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Chem. Res. Toxicol. 1991,4,466-473
(14) Tornqvist, M., Kautiainen, A., Gatz, R. N., and Ehrenberg, L. (1988) Hemoglobin adducts in animals exposed to gasoline and diesel exhausts. I. Alkenes. J. Appl. Tonicol. 8, 159-170. (15) Tomqvist, M., Gustafsson, B., Kautiainen, B., Harms-Ringdahl, M., Granath, F., and Ehrenberg, L. (1989)Unsaturated lipids and intestinal bacteria as sources of endogenous production of ethane and ethylene oxide. Carcinogenesis 10,39-41. (16) Farmer, P. B., Bailey, E., Gorf, S. M., Tornqvist, M., Osterman-Golkar, S., Kautiainen, A,, and Lewis-Enright, D. P. (1986) Monitoring human exposure to ethylene oxide by the determination of haemoglobin adducts using gas chromatography-mass spectrometry. Carcinogenesis 7,637-640. (17) Bailey, E.,Brooks, A. G. F., Dollery, C. T., Farmer, P. B.,
Passingham, B. J., Sleightholm, M. A., and Yates, D. W.(1988) Hydroxyethylvaline adduct formation in haemoglobin as e biological monitor of cigarette smoke intake. Arch. Toxicol. 62, 247-253. (18) Tomqvist, M., Osterman-Golkar, S., Kautiainen, A., Jensen, S., Farmer, P. B., and Ehrenberg, L. (1986b) Tissue d m s of ethylene oxide in cigarette smokers determined from adduct levels in hemoglobin. Carcinogenesis 7, 1519-1521. (19) Blum, W.,and Eglinton, G. (1989) Preparation of high temperature stable glass capillary columns coated with PS-090 (20% diphenyl-substituted CHSO-terminated polysiloxane) a selective stationary phase for the direct analysis of metal-porphyrin complexes. High Res. Chrom. Chrom. Commun. 12, 290-293.
Urinary Excretion and DNA Binding of Coal Tar Components in B6C3F1 Mice following Ingestion Eric H. Weyand,* Yun Wu, and Shruti Pate1 Rutgers, The State University of New Jersey, College of Pharmacy, P.O. Box 789, Piscataway, New Jersey 08855-0789
Barbara B. Taylor and David M. Mauro META Environmental, Inc., 49 Clarendon Street, Watertown, Massachusetts 021 72 Received March 4, 1991
Urinary excretion of polycyclic aromatic hydrocarbon (PAH) metabolites and DNA binding of coal tar components in male mice were investigated following the ingestion of a coal tar adulterated diet. Male B6C3F1 mice were able to tolerate an F0927 basal gel diet which contained from 0.1 to 1% coal tar (tar weight/dry food weight) for 15 days. Mice maintained on a 0.1 and 0.2% coal tar diet had body weight gains similar to those of control animals. However, mice maintained on the 0.5 and 1.0% diet had body weight gains considerably lower than control values. Chemical-DNA adduct formation was detected and quantified in lung and forestomach tissue of animals on 0.1, 0.2, 0.5, and 1% coal tar containing diets. A dose-related effect was observed in lung DNA adduct formation while no dose effect was observed in forestomach tissue. In addition, overall adduct levels in lung tissue were considerably higher than forestomach levels for animals on the 0.5 or 1% diet. In contrast, DNA adduct levels were highest in the forestomach of animals on diets lower in coal tar content (0.1 or 0.2%). Chemical-DNA adducts of coal tar components were also evaluated for four other coal tar samples which varied in chemical composition. Mice were maintained on diets containing 0.25% of each coal tar for 15 days. Chemical-DNA adducts were detected in lung, liver, and spleen for all animals receiving these coal tar diets. DNA adduct patterns were similar while quantitative differences were observed between coal tar samples and tissue sites. Highest adduct levels were detected in lung DNA. Benzo[a]pyrene content in coal tar samples could not account for the DNA adduct levels observed with coal tar ingestion. The urinary excretion of select PAH metabolites following coal tar ingestion was evaluated by using urine collected on days 1 and 14 of diet administration. The levels of l-hydroxypyrene in urine, the major PAH metabolite detected, correlated with the pyrene content of these coal tars. These data demonstrate that coal tar components are readily bioavailable following ingestion. Introduction . _ Polycyclic aromatic hydrocarbons (PAHs)' are a large class of ubiquitous environmental pollutants that have been implicated in the etiology of several human cancers ('-3)* Human exposure to occurs through inhalation, ingestion, or topical absorption and is most often occupationally related. Since several PAHs have been determined to be potent chemical carcinogens in laboratory animals, monitoring human exposure to PAHs is essential
* To whom correspondence should be addressed.
