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.