Covalent Binding of Polycyclic Aromatic Hydrocarbon Components of

Jul 15, 1995 - Components of Manufactured Gas Plant Residue to ... College of Pharmacy, Department of Pharmaceutical Chemistry, Rutgers, The State ...
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Chem. Res. Toxicol. 1996,8, 955-962

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Covalent Binding of Polycyclic Aromatic Hydrocarbon Components of Manufactured Gas Plant Residue to Mouse Lung and Forestomach DNA Eric H. Weyand" and Yun Wu College of Pharmacy, Department of Pharmaceutical Chemistry, Rutgers, The State University of New Jersey, P.O. Box 789, Piscataway, New Jersey 08855-0789 Received January 13, 1995@

The present study characterized the DNA adducts induced by manufactured gas plant residue (MGP) and benzo[alpyrene (B[alP) in mouse lung and forestomach. The dose levels used in the present study were comparable to the levels used in a previous animal bioassay. Adduct formation was evaluated in female A/J mice (7weeks old) fed MGP (0.25%) or B[alP (16 and 98 ppm) for 14 days. In addition, adduct formation was also evaluated in mice 24 h after the ip administration of 1.8 mg of B[alP in 0.5 mL of tricaprylin. 32P-Postlabelingcombined with multidimensional TLC and reverse phase HPLC was used to evaluate hydrocarbon-DNA adducts. HPLC separation of chemical-DNA adducts formed in lung following MGP ingestion resulted in three distinct peaks of radioactivity eluting at 22,32.4,and 33.5min. These peaks accounted for 13, 10, and 41% of the total adducts detected. The adducts isolated from forestomach eluted as a series of minor peaks with two more distinct peaks of radioactivity a t 32.4 and 33.5 min. These peaks accounted for 47 and 32% of the total adducts detected in forestomach, respectively. Ingestion of B[ulP (16or 98 ppm) and the ip administration of B[alP resulted in a single major adduct with a retention time of 32.4 min. The DNA adducts formed from MGP administration were further characterized by comparison with adducts formed following the administration of individual hydrocarbons and a mixture of hydrocarbons. Comparison of adduct retention times formed by MGP and pure hydrocarbons indicates that two of the three adducts formed in lung (22 and 32.4 min peaks) are derived from benzo[blfluoranthene and B[alP, respectively. However, the major adduct in lung could not be attributed to any of the PAH identified as constituents of MGP. In contrast, B[alP is responsible for the major adduct formed in forestomach. These results indicate that several PAHs may play a role in the mechanism by which MGP induces tumors in mouse lung.

Introduction Humans are exposed to a number of complex organic mixtures that have been widely implicated in human carcinogenesis(1,2). Mixtures that are primarily derived from incomplete combustion processes often contain high levels of polycyclic aromatic hydrocarbons (PAHs)l(3,4). It is generally assumed that the four- to six-ring hydrocarbon components are responsible for the biological activity of these types of complex mixtures. Although a lot is known about the mechanism by which individual hydrocarbons initiate carcinogenesis, very little is known about the mechanism when these hydrocarbons are constituents of complex mixtures. A critical step in PAH carcinogenesis is metabolic activation and binding to cellular DNA. The development of the 32P-postlabelingtechnique for investigating chemical-DNA adduct formation has greatly advanced our understanding of the mechanism of DNA adduct formation, particularly with individual PAHs. More recently, the development of HPLC separation procedures for the 32P-postlabelingtechnique has provided investigators with the ability to better evaluate hydrocarbons

* To whom correspondence and requests for reprints should be addressed. @Abstractpublished in Advance ACS Abstracts, July 15, 1995. 1 Abbreviations: B[alP, benzo[alpyrene; B[blF, benzo[blfluoranthene; MGP, manufactured gas plant residue; PAH, polycyclic aromatic hydrocarbons, PEI-cellulose, poly(ethy1enimine) cellulose.

