7H-benzo[c]fluorene: a major DNA adduct-forming component of coal tar

Aruna Koganti, Renu Singh, Kimberly Rozett, Nehal Modi, Lawrence S. Goldstein1, Tim A. Roy2, Fang Jie Zhang3, Ronald G. Harvey3 and Eric H. Weyand4

Rutgers, The State University of New Jersey, College of Pharmacy, Department of Pharmaceutical Chemistry, 160 Frelinghuysen Road, Piscataway, NJ 08854-8020,
1 Environment Sector, Electric Power Research Institute, 3412 Hillview Avenue, PO Box 10412, Palo Alto, CA 94303,
2 Petrotec Inc., 527 East Ravine Avenue, Langhorn, PA 19047 and
3 Ben May Institute for Cancer Research, University of Chicago, 5841 South Maryland Avenue, MC 6027, Chicago, IL 60637, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Coal tar is a complex mixture that exhibits high carcinogenic potency in lungs of animals when administered in the diet. Studies have noted that lung tumor induction does not correlate with the benzo[a]pyrene content of coal tar, suggesting that other hydrocarbons may be involved in the observed tumorigenicity. Our previous studies have demonstrated that a major `unknown' chemical–DNA adduct is formed in the lung of mice exposed to coal tar. We have used an in vitro rat microsomal activation system to generate the `unknown' adduct with neat coal tar and fractions of coal tar obtained by chemical fractionation and HPLC. Chemical–DNA adduct formation was evaluated by 32P-postlabeling using both multi-dimensional TLC and HPLC. GC–MS analysis of the coal tar fractions obtained from HPLC, which produced the `unknown' adduct in vitro, demonstrated that the adducting hydrocarbon had a mass of 216. A careful evaluation of candidate hydrocarbons led to the conclusion that a benzofluorene derivative may be responsible for forming the `unknown' chemical–DNA adduct. Comparative in vitro and in vivo studies on the adducting properties of all three isomers of benzofluorene indicated that 7H-benzo[c]fluorene is responsible for producing the `unknown' adduct observed in the lung of mice ingesting coal tar. Animal feeding studies also demonstrated that 7H-benzo[c]fluorene formed considerably more lung DNA adducts than 11H-benzo[a]fluorene and 11H-benzo[b]fluorene. These data indicate that the four-ring polycyclic aromatic hydrocarbon 7H-benzo[c]fluorene, a hydrocarbon not previously shown to form DNA adducts in lung, is in fact a potent lung DNA adductor and is a candidate PAH for causing lung tumors in animals treated with coal tar.

Abbreviations: B[a]P, benzo[a]pyrene; DMSO, dimethylsulfoxide; M-3, mix of 3; PAH, polycyclic aromatic hydrocarbons; PEI, polyethyleneimine.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Coal tars, by-products of incomplete combustion of organic material, consist of complex mixtures of aliphatic and polycyclic aromatic hydrocarbons (PAHs), phenols, heterocyclic oxygen-, nitrogen- and sulfur-containing compounds, volatile aromatics, inorganics and several trace metals (1). PAHs are believed to be major carcinogenic components of coal tars.

Studies have evaluated the carcinogenic potential of coal tars from a few representative coal gasification sites. Weyand and co-workers demonstrated high lung tumor incidences when coal tar was administered in the diet of female A/J mice for 260 days (2). A small, but not statistically significant, increase in lung tumors was found when benzo[a]pyrene (B[a]P) was ingested. In addition, the grade and multiplicity of tumors formed with coal tar were considerably higher compared with the tumors induced by B[a]P alone. In an infant mouse model system, Rodriguez et al. (3) demonstrated liver tumors after a single exposure of male B6C3F1 mice to coal tars. More recently, a 2 year toxicological bioassay with female B6C3F1 mice maintained on diets containing coal tar found an increased incidence of lung tumors in mice ingesting coal tars but no increase in lung tumors in mice ingesting only B[a]P (4). These results indicate that coal tars are highly selective for inducing lung tumors and that the observed differences between coal tars and B[a]P may be due to the collective effect of the mixture and/or chemicals other than B[a]P within coal tar.

Studies have attempted to predict the carcinogenic potency of a few coal tars from the known toxicological properties of constitutive PAH found in these coal tars (5). These studies indicate that the carcinogenic potency of coal tars is much higher compared with the potency of B[a]P alone or the added potencies of all of the known carcinogenic PAHs within the mixture. There are at least two possible explanations for the observed differences in the empirical and predicted carcinogenicity of mixtures: (i) unidentified carcinogenic PAHs are present within the coal tars and/or (ii) known carcinogens are synergistically activated by other compounds present within the mixture. Coal tars contain several thousand compounds of which only a few hundred have been identified. Thus, it is reasonable to expect that all of the carcinogenic PAHs have not been identified. Likewise, several studies have shown that B[a]P biological activity is modified in the presence of other compounds (6,7). Studies have also shown that non-carcinogenic PAHs (less than four-ring compounds) present in coal tars are potent inducers of cytochrome P450 enzyme systems involved in the activation and detoxification of carcinogenic PAHs (8,9). Therefore, non-carcinogenic compounds can modulate the biological effects of carcinogenic PAHs within complex mixtures.

