Hepatic injury induced by various toxic agents, including acetaminophen (APAP), has been attributed, in part, to the production of proinflammatory cytokines and other mediators by resident Kupffer cells within the liver. However, recent evidence from our laboratory has demonstrated that hepato-protective factors, such as interleukin (IL)-10 and cyclooxygenase-derived mediators, are also upregulated in response to hepatic damage to help protect against exacerbated injury, and Kupffer cells have been suggested to be a source of these modulatory factors. In other models, Kupffer cells also serve important regulatory functions in pathophysiological states of the liver. Therefore, we reevaluated the role of Kupffer cells in a murine model of APAP-induced liver injury using liposome-entrapped clodronate (liposome/clodronate) as an effective Kupffer cell-depleting agent. We show that in contrast to pretreatment of mice with a widely used macrophage inhibitor, gadolinium chloride, which did not deplete Kupffer cells but moderately protected against APAP-induced hepatotoxicity as reported previously, the intravenous injection of liposome/clodronate caused nearly complete elimination of Kupffer cells and significantly increased susceptibility to APAP-induced liver injury as compared with mice pretreated with empty liposomes. This increased susceptibility was apparently unrelated to the metabolism of APAP since liposome/clodronate pretreatment did not alter APAP-protein adduct levels. Instead, Kupffer cell depletion by liposome/clodronate led to significant decreases in the levels of hepatic mRNA expression of several hepato-regulatory cytokines and mediators, including IL-6, IL-10, IL-18 binding protein and complement 1q, suggesting that Kupffer cells are a significant source for production of these mediators in this model. Our findings indicate that, in addition to their protoxicant activities, Kupffer cells can also have an important protective function in the liver through the production of a variety of modulatory factors which may counteract inflammatory responses and/or stimulate liver regeneration.

Mechanistic study of idiosyncratic drug-induced hepatitis (DIH) continues to be a challenging problem because of the lack of animal models. The inability to produce this type of hepatotoxicity in animals, and its relative rarity in humans, may be linked to the production of antiinflammatory factors that prevent drug-protein adducts from causing liver injury by immune and nonimmune mechanisms. We tested this hypothesis by using a model of acetaminophen (APAP)-induced liver injury in mice. After APAP treatment, a significant increase was observed in serum levels of interleukin (IL)-4, IL-10, and IL-13, cytokines that regulate inflammatory mediator production and cell-mediated autoimmunity. When IL-10 knockout (KO) mice were treated with APAP, most of these mice died within 24 to 48 hours from liver injury. This increased susceptibility to APAP-induced liver injury appeared to correlate with an elevated expression of liver proinflammatory cytokines, tumor necrosis factor (TNF)-␣, and IL-1, as well as inducible nitric oxide synthase (iNOS). In this regard, mice lacking both IL-10 and iNOS genes were protected from APAP-induced liver injury and lethality when compared with IL-10 KO mice. All strains, including wild-type animals, generated similar amounts of liver APAPprotein adducts, indicating that the increased susceptibility of IL-10 KO mice to APAP hepatotoxicity was not caused by an enhanced formation of APAP-protein adducts. Epidemiologic studies reveal that as many as 25% of fulminant hepatitis cases in intensive care units are caused by DIH. 1 It is also the major reason for removal of new drugs from clinical development and widespread use. 2 Although DIH can be very severe, the incidence for any given drug is usually low. It has been hypothesized that this idiosyncratic nature of DIH is either caused by a metabolic idiosyncrasy, in which patients who develop toxicity metabolize the drug to reactive metabolites to a greater extent than most others, 3 or an immunologic idiosyncrasy (druginduced allergic hepatitis [DIAH]), in which the immune system of patients who develop DIH react against protein adducts of drugs or other neoantigens formed in the liver. 3,4 These 2 mechanisms are not mutually exclusive and might both contribute to the initiation of DIH. 3 However, the demonstration of specific antibodies and sensitized T cells that react with drugs, their metabolites, or specific protein neoantigens and/or autoantigens supports the immunologic basis of DIH. [2][3][4][5][6] The host-dependent, idiosyncratic nature of DIH is poorly understood, but may be related to a variety of factors that in most individuals either fail to promote or, in fact, protect against DIH. For example, genetic polymorphisms in the specific repertoire of major histocompatibility complex class I and II antigens and/or Band T-cell receptors may render the immune cells from most individuals incapable of satisfactorily binding or presenting protein adducts of drugs or their derived peptides and, therefore, unable to lead to immunopatholog...

