- Email: [email protected]
Isolated rat hepatocytes as a model to study drug metabolism: Dose-dependent metabolism of diphenylhydantoin
Pergamon Press
Life Sciences Vol . 16, pp " 1227-1232 Printed in the U .S .A .
ISOLATED RAT HEPATOCYTF~ AS A MODEL TO STUDY DRUG METABOLISM : D03E-DEPEAiDENT MisTAHOLISM OF DIPHEAYLHYDAATOIN Tadaaobu Inaba, Teruhiea Umeda, William A . Mahoa, Joseph Ho and Khuraheed N . Jeejeebhoy Departments of Pharmacology and Medicine University of Toronto, Toronto M5S 1A8, Canada (Received in final form February 10, 1975)
The metabolism of diphenylt~ydantoin (DPH) was studied using isolated rat hepatocytee . The Y~ of DPH metabolism xhen calculated from the disappearance of DPH in the medium xas 0 .39 to 0 .48 umole/g cell/hr which is comparable to that in microsomal preparations . Disappearance rate of DPH was concentration-dependent but not of a simple Michaelas-hSenten type . The formation of a DPH metabolite (glucuronide of hydrorylated DPH, HPPH~) has an apparent Ymnr of 0 .29 to 0 .32 Umole/g cell/hr . DPH binds extensively to hepatocytes and its distribution between the hepatocytes and the suspension medium xas independent of DPH concentration . Liver is the primary Bite of drug transformation . target organ for studies of drug metabolism .
Therefore this is the
Several systems starting from
the xhole animal to the perfused liver system, liver slices, homogenates and subcellular fractions have been utilized to investigate the metabolism of drugs (1) .
Isolated hepatocytes, in spite of obvious advantages, have not
been widely employed, because of poor yields in the isolation of the cells and their abort viability . Recently, Berry and Friend developed an efficient method to isolate rat hepatocytes which has been used successfully in the study of the metabolism of alcohols (2,3) .
Jeejeebhoy et al modified the method of Berry and Friend
and improved the yield of cells considerably (4) .
The rat hepatocytes thus
obtained xere viable and synthesis of several proteins continued for 48 hours following the isolation .
The present xork xas conducted to study drug
metabolizing capacities of isolated rat hepatocytee prepared by the above 1227
122 8
Druq Metabolism by Isolated Hepatocytes
modification .
Vol . 16, No . 8
5,5-diphenylhydantoin (DPH) was chosen for this work and
varying amounts of DPH were added to the hepatocyte suspension to determine if a dose-dependent metabolism (5,6,7) can be demonstrated in this type of preparation . Methods The isolated hepatocytes (4) were suspended in 150 ml of Weimouth incubating medium at a concentration of 1-2 x 10
6
cells/ml (12 to 20 mg wet wt/ml) .
In
addition, the suspension contained 17 .52 (v/v) of inactivated horse serum, The hepatocyte suspension was
50 ug/ml of gentamicin and 7 i .u . heparin/ml.
°
incubated in a water bath at 37 C "
The ozygen tension of the suspension was
kept at 140-200 mmHg sad the pH was maintained at 7 .3-7 " 4 "
14
of preliminary incubation, a prepared amount of DPH 5 .21 m0i/mmole) was added to the suspension .
After 20 minutes
C (14ew Eagland Nuclear,
Following the addition of the
drug solution, 5 ml aliquots of the incubating cell suspension were taken at intervals .
Each 5 ml sample was immediately centrifuged to separate the cells
from the suspension .
The cell pellet was mixed with 4 ml of water and sonicated
for 6 min to yield clear solution .
DPH and HPPH-G were assayed by a method
similar to that described by Gerber et al (5,8) .
Aliquots of 1-2 ml of the
supernatant and the solution of soaicated cells were mixed with 2 ml of 1 M phosphate buffer
(pH 6 .8) and DPH was extracted with 15 ml of 1-chlorobutaae .
The aqueous phase was then washed with 10 ml of ethyl ether and was subjected to Gluaulase (Eado Laboratories) treatment .
The extraction with 20 ml of ethyl
ether yielded HPPH from the conjugated metabolite . pellet by the medium was checked using albumin
125
Contamination of the cell I and found to be a small
contribution of less than 2x and was not corrected. Results and Discussion Table 1 s»~Ar izes the experimental conditions . of the rat hepatocytes were divided into 5 flasks .
Two separate preparations DPH in the medium is
expressed as initial concentration ug/ml medium or as VM of DPH.
Vol . 16, No . 8
1229
Druq Metabolism by Isolated Hapatocytes TART.R 1
Dipheaylbyydsatoin Metabolism in Isolated Rat Hepatocytes (Experimental Conditions)
Experiment No .