responsible for the genotoxicity of complex mixtures. Hughes et al. (5) evaluated chemical-DNA adduct formation in mouse skin following the topical administration of pharmaceutical grade coal tar. King and co-workers (6)used 32P-postlabelingand HPLC analysis to separate PAH and nitro-PAH DNA adducts formed from organic extracts of diesel emission particles. The emphasis of these and other related studies (7, 8) has been on identifying constituents that are most responsible for the genotoxicity induced by complex mixtures. It is anticipated that this approach will help to identify hydrocarbons that play a major role in the tumorigenicity exhibited by complex mixtures. In addition, once characterized, the presence and levels of these hydrocarbons may potentially serve as markers for better estimating the carcinogenic potential of similar complex mixtures as well as be useful in developing more practical means in determining human risk associated with exposure to complex mixtures. The objective of the present study was to characterize the chemical-DNA adducts formed from manufactured gas plant residue (MGP), a complex mixture previously determined to be tumorigenic in AIJ mice. Since the ingestion of MGP only induced tumors in mouse lung, the DNA adducts formed in lung were compared with those formed in forestomach tissue, a site not responsive to tumor induction by MGP. In addition, the chemicalDNA adducts formed with MGP were compared with

Q893-228x/95/27Q8-Q955$09.00/00 1995 American Chemical Society

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Table 1. Composition of MGP Adulterated Diets and PAH Mixtures

compound indan naphthalene 2-methylnaphthalene 1-methylnaphthalene acenaphthylene acenaphthene dibenzofuran fluorene phenanthrene anthracene fluoranthene pyrene benz[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[alpyrene indeno[l,2,3-cdlpyrene dibenz[a,h]anthracene benzo[ghilperylene total amount

manufacture gas plant residue MGP" mgkg dietb mgkg ingestedc of MGP of diet (mgimouse) 490 32300 10700 5660 5710 1270 1810 4770 10100 2900 6370 7220 3340 2960 2890 1010 2760 1990 370 2290

0.47 31.0 10.3 5.4 5.5 1.2 1.7 4.6 9.7 2.8 6.1 6.9 3.2 2.8 2.8 0.97 2.6 1.9 0.36 2.2

0.05 2.97 0.99 0.52 0.53 0.11 0.16 0.44 0.93 0.27 0.58 0.66 0.31 0.27 0.27 0.09 0.25 0.18 0.03 0.21 9.82

polycyclic aromatic hydrocarbons dietd (mgkg ingested' injectedf of diet) (mg/mouse) (mgimouse)

29.8 13.7 12.2 11.9 4.1 11.4 8.2 1.6 9.5

4.8 2.2 2.0 1.9 0.7 1.9 1.3 0.3 1.5 16.3

0.58 0.27 0.24 0.23 0.08 0.22 0.16 0.03 0.18 1.99

a Amount of each hydrocarbon in neat MGP. Amount of each hydrocarbon in a 0.258 MGP-adulterated basal gel diet. Amount of each hydrocarbon ingested during 14 days of MGP diet administration. Amount of each hydrocarbon incorporated into a basal gel diet (PAH mixture). e Amount of each hydrocarbon ingested by mice after 14 days of consuming a diet containing a mixture of pure PAH. f Amount of each hydrocarbon administered to mice in 0.5 mL of corn oil by ip injection.

adducts formed by individual and mixtures of PAH in order to determine which hydrocarbons are responsible for the DNA binding observed with MGP.

Materials and Methods Chemicals. Caution: The hazards of manufactured gas plant residue have not been fully evaluated. Hence, protective clothing a n d appropriate safety procedures should be followed when working with this material. Manufactured gas plant residue (MGP) was supplied by the Electric Power Research Institute (Palo Alto, CAI. This MGP was the same sample evaluated for tumorigenic activity in the previously described NJ mouse bioassay (9). The chemical composition of this MGP sample has been previously reported (10) and is partially summarized in Table 1. The F0927 basal gel diet was purchased from Bio-Serv, Inc. (Frenchtown, NJ). Tricaprylin (99% pure) was purchased from Sigma Chemical Co. (St. Louis, MO). Pyrene, benz[alanthracene, chrysene, benzo[blfluoranthen (B[b]F or benz[elacephenanthrylene),benzo[alpyrene (B[a]P), dibenz[a,h]anthracene, and benzo[ghilpyrylene were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). Benzo[k]fluoranthene and indeno[l,2,3-cdlpyrene were purchased from the Commission of the European Communities Bureau of Reference (Brussels, Belgium). Adulterated Diets. Adulterated basal gel diets were prepared as previously described (11). In the case of MGPadulterated diets, the amount of MGP incorporated was based on amount of dry food used in preparing diets. Thus, a 0.25% MGP-adulterated diet contained 4.87 g of MGP/1948 g of dry food wt (gel diet total wt, 5068 g), respectively. The level of B[a]P in a 0.25% MGP diet is approximately 7 ppm. In the case of B[alP adulterated diets, the amount incorporated was 31 and 190 mg/1948 g of dry food wt for 16 or 98 ppm B[alP diets, respectively. A gel diet containing nine-hydrocarbons was also prepared by dissolving pyrene (30.2 mg), benz[alanthracene (13.9 mg), chrysene (12.4 mg) B[b]F (12.1 mg), benzo[klfluoranthene (4.2 mg), B[alP (11.6 mg), indeno[l,2,3-cdlpyrene (8.3 mg), dibenz[a,h]anthracene (1.6 mg), and benzo[ghilperylene (9.6 mg) in a small volume of acetone and incorporating this solution into a basal gel diet (22 g of agar, 604 g of water, and 389 g of food for a total gel diet weight of 1015 g).

