Review of Literature - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/35195/7/07_chapter2.pdf · Review of Literature ... • Nomenclature and classification • Subcellular

Review of Literature

• Glutathione S-transferase (GS'D

• Nomenclature and classification

• Subcellular localization of GST

• Catalysis and structure

• GST as intracellular binding proteins

• GST and physiology

• GST expression

• GST supergene family

• GST substrates

• Post-translational modifications of GST

• Other functions

• Sex-specific expression of GST

• GST mu gene family

• GST and testis

PiGST

MuGST

• GST and cancer

• GST induction and its applications

• GST as therapeutic targets in disease

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Review of literature

Glutathione S-transferase (GST)

Glutathione S-transferases represent a major group of

detoxification enzymes. They are known to be enzymes that catalyze the

nucleophilic attack of the sulfur atom of glutathione on electrophilic

groups in a second substrate (Daniel, 1993). They are believed to play an

important role in the protection of cellular macromolecules from attack by

reactive electrophiles. Their main function is the intracellular

detoxification of mutagens, carcinogens and other noxious chemical

substances. In addition, GSTs, via their glutathione-dependent

peroxidase activity, may play an important role in protecting tissues from

endogenous organic hydroperoxides produced during oxidative stress.

They bind reversibly, usually with high affinity and high capacity, to

certain hydrophobic organic compounds such as heme, bilirubin,

hormones and drugs and thereby act as intracellular carrier proteins for

the transport of various ligands. They have also been known to serve a

protective role by binding covalently with certain reactive electrophilic

molecules with consequent inactivation and immobilization. The

biochemical basis for protection by GST includes not only conjugation

reactions, but also drug sequestration.

Different GSTs may exhibit different activities for either a specific

compound or metabolites formed from the particular compound. In this

manner, the GST supergene family provides several tiers of defense

against toxic chemicals through the concerted actions of several

isoenzymes.

GSTs are widely distributed in nature and in addition to mammals,

are also found in fish, insects, plants, parasites, yeast, fungi, and bacteria.

They differ in their expression from one tissue to another and they may be

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. Review of literature

activated by inducers of drug metabolism (Mannervik., 1985). In view of

the putative detoxification functions of the proteins, GSTs may participate

in adaptive responses of these organisms to insecticides, fungicides, and

antimicrobial agents (Listowsky, 1993).

Nomenclature and classification

Several nomenclatures have been proposed for rat GST subunits

over the years. In an early attempt by Boyland and Chasseaud (1969) to

classify different forms of glutathione transferase based on their

specificities towards electrophilic substrates, they introduced the terms

aryltransferase, epoxide transferase, alkyltransferase, aralkyltransferase

and alkenetransferase. Later, separation and purification of several forms

of the enzyme demonstrated, that these enzymes display overlapping

substrate specificities. Consequently, the original nomenclature was

replaced by designations based on the physical or structural properties of

the proteins rather than on their enzymatic properties. Bass et al (1977)

resolved rat hepatic cytosol enriched for GST (referred to as the "Y"

fraction or a "ligandin-containing" fraction) into three bands which were

designated as Ya, Yb and Yc according to their decreasing anodal

mobility. It was later found that the Ya and Yc bands represent class

alpha, whereas the Yb band represents class mu GST (Hayes et al, 1979).

Jakoby and coworkers (1984) identified six different forms of GST

in rat liver which they empirically named as GST E, D, C, B, A and AA, in

the order of their elution from a carboxymethykellulose ion exchange

matrix. A seventh form identified earlier by Gillham (1973) was named,

GST M. The Arabic numeral nomenclature for GST, devised by Jakoby et

8


al (1984) is unambiguous for identification of the subunits but does not

clearly indicate the gene family to which each subunit belongs.

Mannervik and Jensson (1982) showed that six major enzyme

forms in rat liver can be regarded as homo or heterodimeric combinations

of four different subunits with distinct substrate specificities. Since the

enzymatic properties of a protein dimer would reflect its subunit

composition, it was suggested that GST should be named on the basis of

its constituent subunits.

