Environmental Toxicology and Chemistry Laboratory, Great Lakes Center for Environmental Research and Education, State University of New York College at Buffalo, 1300 Elmwood Avenue, Buffalo, New York 14222
Received July 10, 2002; accepted September 27, 2002
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ABSTRACT |
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Key Words: fish; brown bullhead; Ameriurus nebulosus; rat; chrysene; metabolism; liver microsomes; regioselectivity; stereoselectivity.
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INTRODUCTION |
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In order to exert their carcinogenic effects, PAHs must first be bioactivated to bay-region diol epoxides via dihydrodiols with a bay-region double bond by the combined action of cytochrome P450-dependent monoxygenases and epoxide hydrolase (Conney, 1982). The cytochrome P450-dependent monoxygenase system oxygenates a PAH at several different sites. Therefore, the position of the molecule where the primary oxygenation occurs (regioselectivity) is an important determinant of the carcinogenic activity of PAH metabolites.
There is considerable evidence showing that teleost fish biotransform PAHs to metabolites that are similar to those reported for rodents (Egass and Varanasi, 1982; Pangrekar et al., 1995
, in preparation; Sikka et al., 1990
; Swain and Melius, 1984
; Varanasi et al., 1986
; Yuan et al., 1999
). Our previous studies with benzo[a]pyrene (BaP; a five-ring PAH; Sikka et al., 1990
), dibenzo[a,l]pyrene (DB[a,1]P; a six-ring PAH; Yuan et al., 1999
), and phenanthrene (a three-ring PAH; Pangrekar et al., 1995
, in preparation) have shown that fish liver microsomes, in contrast to rat liver microsomes, metabolize the PAHs predominantly in the benzo-ring with much less oxidation at the K-region of the molecule, suggesting differences in the regioselectivity of the fish and rat liver cytochrome P450 system in the metabolism of these molecules. Our studies have also shown that among benzo-ring diols, brown bullhead (Ameriurus nebulous) and trout liver microsomes produce a higher proportion of the dihydrodiols with a bay-region double bond (putative proximate carcinogenic metabolites) than they do of bay-region diols. The regioselectivity in the metabolism of PAHs by rat liver microsomes varies considerably with the size and shape of the molecule (Thakker et al., 1985
). In order to investigate whether molecular features influence the regioselective metabolism of PAHs by fish, we extended our studies to the metabolism of chrysene (a four-ring symmetrical carcinogenic PAH that differs from BaP, DB[a,1]P, and phenanthrene with respect to molecular shape and size) by brown bullhead liver microsomes. Significant levels of chrysene have been found in sediments from the Great Lakes region (Bauman et al., 1987
; Black, 1982
) and from coastal regions in the U.S. (Gardner et al., 1991
; Malins et al., 1985
).
Stereochemical factors, including absolute and relative configuration of the diols and bay-region diol epoxides, also play a critical role in the expression of the carcinogenic effects of these compounds, because among various possible stereoisomers, only R,R-diols and R,S-diolS,R-epoxides exhibit exceptionally high carcinogenic activity (Thakker et al., 1985). Our earlier studies showed that brown bullhead liver microsomes metabolize BaP to 7R,8R-diol with a high degree (90%) of stereoselectivity (Sikka et al., 1990
), while phenanthrene is metabolized to 1R,2R-diol with less than 60% stereoselectivity (Pangrekar et al., in preparation). On the other hand, rat liver microsomes metabolize BaP, phenanthrene, and several other PAHs to their corresponding R,R-diols with a bay-region double bond with a high degree of stereoselectivity (Thakker et al., 1985
). Our limited data suggest that the stereoselectivity of the fish liver microsomal enzyme system, in contrast to that of the rat liver microsomal enzyme system, varies with the hydrocarbon. In order to provide further support to this observation, we have examined the stereoselective metabolism of chrysene by fish liver microsomes.
