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Conjugation reactions in primary cultures of rat hepatocytes
Fd Chem, Toxic. Vol. 24, No, 6/7, p. 758, 1986 Printed in Great Britain
0278-6915/86 $3.00 + 0.00 Pergamon Journals Ltd
CONJUGATION REACTIONS IN PRIMARY CULTURES OF RAT HEPATOCYTES M. H. GRANT a n d G. M. HAWKSWORTH Department of Therapeutics and Clinical Pharmacology, University Medical Buildings, ForesterhiU, Aberdeen AB9 2ZD, Scotland
T h e use o f p r i m a r y cultures of hepatocytes for studies o f xenobiotic-induced toxicity requires the maintenance of b o t h drug c o n j u g a t i o n a n d c y t o c h r o m e P - 4 5 0 - m e d i a t e d mixed-function oxidation ( M F O ) . Very little is k n o w n a b o u t the stability o f c o n j u g a t i o n reactions d u r i n g culture, whereas M F O declines rapidly over the first 24-48 hours. We have m e a s u r e d the activity o f glucuronosyl transferase (GTase) a n d sulphotransferase (STase) in hepatocytes cultured from male S p r a g u e - D a w l e y rats using the m o d e l c o m p o u n d s 1-naphthol a n d phenolphthalein, which are respectively, substrates for a 3-methylcholanthrene-inducible a n d a p h e n o barb±tone-inducible isozyme of G T a s e ( M o r r i s o n & H a w k s w o r t h , 1984). In addition, the levels o f reduced glutathione ( G S H ) a n d G S H c o n j u g a t i o n o f 1-chloro-2,4-dinitrobenzene were measured. T h e hepatocytes were cultured o n collagen-coated Petri dishes using WiUiams's E m e d i u m with 5 % foetal calf serum. T a b l e 1 shows t h a t the activity of G T a s e decreased to a b o u t 50% o f fresh cell values by 24 h o u r s in culture. By 72 hours, however, the levels o f activity with b o t h substrates were twice the initial values. P h e n o l p h t h a l e i n was not sulphated to a significant extent even in freshly isolated cells, so s u l p h a t i o n was only quantified with 1-naphthol. The results show t h a t in c o n t r a s t to the activity of GTase, s u l p h a t i o n declined slowly t h r o u g h o u t the 72 h o u r s in culture. As a result o f these changes, the ratio o f
g l u c u r o n i d a t i o n to sulphation of l - n a p h t h o l increased f r o m 3.6 in fresh cells to 14.3 after 72 hours in culture. B o t h the c o n c e n t r a t i o n of G S H in the cells a n d the activity o f G S H transferase a p p e a r to be m a i n t a i n e d for at least 72 h o u r s in the cultured hepatocytes. T h e increase in G T a s e activity was prevented by the inclusion of 10-sM-cycloheximide, a proteinsynthesis inhibitor, in the m e d i u m , thus indicating t h a t the increase in G T a s e was due to protein synthesis. A t this c o n c e n t r a t i o n , cycloheximide did n o t cause a loss of cell viability a n d did n o t alter either the G S H or c y t o c h r o m e P-450 c o n t e n t o f the cells. T h e increase in G T a s e activity is t h o u g h t to reflect de-differentiation of the hepatocytes a n d develo p m e n t of a preneoplastic p a t t e r n of drugmetabolizing activities (Toribara, Kirkpatrick, Falany et al. 1984).
REFERENCES
Mort±son M. H. & Hawksworth G. M. (1984). Glucuronic acid conjugation by hepatic microsomal fractions isolated from streptozotocin-induced diabetic rats. Biochem. Pharmac. 33, 3833. Toribara N. W., Kirkpatrick R. B., Falany C. N., LaBrecque D. R. & Tephly T. R. (1984). Sequential changes in primary rat hepatocyte monolayer culture UDPglucuronyltransferase activities show a preneoplastic-like pattern. Hepatology 4, 1054.
Table 1. Maintenance of conjugation in primary cultures of rat hepatocytes maintained for up to 72 hours Conjugation activity [or concnl (% of initial Substrate fresh cell valuer) at: conch
Reaction* (#ra) 24 hr 48 hr 72 hr P-GTase 100 56 + 8 (6) 135 __.19 (6) 154 + 19 (8) N-GTase 100 48 + 4 (5) 124 ± 14 (4) 161 + 22 (7) N-STase 100 53 + 3 (3) 36 ± 2 (3) 28 ± 4 (4) GSH content -[85 ± 2] (3) [71 ± 9] (3) [80 + l 1] (3) GSH-CDNB 50 153 ± 25 (4) 161 + 26 (4) 118 ± 15 (4) P = Phenolphthalein N = l-Naphthol GSH = Reduced glutathione GTase = Glucuronosyl transferase STase= Sulphotransferase CDNB = l-chloro-2,4-dinitrobenzene *All of the conjugation reactions were carried out in intact cells. The incubation buffer was Krebs-Henseleit buffer, pH 7.4, containing 10mM-HEPES. tFresh cell values were: P-GT 0.41 + 0.04 nmol/mg cell protein/rnin; N-GT 1.08 ± 0.23 nmol/mg cell protcin/min; N - S T 0.34± 0.01 nmol/mg cell protein/min; GSH content 42.3 ± 2.4 nmol/mg cell protein; GSH-CDNB formation 1.1 ± 0.1 nmol/mg cell protein/rain. Results are means + SEM for the numbers of experiments indicated in brackets.
