Quantification of DNA Adducts Formed in Liver, Lungs, and Isolated Lung Cells of Rats and Mice Exposed to 14C-Styrene by Nose-Only Inhalation

Abstract

Bronchiolo-alveolar tumors were observed in mice exposed chronically to 160 ppm styrene, whereas no tumors were seen in rats up to concentrations of 1000 ppm. Clara cells, which are predominant in the bronchiolo-alveolar region in mouse lungs but less numerous in rat and human lung, contain various cytochrome P450s, which may oxidize styrene to the rodent carcinogen styrene-7,8-oxide (SO) and other reactive metabolites. Reactive metabolites may form specific DNA adducts and induce the tumors observed in mice. To determine DNA adducts in specific tissues and cell types, rats and mice were exposed to 160 ppm [ring-U-14C]styrene by nose-only inhalation for 6 h in a recirculating exposure system. Liver and lungs were isolated and 42 h after exposure. Fractions enriched in Type II cells and Clara cells were isolated from rat and mouse lung, respectively. DNA adduct profiles differed quantitatively and qualitatively in liver, total lung, and enriched lung cell fractions. At and 42 h after exposure, the two isomeric N7-guanine adducts of SO (measured together, HPEG) were present in liver at 3.0 ± 0.2 and 1.9 ± 0.3 (rat) and 1.2 ± 0.2 and 3.2 ± 0.5 (mouse) per 108 bases. Several other, unidentified adducts were present at two to three times higher concentrations in mouse, but not in rat liver. In both rat and mouse lung, HPEG was the major adduct at ∼1 per 108 bases at h, and these levels halved at 42 h. In both rat Type II and non-Type II cells, HPEG was the major adduct and was about three times higher in Type II cells than in total lung. For mice, DNA adduct levels in Clara cells and non-Clara cells were similar to total lung. The hepatic covalent binding index (CBI) at and 42 h was 0.19 ± 0.06 and 0.14 ± 0.03 (rat) and 0.25 ± 0.11 and 0.44 ± 0.23 (mouse), respectively. The pulmonary CBIs, based on tissues combined for and 42 h, were 0.17 ± 0.04 (rat) and 0.24 ± 0.04 (mouse). Compared with CBIs for other genotoxicants, these values indicate that styrene has only very weak adduct-forming potency. The overall results of this study indicate that DNA adduct formation does not play an important role in styrene tumorigenicity in chronically exposed mice.

Article

Styrene is an important industrial chemical used for the production of reinforced plastics and various polymers. Review of 11 long-term toxicity studies to evaluate the carcinogenic potential of styrene led to the conclusion that there was no convincing evidence of carcinogenic activity of styrene in animals, although some of the studies were considered inadequate (McConnell and Swenberg, 1994). Recent state-of-the-art carcinogenicity studies in CD-1 mice and Sprague-Dawley rats exposed to styrene for 2 years via inhalation revealed an increased incidence of masses in the bronchiolo-alveolar region in the lungs of mice exposed to 160 ppm styrene, but no increases in tumor incidence in rats exposed to up to 1000 ppm styrene (Cruzan et al., 1998, in press).

In 1994, IARC concluded that there was inadequate evidence in humans and limited evidence in experimental animals for the carcinogenicity of styrene. Nevertheless, styrene was classified as a Type 2B carcinogen, i.e., a possible human carcinogen, because it is extensively metabolized in human and animal tissues to the genotoxic metabolite styrene-7,8-oxide (SO) (IARC, 1994a,IARC, 1994b). However, the concentration of SO or other reactive metabolites available for macromolecular binding will be the net result of formation and subsequent detoxification reactions. SO is rapidly detoxified to more water-soluble compounds and excreted in the urine. In this study, approximately 95% and 85% of the styrene retained upon inhalation was excreted as urinary metabolites by rats and mice, respectively (Boogaard et al., 2000). As depicted in Figure 1, important routes of detoxification of SO are glutathione conjugation, eventually leading to the urinary excretion of the corresponding mercapturic acids, and epoxide hydrolysis, eventually leading to the urinary excretion of mandelic, phenylglyoxylic, and hippuric acid. Quantitatively, the glutathione pathway was equally important in rats and mice, accounting for about a quarter of the total urinary metabolites in the present study (data not shown), whereas the EH pathway accounted for 63% and 31% of total urinary metabolites in rats and mice, respectively. The excretion of phenylaceturic acid, probably derived from phenylacetaldehyde, was also an important pathway in mice, accounting for 22% of the total urinary metabolites, whereas in rats phenylaceturic acid accounted for only 3% of the total urinary metabolites.

In some cases lung selective injury is simply related to the levels of toxicant reaching the respiratory tract. The apparent levels of microsomal cytochrome P450 monooxygenases in both animal and human lungs are less than 10% of those present in the liver (Bond, 1993). Nevertheless, the highly focal localization of these enzymes to only a few of the many cell types within the lung predisposes these cells to a disproportionate susceptibility to chemicals that undergo metabolic activation. This may also apply to styrene, as metabolic activation to SO has often been considered to be responsible for its carcinogenic effects (Phillips and Farmer, 1994). Alternatively, ringoxidation of styrene or SO (see Fig. 1), catalyzed by specific isoforms of cytochrome P450, which might subsequently lead to reactive ringopened products, might explain the susceptibility differences. Evidence for pathways involving ringopening of styrene, accounting for up to 10% of the metabolism of styrene in mice but almost absent in rats, has been reported (Sumner et al., 1995).

Clara cells in mouse lung and Type II cells in rat lung are considered to be responsible for a significant proportion of the oxidative metabolizing capacity of this tissue (Pinkerton et al., 1997). Clara cells, the target cells for styrene-induced pneumotoxicity (Cruzan et al., 1997), are a predominant cell type in mouse lung but not in rat lung. In human lung, nonciliated cells are present in the bronchiolar epithelium, but the morphology of these cells differs significantly from the Clara cell morphology seen in rodents. The most striking difference is the low proportion of agranular endoplasmatic reticulum, which accounts for 55 ± 8% and 66 ± 10% of the cellular components in mouse and rat Clara cells, but for only 3.1 ± 3.5% in human nonciliated bronchiolar epithelial cells (Pinkerton et al., 1997). Therefore, an important question to answer is whether the effects of styrene observed in mouse lungs are relevant for humans. In the mechanistic studies described here, we investigated the covalent binding of styrene metabolites to DNA in mouse Clara cells and in rat Type II cells to provide information to assist in understanding the causes of the sensitivity of the mouse to the effects of styrene and help determine the relevance of the effects in mice for human risk assessment. In the past, in vivo experiments with radiolabeled styrene and SO demonstrated in rodents that both compounds cause a low level of DNA binding (reviewed by Phillips and Farmer, 1994). Following ip administration of styrene, the two regio-isomers of the N7-guanine alkylation product of SO, N7-(2-hydroxy-1-phenylethyl)guanine and N7-(2-hydroxy-2-phenylethyl)guanine (HPEG), were the main DNA adducts formed, whereas the corresponding N2- and O6-guanine adducts were formed in much lower concentrations (Byfält-Nordqvist et al., 1985; Pauwels et al., 1996). In vitro reactions of SO with DNA or nucleosides also results in HPEG as the major adduct, but several other minor adducts, such as the N2- and O6-guanine and the N6-adenine adducts of SO, were also found (reviewed by Phillips and Farmer, 1994, and Segerbäck, 1994). To the best of our knowledge, adducts from styrene metabolites other than SO have never been reported. In the present study, styrene with a high specific radioactivity was used, which enabled detection of DNA adducts without any preselection at a level of approximately one adduct per 108 nucleotides in a sample of 1 mg DNA.

MATERIALS AND METHODS

Safety

Styrene (CAS no. 100-42-5) is a flammable, toxic agent. Ventilation, both local and general, must be utilized as necessary to minimize exposure to styrene. Minimum standard protective apparel and equipment for work with styrene includes disposable, chemical-resistant gloves, laboratory coats, and safety glasses with side shields. Personnel transferring styrene must also wear a respirator and must use containers that are electrically grounded to transfer the styrene.