in establishing reliable risk assessments. Biological monitoring methds such as mutagenicity of urine extracts and the detection of PAH in urine have been used to evaluate exposure to PAH using both laboratory animals and humans (4-13). Rscently, studies have evaluated the covalent binding of aromatic chemic& to of exposure to complex DNA as a mixtures, &hoket and co-workers have demonstraM that Abbreviations: B[o]P, benzo[o]pyrene;TLC,thin-layer chrometography; HPLC,high-pressure liquid chromatography; PAH,polycyclic aromatic hydrocarbon;PEI-cellulose, poly(ethylenimine)-cellulose.
0S93-228~/91/2104-0466~02.50~0 0 1991 American Chemical Society
Bioavailability of Coal Tar Components
significant levels of chemical-DNA adducts were formed in mouse skin following the topical application of coal tar, creosote, or bitumen (14). Interestingly, chemical-DNA adducts were also detected in lung tissue, which demonstrated significant systemic availability of coal tar components. Chemical-DNA adducts have also been detected in DNA of white blood cells isolated from humans following exposure to coal tar, tobacco smoke, or coal burning environments (14-18). High levels of PAHs are known to be present in crude coal tar and coal tar products used to prevent corrosion, construction materials, and medicinal agents (19). In addition to its commercial facet, coal tar is also produced as a waste byproduct of coal-conversion processing to synthetic fuels and chemicals. For example, coal gasification, a process used as early as the late 1700s to generate methane gas from coal, produced coal tar as a waste byproduct. Although this manufacturing process is no longer practiced commercially in the Unites States, the coal tar byproduct generated during coal gasification remains a potential concern as an environmental pollutant and human health issue. The carcinogenic potential of coal tar and its components has been well documented by use of coal tar extracts and mouse skin initiation and promotion studies (19). There are only limited studies on the effects of coal tar exposure associated with chronic ingestion. Robinson et al. (20)determined the carcinogenic effects of a particulate fraction isolated from crude coal tar paint. This material was tumorigenic in both lung and forestomach of female A / J mice when administered by gavage in 2% Emulphor. Studies have not investigated the biological effects associated with exposure to coal tar generated from coal gasification. In the present study we have evaluated the relationship between coal tar ingestion and systemic availability of coal tar components in male mice. These studies were performed with tar samples collected from four separate coal gasification sites. In contrast to previous investigations, these studies were performed with whole (unfractionated) crude coal tar. These coal tar samples were incorporated into a basal gel diet, and the effects of ingestion on animal body weight and food consumption were evaluated. The urinary excretion of PAH metabolites and the quantification of chemical-DNA adducts were used to monitor systemic availability.
