32P-Postlabeling analysis of DNA adduct formation and persistence in English sole (Pleuronectes vetulus) exposed to benzo[a]pyrene and 7H-dibenzo[c,g]carbazole

Chem.-Biol. Interactions, 88 (1993) 55-69 Elsevier Scientific Publishers Ireland Ltd.

55

32p-POSTLABELING ANALYSIS OF DNA ADDUCT FORMATION AND PERSISTENCE IN ENGLISH SOLE (Pleuronectes vetulus) EXPOSED TO BENZO[a]PYRENE AND 7H-DIBENZO[c,g]CARBAZOLE

JOHN E. STEIN, WILLIAM L. REICHERT, BARBARA FRENCH and USHA VARANASI

Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Boulevard East, Seattle, WA 98112-2097 (USA) (Received December 15th, 1992) (Revision received February 17th, 1993 ) (Accepted February 17th, 1993)

SUMMARY

The formation and persistence of benzo[a]pyrene (BaP)- and 7H-dibenzo[c,g]carbazole (DBC)-DNA adducts in liver of English sole (P~uronectes vetulus) were investigated. BaP is a putative hepatocarcinogen in English sole based on its ability to induce formation of preneoplastic foci. while DBC is a hepatocarcinogen in mammals but whose carcinogenicity in fish is not known. English sole liver was sampled from 2 h through 84 days after a single intermuscular injection of a BaP and DBC mixture (100/zmol of each/kg body wt.), and DNA adduct levels were measured by the nuclease P1 version of the 32p-postlabeling assay. The major BaP adducts detected were from binding of BaP-7,8-diol-9,10-epoxide to DNA, whereas multiple uncharacterized DBC-DNA adducts were detected. Total adduct levels for both BaP and DBC reached a maximum at 2 days post exposure. The levels of DBC-DNA adducts were greater than the levels of BaP adducts at all time points and increased more rapidly than did the levels of BaPDNA adducts. The DBC to BaP adduct ratio was 33 ± 8.8 at 2 h and declined to 4.2 ± 0.48 by 12 h post exposure. From 2 to 28 days, the levels of both BaP and DBC adducts declined with apparent half-lives of 11 and 13 days, respectively. There was no apparent decline from 28 to 84 days in the levels of the remaining BaP or DBC adducts; these persistent adducts represented 32 and 36% of maximum levels, respectively. These results provide the first data on the kinetics of adduct formation and removal of a carcinogenic nitrogen-containing polycyclic aromatic compound in fish. The results showing greater binding and similar persistence of DBC-DNA adducts compared to BaP-DNA adducts suggest that DBC may be hepatotoxic and potentially carcinogenic in English sole. In a separate Correspondence to: John E. Stein, Environmental Conservation Division, Northwest Fisheries Science Centre, 2725 Montlake Blvd. East., Seattle, WA 98112-2097, USA. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

56

experiment, the effect of multiple doses of BaP (30 t~mol/kg body wt.) on the levels of hepatic BaP-DNA adducts showed that adduct levels increased linearly (r = 0.815, P = 0.0007) with 5 successive doses administered at 2 day-intervals and sampled 2 days after the last dose. The persistence of both BaP-DNA and DBC-DNA adducts in liver, together with the increase in BaP-DNA adducts in English sole exposed to successive doses of BaP, suggest that hepatic xenobioticDNA adducts in English sole are molecular dosimeters of relatively longterm environmental exposure to genotoxic polycyclic aromatic compounds.

Key words: DNA-adducts -- Benzo[a]pyrene -- 7H-dibenzo[c,g]carbazole -- 32p. postlabeling

