Differential expression of glutathione-S-transferase isoenzymes in various types of anemia in Taiwan

Clinica Chimica Acta 375 (2007) 110 – 114 www.elsevier.com/locate/clinchim

Differential expression of glutathione-S-transferase isoenzymes in various types of anemia in Taiwan Whei-Ling Chiang a , Yih-Shou Hsieh b , Shun-Fa Yang b , Tsang-An Lu c , Shu-Chen Chu d,⁎ a

d

School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan b Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan c Department of Clinical Laboratory, Chu Shang Show Chwan Hospital, Natou, Taiwan Department of Food Science, Central Taiwan University of Sciences and Technology, No. 11 Pu-tzu Lane, Pu-tzu Road, Taichung 406, Taiwan Received 16 May 2006; received in revised form 20 June 2006; accepted 22 June 2006 Available online 27 June 2006

Abstract Background: Published reports concerning the expression of GST in various anemias including aplastic, hemolytic, iron deficiency and thalassemia anemia has been insufficient. We improved the conventional GST assay by incorporating a chloroform treatment to remove the interference of hemoglobin and evaluated the altered expression of GSTs in various anemias in Taiwan. Methods: We incorporated a chloroform treatment to eliminate the interference of hemoglobin. Erythrocyte total GST and isoenzymes activities from 35 control subjects and 125 subjects of various anemias, including aplastic, hemolytic, iron deficiency and thalassemia anemias were measured spectrophotometrically. Results: Chloroform treatment did not significantly affect GST activities in erythrocytes of control subjects while the activities of erythrocyte total GST and α-GST were significantly increased in all anemic patients (P b 0.001). The expression of μ-GST was significantly decreased, although at a less extent, in cases of aplastic, iron deficiency and thalassemia anemia (P b 0.05), but π-GST was not physiologically different in various types of anemia. Conclusion: The determination of changes in erythrocyte GST activity is a promising indicator of oxidative stress conditions that occur in various types of anemia. Measurement of GST activity might be useful for the evaluation of prophylactic treatment in trials of antioxidant strategies. © 2006 Elsevier B.V. All rights reserved. Keywords: Glutathione-S-transferase; Aplastic anemia; Hemolytic anemia; Iron deficiency anemia; Thalassemia anemia

1. Introduction Anemia is one frequently encountered medical problem and can be classified as microcytic, macrocytic, or normocytic, using the mean cell volume (MCV) and mean cell hemoglobin concentration (MCHC) from the hemogram. Normocytic anemia is characterized with a decreased erythrocyte cell production and increased erythrocyte cell destruction and occurs in primary hematopoietic disorders, such as myelocytic leukemias, hemolytic anemia and aplastic anemia. Microcytosis occurs when there is insufficient hemoglobin production in the developing erythrocyte. The most common causes of microcytic anemia include iron deficiency, anemia of chronic disease, and ⁎ Corresponding author. Tel.: +886 4 22391647x7500; fax: +886 4 2239 6771. E-mail address: [email protected] (S.-C. Chu). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.06.023

thalassemia trait [1]. In erythrocytes, the glutathione-S-transferase (GST; EC 2.5.1.18) could act intracellularly to prevent superoxide-induced hemolysis [2]. The ubiquitous GSTs are a group of enzymes involved in the initial step of mercapturic acid synthesis [3]. The functions of GST have been classified into 2 general categories [4]. GSTs may act as binding proteins, which are primarily involved in the neutralization of harmful exogenous or endogenous compounds by enzymatic conjugation with the scavenger peptide glutathione (GSH) and/or by direct binding of nonsubstrate ligands [5,6], such as bilirubin [7,8]. The other major function is to protect against oxidative damage to lipids and nucleic acids and participate in the metabolism of some steroids and leukotrienes [6]. Four major subfamilies of GSTs including the alpha (α), mu (μ), pi (π), theta (θ) can be distinguished in humans while over 20 distinct soluble GST isoenzymes have been identified in

