Mutation Research, 244 (1990) 157-161
157
Elsevier
MUTLET 0354
High sensitivity of Chinese hamster epithelial liver cells to toxic analogues of purines M. Simili, P. Tartaglia and G. Turchi Institute of Mutagenesis and Differentiation, CNR, 1-56100 Pisa (Italy) (Accepted 29 December 1989)
Keywords." Chinese hamster epithelial liver cells; Resistance to purine analogues; Enzyme activities
Summary The resistance of Chinese hamster epithelial liver cells (CHEL) and Chinese hamster fibroblasts (V79) towards toxic purine analogues has been determined. The liver ceils are more sensitive than fibroblasts to 6-thioguanine (6-TG), 8-azaguanine (8-AZ) and 2,6-diaminopurine (DAP). The hypoxanthine-guanine (HGPRT) and adenine phosphoribosyl transferase (APRT) activities of extracts of CHEL cells were lower than those of corresponding extracts of V79. The level of 5'-nucleotidase was about 5-fold higher in the epithelial cells. It appears that HGPRT and APRT activities of extracts of liver epithelial cells are masked or reduced by 5 '-nucleotidase activity and other inhibitors. The significance of these findings is discussed.
The liver constitutes the major organ for detoxification of xenobiotics in animals (Gillette and Jollow, 1974; Thurman et al., 1979). However the reactions which degrade foreign compounds often lead to the production of 'active intermediates', which can be effective toxic substances or mutagens and/or carcinogens (Miller and Miller, 1977). It has been common in the past to incubate test substances for toxicity with a microsomal $9 fraction from rat liver to obtain metabolic intermediates before screening these compounds for biological activity on indicator lines (Bradley et al., 1981; Bartsch et al., 1982). The availability of cell
Correspondence: Dr. M. Simili, Institute of Mutagenesis and Differentiation, CNR, Via Svezia, 10, 1-56100 Pisa (Italy).
lines of hepatic origin raises the possibility of single-step screening for mutagens and/or carcinogens in which liver-derived cells would both metabolize the test compound and act as its target, i.e., the metabolizing cells would be the target of the ultimate mutagen/carcinogen. Moreover, as the vast majority of human tumors occur in cells of epithelial or lymphoblastic origin (Cairns, 1975), epithelial liver cells could possibly offer a more representative target than the commonly used fibroblasts. To this end we have punt into culture epithelial liver cells from Chinese hamster (CHEL) which do not appear to senesce, have a nearly diploid chromosome number, retain numerous enzymatic activities of liver cells and the capacity to activate promutagens (Turchi et al., 1987). Since this cell
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
158 system could be used as a single-step screen for both direct and indirect mutagens, resistance to toxic analogues of purines was investigated.
Materials and methods
TABLE 1 CYTOTOXICITY OF PURINE ANALOGUES TO CHEL AND V79 CELLS CHEL V79
6-TG
8-AZ
DAP
0.02 #M 0.68 ~M
2.3 #M 5.4 ttM
5.3 #M 9.9 #M
Cell culture Chinese hamster liver epithelial cells (CHEL), obtained as described by Turchi et al. (1987), were grown as monolayers in Williams medium (Flow) containing 10070 fetal calf serum (FCS, Flow). A Chinese hamster fibroblastoid cell line (V79), used extensively in mutagenic studies, was obtained from Dr. C.F. Arlett. This line was grown as monolayers, in Dulbecco's modified Eagle's medium (DMEM, Flow) containing 5°70 FCS. Both cell lines were grown at 37°C in at atmosphere of 5070 CO2. Resistance to 8-azaguanine (8-AZ), 6-thioguanine (6-TG) and 2,6-diaminopurine (DAP) was determined as previously described (Colella et al., 1983).
Enzymatic activity Hypoxanthine-guanine (HGPRT) and adenine phosphoribosyl transferase (APRT) activities were determined in cell extracts according to the procedure of Simili et al. (1983) which consists briefly of a preferential desorption of hypoxanthine or adenine relative to the corresponding nucleotides from polyethyleneimine-cellulose ion exchanger. 5' -Nucleotidase was assayed by the procedure of Arkesteijn (1976). Enzymatic activity was monitored with a Perkin Elmer 570 spectrophotometer by following the change of absorbance at 340 nm for 2 min. Protein concentrations were determined by the method of Lowry et al. (1951) with bovine serum albumin (Sigma) as standard.
