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Cytochrome P-450 content and aldrin epoxidation to dieldrin in isolated rat hepatocytes
PESTICIDE
BIOCHEMISTRY
Cytochrome
AND
PHYSIOLOGY
21, 63-73 (1984)
P-450 Content and Aldrin Epoxidation Isolated Rat Hepatocytes NORIO KURIHARA,~NOBUAKI Radioisotope
Research
Center.
Kyoto
to Dieldrin
in
HORI, AND REIJI ICHINOSE Universiiy,
h’yoto
606. Jtrpan
Received May 27. 1983: accepted July 22. 1983 A rat hepatocyte suspension effectively epoxidized aldrin to dieldrin with a V,,, of 7.19 mol/ mol P-450/min and a K,,, of 9.27 p,,W. Viability and metabolic activity were stable for 6 hr after isolation when cells were maintained at room temperature (20°C) with the gentle introduction of Oz/COZ onto the surface of the suspension. The cytochrome P-450 content of the suspension was 303 pmol/106 cells. Primary maintenance culture of the cells also epoxidized aldrin. During culture for 3 days, metabolic activity decreased slowly day by day. Metabolic activity of microsomal fraction from rat liver was also examined. Microsomes epoxidized aldrin with a V,,, of 5. I I moli mol P-450/min and a K, of 1.64 FM. Significant loss of some subspecies of cytochrome f-450 during fractionation of liver homogenate was indicated.
INTRODUCTION
sessment of in vivo metabolic activity from hepatocyte suspension-metabolism studies isolated rat hepatocytes are now widely is another important problem. used in xenobiotic metabolism studies. We here describe the metabolism of They retain both the compositions of their aldrin by an isolated rat hepatocyte suspenenzyme systems and cofactors, as well as sion and by a primary maintenance rat hemembranes and various particles related to patocyte culture. In the initial metabolic xenobiotic metabolism. Thus, they should phase of this substrate, an epoxidized be a better model of in Go metabolic sys- product, dieldrin, was almost the only tems than are liver homogenates or enzyme product. We compared the reaction rates in preparations from liver (1, 2). Although we these systems with the rates of a liver mimust carefully consider the spatial hetero- crosomal fraction, and examined the relageneity of cells in the whole organ (2) and tions between these rates and cytochrome compound transport within the organ (3- P-450 content which should have the most 5), use of isolated hepatocytes still is a important role in epoxidation. The uptake better model and a simpler system for pre- and release of these compounds by hepadicting in vivo xenobiotic metabolism from tocytes will be dealt with elsewhere. in vitro results. MATERIALS AND METHODS Much information on xenobiotic metabolism has been accumulated by use of the Chemicals. Aldrin and dieldrin were purisolated cell technique ((1, 2, 6) and refer- chased from Wakenyaku Company Ltd. ences given there). Some general problems Their purities were more than 99.5% on gas remain, however, in application of this chromatography. Organic solvents were of technique; the relation between overall reagent grade. Clostridium histolyticum metabolic activity and the enzyme content collagenase Class IV (Worthington) was of the cells, and the rate of uptake and re- purchased from Millipore Ltd., and Type lease of the compound by the cells. As- IV from Sigma Chemical Company Ltd., both through Wakenyaku Company. Fraction V of bovine serum albumin (BSA) was ’ To whom correspondence and reprint requests should be addressed. obtained from Armour Pharmaceutical 63 0048-3575/84 $3.00 Copyright
8 1984 by Academic
Press, Inc.
64
KURIHARA,
HORI,
Company. The rest of the chemicals used were of the highest purity commercially available.
AND
ICHINOSE
sion or introduced onto the surface of the suspension without bubbling. Method 121: Hepatocytes were isolated Preparation of hepatocyte suspension. with the Hanks- Wallace solution-based Male Wistar rats (ZOO-250 g) fed ad libitum perfusate. The liver was perfused with were used. Hepatocytes were obtained by Ca2+-free Hanks-Wallace solution cona collagenase perfusion method (1). taining 0.3 mM EGTA for 10 to 15 min at Method [l]: The liver was perfused in situ 30 to 50 ml/min, followed by brief perfusion for 5 to 10 min with Caz+-free Krebs-Henwith EGTA-free Hanks- Wallace solution. seleit solution containing 0.5 mM EGTA Recycling perfusion with collagenase solu(ethylene glycol bis(P-aminoethyl ether)tion was done for I5 to 20 min at 70 ml/min. N,N’-tetraacetic acid) and 2% BSA, at 30 This enzyme solution (Hanks- Wallace, 80 to 40 ml/min. After the liver had been freed ml) contained CaC12 (33.2 mg), dextran from the body, perfusion was continued for (MW 58,000, 3.9 g), and collagenase (40 to 1 min with EGTA-free Krebs-Henseleit so- 80 mg). Subsequent procedures were the lution (1) containing 1.5 mM Ca*+ and same as those described above with Krebs2.34% BSA (solution A) at 80 ml/min. Then Henseleit solution. Hanks-Wallace solution is said to be in recycling was done for 10 to 20 min with Krebs-Henseleit solution containing equilibrium with CO2 in ordinary air 0.08% collagenase, 1 n&f Ca2+, and 2.5% (0.03%) and as such was convenient for cell dextran at 80 to 90 ml/min. All perfusion isolation under sterilized conditions, but we media were maintained at 37°C and pH 7.4 frequently observed a large pH change during the isolation steps. Adjustment of with 95% 02/5% CO2 bubbled continuously pH, monitored by the dissolved indicator through them. When the liver started to swell and to phenol red, was made during the procedure by the injection of an appropriate concendisintegrate, it was perfused again briefly with solution A, after which it was trans- tration of aqueous NaHCOj solution. ferred to a small dish. The capsula was cut Primary culture of hepatocytes. All the open in 20 ml of solution A, and its cells isolation steps were done in a clean bench were dispersed by gentle stirring with a pair with sterilized Hanks- Wallace solution by of tweezers. At an ambient temperature method [2]. After the second centrifugation at 4Og, cells were resuspended in an appro(20-25”C), the dispersed cells were filtered through four layers of cotton gauze to re- priate volume of William’s medium (7) fortified with fetal calf serum (IO%), insulin move connecting tissue and nondispersed cells. The filtrate was centrifuged for 60 set ( 10e6 M), testosterone ( 10P6 M), estradiol (10 mg/liter), hyat 40g. The collected cells were resus- ( 10M6 M), DL-a-tocopherol pended and centrifuged again at the same drocortisone 21-p-acetate (lops M), and 6acid (10V6 M). The comspeed, and then resuspended and diluted to aminolevulinic pounds were added in ethanol solution, the a concentration of IO6 cells/ml in solution volume of the ethanol being less than 3 ml/ A (BSA was 2.34% unless otherwise noted). Cell numbers were counted in a liter of medium. A suspension of IO6 cells/ml medium was Buerker chamber. The trypan blue-exclusion test was used to estimate cell viability. prepared, and l-ml portions of it were In the metabolic study, cells of 90 to 95% transferred to plastic dishes 35 mm in diameter (or 3 ml to a 60-mm dish). After the viability were used. During the isolation processes 95% 02/5% CO2 gas was contin- dishes had set for 1 hr, the viable cells aduously bubbled through the buffer solution hered to the bottom of each dish (Fig. 1). to maintain a pH of 7.3 to 7.5. The mixed Floating dead cells were removed with the gas also was bubbled into the cell suspen- medium, and new medium was added.
P-450
FIG.
AND
ALDRIN
1. (A) Rat heparocytes
EPOXIDATION
I hr after
IN RAT
isolation;
HEPATOCYTES
(B) I day after
isolution.