The lung has also been shown to be a target organ for chemical–DNA adduct formation by coal tars (10). Culp and co-workers demonstrated a dose-dependent increase in both coal tar-induced total lung DNA adducts and tumorigenicity in B6C3F1 mice (11). More recently, Culp and co-workers evaluated DNA adduct formation, cell proliferation and tumor mutation induction in mice fed coal tar or B[a]P. These investigators concluded that the coal tar-induced lung tumors were likely derived from a component in coal tar other than B[a]P (12). Since characterization of lung chemical–DNA adducts could lead to the identification of new lung tumor-inducing agents present in coal tars, studies have attempted to characterize DNA adducts formed in lungs of mice treated with coal tars (13). Three major chemical–DNA adducts were detected. Two of the lung DNA adducts were determined to be derived from benzo[b]fluoranthene and B[a]P by virtue of identical chromatographic properties when compared with adduct standards. The third adduct, which was predominant, could not be identified by comparison with known PAH–DNA adducts. The predominance of the `unknown' adduct along with the inability to fully account for the tumorigenicity of coal tar suggests an important role for the chemical component responsible for forming the `unknown' adduct in the ultimate biological effects of coal tar in vivo.

In the present study, the types of chemical–DNA adducts formed in an in vitro metabolic activation system from coal tar and isolated fractions of coal tar were evaluated to gain insight into the hydrocarbon responsible for forming the major `unknown' chemical–DNA adduct previously observed in mouse lung. The responsible hydrocarbon was identified by GC–MS analysis of coal tar fractions, followed by in vitro activation of candidate hydrocarbons to form the `unknown' adduct. The ability of this newly identified hydrocarbon to form DNA adducts in vivo was verified by feeding it to mice and evaluating the chromatographic properties of adducts on TLC and HPLC. Our data indicate that the four-ring PAH 7H-benzo[c]fluorene, a hydrocarbon not previously shown to form DNA adducts in lung, is in fact a potent lung DNA adductor. 7H-benzo[c]fluorene is therefore a candidate PAH for causing lung tumors in animals treated with coal tar.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Coal tar derived from coal gasification processes and polycyclic aromatic hydrocarbons described within this paper have been determined to be carcinogenic to laboratory animals. Hence, protective clothing and appropriate safety procedures should be followed when working with this material. The Electric Power Research Institute collected coal tars from different coal gasification sites. Tar samples were labeled sites 1–9. Equal volumes of tars collected from sites 5, 7 and 9 were combined to make a generic sample termed the mix of 3 (M-3). A detailed chemical analysis of the coal tars and mixtures has been previously published (14). The carcinogenic potential of M-3 has been previously demonstrated (4). All experiments described in this paper were performed with M-3 tar. Benzo[a]pyrene was purchased from Sigma Chemical Co. (St Louis, MO). 11H-benzo[a]fluorene and 11H-benzo[b]fluorene with purities of >=98% were obtained from Fluka Chemical Co. (Ronkonkoma, NY). 7H-benzo[c]fluorene was synthesized by the method previously reported and purity was >=98% as determined by HPLC analysis (15). The nomenclature employed in this paper for naming PAHs is in accordance with the IUPAC rules used by Chemical Abstracts and recommended by the American Chemical Society. These rules have been previously reviewed (16).

Microsome preparation
Coal tar (2 g) was extracted with dimethylsulfoxide (DMSO) (9.5 ml) by vortexing intermittently over 3 days. The sample was centrifuged at 4000 g at room temperature for 10 min and the DMSO supernatant fraction was collected. Two Sprague–Dawley rats (250–300 g) were administered 0.5 ml of the DMSO fraction daily by i.p. injection for a total of 3 days. Rats were killed by decapitation 24 h after the last injection and livers were quickly excised, rinsed in cold 1.15% KCl, weighed and homogenized with a Brinkman Polytron in 2–3 vol (w/v) of 0.05 M Tris–HCl, pH 7.1, containing 1.15% KCl. Homogenates were centrifuged at 11 000 g for 20 min at 4°C. The supernatant was decanted and further centrifuged at 100 000 g for 90 min at 4°C. The pellet containing the microsomal fraction was resuspended in a buffer containing 1.15% KCl and 10 mM EDTA (pH 7.0–7.5) and once again centrifuged at 100 000 g for 60 min at 4°C. The microsomal pellet was then resuspended in a minimal volume of cold 0.25 M sucrose containing 0.1 mM EDTA. Protein content was determined using the Lowry protein assay and microsomes were stored at –80°C.

Chemical–DNA adduct formation in vitro
Chemical–DNA adducts from coal tar and subfractions were generated in vitro based on a procedure reported by Shaw and co-workers (17). All reactions were performed away from bright light and in 15 or 30 ml corex tubes in a total volume of 2.5 ml. Each reaction contained 200 µg calf thymus DNA, 2 ml of 0.1 M Tris, pH 7.4, 400 µg NADPH and 500 µg microsomal protein. Samples were placed in a shaking water bath at 37°C and reactions were started by addition of test material dissolved in 5 µl of DMSO. Reactions were incubated for a total of 1 h, after which 330 µl of a solution containing 1 mg proteinase K in 0.5 ml of 1% SDS, 2 mM EDTA and 130 µl of 10% SDS were added to each sample. Reactions were again incubated at 37°C overnight. A 1.0 ml volume of 5 M sodium chloride was added to each reaction and samples were mixed well and centrifuged at 4000 g for 10 min at room temperature. Supernatants were collected and chilled ethanol (2:1 vol) was added and samples were stored overnight at –20°C. Precipitated DNA was pelleted by centrifugation at 4000 g for 10 min at 4°C. DNA pellets were resuspended in 0.1x SSC buffer. DNA concentration was determined using A260 values while DNA purity was confirmed by A260:A280 ratios ranging from 1.75 to 1.85.

