Mechanisms Of Drug-induced Toxicities

Heart, Lung, And Blood Institute

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Abstract

We have continued our studies of the molecular basis of drug-induced liver disease (DILD), which is a rare but often life-threatening toxicity. It is the major cause of acute liver failure and a principal reason drugs are withdrawn from clinical use. We have hypothesized that the idiosyncratic nature of this disease is due in part to a deficiency in hepatoprotective factors. This idea has been explored in the following ways this year: 1. Last year we reported that endogenous IL-13 can protect mice from acetaminophen-induced liver disease (AILD), which is a leading cause of drug-induced liver failure. This year with the use of IL-13 knockout mice and multiplex protein chips we have discovered that IL-13 deficiency leads to the over-expression of several proinflammatory cytokines and chemokines, which may have a role in determining the susceptibility of mice and possibly humans to liver disease caused by acetaminophen and other drugs. 2. Last year we reported that one of the ways the antidote N-acetyl-L-cysteine (NAC) may protect mice and possibly humans from AILD is by up-regulating several potential hepatoprotectic factors including heat shock proteins, acute phase proteins, and cell cycle regulation genes. This year we have found that one of the acute phase proteins induced by NAC in the liver is lipocalin 2. We are currently determining whether deficiency in this acute phase protein, produced by administering neutralizing antibodies to wild type mice or by the use of lipocalin 2 knockouts, makes mice more susceptible to AILD. 3. Last year we investigated the multiplicity of hepatoprotectant factors in DILD by comparing the liver proteomes of resistant (SJL) and susceptible (C57Bl/6) strains of mice to AILD with the use of isotope-coded affinity tag (ICAT) mass spectrometry. The results revealed that SJL mice had higher levels of several proteins that could potentially protect the liver from injury. This year we have explored the polygenicity of regulatory factors by comparing hepatic mRNA expression profiles of these two strains of mice with that of SJLxB6-F1 hybrid mice, which were found to be of intermediate susceptibility to AILD. Global hepatic gene expression profiling over a 24 h period following APAP treatment revealed a unique elevated pattern in the mRNA expression of several cytoprotective genes in resistant SJL mice compared to susceptible B6 mice, while SJLxB6-F1 mice had intermediate mRNA expression levels of these genes. One of these genes encoded for heat shock protein (HSP) 70 whose relative protein expression among the three strains of mice was found to parallel that of their mRNA levels, suggesting that this protein had a protective role against AILD. However, there was no difference in the susceptibility of HSP70 knockout mice to AILD compared to wild-type mice. There were also a number of pro-inflammatory genes, such as osteopontin, with elevated mRNA expression levels in the B6 mice compared to the SJL mice, and with intermediate levels in the SJLxB6-F1 mice, suggesting that they may play a role in exacerbating liver injury after APAP treatment. In support of this idea, osteopontin knockout mice were found to be more resistant to AILD than wild-type mice. Additionally, the results from both the proteomic and genomic studies were compared and found to be complementary to each other and not simply overlapping. 4. Current evidence indicates that DILD is often caused by an allergic response (drug-induced allergic hepatitis, DIAH) induced by hepatic drug-protein adducts. The low incidence of DIAH and inability to reproduce it in animals suggests that tolerogenic mechanisms may prevent DIAH from occurring in most people and animals. In this regard, last year we discovered that hepatotoxic doses of APAP were associated with marked lymphocytolysis in the thymus, spleen, and hepatic lymph nodes of mice. This year we have found that CD4 and CD8 single positive T cells as well CD4CD8 double positive T cells and B cells are diminished by this process, resulting in the inhibition of the adaptive immune system. Moreover, the degree of lymphocytolysis correlated with serum corticosterone levels, suggesting a role of this stress hormone in the mechanism of lymphocytolysis. Similar processes may occur in humans and help explain the relatively low incidence of DIAH.