- Email: [email protected]
Uptake of dieldrin, lindane, and DDT by isolated rat hepatocytes
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
23, 116-122 (1985)
Uptake of Dieldrin, Lindane, and DDT by isolated Rat Hepatocytes REIJI ICHINOSE Radioisotope
Research
AND NORIO
Center,
Kyoto
KURIHARA’
University,
Kyoto
606, Japan
Received March 16, 1984; accepted May 22, 1984 Studies of the uptake of dieldrin, lindane, and DDT by isolated rat hepatocytes in KrebsHenseleit salt solution containing 2.34% bovine serum albumin have shown that the partition equilibrium of these hydrophobic organochlorine compounds is established very rapidly between the hepatocytes and the extracellular medium by reversible uptake and release processes. In the hepatocyte suspension system, these compounds are in dynamic partition equilibrium between the hepatocytes and the Krebs-Henseleit salt solution, and also between the bovine serum albumin molecules and the solution. The respective rate constants for the uptake and release processes were 0.83 and 2.52 x 10m2 set-t in a 2-ml suspension containing lo6 cells. It has thus been demonstrated that the transport of hydrophobic organochlorine compounds between the hepatocytes and the extracellular medium is a much faster process than the metabolic transformation reaction in hepatocytes. 0 1985 Academic Press. Inc INTRODUCTION
Isolated hepatocytes recently have been introduced for the study of the xenobiotic metabolism (l-3). The technique using cells has the advantage of retaining the biochemical composition and structural organization of the cells intact. When we study xenobiotic metabolism using isolated hepatocytes, it is necessary to investigate both biotransformation reactions and the uptake and release processes of the substrate and metabolites. There have been only a few pesticide studies that have investigated possible interactions between pesticide biotransformation in hepatocytes and the transport of the substrate and metabolites through the cell membrane (4-6). In a previous paper we described aldrin epoxidation to dieldrin in isolated rat hepatocytes (7). Here we report the partition equilibrium of dieldrin, which is much more resistant to biotransformation than aldrin, as well as the partition equilibria of some other organochlorine compounds in hepatocyte suspension. Factors that affect the partition equilibrium between the hepatot To whom correspondence should be addressed.
and reprint
requests
cytes and the extracellular cussed. MATERIALS
Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Compounds. Aldrin and dieldrin were purchased from Wakenyaku Company Ltd. Lindane and DDT were prepared as described (8, 9). These compounds were recrystallized until their purities were 99.5% or more by gas chromatography. Other compounds were the highest purity commercially available. Determination of octanollwater partition coefficient. Partition coefficients by using the 1-octanoywater system for the insecticides were determined experimentally according to the usual flask-shaking method (8). Isolation of rat hepatocytes. Hepatocytes were prepared from male Wistar rats weighing 200 to 250 g that had been maintained on laboratory chow (Oriental Yeasts Co. Ltd.). The method of hepatocyte isolation has been described elsewhere (7). Hepatocytes were suspended in KrebsHenseleit salt solution (1) containing 2.34% bovine serum albumin, which was used throughout the present study as the standard incubation medium. The cell suspen116
0048-3575185 $3.00
medium are dis-
UPTAKE
OF
DIELDRIN
sion at a concentration of IO6 cells/ml was stored with gas (95% O,, 5% CO,) gently introduced onto its surface at room temperature before use. Cell viability, determined by trypan blue exclusion, was more than 90%. Compound uptake by hepatocytes. The suspension of lo6 isolated hepatocytes in 2 ml of medium was shaken gently in a glassstoppered tube for 5.5 min, and then centrifuged at 40g for 1 min to separate the cells. The incubation temperature was 37°C throughout the experiments. After the cell pellet had been resuspended in medium (4 ml unless otherwise noted), the substrate (1.8 to 18.0 nmol), in 10 ~1 ethanol, was added to the suspension and shaking was begun. Incubation was performed for the various periods specified. At the end of incubation, the cell suspension was centrifuged at 8Og for 15 sec. The distribution of the compound between the hepatocytes and the extracellular medium was determined as described below. Compound
release by hepatocytes.
Iso-
lated hepatocytes (106) were incubated for 0.5 min as in the above experiments in a glass-stoppered tube containing 2 ml of medium, and then the substrate (3.2 to 40.0 nmol), in 10 ~1 ethanol, was added to the cell suspension. After the 5-min incubation (uptake of the compound), the cells were separated by centrifugation at 40g for 1 min, resuspended in 4 ml of medium, and incubated again for the various periods specified (release of the compound). At the end of this incubation, the cell suspension was centrifuged at 8Og for 15 sec. The distribution of the compound between the hepatocytes and the extracellular medium was determined as described below. Analytical procedures. The compound present in the medium after separation of the cells was extracted with a mixture of hexane (4 ml) and ethyl acetate (1 ml). The compound present in the cells was extracted as above after the cell pellet had been resuspended in water. The extract produced was diluted with hexane and then
BY
RAT
117
HEPATOCYTES
analyzed by gas chromatography. Recovery of the compound after incubation was more than 90%. The GC used 1% OV-17 on Chromosorb W AW DMCS (1.5 m). The column temperature was between 170” and 210°C. The carrier gas was N, of high purity, and the detector was an electron capture device (63Ni). Pharmacokinetic analysis. Using the data obtained in uptake experiments, a pharmacokinetic analysis with dieldrin was made based on a closed two-compartment model which includes a cellular compartment, 1, and an extracellular medium compartment, 2. The rate constant k,2 represents cellular release and kZ1 cellular uptake. Q(t) is the amount of compound at time t. The differential equation for compartment 1 (cellular compartment) is dQ,ldt
= - k,,Ql (t) + k2,Q2 (t).
