The development of glutathione S-transferase and glutathione ...

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Biochimica et Biophysica Acta 883 (1986) 448 453 Elsevier

BBA 22439

The development of glutathione S-transferase and glutathione peroxidase activities in human lung Anthony A. Fryer a, Robert Hume b and Richard C. Strange a u Clinical Biochemistry Research Laboratory, University of Keele, Central Pathology Laboratory, North Staffordshire Hospital Centre, Hartshill Road, Stoke-on-Trent, ST4 7PA and ~ Department of Child Life and Health, University of Edinburgh, Edinburgh, EH8 1 UD (U.K.) (Received 9 April 1986)

Key words: Glutathione S-transferase; Glutathione peroxidase; Development; (Lung)

The development of glutathione S-transferase and glutathione peroxidase activities has been studied in human lung cytosols. Whilst no clear change in glutathione peroxidase activity was identified, expression of the acidic glutathione S-transferase isoenzyme decreased markedly after 15 weeks of gestation so that at birth the level of activity of this isoenzyme was only about 20% of that in samples obtained during the first trimester. Basic glutathione S-transferase isoenzymes were weakly expressed during development and usually comprised less than 10% of cytosolic activity. Ion-exchange studies identified several basic isoenzymes that may correspond to the a, fl, ~, 8 and e set previously identified in liver. Weak expression of apparently near-neutral isoenzymes was also detected; they were detected in only a few cytosois.

Introduction The glutathione S-transferases (EC 2.5.1.18) are a group of dimeric enzymes found in all human tissues. They catalyse the conjugation of GSH with various xenobiotics including organic peroxides [1,2] but unlike the selenoenzyme, glutathione peroxidase (EC 1.11.1.9), cannot catalyse the detoxication of hydrogen peroxide [3]. A variety of glutathione S-transferase isoenzymes has been identified and although the isoenzyme composition of human tissues is generally complex [4,5], the enzymes can be readily classified on the basis of their isoelectric points as basic, near-neutral or acidic [6]. Five isoenzymes with basic isoelectric points (pI values 7.8-8.8) Correspondence address: Dr. Richard C. Strange, Clinical Biochemistry Research Laboratory, University of Keele, Central Pathology Laboratory, North Staffordshire Hospital Centre, Hartshill Road, Stoke-on-Trent, ST4 7PA, U.K.

were first identified in liver and termed a, fl, T, 8 and e [7]. This isoenzyme set appeared to comprise homodimers and it was suggested that the enzymes were products of a single locus, GST2 [4]. More recently, however, two monomers (B 1 and B2) that may be the products of separate genes have been identified by Stockman et al. [8]. A near-neutral isoenzyme (pI value 6.5) termed /~ has also been identified in liver [9]. This enzyme appears to be the product of a further locus, GST1 [4], but since combinations of three alleles, GSTI*I, GSTI*2, GSTI*0, can be present at this polymorphic locus it is not clear whether/~ corresponds with the isoenzyme found in individuals with the GST1 1 (GSTI*I/GSTI*I; G S T I * I / GSTI*0) or GST1 2 ( G S T I * 2 / G S T I * 2 ; GSTI*2/GSTI*0) phenotypes. Individuals with the GST1 0 phenotype (GSTI*0/GSTI*0) appear to express no near-neutral activity. Acidic isoenzymes (pI values approx. 5.0) have been identified in several tissues including erythrocytes, lung,

0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

449 placenta and retina but it is not clear whether these are the products of a single locus [10-14]. Only the basic isoenzymes exhibit peroxidase activity. Although the ontogeny of this multi-locus set of isoenzymes is of interest because of their putative importance in the detoxication of xenobiotics, there is little information concerning their development in lung. The levels of glutathione S-transferase and glutathione peroxidase activity in this tissue are of particular interest, since recent work suggests that the premature lung is particularly sensitive to oxygen radical-induced damage because of its failure to induce key detoxicating enzymes at birth [15,16]. Whilst adult lung expresses acidic as well as small amounts of basic glutathione S-transferase activity, the near-neutral enzyme has not so far been identified in this tissue [13,17]. We now describe experiments to, firstly, define the changes in glutathione S-transferase and glutathione peroxidase activities during development, secondly, use chromatofocusing to separate the different glutathione S-transferase isoenzyme sets and quantify their contribution to total activity, and thirdly, use ion-exchange chromatography to determine whether the basic glutathione S-transferase set comprises various isoenzymes that might correspond with a, /3, T, 8 and e. Materials and Methods

Chemicals Polybuffer exchanger, PBE 94 and Polybuffer, PB 74 were obtained from Pharmacia Fine Chemicals and DEAE- and CM-cellulose were from Whatman Lab. Ltd. All other chemicals were obtained from Sigma Chemical Co. Ltd.

