Comparative Metabolism and Excretion of Benzo(a)pyrene in 2 Species of Ictalurid Catfish

Kristine L. Willett*,1, Piero R. Gardinali{dagger}, Laila A. Lienesch* and Richard T. Di Giulio*

* Ecotoxicology Laboratory, Nicholas School of the Environment, Duke University, Durham, North Carolina 27708–0328; and {dagger} Department of Chemistry and Southeast Environmental Research Program, Florida International University, University Park Campus, Miami, Florida 33199

Received June 6, 2000; accepted July 31, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Differential susceptibility of polycyclic aromatic hydrocarbon (PAH)-mediated liver cancer exists in two related species of Ictalurid catfish. Two hypotheses are addressed in this study to explain this difference. Specifically, the relatively insensitive channel catfish 1) do not produce mutagenic PAH metabolites, and/or 2) they more quickly eliminate PAHs due to greater Phase II enzyme activities than the more sensitive brown bullhead. Livers and bile were collected from each species 6, 24, 72, and 168 h after a single 10 mg/kg i.p. benzo(a)pyrene (BaP) exposure. BaP treatment had no significant effect on cytosolic 1-chloro-2,4-dinitrobenzene or ethacrynic acid (EA)-glutathione-S- transferase (GST) and cis-stilbene oxide-microsomal epoxide hydrolase (EH) activities of either species. Channel catfish EH and GST activities were 1.2-fold higher than brown bullhead activities (p = 0.058 and p < 0.002, respectively). HPLC-APCI-MS of extracted bile and bile enzymatically digested to detect glucuronyl transferase (GT), GST, and sulfotransferase (ST) conjugated metabolites indicated no species differences in elimination or profiles of total biliary metabolites. GT conjugates predominated; ST and GST conjugates were minimal. BaP-diones accounted for the majority of metabolites in both species. Overall, these results indicated that brown bullhead preferentially formed BaP-7,8-dihydrodiol, a precursor to the DNA-reactive BaP-7,8-dihydrodiol-9,10-epoxide (BPDE), which may be linked to the increased PAH susceptibility in this species.

Key Words: benzo(a)pyrene; bile, metabolism; catfish; PAH; GST; epoxide hydrolase; HPLC-MS.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Currently, identification of the genotypic and biochemical reasons for disease susceptibility comprises a major concern in both human and environmental health research. Evidence exists for species differences in cancer susceptibility in environmentally exposed wildlife. Hudson River tomcod (Microgadus tomcod) have much higher incidences of tumors compared to hogchoker (Trinectes maculatus) collected at the same locations (Wirgin and Waldman, 1998Go). Similarly, in the highly polycyclic aromatic hydrocarbon (PAH)-polluted Elizabeth River, oyster toadfish (Opsanus tau) were tumor resistant compared to killifish (Fundulus heteroclitus) (Collier et al., 1993Go). PAH-contaminated environments have also been implicated in liver tumor formation in brown bullhead (Ameriurus nebulosus) (Baumann and Harshbarger, 1995Go), whereas neoplasms in the related species, channel catfish (Ictalurus punctatus), have not been reported (Harshbarger and Clark, 1990Go). Our laboratory has been investigating the mechanistic reasons for the differential susceptibility between these two related Ictalurid catfish species.

Previous work has indicated that the two species have differences in both Phase I and Phase II biotransformation enzymes and ability to form PAH-DNA adducts. Consistently, channel catfish, the resistant species, have shown higher CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activities. Both control and induced channel catfish typically have 5- to 10-fold higher liver microsome EROD activities compared respectively to control and BaP or ßNF-induced brown bullhead (Ploch et al., 1998Go; Watson and Di Giulio, 1997Go). In comparative studies of the Phase II glutathione-dependent defense system, Hasspieler et al. (1994b) determined that channel catfish were better poised to deal with oxidative stressors due to higher {gamma}-glutamylcysteine synthetase, glutathione reductase, and GST activities. Consistent with these differences, GSH:GSSG ratios were less affected and more rapidly recovered in the channel catfish when challenged by tert-butyl hydroperoxide (t-BOOH) (Hasspieler et al., 1994aGo). However, neither species exhibited an increase in hepatic concentrations of 8-hydroxy-2-deoxyguanosine (8-OHdG, a marker of oxidative DNA damage) following in vivo exposures to t-BOOH (Ploch et al., 1999Go). Furthermore, when brown bullhead hepatocytes were treated with BaP or BaP quinone, neither changes in 8-OHdG levels nor apurinic site formation occurred relative to controls (Ploch, 1997Go). Collectively, these data suggest that although there are differences in the Phase II and redox enzymes between species, neither oxidative injury nor apurinic site DNA damage (both associated with free radical intermediates) account for the differences in cancer susceptibility between these species.