[II
Under conditions Q,(O) = 0 and Qtotal = Q, (t) + Q2 (t), the solution for Eq. [I] is given by
lnklQtot,~ - (4, + MQ1 (t)l = -WI2 + k2,)t + ln[k21Q,,,,~l. VI After compound distribution equilibrium between the cells and the extracellular medium, Eq. [3] is obtained.
dQ, trnYdt
= k21Qtota1 - (k,, + k2,)QI (~1 = 0 [31
Combining
Eqs. [2] and [3],
ln[Q, (~0) - Q,
(t)] = -
(k,? + k21)t
+ ln[Q, (=)I. [41 By substituting experimental values into Eq. [4], (k,, + k,,) can be calculated and, as Q, (m)/Q, (x) = k2,1k12, kzl and k12 can be determined. RESULTS
AND
DISCUSSION
General features. When dieldrin was incubated for a few minutes in medium with rat hepatocytes, it was resistant to biotransformation and no metabolites were detected. Uptake of dieldrin into the hepato-
118
ICHINOSE
Time
AND
(min)
Time course of the partition of dieldrin between the hepatocytes and the extracellular medium. Dieldrin (I.8 nmo/) in ethanol (10 +I) was incubated at 37°C with 106 cells in 4 ml of medium. The amount of dieldrin in the hepatocytes (O), in the extracellular medium (A), and the incubation period, are indicated in the figure. Each point represents the average triplicate runs with a standard deviation of less than 6% of the value; one typical experiment out of six is shown. For each of the other concentrations of dieldrin (4.6, 9.0, 10.0, and 18.0 nmol in 4 ml), several runs were conducted which gave time courses similar to that in the figure. FIG.
1.
KURIHARA
FIG. 2. Plot of IIP,,, values against the concentration of bovine serum albumin. Dieldrin (10 nmol) in ethanol (10 (.~l) was incubated for 10 min at 37°C in 2 ml of medium containing lo6 cells. Each point represents the average of triplicate runs with a standard deviation of less than 8% of the value. The value 69,000 was adopted as the molecular weight of bovine serum albumin. Pcim is the partition coefficient value between the cells and the medium.
the whole hepatocyte as one phase in our experiments. This treatment is supported in our later discussion and by the data shown in Fig. 2. To calculate the concentration of organochlorine compound in the hepatocytes, we used the average hepatocyte cytes was very rapid (Fig. 1). In 30 set volume of 6186 km3 (10). about 60% of the cellular content at equiDependence of the partition coefficient librium was reached. Equilibration of the between the hepatocytes and the extracelpartition of dieldrin between the hepatoMar medium on the concentration of bocytes and the extracellular medium was vine serum albumin. We used Krebs-Hensereached in less than 5 min. leit salt solution containing 2.34% bovine We defined the partition coefficient (PC,,,,) serum albumin as the standard medium. as the concentration ratio between the he- Hydrophobic compounds in blood are patocytes and the extracellular medium at known to bind to albumin molecules by equilibrium. The value PC,,,, has the same nonspecific, reversible processes. The meaning as the distribution quotient de- binding of dieldrin to bovine serum albumin fined by Nakatsugawa et al. (4, 5). We appears to be an important factor that afprefer the name “partition coefficient” in fects the distribution of the pesticide bethis uptake-release equilibrium because tween the hepatocytes and the extracellular this equilibrium can be considered a purely medium. When the concentration of bovine physicochemical partition equilibrium, at serum albumin was increased in the extraleast in the range of compound concentracellular medium, the Pclm value decreased tions used in this study. (Fig. 2 and the discussion that follows Although the intracellular compound here). This decrease in the PC,,,,value must should be in partition equilibrium among be due to an increase in the albumin-bound dieldrin fraction. Nakatsugawa et al. rethe various intracellular particles, macromolecules, and the cytosol, we could treat ported that the ratio of the intra- and extra-
UPTAKE
OF DIELDRIN
cellular concentrations of parathion decreased when horse serum albumin was added to the hepatocyte suspension (4, 5). Partition equilibrium among the three components; hepatocytes, bovine serum albumin, and Krebs-Henseleit salt solution.
We examined the partition of dieldrin with a model composed of three components; hepatocytes, bovine serum albumin, and Krebs-Henseleit salt solution. Let V, be the volume of the extracellular medium, V, the total volume of the cells, Q, the amount of dieldrin in the cells, Qr, the amount of dieldrin bound to the protein, bovine serum albumin, Q, the amount of free dieldrin bound neither to bovine serum albumin molecules nor to the hepatocytes, C, the average concentration of dieldrin bound to bovine serum albumin in the medium, C, the concentration of free dieldrin, K, the apparent association constant between free dieldrin and that bound to bovine serum albumin molecules, and [BSA] the concentration of bovine serum albumin in the medium. If the simple partition equilibria of the compound exist between the hepatocytes and the Krebs-Henseleit salt solution and between the bovine serum albumin molecules and the Krebs-Henseleit salt solution, Eqs. [5] and [6] apply: K, =
C&J
C, x [BSA]
119
BY RAT HEPATOCYTES
151
TABLE I Coefficients between the Hepatocytes and the Extracellular Medium at Various Dieldrin Concentrations” Initial concentration of dieldrin b ((LM PC/III
Partition
0.45 1.15 2.25 2.50 4.50
287.5
i 14.5’ 322.5 271.6 IT 49.3’ 293.7 261.7
(n = 6)” (n = I)
(n = 3) (n =
1)
(n = 1)
(1Dieldrin (various amounts) in ethanol (10 ~1) was incubated for 10 min at 37°C in 4 ml of medium with lo6 cells. b PC/, is the coefficient value between the cells and the medium. c Average value of independent runs k SD. d n is the number of independent experimental runs.