INTRODUCTION

Epizootiological studies have established a correlation between exposure to polycyclic aromatic hydrocarbons (PAHs) and the prevalence of hepatic tumors in several benthic fish species [1-3]. Laboratory studies have substantiated these findings by showing that individual PAHs, such as benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene, and organic-solvent extracts of contaminated marine sediments containing polycyclic aromatic compounds are hepatocarcinogens in fish or induce presumptively preneoplastic focal lesions [4-7]. The covalent modification of cellular DNA by genotoxic compounds resulting in the formation of DNA adducts is a necessary but not a sufficient step in chemical carcinogenesis. The carcinogenicity of a compound appears to be related to both the level of binding to DNA and the relative persistence of the adducts, which reflects the balance between the formation and repair of DNA adducts and the extent of cell replication [8-11]. Recent studies with marine [12,13] and freshwater [14] fish have shown that DNA adducts detected in liver by the 82P-postlabeling assay [15] are related, in part, to exposure to environmental PAHs. Moreover, recent laboratory studies in fish have shown that DNA adducts arising from the binding of large hydrophobic xenobiotics tend to persist in fish tissues. Bailey et al. [16] found that aflatoxin B1-DNA adducts in liver of coho salmon (Oncorhynchus kisutch) and rainbow trout (0. mykiss, formerly Salmo gairdneri) were persistent for at least 3 weeks following a single exposure to [3H]aflatoxin B1 whereas in the rat the half-life of aflatoxin B1-DNA adducts is ca. 8 h [17]. In the brown bullhead (Ictalurus nebulosus) exposed to a single dose of BaP, 26% of BaP-DNA adducts still remained after 70 days [18]. We observed in a previous study [19] with juvenile English sole (Pleuronectes vetulus, formerly Parophrys vetulus) that hepatic BaP-DNA adducts were persistent for at least two months after a single exposure to BaP; however, the English sole were sampled at only three time points, thus providing limited information on the time course of formation and removal of hepatic BaP-DNA adducts. In contrast, there is no information on the formation and persistence of adducts from N-heterocyclic polycyclic aromatic corn-

57

pounds in fish. These compounds were detected in an organic-solvent extract of a contaminated marine sediment that induces preneoplastic lesions in the liver of English sole [20]. Additionally, the N-heterocyclic polycyclic aromatic compound 7H-dibenzo[c,g]carbazole (DBC), which is an hepatocarcinogen in mammals [21] but whose carcinogenicity in fish is unknown, has been detected in sediments from contaminated rivers [22]. Because the levels of xenobiotic-DNA adducts in wild fish provide a measure of a biologically effective dose of environmental genotoxins reaching a critical target site in the cell, there is considerable interest in the use of DNA adducts as molecular dosimeters of exposure of fish to genotoxic compounds. Knowledge of the formation and persistence of model xenobiotic-DNA adducts in fish tissues is needed to determine whether in wild fish DNA adducts detected by ~2p-postlabeling are markers of either relatively short- or longterm exposure to genotoxic compounds. Accordingly, in the present study the formation and persistence of hepatic DNA adducts in English sole exposed to the model hepatocarcinogens, BaP and DBC, were determined. Further, to simulate environmental conditions in which benthic fish are often continually exposed to genotoxic compounds, the effect of multiple doses of BaP on the accumulation of hepatic BaP-DNA adducts was assessed. MATERIALS AND METHODS

Chemicals The BaP was obtained from Sigma Chemical Co., St. Louis, MO. The DBC (H0258) (purity 99.9%) and 7R, 8S, 9S-trihydroxy-10R-(N2-deoxyguanosyl-3 'phosphate)-7,8,9,10-tetrahydro-BaP (AD0884) were purchased from the Midwest Research Institute, Kansas City, KS. The BaP was purified by the method of Varanasi and Gmur [23]. High purity phenol (99%) was obtained from Aldrich Chemical Co., Milwaukee, WI. The solvent vehicle, Emulphor 620* (polyoxyethylated castor oil), used for administering the BaP and DBC, was a gift from the GAF Corp., New York, NY. Salmon sperm DNA, used as a negative control, was extracted from testes of mature, laboratory-raised Atlantic salmon (Salmo salar) as described below. All other chemicals were analytical grade and used without further purification. Fish collection and exposure English sole were collected by otter trawl from a reference site (48 ° 16' N, 122 ° 33' W) in Puget Sound, WA, and transferred to our seawater facility. The fish were held in circular tanks (750 L) supplied with flowing seawater (12 ± 1°C) and acclimated for two weeks before initiation of the study. Fish were injected (2 ml/kg body wt.) intermuscularly between the dorsal epaxial and axial muscles with a binary mixture of BaP (100 ~mol/kg body wt.) and DBC (100 ~mol/kg body wt.), or with solvent vehicle (Emulphor 620:acetone, 1:1). The fish were sampled at times from 2 h to 84 days post exposure to the BaP/DBC mix*Mention of tradenames is for information only and does not constitute endorsement by the U. S. Department of Commerce.

