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.

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.

Covalent modification of rat liver dipeptidyl peptidase IV (CD26) by the nonsteroidal anti-inflammatory drug diclofenac.

Diclofenac is a nonsteroidal anti-inflammatory drug that has been implicated in several cases of severe hepatotoxicity. Our previous study showed that diclofenac metabolites bound covalently and selectively to rat liver plasma membrane proteins with estimated monomeric masses of 110, 140, and 200 kDa. We report here that we have identified the 110 kDa diclofenac-labeled protein in rat liver as dipeptidyl peptidase IV, also known as CD26. In addition, we found that the activity of dipeptidyl peptidase IV in liver plasma membrane fractions was lowered after diclofenac treatment of rats. These results suggest that the hepatotoxicity associated with diclofenac might be due, in part, to the covalent modification of dipeptidyl peptidase IV.

Metabolic activation and immunochemical localization of liver protein adducts of the nonsteroidal anti-inflammatory drug diclofenac.

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)

Stereochemistry of the microsomal glutathione S-transferase catalyzed addition of glutathione to chlorotrifluoroethene.

The stereochemistry of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation was studied in rat liver cytosol, microsomes, N-ethylmaleimide-treated microsomes, 9000g supernatant fractions, purified rat liver microsomal glutathione S-transferase, and isolated rat hepatocytes. The absolute configuration of the chiral center generated by the addition of glutathione to chlorotrifluoroethene was determined by degradation of S-(2-chloro-1,1,2-trifluoroethyl)glutathione to chlorofluoroacetic acid, followed by derivatization to form the diastereomeric amides N-(S)-alpha-methylbenzyl-(S)-chlorofluoacetamide and N-(S)-alpha-methylbenzyl-(R)-chlorofluoroacetamide, which were separated by gas chromatography. Native and N-ethylmaleimide-treated rat liver microsomes, purified rat liver microsomal glutathione S-transferase, rat liver 9000g supernatant, and isolated rat hepatocytes catalyzed the formation of 75-81% (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione; rat liver cytosol catalyzed the formation of equal amounts of (2R)- and (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione. In rat hepatocytes, microsomal glutathione S-transferase catalyzed the formation of 83% of the total S-(2-chloro-1,1,2-trifluoroethyl)glutathione formed. These observations show that the microsomal glutathione S-transferase catalyzes the first step in the intracellular, glutathione-dependent bioactivation of the nephrotoxin chlorotrifluoroethene.