The developmental expression of alpha-, mu- and pi-class glutathione S-transferases in human liver

186

Bior'himica et Biophystca Aeta, 993(1989) 186-190

Elsevier BBAOEN 23210

The developmental expression of alpha-, mu- and pi-class glutathione S-transferases in human liver R.C. S t r a n g e 1, A.F. H o w i e 2, R. H u m e 3, B. M a t h a r o o 1, j . Bell 4, C. H i l e y ~, P. J o n e s 5 a n d G A . B e e k e t t 2 I Chnical Biochemistry Research LaOorawry, School of Postgraduate Medicine and Biologi~a/Sciences, Uni.,errlty of Keele, Central Pathology Laboratory. Hartsbill. Stoke-on-Trent, 2 Dapartmem of Clinical Choral#try, University of Edinburgh and ~ Special Care Baby Umt, Simpson Memorial Maternity Pavihon. Royal lnf#rmary and # Neuropatholog~: Laboratory, Western General Hospital. Edinburgh and ~ Department of Mathematics. University of Keele, Keele ( U K )

(Received23 June 1989) Key words: GlutathaoneS-transferase; Development;(Human liver) The developmental expression of the alpha, mu and pl class glutathione S-transferases has been defined in humatl liver using radinimmunoassay and immunohistochemistry. Expression of alpha and mn class isoenzymes increased significantly at birth, while that of the pi isoenzyme declined during the first trimester. Mu-class isoenzymes (GSTI 1, GSTI 2, GST1 2-1) were expressed in hepatocytes but not in other liver cell types. Introduction The glutathione S-transferases (EC 2.5.1.18) are dimerle enzymes that eatalyse the conjugation of GSH with various electrophiles [1]. The wide variety of isoenzymes found in human tissues has been conveniently classified as belonging to either the alpha, mu, or pi classes [2]. The alpha class comprises proteins with basic isoelectric points that possess peroxidase activity and are encoded by at least two closely related genes, BI and ~ [3,4] situated on chromosome band 6p12 [5]. The mu class enzymes are the products of a polymorphic locus, GST1, and the phenotypes identified GST1 0, GST1 1, GST1 2 and GST1 2-1, the result of homo- and heterozygotic combinations of the G S T I • 0, and G S T 1 . 1 and G S T 1 . 2 alleles [6]. It is not clear which of the mu-type isoenzymes identified in human tissues corresponds with /1, the first enzyme of this class to be characteriscd [7] although, on the basis of isoelectric points, it is likely that ~ corresponds to the GST1 2, and ff [8,9] to the GST1 1 isoforms. Pi-class isoenzymes are encoded by agene situated on chromosome II [10], they demonstrate wide tissue expression ]11]. The deve!opmental expression of these enzyme classes is of interest bee~.t~se they demonstrate time- and

Abbreviation:GSH.glutathione. Correspondence: R.C. Strange, Department of ClinicalBiochemistry, Central Pathology Laboratory, North StaffordshireHospital Center, Stoke-on-Trent ST4 7QB, U.K.

tissue-specific expression during fetal, neonatal and infant life. So far, comparisons of the relative levels of expression of these genes during development have been based on measurements of enzyme activity using chlorodinitrobenzene as electrophile [11,12]. The mu isoenzymes, however, demonstrate a markedly higher specific activity for this subslrate than do the alpha and pi isoforms [1], and studies based on such activity measurements will consequently overestimate the contribution of this locus to total activity. We now describe further studies of the developmental expression of the alpha, mu and pi isoenzymes in human liver using a sensitive radioinununoassay approach. Our aims were, firstly, to define the expression of the B1 and Bz genes. These data are difficult to obtain using activity measurements, since the BIBz and BaB1 dimers are only partially resolved by ion-exchange chromatography, and the elution profiles demonstrate marked between-subject variation [13]. Our second a2m was to compare the reactivities of the GSTI 0, GST~ 1 and GST1 2 isoenzymes with anti-mu serum. Although population studies indicate that these enzymes are polymorphic variants [6,12], study of their reaction with appropriate antibody ~hould provide supporting data. Our third aim was to define the expression of the mu isee.n!ymes during development. Expression of these :,~oenzymes is weak during fetal life, and it is difficult, using activity measurements, to determine with confidence whether the locus is expressed during the first trimester [11,13]. The availability of antiserum also all~wed us to determine the cellular distribution of the mu isoeuzymes in developing liver. These glutalhione

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187 S-trausferase loci demonstrate lime-specific changes in expression, and our fourth aim was to compare the masses of the different isoenzymes, to determine whether theil exp, ~.ss[on is co-ordinated. Materials and

subunits, which showed a cross-reactivity of 17o in the assay for B 2 subunits, and B~ subunits, which demonstrated a cross-reactivity of 2~, in the assay for 1~i ~,~bu~,iL. The coefficient of v:~r!:tti~ ef each assay w,~s less than 10%.

