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Age and gender dependent levels of glutathione and glutathione S-transferases in human lymphocytes

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Abstract

Glutathione S-transferases (GSTs) are a family of enzymes involved in the detoxification of a wide range of chemicals including chemical carcinogens. Human cytosolic GSTs are divided into four major classes; alpha, mu, pi and theta. This study was performed to evaluate the influence of age and gender on the GST isoenzyme expression and glutathione (GSH) content in lymphocytes. Blood was collected from 124 healthy controls, which were divided into age groups of 20-40 years (21 females, 20 males), 40-60 years (20 females, 21 males) and 60-80 years (20 females, 22 males). Lymphocytes were isolated by density centrifugation on Histopaque-1077. After homogenization, cytosolic fractions were isolated. Herein, GST isoenzyme levels were determined by densitometrical analysis of western blots after immunodetection with monoclonal antibodies. Total GSH content was determined by high performance liquid chromatography after conjugation with monobromobimane. Spearman rank correlation and Wilcoxon rank sum tests were used for statistical evaluation. Lymphocytic GSTmu and pi levels were not correlated with age or gender. GSTalpha was not detectable in lymphocytes. GSH contents were not different in males and females, but decreased with age in both males and females. In age group 60-80, GSH content was significantly lower as compared with age groups 20-40 and 40-60 in both sexes. Since high GSH is an essential factor in the detoxification of many compounds, these data indicate that the detoxification potential of the GSH/GST system in lymphocytes may decrease with age in man.
Carcinogenesis vol.19 no.10 pp.1873–1875, 1998
SHORT COMMUNICATION
Age and gender dependent levels of glutathione and glutathione
S-transferases in human lymphocytes
Esther M.M.van Lieshout and Wilbert H.M.Peters
1
Department of Gastroenterology, University Hospital St. Radboud, PO Box
9101, 6500 HB Nijmegen, The Netherlands
1
To whom correspondence should be addressed
Glutathione S-transferases (GSTs) are a family of enzymes
involved in the detoxification of a wide range of chemicals
including chemical carcinogens. Human cytosolic GSTs are
divided into four major classes; α, µ, π and θ. This study
was performed to evaluate the influence of age and gender
on the GST isoenzyme expression and glutathione (GSH)
content in lymphocytes. Blood was collected from 124
healthy controls, which were divided into age groups of
20–40 years (21 females, 20 males), 40–60 years (20 females,
21 males) and 60–80 years (20 females, 22 males). Lympho-
cyteswere isolatedbydensitycentrifugationonHistopaque-
1077. After homogenization, cytosolic fractions were isol-
ated. Herein, GST isoenzyme levels were determined by
densitometrical analysis of western blots after immuno-
detection with monoclonal antibodies. Total GSH content
was determined by high performance liquid chromato-
graphy after conjugation with monobromobimane. Spear-
man rank correlation and Wilcoxon rank sum tests were
used for statistical evaluation. Lymphocytic GSTµ and π
levels were not correlated with age or gender. GSTα was
not detectable in lymphocytes. GSH contents were not
different in males and females, but decreased with age in
both males and females. In age group 60–80, GSH content
was significantly lower as compared with age groups 20–
40 and 40–60 in both sexes. Since high GSH is an essential
factor in the detoxification of many compounds, these data
indicate that the detoxification potential of the GSH/GST
system in lymphocytes may decrease with age in man.
Human cytosolic glutathione S-transferases (GSTs) are a family
of dimeric enzymes, divided into the main classes α, µ, π and
φ (1–3). GSTs catalyse the binding of a large variety of
electrophiles to the sulphydryl group of glutathione (GSH),
generally resulting in less harmful and more water soluble
molecules (1). The GSH/GST system may be a critical factor
in protecting cells and organs against toxicity and disease.
GSH, an important factor in the normal functioning of the GSH/
GST system, is involved in the detoxification of xenobiotics,
carcinogens, free radicals and peroxides (1). Low GSH contents
have been found in several pathological conditions, including
alcoholic liver disease (4), acquired immunodeficiency syn-
drome (5), xenobiotic-induced oxidative stress and toxicity (6)
and (pre)cancerous lesions (7). From these studies it may be
concluded that the availability of GSH might be a key factor
in the maintenance of health, and that GSH concentration may
serve as a useful indicator of disease risk in humans.
Abbreviations: GSH, glutathione; GST, glutathione S-transferase.