58

ture or solvent vehicle. Additionally, in a separate experiment, fish were treated with BaP and DBC (100 ~mol/kg body wt. of each compound) separately and in combination and sampled at 3 days. English sole were also administered 1, 3, or 5 doses of BaP (30 #mol/kg body wt.) at 2 day-intervals and sampled at 2 days after the last dose. Livers excised from the fish were immediately frozen in liquid nitrogen and then stored at -80°C until analyzed.

s2p-postlabeling analysis of adducts Hepatic DNA was isolated from tissue using the phenol extraction method previously described in Varanasi et al. [19]. The 32P-postlabeling assay was conducted essentially according to Gupta and Randerath [24], and salmon sperm DNA was used as a negative control in each set of analyses. Briefly, DNA was enzymatically hydrolyzed to deoxyribonucleoside 3'-monophosphates, and nuclease P1 was used to hydrolyze normal nucleotides to nucleosides, thereby enriching the mixture in adducted 3'-monophosphates [25]. The S2p-labeling was initiated by adding a buffer mix (100 mM bicine, 100 mM magnesium chloride, 100 mM dithiothreitol and 10 mM spermidine, pH 8.75) containing 100 t~Ci of [~-32p]ATP and 8 U of T4-polynucleotide kinase. The [~-32p]ATP was prepared according to Gupta and Randerath [24] and the range in specific activities was 350-1540 Ci/mmol. Polyethyleneimine-cellulose thin-layer chromatography (TLC) of the 32p_ labeled adducts [26] was carried out using TLC sheets prepared according to Gupta and Randerath [24]. The solvent systems used in the multi-directional chromatography were as follows: D1 - 1.0 M sodium phosphate, pH 6.0; D2 was omitted; D3-7.65 M urea and 4.32 M lithium formate, pH 3.5; and D4-7.65 M urea, 1.44 M lithium chloride and 0.45 M Tris, pH 8.0. Elution of the chromatograms in D5 was not done. The ~2p-labeled DNA-adducts were located and quantitated using storage phosphor imaging technology [27]. Briefly, storage phosphor plates were exposed to the chromatograms for 8 h and then scanned using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The location and intensity of images on the plates corresponding to the distribution of radioactivity on the chromatograms was recorded. The data obtained from the imaging plates were processed with lmageQuant (version 3.0) software (Molecular Dynamics, Sunnyvale, CA) and were then converted to 32p disintegrations per minute (dpm) by using a conversion factor determined from processing a TLC sheet spotted with a serial dilution of known concentrations of [~-32P]ATP with each set of sample chromatograms [27]. Total nucleotides were determined by one-dimensional TLC of 5'-labeled nucleotides using 0.24 M ammonium sulfate in 8 mM sodium phosphate, pH 7.4, as a solvent and quantitation of deoxyguanosine 3',5'-bisphosphate. Adduct levels (nmol adducts/mol nucleotides) were calculated from the storage phosphor readings, the 32p dpm conversion factor, the specific activity of the [~-32p]ATP, and the number of total nucleotides analyzed. In our previous studies, using the n-butanol version of the 32P-postlabeling method [19] two spots in the upper right-hand corner of the chromatograms were present in all fish irrespective of treatment or site of capture. The absence

59 of these spots in the chromatograms from the present study is attributed principally to slight modifications to the DNA isolation procedure to shorten the time between initial homogenization of the tissue and the final precipitation of DNA [28] and from the finding that the storage of tissue samples and isolated DNA at -80°C rather than at -20°C resulted in the virtual elimination of the spots. Additionally, the absence of the spots was further attributed to the use of the nuclease P1 version of the 32p-postlabeling assay, in which only one of these spots is usually detected in chromatograms of DNA of mammals [29].