The cytotoxic effect of 6-thioguanine (6-TG), 8-azaguanine (8-AZ) and 2,6-diaminopurine (DAP) to CHEL and V79 cells is expressed as LDs0, i.e., the dose required to kill 50% of the cells.
be toxic, although the exact mechanisms of toxicity are unknown. When H G P R T or A P R T activities are reduced or eliminated, cells become more resistant to the toxic analogues of purines. Chinese hamster epithelial liver cells were assayed for suitability in the mutagenic assay by measuring the sensitivity to 8-AZ, 6-TG and D A P of this cell line and of the reporter cell line V79. As seen in Table 1, C H E L cells were more sensitive to these compounds than the fibroblasts. While they are twice as sensitive towards the toxic effect of 8-AZ and D A P , C H E L cells are about 30 times more sensitive to 6-TG than V79.
0.5
o
0.~
-6 E ,--
0.3
13_
-~
0.2
0.1
I0
20
30
Results Toxic purine analogues such as 8-AZ, 6-TG and D A P have been widely used as selective agents in mutagenic studies. The drugs are metabolized by H G P R T and A P R T to the corresponding nucleoside monophosphates which are supposed to
t (rain) Fig. 1. HGPRT activity was determined as described in Materials and methods. The protein concentration in the assay was 0.5 mg/ml, the total volumeof the incubation mixture was 50/~1, aliquots of 10 #1were taken every 10 rain. On the ordinate the absolute amount in nmole of IMP formed in 10 ml is reported. • V79 HGPRT; • CHEL HGPRT; [] CHEL HGPRT in the presence of TTP (1 mM).
159 T h e H G P R T / A P R T activities o f the 2 cell lines were d e t e r m i n e d to see if the relative sensitivities to t o x i c p u r i n e a n a l o g u e s were due to differences in t h e activities o f p u r i n e salvage e n z y m e s . F r o m Fig. 1 it c a n be seen t h a t H G P R T activity was m u c h l o w e r in extracts o f C H E L cells t h a n in extracts o f V79 cells. I n the case o f C H E L extracts d i m i n i s h i n g t h e c o n c e n t r a t i o n o f extract in the assay resulted in h i g h e r a p p a r e n t specific activity. T h e p h e n o m e n o n was less m a r k e d with V79 extracts ( T a b l e 2). This t y p e o f b e h a v i o r is suggestive o f the presence o f inh i b i t o r s o f H G P R T activity in the extracts. A s the i n h i b i t o r is d i l u t e d so the a p p a r e n t specific activity o f t h e extracts increases. Since b o t h the H G P R T a n d A P R T assays d e p e n d o n the c o n v e r s i o n o f a l a b e l l e d base to a n u c l e o t i d e which can, b y w a y o f t h e i n c r e a s e d net charge, be r e t a i n e d o n an ione x c h a n g e r u n d e r c o n d i t i o n s which r e m o v e the unc h a r g e d p r e c u r s o r , it was possible t h a t 5 ' - n u c l e o t i d a s e activity c o u l d interfere with b o t h assays. N u c l e o t i d e t r i p h o s p h a t e s are k n o w n i n h i b i t o r s o f 5 ' - n u c l e o t i d a s e activity a n d the effect o f a d d i t i o n o f 1 m M t h y m i d i n e t r i p h o s p h a t e ( T T P ) to the e n z y m e assays was i n v e s t i g a t e d ( B e r m a n et al., 1980). It was f o u n d t h a t a d d i t i o n o f T T P enhances the H G P R T activity in C H E L extracts (Fig. 1) a l t h o u g h the activity d i d n o t reach the level o b s e r v ed in extracts o f V79 cells. S o m e w h a t d i f f e r e n t results were o b t a i n e d w h e n A P R T activity was a s s a y e d (Fig. 2). In this case A P R T activity in C H E L extracts was a b o u t h a l f o f t h a t f o u n d in V79 a n d t h e effect o f T T P was less p r o n o u n c e d in the
TABLE 2
0.5 0.~ E
0.3
13_
0.2 0.1
0
10
20
30
t (rnin)
Fig. 2. APRT activity was determined as described in Materials and methods. The protein concentration in the assay was 0.5 mg/ml, the total volume of the incubation mixture was 50/~1, the amount of AMP formed (in nmole) in 10/~1 of incubation mixture is reported on the ordinate. • V79 APRT; • CHEL APRT; ta CHEL APRT in the presence of TTP (1 mM). A P R T a s s a y t h a n in the H G P R T assay. O n l y a slight increase in the specific activity o f A P R T was o b s e r v e d b y d i m i n i s h i n g the c o n c e n t r a t i o n o f p r o tein in the a s s a y ( T a b l e 2). A d d i t i o n o f 1 m M T T P d i d n o t e n h a n c e H G P R T o r A P R T activity in V79 extracts ( d a t a n o t shown). W e t h e n d e t e r m i n e d 5 ' - n u c l e o t i d a s e activity in C H E L a n d V79 cell extracts. T r i t o n X-100 was u s e d to solubilize the cell m e m b r a n e , so t h a t b o t h the soluble and membrane-associated 5'-nucleot i d a s e c o u l d be d e t e r m i n e d . T h e results in T a b l e 3 s h o w t h a t the 5 ' - n u c l e o t i d a s e activity f o u n d in
