124
Biochimica ct Biophysica Acta, 500 (1977) 124--131
© Elsevier/North-Holland Biomedical Press
BBA 28341 THE E F F E C T OF AN N A D P H - R E G E N E R A T I N G SYSTEM ON BIPHENYL METABOLISM IN I SO L A T E D R A T H E P A T O C Y T E S
ROBIN S. JONES, DILSHAD MENDIS * and DENNIS V. PARKE Department o f Biochemistry, University o f Surrey, Guildford, G U2 5XH (U. K.)
(Received April 29th, 1977)
S u mmar y Biphenyl 4 - h y d r o x y l a t i o n was studied in isolated rat hepatocytes. It was found that there was an inter-relationship between 4-hydroxylase activity and glucuronidase activity, removal o f 4 - h y d r o x y b i p h e n y l by conjugation being necessary to stimulate a second phase of h y d r o x y l a t i o n . Addition of an NADPHregenerating system resulted in an initial depression of both processes, but later their activities were enhanced. This action could n o t be explained by the presence o f non-viable cells.
Introduction The activity o f hepatic microsomal mixed function oxidase is d e p e n d e n t on the supply o f electrons provided by NADPH. For this reason it is customary to add either NADPH or an NADPH-regenerating system to microsomal preparations which normally would be deficient in the cofactor. In contrast, under most experimental circumstances, isolated rat h e p a t o c y t e s are able to maintain a sufficiently high NADPH/NADP ÷ ratio to support drug oxidations [1] and it is generally t h o u g h t unnecessary to add exogenous NADPH to such preparations. However, some workers do so routinely [2] even though it is difficult to envisage the reagent entering intact cells to exert an effect. Indeed, Wiebkin et al. [3] have suggested that e n h a n c e m e n t of drug metabolism by exogenous NADPH may be an indication of cellular damage. In the study to be described, biphenyl 4 - h y d r o x y l a t i o n (and subsequent glucuronidation) was followed in isolated rat hepatocytes. Experiments were cond u c t ed using viable and non-viable hepatocytes, each in the absence and presence o f an NADPH-regenerating system, in order to investigate the possible relationship between the effects of exogenous NADPH and cell viability. * Present address: North East Surrey Collage of Technology, Ewell, Epsom, Surrey, KT17 3DS, U.K.
125
Methods and Materials
Chemicals. Biphenyl (m.p. 70°C), 4-hydroxybiphenyl (m.p. 162°C), NADP and glucose 6-phosphate were all purchased from B.D.H. Chemicals Ltd., Poole, Dorset, U.K. Bovine serum albumin (fraction V) and glucose-6-phosphate dehydrogenase were obtained from Sigma (London) Chemical Co. Ltd., Kingstonupon-Thames, Surrey, U.K. Other materials used were fetal calf serum and Eagle's minimum essential medium (Flow Laboratories Ltd., Irbine, Ayrshire, U.K), tissue culture medium 199 without phenol red (Difco, West Molesey, Surrey, U.K.), Ketodase (William and Warner Co. Ltd., Eastleigh, Hampshire, U.K) and collagenase (Worthington Biochemical Corp., N.J., U.S.A.). Animals. Male Wistar albino rats (200--250 g) were used as liver donors. They were allowed to feed and drink ad libitum prior to experiments. Hepatocyte preparation. The technique used for the isolation of rat hepatocytes was an adaptation of the method of Berry and Friend [4]. Each rat was anaesthetised by intraperitoneal sodium pentobarbitone (Nembutal; 60 mg/kg) and after cannulation of the hepatic portal vein with polypropylene tubing (pp 60; Portex Ltd., Hythe, Kent, U.K.) an infusion of calcium-free Dulbecco's phosphate buffer (50 ml containing 4 mM ethanedioxybis(ethylamine)N,N,N',N'-tetraacetate) in situ was initiated at a rate of 10 ml/min. The infusion was continued throughout hepatectomy, after which the liver was transferred to a constant temperature cabinet (37°C) and flushed with Dulbecco's phosphate buffer (150 ml; 10 ml/min) to remove traces of ethanedioxybis(ethylamine)tetraacetate. The liver was then perfused with oxygenated (O2/CO2; 9 5 : 5 , v/v) recirculating Hanks buffered saline (150 ml) containing bovine serum albumin (2.25 g), glucose (0.15 g), NaHCO3 (75 mg) and collagenase (75 mg). After 30--40 rain the soft liver was removed from the recirculating system and disrupted by gentle shaking for 10 min with some of the collagenase buffer (20 ml) in a conical flask at 37°C. The resulting suspension was passed through 100 mesh nylon screen and centrifuged at 200 x g for 2 min, the pellet being resuspended in fresh medium and the.washing process repeated three times. Finally, the cells were suspended in an appropriate volume of incubation medium (see below). Assessment o f cell viability. Viability was assessed by the ability of cells to exclude the vital stain Trypan