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mammals [9,10]. According to previous studies, the three evolutionary classes of GST identified were designated γ, μ, and π, which represent the human basic, natural, and acidic GST on isoelectric point, respectively [6]. The majority of GSTs are homo- or heterodimer subunits of about 26 kDa, and are mainly found in the cytosol. The polymorphic expression of which has been widely studied in human tissues and in various cells [6,11,12]. On the other hand, a distinct microsomal family of membrane-bound GSTs has also been reported [13–15]. 2. Methods 2.1. Subjects and specimen collection Venous blood samples were obtained via routine venipuncture from anemic patients of Veteran General Hospital-Taichung, Taichung, Taiwan. A total of 160 patients from 5 groups, including control (n = 35), aplastic anemia (n = 25), hemolytic anemia (n = 25), iron deficiency anemia (n = 60) and thalassemia anemia (n = 15), were voluntarily recruited into this study. The taken EDTA anti-coagulated blood samples were centrifuged at 1000×g for 10 min and then the pelleted red blood cells were washed 3 times with normal saline. Thereafter, red blood cells were frozen in −20 °C to break cell membranes to induce hemolysis. The hemolysates were centrifuged again at 11,600×g for 5 min and then the supernatants were collected for GST activity assay. Clinical characteristics and laboratory findings of patients were summarized in Table 1. 2.2. Pretreatment of hemolysate specimens Hemoglobin was removed from erythrocyte lysates according to the following procedures. Initially, 1 ml of lysate was mixed with 1 ml 40% (v/v) ethanol and 0.5 ml chloroform for 1 min and then centrifuged at 12,000×g for 5 min. The chloroform layer and membranous interface were discarded; supernatants were recovered and kept at −20 °C [16,17]. 2.3. Determination of erythrocyte total GST activity In both the hemolysates and hemoglobin-free lysates, GST activity and kinetic parameters, i.e., maximal activity (Vmax),

Fig. 1. Comparison of total GST activity, with or without chloroform treatment, of controls and subjects of hemolytic anemia.

apparent Michaelis constants (Km) for GSH and 1-chloro-2, 4dinitrobenzene (CDNB), and pH 6.5, were determined by following the method of Habig et al. [5] with minor modifications using a potassium phosphate buffer (0.1 mol/l, pH 6.25) containing EDTA (1 mmol/l), CDNB (45 mmol/l in 95% ethanol), and GSH (15 mmol/l in deionized water). CDNB and reduced glutathione (GSH) were from Sigma (St. Louis, MO). All other chemicals were of analytical quality. The GST activity was expressed as U/g. 2.4. Determination of erythrocyte GST isoenzyme (α, μ, π) activities The GST isoenzyme activities were measured spectrophotometrically using 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBDCl) [18], 1,2-dichloro-4-nitrobenzene (DNCB) and ethacrynic acid (EA) [3] as broad spectrum and α-, μ-, and π-GST specific substrates, respectively. All assay procedures were the same as total GST activity. 2.5. Statistical evaluation Values were expressed as means ± S.E. The statistical significance of the means for cytosolic GST isoenzyme was determined by Mann–Whitney Rank sum test between groups. SigmaStat software (Jandel Scientific Software, USA) was used

Table 1 Clinical characteristics and laboratory findings of subjects Clinical characteristics

Controls (n = 35)

Aplastic anemia (n = 25)

Hemolytic anemia (n = 25)

Iron deficiency anemia (n = 60)

Thalassemia anemia (n = 15)

Age (years) Sex Male Female WBC (×103) Reticulocyte (%) Hb (g/dl) HCT (%) RBC (×107) MCV MCHC

50.71 ± 10.49

35.24 ± 15.13

57.4 ± 17.07

53.84 ± 18.43

27.2 ± 12.32

16 (45.7%) 19 (54.3%) 6.77 ± 1.99 0.985 ± 0.04 13.75 ± 1.52 41.79 ± 4.24 4.67 ± 0.66 90.32 ± 8.85 29.72 ± 3.16

