* Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas; and
Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received September 9, 1999; accepted March 10, 2000
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ABSTRACT |
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Key Words: bioavailability; 32P-postlabeling; coal tar; DNA adducts; genotoxicity; polycyclic aromatic hydrocarbons; soil ingestion; coal tar; contaminated soil.
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INTRODUCTION |
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CT is a complex mixture of thousands of compounds, most of which have not been identified. Compounds in CT extracts are mutagenic in the Salmonella histidine reversion assay (Donnelly et al., 1993). Other studies have demonstrated that CT forms DNA adducts and is carcinogenic in vivo under a number of treatment protocols (Culp et al., 1998
; Randerath et al., 1996
; Rodriguez et al., 1997
; Weyand et al., 1995
; Weyand and Wu, 1995
). It generally is accepted that the mutagenic and carcinogenic potential of CT is due in part to the activity of the PAH fraction. PAHs are metabolized to reactive intermediates that covalently bind to DNA, resulting in the formation of DNA adducts. This is hypothesized to be an initiating event in PAH-induced mutagenesis and carcinogenesis (Miller and Miller, 1971
).
A potential route of exposure to CT is through ingestion of contaminated soils, inhalation of soil particles, and skin absorption. However, soil appears to alter the biological availability of PAHs. Several studies have reported that both the genotoxicity and metabolite concentration in tissues and urine is reduced for soil-bound pollutants (Bonaccorsi et al., 1984; Koganti et al., 1998
; McConnell et al., 1984
; Umbreit et al., 1986
; Van Schooten et al., 1997
). As CT and PAHs are common soil pollutants, it is important to determine how the presence of chemicals in soil influences their bioavailability.
An accurate method for determining the percentage of pollutant that is desorbed from the soil and absorbed into the systemic circulation (bioavailability) is essential for risk assessment. Recently, the Agency for Toxic Substances and Disease Registry (ATSDR) proposed an acute oral minimum risk level (MRL) for tetrachlorodibenzo-p-dioxin (TCDD) based on a study in mice exposed to TCDD in corn oil by gavage. However, because TCDD exposure primarily results from ingestion of contaminated foods and soil, the MRL was adjusted to account for the greater bioavailability from corn oil compared to that from food or soil (De Rosa et al., 1998). Consequently, lack of data regarding the impact of soil on bioavailability of PAHs and CT, common soil pollutants, has forced stringent regulations that will continue until more data are obtained to determine a reliable soil-based MRL.
The goal of this study was to determine if the genotoxicity of CT is decreased due to the presence of soil or aging of PAHs in soil. A 17-day feeding study was performed to assess DNA adduct levels in liver and lung tissues of Fischer 344 rats. 32P-postlabeling was coupled with TLC and HPLC analysis to separate and identify specific adducts formed from exposure to CT. Histopathological changes in the liver also were investigated.
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MATERIALS AND METHODS |
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Soil Preparation
It was critical that a well-characterized soil free of contaminants be used. Because the facility at which the CT was obtained could not guarantee that the background soil was free of contaminants, a Weswood soil was chosen. This soil is readily available and known to be free of chemicals that might have a toxic affect on animals. Weswood soil (pH 7.8, 1.7% organic content) was characterized as a silty, clay loam with 12% sand, 49% silt, and 39% clay by the Soil and Crop Sciences Department at Texas A&M University, College Station, TX. Soil was ground and sieved to 2 mm, autoclaved, and dried. The soil was rehydrated with water to a capacity of 18%. Six kilograms of soil was prepared and stored in stainless steel pans and covered with foil. The soilpreparations were tilled and watered as needed. Soil was aged in a plastic greenhouse and exposed to seasonal humidity and temperature fluctuations in College Station, TX.
Two hundred milliliters of a CT/methylene chloride mixture was added for every 1 kg soil to achieve 5% CT by weight. Unaged CT/soil was prepared the week before dosing began, and aged CT/soil was prepared 9 months prior to dosing. To achieve a final concentration of 0.35% CT in the diets, rodent chow was prepared with 7% clean soil, CT, and CT/soil. A concentration of 7% soil in the diet was prepared as follows: 140 g of soil was combined with 1810 g of rodent chow (Purina Mills, St. Louis, MO) and 50 g of binder mixture (1:1:1 mixture by weight of sodium alginate, methylcellulose, and gum tragacanth). Approximately 1 L of sterile distilled water was added to 2 kg of dry mix in an autoclaved stainless steel bowl to form a thick paste. An autoclaved glass tube (1.5 x 40 cm) was inserted into feed and connected to a vacuum pump. Food was collected in a glass column, and when collection was complete the column was inverted and food extruded onto a tray. Food was cut into 3-cm pellets and dried.