Chemicals

For the pilot studies, [ring-U-14C]-styrene with a specific radioactivity of 55.2 Ci/mol (2.04 TBq/mol) and a radiopurity of 96% (major contaminant 4% 14C-benzaldehyde) was prepared as described elsewhere (Boogaard et al., 2000). For the inhalation studies [ring-U-14C]-styrene with a specific radioactivity of 52 Ci/mol (1.92 TBq/mol) was prepared in two batches as described elsewhere (Boogaard et al., 2000). Chemical purity was > 96%. Radiochemical purities were 96.8 and 96.7%, the major contaminant being 3.0 and 3.2% [ring-U-14C]-benzaldehyde for the first and second batch, respectively.

The diastereomers of N7-2-hydroxy-1-phenylethyl-2`-deoxyguanosine 3`-monophosphate were a gift from Dr. K. Hemminki (Finnish Institute of Occupational Health, Helsinki, Finland). Guanidinium hydrochloride, N-[2-hydroxyethyl]piperazine-N`-[2-ethanesulfonic acid] (HEPES), 3-[N-morpholino]propanesulphonic acid (MOPS), sodium chloride, sodium edetate dihydrate, sodium hydroxide, TRIS base, Triton-X100, and Tween-20 were all molecular biology grade (DNase-free) and purchased from Sigma (Sigma-Aldrich, Zwijndrecht, The Netherlands). Acid phosphatase (Type I, from wheat germ), adenosine, adenosine 5`-monophosphate monohydrate (AMP), alkaline phosphatase (Type III, from Escherichia coli), benzaldehyde (BA), calf thymus DNA (Type I), corn oil, crystalline trypsin (Type I, from bovine pancreas), guanosine, Harris hematotoxylin, HEPES and phosphate-buffered saline (HEPES-PBS), methylene green, N7-methylcytosine, N7-methylguanine, Percoll, reduced β-nicotinamide adenine dinucleotide phosphate tetrasodium salt (NADPH), and venom phosphodiesterase I (Type VII, from Crotalus atrox), were purchased from Sigma. Benzoic acid, mandelic acid, N3-methyladenine, 1-phenylethanol, 2-phenylethanol, 1-phenyl-1,2-ethanediol were from Aldrich (Sigma-Aldrich). Nuclease P1 (from Penicillium citrinum) was purchased from ICN Biomedicals (Zoetermeer, The Netherlands). Nitrotetrazolium blue and methanol (Lichrosorb, HPLC grade) were obtained from Merck (Darmstadt, Germany), deoxyribonuclease I (DNase I, Grade II, from bovine pancreas) was obtained from Boehringer Mannheim (Mannheim, Germany), n-pentane (> 99.5%) and lithium heparin were obtained from Fluka (Buchs, Switzerland) and styrene oxide (SO, > 97%) was obtained from Riedel de Haën (Seelze, Germany). All other chemicals were of the highest purity available.

Adduct Syntheses

Reaction of styrene-7,8-oxide (SO) with guanosine.

N7-(2-hydroxy-1-phenylethyl)guanine and N7-(2-hydroxy-2-phenylethyl)guanine were prepared as a mixture of two regio-isomers (HPEG; see Fig. 2) from guanosine and SO as described earlier (Pauwels and Veulemans, 1998). HPLC analysis (System A) showed two adducts, with retention times of 25.9 and 26.9 min, for which the UV spectra were typical for N7-guanine adducts: λmax = 284 nm, λmin = 260 nm (first peak, diastereomeric N7-(2-hydroxy-1-phenylethyl)guanines) and 262 nm (second peak, diastereomeric N7-(2-hydroxy-2-phenylethyl)guanines), λshoulder = 248 nm. The two diastereomeric adduct pairs were isolated and purified by semipreparative HPLC (System B). The UV spectra (Lambda 16, Perkin Elmer, Norwalk, CT) showed λmax 280 nm, λmin 258 nm in 0.1 M NH4OH, λmax 282 nm, λmin 263 nm in 0.1 M TRIS/HCl pH = 7.4, and λmax 251 nm, λmin 233 nm in 0.1 M HCl, which are strongly indicative for HPEG (Byfält Nordqvist et al., 1985; Savela et al., 1986). Further identification was by LC-MS and 1H-NMR. LC-MS showed a molecular ion peak at m/z 272 (MH+) for both adducts. The spectrum for N7-(2-hydroxy-2-phenylethyl)guanine showed one additional peak at m/z 152, corresponding to protonated guanine. No further fragmentation details were seen. The adducts were dissolved in perdeuterated dimethylformamide for 1H-NMR: δ 8.09 (1H, s, N1-H), 7.95 (1H, s, C-8 H), 7.58, 7.40 (5H, m, C6H5), 6.69, (2H, br, NH2), 5.21, 5.19 (1H, dd, CH), 4.62, 4.59 (1H, dd, CH2), 4.37, 4.30 (1H, dd, CH2). The observed splitting pattern at δ 5.20, 4.60, and 4.32 ppm was typical for an ABX-system (JAB = 13 Hz, JBX = 9 Hz, JAX = 3.5 Hz) corresponding to the structure of a hydroxyethylphenyl adduct. N7-(2-hydroxy-1-phenylethyl)guanine was also prepared by neutral thermal hydrolysis (vide infra) of the two diastereomeric N7-(2-hydroxy-1-phenylethyl)-2`-deoxyguanine 3`-monophosphate adducts that were obtained from Dr. Hemminki. Both diastereomers co-eluted with the synthesized N7-(2-hydroxy-1-phenylethyl)guanine and showed the same characteristics in UV and MS as the synthesized product. The diastereomeric mixtures of N7-(2-hydroxy-1-phenylethyl)guanine and N7-(2-hydroxy-2-phenylethyl)guanine (HPEG) were used as nonlabeled reference standards throughout the study.

A small portion of the reaction product of guanosine and SO was not hydrolyzed with hydrochloric acid, but directly analyzed by HPLC (System C). The UV trace showed several peaks at λ = 254 nm, representing the various diastereomers of the guanosine adducts of SO. Analysis of the peaks with UV indicated that the peak with a retention time of 30.2 min represented most likely the N2-hydroxyethylphenylguanosine isomers. The peak was isolated and analyzed with UV and LC-MS. The UV spectra for the adduct showed the following characteristics: in 0.1 M NH4OH λmax 257 nm and λmin 281 nm, in 0.1 M TRIS/HCl (pH = 7.4) λmax 269 nm, and in 0.1 M HCl λmax 267 nm. LC-MS analysis showed a single large peak at m/z 272 (MH+), confirming the attribution as the N2-guanine adducts of SO to the isolated product.

Reaction of styrene-7,8-oxide (SO) with adenosine.

Adenosine was suspended in water:ethanol (1:1). The mixture was heated to 50°C; after addition of a 10-fold molar excess of SO, the solution was stirred for 24 h at 50°C. After cooling to room temperature, the solvent was evaporated. The white residue was suspended in 0.1 M HCl and refluxed for 1 h at 100°C. After cooling to room temperature, the mixture was neutralized with 1M KOH and the solvent evaporated. The resulting yellow solid was suspended in acidified water and filtered over a P3 glass filter. The filtrate was applied to a Sep-PAK solid-phase extraction cartridge (Baker, Phillipsburg, NJ), by-products were removed by washing with 10% methanol, and the adducts were eluted with 60% methanol. The solvent was evaporated to give a white solid. HPLC analysis (System C) of this solid showed two sharp peaks at λ = 254 nm, with retention times of 33.0 and 34.6 min. Both adducts were isolated and purified by semipreparative HPLC (System B). Further identification was by LC-MS and UV analysis (diode array spectrometer). The UV spectra for the first peak showed λmax 268 nm, λmin 233 nm (in 0.1 M NH4OH); λmax 275 nm, λmin 235 nm (in 0.1 M TRIS/HCl pH = 7.4); λmax 269 nm, λmin 231 nm (in 0.1 M HCl). LC-MS analysis showed the molecular ion peak at m/z 256 (MH+). Both adducts showed one additional fragment in the spectrum at m/z 136, corresponding to the protonated adenine ion. Based on UV and LC-MS evidence, the two adduct peaks were identified as N6-(2-hydroxy-1-phenylethyl)adenine and N6-(2-hydroxy-2-phenylethyl)adenine.