Statistical analysis Comparisons of multiple means were done using analysis of variance and Fisher's protected least significant difference test, and comparisons between two means were done using the Student's t-test. The half-lives for the initial decrease in BaP- and DBC-adducts were estimated by the method of curve stripping [30], and the relationship between BaP-DNA adduct levels and number of consecutive doses was determined by linear regression analysis. The level of statistical significance was set at P -- 0.05. RESULTS The chromatographic profiles of hepatic DNA adducts in BaP/DBC-exposed fish (Fig. 1A) and in fish exposed to these compounds singly (Fig. 1B and C) showed that treatment of English sole with DBC yielded multiple adducts (spots/areas 1 - 7) and treatment with BaP gave a single major spot (spot a). This same pattern of multiple DBC spots and a major BaP spot was observed at all sampling times. In control fish, no spots were detected on chromatograms of hepatic DNA digests from fish sampled from 1 to 84 days after treatment with solvent vehicle. The major BaP spot comprised two partially resolved adducts having similar chromatographic characteristics as the anti-BaPDE-dG adduct standard (Fig. 1D) and the syn-BaPDE adduct as shown previously [19]. The distribution of radioactivity along a vector (see Fig. 1A) through the major BaP spot indicated that the anti-BaPDE isomer comprised about 30% of the total radioactivity (Fig. IF) at all sampling times. Minor BaP-DNA adducts were detected with longer exposure of the chromatograms to the imaging screens (data not shown). The minor BaP-DNA adducts comprised approximately 10% of the total BaP-DNA adducts. For DBC, the relative proportions of DBC-DNA adducts (spots/areas 1 - 7) were comparable at all times. Currently, the identities of the DBC-DNA adducts have not been determined. Both BaP- and DBC-DNA adducts were detected at 2 h and reached a maximum by 2 days after exposure to the BaP/DBC mixture (Fig. 2). The levels of total hepatic DBCoDNA adducts increased more rapidly than did the levels of BaP-DNA adducts (Table I). The ratio of DBC-DNA adducts to BaP-DNA adducts was 33 ± 8.8 at 2 h post exposure and declined to 4.2 ± 0.48 by 12 h. From 12 h to 84 days after exposure no significant change in this ratio was observed. Following the maximum in adduct levels at 2 days, the levels of both

60 A

O

C

E

F

syn-Ba~

t-

.5 antl-B

I

I

I

]

relativedistance

I

61

10000

1000

BaP -I~

100. "o "0 m

E c

10

I I

0

'

I

I

42 time (days)

84

Fig. 2. Levels of hepatic benzo[a]pyrene (BaP)- and 7H-dibenzo[c,g]carbazole (DBC)-DNA adducts in English sole after a single treatment with a mixture of these compounds. The dosage of each compound was 100 ~mol/kg body wt. Values are means ± S.E.; n = 4.

BaP and DBC adducts exhibited a relatively rapid decrease until 28 days (Fig. 2). The apparent half-lives for BaP- and DBC-DNA adducts, calculated using adduct levels measured between 2 and 28 days, were 11 and 13 days, respectively. The remaining adducts were highly persistent with little evidence of decline in levels of either BaP or DBC adducts. At 84 days post exposure, 32% of the maximum level of BaP adducts and 36% of the maximum level of DBC adducts remained.

Fig. 1.32p-postlabeling analysis of hepatic DNA adduct patterns in English sole sampled 24 h after exposure to a mixture of 7H-dibenzo[c,g]carbazole (DBC) and benzo[a]pyrene (BaP) (A), to DBC (B) or BaP (C) singly, or to the solvent vehicle (E). Corresponding DBC-derived spots and areas of multiple spots were given identical numbers in charts A and B, while corresponding BaP spots were given identical letters in charts A and C. The dosage of DBC and Bap was 100 ~mol/kg body wt. for each compound. CSart D is the chromatographic profile for the anti-benzo[a]pyrene diolepoxide (BaPDE)dG adduct standard. Chart F is the distribution of radioactivity between the anti- and syn-BaPDE dG determined along the arrow in chart A. The origin is located at the bottom left-hand corner of Charts A - D . The hepatic DNA was analyzed by the nuclease P1 version of the 32p-postlabeling assay and the adducts detected by storage phosphor imaging.

