Mutagenesis, Vol. 17, No. 4, 331-336,
July 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Combined treatment with 4-(N-methyl-N-nitrosamino)-1- (3-pyridyl)-1-butanone and dibutyl phthalate enhances ozone-induced genotoxicity in B6C3F1 mice
1 Laboratory of Toxicology, College of Veterinary Medicine and 2 School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea and 3 Department of Public Health, College of Natural Science, Keimyung University, Taegu 705-751, Korea
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Abstract |
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Potential toxicological interactions of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and/or dibutyl phthalate (DBP) with ozone were investigated. Male and female B6C3F1 mice were exposed to ozone (0.5 p.p.m.), NNK (1.0 mg/kg), DBP (5000 p.p.m.) and different combinations of these toxicants 6 h/day for 16, 32 and 52 weeks. Two cytogenetic end-points, determined by the chromosomal aberration (CA) and supravital micronucleus (SMN) assays, were investigated in vivo. Our results show that all treated groups of both sexes showed genotoxic effects when compared with the control group. Additive and/or synergistic responses were observed in the CA assay for all test periods when mice of both sexes were exposed to ozone and NNK, ozone and DBP and the combination of ozone, NNK and DBP. In the SMN assay, additive interactions were noted for both sexes in the 16 and 32 week studies, similar to the results with the CA assay. All combination groups of both sexes showed synergistic interactions in the 52 week study. The results indicate that combined exposure to ozone, NNK and DBP in both sexes of mice has enhanced genotoxic effects compared with exposure to ozone alone.
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Introduction |
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Ozone is a common urban air pollutant to which humans are routinely exposed. This environmental air pollutant, which is generally detected in large cities, can cause damage to the lung when inhaled. Several cities world wide exceed the national ambient air quality standard for ozone, 0.12 p.p.m. for a daily 1 h average (US Environmental Protection Agency, 1986
). Laboratory animal and human clinical studies have demonstrated that ozone causes a reversible decrease in pulmonary function, increased permeability of the epithelium, influx of inflammatory cells, impaired pulmonary defense capacity and tissue damage (Lippmann, 1989). Moreover, ozone is mutagenic and has carcinogenic potential in rodents, causing DNA damage through reactive oxygen species such as hydroxyl radicals, superoxide anion, singlet oxygen and hydrogen peroxide (Lippmann, 1989).
Several nitrosamines derived from tobacco alkaloids are carcinogenic to laboratory animals (Hoffmann and Hecht, 1985; Hecht and Hoffmann, 1989). Among these, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is not only a potent lung carcinogen in rodents, but is also a likely causative factor in human lung carcinogenesis (Hoffmann and Hecht, 1985; Hecht and Hoffmann, 1989). A wide range of uses has been found for various phthalic acid esters (PAEs), with the largest portion of these esters being used as plasticizing agents for polyvinyl chloride products (Autian, 1973). Dibutyl phthalate (DBP) has been used as a plasticizer in explosives and elastomers, such as polyvinyl, a textile lubricating agent, a resin solvent, a safety glass, in printing inks, in paper coatings and in adhesives (Cosmetic Ingredient Review Committee, 1985). It is also used in cosmetics as a perfume solvent and a fixative, a suspension agent for solids in aerosols, a lubricant for aerosol valves, an antifoamer, a skin emollient and a plasticizer in nail polish, fingernail elongators, and hairspray (Cosmetic Ingredient Review Committee, 1985). Recently, DBPs were reported to be estrogenic in estrogen-responsive human breast cancer cells (Jobling et al., 1995; Sonnenschein et al., 1995; Harris et al., 1997). The possibility of these compounds entering biological systems has caused great concern in terms of their reproductive and developmental toxicities.