Initial Concentration of DPH va ml medium
Ia Ib Ic IIs IIb
40 10 1 10 1
uM 177.0 44 .2 4 .42 44 .2 4.42
Semilogarithmic plots of DPH concentrations is the hepatocyte suspension medium ve . time .
Hepatocy-te Concentration
Incubation Period
mg xet vt ml medium 12 .0 12 .0 12 .0 19 .7 19 .7
4 4 2 4 2
Linear plots of DPH concentrations in the hepatocy-te suspension medium vs . time .
The decline of DPH concentration in the medium vs . time in the form of semilogarithmic plot is shosm in Figure 1.
It is clear that the decline of
1230
Druq Metabolism by isolated Hepatocytes
Vol . 16, No . 8
DPH levels is concentration-dependent and that the rate of decline decreases with high initial concentration .
These data are consistent with the results
of dose-dependent elimination of DPH in the rat reported by Gerber et al (5) and Ashley and Levy (9) .
In order to determine if DPH metabolism in the rat
hepatocytea can be described by a simple Michaelis-Menten kinetics, several plotting techniques (1,7,10) were adopted.
However, the plotting showed no
obvious linear relationship, suggesting DPH metabolism in the isolated rat hepatocytes was not a simple Michaelis-Menten type .
Further investigation is
necessary to examine what ie the main contributing factor of dose-dependent metabolism of DPH . The linear plot of concentration vs . time ie shown in Figure 2 .
With the
initial concentration of DPH of 40 uB/~ (~P " Ia) the decline of concentration vs . time is linear throughout 4 hours .
In contrast, the lower concentration
10 u8/ml (Exp . Ib) is linear for only the first 1 .5 hours and thereafter folloxs a non-linear decline . non-linear
Plots of data from Exp . Ic, IIa, and IIb are
(only IIa is shown because of scaling) .
From the linear decline in
concentration (Exp . Ia, Ib)V~ of DPH metabolism was calculated and found to be 0 .39 and 0 .48 mmole/g cell/hr, respectively .
The values obtained were in
good agreement with the data of DPH metabolism in microsomal enzymes .
From the
data of Kutt and Verebely (11) it can be calculated that the rat microsomal enzymes metabolized DPH at a rate of 0 .50 mole/g liver/hour . The end product of DPH metabolism in the rat is a glucuronide of 5-(phydro~grpher~yl)-5-phenylhydantoin (HPPH-G)
(5) .
It has been shown in the
isolated rat hepatocytes that DPH is metabolized to HPPH-G ae confirmed by thin-layer chromatography and enzymatic hydrolysis studies, the technique of which has been described elsewhere (8) . xea presented in Table 2 .
Appearance of HPPH-G in the medium
From the linear metabolic appearance in Exp . Ia,
Ib, IIa, an apparent V~ was calculated to be 0 .29 to 0 .32 mmole/g cell/hr . This value is slightly smaller than the V~ calculated from the disappearance of DPH and the difference might be due to the transport or distribution
1231
Drug Metabolism by Isolated Hepatocytea
Vol . 16, No . 8 phenomena.
TABLE 3
TABLE 2
Distribution of Diphenylhydantoin Between Hepatocytea and Medium
Appearance of Glucuronide of 5-(-p-Hydroxyphenyl)-5Phenylhydantoin in the Medium
Fsp . No .
Conc . Ratio rug/g he estoc \ug ml mediam (mean + SD)
Exp . No .
HPPH-G formed (umole/g hepatocyte) 1/4 hr
1 hr
2 hr
4 hr
Ia
O .OÎ
0.21
0 .59
1 .26
Ia
18 + 1* (n= 0)
Ib
o .08
0.26
0 .63
1 .14
Ib
20 + 1* (n= 0)
Ic
0.02
0.05
0 .15
----
Ic
22 + 3**(n= 6)
IIe
o .06
0.32
0 .62
1 .26
IIa
20 + 4* (n= 0)
IIb
o .02
0.10
0 .15
--
IIb
23 + 1**(n= 4)
* 1/4_4 hr, **1/4-2 hr, n : number of observations
Table 3 shows the distribution of DPH and HPPH.1} between hepatocytes and suspension medium .
The results show that DPH binds eztensively to cells and
that the distribution between cells sad medium appears constant for the duration of the incubation and over the DPH concentration range of 0 .01-40 ue/ml .
Thus,
the initial uptake of DPH by cells appears complete by the first sampling . In conclusion, isolated rat hépatocytes prepared by the method of Jeejeebhoy et al (4) metabolize DPH et rates comparable to those in microeomal preparation with as excess of cofactors .
