Pathologic role of stressed-induced glucocorticoids in drug-induced liver injury in mice.

UNLABELLED: We previously reported that acetaminophen (APAP)-induced liver injury (AILI) in mice is associated with a rise in serum levels of the glucocorticoid (GC), corticosterone. In the current study, we provide evidence that endogenous GC play a pathologic role in AILI. Specifically, pretreatment of mice with the GC receptor (GCR) inhibitor, RU486 (mifepristrone), protected normal but not adrenalectomized mice from AILI, while pretreatment with dexamethasone, a synthetic GC, exacerbated AILI. RU486 did not affect the depletion of whole liver reduced GSH or the formation of APAP-protein adducts. It also had no effects on the formation of reactive oxygen species or the depletion of mitochondrial GSH or ATP. While RU486 pretreatment also protected against halothane-induced liver injury, it exacerbated concanavalin A (ConA)- and carbon tetrachloride (CCl(4))-induced liver injury, demonstrating the complexity of GC effects in different types of liver injury. CONCLUSION: These results suggest that under certain conditions, elevated levels of GC might represent a previously unappreciated risk factor for liver injury caused by APAP and other drugs through the diverse biological processes regulated by GCR.

The role of cytokines in the mechanism of adverse drug reactions.

Cytokines are thought to play a role in acute and/or immune-mediated adverse drug reactions (ADRs) due to their ability to regulate the innate and adaptive immune systems. This role is highly complex owing to the pluripotent nature of cytokines, which enables the same cytokine to play multiple roles depending on target organ(s) involved. As a result, the discussion of cytokine involvement in ADRs is organized according to target organ(s); specifically, ADRs targeting skin and liver, as well as ADRs targeting multiple organs, such as drug-induced autoimmunity and infusion-related reactions. In addition to discussing the mechanism(s) by which cytokines contribute to the initiation, propagation, and resolution of ADRs, we also discuss the usefulness and limitations of current methodologies available to conduct such mechanistic studies. While animal models appear to hold the most promise for uncovering additional mechanisms, this field is plagued by a lack of good animal models and, as a result, the mechanism of cytokine involvement in ADRs is often studied using less informative in vitro studies. The recent formation of the Drug-Induced Liver Injury Network, whose goal is collect thousands of samples from drug-induced liver injury patients, has enormous potential to advance knowledge in this field, by enabling large-scale cytokine polymorphism studies. In conclusion, we discuss how further advances in this field could be of significant benefit to patients in terms of preventing, predicting, and treating ADRs.

Pathogenic role of natural killer T and natural killer cells in acetaminophen-induced liver injury in mice is dependent on the presence of dimethyl sulfoxide.

UNLABELLED: Dimethyl sulfoxide (DMSO) is commonly used in biological studies to dissolve drugs and enzyme inhibitors with low solubility. Although DMSO is generally thought of as being relatively inert, it can induce biological effects that are often overlooked. An example that highlights this potential problem is found in a recent report demonstrating a pathogenic role for natural killer T (NKT) and natural killer (NK) cells in acetaminophen-induced liver injury (AILI) in C57Bl/6 mice in which DMSO was used to facilitate acetaminophen (APAP) dissolution. We report that NKT and NK cells do not play a pathologic role in AILI in C57Bl/6 mice in the absence of DMSO. Although AILI was significantly attenuated in mice depleted of NKT and NK cells prior to APAP treatment in the presence of DMSO, no such effect was observed when APAP was dissolved in saline. Because of this unexpected finding, the effects of DMSO on hepatic NKT and NK cells were subsequently investigated. When given alone, DMSO activated hepatic NKT and NK cells in vivo as evidenced by increased NKT cell numbers and higher intracellular levels of the cytotoxic effector molecules interferon-gamma (IFN-gamma) and granzyme B in both cell types. Similarly, when used as a solvent for APAP, DMSO again increased NKT cell numbers and induced IFN-gamma and granzyme B expression in both cell types. CONCLUSION: These data demonstrate a previously unappreciated effect of DMSO on hepatic NKT and NK cells, suggesting that DMSO should be used cautiously in experiments involving these cells.