Pure CT diet was prepared as above, substituting a 3:1 mixture of silicon dioxide and titanium dioxide (SiO2 and TiO2) for the soil to achieve the same concentration. SiO2 and TiO2 are generally recognized as safe (GRAS) and are commonly used as food additives (Furia, 1980). It was necessary to use the SiO2:TiO2 mixture to prevent clumping and allow for even dispersal of the CT in the chow. SiO2 and TiO2 are inert substances, so binding of CT to these compounds is minimal.
Animals and Treatment
Male Fischer 344 rats, 60 days of age and weighing between 210 and 230 g were obtained from Charles River Laboratories (Raleigh, NC). Rats were housed in an Association for the Assessment and Accreditation of Laboratory Animal Care International (AALAC)-accredited facility under the guidance of the National Health and Environmental Effects Research Laboratory Institution Animal Care and Use Committee. Rats were randomly assigned to control or CT treatment groups (five animals per group) and grouped two per cage. Rats were housed in polycarbonate cages containing heat-treated pine shavings (Northeastern Products Corporation, Warrensburg, NY) and acclimated for 2 weeks. Light was maintained on a 12-h light/dark cycle with room temperature at 21 ± 2.2°C and relative humidity at 50 ± 10%.
A pilot study was performed using 0.25% and 0.35% CT to assess palatability and weight gain (data not shown). The results from this study showed that there were no differences in weight gain from the control and no signs of toxicity associated with ingestion of CT. Based on these results and those of Weyand and coworkers (1994) the higher of the two doses was chosen (0.35%).
Rats were fed a diet of 1) control chow, 2) soil, 3) 0.35% CT/SiO2:TiO2, 4) 0.35% unaged CT/soil, and 5) 0.35% aged CT/soil. Food and water were administered ad libitum. Food consumption and body weights were monitored regularly throughout the experiment. After 17 days of treatment, animals were transferred to individual metabolism cages (Nalge Co., Rochester, NY) and provided pelleted food (dustless, 45-mg, rodent grain-base formulation, BioServe, Frenchtown, NJ) and deionized water ad libitum. On day 18 the rats were euthanized via CO2 asphyxiation, at which time the lungs and liver were removed, minced, split, and frozen with liquid nitrogen. Tissues were stored at 80°C prior to analysis.
A replicate experiment with three rats per treatment group was performed to determine the cause of liver enlargement. Animals were similarly euthanized and necropsied. Blood was collected and serum was obtained using sterile Vacutainer® clot tubes (Fischer Scientific, PA). At necropsy, the liver and lungs were examined for gross lesions, fixed in 10% neutral-buffered formalin (Fisher Scientific, PA), and processed for microscopic evaluation.
Clinical Chemistry and Pathology
Plasma was analyzed for alkaline phosphatase (ALP), alanine aminotransferase (ALT), 5'-nucleotidase (5'-ND), total bilirubin, and albumin bilirubin. Commercially available kits (Sigma, St. Louis, MO.) were adapted for use on a centrifugal spectrophotometer analyzer (Cobas Fara II, Roche Diagnostics, Branchburg, NJ). Lung and liver tissues were processed by routine methods to paraffin, sectioned to 5 microns, and stained with hematoxylin and eosin for microscopic examination.
Chemical Analysis
Triplicate samples of CT/SiO2:TiO2, unaged CT/soil, and aged CT/soil prior to and after incorporation in rodent diet were prepared for gas chromatography/mass spectrometry (GC/MS) analysis by passing 10 g through a 2-mm sieve and then extracting three times with a 1:1 mixture of hexane:acetone. The organic phase was dried to a residue and reconstituted in a 3:1 hexane:methylene chloride mixture. The extract was passed through a Bakerbond® silica cartridge (6 ml/g), using hexane as a mobile phase, and concentrated to 1 ml.
