[Rhinophyma : Successful treatment with low-dose oral isotretinoin]. / Rhinophym : Therapieerfolg mit niedrig dosiertem systemischem Isotretinoin.

Rhinophyma is a form of rosacea and is often cosmetically disfiguring. There are various therapeutic ablation modalities. Surgery is often associated with down-time and side-effects. We describe successful treatment with low-dose isotretinoin as a safe alternative with a lower risk of complications. We also discuss the advantages and disadvantages of various therapeutic modalities.

Chitosan encapsulation modulates the effect of capsaicin on the tight junctions of MDCK cells.

Capsaicin has known pharmacological effects including the ability to reversibly open cellular tight junctions, among others. The aim of this study was to develop a strategy to enhance the paracellular transport of a substance with low permeability (FITC-dextran) across an epithelial cell monolayer via reversible opening of cellular tight junctions using a nanosystem comprised by capsaicin and of chitosan. We compared the biophysical properties of free capsaicin and capsaicin-loaded chitosan nanocapsules, including their cytotoxicity towards epithelial MDCK-C7 cells and their effect on the integrity of tight junctions, membrane permeability and cellular uptake. The cytotoxic response of MDCK-C7 cells to capsaicin at a concentration of 500 µM, which was evident for the free compound, is not observable following its encapsulation. The interaction between nanocapsules and the tight junctions of MDCK-C7 cells was investigated by impedance spectroscopy, digital holographic microscopy and structured illumination fluorescence microscopy. The nanocapsules modulated the interaction between capsaicin and tight junctions as shown by the different time profile of trans-epithelial electrical resistance and the enhanced permeability of monolayers incubated with FITC-dextran. Structured illumination fluorescence microscopy showed that the nanocapsules were internalized by MDCK-C7 cells. The capsaicin-loaded nanocapsules could be further developed as drug nanocarriers with enhanced epithelial permeability.

Is non-ablative 1550-nm fractional photothermolysis an effective modality to treat melasma? Results from a prospective controlled single-blinded trial in 51 patients.

BACKGROUND: Melasma is a common benign pigmentary disease and can pose a substantial therapeutic challenge. Although the use of non-ablative fractional photothermolysis (NFP) has gained much popularity, there are still very few evidence-based data supporting NFP in the treatment of melasma. OBJECTIVE: To assess the efficacy and safety of NFP for the treatment of melasma in a controlled observer-blinded parallel-group study. PATIENTS AND METHODS: Fifty-one patients [90.2% women, 9.8% men; mean age 40.3±6.1 (control group) and 41.7±11.4 (treatment group)] received a broad-spectrum sunscreen either alone (n=25; 49.0%) or in combination with a 1550-nm NFP treatment (n=26; 51.0%) [energy: 15 mJ/microthermal zone (MTZ); total density: 1048 MTZs/cm(2); density per pass: 131 MTZs/cm(2); number of passes: 8; total coverage: 20%]. Four sessions of NFP treatment were performed at 3-week intervals on each subject in the treatment group. Patients were evaluated at baseline and 12 weeks after final treatment. The primary efficacy variables were the Melasma Area and Severity Index (MASI) and the physician's global assessment (PGA); secondary efficacy variables were the patients' subjective assessment of improvement and patient satisfaction. Safety was evaluated through the reporting of adverse events. RESULTS: The percentage of subjective improvement was virtually identical in both groups: one-third of the patients reported a 'marked improvement' and another half reported 'some improvement'. Twenty-three patients in each group declared that they were 'satisfied' with the treatment result. The MASI corroborated the patients' subjective estimate, both in terms of the degree of improvement and the lack of a group difference. In both groups, the MASI score and the PGA were reduced significantly after therapy, and the reduction was also clinically relevant. No serious side-effects were reported in either group. CONCLUSION: Our findings do not support the hypothesis of NFP providing a substantial benefit in treating melasma when compared with the lone application of a broad-spectrum sunscreen.

[Recurrent pyogenic granuloma. Treatment at difficult anatomic sites with the long-pulsed Nd:YAG laser (1064 nm)]. / Therapieresistentes Granuloma pyogenicum. Behandlung an schwierigen anatomischen Lokalisationen mit dem lang gepulsten Nd:YAG-Laser (1064 nm).