equilibrium state between the hepatocytes and the Krebs-Henseleit salt solution and between the bovine serum albumin molecules and the Krebs-Henseleit salt solution. Compound binding to bovine serum albumin affected the amount of free dieldrin available for equilibration with the rest of the system. Relation of the partition coefficient between the hepatocytes and the extracellular medium to the hydrophobicity of organochlorine compounds. The partition coeffi-
cient should be an inherent value that is independent of the initial concentration of and the compound used if the partition equilibrium in the suspension medium is based Qc Qf + Qp P c,m = -7 [61 purely on physicochemical processes. vm . vc Table 1 shows that this was the case. Pcim From these equations and the relations values determined with various concentraQJV, = C,, QflV, = C,, and Q,lV,,, = C,, tions of dieldrin were essentially constant. we obtain Hepatocytes did not seem to be saturated with dieldrin up to the initial concentration -=-1 ~Cf + TK&f [BSA]. [71 of 4.50 /JM. n ‘c/m Lc Lc To examine the relation between the parThe plot of l/P,,, values against the con- tition of a hydrophobic compound in a hecentration of bovine serum albumin was patocyte suspension and its physicochemlinear, as expected from Eq. [7]; results are ical properties, we determined the Pcim shown in Fig. 2. Consequently, dieldrin in values of lindane and DDT as well as then this system must be in a dynamic partition octanol/water partition coefficients. The
120
ICHINOSE
AND KURIHARA TABLE
Partition
Coefficients
Compound Lindane Dieldrin DDT
of Organochlorine
2
Compounds in the HeputocyteslExtracellular the I-OctanoliWater System
Medium
System
P c/mu
log PC/m
log P
135.0 2 30.5’(n = 4)d 285.0 e 27.8 (n = 12) 317.5 2 70.5 (n = 2)
2.13 2.45 2.50
3.72’ 5.13 6.29f
and in
0 The Pcim value of each compound was determined by uptake studies. The compound in ethanol (10 ~1) was incubated for 10 min at 37°C in 4 ml of medium with IO6 cells. The initial concentration of the substrate used ranged from 0.45 to 4.50 PM. P,,, is the partition coefficient value between the cells and the medium. b P is the oartition coefficient value between I-octanol and water. c Average -value of independent runs -+ SD. d n is the number of independent experimental runs ’ Data from Ref. (8). f Data from Ref. (9).
partition equilibria of these compounds between the hepatocytes and the extracellular medium were attained in less than 10 min. The average PC,,,, values are listed in Table 2. Lindane and DDT were metabolized very slowly as was dieldrin in the hepatocyte suspension (7), and their PC,,,, values also were easily determined. Log P values of lindane, dieldrin, and DDT with corresponding log Pclm values are shown in Table 2. The compounds studied were very hydrophobic, as seen in the table. Although the data given are limited to only three compounds, a positive correlation was apparent. The partition equilibrium between the hepatocytes and the extracellular medium seems to depend on the physicochemical properties of the hydrophobic compound tested. The variation of log Pclm was small compared to that of log P. Probably this is because hydrophobic binding to bovine serum albumin molecules in the medium offset the PC,, values by reducing the amount of free organochlorine compound available for the hepatocytes. Reversibility. A dilution experiment was performed to examine the reversibility of dieldrin uptake by hepatocytes. A set of three experimental conditions (A, B, and C) was applied to a single preparation of hepatocytes to ensure that the hepatocytes properties would be as identical as possible. In experiment (A), 2 ml of medium containing lo6 cells was incubated with
dieldrin for 5 min, and then the suspension was diluted twofold with the same medium and incubated for another 5 min. In experiment (B), 4 ml of medium containing IO6 cells was incubated with dieldrin for 10 min. In experiment (C), 2 ml of medium containing lo6 cells was incubated with dieldrin for 5 min. The concentration of dieldrin was analyzed for both the hepatocytes and the extracellular medium. We obtained the same Pcim value in each run (Table 3). This shows that the partition equilibrium of dieldrin between the hepatocytes and the extracellular medium is the result of reversible uptake and release processes. A similar reversibility has been reported for parathion by Nakastugawa et al. (4). Rate of uptake
and biotransformation.
The respective rate constants for the uptake and release processes were calculated as 0.83 (? 0.13) x IO-* set-’ (n = 8) and 2.52 (20.68) x IO-* see-’ (n = 8) when dieldrin was incubated with lo6 cells in 4 ml of medium. The rate for dieldrin in the cellular uptake processes calculated on the amount basis was 0.83 x lo-* x (4012) x 4 = 0.664 nmol set- 1 when 40 nmol of the compound was incubated with lo6 cells in 2 ml of medium. The corresponding uptake rate of aldrin could not be determined accurately because of its fairly rapid conversion to dieldrin but it was not much different from that of dieldrin. The rate of aldrin epoxidation was 1.36 nmol mini, that is,
UPTAKE
OF DIELDRIN
TABLE Reversibility
Hepatocyte preparation 1 2
of Dieldrin
3
Uptake
by Hepatocytes P clmO
Dieldrin (nmol) 1.8 10.0
121
BY RAT HEPATOCYTES
Ah 291.2 267.0
B’
t 8.7 ? 4.8
263.9 240.8
k
C” 2.8
270.9 240.4
f 12.1
2 3.0 -t 1.2
u Average value k SD of triplicate runs. b After dieldrin (1.8 or 10.0 nmol) in ethanol (10 ~1) had been incubated for 5 min at 37°C in 2 ml of medium with lo6 cells, the suspension was diluted with fresh medium and incubated for another 5 min. c Dieldrin (1.8 or 10.0 nmol) in ethanol (10 ~1) was incubated for 10 min at 37°C in 4 ml of medium with 10’ cells. ’ Dieldrin (1.8 or 10.0 nmol) in ethanol (10 ~1) was incubated for 5 min at 37°C in 2 ml of medium with IOh cells.