No abstract

Cytochrome P450 2C11 in rats was recently found to metabolize diclofenac into a highly reactive product that covalently bound to this enzyme before it could diffuse away and react with other proteins. To determine whether cytochromes P450 in human liver could catalyze a similar reaction, we have studied the covalent binding of diclofenac in vitro to liver microsomes of 16 individuals. Only three of 16 samples were found by immunoblot analysis to activate diclofenac appreciably to form protein adducts in a NADPH-dependent pathway. Cytochrome P450 2C9, which catalyzes the major route of oxidative metabolism of diclofenac to produce 4'-hydroxydiclofenac, did not appear to be responsible for the formation of the protein adducts, because sulfaphenazole, an inhibitor of this enzyme, did not affect protein adduct formation. In contrast, troleandomycin, an inhibitor of P450 3A4, inhibited both protein adduct formation and 5-hydroxylation of diclofenac. These findings were confirmed with the use of baculovirus-expressed human P450 2C9 and P450 3A4. One possible reactive intermediate that would be expected to bind covalently to liver proteins was the p-benzoquinone imine derivative of 5-hydroxydiclofenac. This product was formed by an apparent metal-catalyzed oxidation of 5-hydroxydiclofenac that was inhibited by EDTA, glutathione, and NADPH. The p-benzoquinone imine decomposition product bound covalently to human liver microsomes in vitro in a reaction that was inhibited by GSH. In contrast, GSH did not prevent the covalent binding of diclofenac to human liver microsomes. These results suggest that for appreciable P450-mediated bioactivation of diclofenac to occur in vivo, an individual may have to have both high activities of P450 3A4 and perhaps low activities of other enzymes that catalyze competing pathways of metabolism of diclofenac. Moreover, the p-benzoquinone imine derivative of 5-hydroxydiclofenac probably has a role in covalent binding in the liver only under the conditions where levels of NADPH, GSH, and other reducing agents would be expected to be low.

Autoantibodies against specific human cytochrome P450s have been found in the sera of patients suffering from a variety of diseases, including those caused by drugs. In the cases of tienilic acid- and dihydralazine-induced hepatitis, patients have serum autoantibodies directed against cytochromes P450 2C9 and P450 1A2, respectively. In the present study, we have found that 25 of 56 (45%) patients diagnosed with halothane hepatitis have autoantibodies that react with human cytochrome P450 2E1 that was purified from a baculovirus expression system. The autoantibodies inhibited the activity of cytochrome P450 2E1 and appeared to be directed against mainly conformational epitopes. In addition, because cytochrome P450 2E1 became trifluoroacetylated when it oxidatively metabolized halothane, it is possible that the covalently altered form of cytochrome P450 2E1 may be able to bypass the immunologic tolerance that normally exists against cytochrome P450 2E1. A similar mechanism may explain the formation of autoantibodies that have been found against other cellular targets of the reactive trifluoroacetyl chloride metabolite of halothane.

No abstract

Diclofenac is a nonsteroidal anti-inflammatory agent that is reported to cause serious hepatic injury in some patients. To investigate the possibility that protein adducts derived from reactive intermediates of diclofenac might be responsible for the hepatotoxicity produced by this drug, we recently developed polyclonal antisera that recognized protein adducts of diclofenac. In the present study, we have characterized further the diclofenac adducts in rat liver. Immunoblotting studies showed that diclofenac-labeled hepatic proteins were formed in a dose- and time-dependent manner in rats given diclofenac. Subcellular fractionation of liver homogenates from diclofenac-treated rats showed that a 50-kDa microsomal protein and 110-, 140-, and 200-kDa plasma membrane proteins were labeled preferentially. Immunofluorescence studies of isolated hepatocytes and immunohistochemical analysis of liver slices from diclofenac-treated mice and rats confirmed that plasma membrane proteins were labeled by diclofenac metabolites and showed that the bile canalicular domain of the plasma membrane was a major site of diclofenac adduct formation. Additionally, we found that cytochrome P-450 and UDP-glucuronosyltransferase, but not acyl-CoA synthase, catalyzed the formation of reactive intermediates of diclofenac that were bound covalently to proteins in vitro. The metabolites catalyzed by cytochrome P-450 in vitro were bound exclusively to a 50-kDa microsomal protein, even in the presence of albumin. In contrast, the 110-, 140-, and 200-kDa plasma membrane proteins as well as others appeared to be labeled when diclofenac was activated by UDP-glucuronosyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)

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 D rug-induced liver injury (DILI) is a serious, often fatal side effect of drug treatment representing a major impediment to drug development. 1,2 Attempts to identify hepatotoxic drugs early in development have been hindered in part by a poor understanding of the underlying mechanism of DILI. Current evidence suggests that reactive metabolites, drug-protein adducts, and glutathione depletion might be common events involved in the initiation of DILI; however, it is currently not possible to predict hepatotoxicity based on these criteria alone. 3 Therefore, there is a great deal of interest in studying the downstream events of these initiating factors in hope of identifying potential markers of hepatotoxicity and pathways leading to DILI. 4,5 The most extensively used model for uncovering fundamental mechanisms of DILI has been acetaminopheninduced liver injury (AILI) in mice. 4,6 In this model, the preponderance of evidence indicates that N-acetyl-p-benzoquinone imine (NAPQI), the reactive metabolite of acetaminophen (APAP); NAPQI-protein adducts; glutathione depletion; oxidative stress; and mitochondria damage all play a role in AILI. 7 More recently, evidence suggests that the innate immune system can contribute to the severity of AILI through the production of proinflammatory cytokines and other protoxicant factors subsequent to the early metabolic events initiated by NAPQI