Drug-induced hepatoxicity is a major concern when developing new therapeutic agents, and a leading cause of acute liver failure. Toxic metabolites can lead to oxidative stress and DNA damage in hepatocytes, while the innate and adaptive immune systems respond with both damaging and hepatoprotective functions (1). In the case of acetaminophen, CYP enzymes such as Cyp2e1 oxidize the drug to N-acetyl-p-benzoquinone imine (NAPQI). When NAPQI levels become high enough to deplete glutathione (GSH), which conjugates and detoxifies NAPQI, liver injury ensues (2). While drug-induced liver injury (DILI) from acetaminophen is dose-dependent, toxicity from other drugs can be idiosyncratic (3). Understanding the mechanisms of hepatotoxicity could not only help in predicting which individuals may be susceptible to DILI, but could also lead to treatments to protect the liver during or after exposure to hepatotoxins.
Recently, new findings have started to shed light on a number of different mechanisms that could be exploited to prevent or ameliorate drug toxicity. For example, Ni et al. demonstrated that autophagy combats acetaminophen-induced hepatotoxicity by removing damaged mitochondria from cells, preventing cell death (4). Meanwhile, Singh et al showed that camphene and geraniol, plant-derived terpenes, could block oxidative stress and mitochondrial dysfunction induced by the anti-inflammatory drug nimesulide, when administered prior to the drug (5).
An exciting study by Hur et al. showed that regulated IRE1-dependent decay (RIDD) of a set of mRNAs involved in drug metabolism ameliorated acetaminophen-induced hepatotoxicity. IRE1-alpha, a transmembrane protein of the endoplasmic reticulum (ER), is well known for its role in the ER stress response/unfolded protein response (UPR). In this study, inducible deletion of XBP1, IRE1-alpha's downstream transcription factor, constitutively activated IRE1-alpha and protected mice from hepatotoxicity after treatment with acetaminophen. Activation of IRE1-alpha drove RIDD directed at the drug metabolism enzymes Cyp1a2 and Cyp2e1, which break acetaminophen down into the toxic metabolites that lead to liver injury. These studies and others suggest that there are many routes to preventing drug toxicity.
In recent years, studies have suggested a role for gap junction intercellular communication in spreading liver damage and immune responses. Cell-cell communication through gap junctions involves connexin protein channels that allow intercellular exchange of ions and metabolites (7). In the liver, studies have suggested that communication through gap junctions is involved in bile secretion and ammonia detoxification among other processes, as well as in regulating cell cycle progression, cell proliferation and differentiation, and cell death (8). Recently, gap junctions have also been proposed as players in transmitting antiviral and apoptotic signals in the context of infection and drug toxicity, respectively.
The role of gap junctions in propagating antiviral signals was defined in 2009, using a hepatocyte-derived ISRE-GFP reporter cell line and double-stranded DNA as a stimulus (9). When treated with dsDNA, cells showed a striking spatial pattern of activation, becoming activated in clusters rather than evenly across the culture. These few activated cells produced most of the cytokines, such as IFN-beta, and activated and nonactivated cells alike reaped the benefits: IFN-beta-inducible antiviral genes were expressed fairly equally between these groups. Contact-dependent cell communication through gap junctions was responsible for the formation of these activated cell clusters, and impairing this communication led to diminished levels of antiviral and inflammatory cytokines.
The following year, gap junction communication was shown to play a role in liver damage in response to acetaminophen toxicity (10). Rats with a mutated gene for connexin 32, one of the primary gap junction proteins in the liver, showed less cell damage and inflammation than wild-types in response to high doses of acetaminophen, and other markers of liver damage such as increased liver weight were also diminished. Additionally, levels of cytochrome P450 mRNAs were constant between wild-type and Cx32 mutants, suggesting that differences in drug metabolism were not responsible for the change. However, many details about gap junctions' role in drug toxicity remained to be clarified.
A new study from Patel et al elaborates on the role of connexin 32 and gap junctions in acetaminophen-induced toxicity, and introduces a potential therapeutic strategy based on these findings (11) (see figure Drug toxicity
). The team first used connexin 32 knockout mice to clarify the role of gap junction communication in spreading liver injury. Upon treatment of the mice with a known hepatotoxin, thioacetamide (TAA), the differences were dramatic. Knocking out connexin 32 protected mice from liver hemorrhage and necrosis, and inflammation. Serum ALT/AST levels were highly elevated in wild-type mice, but not Cx32 knockouts, and while the TAA dose was lethal for the normal mice, every Cx32 knockout mouse survived, showing the vital role of gap junctions in amplifying liver injury to lethal levels.