Methods Starch-gel electrophoresis

['reparation of tissue cyfosols

Horizontal starch-gel electrophoresis, performed as described previously [12] was used to classify cytosols as being of the GST1 0, GST1 1, G S T I 2 or GST1 2-1 phenotypes.

Samples of liver were obtained immediately after death from aborted fetuses (10 22 weeks of gestation) and wit :hLr~ 4 h of death from premature and term infants (2/J 42 weeks of gestation) who died within 24 h of birth. These livers were used to define in utero ontogeny and compared to liver samples from (a) funterm infants who died at 2 - 8 5 weeks postnatal age, the majority as sudden and unexpected deaths usually as cases of sudden infant death syndrome, and (b) adults. T h e aim and methods of the study were approved by the Reproductive Medicine Ethics of Medical Research Sub-Committee of the Lothian Health Board. Liver samples were obtained from 20 adults within 6 h of death, at autopsy, with the permission of Her Majesty's Coroner. Cytosols (150000 × g supematants) were prepared as described previously [13].

lmmunohistochemistry Liver samples were fixed in buffered 10% fort'oalin, processed to paraffin wax and sequential 5/tm sections were cut. The first was stained with haematoxylin and eosin and the second with rabbit anti-mu serum. Mu isoenzymes were identified using the technique previously described for alpha and pi isoforms [18]. Results

Developmental expression of alpha-class isoeneymes Fig. 1 shows the levels of Bi in liver cytosols obtained between 10 and 125 weeks post-menstrual age. Whilst there was no significant change in the level of these enzymes either in the period between 10 and 40 weeks gestation ( r = - 0 . 1 6 ) or between term and 85 weeks postnatal age ( r = - 0 . 1 ) , the levels of enzyme were significantly higher in term postnatal infants compared to in utero ontogeny ( P < 0.001) (Table 1). Adult liver demonstrated the same range of values as postnatal infants but significantly higher levels than those for in utero ontogeny ( P < 0.005). T a b l e I also shows that the levels of the Ba-containing ghitathione S-transferases during development were substantially lower than those of tile Bl-containing ±soenzymes. There was no significant change in the level of the B 2 isoenzymes either in the period between 10 and 40 weeks gestation ( r ~ - 0 . 1 7 ) or between term and 85 weeks postnatal age ( r = - 0 . 3 2 ) . The level of expression of these isoenzymes did change during develop-

Radioimmunoassay Detailed descriptions of the specific glutathione Stransferase radioimmunoassays have been described previously [14-16]. Briefly, total protein concentrations in eytosols were determined using the Coomassie blue dye binding-technique [17] adapted for use on a Cobas F a r a (Roche, U.K.) centrifugal analyser; if necessary for RIA, cytosols were diluted in assay diluent consisting of 25 m m o l / l sodium phosphate b u f f e r / b o v i n e serum albumin ( l g / 1 ) / s o d i u m azide (0.2 g / l ) (pH 7.6, 4 " C ) . The specificity of the radioimmunoassays were such that isoenzymes other than the immunogen used to raise each antiserum usually exhibited a cross-reactivity of less than 0.1%. The exceptions were the G S T 4 ±soform, which showed a cross-reacfivity of less than 0.4% in the radioimmunoassay for mu class isoforms, B t

TABLE [

Expression of alpha, mu and pi glutathione S.transferases in developing liver Tile levels (Itg/mg cytosol protein) of the alpha (BI and B2), rnu and pi glutathionc S-transferases irt liver cytosols were determined using radiointmunoassay. Data from subjects with the GST1 0 phenotype have not been included in that shown for the mu group.

B1 B~ mu pi

[n-utero ontogeny 10-42 weeks mean +S.D. n 9.0 ±3.! 22 0.93+0.76 22 0.10±0.054 10 0.53.t.0.27 22

