PPAR-{alpha}: A Key to the Mechanism of Hepatoprotection by Clofibrate

Harihara M. Mehendale

Department of Toxicology, College of Pharmacy, The University of Louisiana at Monroe, 700 University Avenue, Monroe, Louisiana 71209

ABSTRACT

The article highlighted in this issue is "Peroxisome Proliferator-Activated Receptor Alpha-Null Mice Lack Resistance to Acetaminophen Hepatotoxicity Following Clofibrate Exposure" by Chuan Chen, Gayle E. Hennig, Herbert E. Whiteley, J Christopher Corton, and José E. Manautou (pp. 338–344).

Clofibrate (CFB), a hypolipidemic drug, known to induce peroxisome proliferation, is a member of a large class of diverse exogenous and endogenous chemicals known as peroxisome proliferators (PPs). Peroxisomes are subcellular organelles that are found in most animal cells and perform diverse metabolic functions, including H2O2-derived respiration, ß-oxidation of fatty acids, and cholesterol metabolism. PPs are considered to cause cancer, by altering gene expression and affecting the phenotype of the target cell in a manner similar to steroid hormone receptor ligands such as estrogen. Peroxisome proliferation (PP) response is most notable in the liver and kidney, and shows a strong species difference with rats and mice being sensitive, while humans are relatively refractory. Many of the effects of PPs have been shown to be receptor-mediated. Activation of the peroxisome proliferator activated receptor-{alpha} (PPAR-{alpha}), a member of the nuclear superfamily highly expressed in hepatocytes, cardiomyocytes, enterocytes and renal proximal tubule cells, has been strongly correlated with peroxisome proliferation and liver cancer (Corton et al., 2000Go). PPARs are ligand-activated transcription factors activated by xenobiotic-PPs, endogenous fatty acids, and eicosanoids, and have been cloned from several species, including human, which helps explain the molecular events involved in PP-dependent gene regulation. They control gene expression by interacting with specific DNA response elements located upstream of responsive genes, which contain peroxisome proliferator response element (PPRE) motifs including acyl-CoA, liver fatty acid binding protein, microsomal CYP4A, a fatty acid {omega}-hydroxylase and growth regulatory genes such as c-myc, c-Ha-ras, fos, jun, and egr-1 (Corton et al., 2000Go; Vanden Heuvel, 1999Go). The growth regulatory genes are pivotal in the progression of cell cycle, in particular, the transition from G1 to S phase. The obligatory need for PPAR-{alpha} activation for the expression of PP-induced events is evident in the observations that PPAR-{alpha} knockout mice do not show the morphological and biochemical changes that are typically observed in rodents following acute or chronic administration of PPs.

In 1992, Nicholls-Grzemski and associates (Nicholls-Grzemski et al., 1992Go) demonstrated that PPs of as diverse a chemical composition and properties as fibrates (e.g., CFB), phthalate-ester plasticizers and herbicides were equally effective in blocking acetaminophen (APAP) hepatotoxicity. Soon thereafter, attracted by the possibility that the key to hepatoprotection by PPs such as CFB might lie either in the alteration of major APAP electrophile's (N-acetyl p-benzoquinoneimine, NAPQI) target proteins or CFB-induced modification in APAP metabolism impacting the availability of NAPQI, Manautou and associates undertook a series of investigations. Although selective covalent binding of the APAP electrophile was diminished by prior exposure to CFB, companion in vitro studies revealed that the specific activity in hepatic microsomes for APAP oxidation by cytochrome P-450 was not decreased by CFB pretreatment. Even though hepatic glutathione-S-transferase activity was decreased by 25% after multiple exposure to CFB, hepatic glutathione (GSH) content itself was increased. This suggested the possibility that increased availability of GSH to trap the APAP electrophile nonenzymatically may decrease covalent binding, thereby preventing hepatotoxicity. However, subsequent studies established that pre-administration of a single dose of CFB (in contrast to 5- or 10-day multiple treatments) provided hepatoprotection against APAP. Most interestingly, neither a decrease in covalent binding of the APAP electrophile nor any decrease in the GSH depletion (Table 1Go) accompanied hepatoprotection.


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TABLE 1 Mechanism of Hepatoprotection by Peroxisome Proliferators
 
In contrast to the multiple doses of CFB, a single dose of CFB did not increase hepatic GSH content. Therefore, it is clear that protection against APAP hepatotoxicity by CFB does not require repeated CFB administration. Protection does not depend upon prior elevation of hepatic GSH, and occurs in the absence of any significant decrease in selective covalent binding. In all of the above hepatoprotection studies (Table 1Go) liver injury of APAP was measured only at 24 h or earlier leaving the possibility of a potential shift or delay in the time course of injury. However, the investigators also reported protection against 38% mortality of APAP (800 mg/kg) in the single dose protection experiments. Protection against APAP mortality is significant. This suggests that hepatoprotection by a single dose of CFB and possibly with multiple dose studies cannot be explained by a shift or delay in the time course of liver injury.