© Oxford University Press
1873
Little is known about the differences in GST and GSH
expression between men and women. Also data on GST and
GSH levels with respect to aging in humans are scarce.
Loguercio et al. (8) showed that GSH content in body and
antrum of the stomach decreased with age. They did not find
a sex dependency in GSH content. To obtain information of
the lymphocytic GSH/GST system during aging, we investi-
gated GST isoenzyme levels and GSH contents in human
lymphocytes from 124 healthy subjects, aged 20–80 years.
Blood was collected by venapuncture into sterile siliconized
EDTA K3 10 ml vacutainer tubes (Beckton Dickinson, San
Jose, CA). Controls were divided into three age groups; 20–
40 years (21 females, 20 males), 40–60 years (20 females,
21 males) and 60–80 years (20 females, 22 males). Lympho-
cytes were isolated by density centrifugation on Histopaque-
1077, according to the manufacturers instructions (Sigma
diagnostic, St Louis, MO). Lymphocytes were pelleted and
stored at –20°C until use. For preparation of cytosolic fractions,
lymphocytes were thawed slowly, homogenized in 100 µlof
20 mM Tris–HCl buffer pH 7.4, containing 1 mM dithiothreitol
using a glass/glass potter. Homogenates were centrifuged at
12 000 g (4°C) for 20 min. Aliquots of the supernatant were
stored at –20°C until use. The investigations were approved
by the local ethical committee on human experimentation.
Protein was assayed in triplicate by the method of Lowry
et al. (9), using bovine serum albumin as a standard. Specific
GST isoenzyme levels were determined as described previously
(10). In short, cytosolic fractions were subjected to SDS–
PAGE [11% acrylamide (w/v)], and subsequently to western
blotting, using a semi-dry blotting system (Novablot II, Pharm-
acia, Uppsala, Sweden). Western blots were incubated with
monoclonal antibodies against human GST classes α, µ and
π. Class α antibodies react with human GST A1-1, GST
A1-2 and GST A2-2 (10). Class µ antibodies recognize human
GST M1a-1a, GST M1a-1b and GST M1b-1b (11,12). Class
π antibodies react with human GST P1-1 (13). The specific
binding of the monoclonal antibodies to the isoenzymes was
demonstrated by incubation with peroxidase-conjugated rabbit
anti-mouse immunoglobulins (Dakopatts, Glostrup, Denmark)
andsubsequent stainingwith 4-chloro-1-naphtholand hydrogen
peroxide. Staining intensity on the immunoblots was quantified
using a laser densitometer (Ultroscan XL, LKB, Bromma,
Sweden). Known amounts of purified GSTs were run in parallel
with the experimental samples and served as standards for the
calculation of the isoenzyme levels in the cytosolic fractions.
The detection limit of this assay is ~50 ng/mg protein. Total
GSH was quantified by high performance liquid chromato-
graphy after reaction with monobromobimane, as described
previously (14). In this assay, oxidized GSH present is reduced
by adding sodium borohydride to the reaction mixture. A
Spearman rank correlation test was used to correlate
lymphocytic GSH content and GST isoenzyme expression
with age and gender. A Wilcoxon rank sum test was used to
assess statistical significance of differences between age
E.M.M.van Lieshout and W.H.M.Peters
Table I. GSH content and GSTα, µ and π isoenzyme levels in human lymphocytes
Age group GSH (nmol/mg protein) GSTα (ng/mg protein) GSTµ (ng/mg protein) GSTπ (ng/mg protein)
20–40 Total (n 5 41) 21.5 6 4.1 (4.7–43.7)
**
ND 368 6 76 (0–1766) 883 6 97 (0–2743)
Males (n 5 20) 19.4 6 1.8 (4.7–43.7)
***
ND 278 6 99 (0–1199) 892 6 199 (0–2743)
Females (n 5 21) 23.8 6 3.2 (5.1–25.4)
**
ND 454 6 97 (0–1766) 874 6 95 (0–2377)
40–60 Total (n 5 41) 17.9 6 1.1 (5.5–41.7)
**
ND 383 6 90 (0–2864) 871 6 102 (0–3025)
Males (n 5 21) 16.8 6 1.2 (5.5–26.7)
*
ND 376 6 146 (0–2864) 969 6 182 (0–3025)
Females (n 5 20) 19.0 6 1.8 (8.9–41.7)
**
ND 389 6 96 (0–1230) 769 6 97 (0–1701)
60–80 Total (n 5 42) 12.3 6 0.6 (2.4–26.3) ND 363 6 55 (0–917) 791 6 57 (207–1816)
Males (n 5 22) 13.3 6 0.9 (8.2–26.3) ND 357 6 70 (0–872) 879 6 70 (409–1618)
Females (n 5 20) 11.2 6 0.9 (2.4–18.7) ND 370 6 88 (0–917) 695 6 90 (217–1816)
GSH content and GST isoenzyme levels in lymphocytes were determined as described in the text.
