Partial purification and characterization of the soluble glutathione transferase isoenzymes from cultured Hep G2 cells

Ceil Biology

International

Reports,

585

Vol. 13, No. 7, July 1989

PARTIAL PURIFICATION AND CHARACTERIZATION OF THE SOLUBLE GLUTATHIONE TRANSFERASE ISOENZYMES FROM CULTURED HEP G2 CELLS Paul J. Dierickx Instituut voor Hygiene en Epidemiologie Wytsmanstraat 14 B- 1050 Brussel, Belgium

Abstract Hep G2 cells, an established cell line derived from a human hepatoma, were mass cultured in a cell factory for the isolation of glutathione transferase isoenzymes. These were enriched by affinity chromatography and separated in an anionic and a cationic fraction. They were partially characterized by different kinetic and inhibition parameters. Three different subunits were observed. The results were compared with human liver data. It is concluded that Hep G2 cells can be considered as a valuable alternative tool for in vitro research of human liver phenomena, especially when toxicological interactions are investigated. Introduction The liver is the principal organ involved in the biotransformation and detoxification of xenobiotics. It is also the first organ after the gastrointestinal tract that a drug administered by oral route passes. Therefore, knowledge of hepatic metabolic pathways of xenobiotics is of major importance. Freshly isolated hepatocytes, cultured for up to two weeks, offer a valuable in v$ro model in this context (Le Bigot and Kiechel, 1988). Attempts of establishing permanent cell lines of hepatocytic origin were unsuccesful so far, because important biochemical functions are mostly lost. Hep G2 cells, an established cell line, were derived from a human hepatoma (Aden et al., 1979. They synthesize and secrete 17 of the major human plasma proteins (Knowles et al., 1980) and most of the major apoproteins (Zannis et al., 1981). Several other parameters confirmed later on the promising properties of this cell line. The type I drug metabolism inducers phenobarbital and 3-methylcholanthrene enhance the aldehyde dehydrogenase activity at the same level in Hep G2 cells and isolated human hepatocytes, as in rat liver (Marselos et al., 1987). The presence of microsomal mixed function oxidases was also demonstrated (Eddy et al., 1987). Glutathione (GSH) plays an important role in the detoxication of potentially toxic compounds. Its most widely known biological role is the conjugation with xenobiotics, including carcinogens, mutagens, toxic or pharmacologically active compounds, and their metabolites (Chasseaud, 1979). This conjugation is catalyzed by GSH transferase (GST, EC 2.5.1.18), most recently reviewed by Mannervik and Danielson (1988). GST is also capable of direct binding of toxic compounds, another important detoxication pathway (Smith and Litwack, 1980). Moreover GST has peroxidase activity for organic hydroperoxides and, therefore, the potential to detoxify lipid and DNA hydroperoxides arising from radical damage in the presence of oxygen (Tan et al., 1984). I described previously that Hep G2 cells contain both GST and GSH. The content of the latter can be manipulated by application of L-buthionine-S,R-sulfoximine and L-oxothiazolidine-4-carboxylic acid, proving that

0309-1651189/07058!S91$03.0010

0 1989 Academic

Press Ltd.

