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Metabolism of benzo(a)pyrene by isolated hepatocytes and factors affecting covalent binding of benzo(a)pyrene metabolites to DNA in hepatocyte and microsomal systems
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 204, No. 2, October 15, pp. 511-523, 1980
Metabolism of Benzo(a)pyrene Affecting Covalent Binding DNA in Hepatocyte ANNA
L. SHEN, Department
WILLIAM
by Isolated Hepatocytes and of Benzo(a)pyrene Metabolites and Microsomal Systems’ E. FAHL,’
of Pharmacology, School, Madison, Received
AND
University Wisconsin
January
COLIN
of Wisconsin
Factors to
R. JEFCOATE Medical
53706
17, 1980
Covalent binding of benzo(a)pyrene (BP) metabolites to DNA was investigated in hepatocytes and liver microsomes (MC-microsomes) isolated from 3-methylcholanthrenetreated rats. The major DNA adducts formed during BP metabolism in both hepatocytes and incubations of calf thymus DNA with MC-microsomes were adducts of anti and syn isomers of trans-7,8,-dihydroxy-9,lO-epoxy-7,8,9,lO-tetrahydrobenzo(a)pyrene (diolepoxides) and of epoxide derivatives of BP-g-phenol (phenol-oxides). Diol-epoxide adducts predominated over phenol-oxide adducts in hepatocytes, while the reverse was found in microsomal incubations. In hepatocytes, both diol-epoxide and phenol-oxide adducts increased with increasing BP concentration; the ratio of diol-epoxide adduct to phenol-oxide adduct decreased from 6:l to 3:l between 30 and 100 WM BP. In microsomal incubations, decreases in DNA concentration or addition of the hepatocyte L15 medium produced larger decreases in phenol-oxide adducts than in diol-epoxide adducts. The effects of the inhibitors salicylamide, diethylmaleate, and 3,3,3,-trichloropropene oxide on formation of BP-DNA adducts are interpreted in terms of changes in precursor formation and metabolism and reductions in hepatocyte glutathione levels. Addition of 1.5 mg/ml exogenous DNA to hepatocyte incubations produced no change in covalent binding to cellular DNA, even though extracellular BP-DNA adducts accounted for 97% of the total adducts formed. Both the relative amounts of diol-epoxide and phenol-oxide adducts and the total adducts per milligram of DNA were indistinguishable with respect to extracellular and intracellular DNA. Modification of extracellular DNA by diol-epoxides was at least as efficient as modification of calf thymus DNA in incubations with MC-microsomes. It is concluded that BP diol-epoxides and phenol-oxides can leave the cell or enter the nucleus with equal facility but are more effective in binding to DNA in the cell in which they are generated.
The carcinogenicity of polycyclic aromatic hydrocarbons, such as BP,3 is dependent
upon metabolic activation by microsomal mixed-function oxygenases (1). In liver and in other tissues, BP is metabolized by cytochrome P-450-dependent mixed-function oxygenases to epoxides, which are either converted to the corresponding transdihydrodiols by epoxide hydratase (2), conjugated with glutathione (3-G), or rearrange nonenzymatically to phenols (2).
1 Supported by NIH Research Grant CA-16265 and Research Career Development Award CA-00250 from the National Cancer Institute, Department of Health, Education, and Welfare. 2 Present address: Pharmacology Department, The Medical School, Northwestern University, 303 E. Chicago Ave., Chicago, Ill. 60611. ’ Abbreviations used: BP, benzo(a)pyrene; BP-7, 8-diol-9, lo-epoxide or diol-epoxides, (t)-7o, 8P-dihydroxy-9/3, lOP-epoxy-7,8,9,10-tetrahydrobenzo(a) pyrene (anti-BP-7,8-diol-9,lOepoxide) and (t)-701, 8P-dihydroxy-So, lOol-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (syn-BP-7,8-diol-9,10-epoxide); BP-7, 8-dihydrodiol, trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene; BP-S-phenol, 3-hydroxybenzo(a)pyrene;
BP-H-phenol, 9-hydroxybenzo(a)pyrene; TCPO, 3,3,3trichloropropcne oxide; S-MC, 3-methylcholanthrene; MC-microsomes, 3-methylcholanthrene-induced rat liver microsomes; Hepes, 4-@hydroxyethyl)-l-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate; UDPGA, UDP-glucuronic acid. 511
0003-9861/80/120511-10$02.00/O Copyright 0 1980 by Academic Press. Inc. All rights of reprodurt.ion in any form reserved
512
SHEN,
FAHL,
BP-phenols are also formed by direct oxidation of BP while BP-quinones probably arise via nonenzymatic oxidation of BP-6phenol (7). These metabolites may be removed as conjugates of UDP-glucuronic acid @-lo), sulfate W-13), or glutathione (3-6), or they may be further oxygenated (14, 15). Both phenol and dihydrodiol metabolites of BP are further metabolized to products which bind covalently to cellular macromolecules, including DNA (16-20). Current evidence indicates that the anti- and syn-BP-7,8-diol-9,10-epoxides formed from metabolism of BP-7,&dihydrodiol are the most carcinogenic and mutagenic metabolities of BP (21-23). Liver microsomes, which lack mechanisms for conjugation of reactive metabolites, metabolize BP to primarily organicsoluble metabolites (phenols, quinones, and dihydrodiols) (6, 23, 24). In contrast, intact cells produce large quantities of watersoluble metabolites as a result of conjugation processes (25-27), which provide an effective means for the removal of toxic or reactive metabolites. Metabolism of BP to products which can bind covalently to cellular macromolecules, including DNA, is an essential step in BPinduced carcinogenesis (1, 28) and covalent binding of BP metabolites to DNA has been demonstrated both in whole cells and in incubations of microsomes and DNA (16-20, 26). Nucleosides covalently modified by BP metabolites can be separated by Sephadex LH-20 column chromatography (29, 30); the major nucleoside adducts isolated by this method have been identified as purine base (principally guanine) adducts of syn- and anti-BP-7,8-diol-9,10epoxides (31-33) and an epoxide derivative of BP-g-phenol (16,29). Pelkonen et al. (30) have also identified several further BP metabolites giving rise to BP-DNA adducts separable by Sephadex LH-20 chromatography. In mammalian cells, the diolepoxide adducts are predominant, even though BP-phenols are formed in large proportions (18). Hepatocytes isolated from 3-MC-treated rats metabolize BP to products which bind covalently to DNA (26). This differs from a simple MC-microsome-DNA SYS-
AND
JEFCOATE
tern, however, in several important aspects. For example, conjugation processes operating in the intact cell play a major role in removal of reactive BP metabolites and covalent binding increases as much as fivefold in the presence of inhibitors of conjugation enzymes (26). Other factors, including diffusion of metabolites from the site of generation and trapping of reactive metabolites by cellular proteins, also may determine levels of covalent binding to DNA. The studies described here examine BP metabolism and covalent binding to DNA in hepatocytes from 3-MC-treated rats. Hepatocyte BP metabolism is compared with that of MC-microsomes to identify factors important in determining levels of covalent binding. Comparisons are made between formation of BP-DNA adducts in intact cells and in a MC-microsomeDNA system under conditions approximating those which might be found intracellularly. MATERIALS
AND
METHODS
Chemicals. 3-MC was obtained from Tridom Chemical Company (New York, N. Y.), L15 from Grand Island Biological Company, (Grand Island, N. Y.), BP, diethylmaleate, and TCPO from Aldrich (Milwaukee, Wise.), and albumin (Fraction V from bovine plasma) from Reheis Pharmaceutical (Phoenix, Ariz.). Generally labeled pH]benzo(a)pyrene (27 Ci/ mmol) was obtained from AmershamiSearle (Arlington Heights, Ill). Other biochemicals were purchased from Sigma Chemical Company (St. Louis, MO.). Alzinzals. Male Holtzman rats (175-200 g) were maintained on Wayne Lab-blox and given a single intraperitoneal injection of 3-MC (15 mg) in corn oil 43 to 48 h before preparation of hepatocytes. Hepatocyte prejoarution. Hepatocytes were isolated by the method of Seglen (35), with some modifications. Livers were perfused in situ with Ca’+-free KrebsHensleit buffer containing 15 mM glucose and 0.2% albumin, followed by perfusion for 20 min with the same buffer containing 5 mM CaCl, and 0.05% collagenase. The hepatocytes were filtered once through nylon mesh (253 pm) and washed three times with cold L15 medium (pH 7.6, containing 8.3 mM glucose, 0.2% albumin, 200 units/liter insulin, 100,000 units/liter penicillin, 100 mg/liter streptomycin, and 15 mM Hepes). Cell number was determined using a hemocytometer. An average of 85-90s of the cells were intact when measured by trypan blue exclusion. The hepatocytes were preincubated for 30 min in L15 medium, centrifuged, and resuspended in fresh L15 medium before use.
BENZO(A)PYRENE
METABOLISM
Hepatocyte BP metabolism. Hepatocytes were incubated in L15 medium at 37°C with shaking. [“H]BP was added in acetone (final concentration 2.5%). Total BP metabolism was measured by the method of Van Cantfort et al. (36). For analysis of metabolites by high-pressure liquid chromatography, samples were extracted with ethyl acetate:acetone (El), as described by Fahlet al. (37) and applied to a Whatman Partisil PX5 10125 ODS-2 column. Metabolites were eluted at room temperature with a linear gradient (60% methanol/water-methanol) and 0.3-min fractions collected using an LKB Redirac fraction collector. In studies with inhibitors, the inhibitors were added in propylene glycol (final concentration less than 0.4%) or acetone (final concentration 1.5%) 5 min before addition of [“H]BP. Intra- and extracellular metabolites were separated by centrifuging the cells at 2009 for 3 min. Cytochrome P-450 assay. For measurement of cytochrome P-450 levels, freshly isolated cells were centrifuged at 200g for 3 min, resuspended in 0.25 M potassium phosphate, 0.15 M KCl, pH 7.4, and briefly sonicated. Cytochrome P-450 in the sonicate was assayed by the method of Omura and Sato (38). Glutathione assay. Cells were centrifuged at 2009 for 3 min and resuspended in 0.6 N perchloric acid, 5 mM EDTA, and sonicated. Reduced glutathione was assayed in the protein-free acid extract by the method of Hissin and Hilf (39). DNA binding. For studies of DNA binding, cells were incubated as described above for 45 min. The cells were then placed on ice, centrifuged for 3 min at 2009, and resuspended in 3 ml 5% p-aminosalicylate, 1% SDS. DNA was extracted as described by Kuroki and Heidelberger (20), with some modifications. After vortexing to lyse the cells, the samples were extracted with an equal volume of phenol reagent (phenol:mcresol:8hydroxyquinoline:water, 500:70:0.5:55 by weight), centrifuged, and the aqueous phase removed. The phenol phase was reextracted with 1.5 ml 5% paminosalicylate, 1% SDS, and DNA precipitated from the combined aqueous phases with cold (-20°C) 95% ethanol. The nucleic acids were centrifuged (lO,OOOg, 15 min) and the pellet washed with 95% ethanol, benzene, 95% ethanol, and ether. Residual ether was removed under N, and the pellet dissolved in 0.01 M NaPO,, pH 7.0. The solution was digested for 30 min with ribonuclease A (50 kg/ml), followed by digestion with protease (50 pg/ml) for 60 min. The samples were then extracted with 2 vol CHCl,:isoamyl alcohol (24:l) and precipitated with 2 ~0195% ethanol. The DNA was centrifuged at 10,OOOgfor 15 min and the pellet washed with 95% ethanol and ether. After drying under N,, the DNA was dissolved in 0.01 M Tris, 0.01 M MgCl,, pH 7.0. DNA was assayed by the diphenylamine method (40) and covalent binding of BP was expressed per milligram of DNA. Recovery of DNA averaged about 80%.