Preparation of lung cytosols Lung tissue was obtained immediately after death from, firstly, aborted fetuses (13-21 weeks gestation) following termination of pregnancy, and secondly, premature and term infants (22-39 weeks gestation) who died between 1 h and 49 h after birth. None of the subjects in the second group suffered from bronchopulmonary dysplasia. Samples were also obtained from a third group of infants (2-118 weeks postnatal age) who suffered sudden infant death syndrome. Ethical approval

was obtained from the Ethics Committee of the Simpson Memorial Maternity Pavilion, Royal Infirmary, Edinburgh. To prepare cytosol, 7 ml ice-cold Tris-HC1 buffer (20 raM; pH 7.30) containing 250 mM sucrose, 0.1 mM EDTA and 1 mM GSH was added to pieces of tissue (0.25-2 g). The tissue was cut into small pieces, homogenised and centrifuged (30 min, 4°C, 20000 x g). The supernatant was re-centrifuged (60 min, 4 ° C, 150 000 x g) and the resulting supernatant was termed cytosol.

Chromatofocusing Cytosols (6-12 ml; approx. 8 mg/ml protein) were eluted (30 ml/h, 4 ° C) from columns (40 x 1.0 cm) containing Polybuffer exchanger 94 previously equilibrated with start buffer (25 mM imidazole buffer; pH 7.30). The pH gradient was formed using Polybuffer 74 adjusted to pH 4.0 with HC1 (1 M). Fractions (2.5 ml) were assayed for glutathione S-transferase activity and pH.

Ion-exchange experiments Cytosols were eluted (30 ml/h, 4°C) from columns (30 × 1.6 cm) containing DEAE-cellulose previously equilibrated with 20 mM Tris-HC1 (pH 7.30), and glutathione S-transferase activity in the 'flow-through' fractions was pooled and dialysed for 16 h at 4°C against 2 litres of 10 mM sodium phosphate buffer (pH 6.70) containing GSH (1 mM) and EDTA (0.1 mM). The dialysate was applied to columns (25 x 2.5 cm) containing CMcellulose previously equilibrated in 10 mM sodium phosphate buffer (pH 6.70). After 80 ml had been eluted a NaC1 gradient was established by adding phosphate buffer containing NaC1 (100 mM) to a reservoir containing 450 ml of the phosphate buffer. This reservoir was used to supply the column. The flow-rate was 30 m l / h and fractions (3 ml) were assayed for glutathione S-transferase activity and Na + concentration.

Analytical methods Glutathione S-transferase activity was assayed at 30°C using GSH (1 mM) and 1-chloro-2,4-di-

nitrobenzene (1 mM) [4]. Glutathione peroxidase activity was measured at 30°C using a modified coupled assay with glutathione reductase [18]. The

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assay solution contained 50 mM sodium phosphate buffer (pH 7.0), 5 mM EDTA, 5 mM GSH, 3.75 mM sodium azide, 0.28 mM NADPH, 1-2 units glutathione reductase and 0.54 mM cumene hydroperoxide. Glutathione S-transferase and glutathione peroxidase activities were expressed as /~mol 1-chloro-2,4-dinitrobenzene conjugated/min per mg protein and /~mol GSH oxidised/min per mg protein, respectively. Protein concentrations were determined using the method of Bradford [19]; Na ÷ was measured by flame photometry and C1 using a Corning Chloride Meter. Since lung cytosols contained haemoglobin from contaminating erythrocytes, protein concentrations in cytosols were corrected for haemoglobin content. Similarly, since erythrocytes contain glutathione S-transferase and glutathione peroxidase activities, it was necessary to determine the level of contamination at which lung cytosol activities were affected. Experiments in which erythrocyte-free glutathione S-transferase and glutathione peroxidase standards were spiked with erythrocyte lysate showed that at the haemoglobin level in cytosols lung activities were unaffected. Results