66
KURIHARA,
HORI,
After 1 day of culture, the initially spherical cells became flat and spread in a monolayer over the surfaces of the dishes (Fig. 1). Maintenance culture was made in a CO2 incubator under an atmosphere of 95% air/ 5% CO2 (v/v), In some case, a gas phase of 95% 02/5% CO2 was also used. The appearance of cells under these two gas phases was about the same. The number of viable cells decreased slowly day by day, but during the first 3 days viability was never less than 90% of the initial value. Attempts were made to improve the culture conditions by facilitation of the gas exchange in each dish. Metabolic transformation of pesticides by hepatocyte suspension. Krebs-Henseleit solution (solution A) was used to suspend the hepatocytes. The most frequently used combination was lo6 cells in 2 ml at 37°C. Dilution of cell suspension was made by gassed solution A. After preincubation for 30 set, the reaction was started in a glass-stoppered tube by an addition of 10 to 40 nmol of the substrate in 10 ~1 of ethanol. It has been reported that 10 mM ethanol inhibits alprenol oxidation in isolated hepatocytes (8), and that 1 M ethanol activates aldrin epoxidation by a reconstituted cytochrome P-450 system about threefold (9); therefore we examined the effect of ethanol on the metabolic rate in this reaction. When we added the substrate in 20 p.1 (l%, 0.25 M) of ethanol, the rate increased about 1.3-fold. Rate studies therefore were made in a 2-ml suspension with 10 pl ethanol solution of the substrate. After shaking the suspension for a specified period, 1 N HCl (about 0.5 ml) was added to stop the reaction. For each period, triplicate runs were done. Metabolic transformation of pesticides by hepatocytes in primary maintenance culture. The reaction was started by the addition of 20 to 50 nmol of the substrate in 10 ~1 of ethanol (made in a clean bench) to culture dishes that contained lo6 cells in 1 ml of Williams medium (or 3 x IO6 cells in 3 ml of medium). After keeping the dishes
AND
ICHINOSE
for the specified period in a CO2 incubator, the reaction was stopped by an addition of HCl, and the mixture was transferred to a glass-stoppered tube. Metabolic transformation of aldrin by the microsomal fraction and the 10,OOOg supernatant from liver homogenate. The liver homogenate was fractionated as described elsewhere (10). The composition of the reaction mixture was microsomes (0.3 to 0.5 mg protein, corresponding to 0.03 to 0.05 g of liver) or supernatant (2.5 to 5 mg protein, corresponding to 0.05 g of liver), NADPH (1 pmol), and substrate (1.4 to 40 nmol in 10 p.1 ethanol) in 2 ml of 200 mM phosphate buffer (pH 7.4). The reaction was started by the addition of the substrate and continued for 1 min under shaking at 37°C. To stop it, 1 N HCl was added. Extraction and analysis of the metabolic mixture. The reaction mixture obtained in each procedure was treated with hexane (4 ml), or a mixture of hexane (4 ml) and ethyl acetate (1 ml). The extract was diluted appropriately with hexane then analyzed by gas chromatography. The GC column was 3% OV-17 on Chromosorb W AW DMCS (1.5 m) and was used between 170 and 210°C. The carrier gas was N2 of high purity and the detector ECD.(63Ni). In some typical reactions, cell viability was checked before solvent extraction of metabolic compounds; it was more than 90% of the initial value. Compound (substrate and metabolites) distribution between cells and medium will be described elsewhere. Determination of cytochrome P-450 content. The cytochrome P-450 content was determined according to Omura and Sato (11) by the CO difference spectra of the materials. The instrument was a Shimadzu UV-300 recording spectrophotometer. Intact cell suspensions, microsomes or 10,OOOg supernatants were reduced with sodium dithionite, and the sample cuvette was gentiy bubbled with CO gas. An example of the CO difference spectrum of reduced hepatocyte suspension is shown in Fig, 2.
P-450 AND ALDRIN
EPOXIDATION
IN RAT HEPATOCYTES
67
450 recovered in the microsomal fraction. however, has been reported to be far less than the original value and the recovery percentage to vary with the amount originally present (19). This also applies to the 10,OOOg supernatant fraction (19); therefore, when we estimate the P-450 content in intact liver from the contents in homogenate fractions, we must take into account I I ml-l 4ca 450 the possible loss of P-450 during preparation of the fractions. To avoid these comFIG. 2. COdifferencrspectrumofdirhionire-reduced suspended rut hepatocytes at n concentration of 106 plexities, Paine et al. (18) measured the cells/ml. P-450 content in the unfractionated homogenate. These values are given in Table 1: The cytochrome P-450 content in the pri- they appear to be consistent. mary culture was determined in the 10,OOOg In isolated hepatocytes, determinations supernatant of the cell homogenate; cells of the P-450 content have been made foe, from about 30 dishes (30 mm diameter) cell microsomes and for the 10,OOOg cell howere collected with a silicone-rubber mogenate supernatant. These values arc spatula. The supernatant fraction was pre- presented in Table 2 together with the pared as described before (10). values per 10h cells calculated from related The cytochrome P-450 content in the mi- experimental data. The values per 1Ohcell% crosomes and in the supernatant of the liver varied greatly as shown. Our own deterhomogenate was determined by the con- mination for the lO.OOOg hepatocyte ho.ventional method ( 11). Protein concentramogenate supernatant also gave very di tion was measured for these fractions by verse values for the P-450 content: 68-X’ the Lowry method ( 12). pmol/106 cells. This range reflects the tluc. tuations and variations in the recovery or’ RESULTS P-450 as compared to the original content Paine and his co-workers reported the I>. Cytochrome P-450 content. The cytochrome P-450 content that we determined 450 contents of unfractionated cell homogfor various preparations are given in Tables enates (17. 18, 26). Their values seem mot-c 1-3 together with other reported values. reliable for assessing the initial content brData for liver homogenate fractions are cause of the lower possibility of loss or shown in Table 1. For microsomes, the re- damage of some species of P-450 during ported values and our data are in good preparation. agreement. The amount of cytochrome PA direct method that uses a hepatocylc
‘7
TABLE 1 Cyrochrome P-450 Content in the LiVer Homogenutr 0.74 0.78 0.871 1.02
microsomal microsomal microsomal microsomal
protein protein protein protein
,from un Uninduced Rut Gillette er ul. (13) Present investigation Ghiasuddin and Menzer t 14) Sugiura et 01. (1%
+ 0.02 t 0.08 i 0.022 t 0.09
nmolimg nmol/mg nmolimg nmol/mg
89.7 I?4
-+ 10.8 i 14
pmolimg 104g supernatant protein pmol/mg 104g supernatant protein
Present investigation Guzelian et al. (16)
155 168
i 10 2 28
pmolimg total protein pmolimg total protein
Allen et ul. ( 17) Paine (Jt u/. (18)
68
KURIHARA,
HORI, AND ICHINOSE
TABLE 2 Cytochrome P-450 Content in isolated Hepatocytes 0 to I Hr After Isolation from an Uninduced Rat (Determined after Cell Homogenization)
Reported value (Reference in parentheses) 125 t 5 pmol/mg 104g supematant protein (16) 6.9 to 7.4 pmol/pg DNA (20) ca. 425 pmol/mg microsomal protein (22) 480 +- 24 pmol/mg microsomal protein (25) 150 2 37 pmoVmg total protein (26) 166 ? 29 pmol/mg total protein (17)
Converted value to pmol/106 cells basis 56-76” 83-89’
Although effective mixing just before measurement is necessary to prevent sedimentation of the cells in the cuvettes, this direct method is versatile and reliable and shows good reproducibility. Since our aim was to assess in vivo metabolism with in vitro data, we used this most convenient method throughout the present investigation. Metabolism of some pesticides in primary culture. To check the metabolic ac-
89-l 18’
tivity of the hepatocytes, we added some chlorine-containing pesticides to the pri96-140d mary culture and followed their disappearance. Aldrin disappeared faster than the 157-260’ other pesticides. Permethrin was degraded 190-271’ at a moderate rate, whereas DDT and lindane were much more resistant to degra’ Based on 1.7 x IO8 cells per gram of liver (21); and 80 to 100 mg of the 104g supematant protein cor- dation in this system. responding to 1 g of liver. The main metabolites of these pesticides * Based on Decad’s data: 12 pg DNA/cell (20). are shown in Table 4. All compounds were c Based on the value 0.21 mg microsomal protein tentatively identified by gas chromatogper lo6 cells by Morello and Agosin (23), and 0.278 mg raphy except for dieldrin which was idenmicrosomal protein per lo6 cells assuming that 20% of tified by GC-MS (gas chromatographythe total protein is microsomal (1.39 mg/106 cells by Bock et al. (24)). mass spectroscopy). The metabolic reacd Based on the assumption in c. tions included are dehydrochlorination of ’ Based on Bock’s data 1.39 mg protein per lo6 cells DDT to DDE, ester hydrolysis of per(24). If based on Morello’s assumption of 1.05 mg proof aldrin, and various tein per lo6 cells, the values become about 30% methrin, epoxidation oxidative reactions and conjugations in linsmaller. dane metabolism. Aldrin produced dieldrin almost exclususpension to determine the cytochrome P- sively over a short period. After 1 hr, aldrin 450 content in isolated rat hepatocytes is produced other unidentified metabolites as reported (1). Values obtained by this well as a very small amount of trans-dihymethod are listed in Table 3. These values drodiol (0.01 nmol from 40 nmol of aldrin) and those we obtained are in good agreewhich was identified as the TMS derivative ment. There is also good agreement with by GC. the values for cell homogenates reported by Aldrin epoxidation by the hepatocyte Paine et al. (17, 18, 26) (Table 2). suspension and by primary culture. Aldrin TABLE 3 Cytochrome P-450 Content in isolated Hepatocytes I Hr After Isolation from an Uninduced Rat (Determined for the Cell Suspension) Investigator Mold&us et al. (1978) Morello and Agosin (1979) Present investigation
Reference (1)
(23)
Cytochrome P-450 content 230 f 50 pmol/106 cells 242 or 320 pmol/106 cellsa 303 ? 68.2 pmol/106 cells (n = 22)*
a Calculated from their data of 0.23 nmoVmg of cell protein, and 1.05 or 1.39 mg protein/106 cells (23, 24). * n is the number of independent experimental runs using different rats.
