PRINCIPLES OF TOXICOLOGY - Biology East Borneo

64 BIOTRANSFORMATION: A BALANCE BETWEEN BIOACTIVATION AND DETOXIFICATIONTABLE 3.3 Preparation of Subcellular Fractions for Xenobiotic Metabolism StudiesStepProcedureResult1 Liver pieces homogenized in 4 volumes of 0.25 M Tissue structure disrupted and hepatocytes sheared.sucrose in Potter Elvehjem glass–Teflonhomogenizer2 Homogenate centrifuged at 2000g for 10 min Unbroken cells, connective tissue, and nucleiisedimented3 2000g supernatant centrifuged at 10,000g for 15 min Heavy mitochondria sedimented as pellet4 10,000g supernatant centrifuged at 18,000g for 15 Light mitochondria sedimented as pelletmin5 18,000g supernatant centrifuged at 105,000g for 60minMicrosomes sedimented as pellet leaving nonturbidcytosol in 0.2 M sucrose supernatantWithout exception, the xenobiotic metabolizing enzymes occur in multiple forms (isozymes), oftenwith differing substrate selectivities. The presence of specialized isozymes, which can more efficientlymetabolize a specific range of chemicals, may enable those specific chemical challenges to be metmore effectively. With differing substrate selectivities, often comes different sensitivity to inhibitors.The presence of multiple forms thus carries the advantage of not having all the metabolism of allcompounds metabolized by that route or chemical reaction being subject to inhibitory influences atthe same time. It has also been found that the synthesis of individual isozymes can be under differentregulatory influences. The body can thus meet a chemical challenge with a finely tuned response toincrease the production of only that enzyme best equipped to counter or neutralize the challenge.Abbreviations (clockwise) are ST = sulfotransferase; PAPS = adenosine 3′-phosphate 5′-phosphosulfate; GST = glutathioneS-transferase; GSH = glutathione; AlcDH = alcohol dehydrogenase; ES = esterase; FP 1 = NADH cytochrome b 5 reductase; b 5 =cytochrome b 5; P450 = cytochrome P450; mEH = microsomal epoxide hydrolase; FP 2 = NADPH-cytochrome P450/c reductase;UGT = UDP-glucuronosyltransferase; UDPGA = uridine 5′-diphosphoglucuronic acid; FP 3 = flavin-dependent monooxygenase.Figure 3.6 Diagram of the subcellular localization and organization of major xenobiotic metabolizing enzymesand necessary cofactors.