Blue [5]. Aliquots (0.25 ml) of hepatocyte suspensions were mixed with 0.5% (v/v) Trypan Blue solution (0.1 ml) and the total and viable cell populations determined using an Improved Neubauer counting chamber. The viable cell population was expressed as a percentage of the total cell population. Production o f non-viable cells. Suspensions of viable hepatocytes were sonicated for 2 min in a Kerry sonieator immediately before use. After this process all cells were incapable of excluding Trypan Blue. Incubation media. Three media were assessed for their ability to maintain isolated hepatocytes in a viable state. They were (a) Hanks buffered saline solution supplemented with bovine serum albumin, fraction v (1.5%, w/v), glucose (0.1%, w/v) and NaHCO3 (0.05%, w/v), (b) Eagle's minimum essential medium containing fetal calf serum (10%, v/v) and (c) tissue culture medium 199 (witho u t phenol red) supplemented with fetal calf serum (10%, v/v). The washed
126 hepatocytes were suspended in appropriate volumes (106 cells/ml) of each medium, and then aliquots (2 ml) incubated at 37°C with gentle oxygenation (O2/CO2, 95 : 5, v/v) for periods up to 2 h. Cell viability was assessed at regular intervals. Biphenyl toxicity. Wiebkin et al. [3] have previously reported that biphenyl is toxic to isolated rat hepatocytes suspended in medium b at concentrations of 1.4 mM and above, but not in the range 14--140 pM. In our study, the range 120--400 pM was investigated more thoroughly using medium c. Biphenyl 4-hydroxylase activity. Aliquots (1.75 ml) of cell suspensions in medium c (2 • 106 hepatocytes) were placed in 20-ml stoppered tubes and incubated with gentle oxygenation (O2/CO2, 95 : 5, v/v) at 37°C in a shaking water bath. Reactions were initiated by the addition of biphenyl (60 nmol) in Tween (4%, v/v in medium c). Where cofactors were to be added, the volume of the cell suspension was 1.5 ml (2 • 106 cells), and 0.25 ml of the regenerating system (5 mM NADP; 60 mM glucose 6-phosphate; 1.5 units glucose-6-phosphate dehydrogenase, in medium c) added. Tubes were incubated for various time intervals and the reaction stopped by plunging into ice. Hydroxylated products were extracted into 7 ml heptane (containing 1.5% (v/v) isoamylalcohol) and then back extracted into 0.1 M NaOH (2 ml). 4-Hydroxybiphenyl was then determined by measuring the fluorescence intensity of the alkaline layer at 400 nm with excitation at 311 nm [6]. Glucuronide conjugates were hydrolysed by addition of 0.2 M acetate buffer pH 5.0 (1 ml) and glucuronidase (300 Fishman units) to aliquots (1.5 ml) of the cell free incubates, which were then further incubated at 37°C for 16 h. The free 4-hydroxybiphenyl was then determined as described above. Results
Cell viability. The isolation procedure described yielded cell populations with 80--90% viability. Of the three media used, tissue culture medium 199 (medium c) proved to be marginally better than Eagle's minimum essential medium {medium b) in maintaining cell viability, but both were far superior to Hanks buffered saline solution {medium a). After 40 min the viable cell population dropped from 87 to 86% in medium c, to 83% in medium b, and to 62% in medium a. After 2 h only 28% of the cells remained viable in medium a, whereas for medium c and medium b 85% and 79%, respectively, remained viable. It was found that an increase in biphenyl concentration led to increased toxicity (Fig. 1). However, at 120 pM, cell viability remained constant for 40--50 rain after which it declined gradually. The substrate concentration used routinely (30 pM) was therefore far below any toxic level. Biphenyl 4-hydroxylation and glucuronidation in viable (85%) cells In absence o f cofactors. During the first 5 min of incubation very little free 4-hydroxybiphenyl appeared in the incubation medium. However, over the next 12.5 min the concentration increased rapidly so that after 17.5 min of incubation 4.5 nmol (7.5% dose) had been formed. At this point it began to disappear from the medium and by 30 min the a m o u n t was scarcely detectable (Fig. 2). This drop was u n d o u b t e d l y due to glucuronide formation because
127 100
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in v i a b l e a n d n o n - v i a b l e h e p a t o c y t e s in the absence and •, viable (85%) hepatocytes alone; • ...... •, viable
(85%) hepatocytes in presence of NADPH-regenerating system; ~ "~, n o n - v i a b l e h e p a t o c y t e s a l o n e ; ,3. . . . . . ~, n o n - v i a b l e h e p a t o c y t e s i n t h e p r e s e n c e o f N A D P H - r e g e n e r a t i n g s y s t e m . V e r t i c a l b a r s r e p r e s e n t + S . D . (n = 3 ) .