P-450 AND ALDRIN
EPOXIDATION TABLE
Pesticide
Substrate
Initial amount (nmolR ml)
DDT Lindane Permethrin
50 16.6 40
Aldrin
40
-
Metabolism
IN RAT HEPATOCYTES 4
by Cultured
Reaction time (hr)
Hepatocytes”
--~.
Metabolized amount (nmol/106 cells)
24 24 1
9.8 1.2 10.6
1
17.3
-. ~.
Main metabolites __DDE Various metabolitesh Dichlorovinylchrysanthemic acid Dieldrin
0 A I-day-old maintenance culture was used. See Materials and Methods for experimental details. h 2.4,6-Trichlorophenol and its sulfate, 2,3,4,&tetrachlorophenol and its sulfate, (36/45) hexachlorocyclohexene, 2,4-dichlorophenyl-glutathione, and others. Conjugated metabolites were hydrolyzed and derivatized prior to GLC analysis according to the reported method (27).
was epoxidized to dieldrin at a high rate. Thus, this reaction was examined in various states of isolated hepatocytes and liver fractions. A fresh cell suspension (1 to 6 hr after isolation) epoxidized aldrin most effectively (Table 5). Because of experimental convenience, a reaction of less than 1 hr during culture was not used. After 1 day of culture, the metabolic activity of the hepatocytes decreased slightly, but the decrease between the second and third day (24- to 4%hr cells) was not significant. Rates of aldrin epoxidation. The rates of aldrin epoxidation listed in Table 5 are tentative; therefore we examined the reaction more closely. Dieldrin formation was linear for at least 5 min when 40 nmol of aldrin was metabolized by 2 ml of a suspension of lo6 cells (see Fig. 3). The rate was proportional to the cell numbers at least in the given range (Fig. 4). The 2- or 5-min reacTABLE AI&in
Epoxidation
to Dieldrin
tion was repeated for several preparations of isolated hepatocytes. When hepatocytes were used for the metabolism experiments within 1 hr of isolation, the average value for the rate of dieldrin formation per unit amount of cytochrome P-450 was fairly high, although the values were rather scattered: 10.82 t 3.55 mol/mol P-4501min. Before the reaction. the hepatocytes were maintained in a subpension through which mixed 02/COZ was continuously bubbled at 20-25°C. Some deviations for each determination of triplicate runs were very large and the values found are not considered very reliable. To determine the origin of these variations, the dependency of the rate values on the time after isolation and on maintenance conditions was examined. Results are shown in Table 6. Changes in cell viability Die$drin(nmol)
,A:’
t
5
10 1 by Rat Hepatocytes” p’
Reaction
system
Suspension of I-hr cells Primary culture
Reaction time 5 min
Formed
dieldrin
(nmol)
6.25
r
1.85
(n = 7) Reoctik
in dishes
I-hr cells 24-hr cells 4%hr cells
1 hr 1 hr I hr
11.4
-t 0.6
(n =
8.3 8.4
r 1.96 -c 0.26
(n = 19) (n = 2)
6)
* Initial amount of aldrin was 40 nmoV106cells. See Matenals and Methods for experimental details. * Phenobarbital
treatment
10 0.5 PM) did not affect the
of cells for 24 hr beforehand rate
significantly.
(0.05
FIG. 3. Time course
Time
(min)
of aldrin epoxidation by a rar hepatocyte suspension. Inirial substrate 40 nmol. I@ cells containing 378 pmol of cytochrome P-450 in 2 ml of Krebs-Henseleit solution. Preincubation was .fu?30 set without substrate. Reaction ar 37°C. Each poinr represents the average of triplicate runs M*itk a slondard deviation of less than 3% qf the value.
KURIHARA,
70
HORI, AND ICHINOSE
Di%drin(nmol)
Cell
Number
FIG. 4. Effect of cell concentration on the aldrinepoxidation rate. Initial substrate 40 nnol in 2 ml at 37°C. The amount of dieldrin formed (nmol) in 2 min is shown. Each value is the average of triplicate runs with a standard deviation of less than 5% of the value.
and in P-450 content also were examined. These are given in Table 7. Hepatocytes maintained in a suspension through which 02/C02 was bubbled (condition B) showed a dieldrin-formation rate (Table 6) that varied considerably with time. During the early stage after isolation, the rate was abnormally high; this corresponds to the above-mentioned results. Later the rate decreased rapidly, then became’ constant for several hours although slightly decreased. When the cells were maintained in a suspension with gas gently introduced onto the surface (condition A), the dieldrin-formation rate was constant during the entire period examined. Viability and P-450 content
also were stable under condition A and in the later stages of conditions B (Tables 6 and 7). The bubbling of gas into the cell suspension at 37°C (conditon C) caused a rapid loss of metabolic activity, viability being almost zero after 6 hr. Taking into account these results, we selected cells 1 to 4 hr after isolation and maintained them at condition A in order to determine the rate of dieldrin formation per unit amount of cytochrome P-450. The average values for these conditions are shown in Table 8. Although the value is smaller than that of the early stage in condition B, it is more reliable and reproducible because of the stable metabolic activity and the viability of the cells. The apparent & and Vmax values determined for this condition also are listed in Table 8. The rate values were similar to those found for the reaction by the liver microsomes (Table 9>, though differ from the values of the liver 10,OOOg supernatant. The apparent K, and V,,,,, values for the reactions by microsomes were different from those by hepatocytes (Tables 8 and 9). The results suggest that the hepatocytes and the microsomes have different compositions of cytochrome P-450 subspecies. Rate comparison on a cell-number basis and on a liver-weight basis. Aldrin epoxi-
dation by rat hepatocytes on a cell-number basis was 1.36 _’ 0.31 nmol/min/106 cells in
TABLE 6 Dependence of the Aldrin Epoxidation Rate on the Time after Cell Isolation and on the Maintenance Condition” Time (mm) Conditionb
SO-55
70-75
90-95
200-210
350-360
A
1.45 * 0.04 *2..51 c 0.51 1.34 k 0.10
1.41 I 0.03 1.54 + 0.24 1.16 ? 0.11
1.38 -c 0.07 1.25 k 0.04 1.14 2 0.05
1.31 c 0.03 1.17 k 0.13 0.19 + 0.00
1.31 i 0.05 0.91 2 0.03 0.09 * 0.01
B
C
a The reaction took place at 37°C in 2 ml of a suspension of 8.4 x IO5 cells, which contained 185 pmol of cytochrome P-450, 50-55 min after isolation. The total amount of dieldrin formed (nmol) in 2 min together with the standard deviation of triplicate runs is shown. Turnover rate can be calculated from each value. For example, the value with an asterisk (*) corresponds to 2.51/0.185/2 = 6.78 (mol/mol P-450/min). b Condition (A) Maintained at room temperature (20°C) with gentle introduction of Oz/COz onto the surface of the cell suspension; (B) maintained at room temperature with bubbling of the same gas mixture; (C) maintained at 37°C with bubbling of the same gas mixture.
P-450
Changes
AND
ALDRIN
TABLE I in Viability during Maintenance Different Conditions Time
Condition” A B’ C
-
90-95 (I) 93.0% (2) 80.8%~ 71.2% 78.5%
EPOXIDATION
under
(min) 210
360
-b 77.9% -
95.0% 80.4% 68.4% 2.0%
u For conditions, see footnote h to Table 6. ir -shows not determined. ( Under this condition, variations in the P-450 content were determined at each stage: at 90-95 min. 185 pmollml: at 210 min. 191 pmoliml: and at 360 min. 178 pmoliml.
experiments with cells maintained under condition A. If we assume that 1 g of liver corresponds to 1.5 x 10” cells, the rate would be 204 & 46.5 nmol/min/g liver (see Table 8 footnote (I). In contrast, the rate for the microsomes was 49.9 to 69.9 (Table 9. footnote) and for the 10,OOOg supernatant was about 67 nmol/min/g liver. Thus, when we compare these values, that of isolated hepatocytes is much larger than the others. The P-450 content on a liver weight basis is calculated as 0.303 x 150 = 45.45 nmoli g liver from hepatocyte data, if we adopt the above assumption of I .5 x lo* cells/g liver. In contrast. 10 to 14 nmol P-450 was recovered in microsomes from 1 g of liver. At most, 21.5 nmol of P-450 is recovered in microsomes from 1 g of liver (29). The low TABLE
Rate”
8
4.50 5 1.01 molimol P-450imin (n = 6Jh 7.19 t- 1.49 molimol P-450imin (n = 4) VI”‘,, 9.27 k 1.17 p” (n = 4) Kt,, -_ (’ Aldrin (40 nmol) in ethanol (10 ~1) was incubated in 2 ml of Krebs-Henseleit solution, after a 30-set preincubation. with IO6 cells at 37°C. An addition of glucose (2 mg) did not change the rate. The rate on a cell number basis is calculated as I .36 nmol/lOh cells/ min from P-450 content of 303 pmol/lOh cells (Table 3). Recovery of the compounds after the reaction was oker 95%. For other details. see Materials and Methods. ” n is the number of independent experimental runs.