The toxicological actions of ozone, NNK and DBP have been extensively studied. However, relatively little is known about toxicological interactions between these toxicants. Studies examining the effects of air pollutants often use a single compound. However, because actual exposures involve more than one chemical, it is necessary to assess responses following exposure to various chemical combinations. The purpose of this study was thus to determine the time course of additive and synergistic effects of NNK, DBP and NNK/DBP co-exposure on the genotoxic capacity of ozone.
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Materials and methods |
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Chemicals
NNK (CAS no. 64091-91-4) was obtained from Chemsyn Science Laboratories (Lenexa, KS) (>99% pure by HPLC analysis). Trioctanoin, obtained from Wako (Japan), was redistilled before use. DBP (CAS no. 84-74-2) was acquired from Sigma (St Louis, MO). Diet containing DBP was freshly prepared every week. A predetermined amount of DBP was weighed, added to a small aliquot of ground basal diet and hand blended. This premix was then added to preweighed ground basal diet and blended in a mill for 30 min.
Animals
Male and female B6C3F1 mice, 4–5 weeks old, were purchased from Seoul National University (SNU) Laboratory Animal Facility (Seoul, Korea) and were acclimated for ~7 days prior to the initiation of chemical exposure. Before and after ozone exposures the mice were housed five per cage in polycarbonate cages with wire net bottoms. Food and water were provided ad libitum except during the period of ozone exposure. Rooms were maintained at 23 ± 2°C, with a relative humidity of 50 ± 20% and a 12 h light/dark cycle. All methods used in this study were approved by the Animal Care and Use Committee at SNU and conform to the NIH guidelines (NIH publication no. 86-23, revised 1985).
The experimental groups were as follows: (a) unexposed (control); (b) exposed to 0.5 p.p.m. ozone (ozone group); (c) exposed to 1.0 mg NNK/kg body wt (NNK group); (d) exposed to 5000 p.p.m. DBP (DBP group); (e) exposed to 0.5 p.p.m. ozone + 1.0 mg/kg NNK (ozone + NNK group); (f) exposed to 0.5 p.p.m. ozone + 5000 p.p.m. DBP (ozone + DBP group); (g) exposed to 0.5 p.p.m. ozone + 1.0 mg/kg NNK + 5000 p.p.m. DBP (three-combination group).
Exposures
Mice were exposed to ozone (0.50 ± 0.02 p.p.m.) for 6 h/day (between 9:00 a.m. and 3:00 p.m.), 5 days/week for 16, 32 or 52 weeks in 1.5 m3 whole body inhalation exposure chambers (Air-Dynamics, USA). Ozone (CAS no. 10028-15-6) was generated from pure oxygen using a silent arc (corona) discharge ozonator (model KDA-8; Sam-Il Environment Technology, Pusan, Korea) and was monitored with a gas detection system with O3 gas sensor (Analytical Technology, USA). Measurements were taken from 12 locations in each chamber to ensure uniformity of ozone distribution, which was enhanced with a recirculation device. Airflow in the chambers was maintained at 15 changes/h. Ambient ozone was removed from air entering all chambers using potassium permanganate, charcoal and HEPA filters.
During the test periods, mice were s.c. injected with 1.0 mg NNK/kg body wt in trioctanoin three times weekly. They also received diets containing DBP at a concentration of 5000 p.p.m. for 16, 32 or 52 weeks. The concentration of each test material was determined based on the National Toxicology Program carcinogenesis study (National Toxicology Program, 1994, 1995).
Chromosomal aberration (CA) assay in splenic lymphocytes
Mice were killed after 16, 32 or 52 weeks exposure by sodium pentobarbital overdose. Spleens were dissected and splenocytes were collected by passing the spleens through a sterilized nylon filter and washing the filter in phosphate-buffered solution (PBS) to collect the cells. The cell suspension was then gently layered on the same volume of Histopaque 1077 (Sigma) and centrifuged at 400 g for 30 min. The interface was carefully collected, resuspended in PBS and centrifuged at 200 g for 15 min. The cell pellet was once again suspended in PBS, centrifuged at 200 g for 10 min and finally resuspended in 1 ml of PBS. The cell concentration was determined using a hemocytometer.