14(56%) 11(44%) 3.64 ± 1.79 1.073 ± 0.14 10.89 ± 2.62 31.81 ± 7.33 3.24 ± 0.86 99.26 ± 9.23*** 33.92 ± 3.19***

11 (44%) 14 (56%) 6.85 ± 3.39 6.891 ± 1.94*** 11.38 ± 2.37 33.47 ± 7.67 3.83 ± 1 89.38 ± 12.73 32.28 ± 10.78

27 (45%) 33 (55%) 6.37 ± 2.03 1.163 ± 0.07 11.48 ± 2.04 35.41 ± 5.15 4.36 ± 0.75 82.04 ± 9.87*** 26.64 ± 4.44***

6 (40%) 9 (60%) 8.76 ± 3.52 1.4 ± 0.06*** 10.14 ± 3.82 34.02 ± 8.16 4.86 ± 0.54 69.52 ± 10.72*** 21.64 ± 5.34***

Student's t-test: ***P b 0.001.

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for all statistical analyses. A P b 0.05 was considered statistically significant. 3. Results 3.1. Clinical characteristics and laboratory findings of studied subjects The demographic, hematologic and clinical characteristics of controls and patients (patients with anemias including aplastic, hemolytic, iron deficiency or thalassemia anemia) are shown in Table 1. No significant difference was observed for any studied parameter between controls and study groups. However, reticulocyte counts were significantly increased in both groups of hemolytic anemia and thalassemia anemia, compared to the control subjects (P b 0.001), while MCV and MCHC were significantly decreased in iron deficiency and thalassemia anemia (P b 0.001). On the contrary, MCV and MCHC were significantly increased in the groups of aplastic anemia, compared to the control subjects (P b 0.001). 3.2. Total GST activity in the absence or presence of hemoglobin in control subjects To assume whether the GST activity was interfered by hemoglobin and chloroform treatment, we compared GST activities of control subjects with or without a pretreatment with chloroform and results shown in Fig. 1 have indicated that a pretreatment with chloroform has no physiologically significant effect on GST activity. 3.3. Erythrocyte total GST activity and its isoenzymes (α, μ, π) in various types of anemia The results of erythrocyte total GST activity and its isoenzymes (α, μ, π) in various types of anemia are shown in Table 2. Statistical analysis showed that the activities of erythrocyte total GST and α-GST were significantly increased in patients with various types of anemia (P b 0.001). Furthermore, the expression of μ-GST was also significantly decreased, although at a less extent, in patients except those with hemolytic anemia (P b 0.05), as compared to the controls. However, no physiologically significant difference in π-GST was observed in all comparisons (Table 2).

4. Discussion Fundamentally, the causes for anemia may involve loss of blood, excessive destruction of mature erythrocytes, or impaired erythrocytes. In addition to certain clinical assessments, the differential diagnosis is based on morphologic examination of erythrocytes and on kinetic considerations. Characteristic changes in the size and hemoglobin content of the red corpuscles occur in some types of anemia. One of them is termed “microcytic anemia” and characterized by a low MCV. In such cases, in addition to alterations in the size of erythrocytes, there usually is a proportionately greater reduction in the total hemoglobin than in the total number of erythrocytes. This class includes iron deficiency anemia and thalassemia anemia (Table 1). The reticulocyte count or index provides the most simple and practical estimate of effective erythrocyte production. The continuously increasing number of reticulocytes, as revealed by serial measurements, indicates that the patient is recovering from the anemia. In this study, reticulocyte counts were significantly increased in both hemolytic anemia and thalassemia anemia compared to that of the control subjects (P b 0.001). In addition, preanalytical factors are the main source of variation in clinical testing, including intracellular enzymes [19,20]. Sample hemolysis can exert a strong influence on result reliability. Erythrocyte and hepatic forms of GST have been shown to bind hemin with variable affinities [21,22]. Vincent et al. suggested that rat and human albumin and rat GST, proteins with moderate affinities for heme, decreased heme-catalyzed lipid peroxidation in a dose-dependent manner but were subjected to oxidation [23]. Harvey and Beutler also suggested that human erythrocyte GST activity was inhibited, probably competitively, by hemin with a Ki of 10− 7 mol/l. It was postulated that GST functions physiologically as a hemin-binding and/or transport protein in developing erythrocyte cells [4]. It was reported that the mean activity level of erythrocyte GST was significantly raised in subjects with HbAS or HbSS (P b 0.001) when compared with that of subjects with HbAA [24]. However, GST expression was inconsistent in various erythrocyte disorders. Development of anemia may be related to a decrease in hemoglobin or pathological changes of hemoglobin. Hemoglobin is an influence factor, we hereby suspected that hemoglobin may interfere the function of GST involved in oxidation which has been well known to contribute to various

