Other hydrolases

Glycosidases and sulfatases are further hydrolases of importance for drug metabolism. They primarily metabolize endogenous substrates including glycosaminoglycans and steroids, but they also accept some xenobiotic substrates; this is particularly true for the beta-glucuronidases. The gut flora can deconjugate compounds that are excreted via the bile as glucuronides or sulfates. The deconjugation products are often reabsorbed from the gut leading to enterohepatic circulation. Frequently, the toxicity and mutagenicity of natural products is masked by sugar moieties in glycosides and deglycosylation leads to their toxic effects. Thus, glycosidases are toxifying in many cases. The procarcinogenic glycoside cycasin of the cycad plant, for example, needs glycosidase-mediated toxification. After oral treatment with cycasin, rats with a normal gut flora develop tumors in the liver, kidney, and intestine as a consequence of the bacterial glycosidase-mediated liberation of the genotoxic aglycon methyl azoxymethanol. However, germ-free rats do not suffer from any long-term effects of cycasin.1 Phase II Enzymes: Transferases Glutathione S-transferases (E.C.

Glutathione (GSH) and glutathione S-transferases (GSTs)48 represent the prime defense system against elec-trophile-mediated drug toxicity in mammals However, as is the case with all adequately investigated drug-metabolizing enzymes, GSTs in some predictable cases act as toxifying enzymes and glutathione is itself mutagenic when activated by rat kidney homogenates (S9).49 The 'normal' detoxifying role of GSTs is due to the fact that they detoxify lipophilic and structurally very diverse electrophiles by conjugating them with the endogenous hydrophilic nucleophile glutathione. This neutralizes the electrophilic reactivity and renders the compound easily excretable.1

The structure and catalytic mechanism of GST are described elsewhere in this book (5.06 Principles of Drug Metabolism 2: Hydrolysis and Conjugation Reactions). Almost all GSTs are soluble, dimeric drug-metabolizing enzymes. Subunit dimerization is necessary for their catalytic activity. The substrate specificity is dictated by the subunit and the substrate specificity of the dimer is purely additive of the specificities of the two individual subunits. In addition, there are at least two membrane-bound glutathione-dependent enzymes, one of which is called the microsomal GST, also a drug-metabolizing enzyme.

Glutathione conjugates are released from the cells by an active transport system that belongs to the multidrug resistance (MDR) protein complex. Since the GSTs are often strongly product-inhibited this active removal of the products is important for the efficiency of GST-mediated detoxification.50

Glutathione conjugates, predominantly formed in the liver, are usually further processed in the kidney. The glutamate moiety is cleaved off by the g-glutamyltranspeptidase. Then glycine is released by aminopeptidase M. The resulting cysteinyl conjugate is usually N-acetylated to form a mercapturic acid. In competition with the N-acetyltransferase, beta-lyase can convert some cysteinyl conjugates to the sulfhydryl metabolite. This reaction can lead to highly toxic metabolites and hence represents a toxification. In these cases the GST-mediated reaction has ultimately led to a highly toxic metabolite.1

The superfamily of cytosolic GSTs (see 5.06 Principles of Drug Metabolism 2: Hydrolysis and Conjugation Reactions) composed of families (also called classes): the a (alpha), m (mu), p (pi), 9 (theta), a (sigma), and k (kappa) families (Table 3). The cytosolic GSTs are homodimers or heterodimers of subunits belonging to the same family. The recommendation for a nomenclature system proposes to use 'GST' for the enzyme, preceded by a lower case letter for the species (h for human, r for rat, etc.) followed by a capital letter for the family (A for a, M for m, P for p, T for 9, K for k, S for a) followed by arabic numbers for the subunits (i.e., mGSTA1-1 is the homodimer of the mouse a family subunit 1). For extrapolations of metabolism-controlled toxicities between species it is important to note that, in contrast to the system used for the CYPs, the use of the same number of GST subunits of different animal species does not (necessarily) indicate that they are orthologous; they are usually used in sequence of their discovery.1 A number of reactions are catalyzed by GSTs:

• Nucleophilic substitution of electron-withdrawing substituents by GSH such as the formation of S-(2,4-dinitrophenyl)glutathione from the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene (CDNB), which is conjugated by almost all GSTs (the exception being the 9 family).

• Nucleophilic addition of GSH to epoxides by opening of the oxirane ring and to a,b-unsaturated carbonyl compounds by Michael addition, both detoxification reactions. Many epoxides are good substrates for several GSTs. GSTP1 from most species has been shown to possess significant activity to detoxify the ultimate carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (Figure 3). Also aflatoxin-8,9-epoxide is detoxified by GST. The glutathione conjugate of the endogenous epoxide leukotriene A4 is leukotriene C4 formed by GST and further metabolized to leukotriene D4 both of them being active components of the slow reacting substance of anaphylaxis (SRS-A), which plays an important role in the genesis of anaphylaxis and bronchial asthma. rGSTA4-4 (in older literature 'rat GST 88' or 'Yk-Yk') has a particularly high Michael addition rate with the toxic lipid peroxidation product 4-hydroxynon-2-enal, implying an important role for GST in the protection against oxidative stress.

Table 3 Rat cytosolic glutathione S-transferases and typical substrates

Glutathione S-transferases

Selective substrate

Alpha family

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