IN RAT
7!
HEPATOCYTES
yield of cytochrome P-450 in this fraction (and in the supernatant) explains the much lower rate values of microsomes and supernatant on a liver-weight basis. DISCUSSION
The kinetic parameters of aldrin epoxldation based on the cytochrome P-450 content differed for isolated hepatocytes and liver homogenate fractions. V,,, value fotthe hepatocytes was greater than that for the microsomes on a cytochrome P-450 basis (Tables 8 and 9). Considerable differences of apparent K,?, values were observed between cells and microsomes. The ay?. parent K, ‘s for purified rat liver cytochrome P-450 and P-448 are reported, being 7 ? 2 and 27 t 7 pM, respectively (9). The K,,, for the hepatocytes was similar to the former value. When the rates of aldrin epoxidation were compared on a cell-number or liver weight basis, the rate for isolated hepatocytes was much greater than that for the microsomal fraction and the 10,OOOg super natant from the liver homogenate. 01-r* viously. this discrepancy is caused by sip nificant loss of P-450 during preparation o!‘ these fractions. McLean and Day reportetf
TABLE
Y
4.99 t 0.09 mol/mol P-4SOimin 01 3)” 5.1 I k 0.28 molimol F-45Olmin (II :I I .64 + 0.08 phi i ,i 3) ~____-___--~~-. ~.. ClThe 10.000~ supernatant from liver homogenate showed the rate 6.68 it 0.83 mollmol P-45Oimin (II 2). Aldrin (40 nmol) m ethanol (10 ~1) was incubated at 37°C with a preincubated (30 set) mixture of lhc microsomes (or supernatant) from the liver homopcnate and NADPH (I kmol) in 2 ml of phosphate buffei Recovery of the compounds after the reaction \n a liver weight basis hould be 49.9 to 69.86 nmolip II\,erimin h ,I is the number of independent experimcntnl r~!n\. Rate” c’m Km
72
KURIHARA,
HORI, AND ICHINOSE
that the amount of cytochrome P-450 recovered in the microsomal fraction varies with the amount originally present (19). The apparent K,,, value for the reaction by microsomes was much smaller than that for the hepatocytes. Although the K, values for very hydrophobic substrates such as aldrin have doubtful meaning in some experimental conditions (28) and some corrections for nonspecific binding of aldrin to biomolecules may significantly modify the Km value (29), the present great difference between the two apparent K, values suggests that the selective loss of some P-450 subspecies that have low affinity to aldrin takes place during preparation of the liver microsomal fraction. Therefore, microsomes seem to retain P450 of high affinity to aldrin. Interestingly, for ethylmorphine (30) and antipyrine (31, 32), great differences in K,,, values for the reactions by microsomes and hepatocytes are reported, whereas alprenol (33) and ethoxybenzamide (29) show similar K, values in both metabolic systems. The exact reason for the differences in apparent K,,, values is a future problem. Hepatocytes could be maintained in a good viability for at least 6 hr in Krebs.Henseleit solution with no loss of cytochrome P-450 content and aldrin-epoxidizing activity. They required an oxygen supply which was provided by the gentle introduction of 02/C02 gas onto the surface of the suspension. Bubbling the gas also was effective at 20°C but in the early stage of this treatment epoxidizing activity varied considerably from tube to tube. Just after isolation, hepatocytes suffer damage to their membranes from the collagenase and proteinase present. Recovery from this damage usually seems to be rapid, but may be retarded by the vigorous bubbling of the gas. Although the values found varied greatly in the early stages when 02/ CO2 was bubbled through at 20°C (condition B), hepatocytes showed high epoxidizing activity (Table 6), which has yet to be explained. The penetration rate of the
substrate may affect the rate of metabolism. Penetration into whole cells was a very rapid process (unpublished data); therefore the difference in the rates of penetration to the site of metabolism in the cells rather than into the cell itself may account for the variations found. In summary, isolated hepatocytes retained good aldrin-epoxidizing activity and a constant cytochrome P-450 content as well as good viability for the first 6 hr after isolation. Hepatocytes are a better model system for in viva metabolism than are liver microsomes or liver 10,OOOg supernatant, because during preparation these fractions lose some of the several cytochrome P-450s they contain. ACKNOWLEDGMENTS We are grateful to Professor Minoru Nakajima for his expert advice and encouragement throughout this study. Thanks also are due to Dr. Yukiaki Kuroda for his helpful suggestions on the technique used for the primary maintenance culture of hepatocytes. REFERENCES 1. P, Mold&s, J. Hogberg, and S. Orrenius, Isolation and use of liver cells, in “Methods in Enzymology” (Fleischer, S., and Packer, L., Eds.), Vol. 52, p. 60, Academic Press, New York, 1978. 2. J. R. Fry and J. W. Bridges, Use of primary hepatocyte cultures in biochemical toxicology, in “Reviews in Biochemical Toxicology” (E. Hodgson, J. R. Bend, and R. M. Philpot, Eds.), p. 201, Elsevier/North-Holland, Amsterdam/ New York, 1979. 3. T. Nakatsugawa, W. L. Bradford, and K. Usui, Hepatic disposition of parathion. Uptake by isolated hepatocytes and chromatographic translobular migration, Pesric. Biochem. Physiol. 14, 13 (1980). 4. 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). 5. A. Blom, A. H. J. Scaf, and D. K. E Meijer, Hepatic drug transport in the rat; A comparison between isolated hepatocytes, the isolated perfused liver and the liver in vivo, Biochem. Pharmacol. 8, 1553 (1982). 6. R. G. Thurman and F. C. Kauffman, Factors regulating drug metabolism in intact hepatocytes, Pharmacol. Rev. 31, 229 (1980).
P-450
AND ALDRIN
EPOXIDATION
7. G. M. Williams. E. K. Weisburger. and J. H. Weisburger, Isolation and long-term cell culture of epithelial-like cells from rat liver, Exp. Cell Res. 69, 106 (1971). 8. R. Grundin, P. Moldeus. H. Vadi, S. Orrenius, C. von Bahr, D. Backstrom and A. Ehrenberg, Drug metabolism in isolated rat liver cells. Advan. Exp. Med. Biol. 58, 251 (1975). 9. T. Wolff, H. Greim, M.-T. Huang, G. T. Miwa, and A. Y. H. Lu, Aldrin epoxidation catalyzed by purified rat liver cytochrome P-450 and P448; High selectivity for cytochrome P-450. Eur.
J. Biochem.
10, 79 (1979).
239,
2370 (1964).
I?. 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with Folin phenol reagent, J. Biol. Chem. 193, 265 (1951). 13. J. R. Gillette. J. J. Kamm, and H. A. Sasame, Mechanism of p-nitrobenzoate reduction in liver: The possible role of cytochrome P-450 in liver microsomes. Mol. Pharmacol. 4, 541 (1968). 14. S. M. Ghiasuddin and R. E. Menzer, Microsomal epoxidation of aldrin to dieldrin in rats, Bull. Environ.
22. 23. 24.
-
11. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes. 1. Biol. Chem.
21.
111, 545 (1980).
IO. K. Tanaka, N. Kurihara, and M. Nakajima, Oxidative metabolism of tetrachlorocyclohexenes, pentachlorocyclohexenes and hexachlorocyclohexenes with microsomes from rat liver and house fly abdomen, Pestic. Biochem. Physiol.
Contam.
Toxicol.
1.5, 324 (1976).
15. M. Sugiura. K. Iwasaki. and R. Kato, Reduction of tertiary amine N-oxides by liver microsomal cytochrome P-450, Mol. Pharmacol. 12, 322 (1976).