Treatment of pyogenic granuloma is still difficult as lesions tend to recur. There are various means of treatment: surgical, cryotherapy, topical agents and various lasers. We successfully treated two recurrent pyogenic granulomas at difficult sites (on the tip of the finger and ear) using the Nd: YAG laser (1064 nm). Several treatments under local anesthesia were needed. The energy was increased until coagulation was visible. The laser treatment led to complete resolution.

Lamotrigine extended-release as adjunctive therapy for partial seizures.

OBJECTIVE: To evaluate the efficacy and tolerability of once-daily adjunctive lamotrigine extended-release (XR) for partial seizures in epilepsy. METHODS: Patients more than 12 years old diagnosed with epilepsy with partial seizures and taking one to two baseline antiepileptic drugs were randomized to adjunctive once-daily lamotrigine XR or placebo in a double-blind, parallel-group trial. The study comprised a baseline phase, a 7-week double-blind escalation phase, and a 12-week double-blind maintenance phase during which doses of study medication and concomitant antiepileptic drugs were maintained. RESULTS: Of the 243 randomized patients, 239 (118 lamotrigine XR, 121 placebo) entered the escalation phase and received study medication. Lamotrigine XR was more effective than placebo with respect to median percent reduction from baseline in weekly partial seizure frequency (primary endpoint-entire 19-week treatment phase: 46.6% vs 24.5%, p = 0.0001 [corrected] via Wilcoxon test; escalation phase: 29.8% vs 15.6%, p = 0.027; maintenance phase: 58.4% vs 26.8%, p [corrected] < 0.0001). The percentage of patients with >or=50% reduction in partial seizure frequency (44.0% vs 20.8%, p = 0.0002) [corrected] and time to >or=50% reduction in partial seizure frequency (p = 0.0001) [corrected] also favored lamotrigine XR over placebo. A similar pattern of results was observed for secondarily generalized seizures. The most common adverse events were headache (lamotrigine XR 16%, placebo 18%) [corrected] and dizziness (lamotrigine XR 19%, [corrected] placebo 5%). Differences between lamotrigine XR and placebo on health outcomes measures were not significant. CONCLUSIONS: Once-daily adjunctive lamotrigine extended-release compared with placebo effectively reduced partial seizure frequency and was well tolerated in this double-blind study. Results support the clinical utility of this new once-daily formulation.

A protective role for cyclooxygenase-2 in drug-induced liver injury in mice.

Despite the utility of cyclooxygenase (COX) inhibition as an antiinflammatory strategy, prostaglandin (PG) products of COX-1 and -2 provide important regulatory functions in some pathophysiological states. Scattered reports suggest that COX inhibition may also promote adverse drug events. Here we demonstrate a protective role for endogenous COX-derived products in a murine model of acetaminophen (APAP)-induced acute liver injury. A single hepatotoxic dose caused the selective induction of COX-2 mRNA and increased PGD2 and PGE2 levels within the livers of COX(+/+) male mice suggesting a role for COX-2 in this model of liver injury. APAP-induced hepatotoxicity and lethality were markedly greater in COX-2(-/-) and (-/+) mice in which normal PG responsiveness is altered. The significantly increased toxicity linked to COX-2 deficiency could be mimicked using the selective COX-2 inhibitory drug, celecoxib, in COX(+/+) mice and was not due to alterations in drug-protein adduct formation, a surrogate for bioactivation and toxicity. Microarray analyses indicated that increased injury associated with COX-2 deficiency coincided, most notably, with a profoundly impaired induction of heat shock proteins in COX-2(-/+) mice suggesting that PGs may act as critical endogenous stress signals following drug insult. These findings suggest that COX-2-derived mediators serve an important hepato-protective function and that COX inhibition may contribute to the risk of drug-induced liver injury, possibly through both nonimmunological and immunological pathways.

A new concept of cellular uptake and intracellular trafficking of long-chain fatty acids.