Fractionation of coal tar
Coal tar was fractionated based on the method of Grimmer (18), as illustrated in Figure 1Go. Coal tar (1.0 g) was combined with 15 ml of cyclohexane/water/methanol (7:3:5 v/v/v) and the resulting mixture was sonicated for 20 min. The mixture was allowed to stand for 5 min, after which the cyclohexane and water/methanol layers were collectively removed from insoluble material and centrifuged at 4000 g for 10 min at room temperature to pellet suspended insoluble material. The cyclohexane and water/methanol fractions were removed and placed into separate containers while the insoluble material remaining at the bottom of the centrifuge tube was combined with the original insoluble material and further extracted with 15 ml of cyclohexane/water/methanol (7:3:5 v/v/v) by sonication. This material was again centrifuged and the cyclohexane and water/methanol fractions were separated and pooled with previous fractions. This process was repeated a total of four times with the insoluble material. The cyclohexane and water/methanol fractions were taken to dryness under vacuum and labeled fractions II and I, respectively (see Figure 1Go). The mass of material present in fraction I was ~30 mg while the amount in fraction II was ~300 mg.



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Fig. 1. Separation scheme for coal tar.

 
Fraction II was further fractionated using DMSO and Sephadex LH-20 chromatography. DMSO fractionation was performed by dissolving 30 mg fraction II in 2 ml of cyclohexane. This was then combined with 2 ml of DMSO and the sample was vigorously mixed by vortexing. The sample was allowed to stand for 5–10 min and the cyclohexane and DMSO layers were carefully separated and labeled fractions II-1 and II-2, respectively. The mass of material in fraction II-1 was ~6 mg while the mass of material in Fraction II-2 was ~24 mg.

Sephadex LH-20 fractionation was performed as follows, Sephadex LH-20 (1.5 g) suspended in isopropanol was poured into the barrel of a 10 ml disposable syringe blocked at the narrow end with cotton. Excess isopropanol was allowed to drain from the barrel through the cotton forming a column of Sephadex beads with a bed volume of 1.5 ml. A 30 mg aliquot of fraction II was reconstituted in isopropanol (2 ml) and loaded onto the column and isopropanol (45 ml) was passed through the column. The first 8 ml of isopropanol was collected and labeled fraction IIA while the remainder of the isopropanol was collected and labeled as fraction IIB. The column was then eluted with 20 ml of acetone followed by 20 ml of methanol. The acetone and methanol collections were pooled and labeled fraction IIC. All fractions were taken to dryness under vacuum.

HPLC separation of fraction II-1
Reverse phase HPLC was used to separate the PAH components of fraction II-1. Separations were performed on a Hewlett-Packard Model 1050 system equipped with a variable wavelength detector (HP79853A) and a fluorescence detector (Shimadzu RF 5000U). An Envirosep-pp column (125x4.5 mm; Phenomenex) was used with a flow rate of 1 ml/min and a column temperature of 40°C. Acetonitrile (solvent A) and water (solvent B) were used with the following gradient system: 40–100% solvent A over 30 min and isocratic 100% A for 10 min. HPLC effluent was collected in liquid scintillation vials under minimum light at 5–10, 10–15, 15–20, 20–25, 25–26, 26–30, 30–35 and 35–40 min time intervals. Collected fractions from multiple HPLC analyses were pooled according to time interval and taken to dryness under vacuum. In general, collections from four or five HPLC separations were used in the in vitro activation systems described above. The retention times of phenanthrene, anthracene, pyrene, fluoranthene, benz[a]anthracene, 11H-benzo[a]fluorene, 11H-benzo[b]fluorene, 7H-benzo[c]fluorene, chrysene, benzo[b]fluoranthene and benzo[a]pyrene using the above chromatography system were confirmed using synthetic reference standards.

32P-post-labeling analysis
Chemical–DNA adducts were analyzed by 32P-postlabeling and multi-dimensional TLC using chromatography conditions slightly different from those previously described (13). In brief, 20 µg DNA were hydrolyzed with micrococcal nuclease enzyme and spleen phosphodiesterase. Hydrolyzed DNA was subjected to nuclease P1 treatment to enrich PAH–DNA adducts. The enriched adducts were labeled with 32P by T4 polynucleotide kinase and the 32P-labeled chemical–DNA adducts were separated using 20x20 or 10x20 cm polyethyleneimine–cellulose (PEI–cellulose) TLC plates. A five solvent system was used for adduct separation (13). However, the application of these solvents was modified in order to adequately separate the multiple adducts formed from coal tar. In the case of 20x20 cm TLC plates (see Figure 2Go) a predevelopment step was performed prior to placing TLC plates into each solvent system. Predevelopment consisted of developing TLC plates with water to the top of the sample origin, removing the plate and blotting off excess water at the bottom of the plate and immediately placing the plate into the appropriate solvent system for adduct separation. This approach allowed the more viscous solvents to reach the top of the 20x20 cm TLC plates. In the case of 10x20 cm TLC plates (see Figures 4, 6 and 7GoGoGo) a predevelopment step was not necessary. However, TLC plates were over-developed when plates were developed in the D3 and D4 solvents. Over-development was performed by opening the tops of the TLC tanks (1 cm on each side) after the solvent (D3 and D4) reached the top of the TLC plate. The TLC plates were then allowed to further develop, in the same buffer, for an additional 1 h, after which the plates were washed with water, dried and developed in the next buffer as appropriate. The over-development maximized adduct separation in D3 and D4, which was critical for resolving the `unknown' and B[a]P-derived DNA adducts. Separation of adducts using 10x20 cm TLC plates offered the advantage of being able to analyze more samples in a single labeling experiment when compared with using 20x20 cm TLC plates.



