligible to greater than 17-fold and there was good agreement with the structure-activity relationships found in cultured hepatocytes. N-methylation of the acidic nitrogen of the tetrazole blocked the peroxisomal effects, indicating that the free acid was required for activity. The length of the alkyl chain linked to the tetrazole also influenced the activity of the compounds. However, the more important determinant of peroxisomal activity may be the spatial orientation of the acidic tetrazole with respect to the planar backbone of the molecule. The data indicate there is a target site for peroxisome proliferation in the liver that is able to distinguish between structurally similar analogs. This site appears to be distinct from the leukotriene receptor since both inducers and noninducers of peroxisomal beta-oxidation were shown previously to be potent leukotriene antagonists.

Inhibition of hepatic fatty acid oxidation by bezafibrate and bezafibroyl CoA

The acute effect of the hypolipidemic agent bezafibrate on fatty acid oxidation was studied in rat hepatocytes and mitochondria. Bezafibrate caused a concentration-related inhibition of oleate oxidation in liver cells. In mitochondria bezafibrate inhibited the oxidation of palmitoyl CoA but had no effect on palmitoylcarnitine oxidation, suggesting the site of inhibition was the formation of the carnitine derivative. Bezafibrate and bezafibroyl CoA inhibited the overt carnitine palmitoyltransferase (I) in rat liver mitochondria with comparable potency but with distinct kinetics. The inhibition caused by bezafibrate was not prevented by omission of Mg++-ATP from the assay mixture, indicating activation of bezafibrate to bezafibroyl CoA was not required for inhibition. The data demonstrate that bezafibrate, like several other peroxisome proliferating agents, inhibits mitochondrial fatty acid oxidation in rat liver. The inhibition may be relevant to the mechanism of peroxisome proliferation.

Genetic ablation of IRAK4 kinase activity inhibits vascular lesion formation.

Inflammation is critically involved in atherogenesis. Signaling from innate immunity receptors TLR2 and 4, IL-1 and IL-18 is mediated by MyD88 and further by interleukin-1 receptor activated kinases (IRAK) 4 and 1. We hypothesized that IRAK4 kinase activity is critical for development of atherosclerosis. IRAK4 kinase-inactive knock-in mouse was crossed with the ApoE-/- mouse. Lesion development was stimulated by carotid ligation. IRAK4 functional deficiency was associated with down-regulation of several pro-inflammatory genes, inhibition of macrophage infiltration, smooth muscle cell and lipid accumulation in vascular lesions. Reduction of plaque size and inhibition of outward remodeling were also observed. Similar effects were observed when ApoE-/- mice subjected to carotid ligation were treated with recombinant IL-1 receptor antagonist thereby validating the model in the relevant pathway context. Thus, IRAK4 functional deficiency inhibits vascular lesion formation in ApoE-/- mice, which further unravels mechanisms of vascular inflammation and identifies IRAK4 as a potential therapeutic target.

Changes in hepatic lipid metabolism associated with lipid accumulation and its reversal in rats given the peroxisome proliferator LY171883

Dietary administration of 0.05, 0.1, and 0.3% LY171883 to rats for 1 day caused a dose-related increase in hepatic triglycerides. When added to rat liver mitochondria in vitro, LY171883 caused competitive inhibition of carnitine palmitoyltransferase 1 (CPT-1), the rate-limiting enzyme for mitochondrial fatty acid oxidation. This effect appears to be involved in the lipid accumulation. The hepatic triglycerides in rats given 0.1% LY171883 increased progressively through 3 months of treatment. In contrast, hepatic triglycerides in high-dose rats returned to control levels by Day 3 and remained there throughout the study. The regression of the lipid corresponded with increases in hepatic peroxisomal beta-oxidation, mitochondrial beta-oxidation, and CPT-1 activity of up to 13-, 7-, and 3.2-fold, respectively. The 0.1% dose increased these parameters modestly compared to those of high-dose rats (2-, 3-, and 1.6-fold, respectively). Addition of LY171883 to mitochondria from rats given dietary treatment for 2 weeks inhibited CPT-I by the same percentage as in control mitochondria. In mid-dose rats, the induction of CPT-I was largely negated by LY171883 in vitro. Even with the inhibition, CPT-I activity in mitochondria from high-dose rats remained 2-fold higher than that in untreated controls. The data suggest that the induction of CPT-I in high-dose rats was sufficient to overcome the inhibitory action of LY171883. The increased oxidative capacity in peroxisomes and mitochondria led to the regression of the lipid in high-dose rats. The more modest increases in fatty acid oxidation in rats given 0.1% LY171883 were not sufficient to reverse the lipid accumulation.