AND COVALENT
BINDING
TO DNA
513
Digestion was carried out as described by Alexandrov et al. (41). The digested DNA was chromatographed on 18 x l.O-cm LH-20 columns, as previously described (10). Studies of BP binding to calf thymus DNA in the presence of MC-microsomes were carried out as described previously (10). RESULTS
BP Oxidation
in Hepatocytes
Hepatocytes isolated from 3-MC-treated rats metabolized BP in a dose-dependent manner (Fig. 1). At 60 PM BP and a cell concentration of 2.5 x lo6 cells/ml, BP was metabolized at a rate of 15.6 2 3.8 nmol BP metabolized/lO’j cells/l5 min. Since there are 0.4 nmol P-450/106 cells, this corresponds to 37.2 + 9.1 nmol BP metabolized/nmol P450/15 min, which is similar to the rate observed in MC-microsomes (28.9 nmol BP metabolized/nmol P450/15 min) (42). V for BP metabolism in 3-MC-induced hepatocytes was 2.48 + 0.6 nmol BP metabolized/nmol P-450/min, compared with 2.62 nmol BP metabolized/nmol P-450/min for MC-microsomes. At 2.5 x lo6 cells/ml, the ED,, for BP in hepatocytes was 30-40 PM, which is much higher than the K, for microsomal BP metabolism (0.79 PM). These values were obtained using hepatocytes incubated for either 5 min (17% conversion) or 15 min at 37°C.
[BP] OJM)
FIG. 1. Dependence of hepatocyte BP metabolism on BP concentration. Hepatocytes were incubated in L15 at 2.5 x 10” cells/ml for 15 min at 37°C. Total BP metabolism was assayed by the method of Van Cantfort et al. (36) and results expressed as nmol metabolizedinmol P-450/15 min * SEM. P-450 content of the cells was 1.0 nmol/2.5 x lo6 cells.
514
SHEN,
IYB
1.
5
IO
I5
a
20
h
FAHL,
AND
during incubations at high cell concentrations. The ED,, for BP was not determined at high cell densities but could be higher than that determined at 2.5 x lo6 cells/ml. BP metabolites produced by hepatocytes were qualitatively similar to those produced by microsomal BP incubations (Table I). However, hepatocyte incubations produced increased amounts of water-soluble metabolites and decreased amounts of organicsoluble metabolites. A large fraction of the organic-soluble metabolites was composed of relatively polar metabolites eluting near the beginning of the 60-100% methanol gradient. These metabolites and an unknown Peak “II” have been designated “Fraction I” by Burke et al. (26) and include polyhydroxylated BP metabolites (14) and sulfate conjugates (11). Peak “II” eluted as if it were more polar than BP-9,10dihydrodiol and included BP-tetrols and other metabolites.
*
25
JEFCOATE
30
Time (mid FIG. 2. Time course of hepatocyte BP metabolism at varying cell densities. Hepatocytes were incubated in L15 in the presence of 60 pM [3H]BP at cell densities of 1.25 (O), 2.5 (O), 5 (X), and 7.5 (A) x IO6 cells/ml and aliquots taken for total BP metabolism at the times indicated. Total BP metabolism was assayed as described by Van Cantfort et al. (36). Values represent duplicate determinations.
Figure 2 shows the time course of BP metabolism (60 PM BP) at various cell densities. At 1.25 x 10” cells/ml, metabolism was nearly linear for up to 30 min, while at higher cell densities linearity was maintained for progressively shorter periods. The decrease in the duration of linear metabolism may be due to a decrease in the concentration of BP available to cytochrome P-450 or to the slow decline in pH
Inhibition
Salicylamide, diethylmaleate, and TCPO effected large changes in the distribution of BP metabolites (Table I). Salicylamide and diethylmaleate had no effect on total BP metabolism, while TCPO had a slight
TABLE
I
nmoU10” Control
Metabolite Fraction I Peak II BP-9,10-dihydrodiol BP-4,5-dihydrodiol BP-7,8-dihydrodiol BP-quinones BP-g-phenol BP-3-phenol Total organic H,O phase Total
phase
metabolites
of Conjugation
Diethylmaleate
‘7.15 1.39 0.65 0.25 0.99 0.51 0.24 0.49 11.67 5.98
k _f k k k 2 rfk 2 2
0.32 0.20 0.06 0.16 0.52 0.13 0.06 0.12 0.69 0.41
5.40 1.72 2.09 0.71 2.44 0.84 0.80 0.97 14.97 4.30
+ + t + + + + k + +
0.69 0.08 0.19 0.02 0.03 0.32 0.08 0.09 1.19 0.18
17.65
2 0.80
19.27
* 1.20
cells/l5
min
Salicylamide (76) (124) (323) (282) (246) (164) (333) (198) (128) (72)
1.64 0.54 3.05 1.12 3.09 1.98 0.99 1.69 14.10 3.78
2 + + k k + + 2 + 2
0.35 0.14 0.21 0.16 0.40 0.46 0.19 0.33 0.85 0.42
17.88
2 0.95
TCPO (23) (39) (471) (444) (312) (386) (412) (344) (121) (63)
0 Hepatocytes were incubated at 2.5 x 10” cells/ml and 60 pM [3H]BP and metabolites under Materials and Methods. Concentrations of inhibitors used were: diethylmaleate, 2.6 mM; and TCPO, 2.3 mM. Results are expressed as mean + SD. Values in parentheses of control levels.
5.40 0.10 0.23 0.46 0.57 0.69 2.10 0.99 10.54 4.23
k 2 2 -I-rt t k 2 iz
2.29 0.04 0.04 0.16 0.21 0.24 0.08 0.04 2.32 0.17
14.77
c 2.33
(76) (7.5) (35) (181) (57) (135) (875) (202) (90) (71)
analyzed as described 2.1 mM; salicylamide, represent percentage
BENZO(A)PYRENE
METABOLISM
AND
inhibitory effect. In the presence of salicylamide, water-soluble and Fraction I metabolites were decreased by 37 and 77%, respectively, while Peak II was decreased by 61%. In contrast, all other organic-soluble metabolites were increased by a surprisingly constant amount (3.1-4.7 times). Diethylmaleate produced a 28% drop in water-soluble metabolites. Fraction I metabolites decreased 24% while all other organic-soluble metabolites were increased (124-333%). TCPO inhibited formation of dihydrodiols, increased formation of BPphenols and quinones, and decreased levels of water-soluble metabolites. Peak II was almost completely absent in the presence of TCPO, indicating that hydration of an epoxide is required for formation of this metabolite. Freshly-isolated hepatocytes had a reduced glutathione content of 7.7 ? 1.1 pg/lO” cells, which was unchanged after incubation in L15 for 30 min at 37°C. Further incubation for 45 min with or without BP did not significantly decrease intracellular GSH levels. Addition of salicylamide to incubations containing BP did not affect reduced glutathione levels; however, 2.1 mM diethylmaleate and 2.3 mM
20
40 Fraction
COVALENT
BINDING
515
TO DNA
TCPO decreased intracellular reduced glutathione levels to 5.2 ? 0.6 and 5.0 ? 1.6 pg/106 cells, respectively (-70% of controls). In these incubations, BP metabolites distributed inside and outside the cells in a ratio of 1.4:1 (data not shown). Salicylamide and diethylmaleate increased the proportion of intracellular metabolites by a small but statistically insignificant amount. Covalent ModiJcation Hepatocytes
of DNA
in
Covalent binding of BP metabolites to DNA was analyzed by LH-20 column chromatography (Fig. 3). The predominant fraction of BP-nucleoside adducts eluting from LH-20 was due to syn- and antiBP-7,8-diol-9,10-epoxides bound to (primarily) guanine (Peak A) (43), although the bulk of the radioactivity eluting from the LH-20 columns was found in the flowthrough peak (F.T.). Peak D, a BP-phenoloxide adduct (16, 43), was always a relatively small fraction of the total BP-DNA adducts. This is in contrast to the MC-microsomes-calf thymus DNA system where BP-phenol metabolite(s) form the
60 Number
eo
loo
FIG. 3. Sephadex LH-20 column chromatography of [“H]BP metabolites covalently bound to nucleosides. Hepatocytes were incubated in L15 at 2.5 x 10” cells/ml and 60 PM [“H]BP for 45 min at 37°C. DNA was isolated, digested, and chromatographed as described under Materials and Methods. Peaks were assigned according to King et al. (29).