Splenocyte cultures were initiated at a concentration of 1x106 cells/ml in complete medium. The growth medium consisted of RPMI 1640 (Gibco, UK) supplemented with 15% fetal bovine serum (Life Technology, Sweden), 2 mM L-glutamine and antibiotics. Concanavalin A (Sigma) at a final concentration of 5 µ/ml was used as a mitogen. At least two separate cultures were prepared from each animal for each experiment. Cultures were allowed to grow at 37°C in a 5% CO2 atmosphere with 95% humidity for 72 h. Mitotic cells were blocked with colcemid and harvested by centrifugation at 400 g for 10 min. The supernatant was removed and the cell pellet suspended in 10 ml of prewarmed hypotonic KCl solution (0.075 M) for 20 min at 37°C. The tubes were centrifuged for 8 min at 400 g, after which the supernatant was removed. The cell pellet was suspended in 10 ml of fixative (acetic acid:methanol, 1:3 v/v), which was changed four times, and the cells were resuspended in 0.3–0.5 ml of fixative prior to slide preparation. Samples of the cell suspension were added to precleaned slides and air dried. The chromosomes were stained with Giemsa and evaluated by a single observer.
A total of 100 well-spread metaphase cells (50 cells/tube) with 40 ± 2 chromosomes per animal were scored for gaps, breaks, exchanges, chromatid breaks and chromatid exchanges. Chromosome and chromatid aberrations were scored separately, and total percentages of the abnormal cells were expressed for statistical analysis. Gaps were recorded, but not included in the total chromosome aberration frequency.
Data are presented as means ± SD. Statistically significant differences among groups were assessed using the 
2 test (P < 0.05 and 0.01). Analyses were done with SPSS v.8.0 software (SPSS, Chicago, IL).
Supravital micronucleus (SMN) assay
Ten microliters of 1 mg/ml acridine orange (AO) (Sigma) was spread on a 70°C prewarmed glass slide and stored at room temperature until use. An adapted version of the method described by Hayashi et al.(1990) was used. Briefly, blood samples were taken, via a small cut in the lateral tail vein, from five male and five female animals per group after the termination of exposure. Five microliters of blood was dropped onto the center of AO-coated slides and clean coverslips were carefully placed over the drops. The slides were subsequently incubated at 4°C for 2 h. They were examined under oil immersion optics using a fluorescence microscope (HBO100W/Z; Carl Zeiss, Germany) fitted with blue excitation and yellow barrier filters. Only type I, II and III erythrocytes were observed. At least 1000 erythrocytes/animal were examined for micronuclei. Frequencies of micronucleated reticulocytes provided indices of the induced genetic damage.
Statistical significance of the differences in micronucleus occurrences among various treated groups was checked by the method of Kim et al.(2000) using a software program for easy use of the statistical procedure. The level of significance was established at P < 0.05.
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Results |
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CA assay in splenic lymphocytes
The frequencies of cells with chromatid/chromosome breaks and exchanges in male and female mice administered different combinations of NNK, DBP and ozone for 16, 32 and 52 weeks are shown in Tables I and II
and Figures 1 and 2. All treated groups showed significant increases in CA compared with the control groups. Significant additive interactions were noted from all combination groups compared with the ozone group for both sexes. The two- and three-combination male mice groups showed significant increases in the percentage of abnormal cells for all test periods, while no differences could be observed in the ozone + NNK and ozone + DBP groups compared with the ozone group in the 16 week study (Table I and Figure 1). In addition, the ozone + NNK group did not show significant increases in CA compared with male mice in the NNK and DBP groups at 16 and 32 weeks exposure, respectively (Table I and Figure 1). The results for female mice were similar to those obtained for male mice. The three-combination group showed a significant increase in CA compared with the ozone group in the 16 and 32 week studies (Table II and Figure 2). Significant increases in CA were observed in the three-combination group in both sexes compared with the other treated groups for 52 weeks exposure (Tables I and II and Figures 1 and 2). Furthermore, the male ozone + DBP and female ozone + NNK mice showed synergistic effects compared with ozone alone in the 52 week study (Tables I and II and Figures 1 and 2).