Values are given as mean 6 SEM. Ranges are indicated in parentheses. ND, not detectable.
Wilcoxon rank sum test was used for statistical evaluation: *P , 0.02, **P , 0.005 and ***P , 0.002 as compared with age group 60–80 years.
groups. Table I shows the GSH contents and GST isoenzyme
levels of all age groups studied.
GSTα was not detectable in lymphocytes. In 54 (44%) of
the samples, no GSTµ protein was found, in 51% (60% males,
43% females) of the age group 20–40 years, in 34% (38%
males, 30% females) of the age group 40–60 years and in
45% (41% males, 50% females) of age group 60–80 years.
GSTµ null phenotype was not related to gender or age. GSTπ
expression was equally distributed among the different age
groups, as well as in males and females. GSH content in
lymphocytes was similar in male and female controls, but
expression declined with age: females R
s
5 –0.36 (95%
CI –0.56–0.12; P 5 0.004); males R
s
5 –0.42 (95%
CI –0.61–0.20; P 5 0.0005). This significant decline in GSH
content with age persisted when males and females were
combined: R
s
5 –0.39 (95% CI –0.53–0.23; P , 0.0001).
GSH content in age group 60–80 years was significantly
reduced as compared with the age groups 20–40 and 40–60
years in both sexes, whereas no difference was observed when
comparing GSH contents in age groups 20–40 and 40–60 years.
GSTs are involved in the protection against potentially
harmful chemical compounds (1). Little data are available
concerning age-related changes in the levels of GSH and GST
isoenzymes in humans. This may be relevant with respect to
the possible role of the GSH/GST system in the enhanced
cancer rates at increased age. We now showed that the
expression of the GSTα, µ and π isoenzymes in human
lymphocytes did not change with age, although in females
there was a tendency for lower levels of GSTπ at older age
(P 5 0.06; 20–40 versus 60–80 year age group). No gender-
related effects were found. Unfortunately, due to lack of
sufficient material we were unable to measure GST enzyme
activity and levels of the most recently discovered GSTφ
forms. Cebalos-Picot et al. (15) demonstrated a negative
correlation between age and GST activity (R 5 0.58, P ,
0.001) in human erythrocytes (120 females, 65 males), but
they found no gender-related differences.
Several studies have suggested that GSH might be a critical
factor in protecting cells and organs against toxicity and
disease, since GSH as an important factor in the GST/
GSH peroxidase systems is involved in the detoxification of
xenobiotics, carcinogens, free radicals and peroxides (1).
However, no information is available on the variation of GSH
in lymphocytes in relation to gender and age in healthy
subjects. We noticed no differences in GSH levels of human
lymphocytes between males and females. Similar results were
found previously in human blood by Richie et al. (16) (484
1874
males, 231 females) and Michelet et al. (17) (107 males, 94
females), in plasma by Yang et al. (18) (125 males, 157
females), and in body and antrum of the stomach by Loguercio
et al. (8) (12 males, 10 females). We showed that GSH levels
in human lymphocytes decreased with age in both males and
females. Similar results were found by Lang et al. (19), who
found a significant increase (P , 0.001) in the proportion of
elderly individuals with low blood GSH values compared with
younger adults. Also plasma GSH levels in Chinese male and
female volunteers were found to decrease with increasing age
(18). Loguercio et al. (8) showed that GSH content in body
and antrum of the stomach decreased with age.
In summary, we demonstrated that GST expression in human
lymphocytes was not related to sex or age. GSH content was
similar in males and females and decreased with age in both
sexes. Since GSH is correlated with protection against cellular
or cytogenetic damage, reduction of GSH content during aging
may be a factor of relevance for the increased risk of developing
diseases such as cancer at an older age.
Acknowledgement
This work was supported by grant 94-715 (EMMvL) from the Dutch
Cancer Society.