TABLE 3
HGPRT'AND APRT ACTIVITIES OF V79 AND CHEL EXTRACTS Protein concen-
HGPRT
tration (mg/ml)
CHEL
V79
APRT CHEL
V79
0.5 0.25 0.125
4 10 35
42 48 55
15 20 32
15 20 22
HGPRT and APRT activities were determined as described in Materials and methods varying the amount of extract present in the assay. The values reported represent ttrnole of IMP or AMP formed per mg of protein after 10 min of incubation at 30°C.
5'-NUCLEOTIDASE ACTIVITY IN CHEL AND V79 EXTRACTS Cells
mU/mg DS
CHEL 7th passage CHEL 20th passage V79
11.44 + 1.34 13.89 + 5.02 1.95 + 0.86
5'-Nucleotidase activity was determined as described in Materials and methods. The values reported represent the average of 3 experiments and are expressed in mU per mg protein present in the assay. One unit corresponds to the amount of enzyme able to convert 1 t~moleof substrate per min.
160
C H E L extracts is more than 5 times higher than in V79, this relatively high 5'-nucleotidase activity seems to be independent of the cell passage number. Discussion Since liver epithelial cells could provide a useful single-step screen for mutagens/carcinogens requiring metabolic activation, such a line has been characterized for response towards toxic purine analogues, selective agents commonly used in mutagenic studies. Chinese hamster liver cells (CHEL) were found to be more sensitive to toxic purine analogues than the Chinese hamster fibroblast line V79. The C H E L cells are about 30 times as sensitive towards the toxic effect of 6-TG as V79 cells, and twice as sensitive towards 8-AZ and DAP. Historically sensitivity towards purine analogues has been found to be inversely related with the level o f H G P R T and A P R T activities; however, the activities of these 2 enzymes in C H E L extracts were lower than the activities found in V79 extracts. In standard assays we observed that the apparent specific activity of H G P R T and to a lesser extent A P R T activity of C H E L cells were inversely related with the amount of extract present in the assay. The inhibitory effect could be partially reversed by inclusion of TTP, suggesting the presence of 5'-nucleotidase activity. This assumption was confirmed as C H E L extracts show about 5-fold higher nucleotidase activity. However, the inclusion of the nucleotide triphosphate in the reaction mixture did not completely suppress the inhibition suggesting that factors other than 5-nucleotidase may be involved. The presence of high levels of 5'-nucleotidase in extracts of cells has previously been claimed to be associated with cellular resistance to toxic analogues of purines (Berman and Tong, 1980). In the case of the C H E L cells, the high nucleotidase activity observed does not confer drug resistance and indeed none of the enzymatic activities measured accounts for the relatively high sensitivity of these cells to toxic purine analogues. It can only be
postulated that the enzyme activities measured in extracts do not accurately reflect the situation in vivo, or that processes such as drug uptake are involved. The lack of any simple correlation between drug sensitivity and H G P R T , A P R T and 5'-nucleotidase activity in C H E L raises the possibility that purine metabolism may be regulated by different mechanisms in these and V79 cells, as suggested by the presence of uncharacterized inhibitors of H G P R T and A P R T activity in extracts o f liver cells. If such inhibitors have a physiological role it is possible that drug resistance could be conferred on liver cells by mutation at more than one locus. Preliminary experiments indicate that the C H E L cells can be used to screen for promutagens and mutagens using toxic purine analogues as selective agents. A number of resistant clones have been isolated and analysis of these lines should establish the locus or loci involved in drug resistance in liver cells and may thus show the extent to which regulations of purine metabolism in fibroblasts and liver epithelial cells differ.