0.023 nmol set- ’ when 40 nmol of aldrin was incubated in 2 ml of medium containing lo6 cells (7). Obviously the rate for dieldrin in the cellular uptake process and, hence, that of aldrin was much faster than the rate of aldrin epoxidation to dieldrin. The epoxidation of aldrin is one of the fastest metabolic transformation processes among the biotransformations of highly lipophilic xenobiotics; therefore, the transport of the highly lipophilic compounds into hepatocytes does not seem to be a rate-limiting step in the overall biochemical processes in this system. Release experiments. Vonk et al. reported the release of dibromosulfophthalein, indocyanine green, and N4-acetyl procainamide ethobromide from hepatocytes that previously had been incubated with those compounds (11). We made release studies of dieldrin, lindane, and DDT, all of which are much more hydrophobic than the above compounds. The time course of dieldrin release by hepatocytes is shown in Fig. 3. Final equilibrium was reached in less than 10 min. At this equilibrium, we determined the partition coefficient between the hepatocytes and the extracellular medium as described for the uptake studies. Pcim values obtained in the release studies are shown in Table 4. The P c,m value of lindane measured in the release studies agreed with the value found in the uptake studies. For dieldrin and DDT there were differences between the PC,,,,
values from the uptake studies and those from the release studies (Tables 2 and 4), but identical partition equilibrium states between the hepatocytes and the extracellular medium were considered to be attained quickly in both the uptake and release pro-
Time
(mid
3. Release of dieldrin from isolated hepatocytes. Dieldrin (4.6 nmofl in ethanol (10 ~1) was incubated for 5 min at 37°C in 2 ml of medium with IO6 cells. Then, the cells were collected by gentle centrifugation and resuspended in 4 ml of medium, ufter which they were incubated further. The amount of dieldrin in the hepatocyres (0) and in the extracellular medium (A). and the time after resuspension, are indicated in the figure. The cellular amount of the compound at t = 0 was 2.28 nmoll106 cells. Each point represents the average of triplicate runs, with a standard deviation of less than 80/G of the value. FIG.
122
ICHINOSE
AND KURIHARA
TABLE 4 Partition Coefficients between the Hepatocytes and the Extracellular Medium Obtained in Release Studies0 Compound Lindane Dieldrin DDT
P elm 148.1 ? 19.0b 400.6 k 56.3 648.9
(n = 4)c (n = 16) (n = 1)
a Each compound (3.2 to 40.0 nmol) in ethanol (10 ~1) was incubated for 5 min at 37°C in 2 ml of medium with lo6 cells. After this uptake period, the cells were collected by gentle centrifugation and resuspended in 4 ml of medium (time 0 of the release). This suspension was incubated for an additional 10 min. Cells loaded with the compound contained the following amounts at time 0 of the release: lindane between 1.16 and 11.20, dieldrin between 1.07 and 8.84, and DDT 7.04 nmol/106 cells. b Average value of independent runs * SD. c n is the number of independent experimental runs.
cesses. In fact, the reversibility of these processes was proved by the dilution experiments described. The reason for apparent irreversibility with dieldrin and DDT is not clear, although we have conducted experiments using various combinations of uptake and release (uptake-release-uptake, repeated release, etc.). One possible explanation is that a change in the organization of the hepatocyte occurred due to the loading with a very hydrophobic compound (e.g., that of log P > 5) followed by centrifugation, and thus the partition equilibrium shifted. We have shown quantitatively that the rate of transport of hydrophobic organochlorine compounds between the hepatocytes and the extracellular medium is much faster than the rate of their biotransformation by the hepatocytes. Also, that the compound partition equilibrium exists between the hepatocytes and the Krebs-Henseleit salt solution and between the bovine serum albumin and the bulk solution.
REFERENCES 1. P. Mold&s, J. Hogberg, and S. Orrenius, Isolation and use of liver cells, in “Methods in Enzymology” (S. Fleischer and L. Packer, Eds.), Vol. 52, p. 60, Academic Press, New York, 1978. 2. C. D. Klaassen and N. H. Stacey, Use of isolated hepatocytes in toxicity assessment, in “Toxicology of the Liver” (G. Plaa and W. R. Hewitt, Eds.), p. 147, Raven Press, New York, 1982. 3. R. G. Thurman and E C. Kauffman, Factors regulating drug metabolism in intact hepatocytes, Pharmacol. Rev. 31, 229 (1980). 4. T. Nakatsugawa, W. L. Bradford, and K. Usui, Hepatic disposition of parathion. Uptake by isolated hepatocytes and chromatographic translobular migration, Pestic. Biochem. Physiol. 14, 13 (1980). 5. T. Nakatsugawa, and S. Tsuda, Metabolism studies with liver homogenate, hepatocyte suspension and perfused liver, in “Pesticide Chemistry: Human Welfare and the Environment (Proceedings of the 5th International Congress of Pesticide Chemistry)” (J. Miyamoto and P. C. Keamey, Eds.), Vol. 3, p. 395, Pergamon, Oxford, 1983. 6. N. S. E. Sargent, D. G. Upshall and J. W. Bridges, The relationship between binding to cytochrome P-450 and metabolism of n-alkyl carbamates in isolated rat hepatocytes, Biochem. Pharmacol. 31, 1309 (1982). 7. N. Kurihara, N. Hori, and R. Ichinose, Cytochrome P-450 content and aldrin epoxidation to dieldrin in isolated rat hepatocytes, Pestic. Biochem. Physiol. 21, 63 (1984). 8. N. Kurihara and T. Fujita, Composition of partition coefficients of polychlorinated hydrocarbons, Bull. Inst. Chem. Res. Kyoto Univ. 61, 89 (1983). 9. K. Nishimura and T. Fujita, Quantitative structure-activity relationships of DDT and its related compounds, J. Pestic. Sci. 8, 69 (1983). 10. P. Drochmans, J.-C. Wanson, and R. Mosselmans, Isolation and subfractionation on Ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fraction, J. Cell Biol. 66, 1 (1975). 11. R. J. Vonk, P. A. Jekel, D. K. E Meijer, and M. J. Hardonk, Transport of drugs in isolated hepatocytes. The influence of bile salts, Biochem. Pharmacol. 27, 397 (1978).