The signal transmitted through the gap junctions appeared to be an oxidative stress signal. Firstly, livers from TAA-treated wild-type mice displayed substantially more reactive oxygen species than Cx32 knockouts. Moreover, a co-culture experiment showed that, for cells exposed to the reactive TAA metabolite, both exposure to free radicals and expression of Cx32 are required for signaling adjacent cells to produce reactive oxygen species.
Ultimately, if these findings are to translate to the clinic, effective Cx32 inhibitors will need to be developed and tested. Toward this goal, Patel and colleagues screened for small-molecule Cx32 inhibitors, and demonstrated that 2-aminoethoxydipenyl-borate (2APB) inhibited communication through gap junctions both in vitro and in vivo. They proceeded to test its hepatoprotective capacity in 3 situations: pretreatment, coadministration, and post-exposure.
Pretreatment with 2APB protected wild-type mice from TAA- or acetaminophen-induced hepatotoxicity, just as knocking out Cx32 had protected mice from TAA-induced liver injury in the earlier experiments. Coadministering 2APB with each of these drugs also protected mice from signs of liver toxicity such as elevated levels of serum ALT, higher levels of free radicals in the liver, inflammation, and hepatic necrosis. Moreover, mortality was also significantly reduced. Finally, the team tested 2APB's ability to halt liver damage after exposure, such as in cases of overdose, and found that 2APB could prevent damage 1.5 hours afterward, and could still limit its progression after 6 hours. These findings suggest that targeting gap junction proteins could be a broad strategy for preventing drug-induced liver injury at all of these stages.
Drug toxicity continues to be a significant health threat, but several new opportunities for addressing this problem are starting to take shape. Development of treatments targeting various processes, including injury signal propagation, cellular disposal of damaged organelles, and more hold the promise for improved drug safety.
In assessing both the toxicity of new therapeutics and the effectiveness of strategies to ameliorate hepatotoxic effects, it is beneficial to study the expression of genes involved in drug metabolism, cell stress responses, and other related pathways. RT2 Profiler PCR Arrays are panels of the 84 genes most relevant to biological and disease-related pathways, and are a fast, effective way to get an overview of the most important gene expression changes in your system.
- Holt, M.P., and Ju, C. (2006) Mechanisms of drug-induced liver injury. AAPS J. 8, E48.
- Kaplowitz, N. (2004) Acetaminophen hepatotoxicity: what we know, what we don't know, and what do we do next? Hepatology 40, 23.
- Navarro, V.J., and Senior, J.R. (2006) Drug-related hepatotoxicity. New. Engl. J. Med. 354, 731.
- Ni, H.M. et al. (2012) Activation of autophagy protects against acetaminophen-induced hepatotoxicity. Hepatology 55, 222.
- Singh, B.K., Tripathi, M., Chaudhari, B.P., Pandey, P.K., and Kakkar, P. (2012) Natural terpenes prevent mitochondrial dysfunction, oxidative stress and release of apoptotic proteins during nimesulide-hepatotoxicity in rats. PLoS One 7, e34200.
- Hur, K.Y. et al. (2012) IRE-alpha activation protects mice against acetaminophen-induced hepatotoxicity. J. Exp. Med. 209, 307.
- Evans, W.H., and Martin, P.E. (2002) Gap junctions: structure and function (Review).Mol. Membr. Biol. 19, 121.
- Kar, R., Batra, N., Riquelme, M.A., and Jiang, J.X. (2012) Biological role of connexin intercellular channels and hemichannels. Arch. Biochem. Biophys. 524, 2.
- Patel, S.J., King, K.R., Casali, M., and Yarmush, M.L. (2009) DNA-triggered innate immune responses are propagated by gap junction communication. Proc. Natl. Acad. Sci. USA 106, 12867.
- Naiki-Ito, A. (2010) Gap junction dysfunction reduces acetaminophen hepatotoxicity with impact on apoptotic signaling and connexin 43 protein induction in rat. Toxicol. Pathol. 38, 280.
- Patel, S.J., et al. (2012) Gap junction inhibition prevents drug-induced liver toxicity and fulminant hepatic failure. Nat. Biotech. 30, 179.