TOXICOLOGICAL HIGHLIGHT |
Molecular Basis for Adaptive Responses during Chemically Induced Hepatotoxicity
Pharmacology Unit, School of Medicine and Pharmacology, the University of Western Australia, Nedlands, Western Australia 6009, Australia
1 To whom correspondence should be addressed at Pharmacology Unit, School of Medicine and Pharmacology, QEII Medical Centre, M-Block, Monash Ave, Nedlands, WA 6009, Australia. Fax: 61-8-9346 3469. E-mail: pburcham{at}meddent.uwa.edu.au.
Received November 28, 2005; accepted November 29, 2005
Prior exposure to toxic xenobiotics can elicit changes within liver that confer resistance upon subsequent reexposure—a phenomenon sometimes referred to as autoprotection (Dalhoff et al., 2001
; Thakore and Mehendale, 1991). Classically, the mechanisms underlying such responses have been assigned to either of two categories. First, so-called toxicodynamic alterations involve molecular changes within hepatocytes that counteract the deleterious biochemical events whereby hepatotoxicants induce toxicity. An example of this phenomenon is the induction of various antioxidant pathways in rodent liver on exposure to such diverse prooxidants as heavy metals, plasticizers, and hepatotoxic drugs (Bando et al., 2005; Nicholls-Grzemski et al., 2000; O'Brien, et al., 2000). Second, dispositional or toxicokinetic alterations decrease the delivery of chemicals to the liver or otherwise diminish the intracellular concentrations of xenobiotics attained in hepatocytes. In the past, the mechanisms underlying this second class of adaptive changes were often largely unknown. Together with other recent work from the Manautou group and other laboratories, the report by Aleksunes et al. in this issue of Toxicological Sciences shows how our understanding of the molecular basis for these complex phenomena is steadily improving.
Studying changes to the abundance of mRNA transcripts during chemical exposure holds promise for illuminating the biochemical mechanisms underlying a wide range of toxicological syndromes (Hayes and Bradfield, 2005). Use of cDNA or oligo microarrays to characterize thousands of mRNA transcripts simultaneously is a common approach, although technologies that focus on gene subsets of known toxicological relevance present a useful alternative strategy. In a companion study to the present work, Aleksunes et al. (2005) used branched DNA signal amplification to quantify mRNAs for one dozen membrane transporters in the livers of mice 6, 24, and 48 h after they received moderately hepatotoxic doses of acetaminophen or carbon tetrachloride (Aleksunes et al., 2005). These classic hepatoxicants induce liver cell injury following conversion to their respective reactive intermediates, N-acetyl-p-quinoneimine and the trichloromethyl radical. Suggesting an attempt by hepatocytes to minimize uptake of noxious substances from the sinusoidal circulation, toxic doses of acetaminophen decreased mRNA transcripts for various basolateral uptake transporters including organic anion-transporting polypeptides (Oatp1a1, Oatp1b2) and the sodium/taurocholate-cotransporting peptide (Ntcp) (Aleksunes et al., 2005). A concurrent effort by acetaminophen- and CCl4-intoxicated hepatocytes to increase compound efflux was indicated by the elevated transcripts for genes involved in canalicular transport into bile (Mrp2) and also for returning chemicals to the general circulation (the basolateral efflux transporters Mrp 1, 3, 4) (Aleksunes et al., 2005).
Although the power of transcript-profiling is readily apparent, use of this technology to characterize gene profiles in rodent models of chemical toxicity can be confounded by such technical challenges as irreproducibility, unwanted signal variation, and noise (Fielden and Zacharewski, 2001). In a similar manner, the results obtained by Aleksunes et al. (2005) were plagued by an unwanted degree of inconsistency. For example, while hepatic levels of the Oatp1a1 mRNA transcript were strongly suppressed (
90%) in mice 48 h after they received 400 mg/kg acetaminophen, this transcript was unaltered by this dose at the 24 h time point, despite the fact that a lower dose of acetaminophen (300 mg/kg) reduced this marker by 60% at the earlier time (Aleksunes et al., 2005). Furthermore, in mice that received the top dose of acetaminophen, the Mrp2 and Mrp3 genes were strongly induced at 6 and 48 h, yet were at comparable levels to controls at an intermediate time point (i.e., 24 h, see Fig. 4 in Aleksunes et al., 2005). Together with the more fundamental concern of whether altered mRNA levels for a given gene translate to corresponding changes in protein abundance, these frustrating outcomes highlight some of the challenges accompanying the use of transcript-profiling during toxicological research.