A class-based subunit nomenclature has been proposed that groups

subunits by gene family and numbers them according to their order of

discovery (Mannervik et al, 1992). This system was originally devised for

human transferases but it is generally applicable. In this nomenclature,

single capital letter abbreviations are used to signify the alpha (A), the mu

(M), the pi (P), the sigma (S), and the theta (T) classes, and Arabic

numerals are employed for numbering each of the separate gene products

; for example, class alpha subunits are called A1, A2, A3, etc. The dimeric

GST isoenzymes are represented by the single letter suffix (signifying

class) followed by hyphenated Arabic numerals (signifying each of the

two subunits). Through subunit hybridization, more than 15 class alpha,

15 class mu, and 5 class theta GST isoenzymes are formed in the rat

(Hayes and Pulford, 1995). GSTs in the rat have been found to be encoded

by as many as 20 genes or more.

Cytosolic rat GSTs are the most widely studied so far. A

comparison of the proposed systems of nomenclatUre for them is shown

in table a.

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Table a. Rat GST subunits

Class Class-based "Y" SDS-PAGE Number of Original subunit subunit subunit name of nomenclature terminology enzyme

(homodimer)

Alpha rGSTA1 Ya1 1a Ligan din Alpha rGSTA2 Ya2 1b Ligan din Alpha rGSTA3 Yq 2 GSTAA Alpha rGSTA4 Yk 8 GSTK Alpha rGSTAS Yc2 10 Alpha n.i. Ya3

n.i. Ys n.i. GSTA

Mu rGSTM1 Yb1 3 GSTA Mu rGSTM2 Yb2 4 GSTD Mu rGSTM3 Yb3 (Yn1) 6 Mu · rGSTM4 Yb4 Mu rGSTMS Yn2 9 Mu rGSTM6 Yo 11 Pi rGSTP1 Yf (Yp) 7 GSTP Sigma rGSTS1 PGDS Theta rGSTT1 5 GSTE Theta rGSTT2 Yrs (Yrs') 12 GSTM Theta rGSTT3 13 Microsomal

GST

Membrane-bound GST Mitochondrial GST

PGDS, Prostaglandin D synthetase; n.i., not included, indicates that a firm designation cannot be made because of either lack of certainty about the class or lack of proof that the subunit is genetically distinct

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Obvious similarities exist between the rat and mouse GST. The

class alpha, mu, pi and theta GST are all represented in the mouse. A

surprising feature about the mouse enzymes is that two distinct class pi

GST subunits exist (Bammler e~ al, 1994), whereas the rat possesses only

one class pi GST (Okuda et al, 1987). In the mouse, the enzyme forms have

been classified also according to the strain from which they have been

isolated (Warholm et al, 1986). In humans five basic proteins purified from

liver cytosol were named GST a, f3, y, 8, and a, in order of their increasing

isoelectric points (Mannervik and Danielson, 1988). There is evidence for

as many as five different human class alpha genes, namely those

encoding A1, A2, A3, A4 and skin GST 9.9. Certain class mu enzymes that

are not expressed in the liver are found in human muscle, testis, and

brain. In addition to the hGSTM1a and M1b subunits, hGSTM2, M3, M4

and MS subunits have been obtained from extrahepatic tissues and cell

lines. The class pi transferase, hGSTP1-1, has been purified from many

extrahepatic tissues (Berhane et al 1994). Two class theta transferases,

GSTT1-1 and GSTT2-2, have been isolated from human liver (Meyer et al,

1991).

Subcellular localization of GST

GSTs are ubiquitously present largely as cytosolic proteins. The

cytosolic GSTs exist as dimeric proteins held together by noncovalent

interactions, composed of similar or different subunits, ranging from

23,000 to 26500 Mr (Aceto et al, 1989). Each subunit of a heterodimer or

homodimer is kinetically independent of the other subunit and has been

shown to dimerize with other subunits of its class only. In addition to

cytosolic GSTs, at least two membrane-bound GST exist in mammals.

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These are referred to as microsomal GST and leukotriene C4 synthase

(LTC4S) (Hayes and Pulford, 1995). The microsomal GST is involved in

the detoxification of xenobiotics, whereas LTC4S, as the name suggests,

conjugates leukotriene A4 with GSH. As far as is currently known, LTC4S

does not play a role in drug metabolism. Since neither of the two

membrane-bound GSTs share sequence identity with the cytosolic

enzymes, it is assumed that they have each evolved separately. The

microsomal GST is immunologically distinct~ and apparently exists

functionally as a trimer of a 154-amino acid subunit of about 17 kDa

(Dejong et al, 1988). In its 5' untranslated region, its gene has a stop codon

in frame with the AUG initiation codon, so it is unlikely to have a transmembrane signal sequence (Listowsky, 1993).