586

Cell Biology

international

Reports,

Vol. 13, No. 7, July 1989

glutamylcysteine synthetase and 5-oxo-prolinase are also present. Since Hep G2 cells contain a well functioning GSH system, they are also in this respect comparable to hepatocytes (Dierickx, 1987). Meanwhile it was reported that the GST activity in freshly isolated human adult hepatocytes and Hep G2 cells is similar, but Hep G2 cells have not retained the cystathionine pathway (Duthie et al., 1988). Their GST activity depends on the composition of the growth medium (Doostdar et al., 1988) Liver GST plays a central role in different detoxication pathways (Chasseaud, 1979; Smith and Litwack, 1980; Tan et al., 1984). Since cultured Hep G2 cells have many hepatocyte characteristics, I investigated the GST isoenzymes in this hepatoma cell line. Materials and Met/w& Cell culture flasks were purchased from Nunc, and cell culture products from Gibco. GSH and bromosulfophtalein were obtained from Janssen Chimica (Beerse, Belgium), bovine serum albumin from Boehringer, chemicals for SDS-polyacrylamide gel electrophoresis from Bio-Rad, epoxy-activated Sepharose 6B and PD-10 columns containing 10 ml Sephadex G-25 Medium from Pharmacia, and carboxymethyl cellulose (CM52) from Whatman. Amicon furnished the YM-10 ultrafiltration membranes. Gossypol acetic acid, hematin and S-hexylglutathione came from Sigma, and all other chemicals from Merck, including triphenyltin chloride, the GST substrate 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dichlorophenoxyacetic acid (2,4-D). Hep G2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 pg/ml streptomycin (complete medium). They were incubated at 37 “C in a 7% CO, atmosphere. Specific GST activities were determined on cells cultured in tissue flasks with a growth area of 175 cm2. The same results were obtained when the cells were either loosen by trypsinisation or by scraping. For the purification of GST the Hep G2 cells were cultured in a cell factory, consisting of 10 separate chambers, with a total culture area of 6000 cm2. The cells were harvested when nearly reaching confluency. They were washed with 0.5 1 Hanks balanced salt solution, trypsinised with 125 ml 0.25% trypsine, suspended in 450 ml complete medium, and centrifuged at 130 xg for 10 min. About 10 g of cells were obtained. Further manipulations were carried out at O-4 OC. The cells were homogenized in 30 ml 22 mM sodium phosphate buffer, pH 7.0, containing 0.25 M sucrose and 1 mM EDTA.Na4, wiih 10 up-and-down strokes in a motor driven Potter-Elvehjem homogenizer (1,500 rpm), equiped with a teflon pestle. A fatfree supematant was obtained after ultracentrifugation (1 h, 100,000 xg). and applied to a GSH affinity column (1.6 x 9 cm), packed with epoxy-activated Sepharose 6B that had been reacted with GSH as described (Simons and Vander Jagt, 1977). The column was eluted with 22 mM sodium phosphate buffer, pH 7.0, containing 1 mM EDTA.Na+ until no further protein was detected by monitoring the absorbance at 280 nm. GST was then eluted with 15 mM GSH in 50 mM Tris-HCl buffer, pH 9.6 (Trakshel and Maines, 1988). The fractions with GST activity were pooled, concentrated to 2.5 ml by ultrafiltration on an YM-10 membrane, equilibrated with 5 mM sodium phosphate buffer, pH 7.0, containing 1 mM EDTA.Na$ on a PD-10 column, and applied to a carboxymethyl cellulose column (1.6 x 9.5 cm), equilibrated with the same buffer. The anionic proteins are not adsorbrd. After these had passed the column, the cationic proteins were eluted with 100 mM sodium phosphate buffer, pH 7.0, containing 1 mM EDTA.Na4. No further GST was eluted when 1.5 N NaCl

Cell Biology

International

Reports,

Vol. 13, No. 7, July 1989

587

was applied afterwards. SDS-polyacrylamide slab gel electrophoresis was carried out on 10% polyacrylamide gels as described (Laemmli, 1970). The gels were silver stained following the method of Wray et al. (1981). The GST activity was determined according to Habig et al. (1974) using CDNB as substrate. Specific activity is expressed in units of enzyme activity/mg protein, as masured by the Bradford (1979) method with bovine serum albumin as standard.

1.8 h

0.8

I

- 1.4 E 2.E 5 [

0.6 Ca

1.0

.-c > F 3 i= 0.6 2 Li c7 0.2

IT-

10

20

30

40

FRACTION

10 NUMBER

Fig. I (left). Elution of the Hep G2 GST activity from a GSH afJinity column. The column was first rinsed with 22 mM sodium phosphate bufSer,pH 7.0, containing 1 mM EDTA.Na4, and then eluted with 15 mh4 GSH in 50 mM Tris-HCl, pH 9.6 (arrow A). Thereafter the column was eluted with 3 N NaCl (arrow B). Fractions of 3 ml were collected. Fig. 2 (right). Separation of anionic and cationic GST on a carboxymethyl cellulose column. The column was first eluted with 5 mM and next with 100 mM (arrow A) sodium phosphate buffer, pH 7.0, containing 1 mM EDTA.Na4, and 1.5 N NaCl (arrow B). Fractions of 4.5 ml were collected. An : anionic GST, Ca : cationic GST.