The PAHs analyzed are commonly found in CT. Standardized methods developed by the U.S. EPA (1985) were followed to monitor, identify, and quantify PAHs. The analysis was performed using a Hewlett-Packard 5890 Series II gas chromatograph coupled to a 5972 mass selective detector employed in full-scan mode (from 50 to 500 amu). The MS was tuned with decafluorotriphenylphosphine (DFTPP) with 1 µl of the GC/MS tuning standard injected. Each 1-µl test sample injection was made with a HP 7673 autoinjector in splitless mode. The GC column was a 30 m x 0.25 mm ID x 0.25 µm film thickness HP-5MS capillary column (Hewlett Packard, Palo Alto, CA). The injection port was maintained at 300°C and the transfer line was kept at 280°C. The oven temperature program was held at 35°C for 6 min. The temperature was increased 5°C/min to 300°C and maintained at this temperature for 20 min, for a total run time of 79 min. Data were collected with HP MS ChemStation Data Acquisition Software/EnviroQuant. The system was tuned daily and calibration standards at midconcentration containing all PAH analytes of interest were analyzed every 12 h during analysis. The limit of detection for each PAH approximates 13 parts per billion.
DNA Adducts
DNA was isolated from frozen liver and lung as previously described (Gupta, 1985). 32P-postlabeling for nonpolar DNA adducts was performed as described by Reddy and Randerath (1986). Briefly, 6 µg DNA or 50 µg PAH-adducted calf thymus DNA (Savela et al., 1995
) was digested to 3'-monophosphates using calf spleen phosphodiesterase and microccocal endonuclease according to the method of Gupta (1985). The 3'-monophosphates (5 µg) were digested further with nuclease P1 (5 units) to select for adducted nucleotides. Samples were incubated with 50 µCi of [
-32P] ATP (Amersham, 3000 Ci/mmol) and 5 units of T4 polynucleotide kinase (3'-phosphatase free) for 30 min. The number of normal nucleotides in each digest were determined by labeling 1.5 ng of 3'-monophosphates with 2.5 µl of 16.6 µCi [
-32P] ATP and 1.6 units of T4 polynucleotide kinase (3'phosphatase free) for 30 min. Normals were diluted with 195 µl 10 mM Tris/5 mM EDTA (pH 9.5).
The total mixture of adducted nucleotides was spotted onto the origin of PEI-cellulose chromatography sheets and developed with the following solvents: D1 and D5, 1.7 M sodium phosphate (pH 6.0); D2 prewash, 2.5 M ammonium formate (pH 3.5); D3, 7.0 M urea/4.5 M lithium formate (pH 3.4); D4 prewash, 0.5 M Tris (pH 8.0); and D4, 7 M urea/1.1 M lithium chloride/0.5 M Tris (pH 8). Five microliters of diluted normal nucleotides were developed in one direction with 0.3 M ammonium sulfate/10 mM sodium phosphate (pH 7.4). Radioactivity was detected by exposure to Kodak® XARS X-ray film for 16 h at 80°C for adducted nucleotides and 30 min at room temperature for normal nucleotides. The excised spots were counted using 5 ml Optifluor or 95% ethanol (for HPLC analysis), using a Packard scintillation counter.
Calculations
Relative adduct levels (RAL):
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These calculations are based on the assumption that adducts were recovered and labeled quantitatively.
Relative Adduct Levels/Total Ingested PAHs (RAL/TIP)
To obtain the total dose of PAHs ingested, the total of PAHs in diet was multiplied by the mean food consumption per rat to obtain mg PAH ingested. This was then divided by the mean body weights on day 18 (Fig. 1) to obtain mg PAH ingested/kg BW. The total ingested PAH/kg BW (TIP) was then divided into the relative adduct levels (RAL) to obtain RAL/PAH ingested and is presented in the text as RAL/TIP.
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32P-postlabeled TLC profiles from hepatic and lung DNA were compared to migration patterns of PAH-adducted calf thymus DNA standards by orienting and aligning calf thymus DNA standards with 32P-postlabeled liver and lung DNA from treated rats. Comigration of calf thymus DNA standards with DNA adducts from 32P-postlabeled liver and lung DNA were recorded to provide a qualitative estimate of the PAHs present in the CT mixture that form DNA adducts.
Data Analysis
32P-postlabeling data were analyzed for treatment differences using a one-way analysis of variance (p < 0.05). Body weight and time effects were analyzed using a two-way analysis of variance (p < 0.05) by SigmaStat software 2.0 (Jandel Scientific, San Rafael, CA).