I6 P. S. Guzelian. D. M. Bissell, and U. A. Meyer. Drug metabolism in adult rat hepatocytes in primary monolayer culture. Gastroenterology 72, 1232 (1977). 17. C. M. Allen, L. J. Hockin, and A. J. Paine, The control of glutathione and cytochrome P-450 concentrations of primary cultures of rat hepatocytes, Biochem. Pharmacol. 30, 2739 (1981). 18. A. J. Paine, L. J. Williams, and R. F. Legg, Apparent maintenance of cytochrome P-450 by nicotinamide in primary cultures of rat hepatocytes, Life Sci. 24, 2185 (1979). 19. A. E. M. McLean and P. A. Day, The use of new methods to measure the effect of diet and inducers of microsomal enzyme synthesis on cytochrome P-450 in liver homogenates, and on metabolism of dimethyl nitrosamine. Biochem. Pharmacol. 23, 1173 (1974). 20. G. M. Decad. D. P. H. Hsieh, and J. L. Byard. Maintenance of cytochrome P-450 and metabolism of aflatoxin B I in primary hepatocyte cul-
77
IN RAT HEPATOCYTES
25.
tures. Biochem. Biophys. Res. Common 78. 279 (1977). E. R. Weibel, W. Staubli, H. R. Gnagr, F. A Hess, Correlated morphometric and biochemical studies on the liver cell. I. Morphometric model, stereologic methods, and normal morphometric data for rat liver. J. CeI1 Biol. 42, 68 (1969). G. Michalopoulos. F. Russell, and C. Biles. I’rimat-y cultures of hepatocytes on human tibroblasts. In Vitro 15. 796 (1979). A. Morello and M. Agosin, Metabolism ofjuvenile hormone with isolated rat hepatocyte\. B&hem. Pharmacol. 28, 1533 (1979). K. W. Bock. G. van Ackeren. E Larch, and F. W. Birke, Metabolism of naphthalene to naphthalene dihydrodiol glucuronide in isolated hepatocytes and in liver microsomes. Biochc~,r. Pharmacol. 25. 235 1 ( 1976). G. Michalopoulos, G. L. Sattler, and H. C. Pitclt. Maintenance of microsomal cytochrome b, and P-450 in primary culture of parenchymal liver cells on collagen membranes. Li’e Sri. 18, I I AY (1976).
26. A. J. Paine and R. F. Legg, Apparent lack of carrelation between the loss of cyotchrome P-350 in hepatic parenchymal cell culture and the stimulation of haem oxygenase activit v. Biochem.
Biophvs.
Res.
Comnrrrt~.
81.
f17:
(1978). 27. N. Kurihara, K. Tanaka, and M. Nakajima. Mercapturic acid formation from lindane in rat$. Pestic.
Biochem.
Physiol.
10, 137 (1979).
28. G. T. Brooks and A. Harrison, The oxidative metabolism of aldrin and dihydroaldrin by houseflies, housefly microsomes and pig liver microsomes and the effect of inhibitors. Biochc,m Pharmacol.
18, 557 (1969).
29. J. H. Lin. Y. Sugiyama. S. Awazu. and M Hanano, Kinetic studies on the deethylation of ethoxybenzamide: A comparative study with isolated hepatocytes and liver microsome\ of rat, Biochem. Pharmucol. 29, 2825 (1980). 30. R. R. Erickson and J. L. Holtzman, Kinetic: studies on the metabolism of ethylmorphine by isolated hepatocytes from adult rats. Rioch~:rn. Pharmacol. 25, 1501 (1976). 31. A. Rane, G. R. Wilkinson. and D. G. Shand. Pr-ediction of hepatic extraction ratio from in ~,irro measurement of intrinsic clearance. J. P/,rirmacol.
E.vp. Therap.
200,
420 (1977).
32. J. S. Hayes and K. Brendel, N-Demethylation ~15 an example of drug metabolism in isolated rat hepatocytes. Biochem. Pharmacol. 25, IJYY (1976). 33. l? Mold&s, R. Grundin. H. Vadi. and S. Orremus. A study of drug metabolism linked to cyto. chrome P-450 in isolated rat-liver cells. E/o, J Biochem.
46. 351 (1974).
BIOCHEMISTRY
Cytochrome
AND
PHYSIOLOGY
21, 63-73 (1984)
P-450 Content and Aldrin Epoxidation Isolated Rat Hepatocytes NORIO KURIHARA,~NOBUAKI Radioisotope
Research
Center.
Kyoto
to Dieldrin
in
HORI, AND REIJI ICHINOSE Universiiy,
h’yoto
606. Jtrpan
Received May 27. 1983: accepted July 22. 1983 A rat hepatocyte suspension effectively epoxidized aldrin to dieldrin with a V,,, of 7.19 mol/ mol P-450/min and a K,,, of 9.27 p,,W. Viability and metabolic activity were stable for 6 hr after isolation when cells were maintained at room temperature (20°C) with the gentle introduction of Oz/COZ onto the surface of the suspension. The cytochrome P-450 content of the suspension was 303 pmol/106 cells. Primary maintenance culture of the cells also epoxidized aldrin. During culture for 3 days, metabolic activity decreased slowly day by day. Metabolic activity of microsomal fraction from rat liver was also examined. Microsomes epoxidized aldrin with a V,,, of 5. I I moli mol P-450/min and a K, of 1.64 FM. Significant loss of some subspecies of cytochrome f-450 during fractionation of liver homogenate was indicated.
INTRODUCTION
sessment of in vivo metabolic activity from hepatocyte suspension-metabolism studies isolated rat hepatocytes are now widely is another important problem. used in xenobiotic metabolism studies. We here describe the metabolism of They retain both the compositions of their aldrin by an isolated rat hepatocyte suspenenzyme systems and cofactors, as well as sion and by a primary maintenance rat hemembranes and various particles related to patocyte culture. In the initial metabolic xenobiotic metabolism. Thus, they should phase of this substrate, an epoxidized be a better model of in Go metabolic sys- product, dieldrin, was almost the only tems than are liver homogenates or enzyme product. We compared the reaction rates in preparations from liver (1, 2). Although we these systems with the rates of a liver mimust carefully consider the spatial hetero- crosomal fraction, and examined the relageneity of cells in the whole organ (2) and tions between these rates and cytochrome compound transport within the organ (3- P-450 content which should have the most 5), use of isolated hepatocytes still is a important role in epoxidation. The uptake better model and a simpler system for pre- and release of these compounds by hepadicting in vivo xenobiotic metabolism from tocytes will be dealt with elsewhere. in vitro results. MATERIALS AND METHODS Much information on xenobiotic metabolism has been accumulated by use of the Chemicals. Aldrin and dieldrin were purisolated cell technique ((1, 2, 6) and refer- chased from Wakenyaku Company Ltd. ences given there). Some general problems Their purities were more than 99.5% on gas remain, however, in application of this chromatography. Organic solvents were of technique; the relation between overall reagent grade. Clostridium histolyticum metabolic activity and the enzyme content collagenase Class IV (Worthington) was of the cells, and the rate of uptake and re- purchased from Millipore Ltd., and Type lease of the compound by the cells. As- IV from Sigma Chemical Company Ltd., both through Wakenyaku Company. Fraction V of bovine serum albumin (BSA) was ’ To whom correspondence and reprint requests should be addressed. obtained from Armour Pharmaceutical 63 0048-3575/84 $3.00 Copyright
8 1984 by Academic
Press, Inc.
64
KURIHARA,
HORI,
Company. The rest of the chemicals used were of the highest purity commercially available.
AND
ICHINOSE
sion or introduced onto the surface of the suspension without bubbling. Method 121: Hepatocytes were isolated Preparation of hepatocyte suspension. with the Hanks- Wallace solution-based Male Wistar rats (ZOO-250 g) fed ad libitum perfusate. The liver was perfused with were used. Hepatocytes were obtained by Ca2+-free Hanks-Wallace solution cona collagenase perfusion method (1). taining 0.3 mM EGTA for 10 to 15 min at Method [l]: The liver was perfused in situ 30 to 50 ml/min, followed by brief perfusion for 5 to 10 min with Caz+-free Krebs-Henwith EGTA-free Hanks- Wallace solution. seleit solution containing 0.5 mM EGTA Recycling perfusion with collagenase solu(ethylene glycol bis(P-aminoethyl ether)tion was done for I5 to 20 min at 70 ml/min. N,N’-tetraacetic acid) and 2% BSA, at 30 This enzyme solution (Hanks- Wallace, 80 to 40 ml/min. After the liver had been freed ml) contained CaC12 (33.2 mg), dextran from the body, perfusion was continued for (MW 58,000, 3.9 g), and collagenase (40 to 1 min with EGTA-free Krebs-Henseleit so- 80 mg). Subsequent procedures were the lution (1) containing 1.5 mM Ca*+ and same as those described above with Krebs2.34% BSA (solution A) at 80 ml/min. Then Henseleit solution. Hanks-Wallace solution is said to be in recycling was done for 10 to 20 min with Krebs-Henseleit solution containing equilibrium with CO2 in ordinary air 0.08% collagenase, 1 n&f Ca2+, and 2.5% (0.03%) and as such was convenient for cell dextran at 80 to 90 ml/min. All perfusion isolation under sterilized conditions, but we media were maintained at 37°C and pH 7.4 frequently observed a large pH change during the isolation steps. Adjustment of with 95% 02/5% CO2 bubbled continuously pH, monitored by the dissolved indicator through them. When the liver started to swell and to phenol red, was made during the procedure by the injection of an appropriate concendisintegrate, it was perfused again briefly with solution A, after which it was trans- tration of aqueous NaHCOj solution. ferred to a small dish. The capsula was cut Primary culture of hepatocytes. All the open in 20 ml of solution A, and its cells isolation steps were done in a clean bench were dispersed by gentle stirring with a pair with sterilized Hanks- Wallace solution by of tweezers. At an ambient temperature method [2]. After the second centrifugation at 4Og, cells were resuspended in an appro(20-25”C), the dispersed cells were filtered through four layers of cotton gauze to re- priate volume of William’s medium (7) fortified with fetal calf serum (IO%), insulin move connecting tissue and nondispersed cells. The filtrate was centrifuged for 60 set ( 10e6 M), testosterone ( 10P6 M), estradiol (10 mg/liter), hyat 40g. The collected cells were resus- ( 10M6 M), DL-a-tocopherol pended and centrifuged again at the same drocortisone 21-p-acetate (lops M), and 6acid (10V6 M). The comspeed, and then resuspended and diluted to aminolevulinic pounds were added in ethanol solution, the a concentration of IO6 cells/ml in solution volume of the ethanol being less than 3 ml/ A (BSA was 2.34% unless otherwise noted). Cell numbers were counted in a liter of medium. A suspension of IO6 cells/ml medium was Buerker chamber. The trypan blue-exclusion test was used to estimate cell viability. prepared, and l-ml portions of it were In the metabolic study, cells of 90 to 95% transferred to plastic dishes 35 mm in diameter (or 3 ml to a 60-mm dish). After the viability were used. During the isolation processes 95% 02/5% CO2 gas was contin- dishes had set for 1 hr, the viable cells aduously bubbled through the buffer solution hered to the bottom of each dish (Fig. 1). to maintain a pH of 7.3 to 7.5. The mixed Floating dead cells were removed with the gas also was bubbled into the cell suspen- medium, and new medium was added.