Animal Treatment. Female NJ mice 6 weeks old were obtained from Jackson Laboratories (Bar Harbor, ME) and were allowed to acclimate for 1 week. Animals were housed in solid polycarbonate Micro-Isolator cages (Lab Products, Inc., Maywood, NJ) with hardwood bedding from Beta-Chip North Eastern Products (Warrenburg, N Y ) . Mice were housed under controlled conditions with a 12-h light-dark cycle and given food and water ad libitum. At 7 weeks of age mice were divided into five groups of 10 each. Animals in groups 1-3 were fed basal gel diets containing 0.25% MGP, 16 ppm B[a]P, or 98 ppm B[alP, respectively. Animals in group 4 were fed unadulterated control basal gel diet while animals in group 5 were administered B[a]P (100 mgkg) by ip injection in 0.25 mL of tricaprylin. Animals receiving basal gel diets (groups 1-4) were killed after 14 days of diet administration. Animals receiving B[alP by ip administration (group 5 ) were killed 24 h after dose administration. Lung and forestomach of animals were quickly removed, pooled according to group, and DNA was isolated using standard procedures (12, 13). Preparation of DNA Adducts from Pure PAHs. Preliminary experiments demonstrated that female NJ and female CD-1 mice formed similar types of chemical-DNA adducts in lung and forestomach following MGP ingestion. In addition, our extensive previous investigations used CD-1 mice to characterize the 32P-postlabeled PAH-DNA adducts from many individual PAHs. Therefore, CD-1 mice were used in these studies to further characterize the adducts formed from MGP. Female CD-1 mice (18-19 g) were obtained from Charles River Breeding Laboratories, Inc. (Kingston, NY). Groups of female CD-1 mice (five to six mice) were fed basal gel diets containing 0.25% MGP or a mixture of nine-hydrocarbons (see Table 1). Animals were killed after 14 days of diet administration. Lung and forestomach were pooled according to groups, and DNA was isolated. In addition to feeding animals the ninehydrocarbon mixture, mice (three to four animals) were also treated with 2 mg total of the nine-hydrocarbon mixture (see Table 1)by ip injection. Animals receiving ip injections were killed 24 h after dose administration, and DNA was isolated from lung and forestomach tissue. Chemical-DNA Adduct Analysis. DNA isolated from lung and forestomach tissue was 32P-postlabeled and analyzed by multidimensional TLC and HPLC as previously described (1416). In brief, 10 ,ug of each DNA sample was hydrolyzed, 32P-

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DNA Binding of Chemical Components of MGP postlabeled, and subjected to five-dimensional poly(ethy1enimine) cellulose (PEI-cellulose) TLC using standard procedures. DNA adducts were visualized by autoradiography and subsequently removed from the PEI-cellulose by extracting three times with 0.7 mL of 4 M pyridinium formate (pH 4.5). In general, pyridinium formate extracts from a total of three to four TLC maps were combined for HPLC analysis. Samples were evaporated to dryness under vacuum, and residues were redissolved in 100 pL of methanol-0.3 M sodium phosphate, pH 2.0 (1:l vol/vol). Analysis of hydrocarbon-modified 32Ppostlabeled 3’,5’-bisphosphate nucleotides by HPLC were performed with a Hewlett-Packard Model 1050 system equipped with a ,%RAM ( I N N S Systems, Inc.) flow-throughmonitor using a 2.74-mL liquid cell. Separations performed with a Zorbax SBPhenyl reverse phase C18 column were carried out with a flow rate of 1m u m i n using a sodium phosphate-methanol gradient system. Initial solvent conditions were 100% 0.3 M sodium phosphate (pH 2.0) with a linear gradient to 30% methanol in 10 min, a linear gradient to 45% methanol over 38 min, which was followed by a linear gradient to 50% methanol in 7 min, and then a final linear gradient to 100% methanol over 15 min.