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RESULTS |
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Although mild changes in the livers of CT-treated rats were observed after microscopic evaluation, severe toxicity such as necrosis was not observed. Livers from CT-treated rats showed diffuse panlobular hepatocellular swelling, scattered periportal vacuolar change, and mild anisokaryosis (Fig. 2). Livers from animals treated with unaged CT/soil also showed diffuse panlobular hepatocyte swelling and mild anisokaryosis, as well as scattered mitotic bodies and some apoptotic cells (Fig. 2
). Rats treated with aged CT/soil had diffuse hepatocyte swelling and decreased staining intensity, within centrilobular hepatocytes that corresponded with moderate vacuolation in centrilobular hepatocytes (Fig. 2
). These hepatocyte vacuoles were small, discreet, and round, morphologically consistent with cytoplasmic lipid accumulation. Other treatment groups were not morphologically different from controls. ALP and 5'-ND levels were elevated over controls by approximately 25% and 44%, respectively (Table 5
). Lungs from treated rats were not different from controls.
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DISCUSSION |
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A complicating factor in studies dealing with CT is the nature of the material. CT is an extremely viscous and chemically complex material. Thus, obtaining a homogenous mixture is a universal problem. In this study, a SiO2:TiO2 carrier was used in the pure CT treatment group because it is an inert material, PAH binding is minimal, and it aids in the dispersal of CT when mixed in the chow. Without this carrier, the CT clumped and a homogenous distribution could not be achieved. The variation observed between CT diets (Table 3) is due to incorporation of CT in the rodent chow and not from binding to the SiO2:TiO2 carrier, based on GC/MS analysis (Table 2
).
The 32P-postlabeling procedure is a sensitive method to determine DNA adducts formed from unidentified components in CT. This assay works particularly well for the separation of DNA adducts formed from bulky aromatics (Reddy and Randerath, 1986). The assay is a viable method to assess bioavailability of CT-contaminated soil because 90% of CT is composed of PAHs that form nonpolar bulky aromatic DNA adducts. A slight decrease in adduct levels based on RAL values was observed for spot 2 in hepatic DNA from aged CT/soil-treated rats (Table 6
). Lower adduct levels for this spot could be due to several factors: 1) inconsistency in administered dose, 2) variations in the chemical composition of CT between treatment groups, or 3) comigration of various nonpolar adducts that were not present in the aged CT/soil.
Besides spot 2, DNA adduct levels in hepatic and lung tissue were not significantly different between treatment groups based on RAL values. However, total adducts in lung DNA were 3-fold higher than liver (Fig. 5). These results are consistent with other studies in which CT-induced DNA adducts were higher and more persistent in lung than in liver (Culp et al., 1994; Randerath et al., 1996
; Randerath et al., 1999
). Interestingly, a study by Culp and coworkers (1998) showed that CT diet administration to mice induced hepatocellular adenomas and carcinomas as well as alveolar/bronchiolar adenomas and carcinomas. Thus, CT is carcinogenic in both liver and lung tissues, suggesting that the differences in adduct levels do not reflect tumorigenicity outcomes.
Because DNA adducts are the endpoint of interest in this study, it was necessary to correct adduct levels based on the amount of PAHs ingested. Once adduct levels were corrected to obtain RAL/TIP, it was revealed that the PAH fraction of CT binds to soil, making it less bioavailable (Tables 6 and 7). This is based on decreased adduct levels observed in liver and lung DNA for CT/soil-treated rats compared to CT-treated rats. DNA adduct analysis illustrates that soil:chemical interactions play a role in the uptake or metabolism of PAHs in CT. However, in this study, residence time in the soil had no effect of PAH uptake.
It has been demonstrated that both soil and chemical aging does effect the bioavailability of PAHs. Hatzinger and Alexander (1995) showed that aging of phenanthrene and 4-nitrophenol in soil resulted in decreased microbial biodegradation and solvent extractability, possibly a result of partitioning of chemicals in the soil matrix. Other studies have shown a decrease in bioavailability of CT-contaminated soils aged for 100200 years compared to the solvent extracts of these same soils. Koganti and coworkers (1998) demonstrated that chemical:DNA adduct levels in lung and 1-hydroxypyrene levels in urine of B6C3F1 mice treated for 14 days with diets containing MGP contaminated soils were lower than their corresponding organic extracts. Van Schooten (1997) examined absorption and excretion of anthracene, pyrene, and B[a]P and their metabolites in the blood, urine, and feces of rats orally exposed to contaminated soils and pure compounds. It was documented that the soil-treated groups had higher levels of unchanged pyrene and B[a]P and lower levels of metabolized pyrene in fecal extracts compared to controls.