BIOCHEMISTRY
AND
PHYSIOLOGY
23, 116-122 (1985)
Uptake of Dieldrin, Lindane, and DDT by isolated Rat Hepatocytes REIJI ICHINOSE Radioisotope
Research
AND NORIO
Center,
Kyoto
KURIHARA’
University,
Kyoto
606, Japan
Received March 16, 1984; accepted May 22, 1984 Studies of the uptake of dieldrin, lindane, and DDT by isolated rat hepatocytes in KrebsHenseleit salt solution containing 2.34% bovine serum albumin have shown that the partition equilibrium of these hydrophobic organochlorine compounds is established very rapidly between the hepatocytes and the extracellular medium by reversible uptake and release processes. In the hepatocyte suspension system, these compounds are in dynamic partition equilibrium between the hepatocytes and the Krebs-Henseleit salt solution, and also between the bovine serum albumin molecules and the solution. The respective rate constants for the uptake and release processes were 0.83 and 2.52 x 10m2 set-t in a 2-ml suspension containing lo6 cells. It has thus been demonstrated that the transport of hydrophobic organochlorine compounds between the hepatocytes and the extracellular medium is a much faster process than the metabolic transformation reaction in hepatocytes. 0 1985 Academic Press. Inc INTRODUCTION
Isolated hepatocytes recently have been introduced for the study of the xenobiotic metabolism (l-3). The technique using cells has the advantage of retaining the biochemical composition and structural organization of the cells intact. When we study xenobiotic metabolism using isolated hepatocytes, it is necessary to investigate both biotransformation reactions and the uptake and release processes of the substrate and metabolites. There have been only a few pesticide studies that have investigated possible interactions between pesticide biotransformation in hepatocytes and the transport of the substrate and metabolites through the cell membrane (4-6). In a previous paper we described aldrin epoxidation to dieldrin in isolated rat hepatocytes (7). Here we report the partition equilibrium of dieldrin, which is much more resistant to biotransformation than aldrin, as well as the partition equilibria of some other organochlorine compounds in hepatocyte suspension. Factors that affect the partition equilibrium between the hepatot To whom correspondence should be addressed.
and reprint
requests
cytes and the extracellular cussed. MATERIALS
Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Compounds. Aldrin and dieldrin were purchased from Wakenyaku Company Ltd. Lindane and DDT were prepared as described (8, 9). These compounds were recrystallized until their purities were 99.5% or more by gas chromatography. Other compounds were the highest purity commercially available. Determination of octanollwater partition coefficient. Partition coefficients by using the 1-octanoywater system for the insecticides were determined experimentally according to the usual flask-shaking method (8). Isolation of rat hepatocytes. Hepatocytes were prepared from male Wistar rats weighing 200 to 250 g that had been maintained on laboratory chow (Oriental Yeasts Co. Ltd.). The method of hepatocyte isolation has been described elsewhere (7). Hepatocytes were suspended in KrebsHenseleit salt solution (1) containing 2.34% bovine serum albumin, which was used throughout the present study as the standard incubation medium. The cell suspen116
0048-3575185 $3.00
medium are dis-
UPTAKE
OF
DIELDRIN
sion at a concentration of IO6 cells/ml was stored with gas (95% O,, 5% CO,) gently introduced onto its surface at room temperature before use. Cell viability, determined by trypan blue exclusion, was more than 90%. Compound uptake by hepatocytes. The suspension of lo6 isolated hepatocytes in 2 ml of medium was shaken gently in a glassstoppered tube for 5.5 min, and then centrifuged at 40g for 1 min to separate the cells. The incubation temperature was 37°C throughout the experiments. After the cell pellet had been resuspended in medium (4 ml unless otherwise noted), the substrate (1.8 to 18.0 nmol), in 10 ~1 ethanol, was added to the suspension and shaking was begun. Incubation was performed for the various periods specified. At the end of incubation, the cell suspension was centrifuged at 8Og for 15 sec. The distribution of the compound between the hepatocytes and the extracellular medium was determined as described below. Compound
release by hepatocytes.
Iso-
lated hepatocytes (106) were incubated for 0.5 min as in the above experiments in a glass-stoppered tube containing 2 ml of medium, and then the substrate (3.2 to 40.0 nmol), in 10 ~1 ethanol, was added to the cell suspension. After the 5-min incubation (uptake of the compound), the cells were separated by centrifugation at 40g for 1 min, resuspended in 4 ml of medium, and incubated again for the various periods specified (release of the compound). At the end of this incubation, the cell suspension was centrifuged at 8Og for 15 sec. The distribution of the compound between the hepatocytes and the extracellular medium was determined as described below. Analytical procedures. The compound present in the medium after separation of the cells was extracted with a mixture of hexane (4 ml) and ethyl acetate (1 ml). The compound present in the cells was extracted as above after the cell pellet had been resuspended in water. The extract produced was diluted with hexane and then
BY
RAT
117
HEPATOCYTES
analyzed by gas chromatography. Recovery of the compound after incubation was more than 90%. The GC used 1% OV-17 on Chromosorb W AW DMCS (1.5 m). The column temperature was between 170” and 210°C. The carrier gas was N, of high purity, and the detector was an electron capture device (63Ni). Pharmacokinetic analysis. Using the data obtained in uptake experiments, a pharmacokinetic analysis with dieldrin was made based on a closed two-compartment model which includes a cellular compartment, 1, and an extracellular medium compartment, 2. The rate constant k,2 represents cellular release and kZ1 cellular uptake. Q(t) is the amount of compound at time t. The differential equation for compartment 1 (cellular compartment) is dQ,ldt
= - k,,Ql (t) + k2,Q2 (t).