Currently, no information is available on the metabolism of chrysene by fish with the exception of a recent study that reported the presence of 1-hydroxychrysene in the bile of fish collected from areas contaminated with PAHs (Ruddock et al., 2002). We have investigated the regio- and stereoselective metabolism of chrysene (Fig. 1
) by the liver microsomes of brown bullhead, a bottom-dwelling fish species known to be susceptible to the carcinogenic action of PAHs (Bauman et al., 1987
). We have compared our data with those reported for the metabolism of chrysene by rats (Nordqvist et al., 1981
). We have reported part of this work in abstract form (Pangrekar et al., 1995
).
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MATERIALS AND METHODS |
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Treatment of fish and preparation of liver microsomes.
Brown bullheads (125175 g) were obtained from the Zetts Fish Hatchery (Drifting, PA). The fish were held in flowing charcoal-filtered dechlorinated city water at a temperature of 18200C, under a 12:12 h light-dark photoperiod. They were acclimated for a period of 4 weeks and fed laboratory fish chow daily (Ziegler Brothers, Inc., Gardener, PA). The fish were injected (ip) with 3-methylcholanthrene (3-MC) at a dose of 20 mg/kg in corn oil (Pangrekar et al., 1995); control fish received an equal amount of corn oil. The fish were fed ad libitum and maintained in flowing water during the induction period. Groups of six to eight fish were sacrificed by severing the spinal cord five days after treatment. The livers were rapidly excised into ice-cold 0.1 M TrisHCl buffer (pH 7.4) containing 1.15% KCl, gently blotted and weighted. The pooled livers were minced, and immediately homogenized into 4 volumes of ice-cold 0.1 M TrisHCl buffer (pH 7.4) containing 1.15% KCl using a motor-driven Potter Elvehjem type glass-Teflon homogenizer. The microsomal fraction was isolated by differential centrifugation (Sikka et al., 1990
), suspended in 0.1 M TrisHCl buffer (pH 7.4) to a concentration equivalent to approximately 1 g wet weight liver/ml suspension, quickly frozen and stored at 800C. All operations were performed at 040C. Protein determination was carried out according to Lowry et al.(1951)
using crystalline bovine serum albumin as a standard.
Metabolism of chrysene by liver microsomes.
The liver microsomal incubation mixture (total volume 1.0 ml) containing 80 µmol of potassium phosphate buffer (pH 7.4), 3 µmol of MgCl2, 1.1 µmol of NADPH, and 0.5 mg of microsomal protein from control or 3-MC-induced liver microsomes was preincubated for 3 min at 280C. The reaction was initiated thereafter by the addition of 15 or 5 µM of [3H]chrysene (approximately 2.0 µCi in 20 µl of DMSO) for control and 3-MC-induced microsomes, respectively. Incubation mixtures without NADPH served as controls. The reaction was carried out in a shaking water bath for 20 min. The extent of chrysene metabolism was determined according to the procedure of Van Cantfort et al.(1977).
For analysis of chrysene metabolites, the reaction was terminated by the addition of 1 ml of ice-cold acetone after 20 min of incubation. The incubation mixture was extracted three times with two volumes of ethyl acetate. The ethyl acetate layers containing chrysene and its metabolites were pooled, dried over anhydrous sodium sulfate, concentrated, and the residue was stored at 80°C until HPLC analysis. All experiments were conducted under low UV yellow light to minimize photodegradation of the chemicals.
HPLC analysis of chrysene metabolites.
Prior to HPLC analysis, concentrated extracts of incubated samples were dissolved in 0.1 ml of freshly distilled tetrahydrofuran (THF). An aliquot of the extract was mixed with appropriate synthetic reference standards (chrysene 1,2-, 3,4-, and 5,6-diol; 1-,2-, 3-,4- and 6-hydroxychrysene, and chrysene 5,6-quione) and chrysene and its metabolites were resolved on a Varian 5000 HPLC equipped with a Zorbax ODS column (5 µm, 25 cm x 4.0 mm, id), a solvent programmer, and a variable wavelength uv/visible detector, set at 280 nm. The column was eluted with a nonlinear gradient of 60100% methanol in water over a period of 40 min after an initial delay of 5 min, at a flow rate of 1.2 ml/min (Nordqvist et al., 1981). Eluent from the HPLC column was collected every 20 s directly into scintillation vials using an ISCO-Foxy fraction collector, scintillation fluid (Scintiverse E, Fisher Scientific) was added to the fractions and the radioactivity was determined using a Beckman LS 3801 liquid scintillation counter. Chrysene metabolites were identified by comparing their retention times with those of authentic standards. The radiolabeled metabolites were quantitated by summing the radioactivity in fractions corresponding in retention time and peak width to peaks of authentic standards. In calculating the metabolism of chrysene, appropriate corrections were made for the values obtained with blanks. The overall recovery of radioactivity from the HPLC column was >95%.