Results The genotoxicity induced by B[alP and MGP was evaluated in female NJ mice using a dosing protocol similar to that used to evaluate their tumorigenicity following ingestion. Animals were fed adulterated diets for 14 days, and the level of DNA adducts formed in lung and forestomach was determined using 32P-postlabeling and multidimensional PEI-cellulose TLC analysis. MGP ingestion resulted in a diagonal band of 32P-labeled adducts that contained concentrated spots of radioactivity within the band (maps not shown). Adduct patterns were typical of those commonly observed with this and other complex mixtures (5, 7, IO). In contrast, the ingestion of B[alP at 16 or 98 ppm for 14 days or 24 h after an ip dose of B[a]P (1.8 mg) resulted in a single major adduct. This adduct had a chromatographic mobility similar to the B[a]P diol epoxide-deoxyguanosine adduct. Adduct levels were highest in lung of animals ingesting either a 0.25% MGP diet or treated with B[a]P by ip administration (Figure 1). In both cases, lung adduct levels were considerably greater than the levels detected in forestomach of the same animal. In contrast, adduct levels in animals ingesting a 16 or 98 ppm B[alP diet resulted in a low level of DNA adducts (in both lung and forestomach) relative to that observed with MGP ingestion and B[a]P ip administration. The adducts formed in female NJ mice following MGP ingestion were collectively removed from PEI-cellulose TLC plates and further evaluated using reverse phase HPLC. Peaks of radioactivity eluting after 18 min were considered hydrocarbon-DNA adducts while earlier eluting radioactivity was considered 32P-labeled residual normal nucleotides. The DNA adducts isolated from lung were resolved into a large number of small peaks and three predominate peaks of radioactivity with a retention time of 22,32.4, and 33.5 min (Figure 2A). The predominate peaks accounted for 13, 10, and 41% of the total radioactivity eluting between 19 and 60 min, respectively. The large number of smaller adduct peaks accounted for 36% of the total radioactivity associated with adducts. The DNA adducts isolated from forestomach eluted as two predominate peaks of radioactivity with retention times of 32.4 and 33.5 min (Figure 2B). These peaks accounted for 47 and 32% of the radioactivity eluting between 19 and 60 min, respectively. The small peaks of radioactivity in this profile eluted between 18 and 32

3.0

F.S.

Lung

Figure 1. Chemical-DNA adduct levels in the forestomach (F.S.) and lungs of animals ingesting 16 ppm B[alP, 98 ppm B[a]P or 0.25% MGP. Female A/J mice were maintained on diets for 14 days. Animals receiving B[a]P (1.8mg) by ip administration were killed 24 h after dose administration. DNA adduct levels were determined by 32P-labeling and multidimensional TLC as described in Materials and Methods. Values are the mean f SE for three to five determinations.

min and accounted for 21% of the radioactivity associated with adducts. HPLC analysis of adducts formed following BCaIP administration was limited to animals receiving B[alP by ip administration. A single major adduct eluting at 32.4 min (Figure 3A) was detected in both lung and forestomach of animals dosed with BCalP. When cochromatographed on HPLC, the retention time of the DNA adduct formed in lung following B[alP ip administration was identical with the retention time of the 32.4 min MGP DNA adduct (Figure 3B). Thus, one of the minor DNA adducts in lung along with the predominate adduct formed in forestomach following MGP ingestion appears to be derived from B[alP. On the basis of its chromatographic retention on HPLC this peak is presumed to be the B[alP diol epoxide-deoxyguanosine adduct. In order to further investigate the nature of the adducts induced by MGP ingestion, additional feeding studies were conducted using CD-1 mice. A diagonal band of 32P-postlabeledadducts with concentrated spots of radioactivity within the band was observed (Figure 4, maps A and B) when separated by multidimensional PEIcellulose TLC. Adduct levels were highest in lung, which is similar to that observed with A/J mice. A total of 1.28 and 0.77 pmol of addudmg of DNA were detected in lung and forestomach of animals ingesting a 0.25% MGP diet, respectively (Figure 5). HPLC analysis of the DNA adducts isolated from lung and forestomach of CD-1 mice ingesting a 0.25% MGP diet were similar to those observed with A/J mice (Figure 6A,B). Peaks eluting at 22, 32.4, and 33.5 min accounted for 11, 10, and 53% of the radioactivity attributed to lung DNA adducts, respectively. In the case of forestomach tissue, peaks eluting at 26, 32.4, and 33.5 min accounted for 29, 49, and 22% of the radioactivity associated with adducts. In addition to feeding MGP, a diet containing a mixture of nine pure hydrocarbons (pyrene through benzo[ghilperylene; see Table 1)was also fed to CD-1 mice for 14 days. The multidimensional PEI-cellulose analysis of DNA isolated from lung resulted in a complex adduct pattern while DNA from forestomach resulted in two