Overall in vitro DNA binding levels also apparently do not correlate with species susceptibility to carcinogenesis. When comparing in vitro DNA binding levels using 3H-BaP, channel catfish microsomes exhibited nearly six times more BaP-DNA binding than brown bullheads (Ploch et al., 1998Go). This is consistent with the previously mentioned higher CYP1A activities reported from channel catfish and the fact that 3H-BaP binding assay measures both polar BaP adducts (Varanasi et al., 1989Go) and (±)-anti-7ß,8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydro-benzo(a)pyrene (BPDE). However, in in vivo studies where DNA adduct formation was measured over 45 days by the 32P-postlabeling assay, brown bullhead had significantly higher total adducts, the majority of which were BPDE-DNA adducts (Ploch et al., 1998Go). Persistently high in vivo BPDE-DNA adduct formation is consistent with the relatively greater cancer susceptibility of brown bullhead. In order to generate the DNA-reactive BPDE molecule, BaP must undergo oxidation predominately by CYP1A to generate an arene oxide (Guengerich, 1992Go). EH is required to generate the BaP-7,8-dihydrodiol that undergoes another CYP1A oxidation to form BPDE. Competing with the formation of BPDE adducts are conjugation reactions with glutathione, glucuronic acid, or sulfate, which make BaP metabolites more water soluble for biliary and fecal excretion. Even after the BPDE-DNA adduct is generated, nucleotide excision repair can occur before the damage is preserved in the genome (Hess et al., 1997Go).

The differences between brown bullhead and channel catfish in vitro and in vivo BaP-DNA binding suggest that Phase II conjugation, rate of biliary excretion, specific profiles of BaP metabolites, and/or the rate of DNA repair could be involved with cancer susceptibility in these two species. This study was undertaken to characterize the two species' biliary excretion with respect to conjugation and metabolite profiles after a single i.p. injection of BaP. Results suggest that although there were not species differences in overall excretion or conjugation profiles, the brown bullhead generated more of the BaP-7,8-dihydrodiol, an intermediate in the pathway to BPDE. In addition, channel catfish had higher cytosolic EA and CDNB-GST activities. By understanding the mechanisms for differential PAH-induced cancer sensitivity in these two catfish species, we hope to gain insight into reasons for differential cancer susceptibility in other organisms.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Brown bullhead and channel catfish were acquired from Northeastern Biologists (Rhineback, NY) and Foster Lake and Pond Management (Garner, NC), respectively. Fish were maintained in 70-liter aquaria with flow-through dechlorinated city water. Water temperature was approximately 15°C (± 2°C) at the time of the experiments. A 12-h light-dark cycle was maintained. Fish were fed Ziegler catfish chow pellets (Ziegler Brothers, Gardener, PA). Twenty 2- to 3-year-old male fish between 80 and 220 g of each species were dosed i.p. with 10 mg/kg BaP (Fluka Scientific, Ronkonkoma, NY) in corn oil (2.5 mg/ml). Controls received corn oil only. Bile and livers were collected from four animals per treatment at 6, 24, 72, and 168 h after dosing.

Tissue preparation.
Livers were homogenized in 4 volumes buffer (0.25 M sucrose, 0.1 M TrisHCl, 1mM EDTA, pH 7.4) and centrifuged 20 min at 10,000 x g. Supernatant was then spun at 105,000 x g for 1 h. Supernatant from the high-speed spin was collected and flash frozen as the cytosolic fraction. The microsomal pellet was washed with 2 ml 0.15 M Tris base and spun an additional hour at 105,000 x g. The microsomal pellet was resuspended in 400 µl buffer (0.25 M sucrose, 0.1M TrisHCl, 1 mM EDTA, 20% glycerol, pH 7.4) and immediately frozen in liquid nitrogen. Protein concentrations of cytosolic and microsomal preparations were determined by the Bio-Rad Protein Assay kit (Hercules, CA).