128
when the total (i.e. free and glucuronide) 4-hydroxybiphenyl concentration was determined it was found to rise throughout the incubation period, 18 nmol (30% dose) being present after 45 min. The rates of formation of total 4-hydroxybiphenyl and its glucuronide conjugate are shown in Fig. 3a. Two maxima occurred for the hydroxylation process; the first at 13 rain (11 nmol/min) and the other at 25 min (5.5 nmol/min). The conjugation reaction showed one maximum only, which was 11 nmol/min at 23 min. In presence o f cofaetors. Addition of the NADPH-regenerating system to the incubation media resulted in a slight increase in the maximum concentration of free 4-hydroxybiphenyl to 2.5 pM (5.0 nmol/2 ml). The peak was also broadened (Fig. 2), but even so, after 30 min only about 0.5 nmol of free 4-hydroxybiphenyl could be detected, which was a similar value to that detected in the 12 (a)
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F i g . 3. T h e r a t e o f f o r m a t i o n o f 4 - h y d r o x y b i p h e n y l and its glucuronide c o n j u g a t e in v i a b l e h e p a t o c y t e s in the (a) absence, and (b) presence of an NADPH-regenerating system. The rates of formation of 4-hydroxybiphenyl (~::) a ' ] d i t s g l u c u r o n i d e c o n j u g a t e (m) a r e a v e r a g e r a t e s f o r t h e r e s p e c t i v e 5 - r a i n i n t e r v a l s . Each point represents the mean of three values obtained from different experiments (+S.D.).
129 absence of cofactors. The total amount (±S.D.) of this metabolite formed during the 45 min incubation was 18.8 ± 0.4 nmol (32% dose) and again its rate of formation showed two maxima. These were 8.0 and 11.7 nmol/min and occurred at 11 and 30 min, respectively. The maximum observed rate (±S.D.) of glucuronide formation was 16.5 + 1.0 nmol/min which occurred at about 27 min.
Biphenyl 4-hydroxylation and glucuronidation in non-viable cells In absence of cofactors. As with the 85% viable population, very little 4-hydroxybiphenyl was produced during the first 5 min of incubation. The maximum amount detectable was 5.6 nmol (9% dose) and this was observed at 25 min. However, unlike viable cells, the non-viable hepatocytes failed to remove the free metabolite from the medium and after 45 min 5.1 -+ 0.4 nmol was still present. This was due to the very low glucuronidating ability of the preparation, the maximum rate of which was only 1 nmol/min (45 min). The rate of 4-hydroxylation showed only 1 peak (6.5 nmol/min) at 9 min, and the reaction had
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Period of Incubation ( r a i n ) The rate of formation of 4-hydroxybiphenyl a n d its g l u c u r o n i d e c o n j u g a t e in n o n - v i a b l e h e p a t o c y t e s in t h e (a) a b s e n c e a n d ( b ) p r e s e n c e o f a n N A D P H - r e g c n e r a t i n g system. The rates of formation of 4-hydroxybiphenyl ( ) a n d its g l u c u x o n i d e (m) a r c a v e r a g e r a t e s f o r t h e r e s p e c t i v e 5 - m i n i n t e r v a l s . E a c h point represents the mean of three values obtained from different experiments (-+S.D.). Fig.
4.
130 ceased by 35 min (Fig. 4a). The total a m o u n t of 4 - h y d r o x y b i p h e n y l produced in 45 min was only 7 nmol as compared with 18 nmol in the intact cells. In presence o f cofactors. The m a x i m u m a m o u n t of free 4 - h y d r o x y b i p h e n y l d e t ected in the incubation medium occurred at 20 min and was 9.3 nmol as compared with a m a x i m u m of 5.6 nmol in the absence of cofactors. Also, the presence o f the regenerating system aided removal of the metabolite, for after 45 min there were only 7.0 nmol left in solution (Fig. 2). In fact, the maximum rate of glucuronide formation was doubled to 2 nm ol / m i n whereas the m a x i m u m rate of 4 - h y d r o x y l a t i o n was increased to 10.5 nm ol / m i n (Fig. 4b). The total a m o u n t of 4 - h y d r o x y b i p h e n y l f orm ed during the 45 min was 13.5 nmol, and as in the absence of cofactors, 4 - h y d r o x y l a t i o n has ceased by this time. Discussion When viable rat h e p a t o c y t e s were exposed to subtoxic levels of biphenyl the rate o f 4 - h y d r o x yl a t i on reached a m a x i m u m at 13 min after which it decreased. The rate o f glucuronidation was initially much slower than the h y d r o x y l a t i o n process. Consequently, the levels of free metabolite began to increase. However, at 16 min the glucuronidation rate overt ook the h y d r o x y l a t i o n rate and shortly afterwards (17.5 min) the concentration of 4 - h y d r o x y b i p h e n y l decreased. This fall was associated with a no t her phase of h y d r o x y l a t i o n with a n o th er peak occurring at 25 min, but by this time conjugation was proceeding at a much higher rate so the increase was not reflected in free metabolite figures. Following the peak at 23 rain the rate of glucuronidation dropped but continued at a slightly higher rate than 4-bydroxylation. Addition o f cofactors to the system did n ot cause any fundamental changes in the metabolism. However, it did cause an initial depression in the rate of 4 - h yd r o x y latio n , with an associated decrease in glucuronidation. As both processes were affected equally, the changes were not reflected in the a m o u n t of free metabolite formed. Indeed, the m a x i m u m free metabolite concent rat i on was not significantly greater than the value obtained w i t hout cofactors. However, the peak was broader, this being due to the fact that the rate of glucuronidation did n o t exceed the rate of h y d r o x y l a t i o n until 18.5 min (cf. 16 min). As before, there was a delay of approx. 