We noted that 1- and 4-hydroxychrysene and 2-and 3-hydroxychrysene, the two pairs of chrysene-derived phenols, coeluted in the solvent system described above. To separate 1- and 4-hydroxychrysene, the column was eluted isocratically with 62% methanol in water containing 1% n-butylamine (Tjessum and Stegeman, 1979). Because a relatively low amount of radioactivity coeluted with 2- and 3-hydroxychrysene, no further attempt was made to resolve the two phenols.
Resolution of enantiomers of benzo ring diols of chrysene.
Metabolically formed [3H]chrysene 1,2- and 3,4-diols were isolated by reverse phase HPLC as described above and were evaporated to dryness under N2. The 3,4-diol was converted to tetrahydrodiol by dissolving it into 3 ml of THF and bubbling with hydrogen gas for 30 min in the presence of Adams Catalyst (PtO2; Weems et al., 1986). The 1,2-diol and 3,4-tetrahydrodiol were mixed with UV detectable amounts of the corresponding synthetic racemic standards. The enantiomers were resolved by normal phase HPLC on a chiral column (Pirkle 1-A, 4.6 mm id x 25 cm., Regis Chemical Company, Morton Grove, IL) packed with (R)-N-(3,5-dinitrobenzoyl)phenylglycine ionically bonded to
-aminopropyl silicated silica (Weems et al., 1986
). The column was eluted with hexane:ethanol:acetonitrile (27:2:1 v/v) at a flow rate of 1.2 ml/min at room temperature. The eluate was monitored at 280 nm. The R,R and S,S peaks were designated on the basis of their elution order reported in the literature (Weems et al., 1986
). The peaks corresponding with the R,R and S,S enantiomers were collected and counted for radioactivity.
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RESULTS |
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The rate of metabolism of chrysene by liver microsomes and the profile of metabolites formed were examined at a saturating substrate concentration (15 and 5 µM for control and 3-MC-induced microsomes, respectively) under conditions that gave linearity with respect to microsomal protein concentration (0.5 mg/ml) and incubation time (20 min). The metabolism of chrysene by control microsomes was 30.1 ± 2.53 pmol/min/mg of microsomal protein. The rate was nearly 2.7-fold higher (82.2 ± 0.71 pmol/min/mg protein) for liver microsomes from 3-MC-pretreated fish. These values represent ± SD of triplicate determinations.
Profile of Chrysene Metabolites Formed by Bullhead Liver Microsomes
A typical profile of ethyl acetate-soluble chrysene metabolites formed by bullhead liver microsomes incubated with [3H] chrysene for 20 min is shown in Figure 2. The following chrysene metabolites were identified on the basis of co-chromatography with authentic standards: chrysene 1,2-, 3,4-, and 5,6-dihydrodiol, 1- and 4-hydroxychrysene, and 2-/3-hydroxychrysene. Table 1
shows the relative proportion of the metabolites formed by control and 3-MC-induced microsomes. Benzo-ring diols (1,2-diol and 3,4-diol) represented the major chrysene metabolites formed by control liver microsomes. Chrysene 1,2-diol with bay region double bond (putative proximate carcinogenic metabolite) and chrysene 3,4-diol accounted for 58 and 24%, respectively of the total ethyl acetate-extractable metabolites formed by control microsomes. The K-region diol (5,6-diol) was formed in only trace amounts (
1%). The phenol fraction (1-, 2-/3-, 4-, and 6-hydroxychrysene) constituted approximately 13% of total chrysene metabolites.