Materlals and Methods Chemicals, Caution: The hazards of coal tar waste products derived from coal gasification have not been fully evaluated. Hence, protective clothing and appropriate safety procedures should be followed when working with this material. Coal tar samples formed as waste products during coal gasification were supplied by the Electrical Power Research Institute of Palo Alto, CA. All samples were generated by using East Coast coal feed stock. Samples A-C were viscous liquids while sample D was a solid at room temperature. The F0927 basal gel diet was purchased from Bio-Serv, Inc. (Frenchtown, NJ). l-Naphthol, 4chloro-l-naphthol, and 1-hydroxypyrene were purchased from Aldrich Chemical Co. (Milwaukee, WI).3-Hydroxybenzo[a]pyrene was obtained from the NCI Chemical Carcinogen Repository (KansasCity, MO). l-Hydroxyphenanthrene was obtained from Dr. LaVoie. 2-Hydroxyphenanthrene was prepared by BaeyerV i e r oxidation of 2-acetylphenanthrenefollowed by hydrolysis of the resulting ester using procedures similar to those previously described (21). Both hydroxylated metabolites of phenanthrene were determined to be greater than 92% pure by high-pressure liquid chromatography (HPLC). 8-Glucuronidase/arylsulfatase (lO0,OOO Fishman units/mL) was purchased from Boehringer Mannheim Biochemicals (Indianapolis,IN). [ T - ~ P ] A T (sp P act. >5000 Ci/mmol) was obtained from Amersham Corp. (Arlington
Chem. Res. Toxicol., Vol. 4, No. 4, 1991 467 Height, IL). Macherey-Nagel polyethyleneimine-cellulose (PEEcellulose) thin-layer chromatography (TLC) plates were purchased from Bodman Chemicals (Aston, PA) while all biochemicals used for 32P-postlabelingwere obtained as previously described (22). All commercial chemicals were of analytical grade or higher. Analysis of Coal Tar PAH Content. Analytical methods were developed to determine the concentration (in coal tar residues) of 37 compounds, which included monocyclic aromatic hydrocarbons, PAHs, and nitrogen- and sulfur-containing polycyclic aromatic hydrocarbons. The levels of naphthalene, 1-and 2-methylnaphthalene, and 16 other PAHs ranging in molecular weight from acenaphthylene to benzo[g,h,i]perylene were monitored for this study. To analyze for these compounds, approximately 0.20 g of each tar residue was dissolved in 10 mL of benzene. A 100-pL aliquot of the surrogate standard was then added to 1.0 mL of the benzene-solubilized sample. After the volume was reduced to 200 p L by using a gentle stream of nitrogen, the sample was applied to a pretreated alumina column for class separation by eluting first with 4.0 mL of benzene then with 2.0 mL of a mixed solvent (5050 dichloromethane/methanol). The resulting benzene fraction was concentrated or diluted to a given volume with fresh benzene as needed and analyzed by using total ion monitoring GC/MS for the determination and confirmation of the PAH and sulfur-containing PAH compounds. These samples were also analyzed by using GC/FID detection. The dichloromethane/methanol fractions were concentrated and analyzed by selective ion monitoring GC/MS for the determination and confirmation of the nitrogen-containing PAH compounds. Diet Preparation. Basal gel diets containing coal tar were prepared according to manufacturer recommendations. In brief, diets were prepared by blending 3020 mL of boiling hot water with 100 g of gelling agent for 1 min, after which 1948 g of dry food was quickly added and blending continued for an additional 2-3 min. Coal tar was added to diets and blending continued until a homogeneous color was obtained (2-3 min). After thorough blending, the gel mixture was poured into bar molds and allowed to cool at room temperature for 4-6 h. Gel diet bars were packaged according to groups into plastic bags and stored at -20 OC. The amount of coal