Hepatoprotective role of endogenous interleukin-13 in a murine model of acetaminophen-induced liver disease.

Recent evidence suggests that a deficiency in one or more hepatoprotective regulatory mechanisms may contribute to determining susceptibility in drug-induced liver disease. In the present study, we investigated the role of interleukin (IL)-13 in acetaminophen (APAP)-induced liver disease (AILD). Following APAP (200 mg/kg) administration to male C57BL/6 wild-type (WT) mice, hepatotoxicity developed up to 24 h post-APAP, with a concomitant increase in serum IL-13 concentration. Pretreatment of these mice with an IL-13-neutralizing antibody exacerbated liver injury, as did APAP administration to IL-13 knockout (KO) mice in comparison to WT mice. No difference was observed in either overall APAP-protein adduct formation or liver glutathione levels between KO and WT mice following APAP administration, suggesting that the increased susceptibility of IL-13 KO mice to AILD was not due to enhanced APAP bioactivation but rather injurious downstream events. In this regard, multiplex antibody arrays were used to identify potential IL-13-regulated biomarkers, including various cytokines and chemokines, as well as nitric oxide (NO), associated with AILD that were present at higher concentrations in the sera of APAP-treated IL-13 KO mice than in WT mice. Subsequent inhibition studies determined interferon-gamma, NO, neutrophils, natural killer cells, and natural killer cells with T-cell receptors had pathologic roles in AILD in IL-13 KO mice. Taken together, these results suggest that IL-13 is a critical hepatoprotective factor modulating the susceptibility to AILD and may provide hepatoprotection, in part, by down-regulating protoxicant factors and cells associated with the innate immune system.

Lymphocyte loss and immunosuppression following acetaminophen-induced hepatotoxicity in mice as a potential mechanism of tolerance.

Current evidence suggests that drug-induced liver disease can be caused by an allergic response (drug-induced allergic hepatitis, DIAH) induced by hepatic drug-protein adducts. The relatively low incidence of these reactions has led us to hypothesize that tolerogenic mechanisms prevent DIAH from occurring in most people. Here, we present evidence for the existence of one of these regulatory pathways. Following a hepatotoxic dose of acetaminophen in C57Bl/6 mice, lymphocyte loss that appeared to be due at least in part to apoptosis was noted in the spleen, thymus, and draining lymph nodes of the liver. There was no observable lymphocyte loss in the absence of hepatotoxicity. Acetaminophen-induced liver injury (AILI) also led to a functional suppression of the immune system as determined by the inhibition of a delayed-type hypersensitivity response to dinitrochlorobenzene. Further studies with adrenalectomized mice suggested a role for corticosterone in the depletion of lymphocytes following APAP-induced liver injury. In conclusion, these findings suggest that lymphocyte loss and immunosuppression following AILI may prevent subsequent occurrences of allergic hepatitis and possibly other forms of APAP-induced allergies induced by hepatic drug-protein adducts. Similar regulatory pathways may inhibit other hepatotoxic drugs from causing allergic reactions.

Evidence of an immune-mediated mechanism for an idiosyncratic nevirapine-induced reaction in the female Brown Norway rat.