DPH metabolism was ceaeeatratioa-dependent but
not of e simple Michaelis-Meaten type .
DPH binds extensively to hepatocytes
and its distribution of DPH between the hepatocytes and the suspension medium was independent of DPH concentrations . We wish to thank Dr . Yedy Israel for his helpful and constructive criticism. Studies were financed partialltiy by a grant from Hoffmean-La Roche Ltd ., Montreal, Canada and the Medical Research Council (Great l~MA-4263 and MT3204), Ottawa, Canada .
1232
Drug Metabolism by Isolated Hepatocytea
Vol . 16, No . 8
References 1.
J.R . GILLETTE, in Fundamentals of D Metabolism and Dr Dia Bition, (Eds . B.N . LaDu, H.G . Mandel sad E.L . Way , p . 00, Williams and Wilkias, Baltimore (1971) .
2.
M.N . BERRY aad D .S . FRISND, J. Cell Biol ., ~, 506 (1969) .
3.
M.N . BERRY, Biochem . J ., 1~, 40p (1971) .
4.
K.N . JEEJEEBHOY, J .HO ., G .R . GRF~~G, M.J . PHILLIPS, A.A . BRUCEROHERTSON and U. SODTKE, Biochem, J ., 146, 141 (1975) "
5"
N. GERBER, W.L . WELLER, R. LYNN, R .E . RANGNO, B .J . SWEEPMAN and M.T . HUSH, J . Pharmacol . Exp . Ther ., 178 (1971) .
6.
N. GERBER and J.G . WAGNER, Research Comm . Chem . Pathol . Pharmacol ., 455 (1972) .
7.
D . PERRIER, J.J . ASHLEY and G. LEVY, J . Pharmacokia . Biopharm ., 1, 231 (1972) .
8.
T. INABA and T . UMEDA, Drug Metabolism and Disposition, (1975) in press .
9.
J.J . ASHLEY and G. LEVY, J . Pharmacokin . Biopharm ., 1, 99 (1973) .
10 .
L. ENDRENYI and F.H .F . KWONG, in sis aad Simulation of Biochemical S sy tens (Eds . H.C . Henker and B . Hess p .219, North-Holland, Amsterdam (1972) .
11 .
H. KUTT and K. VEREBELY, Biochem . Pharmacol ., 12, 675 (1970) "
Life Sciences Vol . 16, pp " 1227-1232 Printed in the U .S .A .
ISOLATED RAT HEPATOCYTF~ AS A MODEL TO STUDY DRUG METABOLISM : D03E-DEPEAiDENT MisTAHOLISM OF DIPHEAYLHYDAATOIN Tadaaobu Inaba, Teruhiea Umeda, William A . Mahoa, Joseph Ho and Khuraheed N . Jeejeebhoy Departments of Pharmacology and Medicine University of Toronto, Toronto M5S 1A8, Canada (Received in final form February 10, 1975)
The metabolism of diphenylt~ydantoin (DPH) was studied using isolated rat hepatocytee . The Y~ of DPH metabolism xhen calculated from the disappearance of DPH in the medium xas 0 .39 to 0 .48 umole/g cell/hr which is comparable to that in microsomal preparations . Disappearance rate of DPH was concentration-dependent but not of a simple Michaelas-hSenten type . The formation of a DPH metabolite (glucuronide of hydrorylated DPH, HPPH~) has an apparent Ymnr of 0 .29 to 0 .32 Umole/g cell/hr . DPH binds extensively to hepatocytes and its distribution between the hepatocytes and the suspension medium xas independent of DPH concentration . Liver is the primary Bite of drug transformation . target organ for studies of drug metabolism .
Therefore this is the
Several systems starting from
the xhole animal to the perfused liver system, liver slices, homogenates and subcellular fractions have been utilized to investigate the metabolism of drugs (1) .
Isolated hepatocytes, in spite of obvious advantages, have not
been widely employed, because of poor yields in the isolation of the cells and their abort viability . Recently, Berry and Friend developed an efficient method to isolate rat hepatocytes which has been used successfully in the study of the metabolism of alcohols (2,3) .
Jeejeebhoy et al modified the method of Berry and Friend
and improved the yield of cells considerably (4) .
The rat hepatocytes thus
obtained xere viable and synthesis of several proteins continued for 48 hours following the isolation .
The present xork xas conducted to study drug
metabolizing capacities of isolated rat hepatocytee prepared by the above 1227
122 8
Druq Metabolism by Isolated Hepatocytes
modification .