tar incorporated into diets was based on the amount of dry food used in preparing diets. For example, 5068 g of a 0.25% gel diet contains 4.88 g of coal tar and 1948 g of dry food. The palatability experiment was performed with 0.1,0.2, 0.5,1,2,5, and 10% coal tar adulterated diets prepared by using sample C. Five separate 0.25% diets for comparison experiments were prepared by using coal tar samples A-D and a coal tar mix containing equal amounts of the four coal tar samples under evaluation. Since tar sample D was a solid at room temperature, acetone was used to solubilize this coal tar (0.2 g/mL) prior to blending. A benzo[a]ppene (B[a]P) adulterated diet was also prepared at a level comparable to the amount present in a 0.25% sample C adulterated diet. B[a]P was solubilized in acetone and blended into the diet at a concentration of 10 mg/5068 g of gel diet. Animals. B6C3F1 mice were used in all experiments. Mice 49-56 days old were obtained from either Charles River Laboratories (Wilmington, MA) or Harlan Sprague Dawley Inc. (Indianapolis, IN). Animals were housed in solid polycarbonate Micro-Isolator cages (Lab Products, Inc., NJ) with hardwood bedding from Beta-Chip North Eastern Products (Warrenburg, NY). Mice were housed under controlled conditions with a 12-h light-dark cycle and given food and water ad libitum. Palatability Experiments. A maximum tolerated dose of coal tar adulterated gel diet was determined by using nine groups of 5 male mice. Eight groups of mice were maintained on a control basal gel diet for 14 days, after which seven of the groups were switched to an adulterated diet containing 0.1, 0.2, 0.5, 1, 2, 5, or 10% of coal tar sample C. Animals were maintained on either control or adulterated diets for an additional 15 days. The ninth group of five mice waa maintained on an NIH-07 pellet diet during the 29-day study. Animal body weight, food, and water consumption were monitored throughout the study. Animals on control and 0.1,0.2,0.5, and 1 % diets were sacrificed on day 29. Organs of forestomach and lung were quickly excised and stored
468 Chem. Res. Toricol., Vol. 4, No. 4, 1991 a t -20 OC for chemical-DNA adduct analyses. Urinary Excretion Experiments. The urinary excretion of l-hydroxypyrene and 3-hydroxybenzo[a]pyrene following coal tar ingestion was evaluated in animals maintained on five chemically different types of coal tar adulterated diets for 15 days. Diets containing 0.25% of each coal tar were selected for these experiments since animals readily adjusted to the 0.2% coal tar sample C diet. Animals (7-9 mice per group) were maintained on a control or adulterated diets containing 0.25% of coal tar samples A-D or a mix of the four coal tar samples. In addition, another group of animals was maintained on a diet containing only B[a]P (10 mg/5068 g of gel diet). Urine samples were collected overnight on the first and last day of coal tar ingestion with plastic metabolism cages from Nalgene Inc. (Rochester,NY). Urine samples were stored at -20 "C until analyzed by HPLC as described below. Animal body weight, food, and water consumption were monitored throughout the study. Animals were sacrificed after 15 days of coal tar ingestion and organs of forestomach, lung, and spleen were quickly excised and stored a t -20 OC for chemical-DNA adduct analyses. Urinary Metabolite Analyses. The levels of l-hydroxypyrene and 3-hydroxybenzo[a]pyrene excreted in urine of animals maintained on coal tar adulterated diets were determined by using a modified procedure described by Jongeneelen and co-workers (5). Equal volumes of urine (10 mL) and 0.1 M sodium acetate buffer (pH 5.0) were combined and spiked with 50 pg of 4chloro-1-naphthol (internal standard). Buffered urine samples were incubated overnight with 1250 Fishman units of 