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Fig. 2. Representative autoradiographs of the PEI–cellulose TLC maps of calf thymus DNA adducts formed from coal tar-induced rat liver microsomal activation of 1 µg of neat coal tar (A) or fraction II-1 (B) (see Figure 1Go). Adduct separation was performed on 20x20 cm TLC plates and autoradiography was at –80°C for 1 h. Spot 4 corresponds to the mobility of the B[a]P–DNA adduct while spot 3 corresponds to the mobility of the 7H-benzo[c]fluorene–DNA adduct.

 


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Fig. 4. Representative autoradiographs of PEI–cellulose TLC maps of calf thymus DNA adducts from coal tar-induced rat liver microsomal activation of coal tar fractions collected from the HPLC separation of fraction II-1. HPLC effluents collected at 5–10, 10–15, 15–20, 20–25, 25–26, 26–30, 30–35 and 35–40 min (Figure 3Go, bottom) were activated in vitro in the presence of calf thymus DNA. Isolated DNA was subjected to 32P-postlabeling analysis using 5-dimensional PEI–cellulose TLC and 10x20 cm TLC plates. Autoradiography was at –80°C for 1 h. Spot 3 in the 15–20 min map corresponds to the 7H-benzo[c]fluorene-derived DNA adduct, while spot 4 corresponds to the B[a]P–DNA adduct.

 


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Fig. 6. Representative autoradiographs of PEI–cellulose TLC maps of DNA adducts formed in mouse lung following 2 weeks of ingesting adulterated diets. 32P-postlabeling was performed with 20 µg DNA and adduct separation was performed on 10x20 cm TLC plates. Autoradiography was at –80°C for 6 h. Photographs (a)–(d) represent adducts isolated from lungs of mice maintained on control, 11H-benzo[a]fluorene, 11H-benzo[b]fluorene and 7H-benzo[c]fluorene diets, respectively. Photographs (1) and (2) represent adducts isolated from lungs of mice maintained on a diet containing 0.25% coal tar or 0.25% coal tar supplemented with 14 mg 7H-benzo[c]fluorene, respectively. Photographs (3) and (4) represent adducts isolated from lungs of mice maintained on a diet containing the synthetic mix of PAH and the synthetic mix of PAH supplemented with 14 mg 7H-benzo[c]fluorene, respectively.

 


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Fig. 7. Representative autoradiographs of PEI–cellulose TLC maps of DNA adducts and HPLC profiles of 32P-labeled DNA adducts formed in lung of mice ingesting adulterated diets. 32P-postlabeling was performed with 20 µg DNA and adduct separation was performed on 10x20 cm TLC plates. Autoradiography was at room temperature for 17 h. Photographs (1) and (2) represent adducts isolated from lungs of mice maintained on diets containing B[a]P or 0.25% coal tar, respectively. (A) HPLC adduct profile of the B[a]P–DNA adduct formed in vitro. (B) HPLC adduct profile of spot 3 adducts derived from 7H-benzo[c]fluorene ingestion (Figure 6dGo). (C and D) HPLC adduct profiles of spot 4 and spot 3 adducts derived from coal tar (2).

 
32P-labeled chemical–DNA adducts separated on TLC plates were visualized by autoradiography and subsequently removed from PEI–cellulose by extraction with 4 M pyridinium formate (pH 4.5) according to Weyand et al. (19). In general, pyridinium formate extracts from a total of three or four TLC maps were combined for HPLC analysis.

HPLC analysis of 32P-labeled chemical–DNA adducts removed from TLC plates was performed with a Hewlett-Packard Model 1050 system equipped with a ß-RAM (IN/US Systems Inc.) flow-through radioactivity monitor using a 2.74 ml liquid cell. Separations were performed with a Zorbax SB-Phenyl reverse phase column (4.6 mmx25 cm, 5 µm). Solvent flow rate was maintained at 0.5 ml/min and column temperature was 40°C. A solvent system of 2 M ammonium formate (pH 3.5) (solvent A) and MeOH (solvent B) was used for separating 32P-labeled adducts. The following solvent gradient system was employed: 100% A isocratic for 5 min, followed by a linear gradient from 0 to 44% solvent B (56% A) over 40 min, remaining at this percentage for 10 min, followed by a linear gradient from 44 to 45% solvent B (55% A) over 10 min, followed by a linear gradient from 45 to 55% solvent B (45% A) over 10 min, remaining at this percentage for 10 min, followed by a linear gradient to 100% B over 25 min. In order to facilitate the comparison of DNA adduct profiles generated in different experiments and to adjust for minor shifts in peak retention times, standard DNA adducts previously generated with coal tar and B[a]P from in vitro and in vivo sources were used to confirm HPLC retention times.