958 Chem. Res. Toxicol., Vol. 8, No. 7, 1995 65'

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Figure 2. HPLC profiles of 32P-labeled DNA adducts isolated from the lungs (profile A) and forestomachs (profile B) of female NJ mice ingesting a 0.25% MGP diet for 14 days. 32P-Labeled adducts were subjected to five-dimensional PEI-cellulose TLC prior to HPLC analysis as described in Materials and Methods.

distinct adduct spots (Figure 4, maps C and D). HPLC analysis of the DNA adducts isolated from the lungs of these animals separated into a single major peak of radioactivity with several minor peaks. The major radioactive peak had a retention of 22 min while one of the minor peaks had a retention time of 32.4 min (Figure 6C). The retention times of these adducts were similar to the two minor peaks observed in lungs of mice ingesting MGP. However, the relative amount of each adduct was considerably different. The 22 min adduct accounted for 48% of the adducts formed while the 32.4 min adduct only accounted for 9%. Interestingly, the adduct peak at 33.5 min was not present in the adducts isolated from lungs of animals ingesting the ninehydrocarbon mixture. The adducts isolated from forestomach of these animals resolved into two distinct adduct peaks (26 and 32.4 min), which accounted for 59 and 40% of the radioactivity attributed to DNA adducts. These results suggest that the adducts with a retention time of 26 and 32.4 min are derived from hydrocarbons present in the PAH mixture. This is consistent with the cochromatography data identifying the 32.4 min peak as a B[alP-DNA adduct. Likewise, the lack of a peak at 33.5 min may also indicate that this adduct is not derived

Time(min)

Figure 3. HPLC profiles of 32P-labeled DNA adducts isolated from female A/J mice. Profile A represents adducts isolated from lung of mice 24 h after the ip administration of B[alP. Profile B represents the cochromatography of adducts isolated from the lungs of animals injected with B[alP and adducts isolated from the lungs of animals ingesting MGP for 14 days (see Figure 2, profile A). 32P-Labeled adducts were subjected to five-dimensional PEI-cellulose TLC prior t o HPLC analysis as described in Materials and Methods. from the hydrocarbons present in the PAH mixture. An additional feeding study was also conducted with a diet that contained all of the hydrocarbons listed in Table 1 (indan to benzo[ghilperylene). Ingestion of this diet by animals resulted in adduct profiles identical to those observed with the nine-hydrocarbon mixture (data not presented). Thus, the two- to three-ring hydrocarbons appear to have little effect on the DNA adducts formed from the four- to six-ring hydrocarbon mixture. In order to further characterize the 22 min adduct, each of the nine-hydrocarbons was administered individually to female CD-1 mice by ip administration and adduct formation in lung was evaluated after 24 h. Interestingly, B[blF was the only hydrocarbon that formed an adduct with a retention time of 22 min (data not shown). DNA adduct formation in lung was also evaluated 24 h after the ip administration of the ninehydrocarbon mixture and with mixtures that did not contain either B[b]F o r B[alP. The complete ninehydrocarbon mixture resulted in two major adducts eluting a t 22 and 32.4 min (Figure 7A). These adducts had retention times identical to two of the adducts

DNA Binding of Chemical Components of MGP

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detected (Figure 7C). Thus, on the basis of this observation and the chromatographic retention of this adduct on HPLC, this peak is presumed to be derived from trans9,10-dihyroxy-anti-l l 12-epoxy-5-hydroxy-9,10,11,12-tetrahydrobenzo[b]fluoranthene (16). As expected, when B[a]P was removed from the mixture, the 22 min adduct was retained while the 32.4min adduct was not detected (Figure 7B). Again this indicates that the 32.4 min adduct is derived from B[a]P.