8-glucuronidase/arylsdfahe at 37 OC in a shaking water bath. Samples were loaded onto ChemElut (CE1020) extraction tubes and allowed to equilibrate for 30 min. ChemElut tubes were eluted two times with 20 mL of ethyl acetate (water saturated). Eluants were combined and evaporated to dryness under nitrogen, and residues were redissolved in 0.25-0.5 mL of dimethyl sulfoxide. Samples were filtered through a 0.45-pm nylon membrane prior to HPLC analyses. Recoveries of l-naphthol, l-hydroxypyrene, and 3hydroxybenzo[a]pyrenein the above extraction methods were 98, 47, and 78%, respectively. The detection limit for urinary 1hydroxypyrene and 3-hydroxybenzo[a]pyrene is 9.9 and 25.4 nmol/L, respectively. HPLC analyses were performed on a Hewlett-Packard (HP) Model 1090 liquid chromatograph equipped with an HP Model 104OA high-speed spectrophotometric detector (diode array) and an H P Model 1046 fluorescence detector. A Lichrosorb RP-18 10-pm reverse-phase CI8column (E. M. Merck, Darmstadt, FRG), 4.0 X 25 cm, was used for these analyses. Metabolih were profiled with a flow rate of 1 mL/min according to a 65-min gradient elution system as follows: Initial conditions were 50% HzO/ MeOH for 5 min, followed by a 5-min linear gradient to 60% methanol which was maintained for 10 min, followed by a 5-min linear gradient to 70% MeOH which was maintained for 10 min, followed by a 5-min linear gradient to 80% MeOH which was maintained for 10 min, followed by a 15-min linear gradient to 100% MeOH. Fluorescence was monitored by using the following program sequence: 0 min = Ex-229 nm, Em-462 nm; 15 min = Ex-zero order, Em-zero order; 29 min = Ex-235 nm, Em-399 nm; 38 min = Ex-251 nm, Em-441 nm. DNA Adduct Analysis. DNA was isolated from tissue according to standard procedures (23,24)and chemical-DNA adduct formation was evaluated by using a2P-postlabelingas previously described (25, 26). In brief, DNA (20 pg) was hydrolyzed to deoxyribonucleotideswith micrococcal endonuclease and spleen phosphodiesterase followed by digestion with nuclease P, to convert unmodified nucleotides to nucleosides. Hydrocarbondeoxyribonucleotides(6-10 pg) were *-labeled by using 25 pCi of carrier-free [-pa2P]ATP. a2P-Labeleddeoxyribonucleoside 3/,5/-bisphosphateswere chromatographed on 10 X 10 cm PEIcellulose TLC plates using a modified four-solvent developing system. Solvents used were as follows: D1, 1.0 M sodium phosphate (pH 6.5); D2, not performed; D3,5.3 M lithium formate (pH 3.5) containing 8.5 M urea; D4,1.2 M lithium chloride, 0.5 M Tris-HC1 (pH 8.0) containing 8.5 M urea; D5, 1.7 M sodium phosphate (pH 6.0). Following development, spots of radioactivity were located by autoradiography using Kodak XOmat AR film with intemifying screens. Relative adduct levels were determined as previously described and picomoles of adducts were calculated
Weyand et al. Table I. PAH Content of Coal Tar Samples PAH, dka," for coal tar sample A B C D naphthalene 71.0 130.0 43.0 13.0 1-methylnaphthalene 24.0 20.0 24.0 4.9 42.0 7.5 34.0 2-methylnaphthalene 33.0 phenanthrene 33.0 35.0 19.0 5.2 13.0 5.3 1.5 fluoranthene 13.0 pyrene 13.0 18.0 7.8 2.4 5.1 2.6 0.8 benz[a]anthracene 4.9 chrysene 5.1 6.5 2.7 1.0 benzo[blfluoranthene 2.2 3.1 1.5 ND (1.2)e benzo[k]fluoranthene ND (1.1) 3.0 2.9 0.7 6.4 1.7 ND (1.8) benzo[a Ipyrene 3.9 ND (1.1) ND (1.4) indeno[l,2,3-cd]NDb (2.6) 1.3 PYene ND (0.5) ND (0.6) ND (0.1) ND dibenz[a,h]anthracene benzo[g,h,i]perylene ND (3.1) 1.9 ND (1.3) ND (1.6) OValues are the average of three determinations by GC/MS analysis. bND = below the detection limits of about 0.3 g/kg using GC/MS detection. cValues in parentheses are the results of determinations using GC/FID detection. by multiplying relative adduct level values by 0.31 X 10' with the assumption that 1mg of DNA = 0.31 X lo7 pmol of nucleotides.