GC–MS analysis
In order to identify the chemical responsible for forming the `unknown' DNA adduct, the material collected from the HPLC separation of fraction II-1 was analyzed by GC–MS. The HPLC effluent collected between 15 and 20 min (Figure 3Go) as well as subfractions of this area were subjected to GC–MS analysis. An HP 5890 gas chromatograph series II/5972 mass selective detector and HP ChemStation data system were used for analysis. A 0.256 mm i.d.x60 m, 1.0 µm film thickness J&W (Folsom, CA) DB-1 fused silica capillary column with an initial temperature of 60°C and a temperature gradient of 6°C/min up to a final temperature of 300°C was used. Helium was used as the carrier gas at 1.0 ml/min flow rate and a mass range of 50–400 was scanned. Spectra were compared with those of authentic standards.



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Fig. 3. Fraction II-1 separation on HPLC. (Top) Retention times of PAH synthetic reference standards. (Bottom) Separation of PAH components in fraction II-1. Retentions of 7H-benzo[c]fluorene (16.55 min) and 11H-benzo[a]fluorene/11H-benzo[b]fluorene (18.65 min) were determined with synthetic reference standards. Fractions from fraction II-1 (Bottom) were collected at 5–10, 10–15, 15–20, 20–25, 25–26, 26–30, 30–35 and 35–40 min. Each collection was evaporated to dryness and evaluated for their ability to form DNA adducts using the in vitro activation system.

 
Animal feeding study
Female CD-1 mice (18–19 g) were obtained from Charles River Breeding Laboratories (Kingston, NY). Mice (3 per group) were fed basal gel diets containing coal tar (0.25%), 20 PAH mix (see Table IGo), benzo[a]pyrene (14 mg), 11H-benzo[a]fluorene (14 mg), 11H-benzo[b]fluorene (14 mg), 7H-benzo[c]fluorene (14 mg), 20 PAH mix supplemented with 11H-benzo[a]fluorene (14 mg), 20 PAH mix supplemented with 11H-benzo[b]fluorene (14 mg), 20 PAH mix supplemented with 7H-benzo[c]fluorene (14 mg) and coal tar (0.25%) supplemented with 7H-benzo[c]fluorene (14 mg). Diets were prepared as previously described (12). In brief, 0.58 g neat coal tar was added to 0.234 kg dry food and combined with 12 g agar and 362 ml of water. PAH mix diet was prepared by combining the appropriate amount of each hydrocarbon in 15–20 ml of acetone which was then added to 0.234 kg dry food, 12 g agar and 362 ml of water. Diets containing individual hydrocarbons and diets supplemented with a single hydrocarbon were prepared as previously described, with each hydrocarbon being dissolved in 5–10 ml of acetone. The addition of 14 mg hydrocarbon to diets corresponds to a concentration of 60 mg/kg food. Mice were fed diets ad libitum for a total of 14 days. Mice were killed and lungs quickly removed and stored at –20°C. DNA was isolated from tissue by a modification of the Marmur procedure (20,21) and chemical–DNA adduct analysis performed as described above.


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Table I. Composition of coal tar and synthetic PAH mix diets
 

    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The utility of an in vitro metabolic activation system to generate chemical–DNA adducts similar to those previously observed in vivo with coal tar was evaluated. Initial experiments were conducted using neat coal tar at four different concentrations. In vitro reactions were performed with 1, 2, 5 and 10 µg neat coal tar dissolved in 5 µl of DMSO. The level of total adduct formation was greatest with reactions performed with 1 µg neat coal tar. The DNA adducts formed from the 1 µg neat coal tar reaction were evaluated using 32P-postlabeling and 20x20 cm PEI–cellulose TLC plates (Figure 2AGo). The separation of adducts using 20x20 cm plates resulted in several distinct spots of radioactivity that could be individually removed and further evaluated by HPLC. Spot 3 on the TLC map (Figure 2AGo) resulted in a major peak of radioactivity with a retention time that matched the `unknown' lung DNA adduct previously observed in vivo (45.19 min) while spot 4 resulted in a major peak of radioactivity with a retention time that matched the lung adduct derived from B[a]P (44.52 min) when B[a]P is fed to mice (13; data not shown). The HPLC separation of the other spots of radioactivity located on the TLC plate (Figure 2AGo) did not produce a major peak of radioactivity with a retention time similar to the adducts derived from either B[a]P or the `unknown' component of coal tar. These results confirmed that the `unknown' adduct can be formed using an in vitro rat microsome activation system and, furthermore, that the `unknown' adduct can be resolved from B[a]P–DNA adducts using both 20x20 cm TLC plates and reverse phase HPLC.