Enzyme assays.
Cytosolic GST activities were determined as previously described (Habig et al., 1974Go). For 1-chloro-2,4-nitrobenzene (CDNB) assays, each incubation contained 2.5 µg cytosolic protein, 1.2 mM GSH (in 0.1M potassium phosphate buffer pH 7.0) and 1.2 mM CDNB (Aldrich, Milwaukee, WI) for a 100 µl total volume. Absorbance was quantified with a plate reader (HTS 7000, Perkin Elmer, Norwalk, CT); kinetics was monitored over 7 min at 340 nm. GST activities were calculated using the molar absorptivity of 9.6/mM*cm. For assays using ethacrynic acid (EA) as substrate, each incubation contained 91–160 µg cytosolic protein, 0.25 mM GSH (in 0.1M potassium phosphate buffer pH 6.5), and 0.2 mM EA dissolved in ethanol. Absorbance was quantified with a spectrophotometer (Shimadzu, Kyoto, Japan); kinetics was monitored over 2 min at 270 nm. GST activities were calculated using the molar absorptivity of 5.0/mM*cm.

Microsomal EROD activities were also determined with the plate reader as described (Willett et al., 1997Go). Each well contained 200 µl including 1.25 µM ethoxyresorufin (ER) (in 0.1 M HEPES, 120 µM NADH, 100 µM NADPH, 5 mM magnesium sulfate, pH 8.0) and 50 µg microsomal protein. Reactions were initiated by addition of ER. Each plate included a resorufin standard curve containing 50 µg BSA protein instead of microsomes. Fluorescence wavelengths were 530/590 nm.

Microsomal EH activities were measured as described by Lauren et al. (1989). 3H-cis-stilbene oxide (CSO), used as substrate, was kindly provided by Dr. Bruce Hammock (University of California, Davis). Reactions (100 µl in 0.1M TrisHCl, pH 8.5) were run for 20 min at 30°C and contained 0.01 mg microsomal protein and approximately 13,000 c.p.m. CSO. Blank samples contained microsomes that had been inactivated by boiling. Reactions were stopped with the addition of 250 µl isooctane and vortexed. After centrifuging the samples for 8 min at 2100 x g, a blunt-end Hamilton syringe was used to sample 30 µl of the aqueous phase for scintillation counting.

Biliary extractions.
Bile samples were extracted using a modification of the method of Steward et al. (1990a). Fifty microliters of bile was combined with 1250 µl sodium acetate buffer (pH 5) and 400 ng 6-hydroxychrysene (AccuStandard, New Haven, CT) was used as an internal standard. Free metabolites were extracted with two volumes ethyl acetate:acetone (2:1) and twice more with ethyl acetate alone. Organic fractions were combined and blown dry under argon. Samples were brought up in 200 µl methanol with 400 ng benzanthracene 7,12-dione (AccuStandard). The aqueous phase was blown down to remove any traces of organic phase. Glucurase (200 µl, 1000 units, ß-D-Glucuronide glucuronosohydrolase, Sigma, St. Louis, MO) was added to the aqueous phase, and tubes were capped, vortexed, and incubated for 6 h at 37°C. The reaction was stopped by the addition of ethyl acetate:acetone (2:1) and the internal standard was added. Two additional extractions were done with ethyl acetate:acetone (2:1) and ethyl acetate alone. As before, the organic phase was blown down to dryness and brought up in methanol with surrogate standard. After the organic phase was removed, aqueous samples were then brought up to 2.1 ml with 0.2 M sodium acetate, pH 5. Nineteen units of aryl sulfatase (Type V from limpets, Sigma) and 20 µmol/ml saccharic acid lactone were added to the tubes and incubated again for 6 h at 37°C. After incubation, the samples were extracted as previously described. The final digestion was with 2.3 units per sample of {gamma}-glutamyltranspeptidase (Type IV, crude from porcine kidney, Sigma) for 6 h and 37°C. Samples were again extracted as previously described.

Chemical analysis.
The four fractionated sample extracts containing both BaP and its metabolites were analyzed by injecting 20 µl on to a C-18 reverse phase high performance liquid chromatography (HPLC) column (250 x 4.6 mm ECQ 5µ S80A C-18 Column, Whatman Inc., Clifton, NJ). Separation of all analytes was achieved at a flow of 1 ml/min using a 2-step gradient elution program (James et al., 1995Go) as follows: 50:50 to 83.5:16.5 methanol:water in 30 min and then to 100% methanol in 60 min. Final optimization of the gradient provided good resolution for both the BaP quinones and the mono-hydroxylated compounds. Detection of the target compounds (Table 1Go) was achieved by connecting the effluent of the HPLC column to a Finnigan (San Jose, CA) Navigator aQa single quadrupole mass spectrometer. The source of the MS was operated under atmospheric pressure chemical ionization in positive ion mode (APCI+). To increase sensitivity, only the most abundant ion for each compound and one confirmation ion were acquired under selected ion monitoring. The specific ions employed for the identification and quantitation of the targeted analytes are also shown in Table 1Go. The optimized source tuning parameters were as follows:


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TABLE 1 Quantitation (M1) and Confirmation (M2) Ions Monitored for the Quantitative Determination of the Targeted Analytes
 

Quantitation of the metabolites present in the samples was conducted by direct comparison of the relative response factors (RRFs) of the analytes with respect to 6-hydroxychrysene, the internal standard. Linear calibration curves were obtained for all compounds in the range of 0.5 to 8 ng/µl. A calibration acceptance criteria of ± 15% RSD was used for all average RRFs.