1.5 min before a fall in free 4-hydroxybiphenyl co n cen tr at i on occurred. After the initial depression the rate of glucuronide f o r matio n was enhanced and a m axi m um of 16.5 nm ol / m i n was observed at 27 min. The sudden removal of free metabolite led to another burst o f 4-hydroxylase activity, the m a x i m u m (11.7 nmol/min, 30 min) being much higher than observed in the absence of cofactors. Despite the changes in the kinetics of biphenyl metabolism which occurred following the addition of the regenerating system, t hey would n o t have been observed had the total 4 - h y d r o x y b i p h e n y l and glucuronide contents been measured at 45 min only. At this time, there was no significant difference between values obtained by incubation in the absence and presence of cofactors. (Total 4 - h y d r o x y b i p h e n y l , 18.0 ~ 0.5 and 18.9 + 0.4; glucuronide, 17.6 + 0.3 and 18.1 * 0.4). When biphenyl was incubated with a 100% non-viable cell population the
131
kinetics of metabolism was very different. 4-Hydroxybiphenyl was produced, but at a much slower rate (6.5 nmol/min, 9 min) than observed in the predominantly viable preparation. Glucuronidation occurred at a very slow rate indeed (1 nmol/min). Subsequently, the level of free metabolite failed to drop significantly, and thus the second phase of h y d r o x y l a t i o n was not stimulated. Therefore, within 35 min 4-hydroxybiphenyl production had ceased. Addition of the regenerating system increased the m a x i m u m rate of 4-hydroxylation to 10.5 nmol/min (10 min). Similarly, glucuronidation was increased, but only to 2 nmol/min which again was not enough to greatly affect the free metabolite concentration and hence the second phase of h y d r o x y l a t i o n was again not stimulated. In the light of these results it becomes clear that the differences encountered by addition of cofactors to an 85% viable population cannot be explained by the presence of the non-viable cells. The mechanism of this observed effect is difficult to envisage as the cell membrane is not known to be permeable to any of the three cofactors and Wiebkin et al. [3], who studied biphenyl metabolism in viable hepatocytes in the presence and absence of NADPH, observed little effect. However, measurements were only taken at 45 min when differences would not be apparent. Temple et al. [7] showed that NADPH (or an NADPH-regenerating system, glucose 6-phosphate or isocitrate) enhanced UDP-glucuronyltransferase activity in microsomal preparations, but no mechanism was suggested. Other workers [8] have suggested that endogenous glucose-6-phosphate dehydrogenase leaks from isolated cells, and that m a x i m u m mixed function oxidase activity can be regained by saturating the medium with the enzyme in an a t t e m p t to prevent leakage. Our results indicate that the cofactors may affect the initial uptake of the biphenyl by the cells in addition to affecting enzyme activities. Also, preliminary findings of more recent experiments indicate that only NADP and glucose 6-phosphate are required for the effect. References 1 2 3 4 5 6 7 8
M o l d e u s , P., G r u n d i n , R., Vadi, H. a n d O r r e n i u s , S. ( 1 9 7 4 ) E u r . J. B i o e h e m . 46, 3 5 1 - - 3 6 0 H e n d e r s o n , P . T h . a n d D e w a i d e , J . H . ( 1 9 6 9 ) B i o c h e m . P h a r m a c o l . 18, 2 0 8 7 - - 2 0 9 4 Wiebkin, P., F r y , J . R . , J o n e s , C.A., L o w i n g , R. a n d Bridges, J.W. ( 1 9 7 6 ) X e n o b i o t i c a 6, 7 2 5 - - 7 4 3 B e r r y , M.N. a n d F r i e n d , D.S. ( 1 9 6 9 ) J. Cell Biol. 4 3 , 5 0 6 C u m m i n g s , H. ( 1 9 7 0 ) in V i r o l o g y - T i s s u e C u l t u r e , 1st e d n . , p. 26, B u t t e r w o r t h , L o n d o n C r e a v e n , P.J., P a r k e , D.V. a n d Williams, R . T . ( 1 9 6 5 ) B i o c h e m . J. 9 6 , 8 7 9 - - 8 8 5 T e m p l e , A . R . , G e o r g e , D.J. a n d D o n e , A . K . ( 1 9 7 1 ) B i o c h e m . P h a r m a c o l . 20, 1 7 1 8 - - 1 7 2 0 H u p k a , A.L. a n d K a r l a r , R. ( 1 9 7 3 ) J. R e t i c u l o e n d o t h e l . Soc. 14, 2 2 5 - - 2 4 1
Biochimica ct Biophysica Acta, 500 (1977) 124--131
© Elsevier/North-Holland Biomedical Press
BBA 28341 THE E F F E C T OF AN N A D P H - R E G E N E R A T I N G SYSTEM ON BIPHENYL METABOLISM IN I SO L A T E D R A T H E P A T O C Y T E S
ROBIN S. JONES, DILSHAD MENDIS * and DENNIS V. PARKE Department o f Biochemistry, University o f Surrey, Guildford, G U2 5XH (U. K.)