Reaction of SO with single-strand calf thymus DNA.

Double-strand calf thymus DNA was slowly heated in TRIS/HCl buffer (pH 7.40) and then rapidly cooled to obtain single-strand DNA, which was subsequently reacted with 10-fold molar excess of SO at 37°C for 48 h. The DNA was extracted, purified, and subjected to neutral thermal hydrolysis. Depurination products were isolated by ultrafiltration and analyzed by HPLC-UV and LC-MS. The two major adducts (accounting for 90% of the total) were the diastereomeric mixtures of N7-(2-hydroxy-1-phenylethyl)guanine and N7-(2-hydroxy-2-phenylethyl)guanine.

Reaction of benzaldehyde (BA) with calf thymus DNA.

Double-strand calf thymus DNA (90.7 mg) was dissolved overnight in 25 ml 10 mM TRIS/HCl buffer (pH 7.40). A 10-fold molar excess of BA (281 μl) was added in 10 ml ethanol, and the mixture was incubated at 37°C for 24 h. The excess BA was extracted twice with 30 ml n-pentane followed by a single extraction with 30 ml diethyl ether, then with 30 ml n-pentane. The DNA was isolated from an aliquot of the extracted reaction mixture by micro-ultrafiltration through a filter with a molecular weight cutoff of 10,000 daltons (Microcon-10, Amicon, Etten-Leur, The Netherlands). The residual DNA was dissolved in purified water and subjected to neutral thermal hydrolysis (NTH) and subsequent micro-ultrafiltration through a filter with a molecular weight cutoff of 3000 daltons (Centricon-3). The filtrate was analyzed by HPLC (System C) and LC-MS.

Reaction of benzaldehyde (BA) with adenosine 5`-monophosphate (AMP).

AMP (125 mg) was dissolved in 10 ml 10 mM TRIS/HCl buffer (pH 7.40) and 10 ml ethanol. A 10-fold molar excess BA (367 μl) was added, and the mixture was incubated for 24 h at 37°C. After the reaction was completed, the mixture was extracted three times with 30 ml n-pentane to remove any remaining BA. The extracted aqueous mixture was then subjected to NTH and subsequently analyzed by HPLC (System C) and LC-MS.

High-Performance Liquid Chromatography (HPLC)

System A.

Apparatus: HP1100 liquid chromatograph with UV detection = 254 nm) (Hewlett Packard, Amstelveen, The Netherlands) and on-line UV scanning over the range λ = 200–300 nm (Rapiscan SA 6508 multiple wavelength detector, Severn Analytical, Shefford, UK). Column: Beckman Ultrasphere (Fullerton, CA), 3 μm ODS, 250 × 4.6 mm. Eluent: purified water (A) and methanol (B). Flow: 1.0 ml/min. Gradient: 0–30 min, linear increase from 6 to 50% B; 30–50 min 50% B.

System B.

Apparatus: HP1100 liquid chromatograph with UV detection = 254 nm) (Hewlett Packard, Amstelveen, The Netherlands). Column: Macherey-Nagel (Düren, Germany), 7 μm ODS, 250 × 10.0 mm. Eluent: purified water (A) and methanol (B). Flow: 3.5 ml/min. Gradient: 0–30 min, linear increase from 6 to 30% B; 30–50 min 30% B.

System C.

Apparatus: Shimadzu Class VP liquid chromatograph equipped with SPD-10Avp variable wavelength detector set at λ = 254 nm, FRC-10A fraction collector (Shimadzu, Kyoto, Japan), and a Ramona 2000 on-line radioactivity detector (Raytest, Straubenhardt, Germany). Column: Beckman Ultrasphere, 3 μm ODS, 250 × 4.6 mm. Eluent: 50 mM ammonium formate pH 5.1 (A) and methanol (B). Flow: 1.0 ml/min. Gradient: 0–30 min, linear increase from to 50% B; 30–40 min 50% B.

Liquid Chromatography-Mass Spectrometry (LC-MS)

LC-MS analyses were performed using a HP 1050 liquid chromatograph with UV detection = 254 nm) coupled to a Micromass Quattro quadrupole mass spectrometer. Electrospray was used as the LC-MS interfacing and ionization technique (positive ions). The scan range was 100–1500 Dalton with 0.5 scan/s. The chromatographic conditions (column, eluent, flow, and gradient) were identical to those of System C.

Liquid Scintillation Counting (LSC)

Radioactivity in solutions was measured using TriCarb 2200 CA liquid scintillation counters (Canberra Packard, Groningen, The Netherlands) in antistatic scintillation vials with 10 volumes of Ultima Gold scintillation cocktail (Canberra-Packard). The machine was calibrated using a commercial 14C internal standard kit for organic solvents (Wallac, Turku, Finland). The calibration was checked daily by counting a set of quenched standards commercially prepared in sealed glass vials. Counting efficiency was determined using the spectral index of the internal standard (SIE), and cpm values were automatically transformed to dpm. Samples were corrected for background. Individual samples were counted for at least 60 min; samples with low activity were counted for up to 360 min each in order to obtain a relative standard deviation of less than 0.05.

Animals

Pilot studies.

Male Sprague-Dawley rats and CD1 mice, 8 and 10 weeks of age, respectively, were purchased from Harlan (Horst, The Netherlands) and acclimatized for at least 1 week. The animals were housed in macrolon cages with hardwood bedding and had free access to food (MRH-B, Hope Farms B.V., Woerden, The Netherlands) and tap water in a climate-controlled room (relative humidity 55%, temperature 21 ± 1°C) on a 12 h light-dark cycle. The pilot studies were conducted according to the Dutch law on Experimental Animals and were approved by the Ethical Committee for Animal Studies (DEC) of Leiden University.

Inhalation studies.

Male Sprague-Dawley rats and CD1 mice were purchased at the age of 9–10 weeks from Charles River Co. (Raleigh, NC). The animals were housed according to standard animal care procedures with free access to food (NIH-07, Zeigler Brothers, Gardner, PA) and tap water in a climate-controlled room (relative humidity 55%, temperature 22 ± 2°C) on a 12 h light-dark cycle. This study was approved by the Institutional Animal Care and Use Committee and was performed in accordance with the declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. Following acclimatization, the rats were used at an age of approximately 12 weeks (∼ 350 g) and the mice at the age of 10–12 weeks (∼ 30 g). The animals were exposed to a target concentration of 160 ppm 14C-styrene (0.34 Ci/m3; 12.6 GBq/m3) using a recirculating nose-only exposure system with concurrent collection of 14C volatile organics and 14CO2 as described in a companion paper (Boogaard et al., 2000). Twelve rats were exposed. The actual exposure concentration was 159 ± 3 ppm 14C-styrene for 6 h. Separate inhalation experiments were conducted on each group of 30 mice. The actual styrene concentrations were 160 ± 3 and 158 ± 5 ppm, respectively. In addition, two rats and five mice served as controls and did not receive treatment. Six rats were killed immediately after the exposure; the other six rats were transferred to metabolism cages and were killed 42 h later. Total lung was isolated from one rat from each of those two groups. Specific lung cells were isolated from the other animals. The two mouse exposure experiments were identical. From each experiment 15 mice were killed immediately after exposure; total lungs were harvested from two animals, whereas specific lungs cells were isolated from the other animals. The other 15 mice were transferred to metabolism cages and killed 42 h later. Total lung was taken from two mice, and specific lung cells were prepared from the remaining animals.