Statistical analysis.
Statistical analysis was performed using the SigmaStat software (SPSS Inc, Chicago, IL) with a level of significance of p < 0.05. Global One Way ANOVA was used to determine species and treatment effects. The Student-Newman-Keuls method was used for pairwise multiple comparisons. Data are reported as means and standard errors, with n = 4 for all channel catfish end points and n = 3 or 4 for brown bullhead end points.


    RESULTS
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EROD activities in control channel catfish were approximately 5-fold higher compared to activities in brown bullhead controls (Fig. 1Go). Likewise, at 72 h and 1 week after a single 10-mg/kg dose of BaP, EROD activities in induced channel catfish were five times higher than the induced brown bullhead.



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FIG. 1. Liver microsomal EROD activities in corn oil-control (depicted as 0 h) and BaP-dosed channel catfish and brown bullhead. Livers were collected 6, 24, 72, and 168 h after a single 10 mg/kg i.p. injection of BaP. Channel catfish EROD activities were significantly higher at all except the 6-h time point (p < 0.05, n = 3 or 4).

 
In contrast to the EROD results, neither cytosolic GST nor microsomal EH activities were induced by BaP treatment (Figs. 2 and 3GoGo). EH and GST activities (using either CNDB or EA substrate) were 1.2-fold higher in channel catfish compared to brown bullhead (p = 0.058 and p < 0.002, respectively).



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FIG. 2. Liver cytosolic 1-chloro-2,4-dinitrobenzene GST (A) and ethacrynic acid GST (B) in corn oil-control (depicted as 0 h) and BaP-dosed channel catfish and brown bullhead. Livers were collected 6, 24, 72, and 168 h after a single 10 mg/kg i.p. injection of BaP. There was not a significant treatment effect. The mean CDNB-GST activities in channel catfish (584 ± 22) were statistically higher than those from brown bullhead (470 ± 23) (p = 0.002). The mean EA-GST activities in channel catfish (65 ± 2.4) were statistically higher than those from brown bullhead (51 ± 2.5) (p < 0.001).

 


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FIG. 3. Liver microsomal cis-stilbene oxide EH activities in corn oil-control (depicted as 0 h) and BaP-dosed channel catfish and brown bullhead. Livers were collected 6, 24, 72, and 168 h after a single 10 mg/kg i.p. injection of BaP. There was no statistically significant difference between species (p = 0.058) or time point (p = 0.356) (n = 3 or 4).

 
To better understand BaP metabolism and excretion in each species, bile was extracted and enzymatically digested, and metabolites were analyzed by HPLC-APCI-MS. HPLC elution profiles (not shown) were consistent with those previously reported (James et al., 1995Go; Michel et al., 1992Go; Steward et al., 1990aGo,bGo) where the tetrols are followed in order by dihydrodiols, quinones, phenols, and the parent BaP.

The total biliary metabolites including those that had been conjugated were not significantly different between species at any time point (Fig. 4Go). Total metabolites ranged from 3.1 ± 1.4 and 9.3 ± 3.6 ng/µl (11 ± 4.9 and 33 ± 13 pmol/µl) for channel catfish and brown bullhead, respectively, at 6 h, to 701 ± 265 and 616 ± 326 ng/µl (2550 ± 970 and 2220 ± 1200 pmol/µl) at 1 week after exposure. The average weight of an individual fish of either species was approximately 140 g. If it is assumed that fish had 300 µl bile (based on random sample of eight fish, range 150 to 600 µl), then the cumulative total BaP metabolites detected over the time course was approximately 24% of the initial dose of BaP the fish received.



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FIG. 4. Total BaP biliary metabolites (means ± standard errors, ng/µl) detected by HPLC-APCI-MS in channel catfish and brown bullhead following a single 10 mg/kg i.p. injection of BaP. There were no detectable metabolites in any control fish (data not shown). There was no significant difference between species at any time point (p = 0.879). a,b,c: Time points not sharing a common letter are significantly different from each other (p < 0.05, n = 3 or 4).