516
SHEN,
FAHL,
AND
predominant BP-DNA adduct and F.T. is only about 20-30% of the total. The F.T. has not been characterized. Digestion of the DNA for longer times and with larger amounts of enzymes did not affect the relative sizes of the F.T., A, or D peaks. Similarly, collection of the F.T. followed by an additional period of digestion did not release any additional Peak A or Peak D material. Rechromatography of the F.T. on G25 indicated several components with the majority of radioactivity associated with a peak of protein separate from the DNA peak. In general, the size of the F.T. correlated with the extent of BP metabolism rather than with covalent binding to DNA. Formation of diol-epoxide and phenol-oxide adducts was rapid, reaching a maximum at between 10 and 15 min (Table II). The ratio of Peak D to Peak A remained constant at about 0.2 from 10 to 45 min, in contrast with the dramatic increase reported by Jernstrom et al. during this period (47). Figure 4 shows the dependence ‘of BPDNA adduct formation on BP concentration. Peak D was undetectable at 15 pM BP, but increased more rapidly than Peak A with increasing BP concentration. The ratio of Peak D to Peak A increased from 0.15 to 0.34 between 30 and 100 pM BP. Covalent binding to hepatocyte DNA (Fig. 1). was more sensitive than BP TABLE
II
TIME COURSE OF COVALENT BINDING OF BP METABOLITES TO HEPATOCYTE DNA” Incubation time (min) 10 15
30 45
Covalent binding (pmol/mg DNA) F.T.
A
D
25.7 35.6 56.6 47.7
28.0 40.3 33.4 28.5
6.8 6.6 6.7 6.4
D/A
0.24 0.16 0.20 0.22
a Hepatocytes were incubated at 2.5 x lo6 cells/ml and 60 PM [“H]BP for the times indicated and [“HIBPDNA adducts isolated as described under Materials and Methods. Values represent duplicate determinations.
JEFCOATE
FIG. 4. Dependence of covalent binding of [“HIBP to DNA on BP concentration. Hepatocytes were incubated in L15 at 2.5 X 10” cells/ml for 45 min at 37°C. DNA was isolated, digested, and chromatographed as described under Materials and Methods. Results are expressed as mean c SD for Peak A (0) and Peak D (0).
metabolism to changes in BP concentration. Between 15 and 60 pM BP, metabolism increased almost eightfold, while covalent binding of BP-7,8-diol-9,10-epoxide increased 14 times. Formation of BP-DNA adducts was also dependent upon cell density (Fig. 5). Peak D was more sensitive to changes in cell number than was Peak A and was undetectable at 5 and 7.5 x lo6 cells/ml. Again, covalent binding was more sensitive than BP metabolism to increasing cell density. At 60 pM BP and cell densities of 5 x lo6 cells/ml, formation of diol-epoxide adducts was only 13% of that observed at 2.5 x lo6 cells/ml, even though BP metabolism was reduced only slightly. Salicylamide, diethylmaleate, and TCPO all increased covalent binding of BP to DNA (Table III). Salicylamide stimulated Peak A and Peak D roughly equally. Diethylmaleate was more effective than salicylamide in enhancing covalent binding and also had a greater effect on Peak D than on Peak A. TCPO decreased Peak A by 90%, while producing the largest (1270%) increase in Peak D. When calf thymus DNA (1.5 mg/ml) was present, 97% of total DNA modification was extracellular (Table IV) and no significant effects on covalent binding of 13H]BP
BENZO(A)PYRENE
METABOLISM
AND COVALENT
BINDING TABLE
517
TO DNA III
EFFECTSOF INHIBITION OF CONJUGATION ENZYMES ANDEPOXIDE HYDRATASEONCOVALENTBINDING OF ["H]BP METABOLITESTO DNA"
Cell
Density (Cells/ml)
FIG. 5. Dependence of covalent binding of [3H]BP to DNA on cell density. Hepatocytes were incubated in L15 in the presence of 60 /.LM[3H]BP for 45 min at 37°C. DNA was isolated, digested, and chromatographed as described under Materials and Methods. Results are expressed as mean 2 SD.
to hepatocyte DNA were observed. Significantly, the ratio of Peak A to Peak D was the same for hepatocyte DNA and extracellular DNA. Covalent binding per milligram DNA was similar for both intraand extracellular DNA, since the concentration of extracellular DNA was 30 times that found intracellularly (0.05 mg/ml). Modifiation of DNA by MicrosomallyGenerated BP Metabolites
The binding BP metabolites
to calf thymus DNA of generated by microsomal TABLE
Additions
Peak A
Peak D
Control Salieylamide (2.6 mM) Diethylmaleate (2.1 mM) TCPO (2.3 mM)
35.1 40.4 (115) 20.6 (201) 3.6 (10)
6.6 8.5 (130) 22.8 (345) 83.8 (1270)
” Hepatocytes were incubated at 2.5 x 10” cells/ml and 60 WM [aH]BP in the presence of the inhibitors indicated and [3H]BP-DNA adducts isolated as described under Materials and Methods. Values represent experiments performed in duplicate. Numbers in parentheses are percentage of control values.
metabolism was examined to determine the relative effects of pH, medium, and DNA concentration on formation of BP-DNA adducts. A drop in pH from 7.5 to 7.0 produced similar decreases in both Peak A and Peak D which were larger than the decrease observed in BP metabolism (Table V). In the presence of L15 medium, which contains a mixture of amino acids, metabolism of BP and formation of Peak A were reduced by about 15% while Peak D decreased by 60%. A 33-fold decrease in DNA concentration decreased formation of Peak A by 13 times, while Peak D decreased 55 times. The total quantity of BP metabolites trapped by DNA in Peaks A and D was saturated at high DNA conIV
EFFECTSOF EXTRACELLULAR DNA ONCOVALENTBINDINGOF
Adduct
Extracellular DNA (q/ml)
pmol bound/ml incubation Cellular
Peak A Peak A
0 1.5
1.5 + 0.4 1.9 * 0.5
Peak D Peak D
0 1.5
0.2 2 0.07 0.2 2 0.05
Extracellular
[SH]BP TO HEPATOCYTE DNA" pmol bound/mg DNA Cellular
62.8 2 18.4
30.8 + 7.5 38.2 k 10.4
7.6 ? 2.0
4.1 2 1.4 4.0 t 1.0
Extracellular 41.9 rt_ 12.3 5.1 lr
1.3
a Hepatocytes were incubated in L15 at 2.5 x lo6 cells/ml in the presence of 60 PM [3H]BP with or without 1.5 mg/ml calf thymus DNA. Cell DNA was isolated as described under Materials and Methods, except that the cells were washed once with phosphate-buffered saline to remove residual DNA. DNA was isolated from the medium as described for DNA from mierosomal incubations (10). Pmol bound/ml incubation was calculated using a value of 0.05 mg DNA/2.5 x lo6 cells. Results are expressed as mean 2 SD.
518
SHEN, FAHL, AND JEFCOATE TABLE
V
EFFECTSOF pH, BUFFER,AND DNA CONCENTRATIONON MICROSOMALBP METABOLISM ANDBINDINGOF [3H]BP TO DNA" pmol bound/ml incubation
[DNA1 Buffer 50 50 50 50
mMKP mMKP mMKP mMKP
50 50 50 50 50
mMKP mMKP mMKP mMKP mMKP
b-w/ml)
PH
BP metabolism (nmo1145 min)
Peak A
Peak D
1.0 1.0 1.0 1.0
7.5 7.25 7.0 7.5
38.3 30.8 25.0 33.0
28.0 21.0 14.6 23.2
219.8 143.0 107.4 88.2
0.03 0.1 0.5 1.0 2.0
7.5 7.5 7.5 7.5 7.5
-
1.5 2.6 15.4 28.0 26.1
2.9 13.3 93.9 219.8 216.3
+ L15
U MC-microsomes (0.3 mg/ml) and calf thymus DNA, in the concentrations indicated, were incubated with 60 PM BP for 45 min as described under Materials and Methods. BP metabolism and formation of [SH]BPDNA adducts were determined as described under Materials and Methods. Values represent results of duplicate determinations.
centrations, although higher DNA concentrations were required for saturation of Peak D binding. Table VI compares BP-DNA adduct formation in hepatocytes with that found in the microsomal system. For purposes of comparison, the concentration of DNA and the rate of BP metabolism are set to
be the same in the microsomal and cellular incubations. This direct comparison indicates similar amounts of Peak A in the two systems but 13 times less Peak D in hepatocytes. When diethylmaleate and salicylamide were added to hepatocytes to decrease conjugation of BP metabolites, Peak A was increased to about three times that
TABLE
VI
MICROSOMALAND HEPATOCYTE ACTIVATIONOF [3H]BPa Covalent binding to DNA
Incubation
(mgiml)
[P-450] (nmol/ml)
BP metabolism (nmoY 45 mm/ml)
MC-microsomes Hepatocytes Hepatocytes + Diethylmaleate + Salicylamide
0.05 0.05 0.05
0.8 1.0 1.0
38.1 47.4 46.1
[DNA1
Total [$H]BP bound (pmoY45 mm/ml)
Proportion of metabolites bound x lo4
Peak A
Peak D
Peak A
Peak D
1.7 1.4 4.7
3.8 0.3 1.6
0.4 0.3 1.0
1.0 0.06 0.3
(1Hepatocytes (2.5 x lo6 cells/ml, containing 0.05 mg DNA/2.5 x lo6 cells) or MC-microsomes and calf thymus DNA at 0.05 mg/ml were incubated with [3H]BP (60 PM) as described under Materials and Methods. Samples were assayed for BP metabolism by the method of Van Cantfort et at. (36). Covalent binding at 45 min was determined as described under Materials and Methods.