Previous studies by these investigators have established that hepatoprotection by CFB is not limited to APAP alone. Protection against liver injury of as structurally and mechanistically diverse hepatotoxicants as bromobenzene, chloroform and carbon tetrachloride was demonstrated using the same 10-day pretreatment protocol for CFB (Manautou et al., 1996Go) as used in the present study. These observations argue against the suggested pivotal role of the higher GSH availability for electrophile trapping as the mechanism of hepatoprotection. Also, hepatoprotection against carbon tetrachloride, which is not GSH-dependent, diminishes the likelihood of this mechanism. The investigators have suggested that the mechanism of hepatoprotection by multiple exposures to CFB might be different from the mechanism of protection by single dose of CFB. The single dose protection experiments are critical in that the findings signal a dissociation of the critical protective mechanism from hepatic GSH content, depletion of hepatic glutathione and any decrease in selective covalent binding. This possibility, based on the assumption that the mechanism of protection is the same regardless of whether a single dose or multiple doses of CFB were used, seems worthy of further consideration. The above observations throw the original question on the mechanism of hepatoprotection by PPs wide open.

In this issue of Toxicological Sciences, Manautou and associates show that the nuclear receptor PPAR-{alpha} is required for hepatoprotection against APAP by 10-day pre-exposure to CFB. They treated female wild type and PPAR-{alpha} knockout mice with 500 mg CFB/kg, ip in corn oil vehicle for 10 days. After overnight fasting (18 h), these mice received a single hepatotoxic oral dose of 400 mg APAP/kg in 50% propylene glycol vehicle. Consistent with earlier findings reported by these investigators, pre-exposure to CFB afforded remarkable protection from APAP-induced liver injury assessed both by elevation of serum sorbitol dehydrogenase (SDH) and liver histopathology at 24 h after the administration of APAP. In contrast, protection against APAP hepatotoxicity was abolished in PPAR-{alpha} knockout mice. Selective arylation of cytosolic proteins measured as an index of APAP bioactivation to electrophilic NAPQI, and hepatic necrosis, were decreased in wild type mice but not in the PPAR-{alpha} knockouts. Concordantly, hepatic GSH depletion was reduced in wild type and not in the knockout mice. Thus, the lack of reduction in hepatic GSH depletion and covalent protein binding of APAP electrophile in the PPAR-{alpha} knockout mice, correlated well with the loss of hepatoprotection in the PPAR-{alpha} knockout mice, consistent with the well established paradigm. These studies provide a very important and significant clue to potential mechanism(s) of hepatoprotection by CFB and other PPs.

Any potential candidate mechanisms must account for two important hallmarks of hepatoprotection by CFB: leakage of marker enzyme (SDH) from hepatocytes; and decreased cell death. In this regard, it is important to consider different forms of cell death that may contribute to tissue injury. APAP liver injury might be accounted for by at least three types of cell death: (1) mechanism-based cell death; (2) cell death by lytic enzymes (death proteins) released by dead or dying cells [as in (1)]; and, (3) apoptosis. Present data are consistent with multiple mechanisms. Although many mechanisms have been advanced to explain APAP toxicity, in view of the present findings any potential mechanism must involve activation of PPAR-{alpha}. Regardless of which cellular mechanism is involved in hepatoprotection by CFB, the finding that hepatoprotection is dependent on PPAR-{alpha} receptor activation is highly significant, helps to discount some potential mechanisms, and points to an exciting set of novel possibilities. Some potential possibilities are:

  1. Increased antioxidants and oxyradical quenching enzymes (e.g., catalase) protect against oxyradical-mediated cell death.
  2. PPAR-{alpha} regulates the expression of many proteins, by decreasing the selective arylation and/or by dampening the impact of covalent binding on cellular integrity.
  3. PPAR-{alpha} activation may prevent cell death by inducing resistance in hepatocytes and/or by preventing cell death from externalized "death proteins" from the dead or dying cells via induction of inhibitors of death proteins.
  4. PPAR-{alpha} activation stimulates cell proliferation through responsive mitogenic genes to obtund the progression of liver injury, thereby paving the way for regression of injury via cell replacement.