Characterization of liver enlargement induced by compound LY171883 in rats.

Compound LY171883 caused dose-related and reversible hepatomegaly in male Fischer 344 rats. Histological examination revealed hepatocellular hypertrophy with no other evidence of liver disease. There were only minor changes in serum glucose, total bilirubin, alkaline phosphatase, and alanine transaminase which were generally unrelated to dose and dissociable from the hepatomegaly. Total liver DNA increased but the DNA concentration decreased, indicating that liver growth involved a combination of hypertrophy and hyperplasia. Total liver protein and RNA increased. Hepatic mitochondrial protein content increased but cytochrome oxidase activity was not changed. There were minor changes in mitochondrial respiratory parameters; however, all the values were in the normal range and there was no indication of mitochondrial toxicity. Microsomal protein, drug-metabolizing activity, and cytochrome P-450 increased, but glucose-6-phosphatase activity was not changed. The induction of drug-metabolizing enzymes and absence of toxicity were evidence that the hepatomegaly was an adaptation to an increased functional load in the liver. An increase in catalase activity suggested that the response may have also involved peroxisomes. In addition to rats, LY171883 administration caused hepatomegaly in mice and hamsters at daily exposures exceeding 100 mg/kg. The response was not observed in guinea pigs, beagle dogs, or rhesus monkeys given maximum tolerated doses, indicating LY171883-induced hepatomegaly is not a response common to all species. The doses required to elicit hepatomegaly greatly exceeded doses that produce pharmacological efficacy in animals and those that are expected to be used clinically. Since humans will not receive doses comparable to those given rodents, and considering that the primate species tested did not experience hepatomegaly, it is unlikely that the effect observed in rodents can be extrapolated to humans.

Effects of chronic treatment with the leukotriene D4 antagonist compound LY171883 on Fischer 344 rats and rhesus monkeys

One-year toxicity studies were done to evaluate potential toxic effects associated with chronic exposure of rats and monkeys to the leukotriene antagonist LY171883. Rats were fed dietary doses of 0.0, 0.01, 0.03, or 0.1%, equivalent to approximately 0, 5, 15, or 50 mg/kg of body weight/day. Monkeys were given daily nasogastric gavage doses of 0, 30, 75, or 175 mg/kg of body weight. No treatment-related effects occurred in physical, behavioral, ocular, food consumption, or urinalysis parameters in either species. Mild dose-related hepatotoxicity occurred in rats given approximately 15 or 50 mg/kg of LY171883. The hepatotoxicity was characterized by liver enlargement associated with induction of hepatic peroxisomal beta-oxidation and microsomal drug metabolism. Male rats also had hepatocellular fatty change, centrilobular hypertrophy of hepatocytes, and increased levels of serum alanine transaminase and total bilirubin. Other effects in rats included minimal decreases in hematocrit values, decreases in serum triglycerides and cholesterol, and increased kidney weight. The monkeys tolerated daily oral doses of LY171883 up to 175 mg/kg with only minor increases in hepatic microsomal enzyme activity and slightly increased liver and kidney weights in males. No effects occurred in monkeys given 30 mg/kg. There was no induction of hepatic peroxisomal enzymes or pathologic abnormalities in monkeys treated with LY171883. The peroxisomal inductive effect was apparently a species-related effect separate from the pharmacologic activity of leukotriene antagonism.