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Received on April 29, 1998; revised on June 5, 1998; accepted on June 5, 1998
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... The activity of enzymes: glutathione peroxidase and gamma-glutamyl-cysteine synthetase is limited with age. It was shown that every cell of an average 20-year-old is exposed to about 100,000 attacks of oxygen free radicals per day, their number at the age of 75 increases three-fold [65]. ...
Preprint
The tripeptide-glutathione (GSH, γ-L-glutamyl-L-cysteinyl-glycine) is one of the most important low molecular antioxidant in human body. Enhancing GSH and associated enzymes represents an aim in the search for cytoprotective strategies against cancer, neurologic degeneration, pulmonary and inflammatory conditions, as well as rheumatoid arthritis (RA).The objective of the study was to agree whether crenotherapy (drinking therapy) with sulfide/hydrogen sulfide (SHS) waters from “Zuzanna” spring located in the area of Busko-Zdrój in Poland leads to increasing of reduced glutathione (GSH) content in human blood. SHS water in distinct from mineral water is characterised by specific pharmacokinetic, invariable content and natural microbiological purity. SHS waters contain at least 1 g of total sulfur per kilogram of water and a treatment effect also depends on other bioelements. The method employing capillary electrophoresis with UV detector for the analysis of glutathione in human blood was developed. The group of 106 volunteers consisted of both women and men, in different age range. The therapy with SHS waters lasted 2 weeks. We recently demonstrated that the administration of hydrogen sulfide (H2S) in SHS waters increases GSH concentration in blood, and therefore crenotherapy could be used in therapeutics.
Preprint
Cysteine-glutathione mixed disulfide (CySSG), a prodrug of glutathione (GSH) --the “Master Antioxidant”, was found to be orally bioavailable in mice, and protected against a toxic dose of acetaminophen. If oral bioavailability can also be demonstrated in humans, this suggests a wide range of applicability for CySSG.
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One of the mechanisms of drug‐induced liver injury (DILI) involves alterations in bile acid (BA) homeostasis and elimination, which encompass several metabolic pathways including hydroxylation, amidation, sulfation, glucuronidation and glutathione conjugation. Species differences in BA metabolism may play a major role in the failure of currently used in vitro and in vivo models to predict reliably the DILI during the early stages of drug discovery and development. We developed an in vitro cofactor‐fortified liver S9 fraction model to compare the metabolic profiles of the four major BAs (cholic acid, chenodeoxycholic acid, lithocholic acid and ursodeoxycholic acid) between humans and several animal species. High‐ and low‐resolution liquid chromatography–tandem mass spectrometry and nuclear magnetic resonance imaging were used for the qualitative and quantitative analysis of BAs and their metabolites. Major species differences were found in the metabolism of BAs. Sulfation into 3‐O‐sulfates was a major pathway in human and chimpanzee (4.8%–52%) and it was a minor pathway in all other species (0.02%–14%). Amidation was primarily with glycine (62%–95%) in minipig and rabbit and it was primarily with taurine (43%–81%) in human, chimpanzee, dog, hamster, rat and mice. Hydroxylation was highest (13%–80%) in rat and mice followed by hamster, while it was lowest (1.6%–22%) in human, chimpanzee and minipig. C6‐β hydroxylation was predominant (65%–95%) in rat and mice, while it was at C6‐α position in minipig (36%–97%). Glucuronidation was highest in dog (10%–56%), while it was a minor pathway in all other species (<12%). The relative contribution of the various pathways involved in BA metabolism in vitro were in agreement with the observed plasma and urinary BA profiles in vivo and were able to predict and quantify the species differences in BA metabolism. In general, overall, BA metabolism in chimpanzee is most similar to human, while BA metabolism in rats and mice is most dissimilar from human. Species differences in bile acid (BA) metabolism may play a major role in the failure of currently used in vitro and in vivo models to predict reliably drug induced liver injury during the early stages of drug discovery and development. We developed an in vitro cofactor‐fortified liver S9 fraction model to compare the metabolic profiles of the four major BAs between humans and several animal species. Major species differences were found in the metabolism of BAs and the relative contribution of the various pathways involved in BA metabolism in vitro were in agreement with the observed plasma and urinary BA profiles in vivo. In general, BA metabolism in the chimpanzee is most similar to human, while BA metabolism in rat and mice is most dissimilar from human.