Table 2 Activity levels of total GST and GST isoenzymes (α, μ, π) in erythrocytes of controls and patients of four types of anemia Controls (n = 35)

Aplastic anemia (n = 25) Hemolytic anemia (n = 25) Iron deficiency anemia (n = 60) Thalassemia anemia (n = 15)

Mean ± S.E.

Mean ± S.E.

Total GST activity (U/l) 2968.75 ± 197.55 7146.38 ± 1220.68 P b 0.001 α-GST activity (U/l) 1512.32 ± 72.03 2313.79 ± 181.9 P b 0.001 μ-GST activity (U/l) 451.56 ± 42.78 316.99 ± 38.67 P b 0.05 π-GST activity (U/l) 3217.14 ± 90.33 3047.36 ± 84.24 P = 0.223

Mean ± S.E.

Mean ± S.E.

Mean ± S.E.

9429.17 ± 1575.53 P b 0.001 2322.76 ± 175.68 P b 0.001 334.22 ± 34.04 P = 0.055 3212 ± 89.13 P = 0.968

12,581.6 ± 845.84 P b 0.001 2358.09 ± 110.32 P b 0.001 297.21 ± 38.85 P b 0.05 3208.47 ± 76.12 P = 0.942

18,614.58 ± 4057.10 P b 0.001 2903.44 ± 293.29 P b 0.001 211.76 ± 51.28 P b 0.05 3300 ± 148.32 P = 0.739

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erythrocyte disorders. However, definitive indications on the GST analysis interference by hemolytic specimens are currently lacking. For the first time, chloroform was employed to remove hemoglobin from erythrocyte lysates to show that GST activities in samples of control subjects with or without a chloroform treatment were not significantly different (Fig. 1). Therefore, we believed that the GST activity was not influence by a chloroform treatment. The main function of the GSTs involves the catalysis of conjugation of electrophilic, hydrophobic compounds with the tripeptide glutathione (GSH). As a family of isoenzyme, the enzyme system is capable of handling an enormous variety of electrophilic compounds, from both exogenous and endogenous origins [25]. Glutathione-S-transferase M1 (GSTM1) and T1 (GSTT1) genes are polymorphic in humans. The GSTT1 null genotype may modulate the metabolism of exogenous pollutants or toxic intermediates. The absence of the GSTT1 enzyme, leading to genetic susceptibility toward certain pollutants, might determine the individual risk for development of acquired aplastic anemia in children [26]. In human erythrocytes, GST is massively expressed in two forms: highly cationic enzymes are the α-GST and θ-GST and the main anionic enzyme corresponding to the π form [27–29]. Based on the study of Schroder et al., we suggest that the amount of θ-GST in human erythrocytes should be abundant enough to be purified [30]. Although the physiological role of the erythrocyte GST is not yet known, it has been suggested that it could be functional in the removal of circulating xenobiotics [27]. Previous studies have identified an erythrocyte GST deficiency in hemolytic anemia, but the underlying cause was unresolved [31]. For examples, in human, the π-class isoenzyme is widely distributed and represents the most thoroughly characterized extrahepatic GST (including placenta and erythrocyte). The α- and μ-GSTare hepatic enzymes, however, αGST is also present in significant amount in testis, kidney, and adrenal glands [32,33]. All 3 classes of GSTs are differentially expressed in various tissues [4]. In erythrocyte, the GST could act intracellularly to prevent superoxide-induced hemolysis [15]. The main anionic GST corresponds to the π form in erythrocytes. Previous studies have reported an erythrocyte GST deficiency in hemolytic anemia, but the underlying cause was unresolved [31]. Our results have shown that the erythrocyte total GST activity was increased in all types of anemia while GST isoenzymes with variable activities were differentially expressed in erythrocytes. This investigation revealed that activities of erythrocyte total GST and α-GST were significantly increased in all anemia patients (P b 0.001). The enzymatic activity of total GST was determined by CDNB. Awasthi et al. demonstrated that the specific activity and Vmax of θ-GST towards CDNB seem to be the highest among GST isoenzymes characterized so far from human tissues [34]. Therefore, the activity of θ-GST should partly contribute to the high total GST activity. The expression of μ-GST was significantly decreased, although at a less extent, in cases of aplastic anemia, iron deficiency anemia and thalassemia anemia (P b 0.05), but π-GSTs was not physiologically different in various types of anemia (Table 2). The cytosolic GSTs in a free form include α- and μ-GST. The activation of cytosolic α-GST may indicate the presence of higher levels of electrophiles/