Multiple forms of GST identified so far have been based on their

substrate specificities, sensitivities to inhibitors, reactions with specific

antisera, physicochemical properties, amino acid composition, peptide

maps, subunit assembly patterns, ligand binding affinities, and functional

properties. GST expression is tissue specific and multiple forms are often

found in the same cell type. Chromatofocussing and immunological

studies have identified at least 16 different GST subunits in human testis,

10 in rat testis and 6 in mouse testis (Fulcher et al, 1995). So far an

invariable feature of GSTs is that no heterodimers exist between subunits

of the three different classes. Also, the sequence homology between

rodent and human GSTs within an individual class is greater than that

between the different classes of GSTs in the same species (Listowsky ,

1993).

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Catalysis and structure

The fundamental basis for the various catalytic activitites of GST is

the ability of the enzyme to lower the pKa of the sulfhydryl group of

reduced glutathione (GSH) from 9.0 in aqueous solution to about 6.5

when bound in the active site (Armstrong ,1994). Glutathione has been

shown to exist as the thiolate (GS-) anion at neutral pH when complexed

with GST (Graminski et al, 1989). X-ray crystallographic studies have also

shown that a conserved tyrosine (in classes alpha, mu, pi, and sigma) or

serine (class theta), found at the N-terminus of most cytosolic GST, is

involved in stabilizing GS- through hydrogen bonding (Hayes and

Pulford, 1995). The glutathione binding site exhibits a high specificity

(Adang et al, 1989), whereas, by contrast, the second substrate- binding

site displays a broad specificity toward hydrophobic compounds. Two

sites are therefore designated on the enzyme, the G-site or the GSH

binding site and the H-site or the hydrophobic substrate-binding site

(Mannervik, 1985). Secondary structures have been predicted for GST

which show that all GSTs should be referred to as a./13 proteins,

characterized by an alteration of a.-helices and 13-strands along the

polypeptide chain. It is therefore possible, that the transferases have an

active-site cavity, which is formed in the region where the C-terminal

ends of two adjacent 13-strands join a.- helices on opposite sides of a 13-

sheet (Mannervik and Danielson, 1988). X-ray crystallographic studies

have revealed that cytosolic GST subunits are folded into two separate

domains of different structure. The smaller N-terminal a./13

domain,domain I, contains most of the amino acids that form the G-site(1

to 78 residues in class alpha GST, 1 to 82 residues in class mu GST, 1 to 74

residues in class pi GST, 1 to 74 residues in class cr GST, 1 to 78 in class

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theta GST) , whereas the larger a domain, domain II contains essentially

all of the H-site (residues 86 to 222 of class alpha GST, residues 90 to 217

of class mu GST, residues 81 to 207 of class pi GST, residues 81 to 202 in

class sigma GST, residues 85 to 208 in class theta GST (Armstrong, 1994).

Within the G-site, there is a conserved tyrosine and a conserved aspartate

which are involved in catalysis. The role of the conserved tyrosine re.sidue

in classes alpha, mu and pi is performed by a serine residue in class

theta GST (Hayes and Pulford, 1995). In class alpha GST, the C-terminal

portion of the protein has been indicated in determining the substrate

specificity (Board and Mannervik, 1991). For all GSTs, the C-terminal

segment seems to be important for the active-site structure (Cooke et al,

1994) as it has been shown to indirectly contribute to enzyme activity by

maintaining the quarternary structure of the protein. The binding of

glutathione to the enzyme molecule appears to involve ionic bonds

(Mannervik and Danielson, 1988). The glutathione molecule has several

functions in the glutathione transferase catalyzed reactions, not only as a

substrate providing the thiol group for different types of chemical

reactions but also as a substrate contributing a carboxylate that acts as a

proton acceptor in the catalytic mechanism and a carboxylate that

modulates binding of the substrate to the enzyme (Widersten et al, 1996).

GSH thus is not only a reactant in the catalyzed reaction but also

contributes functional groups to the catalytic apparatus, and in this

manner, serves as both a substrate and a cofactor for the enzyme .