The latest work by Aleksunes et al. in this issue of Toxicological Sciences addresses some of these issues by using immunohistochemistry to assess the levels of selected membrane transporter proteins in the livers of acetaminophen and CCl4-treated mice. Consistent with regulation at the transcriptional level as a primary determinant of protein abundance, exposure-related changes to the levels of several transporter proteins were observed. Moreover, these trends correlated well with measurements of the corresponding mRNA transcripts in the preceding work. In particular, a striking induction of the sinusoidal ATP-dependent transporter Mrp4 was detected in both acetaminophen and CCl4-treated mouse livers, with the fold increases over controls equal to or exceeding those observed for Mrp4 mRNA in the earlier study. Mrp4 protein was barely detectable in control livers, yet increased strongly in centrilobular hepatocytes 24 and 48 h after mice received acetaminophen and CCl4. At the highest dose of CCl4 (i.e. 25 µl/kg), the degree of induction was, respectively, 7- and 26-fold at the 24 and 48 h time points. Mrp4 induction was typically localized to rings of perivenous hepatocytes, although the diffuse nature of the immunostaining suggested disruption of intracellular Mrp4 trafficking at the top dose of CCl4.
These intriguing observations seem likely to cultivate further fruitful research activity. An obvious issue is the functional significance of the present findings, since while alterations to membrane transporters could conceivably diminish toxicity upon reexposure to hepatotoxicants, the degree to which these changes confer autoprotection needs clarification. A range of mechanisms are thought to contribute to both acetaminophen- and CCl4-induced autoprotection, including elevated tissue repair, mitogenesis, and hepatocellular regeneration (Grunnet et al., 2003; Thakore and Mehendale, 1991). The relative importance of altered transporter expression relative to these other autoprotective mechanisms is an unanswered question that will require careful attention to experimental design during future work.
Second, the strong upregulation of Mrp4 during chemically induced hepatic injury raises the question as to which intracellular substrates are high priorities for removal by this pathway in injured hepatocytes. The list of xenobiotics that are known Mrp4 substrates is quite short but may include various nucleotide or base analogues used in the treatment of cancer or viral infections, although the physiological relevance of Mrp4 to the clearance of these drugs has been questioned (Reid et al., 2003). Given its limited role in sinusoidal xenobiotic export, the overexpression of Mrp4 seems unlikely to represent an effort by the liver to clear the hepatotoxicants used in these experiments (acetaminophen and CCl4). In contrast, a number of endogenous substances do appear to be transported by Mrp4, including various eicosanoids derived from arachidonic acid (e. g., prostaglandins E1 and E2) (Reid et al., 2003). The Mrp4 pathway may also serve as a low-affinity, "overflow" transporter for cyclic nucleotides under conditions where phosphodiesterase activity is limiting (Wielinga et al., 2003).
The recent finding that Mrp4 efficiently transports sulfated bile acids may be particularly relevant to its induction during chemically induced hepatotoxicity. Under cholestatic conditions where apical bile acid export is impaired, compensatory Mrp4 up-regulation accompanied by sulfotransferase induction may offset hepatic accumulation of these toxic species, thereby explaining the strong increases in serum sulfated bile acids during experimental cholestasis in rats (Zelcer et al., 2003). Abnormalities in serum bile acids have long been known to accompany poisoning syndromes involving acetaminophen and CCl4 (Anwer et al., 1976; James et al., 1975). Future studies could seek to clarify the contribution of hepatic Mrp4 induction to the altered plasma profiles of monoanionic and dianionic bile acids seen in hepatotoxicant-treated animals.
Another question raised by the new work concerns the mechanism whereby reactive intermediates as diverse as the quinoneimine derived from acetaminophen and the free radical formed from CCl4 can trigger the common induction of a membrane transporter such as Mrp4. An emerging concept in molecular toxicology is that reactive intermediates attack cysteine residues in "sensor trigger" proteins, producing structural alterations and activating signaling networks that alter patterns of gene expression or commit cells to cell death/cell survival decisions. A classic example of such a trigger protein is KEAP1, a thiol-rich species that upon oxidative damage releases its partner protein NRF2, allowing the latter to activate various genes involved in antioxidant defense and conjugative drug metabolism (Dinkova-Kostova et al., 2002). In contrast, little is known concerning the transcriptional activation of Mrp4, although the recent finding that Mrp4 expression in the basolateral membranes of murine hepatocytes is regulated by constitutive androstane receptor suggests future work could explore the interaction of reactive intermediates with this nuclear receptor and its partner proteins (Assem et al., 2004).
Finally, by providing new insights into the range of molecular alterations occurring in the xenobiotic-challenged rodent liver, the new findings underscore inadequacies in existing classification systems commonly used in toxicology. While the "toxicokinetic versus toxicodynamic" categorization is useful in conceptual terms, the findings of Aleksunes and associates demonstrate that such binary classifications may not fully embrace all of the molecular events occurring in xenobiotic-challenged tissues. Thus, in addition to the toxicokinetic alterations discussed above (i.e., induced membrane transporter expression), strong hepatic induction of the stress protein HO-1 was detected in acetaminophen- and CCl4-intoxicated mice. The latter response clearly belongs to the category of toxicodynamic alterations, since HO-1 is an inducible stress response protein that catabolizes heme groups released from drug-metabolizing enzymes after damage by oxidants and electrophiles (Bauer and Bauer, 2002). By confirming that adaptive responses to chemical hepatotoxicants includes molecular changes that encompass features of both toxicokinetic and toxicodynamic alterations, the work by Aleksunes et al. (2005 and manuscript in this journal) underscores the valuable contributions of mechanistic studies in toxicology.
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