Fatty acids are the main structural and energy sources of the human body. Within the organism, they are presented to cells as fatty acid:albumin complexes. Dissociation from albumin represents the first step of the cellular uptake process, involving membrane proteins with high affinity for fatty acids, e.g., fatty acid translocase (FAT/CD 36) or the membrane fatty acid-binding protein (FABPpm). According to the thus created transmembrane concentration gradient, uncharged fatty acids can flip-flop from the outer leaflet across the phospholipid bilayer. At the cytosolic surface of the plasma membrane, fatty acids can associate with the cytosolic FABP (FABP(c)) or with caveolin-1. Caveolins are constituents of caveolae, which are proposed to serve as lipid delivery vehicles for subcellular organelles. It is not known whether protein (FABP(c))- and lipid (caveolae)-mediated intracellular trafficking of fatty acids operates in conjunction or in parallel. Channeling fatty acids to the different metabolic pathways requires activation to acyl-CoA. For this process, the family of fatty acid transport proteins (FATP 1-5/6) might be relevant because they have been shown to possess acyl-CoA synthetase activity. Their variable N-terminal signaling sequences suggest that they might be targeted to specific organelles by anchoring in the phospholipid bilayer of the different subcellular membranes. At the highly conserved cytosolic AMP-binding site of FATP, fatty acids are activated to acyl-CoA for subsequent metabolic disposition by specific organelles. Overall, fatty acid uptake represents a continuous flow involving the following: dissociation from albumin by membrane proteins with high affinity for fatty acids; passive flip-flop across the phospholipid bilayer; binding to FABP(C) and caveolin-1 at the cytosolic plasma membrane; and intracellular trafficking via FABP(c) and/or caveolae to sites of metabolic disposition. The uptake process is terminated after activation to acyl-CoA by the members of the FATP family targeted intracellularly to different organelles.

Immunohistochemical detection of protein adducts of 2,4-dinitrochlorobenzene in antigen presenting cells and lymphocytes after oral administration to mice: lack of a role of Kupffer cells in oral tolerance.

Although current studies suggest that most drug-induced allergic reactions (DIARS) are caused by immunogenic conjugates formed from the reaction of a reactive metabolite of a drug with cellular proteins, it is not clear why these reactions are relatively rare. One possible pathway that may explain the low incidence of DIARS in many cases is oral tolerance, an antigen-specific immunological hyporesponsiveness induced by oral administration of antigens. The mechanism of oral tolerance, however, is not clearly understood and is difficult to study directly with drugs, because animal models of DIARS have been elusive. We chose 2,4-dinitrochlorobenzene (DNCB) as a model compound to circumvent this problem because animal models of allergic reactions have been established for this compound. DNCB forms immunogenic 2,4-dinitrophenylated (DNP) protein conjugates that can induce immune reactions and it causes oral tolerance when it is fed to animals prior to sensitization. We hypothesized that DNP-protein conjugates may have a role in oral tolerance. To test this idea, we have begun to identify cells bearing these conjugates after the oral administration of DNCB. Female C57BL/6J mice were fed DNCB and tissues were examined after 6 and 24 h. Immunohistochemical analysis indicated the presence of DNP-protein conjugates in enterocytes of the small intestine, in macrophages and lymphocytes of the mesenteric lymph nodes, in dendritic cells and lymphocytes of the spleen, and in Kupffer cells and other sinusoidal cells of the liver. It was found that Kupffer cell depletion did not affect oral tolerance to DNCB. The findings suggest that the cells bearing DNP-protein conjugates, other than Kupffer cells, in the liver and other tissues may be important in the induction of oral tolerance against DNCB. Protein adducts of drugs administered orally may also be present in these cells, and they may have a role in the downregulation of DIARS in many individuals.

Halothane-induced liver injury in outbred guinea pigs: role of trifluoroacetylated protein adducts in animal susceptibility.