62 TABLE I RATIOS OF LEVELS (nmol ADDUCTS/mol N U C L E O T I D E S ) OF TOTAL 7HDIBENZO[c,g]CARBAZOLE-DNA ADDUCTS TO TOTAL B E N Z O [ a ] P Y R E N E - D N A ADDUCTS IN E N G L I S H SOLE a

Length of exposure

DBC-DNA adducts BaP-DNA adducts

2 4 6 12

h h h h

33 21 6.7 4.2

± ± ± ±

8.8 b'c 4.1 d 1.4 e 0.48 e

1 2 5 7 14 28 84

day days days days days days days

3.7 3.8 3.1 4.2 4.0 3.9 3.8

+ ± ± ± ± ± ±

0.33 e 0.54 e 0.12 e

0.90 e 0.47 e 0.50 e 0.37 e

aEnglish sole were exposed, intermuscularly, to a mixture of 7H-dibenzo[c,g]carbazole (DBC) and benzo[a]pyrene (BaP) at a dosage of 100 ~mol/kg body wt. of each compound. bValues (mean ± S.E.; n = 3 - 5) with a common superscript ( c - e) letter were not significantly different as determined by analysis of variance and Fisher's protected least significant difference test.

To assess the effect of simultaneous exposure to BaP and DBC on levels of hepatic DNA adducts of each compound, fish were treated with either BaP and DBC separately or together as a mixture (100 ~mol of each compound/kg body wt.) and sampled at 3 days post exposure. The levels of BaP and DBC adducts in English sole treated separately with these compounds were not significantly different (P > 0.05) from the levels of adducts in fish treated with the mixture of BaP and DBC (Table II). The effect of repeated exposure to BaP on levels of hepatic BaP-DNA adducts was assessed in English sole administered 1,3, or 5 doses of BaP at 2-day intervals and sampled 2 days after the final dose. The results in Fig. 3 show that the levels of hepatic BaP-DNA adducts increased linearly (r = 0.815, P = 0.0007) with cumulative doses of BaP given. DISCUSSION

The 32p-postlabeling assay was used to examine the time course of formation and removal of hepatic DNA adducts in English sole exposed to the carcinogens, BaP and DBC. The results showed rapid formation of hepatic DNA adducts for both compounds, and at all time points the levels of DBC adducts were consistently greater than the levels of BaP adducts. The decrease in levels of both BaP and DBC adducts in the liver of English sole exhibited an initial relatively

63 TABLE II LEVELS OF HEPATIC DNA ADDUCTS IN ENGLISH SOLE EXPOSED TO 7HDIBENZO[c,g]CARBAZOLE (DBC) AND BENZO[a]PYRENE (BaP) SEPARATELY OR AS A MIXTURE a Exposure

DBC

BaP

Separately Mixture

nmol adducts/mol nucleotides 790 ± 160 b 200 ± 49 880 ± 62 280 + 71

aEnglish sole were exposed, intermuscularly, to either a mixture of DBC and BaP or to the compounds separately. The dosage of each compound was 100 gmol/kg body wt. The fish were sampled at 3 days post treatment. The value for total DBC adducts corresponds to the sum of spots/areas 1 - 7 shown in Figs. 1A and B and the value for BaP adducts corresponds to spot a in Fig. 1A and C. bLevels (mean ± S.E., n = 6) of DNA adducts for each compound were not significantly different by Student's t-test between fish treated with the BaPfDBC mixture or with the compounds separately.

600 500 i

1

400

to 3

"o 300 "o (B a.

m 200 0 Er . 100 .1.

0

[

0

3 consecutive doses of BaP

I

5

Fig. 3. Relationship (r = 0.815, P = 0.0007) between hepatic benzo[a]pyrene (BaP)-DNA adducts and number of consecutive doses of BaP. Fish were exposed to BaP (30 ~mol/kg body wt.) every 2 days and sampled 2 days after the last exposure. Values are means ± S.E.; n = 4 - 5 .