BENZO(A)PYRENEMETABOLISMANDCOVALENTBINDINGTODNA
found in microsomal incubations. Peak D was also increased but was still only 42% of microsomally generated Peak D adducts. DISCUSSION Hepatocyte BP Metabolism Microsomal Metabolism
versus
The overall rates of BP metabolism per nanomole P-450 were similar in hepatocytes and microsomes. However, the ED,, for metabolism of BP in hepatocytes was 40 times higher than the K,,, for microsomal BP metabolism. While a part of this difference may be accounted for by nonspecific trapping of BP by cell protein and lipid (hepatocyte incubations contain about 22 times as much protein as the microsomal incubations used for K,,, determinations), other factors may be acting to reduce the concentration of BP in the microsomal membrane. For example, little is known about the rate of BP uptake, which may become limiting at low BP concentrations. Alternatively, some intracellular compartmentalization may act to decrease the microsomal BP concentration in the hepatocytes. We have recently found that microsomal metabolism of BP is activated when inhibitory BP-quinones are removed by glucuronidation (10). In hepatocytes, BPquinones were decreased threefold relative to microsomes (Table I), presumably because of conjugation by UDPGA and sulfate (26). Nevertheless, elevation of BPquinones in the presence of salicylamide, an inhibitor of glucuronide and sulfate conjugation, did not inhibit hepatocyte BP metabolism. It seems, therefore, that inhibition by BP-quinones is less important in hepatocytes than in microsomal metabolism, possibly because of binding to cellular macromolecules. We have recently observed a substantial decrease in the inhibitory effect of BP-quinones on microsomal BP metabolism in the presence of exogenous DNA (G. Keller, unpublished data). of BP Metabolites in Hepatocytes
Conjugation
Conjugation in hepatocytes as measured by water-soluble and polar metabolites
519
(74% of total metabolites) was greater than was found with microsomal metabolism, even when MC-microsomes were supplemented by 2 mM UDPGA (52% of total metabolites). This higher degree of conjugation in cells is to be expected from the presence of cytosolic sulfotransferases and glutathioneS-transferases in the cells. Previous studies have indicated that glucuronidation of BP-dihydrodiols by isolated microsomes was much less effective than glucuronidation of BP-phenols (8). However, the similar stimulatory effect of salicylamide, an inhibitor of both glucuronidation and sulfation, on the recovery of both BP-dihydrodiols and BP-phenols from hepatocytes suggests that, in the hepatocyte, BP-dihydrodiols are conjugated more readily than by the UDP-glucuronyl transferase of isolated microsomes. Burke et al. (26) have shown that BP-4,5-dihydrodiol and BP-7,8-dihydrodiol form glucuronides rather than sulfates in hepatocytes. It seems, therefore, that the specificity of UDPglucuronyl transferase in the hepatocyte is less than in isolated microsomes. Glutathione levels in these hepatocytes were somewhat higher than those reported by Burke et al. and were less readily depleted by diethylmaleate (26). This may in part be due to the presence of cysteine and methionine in the L15 medium which may facilitate the maintenance of high levels of intracellular glutathione. Since the relatively small decline (30%) in intracellular glutathione caused by diethylmaleate produced major changes in BP metabolites and covalent binding to DNA, it is likely that not more than a half of the total intracellular glutathione is readily available for conjugation with these reactive electrophiles. Glutathione conjugation of BP-oxides has been previously established (3, 4). The increase in both BP-dihyrodiols and BP-g-phenol produced by diethylmaleate are consistent with decreased conjugation of intermediate BPoxides by glutathione. The twofold increase in BP-&phenol is more surprising since the intermediate BP-2,3-oxide (6) does not undergo hydration and is probably very shortlived. It is possible but unlikely that half of the BP-2,3-oxide is trapped by glutathione.
520
SHEN,
FAHL,
More likely, changes in glutathione or some direct action of diethylmaleate and TCPO are also linked to the maintenance of UDP-glucuronosyl transferase or sulfotransferase activities. The change in glutathione levels did not have a significant effect on the levels of BP-quinones. The effects of TCPO on BP metabolites from hepatocytes are fully consistent with the dual capability of TCPO to deplete glutathione to the same extent as diethylmaleate and to partially inhibit cellular epoxide hydratase. Similar results have previously been reported by Burke et al. (26). The inhibitory effects of TCPO on dihydrodiol formation in hepatocytes are in the same order (9,lO > 7,8 > 4,5) as has previously been observed for microsomal metabolism of BP (42). Indeed, the slight stimulatory effect on the formation of BP-4,5-dihydrodiol indicates that decreased glutathione conjugation outweighs decreased hydration in this case. Modification of DNA in Hepatocyte and Microsomal Incubations
Covalent
The LH-20 elution profile of BP-modified nucleosides isolated from the DNA of hepatocytes was similar to that obtained from the MC-microsomes-calf thymus DNA system except that nucleosidephenol-oxide adducts (Peak D) were only a minor component as compared to diolepoxide adducts (Peak A). Depletion of glutathione by only 30% in the presence of diethylmaleate enhanced formation of the phenol-oxide adduct 3.5-fold, which can be attributed to the combined effect of both a two- to threefold increase in BP-g-phenol levels and diminished scavenging of BP phenol-oxides by glutathione. The even larger enhancement of covalent binding of phenol-oxides to DNA by TCPO is also due to the combined effects of enhanced BP-gphenol levels and decreased scavenging of phenol-oxides by diminished levels of glutathione. Decreased diol-epoxide modification of DNA caused by TCPO correlated with the decreased level of BP-7,8-dihydrodial, while increased formation of diolepoxide-DNA adducts in the presence of
AND
JEFCOATE
diethylmaleate correlated with increased levels of BP-7&dihydrodiol. Although salicylamide increased BP-7, 8-dihydrodiol and BP-g-phenol levels 3. land 4.1-fold, respectively, DNA modification was enhanced only slightly (Table III). We have previously shown (44) that BP-7, 8-diol metabolism is more sensitive to quinone inhibition than is BP metabolism. Consequently, the enhanced levels of BPquinones in the presence of salicylamide may exert an inhibitory effect on formation of BP-7,8-diol-9,10-epoxides and subsequent DNA modification even though there is no measurable effect on total BP metabolism. The results presented here differ from those of Jernstrijm et al. (46, 47) in that we observe less covalent binding of BP-phenoloxides. It is likely that L15 medium acts both to maintain higher levels of glutathione through the effects of cysteine and methionine on leakage of reduced glutathione from the cells (48, 49) and to directly scavenge extracellular phenol-oxides to a greater extent than the Krebs-Ringer buffer used in their studies. These studies also differ in that a similar time dependence was observed for both diol-epoxide and phenol-oxide binding to hepatocyte DNA (Table II) instead of a sharp peak in phenol-oxide binding after 15 min followed by a rapid decay (47). The total extent of hepatocyte DNA modification in this study was similar to that reported by Burke et al. (26).
Factors affecting microsomal or hepatocyte BP metabolism produced a generally greater effect on covalent binding to DNA and particularly on covalent binding of phenol-oxides. Thus, DNA modification in hepatocytes was more sensitive to increasing BP concentration (Figs. 1 and 4) and cell density (Figs. 2 and 5) while, in the microsomal system, enhanced sensitivity to the effects of pH, L15 medium, and DNA concentration was observed (Table V). The selection between phenol-oxide addition to DNA and diol-epoxide addition increased in the order pH < L15 < DNA concentration. The effect of DNA concentration on the extent of modification of DNA reflects competition between DNA, water, and
BENZO(A)PYRENEMETABOLISMANDCOVALENTBINDINGTODNA
probably microsomal components for the labile intermediates. The saturation of DNA adduct formation at high levels of DNA (Table V) is consistent with a steady-state model for the two intermediates in which the rate of hydration is comparable to the rate of interaction with DNA. The shift in the saturation curve for phenol-oxide adduct formation suggests that phenoloxide hydration and/or rearrangement is faster than diol-epoxide hydration. Quantitative Evaluation of DNA Modijcation in Hepatocytes
The ready export of BP metabolites (42%) found here and by other workers (26,27) suggests that metabolites generated in one cell may enter other cells and undergo further metabolism and DNA binding. Studies of the microsomal system indicate that the concentration of enzymes and DNA is a crucial consideration in these multistep pathways. For a hepatocyte suspension, the effective volume for further oxidation, conjugation, and covalent binding to DNA of BP metabolites must be somewhere intermediate between the volume of the cells (fully intracellular metabolism and adduct formation) and the total incubation volume (cell boundaries insignificant). When the DNA concentration in the MCmicrosomal system is lowered to that found in hepatocytes, total modification of DNA by BP-7,8-diol-9,10-epoxide is quantitatively similar in both systems (Table VI). However, under these conditions, the relative proportion of the precursor BP-7, 8dihydrodiol among the metabolites is lower in hepatocytes (5.6%) than in MCmicrosomes (15%) due to conjugation in the cells. Inhibition of hepatocyte conjugation mechanisms by either diethylmaleate or salicylamide increases the proportion of BP-7,8-dihydrodiol to that found with MCmicrosomes. Nevertheless, modification of hepatocyte DNA by BP-7,8-diol-9,10epoxide in the presence of both diethylmaleate and salicylamide is nearly three times higher than that found in microsomal incubations. It is, however, at least five times less than the maximum rate of DNA modification observed in microsomal
521
incubations containing saturating levels of DNA (about 1 mg/ml, Table V). A major reason for relatively low phenoloxide modification in hepatocytes is the higher reactivity of BP-phenol-oxide with glutathione and other intracellular nucleophiles. On the other hand, DNA modification by both diol-epoxides and by phenoloxides in hepatocytes were similarly proportional to the 15-min levels of the precursors BP-7,8-dihydrodiol (0.04%) and BP-g-phenol (0.06%), irrespective of conjugation activity. Presumably, either formation of phenol-oxide or its reaction with DNA must be more rapid than the equivalent steps from BP-7,8-dihydrodiol to compensate for the greater lability of phenol-oxides. For diol-epoxides, the modification of calf thymus DNA was greater with hepatocyte BP activation (64.7 L 18.4 pmol Peak A/ml, Table IV) than with microsomal activation (28 pmol Peak A/ml incubation, Table V). Indeed, a better comparison is obtained with microsomes supplemented with UDPglucuronic acid, where BP-quinones are removed by conjugation. Formation of diolepoxide-DNA adducts is stimulated up to 2.7-fold (10) while phenol-oxide adducts are decreased, emphasizing the importance of conjugation pathways on BP-DNA adduct formation. When calf thymus DNA was added to hepatocytes, 97% of the BP-DNA adducts were extracellular, strongly suggesting that the vast majority of BP-diol-epoxides and phenol-oxides leave the cells and react with extracellular constituents. The similar Peak A/Peak D ratios, intra- and extracellularly, indicate that the ratios of diolepoxides and phenol-oxides leaving the cell and entering the nucleus are similar. Since DNA modification expressed as picomoles bound per milligram DNA (Table IV) is the same inside and outside the cell, the reactivity and selectivity of hepatocyte DNA must be comparable to that of purified calf thvmus DNA. Finally, the lack of effect of saturating levels of extracellular DNA on the modification of hepatocyte DNA suggests that modification of hepatocyte DNA by BP metabolites is primarily an intracellular process.