References Arkesteijn, C.L.M. (1976) A kinetic method for serum 5-nucleotidase using stabilized glutamate dehydrogenase, J. Clin. Chem. Clin. Biochem., 14, 155-158. Bartsch, H., T. Kuroki, M. Roberfroid and C. Malaveille (1982) Metabolic activation systems in vitro for carcinogen/mutagen screening test, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Vol. 7, Plenum Press, New York, London, pp. 95-148. Berman, J.J., C. Tong and G.M. Williams (1980a) Differences between liver epithelial and fibroblast cells in metabolism of purines, J. Cell. Physiol., 103, 393-398. Berman, J.J., C. Tong and G.M. Williams (1980b) 5 '-Nucleotidase activities in cultured rat liver epithelial and fibroblast cells, J. Histochem. Cytochem., 28, 174-177. Bradley, M.O., B. Bhuyan, M.C. Francis, R. Longenbach, A. Peterson and E. Huberman (1981) Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis of the literature, Mutation Res., 87, 81-142. Cairns, J. (1985) Mutation selection and the natural history of cancer, Nature (London), 255, 197-200. Colella, C.M., G. Rainaldi and A. Piras (1983) 8-Azaguanine versus 6-thioguanine: influence on frequency and expression time of induced HGPRT - mutations in Chinese hamster V79 cells, Mutation Res., 107, 397-411.
161 Gillette, J.R., and D.J. J ollow (1974) Drug metabolism in liver, in: E. Becher (Ed.), The Liver, Normal and Abnormal Functions, Marcel Dekker, New York, pp. 165-183. Lowry, O.H., M.J. Rosebrough, A.L. Far and R.J. Randall (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193,265-275. Miller, J.A., and E.C. Miller (1977) Ultimate chemical carcinogens as reactive mutagenic electrophiles, in: H.H. Hiatt, J.D. Watson and J.A. Winsten (Eds.), Origins of Human Cancer, Book C, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 605-628. Simili, M., C.M. Colella, M. Debatisse and G. Buttin (1983) InCreased inhibition of HGPRT by IMP and GMP and higher levels of PRPP in an 8-azaguanine HAT resistant mutant of Chinese hamster cells, Cell Biol. Int. Rep., 7, 121-128.
Thurman, R.G., L.A. Reinke and F.C. Kauffman (1979) The isolated perfused liver: a model to define biochemical mechanisms of chemical toxicity, in: E. Hodgson, J.R. Bend and R.M. Philpot (Eds.), Reviews in Biochemical Toxicology, Elsevier/North Holland, New York, Amsterdam, Oxford, pp. 249-286. Turchi, G., M.A. Carluccio, F. Oesch, I. Gemperleim and H.R. Glatt (1987) Characterization of an epithelial, nearly diploid liver cell strain, from Chinese hamster, able to activate promutagens, Mutagenesis, 2, 127-135. Communicated by A. Abbondandolo
157
Elsevier
MUTLET 0354
High sensitivity of Chinese hamster epithelial liver cells to toxic analogues of purines M. Simili, P. Tartaglia and G. Turchi Institute of Mutagenesis and Differentiation, CNR, 1-56100 Pisa (Italy) (Accepted 29 December 1989)
Keywords." Chinese hamster epithelial liver cells; Resistance to purine analogues; Enzyme activities
Summary The resistance of Chinese hamster epithelial liver cells (CHEL) and Chinese hamster fibroblasts (V79) towards toxic purine analogues has been determined. The liver ceils are more sensitive than fibroblasts to 6-thioguanine (6-TG), 8-azaguanine (8-AZ) and 2,6-diaminopurine (DAP). The hypoxanthine-guanine (HGPRT) and adenine phosphoribosyl transferase (APRT) activities of extracts of CHEL cells were lower than those of corresponding extracts of V79. The level of 5'-nucleotidase was about 5-fold higher in the epithelial cells. It appears that HGPRT and APRT activities of extracts of liver epithelial cells are masked or reduced by 5 '-nucleotidase activity and other inhibitors. The significance of these findings is discussed.