95% conf. int. %6 -10.3 0.59 - 1.3 0.054- 0.14 0.40 - 0.65

Term post-natal 42-85 weeks mean + S.D. 14.3 ±4.7 2.3 +2.3 0.43±0.33 0.21:[0.22

adults n 20 19 11 20

95% conf. int 12.1 16.5 1.2 - 3.5 0.21- 0.65 0.11 0 . 3 1

mean+S.D, 12.8 5-55 3.7 +21 0.46±0.27 0.01±0013

n 20 20 11 20

95% conf. int. 102 -15.4 2.7 - 4.7 027 0.65 0.002- 0.014

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merit however, since levels for in utero ontogeny were sigaificantly lower than those obtained both posmataly and in adults (P < 0.005; Mann-Whitney U-test). Further, levels in the samples obtained between term and 85 weeks postnatal age were significantly lower than those in adult samples (P < 0.05). Levels of mu enzymes in subjects with different GST1 phenotypes Starch-gel electrophoresis was used to classify liver cytosols from adult samples into GST1 1, GST1 2, GST1 2-1 or GST1 0 phenotypes and the levels of mu isoenzyme in each group were compared (Table II). There were no significant differences between the levels in the samples with GST1 2-1, GST1 1 and GSTI 2 phenotypes (Kruskal-Wallace test). Previous studies have shown the high incidence of the null phenotype (GST1 0) in different human populations [6,7,12]. Since subjects with this p~'tenotype express no mu-set activity, cytosol samples containing no mu isoenzyme detectable by radioimmunoassay were classified as nulls (Table If). In samples obtained between 40 and 125 weeks post-menstrual age, the classification of subjects as either expressors or non-expressors of mu could be confirmed by starch-gel electrophorasis, but before 40 weeks gestation the level of these isoenzymes was often too low to be detected with confidence using the zymogram technique.

TABLE II Levels of glutalhione S-transfer~e mu in hver c),losolswith the GSTI O, GSTI 1, GSTI 2 and GST1 2-I phenotypes The levelof mu isoenzymein livercytasolswas determined by using radioirnmun~ssay. Valuesshown are lag/rag¢ytosolprotein. GSTI0 04-0 15

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GST11 GSTI 2 GST1 2-1 0.33:t:0.08 0.634-0.35 0.55 5 4 2

Fig. 2. Levelsof mu-clas glutathione S-Iransfera.ses in developing human liver.After 40 weeks gestation, sampleshavingno mu isoenzymes detoctable by radioimmunoassay were confirmed as GSTI 0 phenotypes by starch-gelelt~trophoresis.

Developmental expression of mu-class isoenzymes Mu-set isoenzymes were detected using radioimmunoassay in 10 of the 22 samples obtained before 40 weeks gestation, 11 of the 20 samples obtained between term and 85 weeks postnatal age and 11 of the 20 samples obtained from adult samples. The frequency of the null phenotype is similar therefore, in samples obtained over the entire developmental range. The level of the mu isoenzymes in liver cytosols from expressors did not change with time between 10 and 40 weeks gestation, but levels for in utero development were significantly lower than in term postnatal infants (P < 0.005) and in adults (P < 0.01) (Fig. 2). There was no difference between levels in the term postnatal infants and adult subjects (Table I). These data were in agreement with those obtained from immunohistochemistry. Expression of the isoenzyme was too low to allow detection in fetal hver sections or in samples obtained later in development from individuals classified as GST1 0 phenotypes. In individuals classified as GST1 1 or GST1 2 phenotypes, expression of the enzyme was Iocalised to hepatocytes with no evidence of positivity in other hepatic cell types (Fig. 3). Developmental expression of the pi-class isoenzymes Fig. 4 shows the developmental expression of the pi isoenzyme in liver. Inspection of the data suggests a negative exponential relationship between time and the level of pi isoenzymes. However, the best-fit model, even ~,~'ter removal of the two apparent outUers seen in the term postnatal r:lata, (pi~0.68e-°lgt), underestimated the larger values of pi. Consequently a linear decrease of pi with time was assumed for in utero onlogeny. The level of the enzyme in cytosols fell significantly between 10 and 40 weeks of gestation (r22 = -0.84). The best-fit straight line had the equation, y = l . 1 3 - 0 . 0 2 3 t . The levels of the pi isoenzyme in

189 utero development in subjects who expressed the mu locus ( r - 0.69, n ~ 10). Discussion

Fig. 3. Sections were taken from a liver obtained after 30 weeks postnatal age and the mu isoenzymedetected as d~cribed in Materials and Methods. Hepatocytes (H) show positivity.Bile ducts (B) o.rteries(A) and veins(V) are shown.Originalmagnificationx 125. samples obtained between 41 and 125 weeks postmenstrual age did not change with time (r20 = 0.14). However, while the level of pi enzyme fell during development, the total mass of the isoform in liver increased during fetal life. Fetal liver weight increases markedly during gestation [19] and estimating liver weight at 10, 25 and 30 weeks of gestation as 1 g, 100 g and 120 g respectively, we estimate that the hepatic content of the pi isoform at these times is 25, 720 and 1200 ug enzyme, respectively. These data indicate that while the gene is down-regulated, it is not switched off.