522
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FAHL,
AND
JEFCOATE
ACKNOWLEDGMENT We are grateful discussions during
to Dr. Bengt Jernstrom the course of this work.
for helpful
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AND JEFCOATE, C. R. (1979) Cancer Res. 39, 4123-4129. 45. FAHL, W. E., SHEM, A. L., KELLER, G., WRIGHTON, S. A., AND JEFCOATE, C. R. (1980) in Microsomes, Drug Oxidations, and Chemical Carcinogenesis (Coon, M. J., Conney, A. H., Estabrook, R. W., Gelboin, H. V., Gillette, J. R., and O’Brien, P. J., eds.), pp. 11991202, Academic Press, New York. 46. JERNSTR~M, B., ORRENIUS, S., UNDEMAN, O., GRASLUND, A., AND EHRENBERG, A. (1978) Gamer
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Metabolism of Benzo(a)pyrene Affecting Covalent Binding DNA in Hepatocyte ANNA
L. SHEN, Department
WILLIAM
by Isolated Hepatocytes and of Benzo(a)pyrene Metabolites and Microsomal Systems’ E. FAHL,’
of Pharmacology, School, Madison, Received
AND
University Wisconsin
January
COLIN
of Wisconsin
Factors to
R. JEFCOATE Medical
53706
17, 1980
Covalent binding of benzo(a)pyrene (BP) metabolites to DNA was investigated in hepatocytes and liver microsomes (MC-microsomes) isolated from 3-methylcholanthrenetreated rats. The major DNA adducts formed during BP metabolism in both hepatocytes and incubations of calf thymus DNA with MC-microsomes were adducts of anti and syn isomers of trans-7,8,-dihydroxy-9,lO-epoxy-7,8,9,lO-tetrahydrobenzo(a)pyrene (diolepoxides) and of epoxide derivatives of BP-g-phenol (phenol-oxides). Diol-epoxide adducts predominated over phenol-oxide adducts in hepatocytes, while the reverse was found in microsomal incubations. In hepatocytes, both diol-epoxide and phenol-oxide adducts increased with increasing BP concentration; the ratio of diol-epoxide adduct to phenol-oxide adduct decreased from 6:l to 3:l between 30 and 100 WM BP. In microsomal incubations, decreases in DNA concentration or addition of the hepatocyte L15 medium produced larger decreases in phenol-oxide adducts than in diol-epoxide adducts. The effects of the inhibitors salicylamide, diethylmaleate, and 3,3,3,-trichloropropene oxide on formation of BP-DNA adducts are interpreted in terms of changes in precursor formation and metabolism and reductions in hepatocyte glutathione levels. Addition of 1.5 mg/ml exogenous DNA to hepatocyte incubations produced no change in covalent binding to cellular DNA, even though extracellular BP-DNA adducts accounted for 97% of the total adducts formed. Both the relative amounts of diol-epoxide and phenol-oxide adducts and the total adducts per milligram of DNA were indistinguishable with respect to extracellular and intracellular DNA. Modification of extracellular DNA by diol-epoxides was at least as efficient as modification of calf thymus DNA in incubations with MC-microsomes. It is concluded that BP diol-epoxides and phenol-oxides can leave the cell or enter the nucleus with equal facility but are more effective in binding to DNA in the cell in which they are generated.
The carcinogenicity of polycyclic aromatic hydrocarbons, such as BP,3 is dependent
upon metabolic activation by microsomal mixed-function oxygenases (1). In liver and in other tissues, BP is metabolized by cytochrome P-450-dependent mixed-function oxygenases to epoxides, which are either converted to the corresponding transdihydrodiols by epoxide hydratase (2), conjugated with glutathione (3-G), or rearrange nonenzymatically to phenols (2).
1 Supported by NIH Research Grant CA-16265 and Research Career Development Award CA-00250 from the National Cancer Institute, Department of Health, Education, and Welfare. 2 Present address: Pharmacology Department, The Medical School, Northwestern University, 303 E. Chicago Ave., Chicago, Ill. 60611. ’ Abbreviations used: BP, benzo(a)pyrene; BP-7, 8-diol-9, lo-epoxide or diol-epoxides, (t)-7o, 8P-dihydroxy-9/3, lOP-epoxy-7,8,9,10-tetrahydrobenzo(a) pyrene (anti-BP-7,8-diol-9,lOepoxide) and (t)-701, 8P-dihydroxy-So, lOol-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (syn-BP-7,8-diol-9,10-epoxide); BP-7, 8-dihydrodiol, trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene; BP-S-phenol, 3-hydroxybenzo(a)pyrene;
BP-H-phenol, 9-hydroxybenzo(a)pyrene; TCPO, 3,3,3trichloropropcne oxide; S-MC, 3-methylcholanthrene; MC-microsomes, 3-methylcholanthrene-induced rat liver microsomes; Hepes, 4-@hydroxyethyl)-l-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate; UDPGA, UDP-glucuronic acid. 511
0003-9861/80/120511-10$02.00/O Copyright 0 1980 by Academic Press. Inc. All rights of reprodurt.ion in any form reserved
512
SHEN,
FAHL,
BP-phenols are also formed by direct oxidation of BP while BP-quinones probably arise via nonenzymatic oxidation of BP-6phenol (7). These metabolites may be removed as conjugates of UDP-glucuronic acid @-lo), sulfate W-13), or glutathione (3-6), or they may be further oxygenated (14, 15). Both phenol and dihydrodiol metabolites of BP are further metabolized to products which bind covalently to cellular macromolecules, including DNA (16-20). Current evidence indicates that the anti- and syn-BP-7,8-diol-9,10-epoxides formed from metabolism of BP-7,&dihydrodiol are the most carcinogenic and mutagenic metabolities of BP (21-23). Liver microsomes, which lack mechanisms for conjugation of reactive metabolites, metabolize BP to primarily organicsoluble metabolites (phenols, quinones, and dihydrodiols) (6, 23, 24). In contrast, intact cells produce large quantities of watersoluble metabolites as a result of conjugation processes (25-27), which provide an effective means for the removal of toxic or reactive metabolites. Metabolism of BP to products which can bind covalently to cellular macromolecules, including DNA, is an essential step in BPinduced carcinogenesis (1, 28) and covalent binding of BP metabolites to DNA has been demonstrated both in whole cells and in incubations of microsomes and DNA (16-20, 26). Nucleosides covalently modified by BP metabolites can be separated by Sephadex LH-20 column chromatography (29, 30); the major nucleoside adducts isolated by this method have been identified as purine base (principally guanine) adducts of syn- and anti-BP-7,8-diol-9,10epoxides (31-33) and an epoxide derivative of BP-g-phenol (16,29). Pelkonen et al. (30) have also identified several further BP metabolites giving rise to BP-DNA adducts separable by Sephadex LH-20 chromatography. In mammalian cells, the diolepoxide adducts are predominant, even though BP-phenols are formed in large proportions (18). Hepatocytes isolated from 3-MC-treated rats metabolize BP to products which bind covalently to DNA (26). This differs from a simple MC-microsome-DNA SYS-
AND
JEFCOATE
tern, however, in several important aspects. For example, conjugation processes operating in the intact cell play a major role in removal of reactive BP metabolites and covalent binding increases as much as fivefold in the presence of inhibitors of conjugation enzymes (26). Other factors, including diffusion of metabolites from the site of generation and trapping of reactive metabolites by cellular proteins, also may determine levels of covalent binding to DNA. The studies described here examine BP metabolism and covalent binding to DNA in hepatocytes from 3-MC-treated rats. Hepatocyte BP metabolism is compared with that of MC-microsomes to identify factors important in determining levels of covalent binding. Comparisons are made between formation of BP-DNA adducts in intact cells and in a MC-microsomeDNA system under conditions approximating those which might be found intracellularly. MATERIALS
AND
METHODS
Chemicals. 3-MC was obtained from Tridom Chemical Company (New York, N. Y.), L15 from Grand Island Biological Company, (Grand Island, N. Y.), BP, diethylmaleate, and TCPO from Aldrich (Milwaukee, Wise.), and albumin (Fraction V from bovine plasma) from Reheis Pharmaceutical (Phoenix, Ariz.). Generally labeled pH]benzo(a)pyrene (27 Ci/ mmol) was obtained from AmershamiSearle (Arlington Heights, Ill). Other biochemicals were purchased from Sigma Chemical Company (St. Louis, MO.). Alzinzals. Male Holtzman rats (175-200 g) were maintained on Wayne Lab-blox and given a single intraperitoneal injection of 3-MC (15 mg) in corn oil 43 to 48 h before preparation of hepatocytes. Hepatocyte prejoarution. Hepatocytes were isolated by the method of Seglen (35), with some modifications. Livers were perfused in situ with Ca’+-free KrebsHensleit buffer containing 15 mM glucose and 0.2% albumin, followed by perfusion for 20 min with the same buffer containing 5 mM CaCl, and 0.05% collagenase. The hepatocytes were filtered once through nylon mesh (253 pm) and washed three times with cold L15 medium (pH 7.6, containing 8.3 mM glucose, 0.2% albumin, 200 units/liter insulin, 100,000 units/liter penicillin, 100 mg/liter streptomycin, and 15 mM Hepes). Cell number was determined using a hemocytometer. An average of 85-90s of the cells were intact when measured by trypan blue exclusion. The hepatocytes were preincubated for 30 min in L15 medium, centrifuged, and resuspended in fresh L15 medium before use.