Pilot Studies

The methods used for lung cell isolations were based on published methods for Clara cell isolation from mouse lungs (Oreffo et al., 1990) and Type II cells from rat lungs (Clouter and Richards, 1997; Richards et al., 1987). A series of pilot studies was carried out. The first pilot studies were aimed at optimization and simplification of the mouse Clara cell isolation procedure and led to the procedure described below. In a second set of pilot studies, we investigated whether exposure to styrene affected the results of the isolation procedures of Clara and Type II cells in terms of yield and purity. Styrene dissolved in corn oil was administered to five rats (weights varied from 310 to 375 g) and five mice at a concentration of 200 mg/kg body weight. Weights varied from 32 to 39 g. Control animals received only the vehicle (2 and 5 ml corn oil per kg body weight for rats and mice, respectively). Lung cells were isolated 5–7 h after treatment using the procedures described below. A third pilot study was carried out to assess the sensitivity of the detection methods, i.e. to determine the number of animals needed in the main study with respect to the specific radioactivity of the styrene. Two male Sprague-Dawley rats (weight 250 g) and 10 male CD-1 mice (weights from 26–37 g) were given [ring-14C]-styrene with a specific radioactivity of 17.9 Ci/mol (0.66 TBq/mol), prepared from styrene with a specific radioactivity of 55.2 Ci/mol by dilution with nonlabeled styrene, dissolved in corn oil by ip injection. The styrene (150 mg) was dissolved in half a volume DMSO (85 μl) and mixed with 2.75 ml corn oil (final concentration 50 mg styrene/ml). The doses were 120 and 200 mg/kg body weight for rats and mice, respectively, the equivalent of the calculated theoretical uptake during a 160-ppm inhalation exposure for 6 h assuming ventilation rates of 250 and 45 ml/min for rats and mice, respectively, and 50% retention. The animals were subsequently housed in metabolism chambers for 48 h. Rats were housed individually and mice in groups of five animals per chamber. After 48 h the animals were anesthetized with a lethal dose (1 ml/kg body weight) of pentobarbital (60 mg/ml) in saline. Once under deep anesthesia, the abdominal cavity was opened, and the animals were bled by collection of blood from the abdominal aorta. Livers and lungs were harvested for DNA isolation, snap-frozen in liquid N2, and stored at –80°C until analysis.

Necropsy and Lung Cell Isolation of Mice

Mice were anesthetized with a lethal dose (1 ml/kg body weight) of a solution of pentobarbital (60 mg/ml) and lithium heparin (300 IU/ml) in saline. Once under deep anesthesia, the abdominal cavity was opened, and the animals were bled. The liver and other abdominal organs were removed without puncturing the diaphragm. The liver was snap-frozen in liquid N2 and subsequently stored at –80°C. The skin was removed from abdomen to throat and the trachea exposed. A Luer cannula (no. 200/300/030, Portex Ltd., Hythe, UK) was tied into the trachea through a small incision at its top. The diaphragm was punctured carefully by means of a small incision just below the xiphisternum to deflate the lungs and subsequently removed. The rib cage over the lungs and heart was removed, and the lungs were perfused via the pulmonary artery with PBS through the right ventricle using a syringe. Excess fluid was allowed to flow out through a cut in the left atrium. Perfusion of the lungs was performed with concomitant ventilation through the tracheal cannula using a syringe until they were fully perfused from blood. The lungs and heart were then dissected free from the cavity. The heart, esophagus, and excess tissue were removed. The lungs were lavaged five times with PBS to remove macrophages and secretions and once with 0.25% crystalline trypsin in Solution I (133 mM NaCl, 10.3 mM HEPES, 5.6 mM glucose, 5.2 mM KCl, 1.89 mM CaCl2, and 1.29 mM MgSO4 in 2.59 mM phosphate buffer, adjusted to pH 7.40). The cells from the combined lavage fluid fractions were collected by centrifugation (2500 g for 10 min at 4°C), snap-frozen in liquid N2, and stored at –80°C. Subsequently, a clean syringe was attached to the trachea cannula, and the lungs were filled completely with fresh 0.25% crystalline trypsin in Solution I to expand all lobes fully. The lungs were suspended in PBS kept at 37°C in a waterbath for about 20 min, during which time the trypsin solution was constantly topped up. After 20 min, the lungs were transferred into a Petri dish, the trachea and main bronchi were removed, and the parenchymal tissue was chopped into 1- to 2-mm cubes in 1 ml fetal calf serum (FCS) and 3 ml of 0.025% (w/v) DNase I in Solution II (133 mM NaCl, 10.3 mM HEPES, 5.6 mM glucose, and 5.2 mM KCl in 2.59 mM phosphate buffer pH 7.40) per lung using two pairs of scissors. The suspension was transferred using a serum pipette to a 50-ml tube and inverted repeatedly for 2 min by hand. The suspension was then sequentially filtered through a 150-μm and a 30-μm nylon filter. Finally, the filtrate was centrifuged at 32 g for 6 min at 10°C to collect Clara cells at the bottom of the tube. The supernatant was centrifuged twice more at 32 g for 6 min at 10°C. The three Clara cell-enriched fractions were combined, washed with PBS, and snap-frozen in liquid N2 after taking a small sample for staining (Clara cells). The cells that remained in the supernatant were centrifuged at 1000 g for 10 min at 4°C, washed with PBS, and snap-frozen in liquid N2 (non-Clara cells). The frozen cells were stored at –80°C until analysis.

Necropsy and Lung Cell Isolation of Rats

The first part of the procedure was identical to that described for mice, except that a larger Luer cannula (no. 200/300/050) was tied in the trachea. Following lavage, a clean syringe was attached to the trachea cannula, and the lungs were filled completely with fresh 0.25% crystalline trypsin in Solution I to expand all lobes fully. The lungs were suspended in PBS kept at 37°C in a waterbath for about 30 min, during which time the trypsin solution was constantly topped up. After 30 min, the lungs were transferred into a Petri dish, the trachea and main bronchi were removed, and the parenchymal tissue was chopped into 1- to 2-mm cubes in 5 ml fetal calf serum and 15 ml 0.025% (w/v) DNase I in Solution II per lung using two pair of scissors. The suspension was transferred using a serum pipette to a 50-ml tube and shaken by hand for 4 min in a water bath at 37°C. The suspension was subsequently filtered through a 150-μm and a 30-μm nylon filter. Ten milliliters of a heavy Percoll solution (1.089 g/l), prepared by mixing 1.0 ml of a 10× concentrated Solution I, 100 μl of fetal calf serum, 6.5 ml Percoll, and 2.4 ml purified water, was brought into a 50-ml tube. Ten ml of a light Percoll solution (1.040 g/l), prepared by mixing 1.0 ml of a 10 times concentrated Solution I, 100 μl of fetal calf serum, 2.7 ml Percoll, and 6.2 ml purified water, was carefully layered on top. The crude cell suspension was layered on top of the light Percoll solution and centrifuged at 250 g for 20 min at 10°C. Type II cells gathered at the intersection of the heavy and light Percoll solution. The Type II cell-enriched fraction was collected, washed with PBS, and snap-frozen in liquid N2 after taking a small sample for staining (Type II cells). The Percoll solutions were diluted with an equal volume of PBS, and the remaining cells, depleted from Type II cells, were centrifuged at 1000 g for 10 min at 4°C, washed with PBS, and snap-frozen in liquid N2 (non-Type II cells). The frozen cells were stored at –80°C until analysis.