Correlation between different isoforms The possibility that expression of the different isoenzymes was co-ordinated was studied by multivariate analysis. There were no significant correlations between the levels of the enzymes except for Bz-eontaining isoenzymes and the sums of the levels o1 B~, 132, mu and pi (P < 0.05) for in utero ontogeny, ( r = 0.70, n = 22), term postnatal (r=0.52, n = 15) and adult (r=0.51, n = 20) subjects. The levels of the Bt and B2 monomers were also significantly correlated (P < 0.05) during in

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We have described the expression of the alpha, mu and pi glutathione S-translerases in developing liver and several general observations regarding the developmental expression of these enzymes can be made. Firstly, whilst the level of expression of each of the isoenzyme classes changed, isoenzymes containing the B1 monomer always comprised the bulk (about 90%) of the total enzyme mass. Secondly, the ellZytl)¢; ..~iaztstT.S demonstrated different patterns of expression; the levels of BI and mu isocnzymes increased with in utero development but were stable thereafter, whilst the level of B2-containing isoenzymes continued to increase post-hatally. The pi isoenzyme was down-regulated during the first half of gestation. Thirdly, out data support previous observations by Edwards and Hopkinson [20] that human development is often accompanied by a generalised increase in the expression of basic iso.~nzymes and a decreased expression of acidic forms. Previous studies based on measurements of the total activity of the alpha-class glutathione S-transferases showed that this class was expressed in liver th~'oughout development. These data were obtained after chromatographic separation of the various isoenzymes and did not allow precise determination of the levels of BI and B2 [11]. Recent studies by Hiley et al. [181 have shown continuous expression of isoenzymes that cross-react with a,~ti-B~B1 serum in hepatocytes and the epithehum of the large bile ducts. However, since that antiserum recognised both monomers, it was not known if Bi and B2 were differentially expressed in developing liver. The present study has extended these data by demonstrating that, whilst the levels of B1 and Bz are constant during fetal life, expression of both proteins is increased during the perinatal period. Thereafter, B1 remains constant, but ~ expression appears to increase, although the nature of this increase, sudden or gradual, is not known. The mu class isoenzymes are the products of GST1, a lo:us that demonstrates genetically determined varia.tion [6,12]. The GSTi 0 phenotype (GSTI. O/GSTI. O) is associated with an absence of mu activity, and Mannereik [1] has reported that samples from these subjects failed to show a precipitin reaction when probed with anti-p, serum. We have confirmed this observation in adult and neonatal samples using a sensitive radioimmunoassay, and assume therefore that the gene is not transcribed. The findings that our antiserum recognised both GST1 1 and GST1 2 isoforms and that levels of mu enzymes in cytosois with the GST1 1, GST1 2 and GSTI 2-1 phenotypes were similar, support population surveys suggesting that these enzymes are allelic variants [6,12]. Unlike the alpha and pi isoforms, which are also

190 expressed in bile epithelial cells, expression of mu isoenz y m e s was iocalised to hepatocytes. T h e e n z y m e was not detected in sections of fetal livers n o r in those f r o m older subjects classified as G S T I 0 phenolypes. T h e distribution of the e n z y m e was similar in subjects class;.fied as G S T 1 1 a n d G S T 1 2 phenotypes. T h e c o n t r i b u t i o n of the mu locus to the total mass of g l u l a t h i o n e S-transferase protein in liver was small. If suggestions that individuais with the G S T 1 0 p h e n o t y p e have increased susceptibility to some cancers are confirmed, it w o u ld e m p h a s i s e the i m p o r t a n c e of the rcaejinos (e.~., with benzo~alpyrene-40-oxJde) that are specifically catalysed b y these isoenzyrnes. Previous studies based o n activity m e a s u r e m e n t s s h o w e d that the level o f expression of mu i s ofnrms was low in fetal liver samples, a n d mu-set activity was generally undetectable before 30 weeks of gestation [11,13]. T h e present stu d y has s h o w n that m u i s oforms are c o n t i n u o u s l y expressed even before 20 weeks of gestation, a l t h o u g h expression increases m a r k e d l y during the later half o f in-utero liver development. In contrast, the pi isoform was d o w n-regul a t e d d u r i n g the second trimester. T h u s, whilst the different i s o f o r m s s h a r e c o m m o n substrates such as chl orodi hi l robe nz e ne , their varied d e v e l o p m e n t a l expression emphasises that their in vf'vo substrates are likely to be very different. Increased expression of the alp ha class m i g h t be expected d u r i n g the perinatal period, since these i s oforms have peroxidase activity [21], a n d the extra-uterine env i r o n m e n t is m a r k e d ' y hyperoxic c o m p a r e d with that of the fetus. However, the reason for the decreased expression of the pi isoform a n d increased expression of m u i s o f o r m s r e m a i n s unclear. Acknowledgements W e gratefully acknowledge the help of Professor A.A. Calder, Professor O.T. Baird, Dr, J.D. Hayes, D r . G.C. Faulder, Dr. I.A. N i m m o , Dr. A . D . Bain a n d Dr. I.I. Smith. R a d i o i m m u n o a s s a y s were developed with s u p p o r t from a Scottish H o m e alad H e a t h D e p a r t m e n t G r a n t a w a r d e d to G.J.B. a n d Dr. J.D. Hayes.

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