64 rapid decline followed by no apparent decrease in the levels of remaining adducts. The apparent half-life (11 days) for the decrease in levels of hepatic BaP-DNA adducts in English sole was similar to the half-life (13 - 16 days) in rat of hepatic BaP-DNA adducts detected by 32p-postlabeling [31]. Further, in English sole, the minimal change in levels of hepatic BaP and DBC adducts remaining after the initial decrease is similar to that observed in salmonids exposed to the carcinogen, aflatoxin B1 [16]. A biphasic decrease in adduct levels is also observed for other carcinogens in mammals [32] and may, in part, reflect differences in repair rates among regions of DNA [33]. The persistence of DNA adducts, in addition to the level of binding to DNA in a target tissue, appear to be factors in the relative potency of some carcinogens in mammals [8-11,34,35] and may be a contributing factor in fish as well. In studies with rainbow trout and coho salmon, fish species that are susceptible and relatively non-susceptible, respectively, to the hepatocarcinogenicity of aflatoxin B1, hepatic aflatoxin BrDNA adducts were found to be persistent in both species whereas the level of binding was significantly higher in rainbow trout [16]. Our previous studies [7] have shown that BaP is hepatotoxic and is a putative hepatocarcinogen based on its ability to induce preneoplastic loci in English sole; thus, the greater binding of DBC and similar persistence of the DBC adducts to those of BaP suggest that DBC may be a hepatocarcinogen in English sole as well. The rate of formation of hepatic BaP-DNA adducts in English sole was substantially faster than in another fish species, the brown bullhead. In the present study, English sole were exposed to BaP dissolved in Emulphor/acetone (1:1) via intermuscular injection, whereas the brown bullhead were exposed intraperitoneally to BaP (80 ~mol/kg body wt.) in corn oil [18]. In brown bullhead, analysis by 32P-postlabeling of hepatic BaP-DNA adducts showed a slow increase in adducts over the first 7 days, followed by a more rapid increase to a maximum at 30 days. The level of BaP-DNA adducts then declined to 26% of the maximum level by 70 days post exposure [18]. In contrast, in English sole the BaP-DNA adduct level reached a maximum at 2 days, declined to 32% of the maximum by 28 days post exposure and then did not change significantly out to 84 days. The difference between fish species in the rate of formation of BaP adducts may be due to a slower absorption by brown bullhead of BaP dissolved in corn oil. Previous studies [19] with English sole exposed intraperitoneally to radiolabeled BaP dissolved in corn oil or in acetone showed that the binding at 24 h post exposure for BaP administered in corn oil was nearly 100 times less than when BaP was administered in acetone. Comparison of the results for English sole and brown bullhead also revealed a marked difference in the level of maximum binding. Calculation of DNA adduct levels using a covalent binding index [CBI expressed as (#mol adducts/mol nucleotides)/(mmol BaP/kg body wt.)] [36], which corrects for dosage, showed that the level of total BaP adducts at the maximum was substantially greater in English sole (CBI = 5 at 48 h) than in brown bullhead (CBI = 0.7 at 30 days). However, such a comparison must be made with caution because different routes of exposure and solvent vehicles were used in the two studies. Thus, whether this

65

difference between CBI values for English sole and brown bullhead represents an actual species difference in the binding of BaP to hepatic DNA or is due to a significant removal of adducts concomitant with the apparent slow absorption of BaP in the study with brown bullhead remains to be determined. In our previous initial study [19], no significant differences among BaP-DNA adduct levels measured by 82p-postlabeling were found in English sole sampled at 1, 28, and 60 days after a single treatment with BaP. In the present study, the levels of BaP-DNA adducts were found to exhibit an initial decline from the maximum levels reached at 2 days post exposure followed by no significant change in adduct levels from 28 to 84 days. The difference between the two studies is probably due to the small number of sampling points in the previous study, which precluded an accurate assessment of the decrease in hepatic BaP-DNA adduct levels in English sole. Previously, the anti-BaPDE-dG adduct was shown by 32p-postlabeling to be the major hepatic DNA adduct observed in English sole exposed to low dosages (8-60 ~mol/kg body wt.) of BaP [19]. In contrast, at a higher dosage (400 #mol/kg body wt.) both syn-(60%) and anti-(40%) BaPDE-dG were detected [19]. A recent study [37] using fluorescence line-narrowing spectrometry has confirmed the binding of syn- and anti-BaPDE to hepatic DNA of English sole exposed to BaP. In the present study, at a dosage of 100 ~mol/kg body wt., the synBaPDE-dG was also the major (ca. 70% of the total BaP adducts) BaP-DNA adduct, indicating that between 60 and 100 ~mol BaP/kg body wt., there is an increase in the formation of syn-BaPDE-dG adducts. The major adduct formed in intact organisms and cell cultures of target tissues of mammals is predominantly anti-BaPDE-dG [38,39]; however, a recent study showed that in rat lung, a target tissue, a major adduct detected by 32p-postlabeling appears to arise from metabolism of 9-OH-BaP [31]. In brown bullhead exposed intraperitoneally to BaP, the major adduct detected by ~2p-postlabeling was the anti-BaPDE-dG (77% of total adducts at maximum binding to DNA) [18]. The presence or absence of syn-BaPDE-dG in brown bullhead; however, was not reported. A further difference in the profile of BaP-DNA adducts between English sole and brown bullhead was that in brown bullhead multiple minor adducts were detected that represented -23% of total BaP-DNA adducts at the time of maximum binding, whereas the minor BaP-DNA adducts detected in English sole were present at lower levels (-- 10% of total BaP adducts). Further, in a recent study [40] of the metabolism of BaP in English sole hepatocytes in vitro and in fish in vivo, a minor BaP-DNA adduct, chromatographing near the BaPDE-dG adduct was detected, which was present at substantially lower levels in the present study. These findings suggest that there appears to be speciesdifferences in the formation of minor BaP-DNA adducts in fish and that in English sole there may also be differences among individual fish in the formation of these minor adducts. However, loss of minor adducts during the isolation of DNA and the chromatographic analysis could also account for the differences between studies in the relative amounts of minor adducts. A previous study [41] has shown that 7,12-dimethylbenz[alanthracene-dA adducts are more labile than 7, 12-dimethylbenz[a]anthracene-dG adducts during isolation of DNA.