The liver constitutes the major organ for detoxification of xenobiotics in animals (Gillette and Jollow, 1974; Thurman et al., 1979). However the reactions which degrade foreign compounds often lead to the production of 'active intermediates', which can be effective toxic substances or mutagens and/or carcinogens (Miller and Miller, 1977). It has been common in the past to incubate test substances for toxicity with a microsomal $9 fraction from rat liver to obtain metabolic intermediates before screening these compounds for biological activity on indicator lines (Bradley et al., 1981; Bartsch et al., 1982). The availability of cell
Correspondence: Dr. M. Simili, Institute of Mutagenesis and Differentiation, CNR, Via Svezia, 10, 1-56100 Pisa (Italy).
lines of hepatic origin raises the possibility of single-step screening for mutagens and/or carcinogens in which liver-derived cells would both metabolize the test compound and act as its target, i.e., the metabolizing cells would be the target of the ultimate mutagen/carcinogen. Moreover, as the vast majority of human tumors occur in cells of epithelial or lymphoblastic origin (Cairns, 1975), epithelial liver cells could possibly offer a more representative target than the commonly used fibroblasts. To this end we have punt into culture epithelial liver cells from Chinese hamster (CHEL) which do not appear to senesce, have a nearly diploid chromosome number, retain numerous enzymatic activities of liver cells and the capacity to activate promutagens (Turchi et al., 1987). Since this cell
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
158 system could be used as a single-step screen for both direct and indirect mutagens, resistance to toxic analogues of purines was investigated.
Materials and methods
TABLE 1 CYTOTOXICITY OF PURINE ANALOGUES TO CHEL AND V79 CELLS CHEL V79
6-TG
8-AZ
DAP
0.02 #M 0.68 ~M
2.3 #M 5.4 ttM
5.3 #M 9.9 #M
Cell culture Chinese hamster liver epithelial cells (CHEL), obtained as described by Turchi et al. (1987), were grown as monolayers in Williams medium (Flow) containing 10070 fetal calf serum (FCS, Flow). A Chinese hamster fibroblastoid cell line (V79), used extensively in mutagenic studies, was obtained from Dr. C.F. Arlett. This line was grown as monolayers, in Dulbecco's modified Eagle's medium (DMEM, Flow) containing 5°70 FCS. Both cell lines were grown at 37°C in at atmosphere of 5070 CO2. Resistance to 8-azaguanine (8-AZ), 6-thioguanine (6-TG) and 2,6-diaminopurine (DAP) was determined as previously described (Colella et al., 1983).
Enzymatic activity Hypoxanthine-guanine (HGPRT) and adenine phosphoribosyl transferase (APRT) activities were determined in cell extracts according to the procedure of Simili et al. (1983) which consists briefly of a preferential desorption of hypoxanthine or adenine relative to the corresponding nucleotides from polyethyleneimine-cellulose ion exchanger. 5' -Nucleotidase was assayed by the procedure of Arkesteijn (1976). Enzymatic activity was monitored with a Perkin Elmer 570 spectrophotometer by following the change of absorbance at 340 nm for 2 min. Protein concentrations were determined by the method of Lowry et al. (1951) with bovine serum albumin (Sigma) as standard.
The cytotoxic effect of 6-thioguanine (6-TG), 8-azaguanine (8-AZ) and 2,6-diaminopurine (DAP) to CHEL and V79 cells is expressed as LDs0, i.e., the dose required to kill 50% of the cells.
be toxic, although the exact mechanisms of toxicity are unknown. When H G P R T or A P R T activities are reduced or eliminated, cells become more resistant to the toxic analogues of purines. Chinese hamster epithelial liver cells were assayed for suitability in the mutagenic assay by measuring the sensitivity to 8-AZ, 6-TG and D A P of this cell line and of the reporter cell line V79. As seen in Table 1, C H E L cells were more sensitive to these compounds than the fibroblasts. While they are twice as sensitive towards the toxic effect of 8-AZ and D A P , C H E L cells are about 30 times more sensitive to 6-TG than V79.