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To obtain a comprehensive profile of the erythrocyte antioxidant defense potential during aging, we investigated copper-zinc superoxide dismutase (CuZn-SOD), seleno-dependent glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-RD), and glutathione-S-transferase (GSH-S-T) activities in human erythrocytes from 167 apparently healthy subjects, ages one month to 63 years (102 females, 65 males). We found a negative correlation between age and activities of CuZn-SOD (r = 0.362, P less than 0.001), GSSG-RD (r = 0.549, P less than 0.001), and GSH-S-T (r = 0.575, P less than 0.001). In contrast, we found a positive correlation between age and GSH-Px activity (r = 0.401, P less than 0.001). To evaluate aging changes, we divided the subjects into five groups: Group 1 (newborn-age one year), Group 2 (1-11 years), Group 3 (11-25 years), Group 4 (25-40 years), and Group 5 (40-63 years). Significant age-related modifications in erythrocyte enzyme activities appeared in Group 3 for CuZn-SOD, GSSG-RD, and GSH-Px activity, whereas for GSH-S-T activity age-related modifications appeared in Group 2. We found no sex-related differences in erythrocyte CuZn-SOD, GSSG-RD, GSH-Px, and GSH-S-T activities.
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Human hepatic glutathione S-transferase (GST) subunits were characterized and quantified with the aid of a recently developed h.p.l.c. method. In 20 hepatic tissue specimens the absolute amounts of the basic Class Alpha subunits B1 and B2, the near-neutral Class Mu subunits mu and psi and the acidic subunit pi were determined. The average total amount of GST was 37 micrograms/mg of cytosolic protein, with the Class Alpha GST being the predominant class (84% of total GSTs), and pi as the sole representative of the Class Pi GSTs present in the lowest concentration (4% of total GSTs). Large interindividual differences were observed for all subunits, with variations up to 27-fold, depending on the subunit. For the Class Alpha GST-subunits B1 and B2, a biphasic ratio was observed. The genetic polymorphism of the subunits mu and psi was confirmed by h.p.l.c. analysis, and correlated with the enzymic glutathione conjugation of trans-stilbene oxide and with Western blotting of cytosols, using a monoclonal anti-(Class Mu GST) antibody. Of the 20 livers examined, ten contained only mu, whereas the occurrence of psi alone, and the combination of mu and psi, were found in only one liver each.
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Effects of dietary naturally occurring anticarcinogens; quercetin, flavone, ellagic acid, ferulic acid, tannic acid, curcumin, coumarin, alpha-angelicalactone, fumaric acid and Brussels sprouts on male Wistar rat hepatic and intestinal (i) glutathione S-transferases (GST) enzyme activity, (ii) GST isozyme levels and (iii) glutathione (GSH) content were investigated. GST enzyme activity was significantly increased by all anticarcinogens tested, except fumaric acid, at least at one of the five sites investigated: proximal, middle, distal small intestine, large intestine and liver. Only alpha-angelicalactone gave an enhanced GST enzyme activity at all five sites. Large intestinal GST enzyme activity was increased only by quercetin (175%) and alpha-angelicalactone (138%). Concomitant changes in GST isozyme levels occurred. Class alpha GSTs were induced in 50% of the cases, especially in liver and upper parts of the intestine by quercetin, flavone, coumarin and alpha-angelicalactone. GST class pi levels were enhanced only at one site by quercetin, coumarin and alpha-angelicalactone. GST class mu changed in 14% of the cases, most profoundly in proximal and middle small intestine by flavone, coumarin and alpha-angelicalactone. Tannic acid and fumaric acid gave a significant raise in class alpha GSTs at almost all sites, whereas overall GST enzyme activity hardly changed. GSH was increased at various sites in 14% of the cases by Brussels sprouts, quercetin, flavone and alpha-angelicalactone. These data demonstrate that most anticarcinogens, in particular flavone, coumarin and alpha-angelicalactone, enhance GST activity in liver and intestine, mainly by induction of class alpha and mu isozymes.
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The role of various sulphydryl compounds in protective mechanisms against acute ethanol toxicity has been investigated in the mouse. Intraperitoneal administration of varying doses of ethanol (0–6 g/kg as 20 per cent, w/v solutions) produced a linear dose response reduction in hepatic reduced glutathione (GSH) levels. The time scale of this effect suggested that GSH depletion occurred as a consequence of hepatic damage rather than contributing towards it. It was also demonstrated that the sulphydryl compounds β-mercaptoethylamine-HCl, cysteine and methionine significantly increased the survival rate of mice given a lethal dose of ethanol.