Vol . 16, No . 8
5,5-diphenylhydantoin (DPH) was chosen for this work and
varying amounts of DPH were added to the hepatocyte suspension to determine if a dose-dependent metabolism (5,6,7) can be demonstrated in this type of preparation . Methods The isolated hepatocytes (4) were suspended in 150 ml of Weimouth incubating medium at a concentration of 1-2 x 10
6
cells/ml (12 to 20 mg wet wt/ml) .
In
addition, the suspension contained 17 .52 (v/v) of inactivated horse serum, The hepatocyte suspension was
50 ug/ml of gentamicin and 7 i .u . heparin/ml.
°
incubated in a water bath at 37 C "
The ozygen tension of the suspension was
kept at 140-200 mmHg sad the pH was maintained at 7 .3-7 " 4 "
14
of preliminary incubation, a prepared amount of DPH 5 .21 m0i/mmole) was added to the suspension .
After 20 minutes
C (14ew Eagland Nuclear,
Following the addition of the
drug solution, 5 ml aliquots of the incubating cell suspension were taken at intervals .
Each 5 ml sample was immediately centrifuged to separate the cells
from the suspension .
The cell pellet was mixed with 4 ml of water and sonicated
for 6 min to yield clear solution .
DPH and HPPH-G were assayed by a method
similar to that described by Gerber et al (5,8) .
Aliquots of 1-2 ml of the
supernatant and the solution of soaicated cells were mixed with 2 ml of 1 M phosphate buffer
(pH 6 .8) and DPH was extracted with 15 ml of 1-chlorobutaae .
The aqueous phase was then washed with 10 ml of ethyl ether and was subjected to Gluaulase (Eado Laboratories) treatment .
The extraction with 20 ml of ethyl
ether yielded HPPH from the conjugated metabolite . pellet by the medium was checked using albumin
125
Contamination of the cell I and found to be a small
contribution of less than 2x and was not corrected. Results and Discussion Table 1 s»~Ar izes the experimental conditions . of the rat hepatocytes were divided into 5 flasks .
Two separate preparations DPH in the medium is
expressed as initial concentration ug/ml medium or as VM of DPH.
Vol . 16, No . 8
1229
Druq Metabolism by Isolated Hapatocytes TART.R 1
Dipheaylbyydsatoin Metabolism in Isolated Rat Hepatocytes (Experimental Conditions)
Experiment No .
Initial Concentration of DPH va ml medium
Ia Ib Ic IIs IIb
40 10 1 10 1
uM 177.0 44 .2 4 .42 44 .2 4.42
Semilogarithmic plots of DPH concentrations is the hepatocyte suspension medium ve . time .
Hepatocy-te Concentration
Incubation Period
mg xet vt ml medium 12 .0 12 .0 12 .0 19 .7 19 .7
4 4 2 4 2
Linear plots of DPH concentrations in the hepatocy-te suspension medium vs . time .
The decline of DPH concentration in the medium vs . time in the form of semilogarithmic plot is shosm in Figure 1.
It is clear that the decline of
1230
Druq Metabolism by isolated Hepatocytes
Vol . 16, No . 8
DPH levels is concentration-dependent and that the rate of decline decreases with high initial concentration .
These data are consistent with the results
of dose-dependent elimination of DPH in the rat reported by Gerber et al (5) and Ashley and Levy (9) .
In order to determine if DPH metabolism in the rat
hepatocytea can be described by a simple Michaelis-Menten kinetics, several plotting techniques (1,7,10) were adopted.
However, the plotting showed no
obvious linear relationship, suggesting DPH metabolism in the isolated rat hepatocytes was not a simple Michaelis-Menten type .
Further investigation is
necessary to examine what ie the main contributing factor of dose-dependent metabolism of DPH . The linear plot of concentration vs . time ie shown in Figure 2 .
With the
initial concentration of DPH of 40 uB/~ (~P " Ia) the decline of concentration vs . time is linear throughout 4 hours .
In contrast, the lower concentration
10 u8/ml (Exp . Ib) is linear for only the first 1 .5 hours and thereafter folloxs a non-linear decline . non-linear
Plots of data from Exp . Ic, IIa, and IIb are
(only IIa is shown because of scaling) .
From the linear decline in
concentration (Exp . Ia, Ib)V~ of DPH metabolism was calculated and found to be 0 .39 and 0 .48 mmole/g cell/hr, respectively .
The values obtained were in
good agreement with the data of DPH metabolism in microsomal enzymes .
From the
data of Kutt and Verebely (11) it can be calculated that the rat microsomal enzymes metabolized DPH at a rate of 0 .50 mole/g liver/hour . The end product of DPH metabolism in the rat is a glucuronide of 5-(phydro~grpher~yl)-5-phenylhydantoin (HPPH-G)
(5) .
