Drug Metabolism in Drug Design and Development Basic Concepts and Practice

(4) If^14 C-tracers are used inin vivocovalent protein binding studies in rats,

evaluation of the loss of the^14 C-label in rat urine may also be warranted

if the portion of the molecule with the^14 C-label attached is likely to be

cleaved to a significantly smaller molecular weight molecule in the

metabolism processes.

14.3 PROTOCOL FOR MEASUREMENT OF INTRACELLULAR GSH

AND GSSG CONCENTRATIONS IN HEPATOCYTES

14.3.1 Introduction

Bioactivation of drugs and xenobiotics leads to the formation of reactive
intermediates. Most of reactive intermediates are electrophiles (e.g,p-quinones)
and readily react with nucleophiles to form corresponding adducts. Figure 14.7
is a schematic presentation of redox cycling and GSH adduct formation of
reactive p-quinones. Mechanistically, reactive p-quinones formed from
bioactivation of drugs and xenobiotics can be reduced by cytochrome P450
and P450 reductase to semiquinone free radicals through one-electron
reduction mechanism or by quinone oxidoreductases to hydroquinones
(Iskander and Jaiswal, 2005). Semiquinone free radicals can be reoxidized to
the quinone form by molecular oxygen with the concomitant formation of
superoxide anion radicals. Superoxide anion radicals can be converted to
hydrogen peroxide by either enzymatic (superoxide dismutase) or spontaneous
dismutation (Bolton et al., 2000). In the presence of trace amount of transition
metals, hydrogen peroxide can react with superoxide anion radical to form
hydroxyl radical (O’Brien, 1991). Both hydrogen peroxide and hydroxyl

O

O

O

OH

P450

OH

OH

SG

GSH

.

2GSH

H 2 O

H 2 O 2

O GSSG

2

O 2

.-

FIGURE 14.7 Redox cycling and formation of GSH adducts of quinones.

PROTOCOL FOR MEASUREMENT OF INTRACELLULAR GSH AND GSSG 467