 
Only a small percentage (range 0.66–7.7%) of the BaP was found as free metabolites in the bile. The majority of the metabolites were detected following the glucurase digestion (Fig. 5Go). Some glutathione conjugates may also have disassociated in this fraction. Considering the low proportion of GST conjugates detected and the known role of GST in conjugation of BaP metabolites (George, 1994Go; Gallagher and Di Giulio, 1992Go; Leaver et al., 1992Go; Steward et al., 1990bGo), it is likely that some GST conjugates were either released into the initial glucurase digest or they were not disassociated by the {gamma}-glutamyltranspeptidase during the third digest. Confirming results from other fish studies (Leaver et al., 1992Go; Zaleski et al., 1991Go), ST conjugates were minimal.



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FIG. 5. Total BaP biliary metabolites (means ± standard errors, ng/µl) detected in the organic phase after the initial extraction or after the subsequent glucurase, aryl sulfatase, and {gamma}-glutamyltranspeptidase enzyme digestions (n = 3 or 4). Bile was collected 6, 24, 72, and 168 h after a single 10 mg/kg i.p. injection of BaP. Note that the y-axis is broken between 43 and 65 ng/µl so that the smaller bars can be seen.

 
The specific BaP metabolites detected following the glucurase digest are shown in Table 2Go. In the channel catfish samples, the BaP-diones predominated (52–91% of total metabolites); however, there were increasing (up to 45%) amounts of the 3-hydroxy-BaP over the time course. A metabolite of particular interest with respect to the mutagenic/carcinogenic pathway of BaP metabolism was the BaP-7,8-dihydrodiol, a precursor to BPDE. In the channel catfish this metabolite made up approximately 8, 11, 3, and 2 % of the total glucurase generated metabolites for the 6-, 24-, 72-, and 168-h samples, respectively. Actual concentrations of BaP-7,8-dihydrodiol in channel catfish bile ranged from 0.502 to 11.6 ng/µl at 6 and 72 h, respectively. In brown bullhead, concentrations of BaP-7,8-dihydrodiol were 4.47 and 65.4 ng/µl at 6 and 72 h. In brown bullhead samples, diones were also detected (42–61% of total); however, of the total metabolites, BaP-7,8-dihydrodiol was 50, 37, 14, and 12% at the 6-, 24-, 72-, and 168-h time points.


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TABLE 2 Specific BaP Metabolites ± Standard Errors Detected following the Glucurase Digestion (ng/µl)
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The relationship between environmental PAH contamination and liver tumor formation in feral fish has been established repeatedly. Examples include English sole (Parophrys vetulus) from Puget Sound, WA (Malins et al., 1985Go; Myers et al., 1991Go), Fundulus heteroclitus from the Elizabeth River, VA (Vogelbein et al., 1990Go), and brown bullhead from the Black and Cuyahoga Rivers, OH (Baumann et al., 1991Go; Baumann and Harshbarger, 1995Go). For each of these examples, another fish species occupies the same environment but is apparently resistant to liver neoplasia. Although the incidence of tumors in English sole from the Duwamish waterway was 20–30%, there was less than 1% incidence in starry flounder (Collier et al., 1992Go). Likewise, Elizabeth River toadfish are comparatively resistant to hepatic neoplasia (Collier et al., 1993, and reviewed in Wirgin and Waldman, 1998). Of relevance to this study, 4-year-old brown bullhead living in the polluted Black River displayed a greater than 40% cancer incidence in 1982 (Baumann and Harshbarger, 1995Go). On the other hand, there have been no tumors reported in channel catfish (Harshbarger and Clark, 1990Go). The mechanistic reasons for species differences in PAH cancer susceptibility are largely unknown, especially in fish.

BPDE adducts were produced in much higher amounts in brown bullhead exposed to BaP compared to channel catfish (Ploch et al., 1998Go). Higher levels of DNA adducts in bullhead is inconsistent with the observed EROD activities in the two species (Fig. 1Go). Because CYP1A-mediated EROD activities are approximately 5-fold higher in channel catfish, and CYP1A is involved in the generation of BPDE, it could be hypothesized that channel catfish would form more adducts. In fact, when microsomes (without Phase II enzyme cofactors) were incubated with 3H-BaP, there was more BaP-DNA binding in channel catfish (Ploch et al., 1998Go). The discrepancy between in vivo and in vitro DNA adduct results leads to the current hypotheses that channel catfish either more efficiently eliminate BaP metabolites by Phase II conjugation or they do not generate the intermediates necessary for BPDE formation (specifically BaP-7,8-dihydrodiol).

