1200
BIOCHEMICAL SOCIETY TRANSACTIONS
of S-oxidation appears to be removed by alternative substrates for FMO. However, at rug Metah. 11, 2 17-265
I20 I 3. Reese,J. A. & Byard, J. L. (198 I ) In Vitro 17,935-940 4. Strom, S. C., Jirtle, R. L., Jones, R. S., Novicki, D. L., Rosenberg, M. R..Novotny, A., Irons, G., McLain, J. R. tk Michalopoulos, G . (1982)J. Natl. CancerInst. (U.S.) 6 8 , 771-775 5 . Gerding, T. K., Drenth, B. F. H., de Zeeuw, R. A,, Tepper, P. G . & Horn, A. S. ( 1990)Xmohiotica 20,525-536
Received 30 May 1990
Maintenance of differentiated function in cultured rat hepatocytes immortalized by transfection with viral DNA ANDREW NAIRN, BRIAN WILLETT, M. HELEN GRANT, ALEXANDER SCOTT and CAROLINE MAcDONALD Department of Biosrience and Biotechnology, Strathrlyde Universiry, The Todd Centre, 31 Taylor Street, Glasgow G4 ONR, U.K. Primary cultures of rat hepatocytes have been widely used for studying xenobiotic metabolism and toxicity in vitro. However, their use is limited because hepatocytes d o not proliferate in culture and the cells undergo rapid de-differentiation losing many parenchymal functions within the first 72 h [ 1 I. We have recently transfected rat hepatocytes with SV40 viral DNA to produce an immortal hepatocyte cell line (SV40RH1) and this study assesses the extent to which these cells have retained differentiated hepatic functions. The presence of the cystathione pathway for glutathione synthesis, and the activities of y-glutamyltransferase ( yGT) and gluthione-S-transferase (GST ) have been compared in freshly isolated rat hepatocytes, in a rat hepatoma cell line (HTC)and in the SV40RH 1 cells (passages 5-9) as indices of differentiated parenchymal functions. Sprague-Dawley rat hepatocytes (viability > 85%) were prepared as described previously [2]. HTC cells were grown in Dulbecco's medium plus 10% fetal calf serum (v/v). SV40RH1 cells were grown on hydrated collagen gels in a hormonally-defined medium described by lsom & Georgoff [ 3 ] ,and all experiments were carried out on confluent cultures. yGT and GST activities were measured in cell homogenates prepared by sonication in 0.1 M-sodium phosphate buffer, pH 7.6. GST activities were measured in the presence of I mM reduced glutathione (GSH) using either 50 ,UM l-chloro-2,4-dinitrobenzene(CDNB) or 0.2 mM ethacrynic acid (EA) as substrates [4]. yGT activity was measured according to Sigma Technical Bulletin No. 418. GSH synthesis was assessed in the presence of either 0.5 mM-Lmethionine or 0.5 mu-L-cysteine essentially as described by Duthie et a/. [ 5 ] . In certain experiments 5 mM-buthionine sulphoximine (BSO)was present as an inhibitor of GSH synthesis. GSH was quantified by the method of Hissin & Hilf [6]and protein as described by Lowry et al. [7]. Fig. I shows the resynthesis of GSH in the presence of ( a ) L-cysteine and ( b )L-methionine. Hepatocytes are one of only a few cell types that can synthesize GSH equally well from Lmethionine or L-cysteine [8], and the SV40RH1 cells have retained this differentiated hepatic function. In contrast, HTC cells, in common with other de-differentiated tumour Abbreviations used: yGT, y-glutamyltransferase; GST, glutathione-S-transferase: HTC, rat hepatoma cells; GSH, reduced glutathione; CDBN, I -chloro-2,4-dinitrobenzene; EA, ethacrynic acid; BSO, buthionine sulphoximine.
VOl. 18
0
50
100
150
Time (min)
0
50
100
150
Time (min)
Fig. 1. GSH resynthesis In the presence of ( u ) L-cysteine and ( h )L-methionine. The cells used were: ( 0 )HTC; (+)rat hepatocytes; and ( m ) SV40 RHI cells.