GSTs as intracellular binding proteins

The GSTs are known to bind bilirubin, heme, bile acids, fatty acids,

and their metabolites and even certain neurotransmitters (Listowsky et al,

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1988). Physiologically, binding to GSTs could prevent accumulation of the

nonpolar molecules at lipophilic sites such as membranes, and thereby

avert cytotoxic events. Binding may also direct transport of these

compounds or direct them to their site of action. GSTs contain non

substrate binding domains. Therefore, lack of inhibition does not

necessarily denote lack of binding to the protein. The anionic GSTs have

two high affinity binding sites per dimer as opposed to the single site for

the cationic forms. There is a certain degree of specificity observed in the

extent of binding for the different forms of the protein. Ligands for GST

do not serve as substrates for them. GSH does not compete with steroids

for the high affinity binding site on the anionic transferase (Maruyama

and Listowsky, 1984). Steroid binding to Ya and Yc forms has been shown

to be of lower affinity than Yb. In view of the selective high-affinity

binding of steroids to pure Yb forms, these proteins have the potential to

function in the formation, transport, metabolism, and perhaps even in the

action of steroid hormones (Homma et al, 1986 ). It has also been shown

that dissociation rates from the Yb forms appear to be sufficiently fast to

permit exchange of bound steroids between this protein and other

components involved in their metabolism and action.

GST and physiology

Evidence suggests that the level of expression of GST is a crucial

factor in determining the sensitivity of cells to a broad spectrum of toxic

chemicals (Hayes and Pulford, 1995). The qualitative and quantitative

differences in the occurrence of various GSTs in different organs as well

as in the same organs of different individuals are of particular

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toxicological importance and might cause differential susceptibility of

tissues to the toxic effect of particular xenobiotics (Aceto et al, 1989).

It has been proposed that GST mu-class may have an especially

important role in the detoxification of geno- and cytotoxic epoxides. It has

been shown that only 60% of the human population expresses this class

and among smokers, those lacking the protein had a significantly high

incidence of lung cancer in comparison to those with GST mu activity

(Mannervik and Danielson, 1988). Exercise training in female rats

induces significant increase in liver GST (Veera Reddy et al, 1995).

GST expression

Multiple forms of GST have been reported. GSTs are known to

exist both in the cytosol and in nuclei (Hayes and Mantle, 1986). They are

known to be age-, sex-, tissue-, species-, and tumor-specific in expression.

A variety of chemicals have been identified that can induce GST. The

induction of GST represents part of an adaptive response mechanism to

chemical stress caused by electrophiles. Transcriptional activation of the

GST genes has been found to be responsible for the increased levels of

mRNAs (Ding, 1986). The occurrence of the different forms changes

dramatically in an organ-specific manner during transition from the fetal

to the adult state (Mannervik and Danielson, 1988). RNA blot

hybridization studies using GST eDNA probes, suggest that the

regulation of tissue specificity in GST expression may occur before the

maturation of mRNAs (Lai et al, 1988).

In rat, the Yb1 and Yb2 subtypes are found in many cell types but

some tissues preferentially express one another. In contrast, the

expression of Yb3 is largely limited to brain and testis (Abramovitz et al,

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1988). The mRNA for Yb3 is longer than that of Yb1 or Yb2 (Abramovitz

and Listowsky, 1987).

Expression patterns of the different GST isoenzymes vary in the

course of development. In rats, it has been shown that liver GST activity

increased approximately fivefold in the first few weeks of development

(Hales and Neims, 1976). These increases were largely due to increased

expression of alpha-class GSTs since Ya mRNA appears and Yp mRNA

disappears rapidly in neonatal animals (Abramovitz and Listowsky,

1988). A fetal type subunit ( alpha-class ) not found in adult liver, was

also identified (Scott and Kirsch, 1987). Liver acquires specialized

functions in the first weeks after birth; that is the time when alpha-class

GSTs appear and pi-class GSTs disappear (Abramovitz and Listowsky,

1988).

Expression of rat liver GST P1-1 has been shown to have three sites

of regulation in a transient induction assay with lead nitrate, which are

transcription, post-transcription and post-translation (Koo et al_ 1994).

Alternative splicing has been demonstrated in a human J..t-class

GST which may represent either a novel form of regulation in this

multigene family or illegitimate transcription and experimental

alternative splicing as part of the evolutionary process (Ross and Board

,1993).

GST supergene family

All eukaryotic species possess multiple cytosolic and membrane

bound GST -isoenzymes, each of which displays distinct catalytic as well

as noncatalytic binding properties: the cytosolic enzymes are encoded by

at least five distantly related gene families (alpha, mu, sigma and theta

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class) whereas the membrane-bound enzymes, microsomal GST and

leukotriene C4 synthase, are encoded by single genes, and both have

arisen separately from the soluble GST.