In order to gain insight into the properties of the chemical forming the `unknown' DNA adduct, coal tar was fractionated according to the scheme outlined in Figure 1Go. Fractions II-1, II-2, IIA, IIB and IIC were evaluated for their ability to form DNA adducts using the in vitro metabolic activation system. Fraction II-1 resulted in two major spots (labeled spots 3 and 4) of radioactivity when separated on 20x20 cm TLC plates (Figure 2BGo). Both spots were removed and analyzed by HPLC. Spot 3 eluted as a major peak of radioactivity with a retention time that matched the `unknown' DNA adduct, spot 4 eluted as a major peak of radioactivity with a retention time that matched the B[a]P–DNA adduct. The in vitro activation of fraction II-2 resulted in a predominant single adduct spot on TLC (with a mobility similar to spot 4 from fraction II-1; Figure 2BGo) which eluted as a single major peak of radioactivity that corresponded to the B[a]P–DNA adduct (data not shown). Fraction IIA resulted in a TLC adduct map similar to the map illustrated in Figure 2BGo. Two predominant spots of radioactivity were detected and both corresponded to adducts derived from the `unknown' (spot 3) and B[a]P (spot 4). In vitro activation of fraction IIB resulted in a single predominant spot of radioactivity (spot 4) that was determined to be the B[a]P–DNA adduct. No adducts were observed with fraction IIC. These results demonstrate that the compound responsible for forming the `unknown' chemical–DNA adduct from coal tar in vivo is primarily present in fractions II-1 and IIA. Selective retention of the chemical responsible for producing the `unknown' DNA adduct in fractions II-1 and IIA suggests that the adducting compound is smaller than B[a]P and is likely a three- or four-ring PAH.

Fraction II-1 was further separated using reverse phase HPLC (Figure 3Go, bottom). In general, the chemical components of fraction II-1 were separated according to size/lipophilicity, with the lower molecular weight compounds eluting earlier. PAH reference standards were used to determine the retention time of known hydrocarbons (Figure 3Go, A, top). HPLC fractions were collected at timed intervals between 5 and 40 min. Each interval collected was concentrated and evaluated in the in vitro activation system for the ability to form chemical–DNA adducts. Significant levels of chemical–DNA adducts were formed with HPLC coal tar material collected between 15–20, 20–25 and 25–26 min (Figure 4Go). The area on the TLC map for the 15–20 min collection that corresponded to the mobility of the `unknown' adduct (labeled spot 3) was removed and further analyzed by HPLC (Figure 5Go). Spot 3 on the 15–20 min map eluted as one major and several minor peaks of radioactivity, with the majority of the radioactivity eluting at 48.40 min. This retention time corresponded to the retention time of the `unknown' adduct as illustrated in the HPLC profile obtained with spot 3 from fraction II-1 (Figure 5Go). In contrast, the HPLC separation of the adducts observed on the 20–25 min TLC map (Figure 4Go, 20–25 min, spot A) resulted in two major peaks of radioactivity with retention times of 42.00 and 47.27 min. The adduct observed in the 25–26 min collection, as expected, resulted in a single peak of radioactivity on HPLC that corresponded to the B[a]P–DNA adduct (47.27 min). The results of this experiment clearly demonstrated that the 15–20 min fraction from the HPLC separation of fraction II-1 contained the adducting chemical responsible for forming the `unknown' adduct.



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Fig. 5. HPLC profiles of 32P-labeled calf thymus DNA from coal tar-induced rat liver microsomal activation of coal tar fractions collected from the HPLC separation of fraction II-1 (15–20, 20–25 and 25–26 min fractions). Spots 3 and 4 (Figure 4Go) were removed from PEI–cellulose TLC maps and subjected to HPLC analysis, respectively. Peaks of radioactivity eluting at 48.4 and 47.2 min correspond to adducts derived from 7H-benzo[c]fluorene and B[a]P, respectively. The spot 3 and spot 4 HPLC profiles correspond to the assignments presented in Figure 4Go and represent adducts formed from the in vitro activation of fraction II-1. These adducts were used to verify the retention times of adducts derived from 7H-benzo[c]fluorene and B[a]P.

 
The 15–20 min fraction as well as subfractions of this area were collected and subjected to GC–MS analysis. These analyses indicated that hydrocarbons eluting within this area had a mass of 216. A review of the collected mass spectra suggested that methylpyrenes, methylfluoranthenes, benzanthracene and benzofluorenes may be responsible for forming the `unknown' adduct. Therefore, four methylpyrene isomers (1-, 2-, 3- and 4-methylpyrene), two methylfluoranthene isomers (2- and 3-methylfluoranthene) and three benzofluorene isomers (11H-benzo[a]fluorene, 11H-benzo[b]fluorene and 7H-benzo[c]fluorene) were further evaluated. The in vitro activation of these hydrocarbons indicated that only the 7H-benzo[c]fluorene formed an adduct that had a mobility on TLC and HPLC that corresponded to the `unknown' adduct observed in vivo with coal tar (data not shown). In order to further confirm that 7H-benzo[c]fluorene formed the `unknown' DNA adduct in mouse lung in vivo, a 2 week feeding experiment was conducted using the three benzofluorene isomers, coal tar, B[a]P and a synthetic mixture of PAH (Table IGo). Each of the three benzofluorene isomers produced DNA adducts in mouse lung that were detectable by TLC (Figure 6Go). However, the 7H-benzo[c]fluorene isomer formed the greatest number of adducts with between four and six adducts being detected. In addition, the level of overall adduct formation was highest with 7H-benzo[c]fluorene based on the relative intensity of the radioactivity observed on the TLC adduct maps (Figure 6bGo–d). In the case of the adducts observed with 7H-benzo[c]fluorene, multiple DNA adducts were formed and the mobility of one of these adducts (labeled spot 3 in Figure 6dGo) corresponds to the mobility of the `unknown' adduct observed with coal tar. The addition of 7H-benzo[c]fluorene to diets containing coal tar enhanced the amount of the adduct observed to be derived from 7H-benzo[c]fluorene [Figure 6Go(2)]. Likewise, the addition of 7H-benzo[c]fluorene to diets containing a mixture of synthetic hydrocarbons resulted in the formation of the 7H-benzo[c]fluorene–DNA adduct [spot 3 in Figure 6Go(4)]. Animals maintained on diets containing the synthetic PAH mixture without 7H-benzo[c]fluorene did not produce an adduct that corresponded to the `unknown' adduct [Figure 6Go(3)].