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oxidants, such as superoxides and possibly also methemoglobin since the generation of methemoglobin has been associated with increased production of superoxides. The significant difference in the expression of μ-GST may indicate either the actual decrease of μ-GST in anemias or the binding to heme and the subsequent removal by chloroform. In humans, a correlation of 100% has been found with regard to the distribution of μ-GST in liver, lymphocytes and other tissues, strongly suggesting that expression of this isoenzyme is not tissue specific, but is either uniformly distributed in the organism, or not present at all [35]. On the other hand, the π-GSTs was not physiologically different in various types of anemia. It was possible that expression of π-GST is lower than α-GST and bound form could be removed from erythrocyte lysates with hemoglobin. It was also possible that the expression of π-GST was not altered in hematological disorders and therefore no statistically significant difference was observed. However, Kamada et al.'s previous findings indicate that π-GST plays an important role in the defense system against oxidative stress through its function as a regulator of stress kinases. It is interesting that π-GST has at least 2 different functions, to scavenge lipid peroxide and to regulate stress kinases as an antioxidant [36]; and it has been found in the nucleus in uterine cancer cells [37] and glioma cells [38]. The mechanism for the selective expression of GSTs in human tissues and the physiological significance of their differential expression has not been understood yet. Here, it was discovered for the first time that hemoglobin was denatured rapidly by chloroform. This finding may implicate that chloroform treatment was a vital step in analyzing GST activity of erythrocytes by eliminating hemoglobin interference, including the determined hydrophilic intracellular enzymes. Furthermore, the results from this modified method may be feasible for evaluating the levels of electrophiles/oxidants, as well as in assays for other enzymes in erythrocytes. We conclude that the determination of erythrocyte GST activity is a promising indicator of oxidative stress conditions that occur in various types of anemia. Measurement of GST activity might be useful for the evaluation of prophylactic treatment in trials of antioxidant strategies. Acknowledgment This study was supported jointly by grants from National Science Council, Republic of China (NSC 94-2313-B-166-004) and Chung Shan Medical University (CSMU 93-OM-B-020). References [1] Wintrobe MM. Clinical hematology 8th ed. Philadelphia: Lea and Febiger. Inc.; 1981. [2] Van Kuijk FJ, Sevanian A, Handelman GJ, Dratz EA. A new role for phospholipase A2: protection of membranes from lipid peroxidation damage. TIBS 1987;12:31–4. [3] Boyland E, Chasseaud LF. The role of glutathione and glutathione Stransferases in mercapturic acid biosynthesis. Adv Enzymol Relat Areas Mol Biol 1969;32:173–219. [4] Harvey JW, Beutler E. Binding of heme by glutathione S-transferase: a possible role of the erythrocyte enzyme. Blood 1982;60:1227–30. [5] Habig WH, Pabst MJ, Jakoby WB. Glutathione transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130–9.

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