Results PAH Content of Coal Tar Samples. The PAH concentrations for the four coal tars used in this study are listed in Table I. PAH concentration profiles varied considerably between coal tar samples. The most abundant PAHs were naphthalene, 1- and 2-methylnaphthalene, and phenanthrene. These hydrocarbons were present at levels ranging from 4.9 to 130 g/kg of coal tar. PAH concentrations in sample D were about 4-10 times lower than levels determined in the other three tar samples. The physical nature in which sample D was collected may account for the observed lower PAH concentrations. Samples A-C were fluid at room temperature and were collected by pumping the tar from subsurface wells. Tar sample D was not fluid at room temperature and was collected from the ground surface where it had mixed with gravel and soil. With a detection limit of about 0.3 g/kg using GC/MS detection, dibenz[a,h]anthracene was not detected in sample D. Maximum Tolerated Dose of Coal Tar Adulterated Gel Diet. Coal tar (sample C) was readily incorporated into a F0927 basal gel diet in amounts ranging from 0.1 to 10% of the dry food weight. Adulterated bars were brown colored and darkened according to the amount of coal tar. Visual inspection of cross sections of the gel diet indicated that the coal tar was homogeneously distributed. Weight loss associated with dehydration of gel diet bars was evaluated with control gel diets. Diet bars were stored in empty animal cages maintained under standard animal housing conditions, and the weight of bars was recorded daily. Dehydration of gel diets during days 1-4 accounted for an average weight loss of 9%/24 h. Weight loss was considerably less for days 5-10 with an average of 4%/24 h. Animals were maintained on control gel diets for 14 days prior to switching to coal tar adulterated diets. No difference in body weight was detected between animals fed a control gel diet or a standard NIH-07 pellet diet (data not shown). The average amount of control gel diet consumed per mouse ranged from 6.4 to 8.4 g/24 h during the first 14 days of the study (Figure 1). During the same time interval, animals maintained on the NIH-07 pellet diet
Bioavailability of Coal Tar Components
Chem. Res. Toxicol., Vol. 4, No. 4, 1991 469 0 control
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Figure 2. Effect of coal tar ingestion on animal body weight was evaluated in animals fed a coal tar adulterated diet prepared with coal tar sample C. Nine groups of 5 male B6C3F1 mice were maintained on control diets for 0-14 days. On day 14, eight groups of mice were given gel diets containing0.1,0.2,0.5,1,2,5, or 10% coal tar. Adulterated diets were maintained for 15 days. Values for a 0.1% diet were similar to those for a 0.2% diet while the values for a 5 and 10% diet were similar to those for a 2% diet.
consumed 3.8-4.4 g/24 h. Water consumption for mice maintained on the gel diet averaged 1.6 mL/24 h while those on the NIH-07 pellet diet averaged 5.6 mL/24 h. The total mass of water and food consumed by both groups of animals over a 24-h time interval was similar (agar gel diet: 8.4 g of food, 1.6 g of water; NIH-07 pellet diet: 4.4 g of food, 5.6 g of water). Animals acclimated to control gel diets for 14 day were switched to coal tar adulterated diets ranging from 0.1 to 10% coal tar. Animals were maintained on these diets for an additional 14 days. Animals given 2,5, and 10% coal tar adulterated diets refused to eat (Figure 1)and rapidly lost body weight (Figure 2). Animals receiving the 0.5 or 1% adulterated diets refused to eat the diets for 2 and 4 days, respectively. However, these animals eventually tolerated the adulterated diets. Animals on a 0.5% diet regained lost body weight, in contrast to animals on a 1% diet where body weight loss was continued. In contrast, animals fed the 0.1 or 0.2% adulterated diet readily con-
Figure 3. Representative PEI-cellulose TLC maps of chemical-DNA adducts formed in tissues of mice following 14 days of consuming gel diets adulterated with coal tar sample C. Maps 1-4 are representative of 32P-labeleddigests of DNA from lung: 1 = control, 2 = 0.1% diet, 3 = 0.2% diet, 4 = 0.5% diet. Maps 5-8 are representative of 32P-labeleddigests of DNA from forestomach: 5 = control, 6 = 0.1% diet, 7 = 0.2% diet, 8 = 0.5% diet. Autoradiography was at -80 "C for 18 h. The origin is located at the bottom left-hand corner of each chromatogram and was excised prior to autoradiography. Table 11. DNA Adduct Levels in Lung and Forestomach of Mice Ingesting Coal Tar SamDle C" % coal tar diet 0.1 0.2 0.5 1.0 0.18 f 0.03 0.11 f 0.04 0.81 f 0.16 0.92 f 0.40 lung forestomach 0.44 f 0.18 0.28 f 0.06 0.72 f 0.21 0.45 f 0.06 a Lung values represent the met& f range of two determinations while forestomach values represent the mean f SE of three determinations. No evidence of DNA modification was observed on the PEI-cellulose TLC maps of 32P-postlabeleddigests of DNA isolated from lung and forestomach of animals maintained on a nonadulterated control basal gel diet.