GST have been studied widely in three species namely rats, mice

and humans. All the class alpha genes isolated from rats, mice and

humans are 11-12kb in length and comprise seven exons. The class mu

genes isolated from rats, mice and humans are all about 5 kb and are

composed of eight exons; by contrast, a hamster mu class GST comprises

nine exons. Oass pi genes from rats, mice and humans are about 3 kb and

contain seven exons. A rat class theta gene has been cloned, which is 4 kb

in length and contains five exons. In humans, the class alpha, mu, pi and

theta GST genes are located on chromosomes 6, 1, 11 and 22, respectively

(Hayes and Pulford, 1995). Dejong et al (1990) have suggested that the

microsomal GST gene contains at least three exons and spans less than 12

kb (Dejong, 1990) . Within a class, protein-coding regions are highly

homologous (70 to 80% ), while the 5'- and 3'- untranslated regions are

very divergent (Tsuchida and Sato, 1992).

GST Substrates

The single most important substrate used for the demonstration of

multiple forms of GST in various biological species is 1-chloro-2,4-

dinitrobenzene, which is widely recognized as the "general substrate" for

GSTs. But certain forms of the enzyme have been found to express low

activity with this substrate which necessitates the use of several substrates

for screening of new sources of the enzyme (Mannervik and Danielson,

1988). One of the basic structural elements of GST substrates is a carbon

carbon double bond activated by an adjacent electron-withdrawing

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carbonyl group. The glutathione conjugation catalyzed by the GST

appears to be a thiol addition to an aA3-unsaturated carbonyl compound

that involves a nucleophilic attack by the sulfhydryl group on the

electrophilic 13 carbon of the double bond. GSTs use a wide variety of

compounds as their substrates. Model GST substrates that display

selectivity for particular subunits are often used in a "diagnostic sense" to

identify isoenzymes. Examples of compounds used for this purpose are,

~5 androstene-3,17-dione for rGSTA1 and A2, 4-hydroxynonenal for

rGSTA4, 1,2-dichloro-4-nitrobenzene for rGSTMl, trans-4-phenyl-3-

buten-2-one for rGSTM2, 1,2-epoxy 3-(p-nitrophenoxy) propane for

rGSTT1 and 1-menaphthyl sulfate for rGSTT2 (Hayes and Pulford, 1995).

The physiological significance and the toxicological implications of the

differences in the sensitivities to substrates in the different classes of GSTs

are incompletely understood, but it has been proposed that GST mu may

have an especially important role in the detoxification of geno and

cytotoxic epoxides.

Post-translational modifications of GST

Post-translational regulation of GST is suggested by reports that

GSTs are activated by active oxygen species (Murata et al, 1990) and that

alpha class GSTs are substrates for protein kinase C (Pyerin et al, 1987) .

Phosphorylation of GST1-1 results in decreased affinity for bilirubin,

indicating a functional significance for this post-translational modification

(Taniguchi and Pyerin, 1989). Methylation of GST which is dependent on

the tissue-specific expression of cytosolic GST methyltransferase, inhibits

GST activity (Johnson et al, 1992) . Glutathione, an inhibitor of GST

methylation, at the physiological concentration, may be sufficient to

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suppress GST methylation in vitro. Methylation of one subunit of the GST

dimer was found to be sufficient to inhibit GST activity. It is not clear

however, whether these modifications occur in vivo and the biological

significance of modifications remains to be clarified.

The human class pi GST and rat GST 7-7 are glycosylated while

the human LTC4S contains a potential N-linked glycosylation site. It is

suggested that glycosylation may be involved partly in the

microheterogeneity of these subunits observed on isoelectric focusing

(Kuzmich et al, 1991) . Of the three classes of murine glutathione S

transferases alpha. mu and pi, the alpha-class is probably acetylated

(Mitchell et al, 1995).

Other functions

Bennett et al have provided evidence that the nonhistone protein

BA, previously demonstrated to co-localize with U-snRNPs within

discrete nuclear domains, is a GST (Bennett et al, 1986) . The parasitic

helminths of the genus Schistosoma have surface antigens that are GSTs.

Acquired immunity in mice, rats, hamsters, and monkeys against this

antigen from S. japonicum or ~· mansoni has been shown to mediate

significant protection against Schistosomiasis (Veri et al, 1994) . These

enzymes are also known to have isomerase activity and to participate in

leukotriene C .biosynthesis . Ligandin is known to isomerize .1,5-3-

ketosteroids by activating glutathione as a nucleophile, but glutathione

does not get consumed in the reaction (Benson et az; 1977).