Genome-wide screen for modulation of hepatic Apolipoprotein A-I (ApoA-I) secretion

Background: Increasing HDL-c through ApoA-I expression is hypothesized to reduce cardiovascular deaths significantly. Results: Genes that regulate hepatocyte ApoA-I secretion were identified using 21,789 siRNAs. Conclusion: Forty genes of interest were confirmed as regulators of ApoA-I production by hepatocytes. Significance: This study provides functional genomics-based data for exploring new mechanisms by which ApoA-I levels may be regulated. Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25–40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.

Cultured hepatocytes for studies of peroxisome proliferation: Methods and applications

Numerous compounds including pharmaceutical agents, herbicides, and plasticizers have been identitied as peroxisome proliferators in rodents (Each0 and Feller, 1991; Lake et al., 1984; Lalwani et al., 1983; Moody and Reddy, 1978). The hepatic effects of these compounds include increases in the number and size of hepatic peroxisomes, hepatomegaly, hepatocellular hypertrophy, and increased rates of hepatocyte replication. There are also increases in the volume and surface area of the smooth endoplasmic reticulum and mitochondria (Hess et al., 1965; Lipsky and Pedersen, 1982; Svoboda and Azarnoff, 1966). The major biochemical effects are on enzymes associated with lipid metabolism. Mitochondrial and peroxisomal fatty acid B-oxidation, carnitine acetyltransferase, and microsoma1 lauric acid hydroxylase activity (Lazarow, 1977; Lazarow and de Duve, 1976; Moody and Reddy, 1978; Orton and Parker, 1982) increase tento 40-fold. In contrast, the activity of catalase, the most abundant protein of the peroxisome, is generally increased twofold or less. Peroxisome proliferators increase the incidence of hepatocellular carcinomas in rats and mice after chronic administration (Reddy and Lalwani, 1983; Reddy and Rao, 1977; Reddy et al., 1976). In general, the potency of a compound for peroxisome proliferation correlates with its hepatocarcinogenicity. The mechanism of the carcinogenesis is not yet understood. The compounds are consistently negative in short-term genotoxicity tests including the Ames assay (Glauert et al., 1984; Warren et al., 1980), sister chromatid exchange (Linnainmaa, 1984), DNA repair (Butterworth et al., 1984; Cattley et al., 1986; Glauert et al., 1984),

Effect of the peroxisome proliferator LY171883 on triglyceride accumulation in rats fed a fat-free diet.

LY171883 is a leukotriene D4 antagonist that induces peroxisome proliferation in the rodent liver. Like many peroxisome-proliferating agents, it causes transient lipid accumulation and several other changes in hepatic lipid metabolism. The effect of LY171883 on lipid metabolism was studied further in rats maintained on a fat-free diet. Administration of a fat-free diet for 14 days caused a 5.6-fold increase in liver triglycerides associated with a 3.3-fold increase in fatty acid synthetase. Co-administration of 0.1% LY171883 increased liver triglycerides slightly, whereas 0.3% LY171883 prevented the accumulation of triglycerides. Furthermore, treatment with 0.3% LY171883 reversed the fatty liver in rats pretreated with the fat-free diet for 14 days. Fatty acid synthetase activity increased comparably in all treatment groups, indicating that 0.3% LY171883 did not prevent the lipogenic response to a fat-free diet. In rats treated with 0.3% LY171883, peroxisomal beta-oxidation increased 9.5-fold, mitochondrial beta-oxidation 4.8-fold, carnitine palmitoyltransferase I 1.9-fold, and plasma ketones 3-fold. In the 0.1% dose group the increases in these parameters were smaller. The data indicate that 0.3% LY171883 sufficiently increased mitochondrial and peroxisomal beta-oxidation such that fatty acids generated by lipogenesis were preferentially oxidized rather than esterified to triglycerides. In the 0.1% dose group oxidation was only mildly increased, and the excess fatty acids continued to be esterified.