Discussion Our previous investigation demonstrated that significant differences exist in the target organ selectivity and tumorigenic potential of MGP and B[a]P. MGP ingestion induced only lung tumors while B[a]P ingestion induced primarily forestomach tumors in female A/J mice. Since tumor induction by PAHs involves the binding of a reactive metabolite to DNA, it is reasonable to anticipate that characterizing the hydrocarbon-DNA adducts formed will help to identify constituents that play a role in the tumorigenic activity of MGP. Figure 4. PEI-cellulose TLC maps of chemical-DNA adducts formed in tissues of female CD-1 mice following 14 days of ingesting gel diets adulterated with 0.25% MGP or a mixture of nine PAHs (see Table 1).Samples were subjected to 32Ppostlabeling analysis as described in Materials and Methods. Maps A and B represent DNA isolated from the lungs and forestomachs of animals ingesting MGP, respectively. Maps C and D represent DNA isolated from the lungs and forestomachs of animals ingesting a mixture of nine PAHs, respectively. Autoradiography was performed at -80 "C for 20 and 72 h for lung and forestomach samples, respectively. The origin is located at the bottom left-hand corner of each map and was excised prior to autoradiography. D3 solvent development was in the direction from left to right while D4 solvent development was in the direction from bottom to top. 2

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Figure 5. Chemical-DNA adduct levels in the forestomachs (F.S.) and lungs of animals ingesting 0.25% MGP or a mixture of nine PAHs (see Table 1).Female CD-1 mice were maintained on diets for 14 days. DNA adduct levels were determined by 32P-labelingand multidimensional TLC as described in Materials and Methods. Values are the mean f SE for four to six determinations.

induced by MGP (Figure 6A,B, Figure 2). A series of minor adducts were also present between 21 and 34 min. When B[b]F was removed from the mixture, the 32.4min adduct was retained while the 22 min adduct was not

The present study clearly demonstrated that MGP ingestion by mice results in significant levels of three different chemical-DNA adducts in lung. Two of these three adducts, accounting for 10 and 13% of the total adducts formed in lung, are derived from B[a]P and B[b]F, respectively. However, the major DNA adduct in lung, accounting for 41% of the total, could not be attributed to any of the PAHs identified as constituents of MGP. In contrast, B[a]P is responsible for the major adduct formed in forestomach following MGP ingestion. This adduct accounted for 47% of the total adducts formed in forestomach. The unidentified adduct observed in lung was also evident in forestomach. This unidentified adduct accounted for 32% of the total adducts in forestomach. Culp and Beland (17)have also evaluated chemicalDNA adduct formation in mice fed MGP or B[alP. These investigators also used HPLC to further characterize the DNA adducts formed in lung following MGP ingestion. However, their analysis was limited to adducts that had a chromatographic mobility similar to B[alP diol epoxide-deoxyguanosine when separated on PEI-cellulose TLC plates. Thus, a direct comparison of their HPLC results with the present study is problematic since our analysis was performed using all of the adducts detected on PEI-cellulose TLC. Both studies do indicate, however, that B[a]P is partially responsible for chemical-DNA adducts formed in lung following MGP ingestion. In addition, the tissue-specific differences in the ability of MGP constituents and B[a]P to form DNA adducts in lung is apparent in both investigations and is consistent with previous studies (10,18). Other studies have evaluated the DNA binding of chemical components of complex mixtures such as coal tar, creosote, used motor oil, and diesel exhaust following topical administration (5,7,19). These previous investigations, however, were limited primarily to assessing the overall level of chemical binding to DNA. Mukhtar and co-workers (20) evaluated the formation of B[a]P-

960 Chem. Res. Toxicol., Vol. 8, No. 7, 1995

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Figure 6. HPLC profiles of 32P-labeledDNA adducts isolated from CD-1 ingesting a 0.25% MGP diet or a diet adulterated with a mixture of nine-hydrocarbons (see Table 1).Diets were fed to animals for a total of 14 days and adducts were isolated, 32P-postlabeled, and subjected to five-dimensionalPEI-celluloseTLC prior to HPLC analysis as described in Materials and Methods. Profiles A and B represent adducts isolated from the lungs and forestomachs of CD-1 mice ingesting a 0.25% MGP diet for 14 days, respectively. Profiles C and D represent adducts isolated from the lungs and forestomachs of CD-1 mice ingesting a diet containing a mixture of

nine-hydrocarbonsfor 14 days, respectively.