Three enzymes having somewhat competing roles in BPDE formation are GST, GT, and EH. GT and GST can conjugate BaP metabolites, facilitating excretion before adducts can form. In contrast, EH is required to convert the BaP-7,8-dihydrodiol-epoxide into BaP-7,8-dihydrodiol for further P450 oxidation to BPDE.

Using CDNB and EA as substrates for GST, there was no induction of GST by either species over the time course of BaP exposure (Fig. 3Go). Lack of induction was not surprising, because longer times generally are required to detect GST induction. In rainbow trout treated with ß-naphthoflavone (ß-NF), EROD and GT activities were significantly elevated, whereas GST was not significantly elevated until 14 days after exposure (Zhang et al., 1990Go). Despite the lack of induction by BaP, channel catfish did have statistically significant 1.2-fold higher average GST activities. This was consistent with previous studies where channel catfish GST activities were 1.5-times higher than brown bullhead activities (Hasspieler et al., 1994bGo). It was doubtful that this subtle difference in CDNB-GST activities accounted for the species difference in adduct formation and cancer. However, because CDNB is a substrate for multiple GST isoforms and thus is a relatively poor surrogate substrate for BPDE (Gadagbui and James, 2000Go; Gallagher et al., 1996Go), assays were also performed with EA. EA is a relatively good surrogate marker for pi class GST. EA activities averaged 65 and 51 nmol/mg*min for channel catfish and brown bullhead, respectively. Gadagbui and James (2000) have also reported EA-GST activities of 65 nmol/mg*min for channel catfish. The fact that channel catfish had statistically significant higher EA-GST activities (p < 0.001) than brown bullhead, and that EA is a better surrogate for BPDE, indicates that GST may be playing a role in the species differences. Molecular characterization of isolated GST isozymes using BPDE will be necessary to completely characterize the role of GST in catfish BaP metabolism.

Like GST, microsomal CSO-EH was not induced by BaP treatment in either species (Fig. 2Go). Our studies used CSO as a surrogate substrate for BaP metabolism. Induction was also not found by other researchers using styrene oxide-EH following ß-NF and Clophen A-50 exposure in rainbow trout (Andersson et al., 1985Go). Additionally, 3-methylcholanthrene (3-MC) did not induce styrene oxide hydrolase in guppy, medaka, sheepshead minnow, killifish or fathead minnow (James et al., 1988Go). Average EH activities in this study were 1030 ± 63 and 850 ± 68 pmol/mg*min for channel catfish and brown bullhead, respectively. Channel catfish EH activities were on average 1.2-fold higher than brown bullhead (p = 0.058). These activities were consistent with those reported in rainbow trout (889 ± 100 pmol/mg*min) using the CSO substrate (Lauren et al., 1989Go). As with GST, it is unlikely that differences in EH can fully explain species susceptibility differences, as has been suggested by some studies (Sikka et al., 1990aGo; Yuan et al., 1997Go). On the other hand, CSO is a general mEH substrate and more definitive results on the role of EH in BaP metabolism might be found using BaP-7,8-oxide as a substrate.

The selectivity of HPLC-APCI-MS was useful in determining specifically how BaP was metabolized and excreted as biliary metabolites. This detection method offered the following advantages: radioactive analytes were unnecessary, gentle ionization allowed for the detection of [M+H]+ and [M-H2O+H]+ ions with selected ion monitoring, and standard HPLC separation methods could be used without lengthy derivatization steps. Additionally, by using selected ion monitoring with quantitation and confirmation ions, the method identified specific PAH metabolites rather than ring classes which are provided using HPLC with fluorescent detection (Krahn et al., 1984Go) or synchronous fluorometric spectroscopy (Lin et al., 1994Go).

When free and conjugated metabolites were summed at each time point, there were no species differences in rate of excretion over the 1-week time course. This is consistent with the finding by Ploch and coworkers (1998) that there was also no difference in total BaP distribution to the liver up to 7 days after a 10-mg/kg dose of BaP. Therefore, our data suggest that over the 1-week time course, the overall rates of BaP biliary excretion are not a major factor affecting species susceptibility. It is not certain, however, how the species might differ in excretion after 1 week. Brown bullhead BPDE-DNA adducts in liver were maximal at 14 days (Ploch et al., 1998Go) or 25–30 days (Sikka et al., 1990bGo) after a single 20-mg/kg BaP exposure, indicating a latency in adduct formation. However, Ploch et al. (1998) observed that channel catfish had consistently lower adducts than bullhead across the entire 45-day time course, including days 3 and 7. Because overall excretion and liver uptake were not different at these time points, a difference in BaP metabolism or DNA repair could be operative.

