Glutathione conjugation

Glutathione is one of the most important molecules in the cellular defence against toxic compounds. This protective function is due in part to its involvement in conjugation reactions, and a number of toxicological examples of this such as bromobenzene and paracetamol hepatotoxicity are discussed later (see Chapter 7). The other protective functions of glutathione are discussed in Chapter 6. Glutathione is a tripeptide (figure 4.56), composed of glutamic acid, cysteine and glycine (glu-cys-gly). It is found in most cells, but is especially abundant in the liver where it reaches a concentration of 5 mM or more in mammals. The presence of cysteine provides a sulphydryl group, which is nucleophilic and so glutathione will react, probably as the thiolate ion, GS-, with

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Gliriathiona (j-ClltnainYtcvEKHnylolyana)

FIGURE 4.56 Structure of glutathione.

electrophiles. These electrophiles may be chemically reactive, metabolic products of a phase 1 reaction, or they may be more stable foreign compounds which have been ingested. Thus, glutathione protects cells by removing reactive metabolites. Unlike glucuronic acid or sulphate conjugation, however (type 1 conjugation reactions), the conjugating moiety (glutathione) is not activated in some high energy form. Rather the substrate is often in an activated form. Glutathione conjugation may be an enzyme-catalysed reaction or simply a chemical reaction. The glutathione conjugate produced by the reaction may then either be excreted, usually into the bile rather than the urine, or the conjugate may be further metabolized. This involves several steps: removal of the glutamyl and glycinyl groups and acetylation of the cysteine amino group to yield a mercapturic acid or ^-acetylcysteine conjugate. This is illustrated for the compound naphthalene which is metabolized by cytochromes P-450 to a reactive epoxide intermediate, then conjugated with glutathione and eventually excreted as an ^-acetylcysteine conjugate (figure 4.57). This sequence of further catabolic steps has been termed phase 3 metabolism. Naphthalene is toxic to the lung and these metabolic pathways are important in this toxicity (see below). There are many types of substrates for glutathione conjugation including aromatic, aliphatic, heterocyclic and alicyclic epoxides, halogenated aliphatic and aromatic

Conjugation Naphthalene Images
FIGURE 4.57 Conjugation of naphthalene-1, 2-oxide with glutathione and formation of naphthalene mercapturic acid.
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FIGURE 4.58 Conjugation of various epoxides with glutathione.
Glutathione Conjugation
FIGURE 4.59 Displacement of aliphatic and aromatic halogens by glutathione.

compounds, aromatic nitro compounds, unsaturated aliphatic compounds and alkyl halides (figures 4.58 -4.60). In each case the glutathione is reacting with an electrophilic carbon atom in an addition or substitution reaction. With the reactive epoxides the two carbon atoms of the oxirane ring will be electrophilic and suitable for reaction with glutathione. Such a reaction occurs with bromobenzene and the polycyclic hydrocarbon benzo[a]pyrene (see Chapter 7). With aromatic and aliphatic halogen compounds and aromatic nitro compounds, the nucleophilic sulphydryl group of the glutathione attacks the electrophilic carbon atom to which the electron withdrawing halogen or group is attached, and the latter is replaced by glutathione (figure 4.59). With unsaturated compounds such as diethylmal-

FIGURE 4.60 Conjugation of an unsaturated aliphatic compound with glutathione and structure of diethylmaleate, a typical example.

eate, the electron withdrawing substituents allow nucleophilic attack on one of the unsaturated carbon atoms and addition of a proton to the other, leading to an addition reaction (figure 4.60). Diethylmaleate reacts readily with glutathione in vivo and has been used to deplete tissue levels of the tripeptide. The reaction may be catalysed by one of the glutathione-S-transferases. These are cytosolic enzymes, although also detectable in the microsomal fraction, and are found in many tissues but particularly liver, kidney, gut, testis and adrenal gland. There are at least six isoenzymes of glutathione transferase, each having specificity for a particular substrate type and there are three or more non-identical sub-units arranged as dimers, various combinations of which make up the functional isoenzymes. Although a wide variety of substrates may be accepted, there is absolute specificity for glutathione. However, the substrates have certain characteristics; namely they are hydrophobic, contain an electrophilic carbon atom and react non-enzymatically with glutathione to some extent. It appears that as well as catalysing conjugation reactions some of the transferases have a binding function. Thus, transferase B is also known as ligandin and will bind both endogenous substances such as bilirubin and such exogenous compounds as the drugs penicillin and tetracycline. This binding facility is associated with one particular sub-unit but is not directly related to catalytic activity. Thus some of the compounds bound to glutathione transferase B (ligandin) are not substrates for the transferase activity. Thus, the enzyme also has a transport or storage function.

After the conjugation reaction, the first catabolic step, removal of the glutamyl residue, is catalysed by the enzyme y-glutamyltranspeptidase (glutamyltransferase). This is a membrane-bound enzyme found in high concentrations in the kidney. In the second step the glycine moiety is removed by the action of a peptidase, cysteinyl glycinase. The final step is acetylation of the amino group of cysteine by an ^-acetyl transferase which utilizes acetyl CoA and is a microsomal enzyme found in liver and kidney, but is different from the cytosolic enzyme described below. The resultant N-acetylcysteine conjugate, also known as a mercapturic acid, is then excreted. With aromatic epoxides, as in the example of naphthalene shown in figure 4.57, the ^-acetylcysteine conjugate may lose water and regain the aromatic ring structure. This will generally not occur in other types of glutathione conjugation reaction.

Also the intermediate such as the cysteinyl-glycine and cysteine conjugates may be excreted as well as being metabolized to the ^-acetylcysteine derivative. It should be noted, however, that there are now examples of glutathione conjugates being involved in toxicity. For example 1, 2-dibromoethane forms a glutathione derivative catalysed by glutathione transferase which loses a halogen atom and becomes a charged episulphonium ion (figure 4.61). This reacts with DNA to give a guanine adduct which is believed to be responsible for the mutagenicity of the 1, 2-dibromoethane. The diglutathione conjugate of bromobenzene

Glutathione Conjugation

FIGURE 4.61 Glutathione mediated activation of 1, 2-dibromoethane. The addition of glutathione is catalysed by glutathione transferase. Loss of bromide from the glutathione conjugate gives rise to an episulphonium ion. This can react with bases such as guanine in DNA.

is believed to be involved in the nephrotoxicity of bromobenzene after further metabolic activation (figure 7.14).

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