The observations made with the TLC adduct maps were confirmed by removing the adducts from each TLC map and separating the material by HPLC. Figure 7Go illustrates the TLC adduct maps obtained from lung of animals ingesting coal tar [Figure 7Go(2)] or pure B[a]P [Figure 7Go(1)]. In addition, the HPLC adduct profiles derived from B[a]P, coal tar and 7H-benzo[c]fluorene are also illustrated in Figure 7Go. The B[a]P–DNA adduct standard eluted at 45.42 min while the spot 3 adduct derived from 7H-benzo[c]fluorene (Figure 6dGo) eluted as a major peak of radioactivity at 47.12 min. In the case of coal tar-treated animals [Figure 7Go(2)] spot 4 eluted as a single peak of radioactivity at 45.42, which corresponded to the B[a]P–DNA adduct. Spot 3 from coal tar-treated animals eluted as a major peak of radioactivity at 47.12 min, which corresponded to the adduct derived from 7H-benzo[c]fluorene. Spot 3 also contained the adduct derived from benzo[b]fluoranthene, which eluted at 35.04 min. A comparison of both TLC adduct maps and HPLC adduct retention times indicates that the top portion of spot 3 on the TLC adduct map obtained with coal tar [Figure 7Go(2)] represents the location of the benzo[b]fluoranthene–DNA adduct while the lower portion of spot 3 represents the location of the adduct derived from 7H-benzo[c]fluorene.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The identification of genotoxic components within complex organic mixtures is an essential step in understanding the mechanism by which mixtures such as coal tar induce cancers. In the case of coal tars, the PAH components are believed to be responsible for the carcinogenic activity exhibited by these mixtures. The metabolic activation of PAH to reactive intermediates that bind to DNA is generally accepted as a critical step in the multi-step process of PAH chemical carcinogenesis. Therefore, the characterization of PAH–DNA adducts formed by complex mixtures such as coal tar can be used to identify hydrocarbons that may contribute to the overall carcinogenicity of complex mixtures.

Previous studies have demonstrated that three predominant adducts are formed in lungs of mice ingesting coal tar (13). B[a]P and benzo[b]fluoranthene account for two of these adducts while the third adduct could not be attributed to any of the PAHs routinely analyzed for within complex mixtures (Table IGo). In the present study the types of chemical–DNA adducts formed by coal tar were evaluated in order to identify the chemical component of coal tar responsible for forming the previously termed `unknown' adduct. Our initial in vitro experiments using both TLC and HPLC clearly demonstrated that the `unknown' adduct observed in vivo could be produced using an in vitro metabolic activation system. In addition, detailed TLC and HPLC experiments demonstrated that adducts derived from B[a]P and the `unknown' component could be resolved on 10x20 TLC plates. The ability to produce this adduct in vitro combined with a more direct method of separating adducts using TLC provided a mechanism to gain insight into the structure of the hydrocarbon responsible for forming the `unknown' adduct.

GC–MS analysis of coal tar fractions obtained from HPLC indicate that the adducting compound was a four-ring hydrocarbon with a mass of 216. A careful evaluation of candidate hydrocarbons with a mass of 216 led to the conclusion that a benzofluorene derivative may be responsible for forming the `unknown' chemical–DNA adduct. Comparative in vitro and in vivo studies on the adducting properties of all three isomers of benzofluorene indicates that 7H-benzo[c]fluorene is responsible for producing the `unknown' adduct observed in lungs of mice ingesting coal tar. In addition, the ingestion of 7H-benzo[c]fluorene produces several DNA adducts in lung tissue (Figure 6dGo). A comparison of DNA adduct maps obtained from mice ingesting 7H-benzo[c]fluorene (Figure 6dGo) or coal tar [Figure 7Go(2)] also suggests that 7H-benzo[c]fluorene may be responsible for other coal tar-derived DNA adducts in lung. However, additional studies are required to clearly establish that these additional adducts are derived from 7H-benzo[c]fluorene. The lung DNA adduct maps generated from animal feeding studies also demonstrated that 7H-benzo[c]fluorene produced significantly more lung DNA adducts than 11H-benzo[a]fluorene or 11H-benzo[b]fluorene (Figure 6Go). These results tend to suggests that 7H-benzo[c]fluorene may have the ability to induce lung tumors in animals following ingestion. Further studies are needed to clearly establish whether the demonstrated ability of 7H-benzo[c]fluorene to form lung DNA adducts is correlated with the tumorigenic potential of this compound in mouse lung.