588

Cell Biology

International

Reports,

Vol. 13, No. 7, July

1989

Kinetic properties and inhibition kinetics by 2,4-D were examined measuring the initial velocities of GST at GSH and CDNB concentrations varying from 0.25 to 1 mM (Dierickx, 1983). They were analyzed by Lineweaver-Burk plots. The correlation coefficient calculated by linear regression analysis was always higher than 0.98. The other GST inhibitors were tested as decribed (Tahir et al., 1985). Results The specific GST activity in Hep G2 cells is 112 f 4 nmol/min/mg protein. Therefore, a large quantity of cells is needed for the purification of GST. The 100,000 xg supernatant of an extract obtained from about 10 g Hep G2 cells was first enriched by GSH affinity chromatography (Fig. 1). Only a small part (6%) of the GST activity was not adsorbed to the matrix and passed freely through the column. Fractions with this unadsorbed GST were not further investigated. As an alternative method the GST in the 100,000 xg supematant was separated in a low- (20%) and a high-affinity (80%) set of GST, by elution of the adsorbed GST from the affinity column first with 50 mM Tris-HCl, pH 9.6, and then with the same buffer containing 5 mM GSH. This low/high affinity ratio is very close to that observed for human liver GST (Vander Jagt et al., 1985). The GST fractions which were eluted with 15 mM GSH from the affinity matrix were applied on a carboxymethyl cellulose column (Fig. 2). The proteins that were not adsorbed contained the anionic GST (21%), while the cationic GST (79%) was desorbed with a high ion-strength buffer. Because of the relatively low GST concentrations no attempts were made for preparative separation of these peaks. The anionic and cationic GST are furtheron called GST An and GST Ca. The SDS-polyacrylamide slab gel electrophoretogram of HeP G2 GST is shown in Fig. 3. No bands were visible after Coomassie blue staining, but silver staining allowed the detection of the Hep G2 GST subunits between 25 and 30 kDa (Mannervik and Danielson, 1988). These bands were the most intensive ones, although supplementary bands were also observed, as could be expected. GST Ca contained one single subunit with a Mr 27,700 and is therefore a homodimer, or a mixture of homodimers with the same Mr. GST An showed 2 bands migrating slightly

Fig. 3. Silver-stained SDS-polyacrylamia’e electrophoretic gel showing the subunits of Hep G2 GST. Lane A : GST An, lane C : GST Ca.

Cell Biology

International

Reports,

-1

589

Vol. 73, No. 7, July 1989

01 i/

2 [CDNB]

3 pfttil;l

Fig. 4. Lineweaver-Burk plot showing mixed-type function inhibition of GST An (0) towards CDNB by 2 mM 2,4-D (0). The concentration of GSH was kept constant at I mM. V is expressed in jtmollminlmg protein. slower and closely together with Mr 28,200 and Mr 28,900. It is thus probably a heterodimer, or a mixture of 2 different homodimers. Some kinetic parameters of the Hep G2 GST isoenzymes are summarized in Table I. The relatively low specific activity of GST An is partly due to the contamination with some other proteins (Fig. 3). Linear Lineweaver-Burk plots (r>O.98) were always observed in the kinetic experiments. The apparent Km towards GSH is nearly identical for both isoenzymes, but the apparent Km towards CDNB allows the discrimination between both. The GST activity, measured in the standard conditions of 1 mh4 GSH and CDNB, was inhibited by 2 mM 2,4-D : it dropped to 29% (GST Table I. Some kinetic properties of the GST isoenzymes, purified from Hep G2 cells. Property Specific activitya (l.tmol/min/mg protein) Apparent Km towards CDNBb (n-m Apparent Vmaxtowards CDNBb (lrmol/min/mg protein) Apparent Km towards GSHC bw Apparent Vmaxtowards GSHC (l.lmol/min/mg protein)

GST An

GST Ca

5.85

35.49

0.48

1.48

8.2 0.15

6.6

aMeasured with both CDNB and GSH at 1 mM. bMeasured at a faed concentration of I mM GSH. CMeasured at a fied concentration of 1 mM CDNB.

85.6 0.18

40.2

Cell Biology

590

5

10

International

Reports,

Vol. 73, No. 7, July 1989

50

25

75

CONCENTRATION

10; (PM)

