Review of Literature -...
Transcript of Review of Literature -...
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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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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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
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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
Review of literature
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
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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
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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
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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
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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
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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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