Limited studies have evaluated the biological properties of 7H-benzo[c]fluorene in vitro and in vivo on mouse skin. LaVoie and co-workers evaluated the mutagenicity of benzofluorene derivatives towards Salmonella typhimurium TA98 and TA100 with and without S9 activation (22). No mutagenic activity was observed. The three benzofluorene isomers were also evaluated for tumor-initiating activity using the mouse skin initiation/promotion model (23). A low tumorigenic response of 10, 20 and 25% tumor-bearing mice with 0.15, 0.35 and 0.25 tumors/mouse was observed for 11H-benzo[a]fluorene, 11H-benzo[b]fluorene and 7H-benzo[c]fluorene, respectively. An earlier study performed by Bachmann et al. (24) reported that 7H-benzo[c]fluorene did not produce skin tumors when applied topically twice a week to mice for life. A review of these results by the IARC in 1983 (25) concluded that `The available data were inadequate to permit an evaluation of the carcinogenicity of 7H-benzo[c]fluorene to experimental animals'. The ability of a compound to produce a tumorigenic response in lung and not in mouse skin has been previously reported. Fluoranthene is inactive as a tumorigenic agent on mouse skin but induces lung and liver tumors in newborn mice (26). Benzo[c]phenanthrene also exhibits low carcinogenic activity on mouse skin while benzo[c]phenantherene-3,4-diol-1,2-epoxides have been shown to be highly carcinogenic (27). The low carcinogenic activity of benzo[c]phenanthrene on mouse skin is believed to be due to the inability of mouse skin to form significant levels of the diol epoxides (28). These investigators also demonstrated that high levels of benzo[c]phenantherene-3,4-diol-1,2-epoxides are formed in the human mammary carcinoma cell line MCF-7. Thus, the effects previously observed on mouse skin with 7H-benzo[c]fluorene may not be fully representative of the biological potential of this compound in rodent tissue other than skin or in human cells. In view of the lung DNA adducts observed in the present study, further studies are warranted in order to fully characterize the biological potential of 7H-benzo[c]fluorene and to delineate its mechanism of action.

Earlier studies have demonstrated that the types of chemical–DNA adducts formed in mice treated with coal tar varies between tissue sites such as forestomach, lung and skin. The topical application of coal tar results in a single major DNA adduct in mouse skin derived from B[a]P while the major adduct observed in lung is derived from 7H-benzo[c]fluorene (previously termed the `unknown' adduct) (29). Likewise, the ingestion of coal tar by mice results in a diagonal band of adducts in both forestomach and lung tissue. However, the B[a]P–DNA adduct is present in both forestomach and lung tissue, while the 7H-benzo[c]fluorene–DNA adduct is present only in lung tissue. In addition to differences observed in adduct formation, the ability to correlate the chemical content of coal tar with tissue susceptibility to tumor induction has been shown to vary according to tissue site. The B[a]P content of coal tar correlates with forestomach tumor induction, however, it does not correlate with lung tumor induction, suggesting that a hydrocarbon other than B[a]P is responsible for the tumorigenicity of coal tar in lung tissue (2,4). Culp and co-workers (4) demonstrated that mice ingesting 25 ppm B[a]P in the diet for 2 years did not develop lung tumors. In contrast, a robust lung tumor response was observed in mice ingesting 0.1–0.3% coal tar. Similar bioassay results were observed with A/J mice (2). More recently, Culp et al. (12) concluded that K-ras mutations in coal tar-induced forestomach tumors likely result from the B[a]P content of coal tar while K-ras mutations in coal tar-induced lung tumors likely result from coal tar components other than B[a]P. The combination of these previous observations with our current results further supports the concept that, in the case of coal tar, the tumorigenic activity in tissue directly exposed to coal tar (such as forestomach and mouse skin) is likely due to the predominant effects of B[a]P while the tumorigenicity observed at tissues distant from the site of coal tar contact (such as lung) is due to another hydrocarbon, possibly 7H-benzo[c]fluorene.

Previous studies have also observed an unidentified PAH–DNA adduct formed in vivo from the application of complex organic mixtures to animals. Gallagher and co-workers (30) investigated dose-related differences in DNA adduct levels following topical application of complex mixtures from air pollution sources. These investigators observed an unidentifiable adduct with chromatographic properties similar to B[a]P–DNA adducts but which was not formed by B[a]P. This adduct was present in skin, lung and liver tissue and accounted for 12–34% of total adduct formation in animals exposed to coke oven soot or coal soot. In the case of diesel particulate extracts, an unidentified DNA adduct accounted for 49–67% of the observed adducts (31). In another study, Hughes et al. (32) observed an adduct in mouse skin that could not be attributed to any of the 19 PAHs used to identify coal tar that was applied topically to mice. It is anticipated that 7H-benzo[c]fluorene would be present in the complex mixtures evaluated by these investigators, since they are products of incomplete combustion. However, further studies are needed to determine if 7H-benzo[c]fluorene contributes to the unidentified DNA adducts observed with these mixtures. Studies are in progress to unequivocally characterize the structure of the DNA adduct formed with 7H-benzo[c]fluorene in mouse lung and to determine the tumorigenic potential of 7H-benzo[c]fluorene.


    Notes
 
4 To whom correspondence should be addressedEmail: weyand{at}cop.rutgers.edu Back


    Acknowledgments
 
This work was funded by the Electric Power Research Institute, grant WO2963-06 and 041918.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 25, 2000; revised May 15, 2000; accepted May 17, 2000.