* Environmental Toxicology and Chemistry Laboratory, Great Lakes Center, State University of New York College at Buffalo, 1300 Elmwood Avenue, Buffalo, New York 14222; and
American Health Foundation, 1 Dana Road, Valhalla, New York
Received November 1, 2002; accepted December 30, 2002
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ABSTRACT |
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Key Words: Shasta rainbow trout; Oncorhyncus mykiss; rat; liver microsomes; chrysene; 5-methylchrysene; metabolism; regioselectivity.
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INTRODUCTION |
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Methyl substitution in a PAH may influence the metabolism of the methylated hydrocarbon due to the steric effect of the methyl group. In addition to altering the rate of metabolism of the methylated PAH, the steric effect at the site of methyl substitution may alter the regioselectivity of oxidation by directing the metabolism towards other regions of the molecule. Furthermore, X-ray studies of 5-methylchrysene (5-MeCHR) demonstrate a significant distortion of the planarity of the polycyclic ring system (Glusker, 1985; Kashino et al., 1984
). Therefore, 5-methyl substitution is also expected to lead to decreased aromaticity of the angular benzo ring, which may result in an increased metabolism in the 1,2,3,4-benzo ring of 5-methylchrysene, a site involved in the formation of the bay-region diol epoxides. The effect of the methyl substitution on the metabolism of PAHs has been investigated only to a limited extent in mammalian species (Amin et al., 1985
; Hecht et al., 1978
; Nair et al., 1992
). Studies on the metabolism of 5-MeCHR by rat liver S-9 preparation indicate that methyl substitution favors the formation of 5-MeCHR-1,2-diol (proximate carcinogenic metabolite of 5-MeCHR) over 5-MeCHR-7,8-diol, while blocking metabolism at the 3, 4-position (Hecht et al., 1978
). Presently, no data are available on the metabolism of methylated PAHs in fish. It has been shown that fish and rat liver enzymes differ greatly with respect to overall substrate specificity and regioselectivity for the metabolism of PAHs (Pangrekar et al., 2003
; Sikka et al., 1990
; Stegeman, 1981
; Varanasi et al., 1986
; Yuan et al., 1999
), presumably due to differences in the types and relative proportions of multiple forms of cytochrome P-450 isozymes in fish and rat liver. Therefore, we expect that the metabolism of methylated PAHs in fish with respect to the rate of metabolism and metabolite profile will be different from what has been reported in rodents.
In order to obtain information on the metabolism of methylated PAHs (having a methyl substituent in a non-benzo ring bay-region position) in fish, we have selected 5-MeCHR as a model compound (Fig. 1). 5-MeCHR is more carcinogenic than any of the other monomethylchrysenes and, in contrast to the weakly active parent hydrocarbon chrysene (CHR), it is as carcinogenic as benzo[a]pyrene (Hecht et al., 1998). We have chosen Shasta rainbow trout (Oncorhynchus mykiss) as a model fish species for these studies because it is susceptible to the carcinogenic action of PAHs (Hendricks et al., 1985
; Reddy et al., 1998
). We have conducted parallel, comparative studies on the metabolism of 5-MeCHR and CHR, in order to assess the effect of a non-benzo ring, bay-region methyl substituent on the oxidative reactions involved in the metabolism of PAHs by fish. We have also examined the metabolism of the two hydrocarbons by rat liver microsomes to determine whether the effect of the methyl substituent on the metabolism of 5-MeCHR is similar in trout and rat.
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MATERIALS AND METHODS |
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Animal treatment and preparation of liver microsomes.