[II
Under conditions Q,(O) = 0 and Qtotal = Q, (t) + Q2 (t), the solution for Eq. [I] is given by
lnklQtot,~ - (4, + MQ1 (t)l = -WI2 + k2,)t + ln[k21Q,,,,~l. VI After compound distribution equilibrium between the cells and the extracellular medium, Eq. [3] is obtained.
dQ, trnYdt
= k21Qtota1 - (k,, + k2,)QI (~1 = 0 [31
Combining
Eqs. [2] and [3],
ln[Q, (~0) - Q,
(t)] = -
(k,? + k21)t
+ ln[Q, (=)I. [41 By substituting experimental values into Eq. [4], (k,, + k,,) can be calculated and, as Q, (m)/Q, (x) = k2,1k12, kzl and k12 can be determined. RESULTS
AND
DISCUSSION
General features. When dieldrin was incubated for a few minutes in medium with rat hepatocytes, it was resistant to biotransformation and no metabolites were detected. Uptake of dieldrin into the hepato-
118
ICHINOSE
Time
AND
(min)
Time course of the partition of dieldrin between the hepatocytes and the extracellular medium. Dieldrin (I.8 nmo/) in ethanol (10 +I) was incubated at 37°C with 106 cells in 4 ml of medium. The amount of dieldrin in the hepatocytes (O), in the extracellular medium (A), and the incubation period, are indicated in the figure. Each point represents the average triplicate runs with a standard deviation of less than 6% of the value; one typical experiment out of six is shown. For each of the other concentrations of dieldrin (4.6, 9.0, 10.0, and 18.0 nmol in 4 ml), several runs were conducted which gave time courses similar to that in the figure. FIG.
1.
KURIHARA
FIG. 2. Plot of IIP,,, values against the concentration of bovine serum albumin. Dieldrin (10 nmol) in ethanol (10 (.~l) was incubated for 10 min at 37°C in 2 ml of medium containing lo6 cells. Each point represents the average of triplicate runs with a standard deviation of less than 8% of the value. The value 69,000 was adopted as the molecular weight of bovine serum albumin. Pcim is the partition coefficient value between the cells and the medium.
the whole hepatocyte as one phase in our experiments. This treatment is supported in our later discussion and by the data shown in Fig. 2. To calculate the concentration of organochlorine compound in the hepatocytes, we used the average hepatocyte cytes was very rapid (Fig. 1). In 30 set volume of 6186 km3 (10). about 60% of the cellular content at equiDependence of the partition coefficient librium was reached. Equilibration of the between the hepatocytes and the extracelpartition of dieldrin between the hepatoMar medium on the concentration of bocytes and the extracellular medium was vine serum albumin. We used Krebs-Hensereached in less than 5 min. leit salt solution containing 2.34% bovine We defined the partition coefficient (PC,,,,) serum albumin as the standard medium. as the concentration ratio between the he- Hydrophobic compounds in blood are patocytes and the extracellular medium at known to bind to albumin molecules by equilibrium. The value PC,,,, has the same nonspecific, reversible processes. The meaning as the distribution quotient de- binding of dieldrin to bovine serum albumin fined by Nakatsugawa et al. (4, 5). We appears to be an important factor that afprefer the name “partition coefficient” in fects the distribution of the pesticide bethis uptake-release equilibrium because tween the hepatocytes and the extracellular this equilibrium can be considered a purely medium. When the concentration of bovine physicochemical partition equilibrium, at serum albumin was increased in the extraleast in the range of compound concentracellular medium, the Pclm value decreased tions used in this study. (Fig. 2 and the discussion that follows Although the intracellular compound here). This decrease in the PC,,,,value must should be in partition equilibrium among be due to an increase in the albumin-bound dieldrin fraction. Nakatsugawa et al. rethe various intracellular particles, macromolecules, and the cytosol, we could treat ported that the ratio of the intra- and extra-
UPTAKE
OF DIELDRIN
cellular concentrations of parathion decreased when horse serum albumin was added to the hepatocyte suspension (4, 5). Partition equilibrium among the three components; hepatocytes, bovine serum albumin, and Krebs-Henseleit salt solution.
We examined the partition of dieldrin with a model composed of three components; hepatocytes, bovine serum albumin, and Krebs-Henseleit salt solution. Let V, be the volume of the extracellular medium, V, the total volume of the cells, Q, the amount of dieldrin in the cells, Qr, the amount of dieldrin bound to the protein, bovine serum albumin, Q, the amount of free dieldrin bound neither to bovine serum albumin molecules nor to the hepatocytes, C, the average concentration of dieldrin bound to bovine serum albumin in the medium, C, the concentration of free dieldrin, K, the apparent association constant between free dieldrin and that bound to bovine serum albumin molecules, and [BSA] the concentration of bovine serum albumin in the medium. If the simple partition equilibria of the compound exist between the hepatocytes and the Krebs-Henseleit salt solution and between the bovine serum albumin molecules and the Krebs-Henseleit salt solution, Eqs. [5] and [6] apply: K, =
C&J
C, x [BSA]
119
BY RAT HEPATOCYTES
151
TABLE I Coefficients between the Hepatocytes and the Extracellular Medium at Various Dieldrin Concentrations” Initial concentration of dieldrin b ((LM PC/III
Partition
0.45 1.15 2.25 2.50 4.50
287.5
i 14.5’ 322.5 271.6 IT 49.3’ 293.7 261.7
(n = 6)” (n = I)
(n = 3) (n =
1)
(n = 1)
(1Dieldrin (various amounts) in ethanol (10 ~1) was incubated for 10 min at 37°C in 4 ml of medium with lo6 cells. b PC/, is the coefficient value between the cells and the medium. c Average value of independent runs k SD. d n is the number of independent experimental runs.