Halothane causes a mild form of liver injury in guinea pigs that appears to model the hepatotoxicity seen in approximately 20% of patients treated with this drug. In previous studies, it was concluded that the increased susceptibility of some outbred guinea pigs to halothane-induced liver injury is not caused by their inherent ability to metabolize halothane to form toxic levels of trifluoroacetylated protein adducts in the liver. In this study, we reevaluated the role of trifluoroacetylated protein adducts in halothane-induced liver injury in guinea pigs. Male outbred Hartley guinea pigs were treated with halothane intraperitoneally. On the basis of serum alanine aminotransferase levels and liver histology, treated animals were designated as being susceptible, mildly susceptible, or resistant to halothane. Immunoblot studies with the use of anti-trifluoroacetylated antibodies showed that susceptible guinea pigs for the most part had higher levels of trifluoroacetylated protein adducts in the liver 48 h after treatment with halothane than did less susceptible animals. In support of this finding, the level of trifluoroacetylated protein adducts detected immunochemically in the sera of treated guinea pigs correlated with sera levels of alanine aminotransferase activity. In addition, the levels of cytochrome P450 2A-related protein but not those of other cytochrome P450 isoforms, measured by immunoblot analysis with isoform-specific antibodies, correlated with the amount of trifluoroacetylated protein adducts detected in the livers of guinea pigs 8 h after halothane administration. The results of this study indicate that the susceptibility of outbred guinea pigs to halothane-induced liver injury is related to an enhanced ability to metabolize halothane in the liver to form relatively high levels of trifluoroacetylated protein adducts. They also suggest that cytochrome P450 2A-related protein might have a major role in catalyzing the formation of trifluoroacetylated protein adducts in the liver of susceptible guinea pigs. Similar mechanisms may be important in humans.

Expression profiling of acetaminophen liver toxicity in mice using microarray technology.

Drug-induced hepatotoxicity causes significant morbidity and mortality and is a major concern in drug development. This is due, in large part, to insufficient knowledge of the mechanism(s) of drug-induced liver injury. In order to address this problem, we have evaluated the modulation of gene expression within the livers of mice treated with a hepatotoxic dose of acetaminophen (APAP) using high-density oligonucleotide microarrays capable of determining the expression profile of >11,000 genes and expressed sequence tags (ESTs). Significant alterations in gene expression, both positive and negative, were noted within the livers of APAP-treated mice. APAP-induced toxicity affected numerous aspects of liver physiology causing, for instance, >twofold increased expression of genes that encode for growth arrest and cell cycle regulatory proteins, stress-induced proteins, the transcription factor LRG-21, suppressor of cytokine signaling (SOCS)-2-protein, and plasminogen activator inhibitor-1 (PAI-1). A number of these and other genes and ESTs were detectable within the liver only after APAP treatment suggesting their potential importance in propagating or preventing further toxicity. These data provide new directions for mechanistic studies that may lead to a better understanding of the molecular basis of drug-induced liver injury and, ultimately, to a more rational design of safer drugs.

Drug enterocyte adducts: possible causal factor for diclofenac enteropathy in rats.

BACKGROUND & AIMS: Enteropathy is a frequent complication of diclofenac and other nonsteroidal anti-inflammatory drugs, yet little is known about the underlying mechanism. One possibility is that reactive metabolites of diclofenac form adducts with enterocyte macromolecules, as previously shown for liver. We addressed this possibility by using immunohistochemistry to detect diclofenac adducts. METHODS: Rats were treated orally with diclofenac (10-100 mg/kg) and killed after 1-24 hours, and their gastrointestinal (GI) tracts were evaluated for ulcer number and area. Adduct distribution and intensity were assessed by immunohistochemistry by using a technique to simultaneously process and stain multiple intestinal rings. RESULTS: Drug treatment led to dose-dependent formation of both adducts and ulcers only in small intestine and only in animals with intact enterohepatic circulation. Adducts formed within enterocytes by 1 hour, translocated to the brush border, preceded ulceration and vascular protein leakage, and were intense at sites of ulceration. Adducts and ulcers exhibited a parallel distribution within intestinal quintiles: 3rd > 5th >> 1st. CONCLUSIONS: Diclofenac treatment resulted in the formation of drug adducts in enterocytes. Because this molecular change occurred before ulceration, was dose dependent, and exhibited concordant distribution with extent of ulceration, the results suggest a causal role for drug adduct formation in diclofenac enteropathy.

Immunochemical evidence against the involvement of cysteine conjugate beta-lyase in compound A nephrotoxicity in rats.