Free metabolites were at very low concentrations, indicating the importance of Phase II conjugation in both species. In these studies, GT BaP metabolites predominated, followed by ST and GST conjugates. In another study, brown bullhead were treated with 27 µg 3H-BaP, and the fate of the BaP was determined at 24 and 72 h posttreatment (Steward et al., 1990aGo). Biliary BaP (parent and metabolites) amounted to 16% of the radioactivity found in the fish at 72 h (compared to the estimated cumulative 24% in our study). They observed 24% of the BaP metabolites glucuronidated, 14% as free metabolites, and 57% as water soluble conjugates that were unhydrolyzed by ß-glucuronidase or arylsulfatase and therefore attributed to GST conjugates. The discrepancies between this study and ours are not clear, but it could be related to different extraction methods used. In both studies, free metabolites were extracted initially from the bile. Steward et al. (1990a) then took the resulting aqueous phase and split it into three fractions for enzyme digestions and extractions. In contrast to the Steward study, the entire aqueous phase was sequentially digested with glucurase, followed by aryl sulfatase and finally {gamma}-glutamyltranspeptidase. This method was selected to maximize the amount of detectable metabolites and to minimize any error that would be introduced by splitting bile samples. A disadvantage of our method is that it may overestimate the contribution of glucuronidated metabolites in the BaP conjugation profile if GST or ST conjugates are labile and are released in the initial digest. Alternatively, if {gamma}-glutamyltranspeptidase digest conditions were not optimal during the final digest, GST conjugates would not be released. Because only organic extractable metabolites could be introduced into the HPLC-MS, metabolites remaining in the aqueous phase after the three digests could not be quantitated. These methodological differences do not explain the difference in the initial free organic extraction (parent BaP and unconjugated BaP metabolites) between the two studies. Different BaP biliary metabolite profiles have also been reported in oyster toadfish following water-borne BaP exposure (Kennedy et al., 1989Go) and English sole exposed i.m. to BaP (Nishimoto et al., 1992Go). After 24-h exposures, 7, 2, 4, and 87% BaP metabolites quantitated in toadfish bile were free, glucuronidated, sulfated, or other unidentified aqueous metabolites, respectively. In contrast, glutathione, glucuronide, and sulfate conjugates comprised 55, 39, and 5.4% of BaP metabolites in English sole bile. Together, these results indicate that there are species and methodological differences in BaP conjugation profiles following fish BaP exposures. However, it is difficult to make comparative statements with respect to sensitivity differences (i.e., that toadfish and channel catfish are resistant whereas sole and bullhead are sensitive) when different biliary extraction methods have been used. Our study used the same method for both brown bullhead and channel catfish bile samples and no dramatic differences were seen in Phase II conjugation profiles of BaP.

By using mass spectrometry detection, it was possible to characterize specifically which BaP metabolites were formed by the two species. As shown in Table 2Go, BaP-diones (BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione) predominated in both species. Quinone concentrations in our study were higher than those reported in most fish studies. During bile extractions, butylated hydroxytoluene was not added to prevent spontaneous oxidation of phenols to quinones, as some investigators have suggested (Steward et al., 1990). However, when the 3- and 9-hydroxyBaP standards were analyzed for the presence of quinones, less than 2.2% auto-oxidation was detected. High amounts of BaP-diones have been implicated in the one-electron oxidation pathway of BaP metabolism (Cavalieri and Rogan, 1992Go). Depurinating adducts that result could be involved in cancer susceptibility differences. However, analysis of depurinated adducts from microsomal incubations of BaP and DNA revealed that no depurinated adducts were detected in either brown bullhead or channel catfish samples (Ploch, 1997Go). Likewise, when brown bullhead hepatocytes were exposed to BaP or BaP quinone, no changes in 8-OHdG oxidative DNA damage relative to controls were observed (Ploch, 1997Go). Thus, it appears unlikely that quinone metabolites are playing a major role in susceptibility differences.