0.5
o
0.~
-6 E ,--
0.3
13_
-~
0.2
0.1
I0
20
30
Results Toxic purine analogues such as 8-AZ, 6-TG and D A P have been widely used as selective agents in mutagenic studies. The drugs are metabolized by H G P R T and A P R T to the corresponding nucleoside monophosphates which are supposed to
t (rain) Fig. 1. HGPRT activity was determined as described in Materials and methods. The protein concentration in the assay was 0.5 mg/ml, the total volumeof the incubation mixture was 50/~1, aliquots of 10 #1were taken every 10 rain. On the ordinate the absolute amount in nmole of IMP formed in 10 ml is reported. • V79 HGPRT; • CHEL HGPRT; [] CHEL HGPRT in the presence of TTP (1 mM).
159 T h e H G P R T / A P R T activities o f the 2 cell lines were d e t e r m i n e d to see if the relative sensitivities to t o x i c p u r i n e a n a l o g u e s were due to differences in t h e activities o f p u r i n e salvage e n z y m e s . F r o m Fig. 1 it c a n be seen t h a t H G P R T activity was m u c h l o w e r in extracts o f C H E L cells t h a n in extracts o f V79 cells. I n the case o f C H E L extracts d i m i n i s h i n g t h e c o n c e n t r a t i o n o f extract in the assay resulted in h i g h e r a p p a r e n t specific activity. T h e p h e n o m e n o n was less m a r k e d with V79 extracts ( T a b l e 2). This t y p e o f b e h a v i o r is suggestive o f the presence o f inh i b i t o r s o f H G P R T activity in the extracts. A s the i n h i b i t o r is d i l u t e d so the a p p a r e n t specific activity o f t h e extracts increases. Since b o t h the H G P R T a n d A P R T assays d e p e n d o n the c o n v e r s i o n o f a l a b e l l e d base to a n u c l e o t i d e which can, b y w a y o f t h e i n c r e a s e d net charge, be r e t a i n e d o n an ione x c h a n g e r u n d e r c o n d i t i o n s which r e m o v e the unc h a r g e d p r e c u r s o r , it was possible t h a t 5 ' - n u c l e o t i d a s e activity c o u l d interfere with b o t h assays. N u c l e o t i d e t r i p h o s p h a t e s are k n o w n i n h i b i t o r s o f 5 ' - n u c l e o t i d a s e activity a n d the effect o f a d d i t i o n o f 1 m M t h y m i d i n e t r i p h o s p h a t e ( T T P ) to the e n z y m e assays was i n v e s t i g a t e d ( B e r m a n et al., 1980). It was f o u n d t h a t a d d i t i o n o f T T P enhances the H G P R T activity in C H E L extracts (Fig. 1) a l t h o u g h the activity d i d n o t reach the level o b s e r v ed in extracts o f V79 cells. S o m e w h a t d i f f e r e n t results were o b t a i n e d w h e n A P R T activity was a s s a y e d (Fig. 2). In this case A P R T activity in C H E L extracts was a b o u t h a l f o f t h a t f o u n d in V79 a n d t h e effect o f T T P was less p r o n o u n c e d in the
TABLE 2
0.5 0.~ E
0.3
13_
0.2 0.1
0
10
20
30
t (rnin)
Fig. 2. APRT activity was determined as described in Materials and methods. The protein concentration in the assay was 0.5 mg/ml, the total volume of the incubation mixture was 50/~1, the amount of AMP formed (in nmole) in 10/~1 of incubation mixture is reported on the ordinate. • V79 APRT; • CHEL APRT; ta CHEL APRT in the presence of TTP (1 mM). A P R T a s s a y t h a n in the H G P R T assay. O n l y a slight increase in the specific activity o f A P R T was o b s e r v e d b y d i m i n i s h i n g the c o n c e n t r a t i o n o f p r o tein in the a s s a y ( T a b l e 2). A d d i t i o n o f 1 m M T T P d i d n o t e n h a n c e H G P R T o r A P R T activity in V79 extracts ( d a t a n o t shown). W e t h e n d e t e r m i n e d 5 ' - n u c l e o t i d a s e activity in C H E L a n d V79 cell extracts. T r i t o n X-100 was u s e d to solubilize the cell m e m b r a n e , so t h a t b o t h the soluble and membrane-associated 5'-nucleot i d a s e c o u l d be d e t e r m i n e d . T h e results in T a b l e 3 s h o w t h a t the 5 ' - n u c l e o t i d a s e activity f o u n d in
TABLE 3
HGPRT'AND APRT ACTIVITIES OF V79 AND CHEL EXTRACTS Protein concen-
HGPRT
tration (mg/ml)
CHEL
V79
APRT CHEL
V79
0.5 0.25 0.125
4 10 35
42 48 55
15 20 32
15 20 22
HGPRT and APRT activities were determined as described in Materials and methods varying the amount of extract present in the assay. The values reported represent ttrnole of IMP or AMP formed per mg of protein after 10 min of incubation at 30°C.