BACKGROUND: Compound A, a degradation product of sevoflurane, causes renal corticomedullary necrosis in rats. Although the toxicity of this compound was originally hypothesized to result from the biotransformation of its cysteine conjugates into toxic thionoacyl halide metabolites by renal cysteine conjugate beta-lyase, recent evidence suggests that alternative mechanisms may be responsible for compound A nephrotoxicity. The aim of this study was to evaluate these issues by determining whether mercapturates and glutathione conjugates of compound A could produce renal corticomedullary necrosis in rats, similar to compound A, and whether renal covalent adducts of the thionacyl halide metabolite of compound A could be detected immunochemically. METHODS: Male Wistar rats were administered, intraperitoneally, N-acetylcysteine conjugates (mercapturates) of compound A (90 or 180 micromol/kg) or glutathione conjugates of compound A (180 micromol/kg) with or without intraperitoneal pretreatments with aminooxyacetic acid (500 micromol/kg) or acivicin (250 micromol/kg). Rats were killed after 24 h, and kidney tissues were analyzed for toxicity by histologic examination or for protein adducts by immunoblotting or immunohistochemical analysis, using antisera raised against the covalently bound thionoacyl halide metabolite of compound A. RESULTS: Mercapturates and glutathione conjugates of compound A both produced renal corticomedullary necrosis similar to that caused by compound A. Aminooxyacetic acid, an inhibitor of renal cysteine conjugate beta-lyase, did not inhibit the toxicity of the mercapturates, whereas acivicin, an inhibitor of gamma-glutamyltranspeptidase, potentiated the toxicity of both classes of conjugates. No immunochemical evidence for renal protein adducts of the thionacyl halide metabolite was found in rats 24 h after the administration of the mercapturates of compound A or in the kidneys of rats, obtained from a previous study, 5 and 24 h after the administration of compound A. CONCLUSION: The results of this study are consistent with the idea that a mechanism other than the renal cysteine conjugate beta-lyase pathway of metabolic activation is responsible for the nephrotoxicity of compound A and its glutathione and mercapturate conjugates in male Wistar rats.

Metabolic activation of diclofenac by human cytochrome P450 3A4: role of 5-hydroxydiclofenac.

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.

Immunochemical detection and identification of protein adducts of diclofenac in the small intestine of rats: possible role in allergic reactions.

Idiosyncratic adverse drug reactions are unpredictable, target multiple organ systems, and often become life-threatening events. Although the causes of idiosyncratic adverse drug reactions are not known in most cases, evidence suggests that they may be mediated through immunological mechanisms. It is generally thought that for a drug to lead to an immune response, it must first become covalently bound to a carrier protein. Since most drugs are unreactive, it is usually a reactive metabolite that is expected to form covalent adducts. However, it is not clear why more people do not develop immune reactions against drug-protein adducts. One possible explanation is that orally administered drugs may lead to oral tolerance in most individuals through mechanisms similar to that found with orally administered antigens. However, very little is known regarding the interaction of drugs with gut-associated lymphoid tissue of the small intestine, where oral tolerance can develop. As an initial step to test this hypothesis, we have investigated whether diclofenac, a commonly used nonsteroidal antiinflammatory drug, can lead to protein adducts in rat small intestine. Diclofenac was administered to rats by gastric gavage. Immunoblot analysis of small intestine homogenates and isolated enterocyte subcellular fractions with drug-specific antiserum revealed 142-, 130-, 110-, and 55-kDa protein adducts of diclofenac. The 142- and 130-kDa adducts of diclofenac were identified as aminopeptidase N (CD13) and sucrase-isomaltase, respectively, by amino acid sequence analyses and by their reactions with protein-specific antibodies. The adducts were localized by immunohistochemistry and found primarily in the mid-villus and villus-tip enterocytes and also in the dome overlying Peyer's patches. Similar adducts were detected immunochemically in villus-tip enterocytes of animals treated with halothane or acetaminophen. These results show that intestinal protein adducts of drugs can be formed in gut-associated lymphoid tissue where they may lead to the down-regulation of drug-induced allergic reactions in many individuals.

Patients with delayed-onset sulfonamide hypersensitivity reactions have antibodies recognizing endoplasmic reticulum luminal proteins.