When Sikka et al. (1990a) incubated brown bullhead microsomes with BaP, diones were 47% of the total BaP metabolites generated. Microsomes from brown bullhead and channel catfish pretreated with 3-MC only generated 10 and 14% BaP-diones, respectively (Yuan et al., 1997Go). Channel catfish maintained on a ß-NF diet had lower microsomal quinone formation (11%) (James et al., 1997Go). In the latter two studies, BaP-phenol metabolites, particularly the 3-hydroxy-BaP, predominated. Biliary 3-hydroxy-BaP significantly increased from nearly 0 at 6 h to 58% and 34% of total metabolites in channel catfish and brown bullhead, respectively. Higher proportions of phenols are indicative of more efficient, less toxic metabolic excretion pathway.

Conversely, formation of the BaP-7,8-dihydrodiol is indicative of a metabolic pathway capable of ultimately yielding the BPDE-DNA adduct. In our study, biliary concentrations of BaP-7,8-dihydrodiol were 9-, 3-, 6-, and 7-fold higher in brown bullhead compared to channel catfish at the 6-, 24-, 72-h, and 1-week time points, respectively. For example, at 72 h (the time point where BaP-7,8-dihydrodiol was highest in both species) biliary concentrations of BaP-7,8-dihydrodiol were 65.4 ng/µl in brown bullhead and 11.6 ng/µl in channel catfish (Table 2Go). At 6 h, BaP-7,8-dihydrodiol constituted 50% of the brown bullhead GT biliary BaP metabolites compared to only 17% in channel catfish. In both species, the relative contribution of BaP-7,8-dihydrodiol went down with time from exposure (down to 12% for brown bullhead and 2% for channel catfish by 1 week). This result was the most dramatic species difference that could be involved with PAH sensitivity. The observation is supported by an in vitro study where BaP-7,8-dihydrodiol accounted for 26% and 14% of BaP metabolites generated by brown bullhead and channel catfish microsomes, respectively (Yuan et al., 1997Go).

The lack of correlation between EROD activities and the in vivo formation of BaP-7,8-dihydrodiol in brown bullhead and channel catfish indicates that dealkylation of ethoxyresorufin and oxygenation of BaP at the 7,8 position may not be catalyzed by the same CYP isoform. In fact, studies in human recombinant systems have found that CYP1B1 is more efficient than CYP1A in metabolizing BaP to BaP-7,8-dihydrodiol (Shimanda et al., 1999Go). Although CYP1B1 has been identified in some fish species (Godard et al., 1999Go), its role in the metabolism of BaP in these catfish species is unknown.

Sikka and coworkers (1990a) also used a comparative approach to characterize PAH susceptibility differences between mirror carp (Cyprinus carpio, resistant) and brown bullhead. As with our work comparing brown bullhead and channel catfish, the BaP metabolites generated by carp and brown bullhead microsomes were qualitatively similar, but the relative proportions of individual metabolites were different (Sikka et al., 1990aGo). In contrast to our work, the resistant species (carp) produced a much greater amount of (-)-BaP-7,8-dihydrodiol (31% of total metabolites) than did the brown bullhead microsomes (15%). The reasons for the differences between carp and bullhead, and between channel catfish and bullhead are intriguing. It is unclear whether in vivo BaP biliary metabolites in carp and bullhead would be consistent with the microsomal studies. The authors suggested that carp must have higher EH activities in order to generate more BaP-7,8-dihydrodiol. This also contrasts the current study, where channel catfish had slightly higher CSO-EH activities, but less BaP-7,8-dihydrodiol formation.

Our research suggests that at least one factor in the susceptibility difference to PAH-induced carcinogenesis is that brown bullhead generate more of the BaP proximate carcinogen BaP-7,8-dihydrodiol. Our results also suggest that there are not dramatic differences in total BaP excretion or Phase II conjugation patterns between the species. However, channel catfish did have significantly higher CDNB and EA-GST activities. Ongoing research in our laboratory is investigating mechanisms of BaP activation and the potential role of DNA repair in the differential susceptibility of these two species.


    ACKNOWLEDGMENTS
 
This work was supported by U.S. Environmental Protection Agency (EPA) grant R827101-01-1 and the Duke University Marine/Freshwater Biomedical Center. The authors would like to thank Dr. Bruce Hammock from the University of California, Davis for kindly providing the CSO EH substrate. Thanks also to Jamie Rogers, Elizabeth McLean, Michelle Rau, and Catherine Andre for their technical help in the Ecotoxicology Laboratory.


    NOTES
 
1 To whom correspondence should be addressed at The University of Mississippi, Department of Pharmacology, 303 Faser Hall, P.O. Box 1848, University, MS 38677–1848. Fax: (662) 915-5148. E-mail: Kwillett{at}olemiss.edu. Back


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