cell lines [5], have a reduced GSH synthetic ability and do not contain an efficient cystathione pathway to utilize L-methionine for GSH synthesis. In all three cell types the L-cysteine-stimulated GSH synthesis was completely prevented by inclusion of BSO in the incubations (results not shown). The yGT activity of freshly isolated hepatocytes and SV40RH1 cells was too low to be detected whereas HTC cells contained 5.2 nmol/min per mg of protein ( n = 3). yGT is an inducible enzyme, the activity of which is low in normal liver tissue, but rises in liver disease and in de-differentiated hepatoma tissue [9].CDNB is a general substrate conjugated by most isoenzymes of rat liver GST 141, and the activity of GST towards this substrate was similar in freshly isolated rat hepatocytes (5.25 nmol/min per mg of protein) SV40RH 1 cells (4.75 nmol/min per mg of protein) and HTC cells (4.31
BIOCHEMICAL SOCIETY TRANSACTIONS
of S-oxidation appears to be removed by alternative substrates for FMO. However, at rug Metah. 11, 2 17-265
I20 I 3. Reese,J. A. & Byard, J. L. (198 I ) In Vitro 17,935-940 4. Strom, S. C., Jirtle, R. L., Jones, R. S., Novicki, D. L., Rosenberg, M. R..Novotny, A., Irons, G., McLain, J. R. tk Michalopoulos, G . (1982)J. Natl. CancerInst. (U.S.) 6 8 , 771-775 5 . Gerding, T. K., Drenth, B. F. H., de Zeeuw, R. A,, Tepper, P. G . & Horn, A. S. ( 1990)Xmohiotica 20,525-536
Received 30 May 1990
Maintenance of differentiated function in cultured rat hepatocytes immortalized by transfection with viral DNA ANDREW NAIRN, BRIAN WILLETT, M. HELEN GRANT, ALEXANDER SCOTT and CAROLINE MAcDONALD Department of Biosrience and Biotechnology, Strathrlyde Universiry, The Todd Centre, 31 Taylor Street, Glasgow G4 ONR, U.K. Primary cultures of rat hepatocytes have been widely used for studying xenobiotic metabolism and toxicity in vitro. However, their use is limited because hepatocytes d o not proliferate in culture and the cells undergo rapid de-differentiation losing many parenchymal functions within the first 72 h [ 1 I. We have recently transfected rat hepatocytes with SV40 viral DNA to produce an immortal hepatocyte cell line (SV40RH1) and this study assesses the extent to which these cells have retained differentiated hepatic functions. The presence of the cystathione pathway for glutathione synthesis, and the activities of y-glutamyltransferase ( yGT) and gluthione-S-transferase (GST ) have been compared in freshly isolated rat hepatocytes, in a rat hepatoma cell line (HTC)and in the SV40RH 1 cells (passages 5-9) as indices of differentiated parenchymal functions. Sprague-Dawley rat hepatocytes (viability > 85%) were prepared as described previously [2]. HTC cells were grown in Dulbecco's medium plus 10% fetal calf serum (v/v). SV40RH1 cells were grown on hydrated collagen gels in a hormonally-defined medium described by lsom & Georgoff [ 3 ] ,and all experiments were carried out on confluent cultures. yGT and GST activities were measured in cell homogenates prepared by sonication in 0.1 M-sodium phosphate buffer, pH 7.6. GST activities were measured in the presence of I mM reduced glutathione (GSH) using either 50 ,UM l-chloro-2,4-dinitrobenzene(CDNB) or 0.2 mM ethacrynic acid (EA) as substrates [4]. yGT activity was measured according to Sigma Technical Bulletin No. 418. GSH synthesis was assessed in the presence of either 0.5 mM-Lmethionine or 0.5 mu-L-cysteine essentially as described by Duthie et a/. [ 5 ] . In certain experiments 5 mM-buthionine sulphoximine (BSO)was present as an inhibitor of GSH synthesis. GSH was quantified by the method of Hissin & Hilf [6]and protein as described by Lowry et al. [7]. Fig. I shows the resynthesis of GSH in the presence of ( a ) L-cysteine and ( b )L-methionine. Hepatocytes are one of only a few cell types that can synthesize GSH equally well from Lmethionine or L-cysteine [8], and the SV40RH1 cells have retained this differentiated hepatic function. In contrast, HTC cells, in common with other de-differentiated tumour Abbreviations used: yGT, y-glutamyltransferase; GST, glutathione-S-transferase: HTC, rat hepatoma cells; GSH, reduced glutathione; CDBN, I -chloro-2,4-dinitrobenzene; EA, ethacrynic acid; BSO, buthionine sulphoximine.
VOl. 18
0
50
100
150
Time (min)
0
50
100
150
Time (min)
Fig. 1. GSH resynthesis In the presence of ( u ) L-cysteine and ( h )L-methionine. The cells used were: ( 0 )HTC; (+)rat hepatocytes; and ( m ) SV40 RHI cells.
cell lines [5], have a reduced GSH synthetic ability and do not contain an efficient cystathione pathway to utilize L-methionine for GSH synthesis. In all three cell types the L-cysteine-stimulated GSH synthesis was completely prevented by inclusion of BSO in the incubations (results not shown). The yGT activity of freshly isolated hepatocytes and SV40RH1 cells was too low to be detected whereas HTC cells contained 5.2 nmol/min per mg of protein ( n = 3). yGT is an inducible enzyme, the activity of which is low in normal liver tissue, but rises in liver disease and in de-differentiated hepatoma tissue [9].CDNB is a general substrate conjugated by most isoenzymes of rat liver GST 141, and the activity of GST towards this substrate was similar in freshly isolated rat hepatocytes (5.25 nmol/min per mg of protein) SV40RH 1 cells (4.75 nmol/min per mg of protein) and HTC cells (4.31