equilibrium state between the hepatocytes and the Krebs-Henseleit salt solution and between the bovine serum albumin molecules and the Krebs-Henseleit salt solution. Compound binding to bovine serum albumin affected the amount of free dieldrin available for equilibration with the rest of the system. Relation of the partition coefficient between the hepatocytes and the extracellular medium to the hydrophobicity of organochlorine compounds. The partition coeffi-
cient should be an inherent value that is independent of the initial concentration of and the compound used if the partition equilibrium in the suspension medium is based Qc Qf + Qp P c,m = -7 [61 purely on physicochemical processes. vm . vc Table 1 shows that this was the case. Pcim From these equations and the relations values determined with various concentraQJV, = C,, QflV, = C,, and Q,lV,,, = C,, tions of dieldrin were essentially constant. we obtain Hepatocytes did not seem to be saturated with dieldrin up to the initial concentration -=-1 ~Cf + TK&f [BSA]. [71 of 4.50 /JM. n ‘c/m Lc Lc To examine the relation between the parThe plot of l/P,,, values against the con- tition of a hydrophobic compound in a hecentration of bovine serum albumin was patocyte suspension and its physicochemlinear, as expected from Eq. [7]; results are ical properties, we determined the Pcim shown in Fig. 2. Consequently, dieldrin in values of lindane and DDT as well as then this system must be in a dynamic partition octanol/water partition coefficients. The
120
ICHINOSE
AND KURIHARA TABLE
Partition
Coefficients
Compound Lindane Dieldrin DDT
of Organochlorine
2
Compounds in the HeputocyteslExtracellular the I-OctanoliWater System
Medium
System
P c/mu
log PC/m
log P
135.0 2 30.5’(n = 4)d 285.0 e 27.8 (n = 12) 317.5 2 70.5 (n = 2)
2.13 2.45 2.50
3.72’ 5.13 6.29f
and in
0 The Pcim value of each compound was determined by uptake studies. The compound in ethanol (10 ~1) was incubated for 10 min at 37°C in 4 ml of medium with IO6 cells. The initial concentration of the substrate used ranged from 0.45 to 4.50 PM. P,,, is the partition coefficient value between the cells and the medium. b P is the oartition coefficient value between I-octanol and water. c Average -value of independent runs -+ SD. d n is the number of independent experimental runs ’ Data from Ref. (8). f Data from Ref. (9).
partition equilibria of these compounds between the hepatocytes and the extracellular medium were attained in less than 10 min. The average PC,,,, values are listed in Table 2. Lindane and DDT were metabolized very slowly as was dieldrin in the hepatocyte suspension (7), and their PC,,,, values also were easily determined. Log P values of lindane, dieldrin, and DDT with corresponding log Pclm values are shown in Table 2. The compounds studied were very hydrophobic, as seen in the table. Although the data given are limited to only three compounds, a positive correlation was apparent. The partition equilibrium between the hepatocytes and the extracellular medium seems to depend on the physicochemical properties of the hydrophobic compound tested. The variation of log Pclm was small compared to that of log P. Probably this is because hydrophobic binding to bovine serum albumin molecules in the medium offset the PC,, values by reducing the amount of free organochlorine compound available for the hepatocytes. Reversibility. A dilution experiment was performed to examine the reversibility of dieldrin uptake by hepatocytes. A set of three experimental conditions (A, B, and C) was applied to a single preparation of hepatocytes to ensure that the hepatocytes properties would be as identical as possible. In experiment (A), 2 ml of medium containing lo6 cells was incubated with
dieldrin for 5 min, and then the suspension was diluted twofold with the same medium and incubated for another 5 min. In experiment (B), 4 ml of medium containing IO6 cells was incubated with dieldrin for 10 min. In experiment (C), 2 ml of medium containing lo6 cells was incubated with dieldrin for 5 min. The concentration of dieldrin was analyzed for both the hepatocytes and the extracellular medium. We obtained the same Pcim value in each run (Table 3). This shows that the partition equilibrium of dieldrin between the hepatocytes and the extracellular medium is the result of reversible uptake and release processes. A similar reversibility has been reported for parathion by Nakastugawa et al. (4). Rate of uptake
and biotransformation.
The respective rate constants for the uptake and release processes were calculated as 0.83 (? 0.13) x IO-* set-’ (n = 8) and 2.52 (20.68) x IO-* see-’ (n = 8) when dieldrin was incubated with lo6 cells in 4 ml of medium. The rate for dieldrin in the cellular uptake processes calculated on the amount basis was 0.83 x lo-* x (4012) x 4 = 0.664 nmol set- 1 when 40 nmol of the compound was incubated with lo6 cells in 2 ml of medium. The corresponding uptake rate of aldrin could not be determined accurately because of its fairly rapid conversion to dieldrin but it was not much different from that of dieldrin. The rate of aldrin epoxidation was 1.36 nmol mini, that is,
UPTAKE
OF DIELDRIN
TABLE Reversibility
Hepatocyte preparation 1 2
of Dieldrin
3
Uptake
by Hepatocytes P clmO
Dieldrin (nmol) 1.8 10.0
121
BY RAT HEPATOCYTES
Ah 291.2 267.0
B’
t 8.7 ? 4.8
263.9 240.8
k
C” 2.8
270.9 240.4
f 12.1
2 3.0 -t 1.2
u Average value k SD of triplicate runs. b After dieldrin (1.8 or 10.0 nmol) in ethanol (10 ~1) had been incubated for 5 min at 37°C in 2 ml of medium with lo6 cells, the suspension was diluted with fresh medium and incubated for another 5 min. c Dieldrin (1.8 or 10.0 nmol) in ethanol (10 ~1) was incubated for 10 min at 37°C in 4 ml of medium with 10’ cells. ’ Dieldrin (1.8 or 10.0 nmol) in ethanol (10 ~1) was incubated for 5 min at 37°C in 2 ml of medium with IOh cells.