5'-NUCLEOTIDASE ACTIVITY IN CHEL AND V79 EXTRACTS Cells
mU/mg DS
CHEL 7th passage CHEL 20th passage V79
11.44 + 1.34 13.89 + 5.02 1.95 + 0.86
5'-Nucleotidase activity was determined as described in Materials and methods. The values reported represent the average of 3 experiments and are expressed in mU per mg protein present in the assay. One unit corresponds to the amount of enzyme able to convert 1 t~moleof substrate per min.
160
C H E L extracts is more than 5 times higher than in V79, this relatively high 5'-nucleotidase activity seems to be independent of the cell passage number. Discussion Since liver epithelial cells could provide a useful single-step screen for mutagens/carcinogens requiring metabolic activation, such a line has been characterized for response towards toxic purine analogues, selective agents commonly used in mutagenic studies. Chinese hamster liver cells (CHEL) were found to be more sensitive to toxic purine analogues than the Chinese hamster fibroblast line V79. The C H E L cells are about 30 times as sensitive towards the toxic effect of 6-TG as V79 cells, and twice as sensitive towards 8-AZ and DAP. Historically sensitivity towards purine analogues has been found to be inversely related with the level o f H G P R T and A P R T activities; however, the activities of these 2 enzymes in C H E L extracts were lower than the activities found in V79 extracts. In standard assays we observed that the apparent specific activity of H G P R T and to a lesser extent A P R T activity of C H E L cells were inversely related with the amount of extract present in the assay. The inhibitory effect could be partially reversed by inclusion of TTP, suggesting the presence of 5'-nucleotidase activity. This assumption was confirmed as C H E L extracts show about 5-fold higher nucleotidase activity. However, the inclusion of the nucleotide triphosphate in the reaction mixture did not completely suppress the inhibition suggesting that factors other than 5-nucleotidase may be involved. The presence of high levels of 5'-nucleotidase in extracts of cells has previously been claimed to be associated with cellular resistance to toxic analogues of purines (Berman and Tong, 1980). In the case of the C H E L cells, the high nucleotidase activity observed does not confer drug resistance and indeed none of the enzymatic activities measured accounts for the relatively high sensitivity of these cells to toxic purine analogues. It can only be
postulated that the enzyme activities measured in extracts do not accurately reflect the situation in vivo, or that processes such as drug uptake are involved. The lack of any simple correlation between drug sensitivity and H G P R T , A P R T and 5'-nucleotidase activity in C H E L raises the possibility that purine metabolism may be regulated by different mechanisms in these and V79 cells, as suggested by the presence of uncharacterized inhibitors of H G P R T and A P R T activity in extracts o f liver cells. If such inhibitors have a physiological role it is possible that drug resistance could be conferred on liver cells by mutation at more than one locus. Preliminary experiments indicate that the C H E L cells can be used to screen for promutagens and mutagens using toxic purine analogues as selective agents. A number of resistant clones have been isolated and analysis of these lines should establish the locus or loci involved in drug resistance in liver cells and may thus show the extent to which regulations of purine metabolism in fibroblasts and liver epithelial cells differ.
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