These studies have evidenced that the uptake and metabolism of biologically reactive compounds in aged contaminated soils are less than their organic extracts. However, it does not prove that the decrease in availability was due to contaminant aging and not soil presence. The experimental protocol in this study involved comparing a soil sample that was freshly spiked with CT and one that had been aged for 9 months. This study demonstrates that the decrease in bioavailability is due to soil presence and that aging has no effect on the formation of DNA adducts. These results could have changed had a weathered CT/soil sample at the MGP facility been obtained. It is probable that a decrease in availability would be apparent in soil aged for 100200 years compared to a sample aged for 9 months. However, to control for aging, researchers must use organic extracts of the aged soil or substitute another CT material and add this to clean soil obtained from the site of interest. The issue of bioavailability based on soil presence and aging may be best addressed by obtaining pure CT and then incorporating this into a well-characterized soil, allowing researchers to use the same soil type and CT, and control the aging process.
Soil characteristics also may dramatically affect the bioavailability of contaminants in soil. Soil is composed of a complex matrix of substances that can absorb pollutants. Factors such as particle size, pH, levels of pollutants in the soil, and percent sand, organic matter, silt, and clay affect the extent to which a pollutant is absorbed (Duffus, 1979; Ibbotson et al., 1989
; Karickhoff et al., 1979
). The experimental soil in this study was a Weswood soil, typically found in Texas, characterized as a silty, clay loam with 1.7% organic content, 12% sand, 49% silt, and 29% clay (pH 7.8). It was evidenced that this soil does alter the systemic bioavailability of PAHs. These results could have differed had the soil been obtained from a beach (with little or no clay and organic matter) or from an area with soil containing higher clay content and/or organic matter.
Both liver and lung DNA showed genetic damage that was dependent on PAH/soil binding interactions. However, pathological observations did not reflect decreased toxicity in liver based on soil presence. Clinical pathology showed slight elevation in ALP in CT/soil treatment groups, but not in CT-treated rats. 5'-ND levels were slightly increased in all CT treatment groups. Typically, with PAHs, these elevated enzyme levels reflect an increase in P450 content. Histological observations of liver tissue showed mild toxicity associated with CT, but differences in toxicity did not correlate with the presence of soil. Results from this study suggest that a relationship between hepatic DNA damage and liver toxicity exist, although microscopic examination does not support the hypothesis that soil alters the bioavailability of PAHs in CT. Histopathology and clinical chemistry, combined with body weight data, reveal that the MTD was not reached in this study. Had the MTD been reached, necrotic lesions and severe body weight loss would have been observed. Although animals in the CT treatment group lost 15% of their original body weight in the first 4 days of treatment, it is apparent that this is due to failure of rats to consume their diets, and not to toxicity.
The identification of DNA adducts formed from exposure to complex mixtures is difficult because of high background activity from the zone, which is an area of multiple putative DNA adducts. In this study, identification of DNA adducts was attempted using both HPLC and TLC. The peaks eluting at 0.73 and 1.32 most likely correspond with either spot 1, 2 and 3 in hepatic DNA or the intense area of radioactivity close to the origin in the lung (Figs. 3 and 4). Based on HPLC analysis, it is possible that these adducts are formed from anti-B[j]FDE, anti-CPPE, and anti-DB[a,l]PDE, which were identified in all the CT treatment groups (Fig. 6
, Table 3
). More importantly, adducts associated with B[a]P were not observed, although this compound is present in the CT mixture. Prahalad and coworkers (1997) demonstrated that dibenzo[a,l]pyrene and cyclopentapyrene are more potent than B[a]P in DNA adduction and tumorigenicity in the strain A/J mouse lung and induce adducts at concentration as low as 0.3 mg/kg. Goldstein and coworkers (1998) compared tumors and DNA adducts in mice exposed to B[a]P and CT. It was revealed that B[a]P content in CT did not account for the tumor induction or DNA adduction in mice, suggesting that risk assessments based on B[a]P content do not accurately reflect the total risk to humans exposed to CT.
In conclusion, the present study showed that soil decreases the uptake of the genotoxic components in CT. However, aging of CT in soil does not result in a decrease in genotoxicity, as observed by DNA adduct analysis. Bioavailability is an important issue in calculating the risk associated with exposure to contaminated soils. The health risks associated with exposure to contaminated soils are decreased if compounds are less available. This has a major impact on regulatory decisions and may ultimately decrease the time and cost of remediating contaminated sites. Results from this study suggest that assuming 100% bioavailability of CT and PAHs in soil overestimates the potential health risks of exposure to CT-contaminated soils.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be sent at present address: NIEHS, Mail Drop B3-07, P.O. Box 12233, 111 Alexander Drive, Research Triangle Park, NC 27711. Fax: (919) 541-4255. E-mail: bordelo1{at}niehs.nih.gov.
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