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SMN assay
The effects of exposure to ozone, NNK, DBP and their combinations on peripheral blood micronucleus frequencies of male and female mice for 16, 32 and 52 weeks are presented in Tables III and IV
and Figures 3 and 4. All treated groups showed significantly higher levels and frequencies of micronucleated reticulocytes than those of the control group for both sexes. Significant additive interactions were noted in all combination groups for both sexes, similar to the results of the CA assay for the 16 and 32 week studies (Tables III and IV and Figures 3 and 4). Furthermore, the three-combination groups of both sexes showed significantly increased micronucleated reticulocytes than the ozone + NNK and ozone + DBP groups for all test periods. In particular, all combination groups of both sexes showed synergistic effects compared with ozone alone in the 52 week study.
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Discussion |
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Numerous environmental and industrial chemicals can cause cytogenetic damage to animals and humans (Heddle et al., 1983
), which are of grave importance due to the potential genetic disorders that may develop (Ricardi, 1977). The genotoxicity of ozone somewhat contradicts the results of the cytogenetic studies with laboratory animals reported so far. Zelac et al. (1971) noted chromosome aberrations, while Tice et al. (1978) only detected chromatid-type aberrations at similar doses in Chinese hamsters. Sister chromatid exchanges (SCEs) were not induced by the same treatment. In mice, no aberrations of either type were observed (Gooch et al., 1976). In the present study the ozone-treated groups showed significant increases in the percentage of abnormal cells and frequencies of micronucleated reticulocytes compared with the control groups of both mice sexes for all study periods (Tables I–IV and Figures 1–4
). We previously performed several representative mutation assays, such as the chromosomal aberration, supravital micronucleus and hprt mutation assays, in B6C3F1 mice exposed to 0.5 p.p.m. ozone for 12 weeks and discovered that 0.5 p.p.m. ozone was genotoxic to the exposed animals (Kim et al., 2001). The underlying discrepancy between other studies and ours may be related to several factors, including the use of different tissues and cell types and different exposure times for determining aberrant cells.
Relatively few studies have been performed on the genotoxicity of NNK and DBP. Our findings clearly revealed in vivo genotoxic effects of NNK (Tables I–IV and Figures 1–4). NNK is metabolized by cytochrome P450 monooxygenase through 
-hydroxylation of either the -methyl or -methylene carbon of NNK (Abdel-Rahman and El-Zein, 2000). This metabolic conversion results in the formation of reactive intermediates that form adducts with DNA. Furthermore, increasing evidence suggests that free radical reactive intermediates, which can induce oxidative stress and oxidative DNA damage, are also generated during NNK metabolism. Studies with laboratory animals revealed that the formation of DNA adducts and the carcinogenicity of NNK are closely correlated, and similar relationships are likely to exist in humans (Abdel-Rahman and El-Zein, 2000). Since DNA repair plays a critical role in protecting the genome of the cell from NNK-induced DNA damage, reduced DNA repair capacity would constitute a significant risk factor for NNK-induced cancers (Abdel-Rahman and El-Zein, 2000). In general, negative results were reported for DBP in most mutagenicity tests performed with eukaryotic cells. No consistent increase in SCEs was noted in Chinese hamster cells treated with graduated doses of DBP (Abe and Sasaki, 1977). The only positive response noted in mammalian cell mutagenicity assays occurred in a mouse lymphoma cell assay for the induction of trifluorothymidine resistance (Hazleton Biotechnologies Co., 1986). In this assay, DBP was mutagenic only in the presence of S9 mix and at concentrations that were highly toxic. However, in our study DBP caused significant increases in the percentage of abnormal cells and the frequencies of micronucleated reticulocytes compared with the control groups in mice of both sexes (Tables I–IV and Figures 1–4).