Acute nephrotoxicity has been reported to mediate enhanced

expression of GST-P isozyme suggesting a role for the enzyme in

mediating cell repairs or increasing the resistance to subsequent injury

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(Fukuda et al, 1996). GST pi has been shown to be involved in arsenic

detoxification by facilitating excretion of arsenic (Wang and Lee, 1993).

GSTs also catalyze selenium-independent GSH peroxidase

reactions with organic hydroperoxides as substrates. In the peroxidase

reaction GSH acts as a reductant with the concomitant formation of

glutathione disulfide. Alpha-class GSTs usually have greater GSH

peroxidase activities (Listowsky et al, 1988).

, I

Sex-specific expression of GST

In the mouse, a sex-related difference in the hepatic expression of a

specific enzyme form, apparently under testosterone control has been

noted (Hatayama et al, 1986) . GST IT (GST-P) in mouse markedly

increases at puberty in males where as no change occurs in the females.

By castration, the levels in males decreased to those in females, while

those in females increased to those in adult males by administration of

testosterone, indicating that this form is developmentally regulated by

testosterone. Singhal et al (1992) have reported that there are differences in

the specific activity of class pi GST (peak II) from male and female mouse

liver. It has been observed that the two different murine class pi GST

subunits, Pl and P2, although possess 97% identity, have marked

variation in the enzyme activity. Because class pi GST is found in

approximately ten fold greater amounts in the male mouse liver than in

the female mouse liver, and the activity is also greater in hepatic cytosol

from male than from female mice, the enzyme iri the liver of the male

mouse comprises primarily the active subunit Pl whereas the enzyme in

the liver of the female mouse comprises both the active P1 and the


inactive P2 subunits. It would then appear that the P2 subunit is the

male-specific class pi GST in the mouse (Hayes and Pulford, 1995).

· It has been shown in the rat that livers from females contain higher

levels of the Ya subunit than those of males (Hales and Neims, 1976).

Likewise in the mouse Yf was found to be conspicuously present in males

of all strains, but present in much smaller amounts in females (Me Lelia~

and Hayes, 1987). The sex-specific expression of YfYf in male mouse may

explain the occurrence of spontaneous hepatomas with a much higher

frequency in inbred male mice than in females (Smith et al, 1973). Liver

from adult female rats have been found to contain about 10-fold greater

levels of Yc2 than is found in livers from male rats. This sex-specific

expression of Yc2 in adult rat liver may contribute to the relative

insensitivity of female rats to aflatoxin B1 (Hayes et al, 1994) . Zangar et al

(1992) reported that adult male rats which have been exposed neonatally

to diethylstilbestrol express a hepatic alpha-class GST which is absent

from adult male rats which have not been treated with this synthetic

estrogen. It was later suggested that this a-class GST resembles GST Yc2

biochemically and since livers from the estrogen treated rats showed

higher activity toward aflatoxin B1 as compared to the untreated livers,

the subunit expressed under the influence of estrogen is probably Yc2(

Hayes et al, 1994). The increase in the size of the hepatic foci positive for

GST-P in female rat livers initiated with diethylnitrosamine and

promoted with 2-acetylaminofluorene and partial hepatectomy, was

found to be under the control of testosterone and not estrogen (Liao et al,

1996) . The physiological implication of the sex-specific expression is

unclear but is likely to be of pharmacological significance.

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GST mu gene family

The eDNA clones corresponding to subunits Yb1, Yb2 and Yb3 of

the rat GST Yb family have been isolated and· characterized. A sequence

comparison among these clones shows about 80% Identity in their protein

coding region and a very high divergence in the 3'-nontranslated mRNA

regions. There is little sequence homology between Yb cDNAs and the Ya

and Yc nucleotide sequences and no cross hybridizations have been

observed between the two GST families under moderately stringent

conditions. The structure of rat GST Yb1, Yb2 and Yb4 genes is similar:

they span about 5 kb, contain eight exons, and three out of seven introns

are conserved to the extent of more than 88% nucleotide identity. This

latter observation has led to the assumption that a gene conservation

mechanism may have played a role in the evolution of GST Yb genes.