P-450
FIG.
AND
ALDRIN
1. (A) Rat heparocytes
EPOXIDATION
I hr after
IN RAT
isolation;
HEPATOCYTES
(B) I day after
isolution.
66
KURIHARA,
HORI,
After 1 day of culture, the initially spherical cells became flat and spread in a monolayer over the surfaces of the dishes (Fig. 1). Maintenance culture was made in a CO2 incubator under an atmosphere of 95% air/ 5% CO2 (v/v), In some case, a gas phase of 95% 02/5% CO2 was also used. The appearance of cells under these two gas phases was about the same. The number of viable cells decreased slowly day by day, but during the first 3 days viability was never less than 90% of the initial value. Attempts were made to improve the culture conditions by facilitation of the gas exchange in each dish. Metabolic transformation of pesticides by hepatocyte suspension. Krebs-Henseleit solution (solution A) was used to suspend the hepatocytes. The most frequently used combination was lo6 cells in 2 ml at 37°C. Dilution of cell suspension was made by gassed solution A. After preincubation for 30 set, the reaction was started in a glass-stoppered tube by an addition of 10 to 40 nmol of the substrate in 10 ~1 of ethanol. It has been reported that 10 mM ethanol inhibits alprenol oxidation in isolated hepatocytes (8), and that 1 M ethanol activates aldrin epoxidation by a reconstituted cytochrome P-450 system about threefold (9); therefore we examined the effect of ethanol on the metabolic rate in this reaction. When we added the substrate in 20 p.1 (l%, 0.25 M) of ethanol, the rate increased about 1.3-fold. Rate studies therefore were made in a 2-ml suspension with 10 pl ethanol solution of the substrate. After shaking the suspension for a specified period, 1 N HCl (about 0.5 ml) was added to stop the reaction. For each period, triplicate runs were done. Metabolic transformation of pesticides by hepatocytes in primary maintenance culture. The reaction was started by the addition of 20 to 50 nmol of the substrate in 10 ~1 of ethanol (made in a clean bench) to culture dishes that contained lo6 cells in 1 ml of Williams medium (or 3 x IO6 cells in 3 ml of medium). After keeping the dishes
AND
ICHINOSE
for the specified period in a CO2 incubator, the reaction was stopped by an addition of HCl, and the mixture was transferred to a glass-stoppered tube. Metabolic transformation of aldrin by the microsomal fraction and the 10,OOOg supernatant from liver homogenate. The liver homogenate was fractionated as described elsewhere (10). The composition of the reaction mixture was microsomes (0.3 to 0.5 mg protein, corresponding to 0.03 to 0.05 g of liver) or supernatant (2.5 to 5 mg protein, corresponding to 0.05 g of liver), NADPH (1 pmol), and substrate (1.4 to 40 nmol in 10 p.1 ethanol) in 2 ml of 200 mM phosphate buffer (pH 7.4). The reaction was started by the addition of the substrate and continued for 1 min under shaking at 37°C. To stop it, 1 N HCl was added. Extraction and analysis of the metabolic mixture. The reaction mixture obtained in each procedure was treated with hexane (4 ml), or a mixture of hexane (4 ml) and ethyl acetate (1 ml). The extract was diluted appropriately with hexane then analyzed by gas chromatography. The GC column was 3% OV-17 on Chromosorb W AW DMCS (1.5 m) and was used between 170 and 210°C. The carrier gas was N2 of high purity and the detector ECD.(63Ni). In some typical reactions, cell viability was checked before solvent extraction of metabolic compounds; it was more than 90% of the initial value. Compound (substrate and metabolites) distribution between cells and medium will be described elsewhere. Determination of cytochrome P-450 content. The cytochrome P-450 content was determined according to Omura and Sato (11) by the CO difference spectra of the materials. The instrument was a Shimadzu UV-300 recording spectrophotometer. Intact cell suspensions, microsomes or 10,OOOg supernatants were reduced with sodium dithionite, and the sample cuvette was gentiy bubbled with CO gas. An example of the CO difference spectrum of reduced hepatocyte suspension is shown in Fig, 2.
P-450 AND ALDRIN
EPOXIDATION
IN RAT HEPATOCYTES
67
450 recovered in the microsomal fraction. however, has been reported to be far less than the original value and the recovery percentage to vary with the amount originally present (19). This also applies to the 10,OOOg supernatant fraction (19); therefore, when we estimate the P-450 content in intact liver from the contents in homogenate fractions, we must take into account I I ml-l 4ca 450 the possible loss of P-450 during preparation of the fractions. To avoid these comFIG. 2. COdifferencrspectrumofdirhionire-reduced suspended rut hepatocytes at n concentration of 106 plexities, Paine et al. (18) measured the cells/ml. P-450 content in the unfractionated homogenate. These values are given in Table 1: The cytochrome P-450 content in the pri- they appear to be consistent. mary culture was determined in the 10,OOOg In isolated hepatocytes, determinations supernatant of the cell homogenate; cells of the P-450 content have been made foe, from about 30 dishes (30 mm diameter) cell microsomes and for the 10,OOOg cell howere collected with a silicone-rubber mogenate supernatant. These values arc spatula. The supernatant fraction was pre- presented in Table 2 together with the pared as described before (10). values per 10h cells calculated from related The cytochrome P-450 content in the mi- experimental data. The values per 1Ohcell% crosomes and in the supernatant of the liver varied greatly as shown. Our own deterhomogenate was determined by the con- mination for the lO.OOOg hepatocyte ho.ventional method ( 11). Protein concentramogenate supernatant also gave very di tion was measured for these fractions by verse values for the P-450 content: 68-X’ the Lowry method ( 12). pmol/106 cells. This range reflects the tluc. tuations and variations in the recovery or’ RESULTS P-450 as compared to the original content Paine and his co-workers reported the I>. Cytochrome P-450 content. The cytochrome P-450 content that we determined 450 contents of unfractionated cell homogfor various preparations are given in Tables enates (17. 18, 26). Their values seem mot-c 1-3 together with other reported values. reliable for assessing the initial content brData for liver homogenate fractions are cause of the lower possibility of loss or shown in Table 1. For microsomes, the re- damage of some species of P-450 during ported values and our data are in good preparation. agreement. The amount of cytochrome PA direct method that uses a hepatocylc
‘7
TABLE 1 Cyrochrome P-450 Content in the LiVer Homogenutr 0.74 0.78 0.871 1.02
microsomal microsomal microsomal microsomal
protein protein protein protein
,from un Uninduced Rut Gillette er ul. (13) Present investigation Ghiasuddin and Menzer t 14) Sugiura et 01. (1%
+ 0.02 t 0.08 i 0.022 t 0.09
nmolimg nmol/mg nmolimg nmol/mg
89.7 I?4
-+ 10.8 i 14
pmolimg 104g supernatant protein pmol/mg 104g supernatant protein
Present investigation Guzelian et al. (16)
155 168
i 10 2 28
pmolimg total protein pmolimg total protein
Allen et ul. ( 17) Paine (Jt u/. (18)
68
KURIHARA,
HORI, AND ICHINOSE
TABLE 2 Cytochrome P-450 Content in isolated Hepatocytes 0 to I Hr After Isolation from an Uninduced Rat (Determined after Cell Homogenization)
Reported value (Reference in parentheses) 125 t 5 pmol/mg 104g supematant protein (16) 6.9 to 7.4 pmol/pg DNA (20) ca. 425 pmol/mg microsomal protein (22) 480 +- 24 pmol/mg microsomal protein (25) 150 2 37 pmoVmg total protein (26) 166 ? 29 pmol/mg total protein (17)
Converted value to pmol/106 cells basis 56-76” 83-89’
Although effective mixing just before measurement is necessary to prevent sedimentation of the cells in the cuvettes, this direct method is versatile and reliable and shows good reproducibility. Since our aim was to assess in vivo metabolism with in vitro data, we used this most convenient method throughout the present investigation. Metabolism of some pesticides in primary culture. To check the metabolic ac-
89-l 18’