BENZO(A)PYRENE
METABOLISM
Hepatocyte BP metabolism. Hepatocytes were incubated in L15 medium at 37°C with shaking. [“H]BP was added in acetone (final concentration 2.5%). Total BP metabolism was measured by the method of Van Cantfort et al. (36). For analysis of metabolites by high-pressure liquid chromatography, samples were extracted with ethyl acetate:acetone (El), as described by Fahlet al. (37) and applied to a Whatman Partisil PX5 10125 ODS-2 column. Metabolites were eluted at room temperature with a linear gradient (60% methanol/water-methanol) and 0.3-min fractions collected using an LKB Redirac fraction collector. In studies with inhibitors, the inhibitors were added in propylene glycol (final concentration less than 0.4%) or acetone (final concentration 1.5%) 5 min before addition of [“H]BP. Intra- and extracellular metabolites were separated by centrifuging the cells at 2009 for 3 min. Cytochrome P-450 assay. For measurement of cytochrome P-450 levels, freshly isolated cells were centrifuged at 200g for 3 min, resuspended in 0.25 M potassium phosphate, 0.15 M KCl, pH 7.4, and briefly sonicated. Cytochrome P-450 in the sonicate was assayed by the method of Omura and Sato (38). Glutathione assay. Cells were centrifuged at 2009 for 3 min and resuspended in 0.6 N perchloric acid, 5 mM EDTA, and sonicated. Reduced glutathione was assayed in the protein-free acid extract by the method of Hissin and Hilf (39). DNA binding. For studies of DNA binding, cells were incubated as described above for 45 min. The cells were then placed on ice, centrifuged for 3 min at 2009, and resuspended in 3 ml 5% p-aminosalicylate, 1% SDS. DNA was extracted as described by Kuroki and Heidelberger (20), with some modifications. After vortexing to lyse the cells, the samples were extracted with an equal volume of phenol reagent (phenol:mcresol:8hydroxyquinoline:water, 500:70:0.5:55 by weight), centrifuged, and the aqueous phase removed. The phenol phase was reextracted with 1.5 ml 5% paminosalicylate, 1% SDS, and DNA precipitated from the combined aqueous phases with cold (-20°C) 95% ethanol. The nucleic acids were centrifuged (lO,OOOg, 15 min) and the pellet washed with 95% ethanol, benzene, 95% ethanol, and ether. Residual ether was removed under N, and the pellet dissolved in 0.01 M NaPO,, pH 7.0. The solution was digested for 30 min with ribonuclease A (50 kg/ml), followed by digestion with protease (50 pg/ml) for 60 min. The samples were then extracted with 2 vol CHCl,:isoamyl alcohol (24:l) and precipitated with 2 ~0195% ethanol. The DNA was centrifuged at 10,OOOgfor 15 min and the pellet washed with 95% ethanol and ether. After drying under N,, the DNA was dissolved in 0.01 M Tris, 0.01 M MgCl,, pH 7.0. DNA was assayed by the diphenylamine method (40) and covalent binding of BP was expressed per milligram of DNA. Recovery of DNA averaged about 80%.
AND COVALENT
BINDING
TO DNA
513
Digestion was carried out as described by Alexandrov et al. (41). The digested DNA was chromatographed on 18 x l.O-cm LH-20 columns, as previously described (10). Studies of BP binding to calf thymus DNA in the presence of MC-microsomes were carried out as described previously (10). RESULTS
BP Oxidation
in Hepatocytes
Hepatocytes isolated from 3-MC-treated rats metabolized BP in a dose-dependent manner (Fig. 1). At 60 PM BP and a cell concentration of 2.5 x lo6 cells/ml, BP was metabolized at a rate of 15.6 2 3.8 nmol BP metabolized/lO’j cells/l5 min. Since there are 0.4 nmol P-450/106 cells, this corresponds to 37.2 + 9.1 nmol BP metabolized/nmol P450/15 min, which is similar to the rate observed in MC-microsomes (28.9 nmol BP metabolized/nmol P450/15 min) (42). V for BP metabolism in 3-MC-induced hepatocytes was 2.48 + 0.6 nmol BP metabolized/nmol P-450/min, compared with 2.62 nmol BP metabolized/nmol P-450/min for MC-microsomes. At 2.5 x lo6 cells/ml, the ED,, for BP in hepatocytes was 30-40 PM, which is much higher than the K, for microsomal BP metabolism (0.79 PM). These values were obtained using hepatocytes incubated for either 5 min (17% conversion) or 15 min at 37°C.
[BP] OJM)
FIG. 1. Dependence of hepatocyte BP metabolism on BP concentration. Hepatocytes were incubated in L15 at 2.5 x 10” cells/ml for 15 min at 37°C. Total BP metabolism was assayed by the method of Van Cantfort et al. (36) and results expressed as nmol metabolizedinmol P-450/15 min * SEM. P-450 content of the cells was 1.0 nmol/2.5 x lo6 cells.
514
SHEN,
IYB
1.
5
IO
I5
a
20
h
FAHL,
AND
during incubations at high cell concentrations. The ED,, for BP was not determined at high cell densities but could be higher than that determined at 2.5 x lo6 cells/ml. BP metabolites produced by hepatocytes were qualitatively similar to those produced by microsomal BP incubations (Table I). However, hepatocyte incubations produced increased amounts of water-soluble metabolites and decreased amounts of organicsoluble metabolites. A large fraction of the organic-soluble metabolites was composed of relatively polar metabolites eluting near the beginning of the 60-100% methanol gradient. These metabolites and an unknown Peak “II” have been designated “Fraction I” by Burke et al. (26) and include polyhydroxylated BP metabolites (14) and sulfate conjugates (11). Peak “II” eluted as if it were more polar than BP-9,10dihydrodiol and included BP-tetrols and other metabolites.
*
25
JEFCOATE
30
Time (mid FIG. 2. Time course of hepatocyte BP metabolism at varying cell densities. Hepatocytes were incubated in L15 in the presence of 60 pM [3H]BP at cell densities of 1.25 (O), 2.5 (O), 5 (X), and 7.5 (A) x IO6 cells/ml and aliquots taken for total BP metabolism at the times indicated. Total BP metabolism was assayed as described by Van Cantfort et al. (36). Values represent duplicate determinations.
Figure 2 shows the time course of BP metabolism (60 PM BP) at various cell densities. At 1.25 x 10” cells/ml, metabolism was nearly linear for up to 30 min, while at higher cell densities linearity was maintained for progressively shorter periods. The decrease in the duration of linear metabolism may be due to a decrease in the concentration of BP available to cytochrome P-450 or to the slow decline in pH
Inhibition
Salicylamide, diethylmaleate, and TCPO effected large changes in the distribution of BP metabolites (Table I). Salicylamide and diethylmaleate had no effect on total BP metabolism, while TCPO had a slight
TABLE
I
nmoU10” Control
Metabolite Fraction I Peak II BP-9,10-dihydrodiol BP-4,5-dihydrodiol BP-7,8-dihydrodiol BP-quinones BP-g-phenol BP-3-phenol Total organic H,O phase Total
phase
metabolites
of Conjugation
Diethylmaleate
‘7.15 1.39 0.65 0.25 0.99 0.51 0.24 0.49 11.67 5.98
k _f k k k 2 rfk 2 2
0.32 0.20 0.06 0.16 0.52 0.13 0.06 0.12 0.69 0.41
5.40 1.72 2.09 0.71 2.44 0.84 0.80 0.97 14.97 4.30
+ + t + + + + k + +
0.69 0.08 0.19 0.02 0.03 0.32 0.08 0.09 1.19 0.18
17.65
2 0.80
19.27
* 1.20
cells/l5
min
Salicylamide (76) (124) (323) (282) (246) (164) (333) (198) (128) (72)
1.64 0.54 3.05 1.12 3.09 1.98 0.99 1.69 14.10 3.78
2 + + k k + + 2 + 2
0.35 0.14 0.21 0.16 0.40 0.46 0.19 0.33 0.85 0.42
17.88
2 0.95
TCPO (23) (39) (471) (444) (312) (386) (412) (344) (121) (63)
0 Hepatocytes were incubated at 2.5 x 10” cells/ml and 60 pM [3H]BP and metabolites under Materials and Methods. Concentrations of inhibitors used were: diethylmaleate, 2.6 mM; and TCPO, 2.3 mM. Results are expressed as mean + SD. Values in parentheses of control levels.
5.40 0.10 0.23 0.46 0.57 0.69 2.10 0.99 10.54 4.23
k 2 2 -I-rt t k 2 iz
2.29 0.04 0.04 0.16 0.21 0.24 0.08 0.04 2.32 0.17
14.77
c 2.33
(76) (7.5) (35) (181) (57) (135) (875) (202) (90) (71)
analyzed as described 2.1 mM; salicylamide, represent percentage
BENZO(A)PYRENE
METABOLISM
AND
inhibitory effect. In the presence of salicylamide, water-soluble and Fraction I metabolites were decreased by 37 and 77%, respectively, while Peak II was decreased by 61%. In contrast, all other organic-soluble metabolites were increased by a surprisingly constant amount (3.1-4.7 times). Diethylmaleate produced a 28% drop in water-soluble metabolites. Fraction I metabolites decreased 24% while all other organic-soluble metabolites were increased (124-333%). TCPO inhibited formation of dihydrodiols, increased formation of BPphenols and quinones, and decreased levels of water-soluble metabolites. Peak II was almost completely absent in the presence of TCPO, indicating that hydration of an epoxide is required for formation of this metabolite. Freshly-isolated hepatocytes had a reduced glutathione content of 7.7 ? 1.1 pg/lO” cells, which was unchanged after incubation in L15 for 30 min at 37°C. Further incubation for 45 min with or without BP did not significantly decrease intracellular GSH levels. Addition of salicylamide to incubations containing BP did not affect reduced glutathione levels; however, 2.1 mM diethylmaleate and 2.3 mM
20
40 Fraction
COVALENT
BINDING
515
TO DNA
TCPO decreased intracellular reduced glutathione levels to 5.2 ? 0.6 and 5.0 ? 1.6 pg/106 cells, respectively (-70% of controls). In these incubations, BP metabolites distributed inside and outside the cells in a ratio of 1.4:1 (data not shown). Salicylamide and diethylmaleate increased the proportion of intracellular metabolites by a small but statistically insignificant amount. Covalent ModiJcation Hepatocytes
of DNA
in
Covalent binding of BP metabolites to DNA was analyzed by LH-20 column chromatography (Fig. 3). The predominant fraction of BP-nucleoside adducts eluting from LH-20 was due to syn- and antiBP-7,8-diol-9,10-epoxides bound to (primarily) guanine (Peak A) (43), although the bulk of the radioactivity eluting from the LH-20 columns was found in the flowthrough peak (F.T.). Peak D, a BP-phenoloxide adduct (16, 43), was always a relatively small fraction of the total BP-DNA adducts. This is in contrast to the MC-microsomes-calf thymus DNA system where BP-phenol metabolite(s) form the
60 Number
eo
loo
FIG. 3. Sephadex LH-20 column chromatography of [“H]BP metabolites covalently bound to nucleosides. Hepatocytes were incubated in L15 at 2.5 x 10” cells/ml and 60 PM [“H]BP for 45 min at 37°C. DNA was isolated, digested, and chromatographed as described under Materials and Methods. Peaks were assigned according to King et al. (29).