Glutathione S-transferase and glutathione peroxidase activities during development Glutathione S-transferase activity was present in all lung tissue cytosols obtained between 13 weeks gestation and 84 weeks post-natal age. Fig. la shows that cytosol activity decreased approx. 5-fold between 13 and 40 weeks gestation. After this time activities remained relatively constant. Glutathione peroxidase activity was found to be present in all but one of the cytosol samples studied. Fig. lb shows the pattern of glutathione peroxidase development between 13 weeks gestation and 84 weeks post-natal age. Large inter-individual variations were observed but there was no evidence of any trend in activity during the period studied. Resolution of basic, near-neutral and acidic isoenzymes using chromatofocusing Chromatofocusing was used to resolve basic, near-neutral and acidic isoenzymes in lung cytosol (Fig. 2). In all samples studied a major peak of

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Fig. 1. Glutathione S-transferase (a) and glutathione peroxidase (b) activities in lung cytosols obtained during development.

glutathione S-transferase activity was eluted at a pH of approx. 5.40. This isoenzyme may correspond with the major acidic form found in erythrocytes [1-1]. Two smaller peaks were also identified in some samples at pH values of approx. 6.30 and 7.60 corresponding to the near-neutral and basic isoenzymes, respectively. Basic isoenzymes were found in six of the eight cytosol samples that were chromatofocused. Their expression was not dependent on the developmental age 12.0

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Fig. 3. Lung cytosols obtained during development were chromatofocused and the percentage contributions of the basic (©), near-neutral and acidic (zx) isoenzymes to total activity in cytosol were determined. The GST1 phenotype in liver is given by GSTI 1, (I); GST1 2, (li2); GST1 0, ( ~ ) ; and (D) where no phenotype could be assigned.

of the sample. Glutathione S-transferase activity was also eluted at pH values similar to those expected for the hepatic near-neutral isoenzymes in five of the samples studied. Their level of expression was low and was not related to their developmental age. The identity of these isoenzymes is not clear and interestingly they were not detected in one sample obtained about 1 year after birth even though analysis of liver cytosol from this subject demonstrated a GST1 2 phenotype. Fig. 3 shows the percentage contribution of each of these isoenzyme sets during development. About 90% of the 1-chloro-2,4-dinitrobenzene conjugating activity was associated with the acidic isoenzymes, whilst the near-neutral and basic isoenzymes comprised less than 10% of total cytosol activity. Chromatofocusing confirmed the decline in expression of glutathione S-transferase activity in lung cytosol (Fig. la) and showed that it resulted from a reduced expression of the acidic isoenzymes. Since the contribution of the basic and near-neutral isoenzymes was small, it was difficult to determine with confidence whether they dem-

onstrated any developmental trend. The basic but not near-neutral or acidic glutathione S-transferase isoenzymes demonstrated low levels of glutathione peroxidase activity. Peroxidase activity eluted in these fractions, however, accounted for less than 10% of the activity found in lung cytosols.

1on-exchange chromatography of basic glutathione S-transferases To determine whether several basic isoenzymes corresponding to the a, /3, 7, 8 and e set identified in liver are also present in lung, samples of lung cytosol obtained after 9 weeks post-natal age and 118 weeks post-natal age were eluted from DEAE-cellulose. The basic enzymes, eluted in the 'flow-through' fractions, were pooled, dialysed and eluted from CM-cellulose. The low levels of basic glutathione S-transferase activity present in the lung cytosols were resolved by the cation-exchanger into a peak of activity that eluted with the 'flow-through' fractions (Na ÷ 15 mM) and a partially resolved peak that eluted at a Na ÷ concentration of 45 mM. The chromatographic be-

452

haviour of these isoenzymes is similar, therefore, to that of a and t, respectively [8]. Glutathione S-transferase activity was also eluted at Na + concentrations between 27 mM and 40 mM but the levels of activity were very low and the activities were poorly resolved. Similar results were obtained when the second cytosol was similarly eluted from DEAE- and CM-cellulose. Because of the small size of the lung during gestation, insufficient cytosol could be prepared from one subject to allow resolution of the basic isoenzymes before birth. Discussion