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Resistance to chemotherapy is a significant problem in the treatment of colorectal carcinomas. To obtain insight into the mechanism of drug resistance, the expression of P-170 glycoprotein and biotransformation enzymes that are potentially able to contribute to drug resistance were investigated in paired samples of normal mucosa and tumors from 24 patients with colorectal cancer. In the tumors, glutathione S-transferase (GST) enzyme activity and content of GST-pi and P-170 glycoprotein were increased significantly compared with normal mucosa (P less than 0.03, P less than 0.003, and P less than 0.02, respectively). In contrast, GST-alpha and -mu, present in minor amounts compared with GST-pi, were downregulated in the tumor. Cytochrome P-450(4,5,6) and UDP-glucuronyltransferase (towards 4-nitrophenol and bilirubin) levels were significantly lower in the tumors (P less than 0.0001 and P less than 0.0002, respectively). Because decreased expression of cytochrome P-450 and increased levels of GST-pi and the P-170 glycoprotein have been implicated in (multi)drug resistance, these findings strongly suggest that in colorectal tumors the inherent resistance is multifactorial. Research to overcome this resistance should therefore be directed toward a combined treatment that eliminates all of these different mechanisms.
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The objective of this investigation was to test the hypothesis that blood glutathione levels are lower in aging human subjects as previously found in blood and tissues of standard rodent models of aging. Thus a study was conducted with 39 men and 130 women, 20 to 94 years old, who were selected by the criteria of being ambulatory, healthy, and free from diabetes mellitus, thyroid disease, anemias, and cancer. The reference group was comprised of the 20- to 39-year-old subjects, whose blood glutathione levels were 547 +/- 53.5 micrograms/10(10) erythrocytes (mean +/- SD) for 40 individuals and defined the reference range (95% confidence limits) of 440 to 654. Based on the 440 micrograms/10(10) erythrocyte cutoff, the incidence of low blood glutathione content in the older subjects increased significantly, particularly in the 60- to 79-year-old group. Their glutathione levels were 452 +/- 86.8 micrograms/10(10) erythrocytes, 17% lower than the reference group (p < 0.001). These findings demonstrate an increased incidence of low glutathione levels in apparently healthy elderly subjects, who thus may be at risk because of a decreased capacity to maintain many metabolic and detoxification reactions mediated by glutathione.
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Several monoclonal antibodies against human liver glutathione S-transferase mu were developed. One of these monoclonal antibodies, called GST-3H4 was further characterized and used in this study. In hepatic tissue, after immunoblotting, GST-3H4 strains a 27 kDa protein with a pI value of 6.2. GST-3H4 recognizes other human class-mu glutathione S-transferases, but does not detect acidic or basic glutathione S-transferases. By immunodetection with this monoclonal antibody, glutathione S-transferase mu can be demonstrated in human breast, stomach, liver, small and large intestine, mononuclear blood cells, kidney and placenta. A 100% correlation is found in the distribution of glutathione S-transferase mu when different tissues or mononuclear blood cells from the same individuals are investigated. In 62.5% of the mononuclear blood cells from controls, glutathione S-transferase mu is present. In patients with polyposis coli, breast cancer or colon cancer a similar distribution is found. Therefore no important role for glutathione S-transferase mu deficiencies in the aetiology of these diseases is suggested.
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In order to evaluate the role of the placental form of glutathione S-transferase (GST-Pi) as a tumour marker, activity and composition of GSTs from human colon were investigated. GSTs were purified from normal colon mucosa and from colonic tumours by affinity chromatography on glutathione-agarose. After SDS-PAGE or isoelectric focusing these purified preparations revealed only one band that comigrated with GST-Pi from human placenta. A monoclonal antibody (mAb) very specific for GST-Pi was developed and characterized. On immunoblot this mAb stains purified GST from normal and diseased colon tissue. GST activity was significantly higher in most cancerous (247 +/- 38 nmol/min/mg protein; n = 7), compared with the corresponding normal tissues (171 +/- 18 nmol/min/mg protein; n = 7). In colon from patients without large bowel malignancies GST-Pi is also by far the most prominent isoform detectable. In conclusion, both normal and tumorous colon tissue predominantly express GST-Pi and therefore GST-Pi is not suitable as a tumour marker for colonic carcinomas. However, the increased GST-Pi levels in colonic tumours could possibly contribute to the relatively high resistance to anti-cancer drugs.