0.023 nmol set- ’ when 40 nmol of aldrin was incubated in 2 ml of medium containing lo6 cells (7). Obviously the rate for dieldrin in the cellular uptake process and, hence, that of aldrin was much faster than the rate of aldrin epoxidation to dieldrin. The epoxidation of aldrin is one of the fastest metabolic transformation processes among the biotransformations of highly lipophilic xenobiotics; therefore, the transport of the highly lipophilic compounds into hepatocytes does not seem to be a rate-limiting step in the overall biochemical processes in this system. Release experiments. Vonk et al. reported the release of dibromosulfophthalein, indocyanine green, and N4-acetyl procainamide ethobromide from hepatocytes that previously had been incubated with those compounds (11). We made release studies of dieldrin, lindane, and DDT, all of which are much more hydrophobic than the above compounds. The time course of dieldrin release by hepatocytes is shown in Fig. 3. Final equilibrium was reached in less than 10 min. At this equilibrium, we determined the partition coefficient between the hepatocytes and the extracellular medium as described for the uptake studies. Pcim values obtained in the release studies are shown in Table 4. The P c,m value of lindane measured in the release studies agreed with the value found in the uptake studies. For dieldrin and DDT there were differences between the PC,,,,
values from the uptake studies and those from the release studies (Tables 2 and 4), but identical partition equilibrium states between the hepatocytes and the extracellular medium were considered to be attained quickly in both the uptake and release pro-
Time
(mid
3. Release of dieldrin from isolated hepatocytes. Dieldrin (4.6 nmofl in ethanol (10 ~1) was incubated for 5 min at 37°C in 2 ml of medium with IO6 cells. Then, the cells were collected by gentle centrifugation and resuspended in 4 ml of medium, ufter which they were incubated further. The amount of dieldrin in the hepatocyres (0) and in the extracellular medium (A). and the time after resuspension, are indicated in the figure. The cellular amount of the compound at t = 0 was 2.28 nmoll106 cells. Each point represents the average of triplicate runs, with a standard deviation of less than 80/G of the value. FIG.
122
ICHINOSE
AND KURIHARA
TABLE 4 Partition Coefficients between the Hepatocytes and the Extracellular Medium Obtained in Release Studies0 Compound Lindane Dieldrin DDT
P elm 148.1 ? 19.0b 400.6 k 56.3 648.9
(n = 4)c (n = 16) (n = 1)
a Each compound (3.2 to 40.0 nmol) in ethanol (10 ~1) was incubated for 5 min at 37°C in 2 ml of medium with lo6 cells. After this uptake period, the cells were collected by gentle centrifugation and resuspended in 4 ml of medium (time 0 of the release). This suspension was incubated for an additional 10 min. Cells loaded with the compound contained the following amounts at time 0 of the release: lindane between 1.16 and 11.20, dieldrin between 1.07 and 8.84, and DDT 7.04 nmol/106 cells. b Average value of independent runs * SD. c n is the number of independent experimental runs.
cesses. In fact, the reversibility of these processes was proved by the dilution experiments described. The reason for apparent irreversibility with dieldrin and DDT is not clear, although we have conducted experiments using various combinations of uptake and release (uptake-release-uptake, repeated release, etc.). One possible explanation is that a change in the organization of the hepatocyte occurred due to the loading with a very hydrophobic compound (e.g., that of log P > 5) followed by centrifugation, and thus the partition equilibrium shifted. We have shown quantitatively that the rate of transport of hydrophobic organochlorine compounds between the hepatocytes and the extracellular medium is much faster than the rate of their biotransformation by the hepatocytes. Also, that the compound partition equilibrium exists between the hepatocytes and the Krebs-Henseleit salt solution and between the bovine serum albumin and the bulk solution.
REFERENCES 1. P. Mold&s, J. Hogberg, and S. Orrenius, Isolation and use of liver cells, in “Methods in Enzymology” (S. Fleischer and L. Packer, Eds.), Vol. 52, p. 60, Academic Press, New York, 1978. 2. C. D. Klaassen and N. H. Stacey, Use of isolated hepatocytes in toxicity assessment, in “Toxicology of the Liver” (G. Plaa and W. R. Hewitt, Eds.), p. 147, Raven Press, New York, 1982. 3. R. G. Thurman and E C. Kauffman, Factors regulating drug metabolism in intact hepatocytes, Pharmacol. Rev. 31, 229 (1980). 4. T. Nakatsugawa, W. L. Bradford, and K. Usui, Hepatic disposition of parathion. Uptake by isolated hepatocytes and chromatographic translobular migration, Pestic. Biochem. Physiol. 14, 13 (1980). 5. T. Nakatsugawa, and S. Tsuda, Metabolism studies with liver homogenate, hepatocyte suspension and perfused liver, in “Pesticide Chemistry: Human Welfare and the Environment (Proceedings of the 5th International Congress of Pesticide Chemistry)” (J. Miyamoto and P. C. Keamey, Eds.), Vol. 3, p. 395, Pergamon, Oxford, 1983. 6. N. S. E. Sargent, D. G. Upshall and J. W. Bridges, The relationship between binding to cytochrome P-450 and metabolism of n-alkyl carbamates in isolated rat hepatocytes, Biochem. Pharmacol. 31, 1309 (1982). 7. N. Kurihara, N. Hori, and R. Ichinose, Cytochrome P-450 content and aldrin epoxidation to dieldrin in isolated rat hepatocytes, Pestic. Biochem. Physiol. 21, 63 (1984). 8. N. Kurihara and T. Fujita, Composition of partition coefficients of polychlorinated hydrocarbons, Bull. Inst. Chem. Res. Kyoto Univ. 61, 89 (1983). 9. K. Nishimura and T. Fujita, Quantitative structure-activity relationships of DDT and its related compounds, J. Pestic. Sci. 8, 69 (1983). 10. P. Drochmans, J.-C. Wanson, and R. Mosselmans, Isolation and subfractionation on Ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fraction, J. Cell Biol. 66, 1 (1975). 11. R. J. Vonk, P. A. Jekel, D. K. E Meijer, and M. J. Hardonk, Transport of drugs in isolated hepatocytes. The influence of bile salts, Biochem. Pharmacol. 27, 397 (1978).