(Received April 29th, 1977)
S u mmar y Biphenyl 4 - h y d r o x y l a t i o n was studied in isolated rat hepatocytes. It was found that there was an inter-relationship between 4-hydroxylase activity and glucuronidase activity, removal o f 4 - h y d r o x y b i p h e n y l by conjugation being necessary to stimulate a second phase of h y d r o x y l a t i o n . Addition of an NADPHregenerating system resulted in an initial depression of both processes, but later their activities were enhanced. This action could n o t be explained by the presence o f non-viable cells.
Introduction The activity o f hepatic microsomal mixed function oxidase is d e p e n d e n t on the supply o f electrons provided by NADPH. For this reason it is customary to add either NADPH or an NADPH-regenerating system to microsomal preparations which normally would be deficient in the cofactor. In contrast, under most experimental circumstances, isolated rat h e p a t o c y t e s are able to maintain a sufficiently high NADPH/NADP ÷ ratio to support drug oxidations [1] and it is generally t h o u g h t unnecessary to add exogenous NADPH to such preparations. However, some workers do so routinely [2] even though it is difficult to envisage the reagent entering intact cells to exert an effect. Indeed, Wiebkin et al. [3] have suggested that e n h a n c e m e n t of drug metabolism by exogenous NADPH may be an indication of cellular damage. In the study to be described, biphenyl 4 - h y d r o x y l a t i o n (and subsequent glucuronidation) was followed in isolated rat hepatocytes. Experiments were cond u c t ed using viable and non-viable hepatocytes, each in the absence and presence o f an NADPH-regenerating system, in order to investigate the possible relationship between the effects of exogenous NADPH and cell viability. * Present address: North East Surrey Collage of Technology, Ewell, Epsom, Surrey, KT17 3DS, U.K.
125
Methods and Materials
Chemicals. Biphenyl (m.p. 70°C), 4-hydroxybiphenyl (m.p. 162°C), NADP and glucose 6-phosphate were all purchased from B.D.H. Chemicals Ltd., Poole, Dorset, U.K. Bovine serum albumin (fraction V) and glucose-6-phosphate dehydrogenase were obtained from Sigma (London) Chemical Co. Ltd., Kingstonupon-Thames, Surrey, U.K. Other materials used were fetal calf serum and Eagle's minimum essential medium (Flow Laboratories Ltd., Irbine, Ayrshire, U.K), tissue culture medium 199 without phenol red (Difco, West Molesey, Surrey, U.K.), Ketodase (William and Warner Co. Ltd., Eastleigh, Hampshire, U.K) and collagenase (Worthington Biochemical Corp., N.J., U.S.A.). Animals. Male Wistar albino rats (200--250 g) were used as liver donors. They were allowed to feed and drink ad libitum prior to experiments. Hepatocyte preparation. The technique used for the isolation of rat hepatocytes was an adaptation of the method of Berry and Friend [4]. Each rat was anaesthetised by intraperitoneal sodium pentobarbitone (Nembutal; 60 mg/kg) and after cannulation of the hepatic portal vein with polypropylene tubing (pp 60; Portex Ltd., Hythe, Kent, U.K.) an infusion of calcium-free Dulbecco's phosphate buffer (50 ml containing 4 mM ethanedioxybis(ethylamine)N,N,N',N'-tetraacetate) in situ was initiated at a rate of 10 ml/min. The infusion was continued throughout hepatectomy, after which the liver was transferred to a constant temperature cabinet (37°C) and flushed with Dulbecco's phosphate buffer (150 ml; 10 ml/min) to remove traces of ethanedioxybis(ethylamine)tetraacetate. The liver was then perfused with oxygenated (O2/CO2; 9 5 : 5 , v/v) recirculating Hanks buffered saline (150 ml) containing bovine serum albumin (2.25 g), glucose (0.15 g), NaHCO3 (75 mg) and collagenase (75 mg). After 30--40 rain the soft liver was removed from the recirculating system and disrupted by gentle shaking for 10 min with some of the collagenase buffer (20 ml) in a conical flask at 37°C. The resulting suspension was passed through 100 mesh nylon screen and centrifuged at 200 x g for 2 min, the pellet being resuspended in fresh medium and the.washing process repeated three times. Finally, the cells were suspended in an appropriate volume of incubation medium (see below). Assessment o f cell viability. Viability was assessed by the ability of cells to exclude the vital stain Trypan Blue [5]. Aliquots (0.25 ml) of hepatocyte suspensions were mixed with 0.5% (v/v) Trypan Blue solution (0.1 ml) and