Staining

The mouse Clara cell or rat Type II cell fractions were diluted with three volumes of a 4% (w/v) solution of bovine serum albumin in PBS and centrifuged at 600 rpm for 5 min against the surface of a glass slide using a Cytospin (Shandon Instruments, Pittsburgh, PA). The preparations of Clara cells were air fixed and incubated at 37°C with a drop of a 0.1% (w/v) solution of nitrotetrazolium blue in HEPES-buffered PBS and a drop of 0.1% (w/v) NADPH solution in HEPES-buffered PBS for 5 min. The slides were drained and counterstained with 1% (w/v) aqueous methylene green, and blue-purple (formazan) cells were scored as Clara cells (NTB+) (Devereux and Fouts, 1980). The air-fixed preparations of Type II cells were incubated with a drop of Harris hematoxylin for 3 min. The slides were dipped in purified water, incubated with a aqueous lithium carbonate solution (1 volume of a saturated Li2CO3 solution diluted with 84 volumes of purified water) for 5 min, and washed with purified water again. Cells with dark-blue granules (lamellar bodies) were scored as Type II cells (PAP+) (Kikkawa and Yoneda, 1974).

DNA Isolation

Lungs were pulverized in liquid N2 using a hammer mill (6700 Freezer/Mill, Glen Creston Inc., Stanmore, UK), liver was minced with scissors, and cells were processed as such. Tissue or cells were homogenized using a Braun homogenizer with 20–25 strokes at 700 rpm in an aqueous buffer of 0.8 M guanidinium hydrochloride with RNase A (54 U/ml) and T1 (13.8 U/ml), 0.03 M TRIS base, 0.03 M ethhylenediaminetetraacetic acid (EDTA), 5% (w/v) Tween-20 and 0.5% (w/v) Triton-X100, adjusted with NaOH to pH 8.0 (approximately 50 ml/g tissue). After addition of 1 ml proteinase K solution (400 U/ml) per gram of tissue, the homogenized suspension was incubated for 5 h at 37°C in a shaking waterbath. Following the incubation, the suspension was stored overnight at –80°C. Anion exchange columns (Qiagen 500/G genomic tip) were washed with 10 ml equilibration buffer [0.75 M NaCl in 15% aqueous ethanol with 0.15 % (w/v) Triton-X100, buffered with 50 mM MOPS, and adjusted to pH 7.0 with NaOH], and 20 ml of the suspension was eluted through the column by gravity. The columns were subsequently washed twice with 15 ml wash buffer (1.0 M NaCl in 15% aqueous ethanol, buffered with 50 mM MOPS, adjusted to pH 7.0 with NaOH) at room temperature. Benzoic acid, phenyl ethanol (PE), styrene glycol, and various other metabolites of styrene were completely eluted from the column using this procedure. Finally, the DNA was eluted from the columns with 15 ml elution buffer (1.25 M NaCl in 15% aqueous ethanol, buffered with 50 mM TRIS/HCl adjusted to pH 8.5) at 37°C. The DNA was then precipitated by addition of half a volume of isopropanol and collected by centrifugation at 7800 g and 4°C for 15 min. The pellet was washed with ice-cold 70% (w/v) ethanol and centrifuged once more at 7800 g and 4°C for 15 min. The pellet was then lyophilized and dissolved in a small volume of purified water. In contrast to more common DNA isolation methods based on phenol/chloroform extraction or hydroxyapatite column chromatography, which both leave minor impurities in the DNA, the anion exchange resin method used in this study provides highly purified DNA preparations, without any protein or metabolite contamination. DNA concentrations were determined in aqueous solution by UV measurements at λ = 260 and 280 nm, assuming that A260 = 20.4 for a DNA solution of 1 mg/ml. An aliquot of the purified DNA solution was used to determine the total radioactivity by LSC.

Analysis of DNA Adducts

DNA was prepared for analysis by NTH (Fig. 3). The DNA solution was heated to 95–100°C for 1 h in a Thermomixer 5437 (Eppendorf, Hamburg, Germany), and the released adducts were isolated by micro-ultrafiltration through a filter with a molecular weight cutoff of 3000 daltons (Centricon-3, Amicon). The remaining depurinated DNA was washed from the filter and lyophilized (Modulyo, Edwards, Crawley, UK). The dry residue was reconstituted in a small volume of purified water (approximately 5 mg DNA/ml). The radioactivity in an aliquot of this solution was determined by LSC. The depurinated DNA was reconstituted with purified water at a concentration of about 1 mg/ml and hydrolyzed enzymatically by subsequent treatment with an equal volume of nuclease P1 suspension (in 100 mM bis[2-hydroxymethyl]imino-tris[hydroxymethyl]methane (bis-tris) buffer pH 6.5 with 0.25 mM ZnCl2 and 2 mM MgCl2 at a final concentration 50 U/ml) for 4 h at 37°C in a shaking waterbath and a mixture and alkaline phosphatase (final concentration 6.0 U/ ml) and acid phosphatase (final concentration 0.4 U/ml) overnight at 37°C. The released nucleosides were separated from the enzymes by micro-ultrafiltration (Centricon-3) (Fig. 3). The filtrates with released adducts or nucleosides were lyophilized, and the residue was dissolved in the HPLC buffer containing the nonlabeled reference standards and analyzed by HPLC (System C) with on-line UV and radioactivity detection while fractions were collected for subsequent LSC or with MS.

For further identification of adducts, a series of reference compounds was analyzed by the same HPLC system (System C). The methylated base adducts and 1-phenyl-1,2-ethanediol were dissolved in 0.1 N HCl at a concentration of 1 mg/ml. The phenethyl alcohols were dissolved in a 1:1 mixture of 0.1 N HCl and methanol. The standards were analyzed as such and as a 1:1 dilution with a solution of HPEG in the HPLC buffer.

Statistics

Unless otherwise indicated, values are arithmetic mean ± SE. Comparisons for between-time and across-species differences were tested for statistical significance using Student's t-test. A probability of p < 0.05 was considered significant.

RESULTS

Pilot Studies

Published procedures for Clara cell isolation aim at obtaining enriched Clara-cell fractions in which the number of fibroblasts is decreased and the viability increased at the expense of yield. The method by Oreffo et al. (1990) was therefore adapted, with the most important alteration being the omission of the differential attachment procedure to reduce the number of contaminating fibroblasts. This adaptation resulted in higher cell numbers (1.0 ± 0.1 (SE) × 106 cells/lung) than those obtained with the original method (0.6 ± 0.2 × 106 cells/lung) with similar purity. The adapted procedure yielded 62 μg DNA/106 cells. Because styrene is pneumotoxic, there is a possibility that certain cell characteristics such as specific density are altered due to the exposure to styrene. Such cell alterations might seriously affect the cell isolation procedures, as they are based on specific gravity and isopycnic centrifugation. However, identical numbers of Clara cells (1.0 × 106 cells/mouse) with similar purities (60% NTB+) and Type II cells (2.4 × 106 cells/rat; 70% PAP+) were isolated from control and styrene-treated animals, indicating that the exposure to styrene did not affect the isolation procedures. Normal amounts of DNA, 1.0 ± 0.1(SE) mg and 2.3 ± 0.3 mg DNA/g for liver and lung, respectively, were isolated from rats and mice treated with 14C-styrene by ip administration. For both rats and mice, the total adduct level in liver was about an order of magnitude higher than in lungs. From the data it was confirmed that the sensitivity of the method allows detection of adducts in a 1-mg sample of DNA at a concentration as low as 1 adduct/108 nucleotides using 14C-styrene with a specific radioactivity of 50 Ci/mol (1.85 TBq/mol).

Inhalation Studies

Hepatic DNA adducts.