sumed the diet and had weight gains similar to control animals. DNA Binding in Lung and Forestomach. Chemical-DNA adduct formation was evaluated in animals maintained on a 0.1,0.2,0.5, and 1%coal tar diet prepared with coal tar sample C and a nonadulterated control gel diet. Preliminary analysis detected chemical-DNA adduct formation in tissues of forestomach, intestines, liver, lung, spleen, heart, and skin of animals maintained on a coal tar diet. Representative maps of lung DNA adducts detected by the 32P-postlabelingmethod are presented in Figure 3. Adduct separation using 10 cm x 10 cm PEI-cellulose TLC plates resulted in a diagonal band of radioactivity with distinct adducts distributed throughout the zone. Similar patterns were also detected for forestomach, spleen, liver, intestines, heart, and skin DNA samples (data not shown). DNA modification was not detected with DNA isolated from animals maintained on a control gel diet. Quantitation of chemical-DNA adducts in tissues was limited to forestomach and lung since these organ sites are particularly susceptible to PAH chemical carcinogenesis (20). A dose-related increase in DNA adduct levels was observed in lung tissue (Table 11). Adduct levels were 4 times greater in animals fed a 0.5 or 1% diet relative to the levels detected in animals maintained on 0.1 or 0.2% diet. In contrast, the dose-related effect on DNA adduct formation in forestomach was not as apparent. The highest forestomach DNA adduct levels were detected in animals fed a 0.5% diet while the lowest levels were detected in animals on a 0.2% diet. The relative adduct levels between lung and forestomach tissue within groups of animals varied
Weyand et al.
470 Chem. Res. Toxicol., Vol. 4, No. 4, 1991
Table 111. Amount of Coal Tar Ingested by Animalsa coal tar sample, mg day A B C D mix BlalP. mg 0-1 5 4 8 7 6 0.02 111 105 140 116 95 0.25 1-14 8 6 7 0.01 14-15 6 7 total 122 117 156 129 108 0.28 a Values represent the maximum amount of material ingested by animals which was calculated on the basis of the amount of gel diets consumed by groups of mice during the time intervals listed above (see Figure 1). Calculations have not taken into account weight loss associated with gel diet dehydration.
