PRINCIPLES OF TOXICOLOGY

64 BIOTRANSFORMATION: A BALANCE BETWEEN BIOACTIVATION AND DETOXIFICATION
TABLE 3.3 Preparation of Subcellular Fractions for Xenobiotic Metabolism Studies
StepProcedure Result
1 Liver pieces homogenized in 4 volumes of 0.25 M
sucrose in Potter Elvehjem glass–Teflon
homogenizer
Tissue structure disrupted and hepatocytes sheared.
2 Homogenate centrifuged at 2000g for 10 min Unbroken cells, connective tissue, and nucleii
sedimented
3 2000g supernatant centrifuged at 10,000g for 15 min Heavy mitochondria sedimented as pellet
4 10,000g supernatant centrifuged at 18,000g for 15
min
Light mitochondria sedimented as pellet
5 18,000g supernatant centrifuged at 105,000g for 60 Microsomes sedimented as pellet leaving nonturbid
min
cytosol in 0.2 M sucrose supernatant
Without exception, the xenobiotic metabolizing enzymes occur in multiple forms (isozymes), often
with differing substrate selectivities. The presence of specialized isozymes, which can more efficiently
metabolize a specific range of chemicals, may enable those specific chemical challenges to be met
more 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 all
compounds metabolized by that route or chemical reaction being subject to inhibitory influences at
the same time. It has also been found that the synthesis of individual isozymes can be under different
regulatory influences. The body can thus meet a chemical challenge with a finely tuned response to
increase 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 = glutathione
S-transferase; GSH = glutathione; AlcDH = alcohol dehydrogenase; ES = esterase; FP1 = NADH cytochrome b5 reductase; b5 =
cytochrome b5; P450 = cytochrome P450; mEH = microsomal epoxide hydrolase; FP2 = NADPH-cytochrome P450/c reductase;
UGT = UDP-glucuronosyltransferase; UDPGA = uridine 5′-diphosphoglucuronic acid; FP3 = flavin-dependent monooxygenase.
Figure 3.6 Diagram of the subcellular localization and organization of major xenobiotic metabolizing enzymes
and necessary cofactors.