the total and viable cell populations determined using an Improved Neubauer counting chamber. The viable cell population was expressed as a percentage of the total cell population. Production o f non-viable cells. Suspensions of viable hepatocytes were sonicated for 2 min in a Kerry sonieator immediately before use. After this process all cells were incapable of excluding Trypan Blue. Incubation media. Three media were assessed for their ability to maintain isolated hepatocytes in a viable state. They were (a) Hanks buffered saline solution supplemented with bovine serum albumin, fraction v (1.5%, w/v), glucose (0.1%, w/v) and NaHCO3 (0.05%, w/v), (b) Eagle's minimum essential medium containing fetal calf serum (10%, v/v) and (c) tissue culture medium 199 (witho u t phenol red) supplemented with fetal calf serum (10%, v/v). The washed
126 hepatocytes were suspended in appropriate volumes (106 cells/ml) of each medium, and then aliquots (2 ml) incubated at 37°C with gentle oxygenation (O2/CO2, 95 : 5, v/v) for periods up to 2 h. Cell viability was assessed at regular intervals. Biphenyl toxicity. Wiebkin et al. [3] have previously reported that biphenyl is toxic to isolated rat hepatocytes suspended in medium b at concentrations of 1.4 mM and above, but not in the range 14--140 pM. In our study, the range 120--400 pM was investigated more thoroughly using medium c. Biphenyl 4-hydroxylase activity. Aliquots (1.75 ml) of cell suspensions in medium c (2 • 106 hepatocytes) were placed in 20-ml stoppered tubes and incubated with gentle oxygenation (O2/CO2, 95 : 5, v/v) at 37°C in a shaking water bath. Reactions were initiated by the addition of biphenyl (60 nmol) in Tween (4%, v/v in medium c). Where cofactors were to be added, the volume of the cell suspension was 1.5 ml (2 • 106 cells), and 0.25 ml of the regenerating system (5 mM NADP; 60 mM glucose 6-phosphate; 1.5 units glucose-6-phosphate dehydrogenase, in medium c) added. Tubes were incubated for various time intervals and the reaction stopped by plunging into ice. Hydroxylated products were extracted into 7 ml heptane (containing 1.5% (v/v) isoamylalcohol) and then back extracted into 0.1 M NaOH (2 ml). 4-Hydroxybiphenyl was then determined by measuring the fluorescence intensity of the alkaline layer at 400 nm with excitation at 311 nm [6]. Glucuronide conjugates were hydrolysed by addition of 0.2 M acetate buffer pH 5.0 (1 ml) and glucuronidase (300 Fishman units) to aliquots (1.5 ml) of the cell free incubates, which were then further incubated at 37°C for 16 h. The free 4-hydroxybiphenyl was then determined as described above. Results
Cell viability. The isolation procedure described yielded cell populations with 80--90% viability. Of the three media used, tissue culture medium 199 (medium c) proved to be marginally better than Eagle's minimum essential medium {medium b) in maintaining cell viability, but both were far superior to Hanks buffered saline solution {medium a). After 40 min the viable cell population dropped from 87 to 86% in medium c, to 83% in medium b, and to 62% in medium a. After 2 h only 28% of the cells remained viable in medium a, whereas for medium c and medium b 85% and 79%, respectively, remained viable. It was found that an increase in biphenyl concentration led to increased toxicity (Fig. 1). However, at 120 pM, cell viability remained constant for 40--50 rain after which it declined gradually. The substrate concentration used routinely (30 pM) was therefore far below any toxic level. Biphenyl 4-hydroxylation and glucuronidation in viable (85%) cells In absence o f cofactors. During the first 5 min of incubation very little free 4-hydroxybiphenyl appeared in the incubation medium. However, over the next 12.5 min the concentration increased rapidly so that after 17.5 min of incubation 4.5 nmol (7.5% dose) had been formed. At this point it began to disappear from the medium and by 30 min the a m o u n t was scarcely detectable (Fig. 2). This drop was u n d o u b t e d l y due to glucuronide formation because
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(85%) hepatocytes in presence of NADPH-regenerating system; ~ "~, n o n - v i a b l e h e p a t o c y t e s a l o n e ; ,3. . . . . . ~, n o n - v i a b l e h e p a t o c y t e s i n t h e p r e s e n c e o f N A D P H - r e g e n e r a t i n g s y s t e m . V e r t i c a l b a r s r e p r e s e n t + S . D . (n = 3 ) .