Shasta rainbow trout (mean body weight: 299 ± 99g SD) were reared by Whispering Pines Fish Farm (Holland, NY) from eggs supplied by Dr. Jerry Hendricks of Oregon State University (Corvallis, OR). Sufficient number of fish with less variability in their body weight were not available at the time of year we conducted these studies. They were maintained in our laboratory in flowing, charcoal-filtered dechlorinated tap water at a temperature of 1516°C in insulated fiberglass tanks (Frigid Units, Toledo, OH) under a 12:12 h light:dark photoperiod. The fish were fed Ziegler trout chow (Ziegler Brothers, Gardner, PA) commercial pellet food and were acclimated for a period of 12 weeks before use. The fish were injected (ip) with 3-MC at a dose of 20 mg/kg in corn oil; 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 weighed. 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 (Yuan et al., 1999), 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 80°C. All operations were performed at 04°C. Protein determination was determined according to the Bio-rad assay (Bio-rad, Hercules, CA).
Long Evans male rats (Harlan Sprague-Dawley Farms, Altmont, NY; mean initial weight 78 ± 4.4 g) were held in a temperature- and photoperiod-controlled (12 h/day) room. They were acclimated for one week and given Purina rat chow and tap water ad libitum. The animals were treated ip with 3-MC (20 mg/kg body weight) dissolved in corn oil; control rats received corn oil only. A group of five rats was killed three days after treatment and the liver microsomes were prepared as described above. The approximate yield of microsomal protein/g wet weight of liver from trout and rat was 12 and 3 mg, respectively.
Metabolism of CHR and 5-MeCHR by liver microsomes.
The liver microsomal incubation mixture (total volume 0.5 ml) containing 100 µmol of potassium phosphate buffer (pH 7.4), 2.5 µmol of MgCl2, 1.0 µmol of NADPH and microsomal protein from control or 3-MC-induced liver microsomes was preincubated for 5 min at 28°C (fish microsomes) or 37°C (rat microsomes). The reaction was initiated thereafter by the addition of [3H]CHR or [3H]5-MeCHR (dissolved in 2-methyloxyethanol). Incubation mixtures containing boiled microsomes served as controls. The reaction was carried out in a shaking water bath for 10 min. The extent of CHR or 5-MeCHR metabolism was determined as the amount of total metabolites formed according to the procedure of Van Cantfort et al. (1977).
For analysis of CHR and 5-MeCHR metabolites, the reaction was terminated by the addition of 1 ml of ice-cold acetone after 10 min of incubation. The incubation mixture was extracted three times with two volumes of ethyl acetate. The ethyl acetate layers containing the parent hydrocarbon and its metabolites were pooled, evaporated to dryness under nitrogen, 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 CHR and 5-MeCHR metabolites.
Prior to HPLC analysis, concentrated extracts of incubated samples were dissolved in 0.1 ml of methanol. An aliquot of the extract was mixed with appropriate synthetic reference standards of CHR or 5-MeCHR. CHR and its metabolites were resolved on a Varian 5000 HPLC equipped with a Zorbax ODS column (5 µm, 250 cm x 4.6 mm.), a solvent programmer and a variable wavelength uv/visible detector, set at 267 nm. The column was eluted with the following solvent system at a flow rate of 1 ml/min (Nordqvist et al., 1981, with modifications): a linear gradient from 55 to 60% MeOH:H2O for 5 min, then a linear gradient from 60 to 63% MeOH:H2O in 5 min, 63 to 77% in 14 min, 77 to 80% in 6 min, and finally 80 to 100% in 10 min. CHR 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%.
5-MeCHR and its metabolites were separated using a Licrosorb RP-18 column (5 µm, 4.6 x 250 mm) using the conditions reported by Amin et al.(1987). The column was eluted using the following solvent system at a flow rate of 1 ml/min: 50% MeOH:H2O for 30 min, then a linear gradient from 50 to 80% MeOH:H2O in 40 min, and finally from 80 to 100% in 10 min. Eluent from the column was collected every 30 s; metabolite identification and the quantity of radioactivity in each metabolite was determined as discussed above. Standards were not available for trans-9,10-dihydroxy-9,10-dihydro 5-MeCHR (5-MeCHR-9,10-diol); 9-hydroxy- or 7-hydroxy-5-MeCHR. The phenols were identified by comparing UV spectra of peaks from pooled incubations with those of standards (Hewlett Packard photodiode array detector). 5-MeCHR-9,10 diol and 5-MeCHR-3,4-diol coeluted and therefore this fraction of diols was dehydrated using p-toluenesulfonic acid as described by Hecht et al.(1978)
, followed by subsequent chromatography to identify the relative composition of the two diols via identification of the respective 9-OH-5-MeCHR and 4-OH-5MeCHR.