Ybs are known to function as DNA-binding proteins that are

present in interchromatic regions and have been proposed to be involved

in nuclear RNA processing (Bennett et al, 1986) . GST gene mGSTM5 in

the mouse seems to be expressed first in the meiotic phase of

development of spermatogenic cells (Fulcher et al, 1995) .

GST and testis

GSTs are best characterized for their capacity to inactivate cytotoxic

substances via conjugation with glutathione. Because of this and the

critical need to maintain integrity of the DNA in spermatogenic cells to

ensure propagation of the species, it is plausible that their primary

function in the testis is protective. In addition to detoxifying electrophilic

compounds, they can also serve as glutathione peroxidases in the testis,

where majority of the peroxidase activity is·accounted for by GSTs, and a

23


class mu GST is known to have high pyrimidine hydroperoxidase activity

(Koo et al, 1994).

PiGST

Many GST subunits are reported to be present in the testis

however, direct evidences regarding their function are not known.

Presence of pi GST (GST -P) has been investigated in the testis. GSTs have

been shown to be important in normal spermatogenesis and protection of

germ cells from teratogens and carcinogens (Klys et al, 1992). GST-P (a Yf

Yf or a Yp-Yp homodimer) mRNA was detected in cultured rat Sertoli

cells, peritubular cells, as well as in transplantable Ieydig cell tumor,

however no GST-P mRNA was detected in the germ cells. When checked

for the presence of testicular GST-P mRNA across the different stages of

germ cell development represented by animals of different days of age,

the mRNA level was found to go up from day 5 to 20 of age after which

there was a decline, the latter probably represents the relative decrease in

somatic cells of the testis due to the increase in germ cells. GST-P has been

reported to have high catalytic efficiency towards certain carcinogens and

synthetic 5-hydroxymethyl uracil, which indicate that this enzyme may

be involved in the repair of DNA (Tan et al, 1986). The high concentration

of GST-P in Sertoli and peritubular cells may therefore participate in

removing toxic compounds which would otherwise reach the germ cells

inside the blood testis barrier (Yoganathan et al, 1989). In addition to its

catalytic role in the addition of glutathione to reactive compounds, GST-P

has high glutathione peroxidase activity towards fatty acids and thymine

hydroperoxides (Meyer et al, 1985). In rats also, the Yf protein was

localized by immunocytochemistry in the Sertoli and Leydig cells (Veri et

24


al, 1993), where they are speculated to be involved in the intracellular

transport of steroids (Homma et al, 1986). Whereas most members of the

GST protein family are found in the greatest abundance in the liver, GST

P is present at higher levels in the epididymis than in any other tissue of

the adult male rat (Pemble et al, 1986). Veri et al, (1993) have reported by

immunocytochemistry that the Yo subunit is expressed in the testis and

not other tissues such as liver, spleen, or kidney, in the rat It was found to

have relatively low affinity for both glutathione and 1-chloro-2A

dinitrobenzene (Hayes, 1988).

MuGST

In humans, an isoenzyme of the class mu GSTs, having an

isoelectric point of 5.2, was found to be a major form in the testis and

present as well in cerebral cortex (Campbell et al, 1990). It could not be

detected in the liver. It was suggested that the testis-brain class mu GSTs

having distinct catalytic and structural properties may be uniquely

involved in blood-barrier functions common to both organs . Similar

patterns of tissue-specific expression of class mu GST which are missing

in livers but present in testis and brains have now been observed in both

rats (Abramovitz and Listowsky, 1987) and humans.

The activities of GSTs in the epididymis are androgen dependent ;

this androgen dependence is specific to each region of the epididymis

(Robaire and Hales, 1982) . Testicular membranes are rich in

polyunsaturated fatty acids, and thus susceptible to peroxidation injury.

In pro-oxidant states, lipid peroxides are formed from polyunsaturated

fatty acids of biomembranes, causing a chain reaction that leads to

deterioration of the membrane structure and integrity. Glutathione

25


peroxidase and GST function in the detoxification of reactive lipid

peroxides and nicotinamide adenine dinucleotide phosphate (NADPH)

formed in the hexose monophosphate shunt iT-P mRNA is normally

present in a wide range of tissues such as kidney, lung, testis, heart,

spleen, and placenta (Pemble et al, 1986). GSTs are present at high levels

in the adrenal gland. An endogenous regulation of the isozymes has been

found to occur in this organ (Mankowitz et al, 1990). Seven isoforms of rat

testicular GSTs have been reported (Anuradha et al, 1995).