128
when the total (i.e. free and glucuronide) 4-hydroxybiphenyl concentration was determined it was found to rise throughout the incubation period, 18 nmol (30% dose) being present after 45 min. The rates of formation of total 4-hydroxybiphenyl and its glucuronide conjugate are shown in Fig. 3a. Two maxima occurred for the hydroxylation process; the first at 13 rain (11 nmol/min) and the other at 25 min (5.5 nmol/min). The conjugation reaction showed one maximum only, which was 11 nmol/min at 23 min. In presence o f cofaetors. Addition of the NADPH-regenerating system to the incubation media resulted in a slight increase in the maximum concentration of free 4-hydroxybiphenyl to 2.5 pM (5.0 nmol/2 ml). The peak was also broadened (Fig. 2), but even so, after 30 min only about 0.5 nmol of free 4-hydroxybiphenyl could be detected, which was a similar value to that detected in the 12 (a)
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F i g . 3. T h e r a t e o f f o r m a t i o n o f 4 - h y d r o x y b i p h e n y l and its glucuronide c o n j u g a t e in v i a b l e h e p a t o c y t e s in the (a) absence, and (b) presence of an NADPH-regenerating system. The rates of formation of 4-hydroxybiphenyl (~::) a ' ] d i t s g l u c u r o n i d e c o n j u g a t e (m) a r e a v e r a g e r a t e s f o r t h e r e s p e c t i v e 5 - r a i n i n t e r v a l s . Each point represents the mean of three values obtained from different experiments (+S.D.).
129 absence of cofactors. The total amount (±S.D.) of this metabolite formed during the 45 min incubation was 18.8 ± 0.4 nmol (32% dose) and again its rate of formation showed two maxima. These were 8.0 and 11.7 nmol/min and occurred at 11 and 30 min, respectively. The maximum observed rate (±S.D.) of glucuronide formation was 16.5 + 1.0 nmol/min which occurred at about 27 min.
Biphenyl 4-hydroxylation and glucuronidation in non-viable cells In absence of cofactors. As with the 85% viable population, very little 4-hydroxybiphenyl was produced during the first 5 min of incubation. The maximum amount detectable was 5.6 nmol (9% dose) and this was observed at 25 min. However, unlike viable cells, the non-viable hepatocytes failed to remove the free metabolite from the medium and after 45 min 5.1 -+ 0.4 nmol was still present. This was due to the very low glucuronidating ability of the preparation, the maximum rate of which was only 1 nmol/min (45 min). The rate of 4-hydroxylation showed only 1 peak (6.5 nmol/min) at 9 min, and the reaction had
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Period of Incubation ( r a i n ) The rate of formation of 4-hydroxybiphenyl a n d its g l u c u r o n i d e c o n j u g a t e in n o n - v i a b l e h e p a t o c y t e s in t h e (a) a b s e n c e a n d ( b ) p r e s e n c e o f a n N A D P H - r e g c n e r a t i n g system. The rates of formation of 4-hydroxybiphenyl ( ) a n d its g l u c u x o n i d e (m) a r c a v e r a g e r a t e s f o r t h e r e s p e c t i v e 5 - m i n i n t e r v a l s . E a c h point represents the mean of three values obtained from different experiments (-+S.D.). Fig.
4.
130 ceased by 35 min (Fig. 4a). The total a m o u n t of 4 - h y d r o x y b i p h e n y l produced in 45 min was only 7 nmol as compared with 18 nmol in the intact cells. In presence o f cofactors. The m a x i m u m a m o u n t of free 4 - h y d r o x y b i p h e n y l d e t ected in the incubation medium occurred at 20 min and was 9.3 nmol as compared with a m a x i m u m of 5.6 nmol in the absence of cofactors. Also, the presence o f the regenerating system aided removal of the metabolite, for after 45 min there were only 7.0 nmol left in solution (Fig. 2). In fact, the maximum rate of glucuronide formation was doubled to 2 nm ol / m i n whereas the m a x i m u m rate of 4 - h y d r o x y l a t i o n was increased to 10.5 nm ol / m i n (Fig. 4b). The total a m o u n t of 4 - h y d r o x y b i p h e n y l f orm ed during the 45 min was 13.5 nmol, and as in the absence of cofactors, 4 - h y d r o x y l a t i o n has ceased by this time. Discussion When viable rat h e p a t o c y t e s were exposed to subtoxic levels of biphenyl the rate o f 4 - h y d r o x yl a t i on reached a m a x i m u m at 13 min after which it decreased. The rate o f glucuronidation was initially much slower than the h y d r o x y l a t i o n process. Consequently, the levels of free metabolite began to increase. However, at 16 min the glucuronidation rate overt ook the h y d r o x y l a t i o n rate and shortly afterwards (17.5 min) the concentration of 4 - h y d r o x y b i p h e n y l decreased. This fall was associated with a no t her phase of h y d r o x y l a t i o n with a n o th er peak occurring at 25 min, but by this time conjugation was proceeding at a much higher rate so the increase was not reflected in free metabolite figures. Following the peak at 23 rain