diol epoxide DNA adducts following the topical application of therapeutic grade coal tar. B[alP-diol epoxide DNA adducts were greater in lung than skin. These investigators concluded that the lower level of B[alP DNA adducts observed in skin is likely due to the presence of inhibitors of carcinogen DNA adduct formation that may be present in coal tar. Likewise, Springer and co-workers (21) also demonstrated that organic mixtures had an inhibiting effect on B[a]P adduct formation as well as altering the types of B[alP DNA adducts formed. These investigators also demonstrated that BEaIP-diol epoxide DNA adducts were the predominate adducts formed in mouse skin following the topical administration of a complex organic mixture. In the present study, the amount of chemical-DNA adduct formation that could be attribute to BbIP (based on HPLC analysis and total level of adducts formed in each tissue) was significantly lower in forestomach than lung. Thus, it is reasonable to speculate that inhibitors of carcinogen DNA adduct formation are present in MGP and may account for the lower level of DNA adducts (total as well as B[alP-DNA adducts) observed in forestomach. On the other hand, the high levels of DNA adducts observed in lung suggest that inhibitors of carcinogen DNA adduct formation have little o r no effect in tissues that are distant from the site of MGP administration. The pharmacokinetic properties of both anticarcinogens and carcinogenic PAHs present

in MGP are likely responsible for the observed tissuespecific differences in levels and types of chemical-DNA adducts formed. In an effort to identify hydrocarbons that play a major role in the carcinogenic activity of complex mixtures, recent studies have used HPLC methods to characterize hydrocarbon-DNA adducts. Hughes et al. (5)characterized hydrocarbon-DNA adducts formed from pharmaceutical grade coal tar following the topical application to mouse skin. These investigators concluded that B[alP plays a major role in the formation of DNA adducts in mouse skin. They also conclude that B[b]F, benzoulfluoranthene, benzo[klfluoranthene, and benzokhilperylene contribute to DNA adducts induced by pharmaceutical grade coal tar. The topical administration of MGP has been shown to result in one major adduct in mouse skin (21). On the basis of its chromatographic mobility on HPLC, this adduct was presumed to be B[a]P-diol epoxide-deoxyguanosine. In the present study, the types of adducts formed in forestomach following MGP ingestion are similar to the adduct patterns observed in mouse skin following the topical administration of MGP. That is, B[alP appears to be responsible for the predominate DNA adduct formed in tissues that come in direct contact with complex mixtures such as pharmaceutical grade coal tar and MGP. However, this is in contrast to the types of adducts formed in lung, a tissue that is not in direct contact with MGP. It appears

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PAHs, B[b]F has been shown to bind extensively to mouse globin and serum proteins (22). The present study has demonstrated significant differences in the extent and types of c1,emical-DNA adducts formed in mouse lung and forestomach following MGP ingestion. The importance of these differences in the tumorigenic activity exhibited by MGP is not fully understood. However, the formation of significant levels of three different chemical-DNA adducts in lung suggests that several PAHs play a role in the mechanism by which MGP induces tumors in mouse lung. Studies are in progress to identify the major chemical-DNA adduct formed in lung following MGP ingestion.

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Acknowledgment. This work was supported by the Electric Power Research Institute Contract RP 2963-1.

PAH mixture without BIP

References

C PAH mixture without BbF

Time(min)

Figure 7. HPLC profiles of 32P-labeled DNA adducts isolated from the lungs of CD-1 mice injected with a mixture of hydrocarbons (see Table 1).Profile A represents adducts isolated from animals treated with the nine-hydrocarbonmixture. Profile B represents adducts isolated from the lungs of animals treated with the hydrocarbon mixture that did not contain B[alP. Profile C represents adducts isolated from the lungs of animals injected with the hydrocarbon mixture that did not contain B[blF. Animals were killed 24 h after dose administration and adducts were isolated, 32P-postlabeled,and subjected to five-dimensional PEI-cellulose TLC prior to HPLC analysis as described in Materials and Methods.

that B[alP is not responsible for the major DNA adduct induced by MGP in mouse lung. However, both B[alP and B[blF do contribute significantly to DNA adduct formation in lung following MGP ingestion. The involvement of B[b]F in DNA binding in lung may be related to its unique mechanism of activation involving the formation of a phenolic diol epoxide. In comparison to other

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