This study has described the development of glutathione S-transferase and glutathione peroxidase activities in human lung cytosols. Whilst glutathione peroxidase activity is reduced during the pre-natal period in rats and rabbits [15,20], we found no obvious developmental trend in the level of activity of this enzyme in human lung. Glutathione S-transferase activity, however, demonstrated a decline during the first and second trimesters and activities remained at a low level in samples obtained more than a year after birth. Peroxidase activity in tissue cytosols can result from the contributions of the basic glutathione S-transferase isoenzymes and the selenoenzyme, glutathione peroxidase. Polidoro et al. [21] found that all the peroxidase activity in lung cytosols obtained during the first trimester resulted from the selenoenzyme, and since we found only small amounts of peroxidase activity associated with the basic glutathione S-transferase isoenzymes after chromatofocusing, we propose that the activities shown in Fig. l b result primarily from the seleh%enzyme. The glutathione S-transferases can be classified as basic, near-neutral or acidic isoenzymes. More than 90% of the activity in lung cytosol results from the acidic isoenzymes and the chromatofocusing experiments revealed that the level of expression of these isoenzymes fell markedly during gestation. The period between 10 and 30 weeks gestation is characterised by reorganisation of structure and differentiation of lung cells. For example, lung bud formation occurs during the glandular phase (5-15 weeks of gestation) and

type II pneumocytes appear during the canalicular phase (15-26 weeks of gestation). For some reason these changes are associated with a decline in the expression of ghitathione S-transferase. The time-course of this fall was similar to that of the acidic isoenzymes in liver [4], and since both liver and lung are derived from endoderm it is interesting to speculate that other tissues derived from this source might also demonstrate a decline in the acidic glutathione S-transferases. Whilst Partridge et al. [17] identified small amounts of basic glutathione S-transferase isoenzymes in lung cytosol from human adults, Polidoro et al. [21] found only acidic isoenzymes in lung cytosols obtained during the first trimester. We detected weak expression of these isoenzymes after 20 weeks of gestation but since the level of expression was low, it was difficult to quantify them with confidence. Stockman et al. [8] have recently suggested that the basic isoenzymes in human liver are composed of at least two monomers, termed B 1 and B2. The c~, B and 3' enzymes in that tissue are B2Bz homodimers, 8 is a B1B2 heterodimer and t a B~B~ homodimer. The a, fl, 7, 8 and e forms in liver are identified using a combination of DEAE- and CM-cellulose [7], and elution of lung cytosols from these ion-exchangers showed that several isoenzymes that might correspond to the a, fl, 7, 8 and e forms are also present in lung at 9 weeks postnatal age. It is possible, therefore, that B 1 and B2 are present in lung as well as adult liver. It was not possible to use this approach to study this isoenzyme set at an earlier stage of development because only small amounts of tissue were available and the isoenzymes were only weakly expressed. A formal study of the development of these isoenzymes in lung would require immunological assay of the B 1 and B2 monomers. Expression of the near-neutral isoenzymes has not been previously reported in lung. Study of the ontogeny of this isoenzyme set is complicated. Firstly, this locus exhibits polymorphism and many individuals are apparently unable to synthesise these enzymes [4]. Secondly, expression of the locus during development is often variable. For example, whilst they are not usually detected in liver before 30 weeks gestation, studies in adrenal and kidney have shown almost continuous weak

453 e x p r e s s i o n of this i s o e n z y m e set d u r i n g developm e n t [4]. W e i d e n t i f i e d w e a k expression of app a r e n t l y n e a r n e u t r a l isoenzymes in p o s t n a t a l lung b u t since expression was n o t a c o n s t a n t feature in a subject with the G S T 1 2 p h e n o t y p e these activities c a n n o t be a s c r i b e d to G S T 1 with confidence. T h e p h y s i o l o g i c a l significance of the changes in g l u t a t h i o n e S - t r a n s f e r a s e expression in lung is unclear. It is p o s s i b l e that the e n z y m e is i m p o r t a n t in d e t o x i f i c a t i o n in the earliest stages of d e v e l o p ment, a p e r i o d d u r i n g which critical tissue differe n t i a t i o n occurs. H o w e v e r , the reason for the s h a r p decline in the expression of the acidic isoenzymes in b o t h lung a n d liver, a n d also at a later stage of d e v e l o p m e n t in k i d n e y [4], is not clear.

Acknowledgements W e wish to t h a n k Dr. I.A. N i m m o a n d Mr. G . C . F a u l d e r for their help with these studies a n d the staff of t h e h a e m a t o l o g y a n d b i o c h e m i s t r y d e p a r t m e n t s for h a e m o g l o b i n a n d p r o t e i n determ i n a t i o n s . F i n a n c i a l s u p p o r t f r o m the N o r t h Staff o r d s h i r e M e d i c a l I n s t i t u t e a n d the W e s t M i d l a n d s R e g i o n a l R e s e a r c h C o m m i t t e e is gratefully acknowledged.

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