Sulfonamide antimicrobials cause a delayed-onset, hypersensitivity-type syndrome characterized by fever, skin rash and multiorgan toxicity occurring 7 to 14 days after initiation of therapy. The pathogenesis is believed to be immune-mediated. We investigated whether patients with delayed-onset sulfonamide hypersensitivity reactions had antibodies recognizing hapten-microsomal protein conjugates and/or native microsomal proteins. By immunoblotting using rat liver as a source of microsomal protein, 17 of 21 patients had antibodies recognizing one or more of three native endoplasmic reticulum proteins of 55 kDa (14 of 21 patients), 80 kDa (4 of 21 patients) or 96 kDa (3 of 21 patients) in size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No control subjects (n = 11) and only 1 of 18 patients with adverse events not consistent with sulfonamide hypersensitivity reactions had antibodies against these microsomal proteins under the conditions used. Only 1 patient had antibodies that recognized the sulfonamide hapten, sulfamethoxazole. The 55-kDa protein was identified as protein disulfide isomerase. The 80-kDa protein was identified as grp78. The 96-kDa protein was not identified. Delayed-onset sulfonamide hypersensitivity reactions are therefore primarily associated with antibodies recognizing specific protein epitopes and not anti-drug antibodies.

Epidemic of liver disease caused by hydrochlorofluorocarbons used as ozone-sparing substitutes of chlorofluorocarbons.

BACKGROUND: Hydrochlorofluorocarbons (HCFCs) are used increasingly in industry as substitutes for ozone-depleting chlorofluorocarbons (CFCs). Limited studies in animals indicate potential hepatotoxicity of some of these compounds. We investigated an epidemic of liver disease in nine industrial workers who had had repeated accidental exposure to a mixture of 1,1-dichloro-2,2,2-trifluoroethane (HCFC 123) and 1-chloro-1,2,2,2-tetrafluoroethane (HCFC 124). All nine exposed workers were affected to various degrees. Both compounds are metabolised in the same way as 1-bromo-1-chloro-2,2,2-trifluoroethane (halothane) to form reactive trifluoroacetyl halide intermediates, which have been implicated in the hepatotoxicity of halothane. We aimed to test whether HCFCs 123 and 124 can result in serious liver disease. METHODS: For one severely affected worker liver biopsy and immunohistochemical stainings for the presence of trifluoroacetyl protein adducts were done. The serum of six affected workers and five controls was tested for autoantibodies that react with human liver cytochrome-P450 2E1 (P450 2E1) and P58 protein disulphide isomerase isoform (P58). FINDINGS: The liver biopsy sample showed hepatocellular necrosis which was prominent in perivenular zone three and extended focally from portal tracts to portal tracts and centrilobular areas (bridging necrosis). Trifluoroacetyl-adducted proteins were detected in surviving hepatocytes. Autoantibodies against P450 2E1 or P58, previously associated with halothane hepatitis, were detected in the serum of five affected workers. INTERPRETATION: Repeated exposure of human beings to HCFCs 123 and 124 can result in serious liver injury in a large proportion of the exposed population. Although the exact mechanism of hepatotoxicity of these agents is not known, the results suggest that trifluoroacetyl-altered liver proteins are involved. In view of the potentially widespread use of these compounds, there is an urgent need to develop safer alternatives.

Cytochrome P4502C11 is a target of diclofenac covalent binding in rats.

Diclofenac antiserum was previously developed and used to detect protein adducts of metabolites of dichlofenac in livers of mice and rats. In this study, the antibody has been used to facilitate the purification of a major 51 kDa microsomal adduct of diclofenac from the liver microsomes of male rats that were treated with diclofenac. The adduct was identified as male-specific cytochrome P4502C11 based on its N-terminal amino acid sequence, reaction with a cytochrome P4502C11 antibody, and by its absence from liver microsomes of diclofenac-treated female rats. When diclofenac was incubated with liver microsomes of control rats in the presence of NADPH, only the 51 kDa adduct was produced. The formation of the adduct was inhibited by a cytochrome P4502C11 monoclonal antibody, but not by reduced glutathione or N-alpha-acetyl-L-lysine. No adduct was detected when diclofenac was incubated with liver microsomes from female rats. Moreover, adduct formation in vivo appeared to lead to a 72% decrease in the activity of cytochrome P4502C11. The results indicate that cytochrome P4502C11 metabolizes diclofenac into a highly reactive product that covalently binds to this enzyme before it can diffuse away and react with other proteins.