758
0278-6915/86 $3.00 + 0.00 Pergamon Journals Ltd
CONJUGATION REACTIONS IN PRIMARY CULTURES OF RAT HEPATOCYTES M. H. GRANT a n d G. M. HAWKSWORTH Department of Therapeutics and Clinical Pharmacology, University Medical Buildings, ForesterhiU, Aberdeen AB9 2ZD, Scotland
T h e use o f p r i m a r y cultures of hepatocytes for studies o f xenobiotic-induced toxicity requires the maintenance of b o t h drug c o n j u g a t i o n a n d c y t o c h r o m e P - 4 5 0 - m e d i a t e d mixed-function oxidation ( M F O ) . Very little is k n o w n a b o u t the stability o f c o n j u g a t i o n reactions d u r i n g culture, whereas M F O declines rapidly over the first 24-48 hours. We have m e a s u r e d the activity o f glucuronosyl transferase (GTase) a n d sulphotransferase (STase) in hepatocytes cultured from male S p r a g u e - D a w l e y rats using the m o d e l c o m p o u n d s 1-naphthol a n d phenolphthalein, which are respectively, substrates for a 3-methylcholanthrene-inducible a n d a p h e n o barb±tone-inducible isozyme of G T a s e ( M o r r i s o n & H a w k s w o r t h , 1984). In addition, the levels o f reduced glutathione ( G S H ) a n d G S H c o n j u g a t i o n o f 1-chloro-2,4-dinitrobenzene were measured. T h e hepatocytes were cultured o n collagen-coated Petri dishes using WiUiams's E m e d i u m with 5 % foetal calf serum. T a b l e 1 shows t h a t the activity of G T a s e decreased to a b o u t 50% o f fresh cell values by 24 h o u r s in culture. By 72 hours, however, the levels o f activity with b o t h substrates were twice the initial values. P h e n o l p h t h a l e i n was not sulphated to a significant extent even in freshly isolated cells, so s u l p h a t i o n was only quantified with 1-naphthol. The results show t h a t in c o n t r a s t to the activity of GTase, s u l p h a t i o n declined slowly t h r o u g h o u t the 72 h o u r s in culture. As a result o f these changes, the ratio o f
g l u c u r o n i d a t i o n to sulphation of l - n a p h t h o l increased f r o m 3.6 in fresh cells to 14.3 after 72 hours in culture. B o t h the c o n c e n t r a t i o n of G S H in the cells a n d the activity o f G S H transferase a p p e a r to be m a i n t a i n e d for at least 72 h o u r s in the cultured hepatocytes. T h e increase in G T a s e activity was prevented by the inclusion of 10-sM-cycloheximide, a proteinsynthesis inhibitor, in the m e d i u m , thus indicating t h a t the increase in G T a s e was due to protein synthesis. A t this c o n c e n t r a t i o n , cycloheximide did n o t cause a loss of cell viability a n d did n o t alter either the G S H or c y t o c h r o m e P-450 c o n t e n t o f the cells. T h e increase in G T a s e activity is t h o u g h t to reflect de-differentiation of the hepatocytes a n d develo p m e n t of a preneoplastic p a t t e r n of drugmetabolizing activities (Toribara, Kirkpatrick, Falany et al. 1984).
REFERENCES
Mort±son M. H. & Hawksworth G. M. (1984). Glucuronic acid conjugation by hepatic microsomal fractions isolated from streptozotocin-induced diabetic rats. Biochem. Pharmac. 33, 3833. Toribara N. W., Kirkpatrick R. B., Falany C. N., LaBrecque D. R. & Tephly T. R. (1984). Sequential changes in primary rat hepatocyte monolayer culture UDPglucuronyltransferase activities show a preneoplastic-like pattern. Hepatology 4, 1054.
Table 1. Maintenance of conjugation in primary cultures of rat hepatocytes maintained for up to 72 hours Conjugation activity [or concnl (% of initial Substrate fresh cell valuer) at: conch
Reaction* (#ra) 24 hr 48 hr 72 hr P-GTase 100 56 + 8 (6) 135 __.19 (6) 154 + 19 (8) N-GTase 100 48 + 4 (5) 124 ± 14 (4) 161 + 22 (7) N-STase 100 53 + 3 (3) 36 ± 2 (3) 28 ± 4 (4) GSH content -[85 ± 2] (3) [71 ± 9] (3) [80 + l 1] (3) GSH-CDNB 50 153 ± 25 (4) 161 + 26 (4) 118 ± 15 (4) P = Phenolphthalein N = l-Naphthol GSH = Reduced glutathione GTase = Glucuronosyl transferase STase= Sulphotransferase CDNB = l-chloro-2,4-dinitrobenzene *All of the conjugation reactions were carried out in intact cells. The incubation buffer was Krebs-Henseleit buffer, pH 7.4, containing 10mM-HEPES. tFresh cell values were: P-GT 0.41 + 0.04 nmol/mg cell protein/rnin; N-GT 1.08 ± 0.23 nmol/mg cell protcin/min; N - S T 0.34± 0.01 nmol/mg cell protein/min; GSH content 42.3 ± 2.4 nmol/mg cell protein; GSH-CDNB formation 1.1 ± 0.1 nmol/mg cell protein/rain. Results are means + SEM for the numbers of experiments indicated in brackets.
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