DNA was isolated from livers of rats sacrificed immediately after inhalation exposure and 42 h after cessation of exposure. DNA was also isolated from three of the liver pools (each from five animals) from mice killed immediately after exposure and from three of the liver pools from mice killed 42 h after exposure. The radioactivity associated with the hepatic DNA from rats killed immediately after exposure was almost completely released by NTH. The major adducts were the N7-guanine adducts of SO (HPEG) at a concentration of 3.0 ± 0.2 adducts/108 nucleotides in five of the animals (Fig. 4A). In one rat, a different adduct profile was found with very little of HPEG. In the livers of the rats killed 42 h after exposure, HPEG was still the major adduct, but the concentration was lower (1.9 ± 0.3 adducts/108 nucleotides); the other adducts that were observed earlier had decreased to very low levels or were no longer detectable (Fig. 4B). The profile of adducts in mice was different, showing quantitative and qualitative differences from rats. In addition, the time course of the adduct formation was different compared to rats: immediately after exposure, total hepatic DNA adduct levels are higher than in rats (Fig. 4C); 42 h later, the adduct levels have increased considerably (Fig. 4D), whereas in rats the adducts almost disappeared in the same time span. HPEG, the major adduct in rats, was only a minor adduct in mice immediately after exposure, although the concentration 42 h after exposure (3.2 ± 0.5 adducts/108 nucleotides) was higher than in rats after 42 h. In mice, there were two major unidentified adducts immediately after exposure that eluted at ∼31 and ∼37 min at concentrations of 2.6 ± 1.5 and 3.8 ± 1.9 adducts/108 nucleotides, respectively (Fig. 4C). HPEG represented only 1.2 ± 0.2 adducts/108 nucleotides. At 42 h, the adduct eluting at ∼37 min had increased to 8 ± 7 adducts/108 nucleotides, whereas the other major peak had decreased (Fig. 4D). In addition, one of the minor unidentified early eluting peaks had increased to about 11 ± 6 adducts/108 nucleotides. In both rats and mice the concentrations of adducts released upon enzymatic hydrolysis of the recovered depurinated DNA were about an order of magnitude lower than those released and measured upon NTH. Again, the adduct profiles differed between mice and rats both quantitatively and qualitatively. The adducts levels in rats were too low for reliable quantification. The total adduct levels in mice were higher: 2 ± 1 and 3 ± 1 adducts/108 nucleotides at and 42 h after exposure, respectively.

From the total amount of adducts in liver, i.e., the sum of adducts released by NTH and enzymatic hydrolysis (total radioactivity), the covalent binding index (CBI) was calculated. For rats, the CBI was 0.19 ± 0.06 (SE) and 0.14 ± 0.03 at and 42 h after cessation of exposure, respectively. For mice, the CBI was 0.25 ± 0.11 and 0.44 ± 0.23 at and 42 h after exposure, respectively.

Pulmonary DNA adducts.

DNA from whole lung was isolated from one rat killed immediately after exposure and from one rat killed 42 h after exposure (yields 3.1 and 5.6 mg DNA, respectively). For mice, DNA was isolated from the pooled lungs from two animals killed immediately after exposure and from two animals killed at 42 h after cessation of exposure in both inhalation experiments with total yields of 1.67 and 1.76 mg DNA, respectively. NTH of the DNA showed very low adduct levels for both rats and mice (Fig. 5). For most peaks, the levels were too low for reliable quantification. In both rat and mouse lung, the major adduct peak corresponded to HPEG at a concentration of about 1 adduct/108 nucleotides (Figs. 5A and 5C). At 42 h after exposure, this level was halved (Figs. 5B and 5D). Qualitatively, the adduct profiles in lung DNA resembled those for liver DNA. The adduct concentrations in lung DNA that were released upon NTH were low, and, from the results observed in liver, it was expected that the levels detectable upon enzymatic hydrolysis of the DNA backbone would be even lower. Therefore, the recovered, depurinated DNA of both rats and of all mice from the different time points were pooled prior to enzymatic hydrolysis. Indeed, only very low concentrations of adducts were present in the depurinated DNA. From the total amount of adducts in lungs, i.e., the sum of adducts released by neutral thermal and enzymatic hydrolysis, the covalent binding index (CBI) was calculated. Since the depurinated DNA was pooled for both time points, the calculated CBIs are averages from and 42 h after exposure. The CBIs for rat and mouse lung were 0.17 ± 0.04 and 0.24 ± 0.04, respectively.

DNA adducts in pulmonary macrophages.

Only a relatively small number of macrophages was present in the pulmonary lavage fluid from both rats and mice. As a consequence, the amounts of extracted DNA were also small (ranging from 6 to 14 μg per group), thus limiting the sensitivity of the adduct analysis. Although lymphocyte DNA was pooled for rats and for mice to increase sensitivity, the overall adduct level was low, and the profiles give no indication for any specific adduct.

DNA adducts in rat lung cells.

In the first isolation (immediate sacrifice), 65 ± 6 (SE) % of the cells were PAP+, and 0.93 mg DNA was isolated. In the second isolation (42 h postexposure), 73 ± 4 (SE) % of the cells were PAP+, and 1.11 mg DNA was isolated. HPEG was clearly present in the Type II cell DNA at a concentration of about 1 adduct/108 nucleotides immediately after exposure (Fig. 6A) and at 2 adducts/108 nucleotides 42 h later (Fig. 6B). The yield of non-Type II cells was much lower, and only 10 and 210 μg of DNA could be isolated from the rats sacrificed at and 42 h following exposure, respectively. The low yield of DNA from non-Type II cells isolated from rat lungs immediately after exposure made it impossible to accurately determine the concentration of adducts. The profile, however, suggests that HPEG was present (Fig. 6C). No distinct adduct pattern was discernible in the non-Type II cells at 42 h (Fig. 6D).

DNA adducts in mouse lung cells.

Clara cell-enriched fractions isolated immediately after exposure stained 57 ± 2 (SE) % and 61 ± 3% NTB+ for the first and second inhalation experiment, respectively. Clara cell-enriched fractions isolated 42 h postexposure stained 70 ± 3 (SE) % and 63 ± 2% NTB+, for the first and second exposures, respectively. The Clara cell fractions as well as the non-Clara cell fractions from the two inhalation experiments were pooled. The DNA yield was 0.90 mg from the Clara cells and 0.45 mg from the non-Clara cells isolated from the mice killed immediately after exposure, and 1.12 and 0.17 mg from Clara and non-Clara cells isolated 42 h after exposure, respectively. DNA adduct concentrations in the Clara cell fractions were relatively low and comparable with the DNA adduct levels in total mouse lungs (Fig. 7A). At 42 h after exposure, the concentration of adducts was higher than immediately after exposure. The major peak at 42 h, equivalent to a level of approximately 6 adducts/108 nucleotides, eluted at ∼9 min (Fig. 7B). This same peak was very prominent in the non-Clara cell fraction at 42 h (Fig. 7D). Its concentration was measured at approximately 80 adducts/108 nucleotides, but there is a large relative error (approximately 30%) associated with this value, as it was determined using a very small amount of available DNA (170 μg).

Further identification of adducts.

The retention time of the compound eluting at ∼9 min, observed in mouse non-Clara cells and Clara cells, did not match with the retention times of depurinating methylated base adducts such as N3-methylcytosine, N3-methyladenine, or N7-methylguanine, nor with guanine or adenine, nor did these compounds co-elute with any of the other, minor peaks in the chromatograms. A series of styrene metabolite standards was also analyzed using the same HPLC system, and benzoic acid appeared to co-elute with the unknown compound. Large amounts of DNA were isolated from the remaining liver tissue from mice. The DNA was depurinated by NTH, and the adducts were collected by ultrafiltration and concentrated by lyophilization. The concentrated adducts were analyzed by HPLC (System C), and the fractions with the same retention time as the unknown adduct from non-Clara cells were collected. The combined fractions were concentrated by lyophilization and analyzed by LC-MS. Mass fragments strongly indicative for benzoic acid (m/z 121) were found.

Benzoic acid itself was not retained on the extraction columns during the DNA isolation and, if present in the tissue, would not be isolated as an impurity along with the DNA. The most likely source for the benzoic acid is benzaldehyde (BA), which might form a reversible Schiff base adduct with the exocyclic amino groups of the purines in the DNA (Fig. 8). Therefore, we investigated whether BA would form DNA adducts that would be stable enough to be isolated and labile enough to yield benzoic acid during NTH. Calf thymus DNA was reacted with BA. After removal of the unreacted BA, the DNA was isolated and purified using the standard method. The precipitated DNA was lyophilized, dissolved in water, and subjected to NTH. In addition, AMP was reacted with BA. The reaction mixture was thoroughly extracted to remove unreacted benzaldehyde and was then subjected to NTH. From both the DNA and the AMP that had reacted with BA, benzoic acid was released and detected by HPLC-UV and LC-MS.