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Stereoselectivity in the Metabolic Formation of Chrysene Diols
Enantiomeric composition of chrysene 1,2-diol and chrysene 3,4-diol formed by liver microsomes of control and 3-MC-treated bullheads was determined using a chiral column (Weems et al., 1986). Since (±)-chrysene 3,4-diol, unlike its hydrogenated product (±)-chrysene 1,2,3,4-tetrahydro-3,4-diol, can not be resolved into its (+)- and ()-enantiomers on the chiral column (Weems et al., 1986
), the metabolically formed chrysene 3,4-diol was catalytically hydrogenated to chrysene 1,2,3,4-tetrahydro-3,4-diol prior to chromatographic separation. The percentage of each enantiomer of the metabolically formed chrysene 1,2-diol and chrysene 3,4-diol was determined from the amount of radioactivity coeluting with the corresponding R,R and S,S enantiomers of the authentic diols (Figs. 3 and 4
). Chrysene 1,2-diol formed by both control and 3-MC-induced liver microsomes contained the R,R enantiomer to the extent of 7080% (Table 2
). However, the enantiomeric purity of the R,R-enantiomer of metabolically formed chrysene 3,4-diol was >94%. Because of the trace amount of metabolically formed chrysene 5,6-diol, no attempt was made to establish its enantiomeric composition.
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DISCUSSION |
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A comparison of the rate of metabolism of chrysene with that of BaP and phenanthrene by liver microsomes from control and 3-MC-treated bullheads indicates that the substrate specificity of the bullhead liver microsomal enzymes for the three PAHs varies considerably. The control and 3-MC-induced microsomes metabolized chrysene at approximately 0.78 to 0.13 the rate of metabolism of BaP, and 2 to 4 times the rate of metabolism of phenanthrene, respectively (Pangrekar et al., 1995, in preparation). Compared to rat liver microsomes (Nordqvist et al., 1981
), the liver microsomes from control and 3-MC-treated bullheads metabolized chrysene at a considerably lower rate. Furthermore, the liver microsomes from bullhead and rat differ considerably from each other with respect to their relative substrate specificity for chrysene, BaP, and phenanthrene. Compared to phenanthrene, chrysene was metabolized at a higher rate by bullhead liver microsomes (Pangrekar et al., 1995
, in preparation) but at a lower rate by rat liver microsomes (Nordqvist et al., 1981
).
The regiospecific metabolism of chrysene by bullhead liver microsomes is similar to that of two other PAHs, BaP (Sikka et al., 1990) and phenanthrene (Pangrekar et al., 1995
, in preparation). All three hydrocarbons are converted to their K-region diols to a minor extent but are metabolized substantially to benzo-ring diols, which account for as much as 81% of the total metabolites in the case of chrysene. Like BaP and phenanthrene, chrysene is converted to a diol with a bay-region double bond (1,2-diol; proximate carcinogenic metabolite) to a much greater extent than to a bay-region diol (3,4-diol) by control bullhead liver microsomes. The diol with a bay-region double bond represented as much as 57.6% of the total chrysene metabolites compared to 14.8 and 25.3% in the case of BaP and phenanthrene, respectively. These data indicate that among the three PAHs, chrysene is converted to a precursor of a bay-region diol epoxide (the ultimate carcinogenic metabolite) to the greatest extent by bullhead liver microsomes.
Although the types of chrysene metabolites formed by control and 3-MC-induced liver microsomes were similar, there were considerable differences in the relative proportions of the individual metabolites formed by the two types of microsomes. A striking feature of chrysene metabolism by control microsomes was that, in comparison to 3-MC-induced microsomes, the control microsomes produced 3.5 times greater percentage of 1,2-diol. The control microsomes produced a much higher proportion of 1,2-diol plus 1-hydroxychrysene than of 3,4-diol plus 3-hydroxychrysene, indicating that these microsomes are selective in their attack at the 1,2-position of the benzo ring. In contrast, 3-MC-induced microsomes did not exhibit such a regioselectivity since the proportion of chrysene metabolites formed via oxidation at the 1,2-double bond (16.1% of 1,2-diol plus 17.2% of 1-hydroxchrysene) was nearly identical to that of the metabolites formed via oxidation at the 3,4-double bond (28.1% of chrysene 3,4-diol plus 8.5% of 4-hydroxychrysene). These data suggest that the cytochrome(s) P450 in uninduced microsomes is more efficient in oxidizing the chrysene 1,2-double bond than the cytochrome(s) P450 in 3-MC-induced microsomes.