There is ample evidence to suggest that a variety of antioxidants protect against APAP toxicity. PPs such as CFB characteristically induce antioxidant defenses such as catalase. CFB may bolster the ability of hepatocytes to detoxify oxidant injury and thereby prevent cell death. Expression of many proteins by PPAR-{alpha} activation may contribute to a decrease in selective covalent binding of the APAP electrophile. PPs are well known to regulate the expression of over 100 proteins, some of which are involved in fatty acid metabolism and cell proliferation. It is not unreasonable to assume that PPAR-{alpha} activation by PPs may stimulate or decrease the expression of many other proteins. For instance, PPs may decrease the expression of acetaminophen binding proteins (ABPs, 56 and 44 kDa). However, the investigators have confirmed that 58-kDa protein is not decreased after multiple exposure to CFB. Furthermore, other hepatotoxicants such as carbon tetrachloride are not known to express their toxicity by binding to these proteins and yet, are protected by exposure to CFB.

Manautou and associates have already examined several potential hepatoprotective mechanisms. They have shown that pre-exposure to CFB is unlikely to decrease APAP bioactivation. Decreased selective binding of the APAP electrophile may still turn out to be an important factor. However, the underlying mechanism is yet to be identified. Whether it is related to modulation of selective binding proteins by PPAR-{alpha} dependent mechanisms after multiple exposure to CFB remains to be investigated. Higher hepatic GSH content, observed with multiple exposure to CFB may not be the pivotal mechanism in view of hepatoprotection by a single dose of CFB, which does not increase GSH. Although the investigators interpret these observations to signal two separate mechanisms for protection by a single CFB versus multiple CFB exposure, the alternative possibility that the apparent difference may merely stem from an extended effect of PPAR-{alpha} activation after multiple exposure appears viable. A shift or delay in the time course of injury is unlikely in view of the protection against APAP-induced lethality (38% after 800 mg APAP/kg) after single exposure to CFB. Collectively, these observations suggest that hepatoprotective mechanism is unlikely to be related to mechanisms of initiation of liver injury. Instead, hepatoprotection is more likely to be closely related to a combination of cellular resistance against mechanism-based cell death and an early onset of mitogenic stimulation and cell replacement. The following discussion is intended to suggest these latter possibilities, which are dependent on PPAR-{alpha} activation and are consistent with the investigator's present work showing that hepatoprotection is abolished in the absence of PPAR-{alpha}.

PPAR-{alpha} activation by CFB and other PPs is known to stimulate mitogenic genes and the progression of G0 to S phase. Therefore, an obligatory need for nuclear PPAR-{alpha}-ligand activated transcription factor to afford hepatoprotection against APAP by CFB is consistent with the possibility that hepatoprotection may be rooted wholly or partly in the stimulation of mitogenic response through activation of PPAR-{alpha}. Early onset of cell division and cell replacement mediated by PPAR-{alpha} activation would have a distinct advantage in restoring lost tissue and rapid recovery from injury. Thus stimulation of cell proliferation via PPAR-{alpha} activation as a survival strategy is loaded with several advantages, all of which collectively enhance the chances of not only decreasing the progressive phase of injury, but also rapid recovery from mechanism-based injury inflicted during the early hours after the administration of APAP (Soni and Mehendale, 1998Go). Annexins, induced in pre-proliferative and proliferating cells are known to impart resistance against cell death by toxicants. Such a mechanism may explain lack of cell death in the CFB model of hepatoprotection.

Prior to this study, an association between peroxisome proliferation and hepatoprotection was not established in spite of substantial evidence for the hepatoprotective effects of PPs. Evidence presented by Manautou and associates represents a significant milestone towards understanding the mechanism of hepatoprotection. Since a large number of hepatic effects produced by PPs have been attributed to transcriptional regulation of PPAR-{alpha} response genes, this study suggests the possibility that hepatoprotection may also be mediated by this mechanism. With the advent of this finding, a number of novel potential mechanisms including those discussed above and avenues for their experimental validation have come to surface. This development is also likely to spawn additional inquiries into the role of PPAR-{alpha} receptor in additional acute or chronic effects of xenobiotics.

ACKNOWLEDGMENTS

This article was supported by the Louisiana Board of Regents Fund through The University of Louisiana at Monroe for the Kitty DeGree Endowed Chair in Toxicology.

REFERENCES

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Nicholls-Grzemski, F. A., Calder, I. C., and Priestly, B. G. (1992). Peroxisome proliferators protect against paracetamol hepatotoxicity in mice. Biochem. Pharmacol. 43, 1395–1396.[ISI][Medline]

Manautou, J. E., Emeigh, S. G., Khairallah, E. A., and Cohen, S. D. (1996). Protection against acetaminophen hepatotoxicity by a single dose of clofibrate: Effects on selective protein arylation and glutathione depletion. Fundam. Appl. Toxicol. 29, 229–237.[ISI][Medline]

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