Interestingly, the co-elution experiments with metabolite standards showed that two other unidentified peaks had retention times identical to two of the standards. First, in liver DNA from rats killed immediately after exposure, the peak eluting at 19 min co-eluted with 1-phenyl-1,2-ethanediol (styrene glycol). Second, the second-largest peak in liver DNA from mice killed immediately after exposure co-eluted with the phenylethanols (∼31 min). The formation of this compound seems specific to mouse, as it was also present in livers from mice killed at 42 h and in mouse lungs (both at and 42 h) but completely absent in rat tissues.

DISCUSSION

Several adverse effects have been observed in experimental animals following exposure to styrene, including hepato-, pneumo-, neuro-, and reprotoxic effects (Bond, 1989; Gadberry et al., 1996; Sumner et al., 1997). The carcinogenicity of styrene is still under debate. Although the evidence for carcinogenicity in humans was considered inadequate and the evidence in animals limited, IARC classified styrene as a possible human carcinogen (2B), considering that styrene is metabolized in human and animal tissues to SO, an established genotoxicant (IARC, 1994a). In a recent 2-year carcinogenicity study in Sprague-Dawley rats and CD-1 mice exposed to styrene by inhalation mice were more susceptible to styrene toxicity than rats. Mice, but not rats, developed bronchiolo-alveolar adenomas (Cruzan et al., in press). In a subchronic inhalation study of styrene in rats and mice, no increase in cell proliferation was found in liver of rats or mice or in rat lung cells. No increase in labeling index of Type II cells in mouse lung was seen, but an increased cell proliferation of Clara cells was observed after 2 weeks, but not after 5 and 13 weeks, at concentrations of 150 ppm and higher (Cruzan et al., 1997). Phillips and Farmer suggested that the adverse effects of styrene are mediated through SO (Phillips and Farmer, 1994). Because SO concentrations in blood were lower in mice exposed to 160 ppm styrene than in rats exposed to 200–1000 ppm styrene, it is unlikely that differences in hepatic conversion of styrene to SO would be responsible for the observed effects in mouse lungs. However, styrene is also metabolized to SO in the lung. Clara cells and Type II cells are responsible for a significant portion of the oxidative metabolizing capacity of the lung, with Clara cells being the predominant cell type in mouse lung and Type II cells in rat lung. Recently the styrene-metabolizing capacity was shown to be much greater in Clara cells than in Type II cells, which showed little activity towards styrene (Hynes et al., 1999). The high rate of oxidation of styrene to SO in Clara cells might explain the greater susceptibility of the mouse to styrene-induced tumorigenicity. Another explanation for the mouse-specific adverse effects might be given by alternative pathways in the metabolism of styrene or SO, such as ringoxidation of styrene or SO, catalyzed by specific isoforms of cytochrome P450, which might subsequently lead to reactive ringopened products. In the present study we wanted to detect and quantify DNA adducts of styrene in mouse Clara cells and in rat Type II cells. To the best of our knowledge, specific adducts have not previously been identified in isolated lung cells.

The methods used for lung cell isolations in the current study were based on published methods. The methodology for the isolation of rat Type II alveolar cells was applied as reported by Richards and coworkers, as it was expected that sufficient DNA could be obtained from the isolated cells. Indeed, the actual yield was similar to that reported (Clouter and Richards, 1997; Richards et al., 1987). The method for isolation of mouse Clara cells was based on the method by Oreffo and coworkers (1990) which was adapted to increase the yield of Clara cells without compromising purity. Higher yields will result in larger amounts of DNA that can be extracted and, consequently, in an increased sensitivity of the analyses, as the sensitivity is limited by the amount of DNA available for analysis. A number of modifications to the original method resulted in almost a doubling of the yield of Clara cells compared to the original method without a significant deterioration of purity. The DNA yield from the Clara-cell–enriched fractions in the actual inhalation exposure studies, however, were significantly less than in the pilot studies. These relatively low yields may partly be explained by the observed toxicity in the mice exposed to styrene by inhalation. In one of the pilot studies, the effects of styrene on the isolation procedure were investigated. This was deemed essential because the isolation procedures are based on the specific density of the cells, which might be affected by styrene, as it is a pneumotoxicant. In the pilot studies, styrene was administered at relatively high doses as an ip bolus. No obvious signs of toxicity were observed in the animals following the ip administration of styrene until necropsy 5 to 7 h later. In contrast, some of the mice that had been exposed to styrene by inhalation showed clear signs of toxicity immediately after exposure, despite the fact that the actual dose of styrene in the inhalation exposure was substantially less than in the ip exposure study. Although the animals that were kept for an additional 42 h seemed to recover, gross signs of toxicity such as dehydration were observed during necropsy. Cell yields may have been affected by toxicity, thus explaining the lower yields of DNA.

DNA adduct profiles in mice and rats differed, not only quantitatively but also qualitatively, in both liver and lung, suggesting that different reactive metabolites are formed in rats and mice. In both liver and lung, and in both rats and mice, most adducts were purine adducts that could be released by NTH. Enzymatic hydrolysis resulted in release of a few adducts at levels just above the detection limit. HPEG was the major adduct in livers of rats killed immediately after exposure. Reaction of DNA with SO in vitro also resulted mainly in the formation of HPEG (∼90% of all adducts), and the adduct profile observed closely resembles that found in rat liver, indicating that SO is the major DNA-reactive metabolite of styrene in the rat. There were no indications for the formation of the N2-guanine adduct of SO, which is the second largest reaction product of SO with double-stranded DNA in vitro (Savela et al., 1986). Formation of O6-guanine or N6-adenine adducts of SO, both reported as minor adducts in DNA treated with SO in vitro, was not observed in rat or mouse liver. However, based on the ratio of formation of these adducts compared to the formation of HPEG in vitro (Phillips and Farmer, 1994) and on the actual concentration of HPEG measured in the current study, it is expected that even the concentration of the N2-guanine adduct of SO, the second largest adduct of SO, would be around or just below the limit of detection. In rats killed 42 h after exposure, the concentration of HPEG had decreased, but HPEG was still the major adduct. The major adduct in mouse liver was not HPEG, although the levels were higher at 42 h than in rats, but three unknown adducts, suggesting that SO itself is not the major DNA-reactive metabolite from styrene in mice. Preliminary results of studies on the excretion of urinary metabolites by the animals of this study also point to a significant qualitative difference in the metabolism of styrene in rats and mice. Mice excrete at least four mouse-specific metabolites, of which one accounts for 9% of the total amount of radioactivity excreted (unpublished observation). The DNA adduct profiles in lung were similar to those in liver, in both rats and mice, but the adduct levels were significantly lower. An adduct level in lungs that was approximately an order of magnitude lower than in liver was also observed in rats and mice dosed with 14C-styrene by ip injection, suggesting that the route of exposure is not a major determinant of the generation of DNA adduct forming metabolites in the lung. Although many adducts involving alkylation of the N7 position of guanine cause destabilization of the glycosidic bond and lead to depurination, it is not expected that spontaneous depurination has influenced the results of this study to a significant extent, as for the depurination of HPEG a half-life of approximately 10 days was found in double-strand DNA (Vodicka and Hemminki, 1988).

Significant amounts of adducts could not be measured in the lymphocytes isolated from the lung lavage fluid. Although the amount of DNA that could be isolated from the lymphocytes was limited, adduct levels should have been measurable if the reactive metabolites that formed adducts in the specific lung cells had been present in the general circulation. This suggests that if the DNA adducts in the specific lung cells result from reactive metabolites, they must have been generated in situ in these cell types.