tivity of the hepatocytes, we added some chlorine-containing pesticides to the pri96-140d mary culture and followed their disappearance. Aldrin disappeared faster than the 157-260’ other pesticides. Permethrin was degraded 190-271’ at a moderate rate, whereas DDT and lindane were much more resistant to degra’ Based on 1.7 x IO8 cells per gram of liver (21); and 80 to 100 mg of the 104g supematant protein cor- dation in this system. responding to 1 g of liver. The main metabolites of these pesticides * Based on Decad’s data: 12 pg DNA/cell (20). are shown in Table 4. All compounds were c Based on the value 0.21 mg microsomal protein tentatively identified by gas chromatogper lo6 cells by Morello and Agosin (23), and 0.278 mg raphy except for dieldrin which was idenmicrosomal protein per lo6 cells assuming that 20% of tified by GC-MS (gas chromatographythe total protein is microsomal (1.39 mg/106 cells by Bock et al. (24)). mass spectroscopy). The metabolic reacd Based on the assumption in c. tions included are dehydrochlorination of ’ Based on Bock’s data 1.39 mg protein per lo6 cells DDT to DDE, ester hydrolysis of per(24). If based on Morello’s assumption of 1.05 mg proof aldrin, and various tein per lo6 cells, the values become about 30% methrin, epoxidation oxidative reactions and conjugations in linsmaller. dane metabolism. Aldrin produced dieldrin almost exclususpension to determine the cytochrome P- sively over a short period. After 1 hr, aldrin 450 content in isolated rat hepatocytes is produced other unidentified metabolites as reported (1). Values obtained by this well as a very small amount of trans-dihymethod are listed in Table 3. These values drodiol (0.01 nmol from 40 nmol of aldrin) and those we obtained are in good agreewhich was identified as the TMS derivative ment. There is also good agreement with by GC. the values for cell homogenates reported by Aldrin epoxidation by the hepatocyte Paine et al. (17, 18, 26) (Table 2). suspension and by primary culture. Aldrin TABLE 3 Cytochrome P-450 Content in isolated Hepatocytes I Hr After Isolation from an Uninduced Rat (Determined for the Cell Suspension) Investigator Mold&us et al. (1978) Morello and Agosin (1979) Present investigation
Reference (1)
(23)
Cytochrome P-450 content 230 f 50 pmol/106 cells 242 or 320 pmol/106 cellsa 303 ? 68.2 pmol/106 cells (n = 22)*
a Calculated from their data of 0.23 nmoVmg of cell protein, and 1.05 or 1.39 mg protein/106 cells (23, 24). * n is the number of independent experimental runs using different rats.
P-450 AND ALDRIN
EPOXIDATION TABLE
Pesticide
Substrate
Initial amount (nmolR ml)
DDT Lindane Permethrin
50 16.6 40
Aldrin
40
-
Metabolism
IN RAT HEPATOCYTES 4
by Cultured
Reaction time (hr)
Hepatocytes”
--~.
Metabolized amount (nmol/106 cells)
24 24 1
9.8 1.2 10.6
1
17.3
-. ~.
Main metabolites __DDE Various metabolitesh Dichlorovinylchrysanthemic acid Dieldrin
0 A I-day-old maintenance culture was used. See Materials and Methods for experimental details. h 2.4,6-Trichlorophenol and its sulfate, 2,3,4,&tetrachlorophenol and its sulfate, (36/45) hexachlorocyclohexene, 2,4-dichlorophenyl-glutathione, and others. Conjugated metabolites were hydrolyzed and derivatized prior to GLC analysis according to the reported method (27).
was epoxidized to dieldrin at a high rate. Thus, this reaction was examined in various states of isolated hepatocytes and liver fractions. A fresh cell suspension (1 to 6 hr after isolation) epoxidized aldrin most effectively (Table 5). Because of experimental convenience, a reaction of less than 1 hr during culture was not used. After 1 day of culture, the metabolic activity of the hepatocytes decreased slightly, but the decrease between the second and third day (24- to 4%hr cells) was not significant. Rates of aldrin epoxidation. The rates of aldrin epoxidation listed in Table 5 are tentative; therefore we examined the reaction more closely. Dieldrin formation was linear for at least 5 min when 40 nmol of aldrin was metabolized by 2 ml of a suspension of lo6 cells (see Fig. 3). The rate was proportional to the cell numbers at least in the given range (Fig. 4). The 2- or 5-min reacTABLE AI&in
Epoxidation
to Dieldrin
tion was repeated for several preparations of isolated hepatocytes. When hepatocytes were used for the metabolism experiments within 1 hr of isolation, the average value for the rate of dieldrin formation per unit amount of cytochrome P-450 was fairly high, although the values were rather scattered: 10.82 t 3.55 mol/mol P-4501min. Before the reaction. the hepatocytes were maintained in a subpension through which mixed 02/COZ was continuously bubbled at 20-25°C. Some deviations for each determination of triplicate runs were very large and the values found are not considered very reliable. To determine the origin of these variations, the dependency of the rate values on the time after isolation and on maintenance conditions was examined. Results are shown in Table 6. Changes in cell viability Die$drin(nmol)
,A:’
t
5
10 1 by Rat Hepatocytes” p’
Reaction
system
Suspension of I-hr cells Primary culture
Reaction time 5 min
Formed
dieldrin
(nmol)
6.25
r
1.85
(n = 7) Reoctik
in dishes
I-hr cells 24-hr cells 4%hr cells
1 hr 1 hr I hr
11.4
-t 0.6
(n =
8.3 8.4
r 1.96 -c 0.26
(n = 19) (n = 2)
6)
* Initial amount of aldrin was 40 nmoV106cells. See Matenals and Methods for experimental details. * Phenobarbital
treatment
10 0.5 PM) did not affect the
of cells for 24 hr beforehand rate
significantly.
(0.05
FIG. 3. Time course
Time
(min)
of aldrin epoxidation by a rar hepatocyte suspension. Inirial substrate 40 nmol. I@ cells containing 378 pmol of cytochrome P-450 in 2 ml of Krebs-Henseleit solution. Preincubation was .fu?30 set without substrate. Reaction ar 37°C. Each poinr represents the average of triplicate runs M*itk a slondard deviation of less than 3% qf the value.
KURIHARA,
70
HORI, AND ICHINOSE
Di%drin(nmol)
Cell
Number
FIG. 4. Effect of cell concentration on the aldrinepoxidation rate. Initial substrate 40 nnol in 2 ml at 37°C. The amount of dieldrin formed (nmol) in 2 min is shown. Each value is the average of triplicate runs with a standard deviation of less than 5% of the value.
and in P-450 content also were examined. These are given in Table 7. Hepatocytes maintained in a suspension through which 02/C02 was bubbled (condition B) showed a dieldrin-formation rate (Table 6) that varied considerably with time. During the early stage after isolation, the rate was abnormally high; this corresponds to the above-mentioned results. Later the rate decreased rapidly, then became’ constant for several hours although slightly decreased. When the cells were maintained in a suspension with gas gently introduced onto the surface (condition A), the dieldrin-formation rate was constant during the entire period examined. Viability and P-450 content
also were stable under condition A and in the later stages of conditions B (Tables 6 and 7). The bubbling of gas into the cell suspension at 37°C (conditon C) caused a rapid loss of metabolic activity, viability being almost zero after 6 hr. Taking into account these results, we selected cells 1 to 4 hr after isolation and maintained them at condition A in order to determine the rate of dieldrin formation per unit amount of cytochrome P-450. The average values for these conditions are shown in Table 8. Although the value is smaller than that of the early stage in condition B, it is more reliable and reproducible because of the stable metabolic activity and the viability of the cells. The apparent & and Vmax values determined for this condition also are listed in Table 8. The rate values were similar to those found for the reaction by the liver microsomes (Table 9>, though differ from the values of the liver 10,OOOg supernatant. The apparent K, and V,,,,, values for the reactions by microsomes were different from those by hepatocytes (Tables 8 and 9). The results suggest that the hepatocytes and the microsomes have different compositions of cytochrome P-450 subspecies. Rate comparison on a cell-number basis and on a liver-weight basis. Aldrin epoxi-
dation by rat hepatocytes on a cell-number basis was 1.36 _’ 0.31 nmol/min/106 cells in
TABLE 6 Dependence of the Aldrin Epoxidation Rate on the Time after Cell Isolation and on the Maintenance Condition” Time (mm) Conditionb
SO-55
70-75
90-95
200-210
350-360
A
1.45 * 0.04 *2..51 c 0.51 1.34 k 0.10
1.41 I 0.03 1.54 + 0.24 1.16 ? 0.11
1.38 -c 0.07 1.25 k 0.04 1.14 2 0.05
1.31 c 0.03 1.17 k 0.13 0.19 + 0.00
1.31 i 0.05 0.91 2 0.03 0.09 * 0.01
B
C
a The reaction took place at 37°C in 2 ml of a suspension of 8.4 x IO5 cells, which contained 185 pmol of cytochrome P-450, 50-55 min after isolation. The total amount of dieldrin formed (nmol) in 2 min together with the standard deviation of triplicate runs is shown. Turnover rate can be calculated from each value. For example, the value with an asterisk (*) corresponds to 2.51/0.185/2 = 6.78 (mol/mol P-450/min). b Condition (A) Maintained at room temperature (20°C) with gentle introduction of Oz/COz onto the surface of the cell suspension; (B) maintained at room temperature with bubbling of the same gas mixture; (C) maintained at 37°C with bubbling of the same gas mixture.