516
SHEN,
FAHL,
AND
predominant BP-DNA adduct and F.T. is only about 20-30% of the total. The F.T. has not been characterized. Digestion of the DNA for longer times and with larger amounts of enzymes did not affect the relative sizes of the F.T., A, or D peaks. Similarly, collection of the F.T. followed by an additional period of digestion did not release any additional Peak A or Peak D material. Rechromatography of the F.T. on G25 indicated several components with the majority of radioactivity associated with a peak of protein separate from the DNA peak. In general, the size of the F.T. correlated with the extent of BP metabolism rather than with covalent binding to DNA. Formation of diol-epoxide and phenol-oxide adducts was rapid, reaching a maximum at between 10 and 15 min (Table II). The ratio of Peak D to Peak A remained constant at about 0.2 from 10 to 45 min, in contrast with the dramatic increase reported by Jernstrom et al. during this period (47). Figure 4 shows the dependence ‘of BPDNA adduct formation on BP concentration. Peak D was undetectable at 15 pM BP, but increased more rapidly than Peak A with increasing BP concentration. The ratio of Peak D to Peak A increased from 0.15 to 0.34 between 30 and 100 pM BP. Covalent binding to hepatocyte DNA (Fig. 1). was more sensitive than BP TABLE
II
TIME COURSE OF COVALENT BINDING OF BP METABOLITES TO HEPATOCYTE DNA” Incubation time (min) 10 15
30 45
Covalent binding (pmol/mg DNA) F.T.
A
D
25.7 35.6 56.6 47.7
28.0 40.3 33.4 28.5
6.8 6.6 6.7 6.4
D/A
0.24 0.16 0.20 0.22
a Hepatocytes were incubated at 2.5 x lo6 cells/ml and 60 PM [“H]BP for the times indicated and [“HIBPDNA adducts isolated as described under Materials and Methods. Values represent duplicate determinations.
JEFCOATE
FIG. 4. Dependence of covalent binding of [“HIBP to DNA on BP concentration. Hepatocytes were incubated in L15 at 2.5 X 10” cells/ml for 45 min at 37°C. DNA was isolated, digested, and chromatographed as described under Materials and Methods. Results are expressed as mean c SD for Peak A (0) and Peak D (0).
metabolism to changes in BP concentration. Between 15 and 60 pM BP, metabolism increased almost eightfold, while covalent binding of BP-7,8-diol-9,10-epoxide increased 14 times. Formation of BP-DNA adducts was also dependent upon cell density (Fig. 5). Peak D was more sensitive to changes in cell number than was Peak A and was undetectable at 5 and 7.5 x lo6 cells/ml. Again, covalent binding was more sensitive than BP metabolism to increasing cell density. At 60 pM BP and cell densities of 5 x lo6 cells/ml, formation of diol-epoxide adducts was only 13% of that observed at 2.5 x lo6 cells/ml, even though BP metabolism was reduced only slightly. Salicylamide, diethylmaleate, and TCPO all increased covalent binding of BP to DNA (Table III). Salicylamide stimulated Peak A and Peak D roughly equally. Diethylmaleate was more effective than salicylamide in enhancing covalent binding and also had a greater effect on Peak D than on Peak A. TCPO decreased Peak A by 90%, while producing the largest (1270%) increase in Peak D. When calf thymus DNA (1.5 mg/ml) was present, 97% of total DNA modification was extracellular (Table IV) and no significant effects on covalent binding of 13H]BP
BENZO(A)PYRENE
METABOLISM
AND COVALENT
BINDING TABLE
517
TO DNA III
EFFECTSOF INHIBITION OF CONJUGATION ENZYMES ANDEPOXIDE HYDRATASEONCOVALENTBINDING OF ["H]BP METABOLITESTO DNA"
Cell
Density (Cells/ml)
FIG. 5. Dependence of covalent binding of [3H]BP to DNA on cell density. Hepatocytes were incubated in L15 in the presence of 60 /.LM[3H]BP for 45 min at 37°C. DNA was isolated, digested, and chromatographed as described under Materials and Methods. Results are expressed as mean 2 SD.
to hepatocyte DNA were observed. Significantly, the ratio of Peak A to Peak D was the same for hepatocyte DNA and extracellular DNA. Covalent binding per milligram DNA was similar for both intraand extracellular DNA, since the concentration of extracellular DNA was 30 times that found intracellularly (0.05 mg/ml). Modifiation of DNA by MicrosomallyGenerated BP Metabolites
The binding BP metabolites
to calf thymus DNA of generated by microsomal TABLE
Additions
Peak A
Peak D
Control Salieylamide (2.6 mM) Diethylmaleate (2.1 mM) TCPO (2.3 mM)
35.1 40.4 (115) 20.6 (201) 3.6 (10)
6.6 8.5 (130) 22.8 (345) 83.8 (1270)
” Hepatocytes were incubated at 2.5 x 10” cells/ml and 60 WM [aH]BP in the presence of the inhibitors indicated and [3H]BP-DNA adducts isolated as described under Materials and Methods. Values represent experiments performed in duplicate. Numbers in parentheses are percentage of control values.
metabolism was examined to determine the relative effects of pH, medium, and DNA concentration on formation of BP-DNA adducts. A drop in pH from 7.5 to 7.0 produced similar decreases in both Peak A and Peak D which were larger than the decrease observed in BP metabolism (Table V). In the presence of L15 medium, which contains a mixture of amino acids, metabolism of BP and formation of Peak A were reduced by about 15% while Peak D decreased by 60%. A 33-fold decrease in DNA concentration decreased formation of Peak A by 13 times, while Peak D decreased 55 times. The total quantity of BP metabolites trapped by DNA in Peaks A and D was saturated at high DNA conIV
EFFECTSOF EXTRACELLULAR DNA ONCOVALENTBINDINGOF
Adduct
Extracellular DNA (q/ml)
pmol bound/ml incubation Cellular
Peak A Peak A
0 1.5
1.5 + 0.4 1.9 * 0.5
Peak D Peak D
0 1.5
0.2 2 0.07 0.2 2 0.05
Extracellular
[SH]BP TO HEPATOCYTE DNA" pmol bound/mg DNA Cellular
62.8 2 18.4
30.8 + 7.5 38.2 k 10.4
7.6 ? 2.0
4.1 2 1.4 4.0 t 1.0
Extracellular 41.9 rt_ 12.3 5.1 lr
1.3
a Hepatocytes were incubated in L15 at 2.5 x lo6 cells/ml in the presence of 60 PM [3H]BP with or without 1.5 mg/ml calf thymus DNA. Cell DNA was isolated as described under Materials and Methods, except that the cells were washed once with phosphate-buffered saline to remove residual DNA. DNA was isolated from the medium as described for DNA from mierosomal incubations (10). Pmol bound/ml incubation was calculated using a value of 0.05 mg DNA/2.5 x lo6 cells. Results are expressed as mean 2 SD.
518
SHEN, FAHL, AND JEFCOATE TABLE
V
EFFECTSOF pH, BUFFER,AND DNA CONCENTRATIONON MICROSOMALBP METABOLISM ANDBINDINGOF [3H]BP TO DNA" pmol bound/ml incubation
[DNA1 Buffer 50 50 50 50
mMKP mMKP mMKP mMKP
50 50 50 50 50
mMKP mMKP mMKP mMKP mMKP
b-w/ml)
PH
BP metabolism (nmo1145 min)
Peak A
Peak D
1.0 1.0 1.0 1.0
7.5 7.25 7.0 7.5
38.3 30.8 25.0 33.0
28.0 21.0 14.6 23.2
219.8 143.0 107.4 88.2
0.03 0.1 0.5 1.0 2.0
7.5 7.5 7.5 7.5 7.5
-
1.5 2.6 15.4 28.0 26.1
2.9 13.3 93.9 219.8 216.3
+ L15
U MC-microsomes (0.3 mg/ml) and calf thymus DNA, in the concentrations indicated, were incubated with 60 PM BP for 45 min as described under Materials and Methods. BP metabolism and formation of [SH]BPDNA adducts were determined as described under Materials and Methods. Values represent results of duplicate determinations.
centrations, although higher DNA concentrations were required for saturation of Peak D binding. Table VI compares BP-DNA adduct formation in hepatocytes with that found in the microsomal system. For purposes of comparison, the concentration of DNA and the rate of BP metabolism are set to
be the same in the microsomal and cellular incubations. This direct comparison indicates similar amounts of Peak A in the two systems but 13 times less Peak D in hepatocytes. When diethylmaleate and salicylamide were added to hepatocytes to decrease conjugation of BP metabolites, Peak A was increased to about three times that
TABLE
VI
MICROSOMALAND HEPATOCYTE ACTIVATIONOF [3H]BPa Covalent binding to DNA
Incubation
(mgiml)
[P-450] (nmol/ml)
BP metabolism (nmoY 45 mm/ml)
MC-microsomes Hepatocytes Hepatocytes + Diethylmaleate + Salicylamide
0.05 0.05 0.05
0.8 1.0 1.0
38.1 47.4 46.1
[DNA1
Total [$H]BP bound (pmoY45 mm/ml)
Proportion of metabolites bound x lo4
Peak A
Peak D
Peak A
Peak D
1.7 1.4 4.7
3.8 0.3 1.6
0.4 0.3 1.0
1.0 0.06 0.3
(1Hepatocytes (2.5 x lo6 cells/ml, containing 0.05 mg DNA/2.5 x lo6 cells) or MC-microsomes and calf thymus DNA at 0.05 mg/ml were incubated with [3H]BP (60 PM) as described under Materials and Methods. Samples were assayed for BP metabolism by the method of Van Cantfort et at. (36). Covalent binding at 45 min was determined as described under Materials and Methods.