GST and cancer

GSTs have been widely reported in the context of cancer diagnosis.

Over expression of specific isoforms of GST in different types of

neoplasms are well documented. Apart from their possible use as tumor

markers, GSTs have also been shown to be involved in multidrug

resistance that is conferred on patients undergoing cancer chemotherapy.

Some forms of GST isolated from malignant human testis were found to

be present only in the testis seminoma suggesting that they may be

tumor-specific isoenzymes (Aceto et al, 1989). Similar patterns of

expression of GST and of estrogen receptors in vitro as well as in vivo

suggest that estrogen receptor-negative breast cancer cells may have

greater protection against antineoplastic agents conferred by GST than

estrogen receptor-positive tumors (Moscow et al, 1988).

GST expression was found to decrease in the mononuclear cells

of patients with chronic lymphoproliferative disorders such as chronic

lymphocytic leukemia. The low content of GST in B-cells could explain

the frequent sensitivity of this disease to alkylating agents (Marie et al,

1995). The demonstration that GST RNA levels are elevated in some

tumors, when compared with normal tissue, suggests that increased

26


expression of GST may serve as a useful marker clinically either of

carcinogen-induced premalignant changes or of inherent drug resistance.

Rat GST Y c has been shown to confer alkylating drug resistance in

mouse fibroblasts, following retrovirus-mediated gene transfer. This

raises the possibility of using GST Yc somatic gene transfer to confer

protection to the hematopoietic system in a gene therapy strategy

applicable to cancer (Greenbaum et al, 1994).

GST induction and its applications

GST is increased in many organisms following exposure to foreign

compounds. The diversity of organisms in which induction has been

obset:Ved and the spectrum of xenobiotics that can serve as inducing

agents, suggest that GST induction is part of an adaptive response

mechanism to chemical stress. Thus, GST along with other detoxification

enzymes provides protection against a range of harmful of compounds. It

has been reported that in selenium and copper-deficient rats, chronically

exposed to increased intracellular levels of hydrogen peroxide due to lack

of selenium-dependent glutathione peroxidase and superoxide dismutase,

marked overexpression of hepatic GST isoenzymes is observed (Arthur et

·az, 1987) . Besides providing protection against chemicals of foreign

origin, GSTs are involved in protection against oxidative stress.

Both species and strain differences in GST induction have been

observed in rats and mice (Hayes and Pulford, 1995). For instance, trans

stilbene oxide is a better inducer in the rat than in the mouse. The level of

induction can be influenced by the age and sex of the rat or mouse also.

The responsiveness of GST in rat liver toward phenobarbital has been

reported to be greatest in animals of about 4 weeks of age. Sexual

27


dimorphism in the induction of expression of GST in Sprague-Dawley

rats has been observed with phenobarbital, 3-methylcholanthrene and

butylated hydroxyanisole. Phenobarbital administered orally to mice,

caused higher GST induction as compared to intravenous administration

implying that the route of drug administration is also a determinant of

GST induction . Not all GST subunits are induced to the same extent by

drugs. Microsomal GST does not appear to be inducible by xenobiotics.

GST-P is specifically induced in rat liver and kidney by lead

cation, which could be blockeError! Bookmark not defined.d by

actinomycin D, suggesting that GST-P production by lead is regulated at

the transcriptional level (Suzuki et al, 1996). Human hepatocytes which

can be induced for production of GST using a wide variety of chemicals

can be employed for predictive studies of chemoprotection in human

pharmacology.

GSTs as therapeutic targets in disease

In general, GST polymorphisms may reflect host-specific genetic

factors that determine differences among individuals in their response to

drugs, hormones and toxins.

Leukotrienes and peptidoleukotrienes, such as LTD4, have been

shown to be important in the pathogenesis of diseases such as human

bronchial asthma. Since leukotriene C4 synthase has been identified as a

unique glutathione S-transferase · required for the production of

peptidoleukotrienes, it should be considered as an important target in

designing therapy for asthma (Rushmore and Pickett, 1993). Differential

cellular resistance to different alkylating agents used in cancer

chemotherapy has been demonstrated in vitro by transfection experiments

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with specific GSTs. Thus therapeutic strategies aimed at inhibiting

specific GSTs might be useful in extending the efficacy of certain anti

cancer drugs.

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Review of Literature - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/35195/7/07_chapter2.pdf · Review of Literature ... • Nomenclature and classification • Subcellular