Previously, we reported a new animal model of an idiosyncratic drug reaction in which nevirapine causes a skin rash in some rats that has characteristics similar to the reaction that occurs in humans. Strong evidence that the reaction is immune-mediated was found; specifically, low-dose pretreatment induced tolerance, while with rechallenge, the time to onset decreased and the severity increased. Furthermore, splenocytes from rechallenged rats transferred rash susceptibility to naïve recipients. We now report the results of studies to explore the immune aspects of this reaction. T cells were found to play an important role, as demonstrated by their ability to adoptively transfer susceptibility to the skin reaction. Of these T cells, CD4+ cells are the likely effectors because they were capable of transferring susceptibility and the reaction was delayed in rats partially depleted of CD4+ T cells. In contrast, it appears that CD8+ T cells are not essential, as CD8+ T cells were unable to transfer sensitivity to a naïve animal and rats depleted of CD8+ T cells still developed skin rash. Unlike the penicillamine model, where we have demonstrated that the tolerance induced by low-dose treatment is immune-mediated, tolerance induced by low-dose nevirapine appears to be largely due to induction of metabolism as it can be overcome by inhibition of cytochrome P450. Pretreatment with the immunosuppressants, cyclosporine and tacrolimus, prevented the rash and even led to resolution of the rash during nevirapine treatment. These studies reinforce the hypothesis that the reaction in this model is similar to that which occurs in humans. In particular, the finding that CD4+ T cells may play a central role in this model fits with the observation that the incidence of idiosyncratic reactions to nevirapine in humans appears to be lower in patients with low CD4+ counts.

D-penicillamine-induced autoimmunity in the Brown Norway rat: role for both T and non-T splenocytes in adoptive transfer of tolerance.

The D-penicillamine autoimmune syndrome observed in Brown Norway (BN) rats is similar to an idiosyncratic reaction seen in some patients. We have previously shown that 2 weeks of low dose (5 mg/day) D-penicillamine pretreatment completely prevented all clinical signs of autoimmunity normally observed in 60-80% of rats treated with high dose (20 mg/day) D-penicillamine. We demonstrated that this tolerance is immune-mediated; tolerance to D-penicillamine is long lasting, we can adoptively transfer splenocytes from tolerant rats into slightly irradiated naive syngeneic recipients, and rats exposed to low dose D-penicillamine produce tolerogenic cytokines [Masson, M. J., and Uetrecht, J. P. (2004) Tolerance induced by low dose D-penicillamine in the brown norway rat model of drug-induced autoimmunity is immune-mediated. Chem. Res. Toxicol. 17, 82-94]. The aim of this study was to provide further understanding of the cells that are responsible for transferring tolerance and to assess the presence of regulatory T cells in the spleen of tolerant animals. We cotransferred T cells or splenocytes depleted of T cells from tolerant BN rats with splenocytes from naive BN rats into lightly irradiated syngeneic recipients. We found that neither tolerant splenocyte subpopulation could completely prevent clinical signs of autoimmunity. These results demonstrate that immune tolerance to D-penicillamine-induced autoimmunity may require both antigen presenting cells and T cells.

Quality assessment of meta-analyses of RCTs of pharmacotherapy in major depressive disorder.

BACKGROUND: Meta-analyses (MAs) of randomized controlled trials (RCTs) have the potential to provide the highest level of evidence, but the quality of published MAs has not been systematically assessed. Therefore, we determined reliability was significant (kappa = 0.89; p < 0.05). the quality of reporting in MAs of RCTs of pharmacotherapy for major depressive disorder (MDD) in adults (18-65 years) without comorbidities and examine trends over time. METHODS: MEDLINE, EMBASE, Healthstar, Psychlit and Cochrane databases were searched (1980-2002) by 4 independent reviewers for MAs of RCTs. Articles meeting inclusion criteria were blinded. Inter-rater reliability (kappa) was evaluated using a test-retest strategy on 4 articles. Quality was (p = 0.74) did not detect a difference in quality of assessed using the QUOROM checklist. Time trends were evaluated by calculating Spearman's rho. RESULTS: One hundred articles were identified, 68 were excluded [co-morbidities (9), inappropriate comparator (13), inappropriate outcome (15), article not available (5), inappropriate patient population (15), and inappropriate study design (11)]; 32 were included. Initial kappa was 0.81 (p < 0.05). After resolution of disagreements, the test-retest The mean overall quality score was 50.2% (SD 15.8%, range = 16.7-88.9%). The overall score for Titles was very poor (22%), Abstracts (40%) and Methods (49%) were poor, while overall Results score was minimally acceptable (54%). Good quality scores were found for Introduction (91%) and Discussion (97%). No time trends were identified using Spearman's correlation analysis (rho 0.05; p = 0.79). The Mann-Whitney U test articles published before and after the QUOROM. CONCLUSION: Despite quality guidelines, the average quality of published MAs of antidepressants is barely acceptable (50.2%). A need exists for adherence to standardized reporting and quality guidelines.