Although human contact with environmental toxicants usually involves simultaneous exposure to more than one chemical and biological agents may depend upon the interplay between individual materials, experimental studies have so far mostly examined effects resulting from a single toxicant. Thus, characterization of effects due to exposure to relevant combinations of air pollutants is necessary for adequate assessment of health risks. Interactions of toxicant combinations can take several forms: additive, less than additive (`antagonistic') and more than additive (`synergistic') toxicity. Antagonistic effects occur when the combined effects of two or more chemicals are less than the sum of the effects of each agent given alone. Synergism occurs when the combined effects of two or more chemicals are much greater than the sum of the effects of each agent given alone. Our investigation focused on the in vivo genotoxic effects of 16, 32 and 52 weeks exposure to ozone alone and various combinations (ozone + NNK, ozone + DBP and ozone + NNK + DBP) in B6C3F1 mice. CA and SMN assays were performed to determine whether NNK, DBP and NNK + DBP exposures have potential additive and/or synergistic effects on the genotoxic capacity of ozone alone.
Lymphocytes, especially those of peripheral blood, are the most easily accessible and, as a result, most studies have been performed using circulating lymphocytes. There are, however, lymphocytes that are localized in various tissues, such as spleen, lymph nodes, Peyer's patches and mucosa-associated lymphoids. Maria and Michael (1997) have suggested the possibility that lymphocytes stored in these areas may be longer lived than those in the peripheral circulation and should, consequently, reflect DNA damage based on a reduced turnover of cells and/or perhaps an extended exposure to genotoxicants than circulating peripheral lymphocytes. Thus, we used mouse splenic lymphocytes in the CA assay. Significant additive interactions were noted with all combination groups compared with the ozone group in both sexes of mice in the above-mentioned genotoxicity assays (Tables I–IV and Figures 1–4). In particular, synergistic interactions were clearly observed in all combination groups compared with the ozone group for both sexes in the 52 week study using the MN assay (Tables I and II and Figures 3 and 4).
The genotoxicity of ozone is of crucial environmental concern (Geraldo et al., 1996). The actual biological mechanisms underlying the interactions between ozone, NNK and DBP are not known. The mechanisms of action of ozone, NNK and DBP on the measured end-points are also not completely understood, adding to the difficulty in explaining the interactions on exposure to the combination of toxicants. Focusing on complex exposures will increase our knowledge of the complexities of biological mechanisms involved in producing harmful health effects such as cancer, which will, in turn, certainly increase awareness of the potential for various interactions and combined effects. Major modes of action occur via genotoxic, cytotoxic or mitogenic pathways. Our present knowledge of various, mainly qualitatively different, interactions already found in biological systems argues against the emergence of simple unifying concepts to explain the risk of combined exposures. Nevertheless, mechanistically based classifications of interactions into groups may be helpful in predicting health effects of complex exposure situations and should be promoted.
In conclusion, this study examined the potential additive and/or synergistic genotoxic effects of NNK, DBP, ozone and their various combinations. The results indicate that, under our experimental conditions, combined exposure of mice to ozone, NNK and DBP induces additive and/or synergistic genotoxic effects compared with exposure to ozone alone.
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Acknowledgments |
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This work was supported in part by a Brain Korea 21 Grant. M.Y.K. is the recipient of a Brain Korea 21 graduate student fellowship.
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Notes |
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4 To whom correspondence should be addressed. Tel: +82 31 290 2746; Fax: +82 31 296 1258; Email: mchotox{at}snu.ac.kr
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References |
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Received on January 21, 2002; revised on April 4, 2002; accepted on April 11, 2002.
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