P-450
Changes
AND
ALDRIN
TABLE I in Viability during Maintenance Different Conditions Time
Condition” A B’ C
-
90-95 (I) 93.0% (2) 80.8%~ 71.2% 78.5%
EPOXIDATION
under
(min) 210
360
-b 77.9% -
95.0% 80.4% 68.4% 2.0%
u For conditions, see footnote h to Table 6. ir -shows not determined. ( Under this condition, variations in the P-450 content were determined at each stage: at 90-95 min. 185 pmollml: at 210 min. 191 pmoliml: and at 360 min. 178 pmoliml.
experiments with cells maintained under condition A. If we assume that 1 g of liver corresponds to 1.5 x 10” cells, the rate would be 204 & 46.5 nmol/min/g liver (see Table 8 footnote (I). In contrast, the rate for the microsomes was 49.9 to 69.9 (Table 9. footnote) and for the 10,OOOg supernatant was about 67 nmol/min/g liver. Thus, when we compare these values, that of isolated hepatocytes is much larger than the others. The P-450 content on a liver weight basis is calculated as 0.303 x 150 = 45.45 nmoli g liver from hepatocyte data, if we adopt the above assumption of I .5 x lo* cells/g liver. In contrast. 10 to 14 nmol P-450 was recovered in microsomes from 1 g of liver. At most, 21.5 nmol of P-450 is recovered in microsomes from 1 g of liver (29). The low TABLE
Rate”
8
4.50 5 1.01 molimol P-450imin (n = 6Jh 7.19 t- 1.49 molimol P-450imin (n = 4) VI”‘,, 9.27 k 1.17 p” (n = 4) Kt,, -_ (’ Aldrin (40 nmol) in ethanol (10 ~1) was incubated in 2 ml of Krebs-Henseleit solution, after a 30-set preincubation. with IO6 cells at 37°C. An addition of glucose (2 mg) did not change the rate. The rate on a cell number basis is calculated as I .36 nmol/lOh cells/ min from P-450 content of 303 pmol/lOh cells (Table 3). Recovery of the compounds after the reaction was oker 95%. For other details. see Materials and Methods. ” n is the number of independent experimental runs.
IN RAT
7!
HEPATOCYTES
yield of cytochrome P-450 in this fraction (and in the supernatant) explains the much lower rate values of microsomes and supernatant on a liver-weight basis. DISCUSSION
The kinetic parameters of aldrin epoxldation based on the cytochrome P-450 content differed for isolated hepatocytes and liver homogenate fractions. V,,, value fotthe hepatocytes was greater than that for the microsomes on a cytochrome P-450 basis (Tables 8 and 9). Considerable differences of apparent K,?, values were observed between cells and microsomes. The ay?. parent K, ‘s for purified rat liver cytochrome P-450 and P-448 are reported, being 7 ? 2 and 27 t 7 pM, respectively (9). The K,,, for the hepatocytes was similar to the former value. When the rates of aldrin epoxidation were compared on a cell-number or liver weight basis, the rate for isolated hepatocytes was much greater than that for the microsomal fraction and the 10,OOOg super natant from the liver homogenate. 01-r* viously. this discrepancy is caused by sip nificant loss of P-450 during preparation o!‘ these fractions. McLean and Day reportetf
TABLE
Y
4.99 t 0.09 mol/mol P-4SOimin 01 3)” 5.1 I k 0.28 molimol F-45Olmin (II :I I .64 + 0.08 phi i ,i 3) ~____-___--~~-. ~.. ClThe 10.000~ supernatant from liver homogenate showed the rate 6.68 it 0.83 mollmol P-45Oimin (II 2). Aldrin (40 nmol) m ethanol (10 ~1) was incubated at 37°C with a preincubated (30 set) mixture of lhc microsomes (or supernatant) from the liver homopcnate and NADPH (I kmol) in 2 ml of phosphate buffei Recovery of the compounds after the reaction \
72
KURIHARA,
HORI, AND ICHINOSE
that the amount of cytochrome P-450 recovered in the microsomal fraction varies with the amount originally present (19). The apparent K,,, value for the reaction by microsomes was much smaller than that for the hepatocytes. Although the K, values for very hydrophobic substrates such as aldrin have doubtful meaning in some experimental conditions (28) and some corrections for nonspecific binding of aldrin to biomolecules may significantly modify the Km value (29), the present great difference between the two apparent K, values suggests that the selective loss of some P-450 subspecies that have low affinity to aldrin takes place during preparation of the liver microsomal fraction. Therefore, microsomes seem to retain P450 of high affinity to aldrin. Interestingly, for ethylmorphine (30) and antipyrine (31, 32), great differences in K,,, values for the reactions by microsomes and hepatocytes are reported, whereas alprenol (33) and ethoxybenzamide (29) show similar K, values in both metabolic systems. The exact reason for the differences in apparent K,,, values is a future problem. Hepatocytes could be maintained in a good viability for at least 6 hr in Krebs.Henseleit solution with no loss of cytochrome P-450 content and aldrin-epoxidizing activity. They required an oxygen supply which was provided by the gentle introduction of 02/C02 gas onto the surface of the suspension. Bubbling the gas also was effective at 20°C but in the early stage of this treatment epoxidizing activity varied considerably from tube to tube. Just after isolation, hepatocytes suffer damage to their membranes from the collagenase and proteinase present. Recovery from this damage usually seems to be rapid, but may be retarded by the vigorous bubbling of the gas. Although the values found varied greatly in the early stages when 02/ CO2 was bubbled through at 20°C (condition B), hepatocytes showed high epoxidizing activity (Table 6), which has yet to be explained. The penetration rate of the
substrate may affect the rate of metabolism. Penetration into whole cells was a very rapid process (unpublished data); therefore the difference in the rates of penetration to the site of metabolism in the cells rather than into the cell itself may account for the variations found. In summary, isolated hepatocytes retained good aldrin-epoxidizing activity and a constant cytochrome P-450 content as well as good viability for the first 6 hr after isolation. Hepatocytes are a better model system for in viva metabolism than are liver microsomes or liver 10,OOOg supernatant, because during preparation these fractions lose some of the several cytochrome P-450s they contain. ACKNOWLEDGMENTS We are grateful to Professor Minoru Nakajima for his expert advice and encouragement throughout this study. Thanks also are due to Dr. Yukiaki Kuroda for his helpful suggestions on the technique used for the primary maintenance culture of hepatocytes. REFERENCES 1. P, Mold&s, J. Hogberg, and S. Orrenius, Isolation and use of liver cells, in “Methods in Enzymology” (Fleischer, S., and Packer, L., Eds.), Vol. 52, p. 60, Academic Press, New York, 1978. 2. J. R. Fry and J. W. Bridges, Use of primary hepatocyte cultures in biochemical toxicology, in “Reviews in Biochemical Toxicology” (E. Hodgson, J. R. Bend, and R. M. Philpot, Eds.), p. 201, Elsevier/North-Holland, Amsterdam/ New York, 1979. 3. T. Nakatsugawa, W. L. Bradford, and K. Usui, Hepatic disposition of parathion. Uptake by isolated hepatocytes and chromatographic translobular migration, Pesric. Biochem. Physiol. 14, 13 (1980). 4. 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). 5. A. Blom, A. H. J. Scaf, and D. K. E Meijer, Hepatic drug transport in the rat; A comparison between isolated hepatocytes, the isolated perfused liver and the liver in vivo, Biochem. Pharmacol. 8, 1553 (1982). 6. R. G. Thurman and F. C. Kauffman, Factors regulating drug metabolism in intact hepatocytes, Pharmacol. Rev. 31, 229 (1980).
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AND ALDRIN
EPOXIDATION
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