according to the amount of coal tar in diets. Forestomach DNA adduct levels were higher than lung levels in animals fed a 0.1 and 0.2% diet. In contrast, the opposite was observed in animals maintained on a 0.5 and 1% diet where DNA adduct levels in lung were considerably higher than in forestomach DNA. Comparison of Coal Tar Samples. Chemical-DNA adduct formation and urinary excretion of PAH metabolites were evaluated by using coal tar samples which varied in chemical composition. Four coal tar diets containing 0.25% tar and a mix diet, containing 0.25% tar prepared from mixing equal proportions of the four coal tar samples together prior to diet preparation, were evaluated. In addition, a diet containing a single hydrocarbon (B[a]P) comparable to the amount present in a 0.25% diet prepared from coal tar sample C was also evaluated. Animals were maintained on diets for 15 days with urine collected during the first and last day of diet administration. All adulterated diets were readily consumed by animals, and body weight gains were determined to be similar to those for control animals (data not shown). The estimated amounts of coal tar and B[a]P ingested by animals in this experiment are provided in Table 111. The amount of coal tar ingested ranged from 108 to 156 mg/animal while the amount of B[a]P ingested was 0.28 mg/animal. Adduct formation was evaluated in forestomach, lung, and spleen tissue. Spleen was included in these analyses since preliminary experiments determined that DNA adduct levels following B[a]P ingestion are considerably greater in spleen than liver, heart, or skin tissue sites. Adduct maps were similar to those previously observed with the ingestion of coal tar sample C (Figure 4). DNA modification was not detected in animals maintained on a control gel diet. A single B[a]P-DNA adduct spot was detected in lung, forestomach, and spleen tissue of animals fed a B[a]P-adulterated diet. On the basis of its chromatographic location on the PEI-cellulose, this spot is presumed to be the B [a]P diol epoxide-deoxyguanosine adduct. Adduct levels varied considerably between the type of coal tar diet ingested and the tissue sites examined (Figure 5). The highest adduct levels were detected in lung tissue. The relative order of binding to lung DNA among diets was C 1 mix > D > A > B > B[a]P. The levels of DNA adducts in forestomach and spleen of animals ingesting coal tar sample C or B were considerably higher than the levels observed with the other adulterated diets. In all cases, adduct levels in animals ingesting a B [a ]P-adulterated diet were among the lowest detected. The levels of DNA adducts formed in lung, forestomach, and spleen did not correlate with total PAH content of coal tar samples. With the exception of coal tar sample D, a negative correlation is observed with lung tissue data when chemical-DNA adduct levels are compared with the coal tar content of hydrocarbons containing three or more fused rings (Table I and Figure 6).
Figure 4. Representative PEI-cellulose TLC maps of chemical-DNA adducts formed in tissues of mice following 14 days of consuming gel diets adulterated with coal tar or B[a]P. Maps a-c were obtained from animals ingesting B[a]P: a = lung, b = forestomach, c = spleen. Maps d-f were obtained from animals ingesting coal tar sample B: d = lung, e = forestomach, f = spleen. Autoradiography was at -80 "C for 18 h. The origin is located a t the bottom left-hand corner of each chromatogram and was excised prior to autoradiography.
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Figure 5. Chemical-DNA adduct levels in tissues of mice following a 14-day ingestion of gel diets containing coal tar samples A-D, a mix of the four, or B[a]P. Adduct levels were determined by using the 3T-postlabelingmethod as described under Materials and Methods. Values represent the mean f range for 2 separate determinations except for forestomach sample B, n = 4, and lung/spleen samp!e C, n = 3, which represent the mean f SE, and forestomach mix, which represents a single determination.
A notable difference was detected in the levels of DNA adducts formed in lung tissue between animals maintained on a 0.2 and 0.25% coal tar diet adulterated with coal tar sample C. Animals fed a 0.25% diet had adduct levels similar to those observed with a 0.5 and 1% diet (Table 11). These levels were 8 times higher than the levels observed in animals fed a 0.2% diet. Similar levels of lung DNA adducts were observed from the ingestion of the other 0.25% coal tar diets. The unexpected and dramatic increase in adduct levels between a 0.2% and 0.25% diet observed with sample C suggests the possibility of a dose-related threshold effect for coal tar ingestion. PAH metabolites excreted in urine of animals ingesting a control or adulterated gel diet were determined by using HPLC with fluorescence detection. Urine samples were treated with P-glucuronidase/arylsulfatase,and metabolites were isolated by solid-phase extraction. Representative HPLC chromatograms of reference standards and urine extracts are illustrated in Figure 7. This chroma-
Chem. Res. Toricol., Vol. 4, No.4, 1991 471
Bioauailability of Coal Tar Components
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