Fig. 5. Inhibition of Hep G2 GST by S-hexylglutathione. Remaining enzyme activity of GST An (0) and GST Ca (0) was measured with CDNB and GSH at 1 miki. Ca) or 54% (GST An) of the contol value. In order to investigate the meaning of this inhibition, enzyme kinetic experiments were performed with CDNB as the variable substrate. Increased apparent Km and decreased apparent Vm values were observed for both isoenzymes, representing mixed-type function inhibition kinetics (Fig. 4). This suggests that 2,4-D interacts with Hep G2 GST at another locus than that at which the substrate interacts. In the last series of experiments the influence of a set of GST inhibitors (Tahir et al., 1985) was investigated. This is illustrated for S-hexylglutathione in Fig. 5. The results for the 5 inhibitors are summarized in Table II. GST An was more inhibited by bromosulfophtalein than GST Ca. The other inhibitors had a stronger effect on GST Ca than on GST An. A relatively small difference in inhibition was observed for gossypol acetic acid. The 4 remaining inhibitors allowed a clear discrimination between GST An and GST Ca, because they were 4 to 43 times more effective when comparing their influence to one GST isoenzyme with their influence to the other GST isoenzyme.

Table II. Inhibition parameters (I,,, /.&I) for the two types of Hep G2 glutarhione transferase.

Inhibitor Gossypol acetic acid Triphenyltin chloride Bromosulfophtalein Hematin S-Hexylglutathione

GST An

GST Ca

2.75 2.50 22 0.29 69

1.95 0.09 87 0.02 1.60

The I,, is the concentration of inhibitor giving 50% inhibition of enzymeactivity, measured with I mM concentrations of GSH and CDNB.

Cell Biology International

Reports, Vol. 13, No. 7, July 1989

591

Discussion The presence of at least two different GST isoenzymes in cultured Hep G2 cells was demonstrated : an anionic and a cationic one. Besides their difference in subunit structure, their Km towards CDNB, and their inhibition by 2,4-D and specific GST inhibitors. It should be pointed out here that the so-called near neutral GST isoenzymes have a p1 between 6 and 6.5; they are, therefore, strictly speaking also anionic proteins. Because the cationic GST isoenzymes of human liver can not be rigorously identified, they are often collectively referred to as basic transferases (Mannervik and Danielson, 1988). At least 15 different GST isoenzymes were demonstrated in human liver, although not all forms were detectable in any liver sample (Vander Jagt et al., 1985). The majority of this GST activity is found in the cationic form. Radial immunodiffusion also revealed that 7 1% of the human liver GST is in the cationic form (Corrigall and Kirsch, 1988). This value is in good agreement with that observed in Hep G2 cells. The inhibition of GST by 2,4-D was previously shown to be induced by direct binding of 2,4-D to GST at a different locus than the substrate (Dierickx, 1983). The identical interaction of 2,4-D with Hep G2 GST strongly suggest that these cellular proteins have the same binding capabilities. Three different GST subunits were found in Hep G2 cells. This is the same number as in human liver, in spite of the high number of isoenzymes (Vander Jagt et al., 1985). The heterodimeric composition of the anionic human liver GST seems to be rather characteristic (Singh et al., 1985), and was also observed for Hep G2 GST. However, these data nor the inhibition by specific GST inhibitors allowed a clear-cut identification of the Hep G2 GST isoenzymes. Nevertheless the results reported prove the occurrence of at least two clearly distinct GST isoenzymes in Hep G2 cells. The catalytic function (Chasseaud, 1979) and the ligand complexing properties (Smith and Litwack, 1980) of GST are important for organisms, since they detoxify a large number of chemicals. The multiplicity of GST isoenzymes is assumed to result from the need to conjugate numerous types of substances differing in the nature of their electrophilic centre and their molecular structure (Ketterer et al., 1983). Therefore, and because of the similarity of the GST activity in Hep G2 cells and freshly isolated human hepatocytes (Duthie et al., 1988), Hep G2 cells can be considered as a valuable alternative tool for in vitro research of human liver phenomena, especially when toxicological interactions are studied. Acknowledgements I thank Mrs. J. Ema and F. Huez for their excellent technical assistance. References Aden, D.P., Fogel, A., Plotkin, S., Damjanov, I. and Knowles, B.B. (1979) Controlled synthesis of HBsAg in a human liver carcinoma-derived cell line. Nature, 282, 615-616. Bradford, M.M. (1979) A rapid and sensitive method for the quantitation of microgram quantities of protein, utilizing the principle of protein dye binding. Analytical Biochemistry, 72,248-254. Chasseaud, L.F. (1979) The role of glutathione and glutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents. Advances in Cancer Research, 29, 175-274.