66

The metabolism of DBC has not yet been examined in fish. Studies in mice and rats have shown that several phenolic metabolites (1 through 6-OH-DBC) are formed during the metabolism of DBC by microsomes from 3-methylcholanthrene-induced animals [42- 44]. The major metabolites formed are the 3- and 5OH-DBC with minor metabolites being the 2-OH-DBC [42] and 1-OH-DBC [44]. The 3,4-dihydroxy-3,4-dihydroDBC was also detected as a minor product [42]. The N-OH-DBC has also been identified as a metabolite formed by 3-methylcholanthrene-induced rat liver microsomes [43]. In rats and mice, cytochrome P4501A isoforms are the predominant cytochromes P450 induced by treatment with 3-methylcholanthrene [45]. Studies with fish have shown that an orthologous cytochrome P4501A enzyme is the inducible P450 in teleost liver [46], which suggests potential similarities in the metabolism of DBC between rodents and fish. The present study is, to our knowledge, the first investigation of the binding of an N-heterocyclic polycyclic aromatic compound to DNA in fish. The profile of DBC adducts in English sole, however, was quite similar to the profile reported for mice exposed to DBC [47]. In both studies, DNA adducts were chromatographed in similar solvent systems for D3 and D4. The hepatic DNA adducts in DBC-exposed mice co-chromatographed with the adducts from liver of mice exposed to the metabolite 3-OH-DBC suggesting that 3-OH-DBC is a proximate hepatocarcinogen. Further, the similarities in adduct profiles between English sole treated with DBC and mice treated with either DBC or 3-OH-DBC suggest that the major hepatic DNA adducts detected in English sole also may arise from 3-OH-DBC, but this remains to be determined. The development of the 32p-postlabeling assay has allowed assessment of the use of xenobiotic-DNA adducts as molecular dosimeters of exposure to environmental genotoxins in mammals [48] and in fish [49]. For fish, a recent study [50] in Puget Sound, WA, showed that the levels of xenobiotic-DNA adducts in English sole detected by 32p-postlabeling were significantly higher in fish from sites near PAH-contaminated urban centers than in fish from sites near sparsely populated areas having low concentrations of anthropogenic chemicals in sediments. These findings, together with the results from the present study showing persistence of DNA adducts of the model carcinogens BaP and DBC and the accumulation of BaP-DNA adducts in English sole given multiple doses, indicates that hepatic xenobiotic-DNA adducts would appear to be molecular dosimeters of relatively longterm exposure to genotoxic environmental contaminants in wild fish species. ACKNOWLEDGEMENTS

We thank Herbert R. Sanborn for assistance in capturing the English sole and Gladys Yanagida for skilled technical assistance with the ~2P-postlabeling analyses. We also are grateful to Mark Myers, Drs. Tracy Collier and Marc Nishimoto, and anonymous referees for their critical review of the manuscript. This work was supported, in part, by the National Oceanic and Atmospheric Administration's Coastal Ocean Program.

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