the rate of glucuronidation dropped but continued at a slightly higher rate than 4-bydroxylation. Addition o f cofactors to the system did n ot cause any fundamental changes in the metabolism. However, it did cause an initial depression in the rate of 4 - h yd r o x y latio n , with an associated decrease in glucuronidation. As both processes were affected equally, the changes were not reflected in the a m o u n t of free metabolite formed. Indeed, the m a x i m u m free metabolite concent rat i on was not significantly greater than the value obtained w i t hout cofactors. However, the peak was broader, this being due to the fact that the rate of glucuronidation did n o t exceed the rate of h y d r o x y l a t i o n until 18.5 min (cf. 16 min). As before, there was a delay of approx. 1.5 min before a fall in free 4-hydroxybiphenyl co n cen tr at i on occurred. After the initial depression the rate of glucuronide f o r matio n was enhanced and a m axi m um of 16.5 nm ol / m i n was observed at 27 min. The sudden removal of free metabolite led to another burst o f 4-hydroxylase activity, the m a x i m u m (11.7 nmol/min, 30 min) being much higher than observed in the absence of cofactors. Despite the changes in the kinetics of biphenyl metabolism which occurred following the addition of the regenerating system, t hey would n o t have been observed had the total 4 - h y d r o x y b i p h e n y l and glucuronide contents been measured at 45 min only. At this time, there was no significant difference between values obtained by incubation in the absence and presence of cofactors. (Total 4 - h y d r o x y b i p h e n y l , 18.0 ~ 0.5 and 18.9 + 0.4; glucuronide, 17.6 + 0.3 and 18.1 * 0.4). When biphenyl was incubated with a 100% non-viable cell population the
131
kinetics of metabolism was very different. 4-Hydroxybiphenyl was produced, but at a much slower rate (6.5 nmol/min, 9 min) than observed in the predominantly viable preparation. Glucuronidation occurred at a very slow rate indeed (1 nmol/min). Subsequently, the level of free metabolite failed to drop significantly, and thus the second phase of h y d r o x y l a t i o n was not stimulated. Therefore, within 35 min 4-hydroxybiphenyl production had ceased. Addition of the regenerating system increased the m a x i m u m rate of 4-hydroxylation to 10.5 nmol/min (10 min). Similarly, glucuronidation was increased, but only to 2 nmol/min which again was not enough to greatly affect the free metabolite concentration and hence the second phase of h y d r o x y l a t i o n was again not stimulated. In the light of these results it becomes clear that the differences encountered by addition of cofactors to an 85% viable population cannot be explained by the presence of the non-viable cells. The mechanism of this observed effect is difficult to envisage as the cell membrane is not known to be permeable to any of the three cofactors and Wiebkin et al. [3], who studied biphenyl metabolism in viable hepatocytes in the presence and absence of NADPH, observed little effect. However, measurements were only taken at 45 min when differences would not be apparent. Temple et al. [7] showed that NADPH (or an NADPH-regenerating system, glucose 6-phosphate or isocitrate) enhanced UDP-glucuronyltransferase activity in microsomal preparations, but no mechanism was suggested. Other workers [8] have suggested that endogenous glucose-6-phosphate dehydrogenase leaks from isolated cells, and that m a x i m u m mixed function oxidase activity can be regained by saturating the medium with the enzyme in an a t t e m p t to prevent leakage. Our results indicate that the cofactors may affect the initial uptake of the biphenyl by the cells in addition to affecting enzyme activities. Also, preliminary findings of more recent experiments indicate that only NADP and glucose 6-phosphate are required for the effect. References 1 2 3 4 5 6 7 8
M o l d e u s , P., G r u n d i n , R., Vadi, H. a n d O r r e n i u s , S. ( 1 9 7 4 ) E u r . J. B i o e h e m . 46, 3 5 1 - - 3 6 0 H e n d e r s o n , P . T h . a n d D e w a i d e , J . H . ( 1 9 6 9 ) B i o c h e m . P h a r m a c o l . 18, 2 0 8 7 - - 2 0 9 4 Wiebkin, P., F r y , J . R . , J o n e s , C.A., L o w i n g , R. a n d Bridges, J.W. ( 1 9 7 6 ) X e n o b i o t i c a 6, 7 2 5 - - 7 4 3 B e r r y , M.N. a n d F r i e n d , D.S. ( 1 9 6 9 ) J. Cell Biol. 4 3 , 5 0 6 C u m m i n g s , H. ( 1 9 7 0 ) in V i r o l o g y - T i s s u e C u l t u r e , 1st e d n . , p. 26, B u t t e r w o r t h , L o n d o n C r e a v e n , P.J., P a r k e , D.V. a n d Williams, R . T . ( 1 9 6 5 ) B i o c h e m . J. 9 6 , 8 7 9 - - 8 8 5 T e m p l e , A . R . , G e o r g e , D.J. a n d D o n e , A . K . ( 1 9 7 1 ) B i o c h e m . P h a r m a c o l . 20, 1 7 1 8 - - 1 7 2 0 H u p k a , A.L. a n d K a r l a r , R. ( 1 9 7 3 ) J. R e t i c u l o e n d o t h e l . Soc. 14, 2 2 5 - - 2 4 1