BENZO(A)PYRENEMETABOLISMANDCOVALENTBINDINGTODNA
found in microsomal incubations. Peak D was also increased but was still only 42% of microsomally generated Peak D adducts. DISCUSSION Hepatocyte BP Metabolism Microsomal Metabolism
versus
The overall rates of BP metabolism per nanomole P-450 were similar in hepatocytes and microsomes. However, the ED,, for metabolism of BP in hepatocytes was 40 times higher than the K,,, for microsomal BP metabolism. While a part of this difference may be accounted for by nonspecific trapping of BP by cell protein and lipid (hepatocyte incubations contain about 22 times as much protein as the microsomal incubations used for K,,, determinations), other factors may be acting to reduce the concentration of BP in the microsomal membrane. For example, little is known about the rate of BP uptake, which may become limiting at low BP concentrations. Alternatively, some intracellular compartmentalization may act to decrease the microsomal BP concentration in the hepatocytes. We have recently found that microsomal metabolism of BP is activated when inhibitory BP-quinones are removed by glucuronidation (10). In hepatocytes, BPquinones were decreased threefold relative to microsomes (Table I), presumably because of conjugation by UDPGA and sulfate (26). Nevertheless, elevation of BPquinones in the presence of salicylamide, an inhibitor of glucuronide and sulfate conjugation, did not inhibit hepatocyte BP metabolism. It seems, therefore, that inhibition by BP-quinones is less important in hepatocytes than in microsomal metabolism, possibly because of binding to cellular macromolecules. We have recently observed a substantial decrease in the inhibitory effect of BP-quinones on microsomal BP metabolism in the presence of exogenous DNA (G. Keller, unpublished data). of BP Metabolites in Hepatocytes
Conjugation
Conjugation in hepatocytes as measured by water-soluble and polar metabolites
519
(74% of total metabolites) was greater than was found with microsomal metabolism, even when MC-microsomes were supplemented by 2 mM UDPGA (52% of total metabolites). This higher degree of conjugation in cells is to be expected from the presence of cytosolic sulfotransferases and glutathioneS-transferases in the cells. Previous studies have indicated that glucuronidation of BP-dihydrodiols by isolated microsomes was much less effective than glucuronidation of BP-phenols (8). However, the similar stimulatory effect of salicylamide, an inhibitor of both glucuronidation and sulfation, on the recovery of both BP-dihydrodiols and BP-phenols from hepatocytes suggests that, in the hepatocyte, BP-dihydrodiols are conjugated more readily than by the UDP-glucuronyl transferase of isolated microsomes. Burke et al. (26) have shown that BP-4,5-dihydrodiol and BP-7,8-dihydrodiol form glucuronides rather than sulfates in hepatocytes. It seems, therefore, that the specificity of UDPglucuronyl transferase in the hepatocyte is less than in isolated microsomes. Glutathione levels in these hepatocytes were somewhat higher than those reported by Burke et al. and were less readily depleted by diethylmaleate (26). This may in part be due to the presence of cysteine and methionine in the L15 medium which may facilitate the maintenance of high levels of intracellular glutathione. Since the relatively small decline (30%) in intracellular glutathione caused by diethylmaleate produced major changes in BP metabolites and covalent binding to DNA, it is likely that not more than a half of the total intracellular glutathione is readily available for conjugation with these reactive electrophiles. Glutathione conjugation of BP-oxides has been previously established (3, 4). The increase in both BP-dihyrodiols and BP-g-phenol produced by diethylmaleate are consistent with decreased conjugation of intermediate BPoxides by glutathione. The twofold increase in BP-&phenol is more surprising since the intermediate BP-2,3-oxide (6) does not undergo hydration and is probably very shortlived. It is possible but unlikely that half of the BP-2,3-oxide is trapped by glutathione.
520
SHEN,
FAHL,
More likely, changes in glutathione or some direct action of diethylmaleate and TCPO are also linked to the maintenance of UDP-glucuronosyl transferase or sulfotransferase activities. The change in glutathione levels did not have a significant effect on the levels of BP-quinones. The effects of TCPO on BP metabolites from hepatocytes are fully consistent with the dual capability of TCPO to deplete glutathione to the same extent as diethylmaleate and to partially inhibit cellular epoxide hydratase. Similar results have previously been reported by Burke et al. (26). The inhibitory effects of TCPO on dihydrodiol formation in hepatocytes are in the same order (9,lO > 7,8 > 4,5) as has previously been observed for microsomal metabolism of BP (42). Indeed, the slight stimulatory effect on the formation of BP-4,5-dihydrodiol indicates that decreased glutathione conjugation outweighs decreased hydration in this case. Modification of DNA in Hepatocyte and Microsomal Incubations
Covalent
The LH-20 elution profile of BP-modified nucleosides isolated from the DNA of hepatocytes was similar to that obtained from the MC-microsomes-calf thymus DNA system except that nucleosidephenol-oxide adducts (Peak D) were only a minor component as compared to diolepoxide adducts (Peak A). Depletion of glutathione by only 30% in the presence of diethylmaleate enhanced formation of the phenol-oxide adduct 3.5-fold, which can be attributed to the combined effect of both a two- to threefold increase in BP-g-phenol levels and diminished scavenging of BP phenol-oxides by glutathione. The even larger enhancement of covalent binding of phenol-oxides to DNA by TCPO is also due to the combined effects of enhanced BP-gphenol levels and decreased scavenging of phenol-oxides by diminished levels of glutathione. Decreased diol-epoxide modification of DNA caused by TCPO correlated with the decreased level of BP-7,8-dihydrodial, while increased formation of diolepoxide-DNA adducts in the presence of
AND
JEFCOATE
diethylmaleate correlated with increased levels of BP-7&dihydrodiol. Although salicylamide increased BP-7, 8-dihydrodiol and BP-g-phenol levels 3. land 4.1-fold, respectively, DNA modification was enhanced only slightly (Table III). We have previously shown (44) that BP-7, 8-diol metabolism is more sensitive to quinone inhibition than is BP metabolism. Consequently, the enhanced levels of BPquinones in the presence of salicylamide may exert an inhibitory effect on formation of BP-7,8-diol-9,10-epoxides and subsequent DNA modification even though there is no measurable effect on total BP metabolism. The results presented here differ from those of Jernstrijm et al. (46, 47) in that we observe less covalent binding of BP-phenoloxides. It is likely that L15 medium acts both to maintain higher levels of glutathione through the effects of cysteine and methionine on leakage of reduced glutathione from the cells (48, 49) and to directly scavenge extracellular phenol-oxides to a greater extent than the Krebs-Ringer buffer used in their studies. These studies also differ in that a similar time dependence was observed for both diol-epoxide and phenol-oxide binding to hepatocyte DNA (Table II) instead of a sharp peak in phenol-oxide binding after 15 min followed by a rapid decay (47). The total extent of hepatocyte DNA modification in this study was similar to that reported by Burke et al. (26).
Factors affecting microsomal or hepatocyte BP metabolism produced a generally greater effect on covalent binding to DNA and particularly on covalent binding of phenol-oxides. Thus, DNA modification in hepatocytes was more sensitive to increasing BP concentration (Figs. 1 and 4) and cell density (Figs. 2 and 5) while, in the microsomal system, enhanced sensitivity to the effects of pH, L15 medium, and DNA concentration was observed (Table V). The selection between phenol-oxide addition to DNA and diol-epoxide addition increased in the order pH < L15 < DNA concentration. The effect of DNA concentration on the extent of modification of DNA reflects competition between DNA, water, and
BENZO(A)PYRENEMETABOLISMANDCOVALENTBINDINGTODNA
probably microsomal components for the labile intermediates. The saturation of DNA adduct formation at high levels of DNA (Table V) is consistent with a steady-state model for the two intermediates in which the rate of hydration is comparable to the rate of interaction with DNA. The shift in the saturation curve for phenol-oxide adduct formation suggests that phenoloxide hydration and/or rearrangement is faster than diol-epoxide hydration. Quantitative Evaluation of DNA Modijcation in Hepatocytes
The ready export of BP metabolites (42%) found here and by other workers (26,27) suggests that metabolites generated in one cell may enter other cells and undergo further metabolism and DNA binding. Studies of the microsomal system indicate that the concentration of enzymes and DNA is a crucial consideration in these multistep pathways. For a hepatocyte suspension, the effective volume for further oxidation, conjugation, and covalent binding to DNA of BP metabolites must be somewhere intermediate between the volume of the cells (fully intracellular metabolism and adduct formation) and the total incubation volume (cell boundaries insignificant). When the DNA concentration in the MCmicrosomal system is lowered to that found in hepatocytes, total modification of DNA by BP-7,8-diol-9,10-epoxide is quantitatively similar in both systems (Table VI). However, under these conditions, the relative proportion of the precursor BP-7, 8dihydrodiol among the metabolites is lower in hepatocytes (5.6%) than in MCmicrosomes (15%) due to conjugation in the cells. Inhibition of hepatocyte conjugation mechanisms by either diethylmaleate or salicylamide increases the proportion of BP-7,8-dihydrodiol to that found with MCmicrosomes. Nevertheless, modification of hepatocyte DNA by BP-7,8-diol-9,10epoxide in the presence of both diethylmaleate and salicylamide is nearly three times higher than that found in microsomal incubations. It is, however, at least five times less than the maximum rate of DNA modification observed in microsomal
521
incubations containing saturating levels of DNA (about 1 mg/ml, Table V). A major reason for relatively low phenoloxide modification in hepatocytes is the higher reactivity of BP-phenol-oxide with glutathione and other intracellular nucleophiles. On the other hand, DNA modification by both diol-epoxides and by phenoloxides in hepatocytes were similarly proportional to the 15-min levels of the precursors BP-7,8-dihydrodiol (0.04%) and BP-g-phenol (0.06%), irrespective of conjugation activity. Presumably, either formation of phenol-oxide or its reaction with DNA must be more rapid than the equivalent steps from BP-7,8-dihydrodiol to compensate for the greater lability of phenol-oxides. For diol-epoxides, the modification of calf thymus DNA was greater with hepatocyte BP activation (64.7 L 18.4 pmol Peak A/ml, Table IV) than with microsomal activation (28 pmol Peak A/ml incubation, Table V). Indeed, a better comparison is obtained with microsomes supplemented with UDPglucuronic acid, where BP-quinones are removed by conjugation. Formation of diolepoxide-DNA adducts is stimulated up to 2.7-fold (10) while phenol-oxide adducts are decreased, emphasizing the importance of conjugation pathways on BP-DNA adduct formation. When calf thymus DNA was added to hepatocytes, 97% of the BP-DNA adducts were extracellular, strongly suggesting that the vast majority of BP-diol-epoxides and phenol-oxides leave the cells and react with extracellular constituents. The similar Peak A/Peak D ratios, intra- and extracellularly, indicate that the ratios of diolepoxides and phenol-oxides leaving the cell and entering the nucleus are similar. Since DNA modification expressed as picomoles bound per milligram DNA (Table IV) is the same inside and outside the cell, the reactivity and selectivity of hepatocyte DNA must be comparable to that of purified calf thvmus DNA. Finally, the lack of effect of saturating levels of extracellular DNA on the modification of hepatocyte DNA suggests that modification of hepatocyte DNA by BP metabolites is primarily an intracellular process.
522
SHEN,
FAHL,
AND
JEFCOATE
ACKNOWLEDGMENT We are grateful discussions during
to Dr. Bengt Jernstrom the course of this work.
for helpful
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