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RESULTS |
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The rate of metabolism of CHR and 5-MeCHR by liver microsomes (calculated from the amount of total CHR or 5-MeCHR metabolites measured according to Van Cantfort et al., 1977) and the profile of metabolites formed were examined at a saturating substrate concentration under conditions that gave linearity with respect to microsomal protein concentration and incubation time. The conditions used for determining the rate of metabolism of CHR and 5-MeCHR and the profile of metabolites are shown in Table 1
. The rates of metabolism of CHR and 5-MeCHR by liver microsomes from control and 3-MC-treated trout and rats are presented in Table 2
.
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Profile of CHR and 5-MeCHR Metabolites Formed by Trout and Rat Liver Microsomes
Because of a low rate of metabolism of CHR and 5-MeCHR by control liver microsomes, the profiles of metabolites formed from the two hydrocarbons were examined using liver microsomes from trout and rats treated with 3-MC. Representative profiles of ethyl acetate-soluble metabolites formed by rat and trout liver microsomes incubated with [3H] CHR or [3H]5-MeCHR for 10 min are shown in Figures 2 and 3, respectively.
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DISCUSSION |
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A comparison of the rate of metabolism of CHR and 5-MeCHR by liver microsomes from control and 3-MC-treated trout shows that although microsomes from 3-MC-treated trout metabolize the two PAHs at a significantly higher rate than the control microsomes, each type of microsomes metabolized CHR and 5-MeCHR at an essentially similar rate. A similar observation was made with rat liver microsomes with respect to the metabolism of the two PAHs, although the rat liver microsomes metabolized CHR and 5-MeCHR at a considerably higher rate than the trout liver microsomes. These data indicate that methyl substitution at position 5 of CHR does not alter the substrate specificity of cytochrome P450(s) involved in the metabolism of the two PAHs by trout or rat liver microsomes.
The data on the profile of metabolites of CHR and 5-MeCHR formed by liver microsomes from 3-MC-treated trout showed that both hydrocarbons were converted to similar types of metabolites. However, there were substantial differences in the relative proportions of the individual metabolites formed from each PAH. Dihydrodiols were the predominant metabolites resulting from the biotransformation of chrysene with the phenols representing only a minor proportion of the total metabolites. However, the reverse was true in the case of 5-methylchrysene. In view of the fact that arene oxides are the common precursors for the formation of both dihydrodiols (via epoxide hydrolase-mediated hydration) and phenols (via NIH shift), our data suggest that chrysene epoxides, compared to 5-MeCHR epoxides, are better substrates for epoxide hydrolase in trout liver microsomes. These findings indicate that 5-methyl substitution alters the substrate specificity of trout microsomal epoxide hydrolase for 5-MeCHR epoxides.
A comparison of the relative proportions of diols and phenols formed from CHR and 5-MeCHR by trout liver microsomes with the proportions of these metabolites formed by rat liver microsomes shows that chrysene diols were the major metabolites in the case of both types of microsomes, indicating that the microsomal epoxide hydrolase in the liver of both species has similar substrate specificity for chrysene epoxides. However, rat microsomes, compared to trout microsomes produced almost three times greater percentage of 5-MeCHR diols, indicating that 5-MeCHR epoxides are better substrates for the microsomal epoxide hydrolase present in rat liver than for the enzyme in trout liver.