The DNA adduct levels in rat Type II as well as non-Type II cells were low. The amount of DNA that could be isolated from the Type II cell-depleted fraction isolated from rats killed immediately after exposure was too low to allow quantification of the adducts. However, the radioactivity profiles suggested that HPEG might be present. HPEG was the major adduct in the Type II cells both and 42 h after exposure, and the levels were a few times higher than in total lung. This is in agreement with the fact that Type II cells are capable of oxidation of styrene to SO. In mouse Clara cells, a single unknown peak was observed as a concentration of approximately 6 adducts per 108 nucleotides at 42 h after exposure. Surprisingly, the same peak was also observed in the non-Clara cell fraction, but at a level of about 80 adducts per 108 nucleotides. Because the Clara cell fractions are not completely pure (about 63%), it cannot be ruled out that this unknown adduct in the Clara cell fraction is due to contamination by non-Clara cells. At a first consideration, it seems surprising that this peak was not observed in the total lung. However, the lungs of only two mice were used to prepare DNA to determine the adduct profile of total lung, whereas lungs of 24 mice were used to prepare DNA from the specific lung cell fractions. Preliminary analysis of the urine of the mice indicates that there were substantial quantitative and some qualitative interindividual differences in urinary metabolite patterns (unpublished observation). Considerable variations in toxicokinetics and susceptibility to styrene in mice has also been observed by other investigators (Cruzan et al., 1997; Sumner et al., 1995, 1997). It was therefore considered feasible that the unknown peak is the result of metabolism that occurred in some of the 24 mice used for preparation of the specific lung cells, but not in the 4 mice used for total lung. It should be noted that this unknown peak was also present in the hepatic DNA from some of the mice killed 42 h after exposure, albeit at much lower levels. In calf thymus, DNA reacted with SO; however, this peak was not detectable, indicating that it is not an adduct directly formed from SO.

Several attempts were made to identify the unknown compound. Because some exhaled 14CO2 could be detected, we investigated whether the unknown peak might represent a depurinating methylated base adduct, which might have been formed through degradation of the 14C-styrene and subsequent incorporation of the 14C through the S-adenosylmethionine pathway. It was demonstrated that the unknown peak did not represent a depurinating methylated base. The relatively short retention time of the unknown compound on reverse-phase HPLC suggests that it is rather polar in nature. A series of styrene metabolite standards was tested to see whether they had the same relative retention time as the unknown adduct. It appeared that the unknown compound exactly co-eluted with benzoic acid. A large amount of hepatic DNA, isolated from mice killed 42 h after exposure, was isolated and hydrolyzed. Following semipreparative HPLC (System C) of the hydrolysate, mass fragments indicative for benzoic acid (m/z 121), were identified by LC-MS analysis in the pooled fractions corresponding to the unknown peak. The most likely source for benzoic acid is benzaldehyde (BA), as BA is rapidly oxidized to benzoic acid, and we showed experimentally that BA forms a thermolabile adduct with adenine that releases benzoic acid during NTH. Most probably, BA forms a Schiff base adduct with the exocyclic NH2 group of adenine. This adduct was stable enough to be isolated during the DNA extraction procedure, but so thermolabile that BA would be released during NTH and rapidly converted to benzoic acid (Fig. 8). BA is a putative intermediate in the metabolism of styrene in the route from mandelic acid to hippuric acid (Fig. 1). It would therefore be possible that the adduct is formed from BA escaping the normal route of metabolism to benzoic acid and derived metabolites. However, both mandelic acid and hippuric acid are common metabolites of styrene in rats as well as mice. Although there are differences in metabolism between rats and mice, with rats producing significantly more hippuric acid and mice significantly more of other metabolites derived from benzoic acid, the overall flux from mandelic acid via benzylalcohol and BA to benzoic acid is quantitatively similar (unpublished observations). It is feasible that the local metabolism in the lung differs between rats and mice, with mice either producing more BA or being less efficient in the detoxification of BA than rats. However, the BA adduct was not found after ip administration of 14C-styrene in either rat or mouse lungs. An alternative explanation for the presence in the mouse lung cells is that the BA adduct is an artifact introduced by the styrene used for exposure. [ring-U-14C]-BA was the immediate precursor in the synthesis of the [ring-U-14C]-styrene used for the generation of the exposure atmosphere, and about 3% of the precursor was still present in the final product. The reason that the unknown peak was not found in the pulmonary DNA of animals from the pilot study, despite the fact that the styrene used for the pilot study contained more BA (4%) than the styrene used for the main studies, may be explained by ip administration of the compound. Ip administration ensures rapid hepatic detoxification of BA, either by cytochrome P450-mediated oxidation to benzoic acid or by GSH conjugation, as the material is primarily distributed to the liver, where both the cytochrome P450 and GSH concentrations are high. In mice, the presumed BA adducts were observed in the specific lung cells fractions but also in livers from mice killed 42 h after exposure at low concentrations and only in the second exposure group. The most likely explanation is that the GSH in the pulmonary tissue was depleted in the mice at the end of the exposure period. Such a depletion would occur more rapidly in mice, as the uptake in mice is larger than in rats. Such a GSH depletion would also explain the gross signs of toxicity observed in some of the mice.

1-Phenyl-1,2-ethanediol and phenylethanols were also among the metabolites tested to see whether they had the same relative retention time as the unknown adduct, as these compounds might have been generated through hydrolysis of a phosphate triester, formed from the attack of SO on one of the phosphate groups in the DNA, during NTH. However, 1-phenyl-1,2-ethanediol had a retention time of 18.3 min, and 1-phenylethanol and 2-phenylethanol had retention times of 29.4 and 29.7 min, respectively. In some of the adduct profiles, peaks at the same retention time were observed. Interestingly, the phenethyl alcohols co-eluted exactly with an adduct peak (retention time in HPLC-LSC ∼31 min), which was observed only in mouse, and might be derived from phenylacetaldehyde, a proposed intermediate in the metabolism of styrene (Fig. 1).

From the total amount of radioactivity in the DNA isolated from the whole liver and lungs, the covalent binding indices (CBIs), as defined by Lutz (1979), were calculated. For rat liver, the CBIs calculated at and 42 h after exposure were 0.19 ± 0.06 and 0.14 ± 0.03, respectively. The corresponding values for mouse liver were 0.25 ± 0.11 and 0.44 ± 0.23. The CBIs for rat lungs, 0.17 ± 0.04, and mouse lungs, 0.24 ± 0.04, were similar to the CBIs calculated in the liver of both species. The values calculated for lungs include the presumed DNA adduct of BA and might thus be slightly overestimated. Our results for liver corroborate the findings obtained with [7-3H]-styrene by Cantoreggi and Lutz (1993), who found CBIs for rat and mouse liver of < 0.1 (detection limit) and 0.05–0.18, respectively. Compared to CBIs for other genotoxic compounds (Lutz, 1979) and assuming that these binding values are due to monofunctional metabolites, the evidence indicates that styrene has only very weak genotoxic carcinogenic potency.

In conclusion, DNA adducts were measured in whole liver, whole lung, and specific lung cells of rats and mice following nose-only exposure to 160 ppm 14C-styrene for 6 h. Qualitative differences between the nature of the adducts were observed in lungs and liver for both mouse and rats, which indicates that different reactive metabolites are formed in the two tissues and species. CBIs for rat and mouse liver and lung were similar and with values less than 1, which is similar to the CBIs of compounds with only very weak genotoxic properties. In the Clara cell-depleted fractions from mouse lungs, a single adduct at relatively high concentration was measured, but there are strong indications that this adduct is an artifact caused by a radioactive impurity in the styrene exposure atmosphere. The overall results of this study demonstrate that DNA adduct formation does not play a significant role in the formation of the lung tumors that were observed in mice chronically exposed to styrene. It is more likely that a nongenotoxic or epigenetic mechanism, possibly caused by a cytotoxic metabolite, is involved in this process.

Acknowledgments

We are indebted to Dr. Roy J. Richards, University of Wales, Cardiff, United Kingdom, and Dr. Gary P. Carlson, Purdue University, Lafayette, Indiana, for valuable discussions and guidance in setting up the lung cell isolation techniques. This study was in part sponsored by the Styrene Information and Research Center.

Footnotes

REFERENCES