592

Ceil Biology

International

Reports,

Vol. 13, No. 7, July 1989

Corrigall, A.V. and Kirsch, R.E. (1988) Glutathione S-transferase distribution and concentration in human organs. Biochemistry International, 16,443~448. Dierickx, P.J. (1983) Interaction of chlorophenoxyalkyl acid herbicides with rat-liver glutathione S-transferases. Food and Chemical Toxicology, 2 I, 575-579. Dierickx, P.J. (1987) Inhibition of glutathione-dependent uridine uptake in cultured human hepatoma cells. Medical Science Research, 15, 1349- 1350. Doostdar, H., Duthie, S.J., Burke, M.D., Melvin, W.T. and Grant, M.H. (1988) The influence of culture medium composition on drug metabolism enzyme activities of the human liver derived Hep G2 cell line. FEBS Letters, 241, 15- 18. Duthie, S.J., Coleman, C.S. and Grant, M.H. (1988) Status of reduced glutathione in the human hepatoma cell line, Hep G2. Biochemical Pharmacology, 37, 3365-3368. Eddy, E.P., Howard, P.C., McCoy, G.D. and Rosenkranz, H.S. (1987) Mutagenicity, unscheduled DNA synthesis, and metabolism of 1-nitroppyrene in the hman hepatoma cell line Hep G2. Cancer Research, 47,3 166-3 168. Habig, W-H., Pabst, M.J. and Jakoby, W.B. (1974) Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. The Journal of Biological Chemistry, 249,7 130-7 139. Ketterer, B., Coles, B. and Meyer, D.J. (1983) The role of glutathione detoxication. Environmental Health Perspectives, 49,59-69. Knowles, B.B., Howe, C.C. and Aden, D.P. (1980) Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science, 209,497-499. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature, 227,680-685. Le Bigot, J.F. and Kiechel, J.R. (1988) In vitro models in the study of the fate of drugs in research and development. In : A. Guillouzo (ed.) Liver Cells and Drugs, pp. 2 1l-220. J. Libbey Eurotext, London and Paris. Mannervik, B. and Danielson, U.H. (1988) Glutathione transferases. Structure and catalytic activity. CRC Critical Reviews in Biochemistry, 23,283-337. Marselos, M., Strom, S.C. and Michalopoulos, G. (1987) Effect of phenobarbital and 3-methylcholanthrene on aldehyde dehydrogenase activity in cultures of Hep G2 cells and normal human hepatocytes. Chemico-Biological Interactions, 62,75-88. Simons, P.C. and Vander Jagt, D.L. (1977) Purification of glutathione S-transferase from human liver by glutathione-affinity chromatography. Analytical Biochemistry, 82, 334-341. Singh, S.V., Das, D.D., Partridge, C.A., Theodore, C. and Srivastava, S.K. (1985) Different forms of human liver glutathione S-transferases from dimeric combination of at least four immunologically and functionally distinct subunits. Biochemical Journal, 232,781-790. Smith, G.J. and Litwack, G. (1980) Roles of ligandin and the glutathione S-transferases in binding steroid metabolites, carcinogens and other compounds. Reviews in Biochemical Toxicology, 2, l-47. Tahir, M.K., Guthenberg, C. and Mannervik, B; (1985) Inhibitors for distinction of three types of human glutathione transferase. FEBS Letters, 181,249-252. Tan, K.H., Meyer, D.J., Belin, J. and Ketterer, B. (1984) Inhibition of microsomal lipid peroxidation by glutathione and glutathione uansferases B and AA. Roles of endogenous phospholipase A2. Biochemical Journal, 220,243-252. Trakshel, G.M. and Maines, M.D. (1988) Characterization of glutathione S-transferases in rat kidney. Alteration of composition by cis-platinum. Biochemical Journal, 252, 127- 136. Vander Jagt, D.L., Hunsaker, L.A., Garcia, K.B. and Royer, R.E. (1985) Isolation and characterization of the multiple glutathione S-transferases from human liver.

Cell Biology

International

Reports,

Vol. 73, No. 7, July 1989

593

Evidence for unique heme-binding sites. The Journal of Biological Chemistry, 260, 11603-l 1610. Wray, W., Boulikas, T., Wray, V.P. and Mannock, R. (1981) Silver staining of proteins in polyacrylamide gels. Analytical Biochemistry, 118, 197-203. Zannis, V.I., Breslow, J.L., SanGiacomo, T.R., Aden, D.P. and Knowles, B.B. (198 1) Characterization of the major apolipoproteins secreted by two human hepatoma cell lines. Biochemistry, 20,7089-7096.