Tolerance induced by low dose D-penicillamine in the brown Norway rat model of drug-induced autoimmunity is immune-mediated.

Most patients taking drugs associated with idiosyncratic drug reactions tolerate the drug and do not develop adverse reactions. Understanding the mechanism of tolerance to drugs is important as it could provide insight into why some patients develop idiosyncratic reactions and others do not. The Brown Norway rat model of D-penicillamine-induced autoimmunity was used as a model of idiosyncratic drug-induced autoimmunity. Two weeks of low dose (5 mg/day) D-penicillamine pretreatment completely prevented all clinical signs of autoimmunity normally seen in 60-80% of rats treated with high dose (20 mg/day) D-penicillamine. Low dose pretreatment also prevented the increase in IgE and IL-4 mRNA characteristic of the response to high dose D-penicillamine. Experiments were conducted to determine whether low dose tolerance is metabolic or immunological. It was found that low dose tolerance possesses key characteristics of immune-mediated tolerance: memory, splenocytes that adoptively transfer tolerance, and regulatory cytokine production. To provide an understanding of the factors that can prevent or reverse established tolerance, the conditions for inducing and maintaining tolerance were investigated. Tolerance induction was investigated by manipulating the immune system during the period of low dose exposure. The induction of tolerance was partially prevented by depleting the macrophage subset of antigen presenting cells with clodronate-filled liposomes or by inhibiting T cells with tacrolimus during the period of low dose exposure. As well, the induction of tolerance was completely prevented by repeatedly stimulating the immune system throughout the period of low dose pretreatment with poly I:C. To investigate the permanence of tolerance, the immune system was stimulated after tolerance induction in an attempt to break tolerance. Both LPS and poly I:C reversed tolerance in a dose-dependent manner. These results demonstrate that immune tolerance to D-penicillamine autoimmunity can be induced by short-term low dose pretreatment.

Investigation of the involvement of macrophages and T cells in D-penicillamine-induced autoimmunity in the Brown Norway rat.

The initiating events in drug-induced autoimmunity are poorly understood and difficult to study. We examined the role of macrophages and T-cells in the Brown Norway rat model of D-penicillamine-induced autoimmunity. When activated, macrophages can act as both antigen presenting cells, initiating immune responses, and as phagocytic cells mediating systemic tissue damage. We found that B7(+) macrophages are the major antigen-presenting cell type infiltrating the spleen and caecum early in the response to Dpenicillamine. As well, the increase in splenic B7(+) macrophages correlates with the incidence of autoimmune disease. Treatments that increase the incidence of disease accentuate the increase in splenic B7(+) macrophages, and treatments that prevent disease also prevent the increase in B7(+) macrophages. In vivo depletion of macrophages appeared to decrease, but not totally prevent, autoimmune disease. The role of T-cells in D-penicillamine-induced autoimmunity was also examined using the T-cell inhibitor tacrolimus. Short-term treatment with tacrolimus not only prevented disease onset but also reversed ongoing disease and prevented disease relapse upon re-challenge with D-penicillamine. The results of this study indicate that both macrophages and T-cells could be important immune cell types involved in D-penicillamine-induced autoimmunity. Furthermore, the effects of tacrolimus in this model suggest that short-term tacrolimus treatment may be an effective way to prevent or treat IDRs in high-risk patients.