A comparison of the profile of chrysene metabolites formed by bullhead liver microsomes (this study) and rat liver microsomes (Nordqvist et al., 1981) showed that the corresponding microsomes from the two species differ considerably with respect to the regioselective metabolism of the hydrocarbon. Compared to control rat liver microsomes, control bullhead liver microsomes produced a considerably greater proportion of chrysene 1,2-diol, the putative proximate carcinogenic metabolite of chrysene. Unlike control bullhead liver microsomes, control rat liver microsomes do not exhibit any regioselectivity in the oxidative attack at the 1,2- and 3,4- positions of the benzo ring. On the other hand, 3-MC-induced rat liver microsomes were more efficient at oxidizing the 3,4-double bond than the 1,2-double bond.
The liver microsomes from control and 3-MC-treated bullheads showed a high degree of stereoselectivity in the metabolism of chrysene to 1, 2-diol and 3, 4-diol, with the ()- (R,R) enantiomer predominating in each case. However, the enantiomeric purity was somewhat less for chrysene 1,2-diol (7078%) than that observed for chrysene 3,4-diol (94%). These data are comparable to those reported for the liver microsomes from 3-MC-treated rats that metabolized chrysene to 1R, 2R- and 3R, 4R-diol with 8097% enantiomeric purity (Nordqvist et al., 1981). However, unlike control bullhead liver microsomes, which show a high degree of enantiomeric selectivity in the formation of chrysene 1,2-diol, control rat liver microsomes showed far less enantiomeric specificity in the formation of 1,2-diol. It appears that, in contrast to what was noted with rats, 3-MC treatment of bullheads does not alter the stereoselectivity of the enzymes (cytochrome P450 and epoxide hydrolase) responsible for metabolizing chrysene to its diols.
A comparison of these studies with our earlier studies on the stereoselective metabolism of BaP (Sikka et al., 1990) and phenanthrene by bullhead liver microsomes (Pangrekar et al., 1995
, in preparation) shows that chrysene is metabolized to its benzo-ring diol having a bay-region double bond with a lower degree of stereoselectivity than BaP. However, the degree of stereoselectivity in the metabolism of chrysene to its benzo-ring diols is considerably higher than that noted with phenanthrene. The data indicate that the degree of stereoselectivity in the metabolism of PAHs by bullhead liver microsomes varies with the size and shape of the molecule and follows the order: BaP > chrysene > phenanthrene. These findings suggest that chrysene and phenanthrene, unlike BaP, are metabolized by more than one cytochrome P450 isozyme, presumably with different stereoselectivities. The observed stereoselectivity of fish liver microsomal enzymes in the metabolism of PAHs is of toxicological significance because PAH diols with an [R,R] configuration are considerably more carcinogenic than diols with an [S,S] configuration (Thakker et al., 1985
).
In summary, the results of this study, in conjunction with our previous studies with BaP (Sikka et al., 1990), DB[a,l]P (Yuan et al., 1999
), and phenanthrene (Pangrekar et al., 1995
, in preparation), show that chrysene, like the other two PAHs, is metabolized predominantly to benzo-ring diols by bullhead liver microsomes. These data appear to indicate that regioselectivity in the metabolism of PAHs by fish liver microsomes does not vary greatly with the size and shape of the molecule, whereas the degree of stereoselectivity in the metabolism of PAHs to benzo-ring diols does. Our data on the regioselective metabolism of chrysene, particularly with regard to the metabolic attack at the benzo-ring, and the stereoselective metabolism of the hydrocarbon, are different from what has been reported for rat liver microsomes (Nordqvist et al., 1981
). These differences may be due to variations in the relative amounts of various cytochrome P450 and epoxide hydrolase isozymes involved in the metabolism of the hydrocarbon by the hepatic microsomes of brown bullhead and rats.
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ACKNOWLEDGMENTS |
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NOTES |
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