It has been shown in the
isolated rat hepatocytes that DPH is metabolized to HPPH-G ae confirmed by thin-layer chromatography and enzymatic hydrolysis studies, the technique of which has been described elsewhere (8) . xea presented in Table 2 .
Appearance of HPPH-G in the medium
From the linear metabolic appearance in Exp . Ia,
Ib, IIa, an apparent V~ was calculated to be 0 .29 to 0 .32 mmole/g cell/hr . This value is slightly smaller than the V~ calculated from the disappearance of DPH and the difference might be due to the transport or distribution
1231
Drug Metabolism by Isolated Hepatocytea
Vol . 16, No . 8 phenomena.
TABLE 3
TABLE 2
Distribution of Diphenylhydantoin Between Hepatocytea and Medium
Appearance of Glucuronide of 5-(-p-Hydroxyphenyl)-5Phenylhydantoin in the Medium
Fsp . No .
Conc . Ratio rug/g he estoc \ug ml mediam (mean + SD)
Exp . No .
HPPH-G formed (umole/g hepatocyte) 1/4 hr
1 hr
2 hr
4 hr
Ia
O .OÎ
0.21
0 .59
1 .26
Ia
18 + 1* (n= 0)
Ib
o .08
0.26
0 .63
1 .14
Ib
20 + 1* (n= 0)
Ic
0.02
0.05
0 .15
----
Ic
22 + 3**(n= 6)
IIe
o .06
0.32
0 .62
1 .26
IIa
20 + 4* (n= 0)
IIb
o .02
0.10
0 .15
--
IIb
23 + 1**(n= 4)
* 1/4_4 hr, **1/4-2 hr, n : number of observations
Table 3 shows the distribution of DPH and HPPH.1} between hepatocytes and suspension medium .
The results show that DPH binds eztensively to cells and
that the distribution between cells sad medium appears constant for the duration of the incubation and over the DPH concentration range of 0 .01-40 ue/ml .
Thus,
the initial uptake of DPH by cells appears complete by the first sampling . In conclusion, isolated rat hépatocytes prepared by the method of Jeejeebhoy et al (4) metabolize DPH et rates comparable to those in microeomal preparation with as excess of cofactors .
DPH metabolism was ceaeeatratioa-dependent but
not of e simple Michaelis-Meaten type .
DPH binds extensively to hepatocytes
and its distribution of DPH between the hepatocytes and the suspension medium was independent of DPH concentrations . We wish to thank Dr . Yedy Israel for his helpful and constructive criticism. Studies were financed partialltiy by a grant from Hoffmean-La Roche Ltd ., Montreal, Canada and the Medical Research Council (Great l~MA-4263 and MT3204), Ottawa, Canada .
1232
Drug Metabolism by Isolated Hepatocytea
Vol . 16, No . 8
References 1.
J.R . GILLETTE, in Fundamentals of D Metabolism and Dr Dia Bition, (Eds . B.N . LaDu, H.G . Mandel sad E.L . Way , p . 00, Williams and Wilkias, Baltimore (1971) .
2.
M.N . BERRY aad D .S . FRISND, J. Cell Biol ., ~, 506 (1969) .
3.
M.N . BERRY, Biochem . J ., 1~, 40p (1971) .
4.
K.N . JEEJEEBHOY, J .HO ., G .R . GRF~~G, M.J . PHILLIPS, A.A . BRUCEROHERTSON and U. SODTKE, Biochem, J ., 146, 141 (1975) "
5"
N. GERBER, W.L . WELLER, R. LYNN, R .E . RANGNO, B .J . SWEEPMAN and M.T . HUSH, J . Pharmacol . Exp . Ther ., 178 (1971) .
6.
N. GERBER and J.G . WAGNER, Research Comm . Chem . Pathol . Pharmacol ., 455 (1972) .
7.
D . PERRIER, J.J . ASHLEY and G. LEVY, J . Pharmacokia . Biopharm ., 1, 231 (1972) .
8.
T. INABA and T . UMEDA, Drug Metabolism and Disposition, (1975) in press .
9.
J.J . ASHLEY and G. LEVY, J . Pharmacokin . Biopharm ., 1, 99 (1973) .
10 .
L. ENDRENYI and F.H .F . KWONG, in sis aad Simulation of Biochemical S sy tens (Eds . H.C . Henker and B . Hess p .219, North-Holland, Amsterdam (1972) .
11 .
H. KUTT and K. VEREBELY, Biochem . Pharmacol ., 12, 675 (1970) "