The metabolism of both CHR and 5-MeCHR either by trout or rat liver microsomes occurs predominantly via oxidation at the benzo ring(s) (1,2- and 3,4- double bonds in CHR and 1,2- and 3,4- double bonds and 3,4- and 9,10 double bonds in 5-MeCHR). However, the two hydrocarbons differ from each other with regard to the site of attack in their respective benzo ring(s). Oxidative attack at the benzo ring(s) represents the sum of proportions of 1,2- and 3,4-diol and the corresponding phenols. Both trout and rat liver microsomes produced a higher proportion of CHR 3,4-diol plus 4-hydroxyCHR than of CHR 1,2-diol, indicating that the liver microsomes of both species are more efficient at attacking the bay-region double bond compared to the non-bay-region double bond in chrysene. On the other hand, liver microsomes of both species produced a much greater proportion of 5-MeCHR-1,2-diol, 1-hydroxy-5-MeCHR and 5-MeCHR-7,8-diol than of 5-MeCHR-3,4- and 9,10-diol, indicating that both trout and rat liver microsomes are more efficient at attacking the non-bay-region double bond versus the bay-region double bond of 5-MeCHR. These findings indicate that methyl substitution at 5-position of chrysene alters the regioselectivity of the enzymes involved in the metabolism of the hydrocarbon. The favored formation of diols with a bay-region double bond (5-MeCHR-1,2-diol and 7,8-diol) is of considerable toxicological significance because further oxidation of these diols is expected to lead to the formation of the ultimate carcinogens of 5-MeCHR. This shift in regioselectivity in the metabolism of 5-MeCHR is of toxicological significance because it results in the formation of a higher proportion of diols with a bay-region double bond (1,2-diol and 7,8-diol; proximate carcinogenic metabolites) compared to bay-region diols (3,4-diol and 9,10-diol).
Although all of the microsomes produced nearly equal proportions of 5-MeCHR-1,2-diol and 5-MeCHR-7,8-diol, the proportion of 5-MeCHR-9,10-diol was four-fold greater than that of 5-MeCHR-3,4-diol. These data suggest that 5-methyl substituent not only shifts the metabolism from one benzo ring to the other, but also blocks the metabolism at the 3,4-double bond located in the vicinity of the methyl group, presumably due to a steric effect. It has been suggested that a significant distortion in the bay-region due to the presence of the methyl group in the bay-region makes the 1,2,3,4-benzo ring of 5-MeCHR less aromatic (Harvey et al., 1986). Consequently, the greater olefinic character of the aromatic bonds in this molecular region may result in enhanced ease of enzymatic activation to the ultimate carcinogenic bay-region diol epoxide metabolites. Contrary to this suggestion, our data indicate that the presence of methyl group in the bay-region did not enhance the formation of 5-MeCHR-1,2-diol compared to chrysene-1,2-diol or 5-MeCHR-7,8-diol. No significant difference in the relative amounts of 5-MeCHR-1,2-diol and 5-MeCHR-7,8-diol has also been previously noted in the metabolism of 5-MeCHR by human liver microsomes (Koehl et al., 1996
).
In conclusion, the results of this investigation regarding the effect of a methyl substituent at a non-benzo ring position on the metabolism of a PAH molecule demonstrate that the methyl substituent significantly influences the metabolism of the parent hydrocarbon. The 5-methyl substituent appears to have no effect on the substrate specificity of cytochrome P450(s) involved in the metabolism of CHR and 5-MeCHR. However, it does alter the regioselectivity of both the rat and trout liver microsomal enzymes involved in the metabolism of 5-MeCHR, resulting in a higher proportion of diols with a bay region double bond (proximate carcinogenic metabolites) compared to bay-region diols. The 5-methyl substitution also alters the substrate specificity of the trout microsomal epoxide hydrolase for 5-MeCHR epoxides.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: Department of Medicine/Division of Infectious Diseases, State University of New York at Buffalo, 3435 Main St., Buffalo, NY 14214.
3 To whom correspondence should be addressed. Fax: (716) 878-5400. E-mail: sikkahc{at}buffalostate.edu.
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