National Institute of Public Health and the Environment,
1 Laboratory of Toxicology,
2 Center for Environment and Health Research, Bilthoven, The Netherlands,
3 University of Utrecht, Department of Pathology, Utrecht, The Netherlands,
4 University of Texas Health Science Center, Department of Physiology, Texas Research Park, San Antonio, Texas, USA and
5 Department of Health Risk Analysis and Toxicology, Maastricht University, Maastricht, The Netherlands
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Abstract |
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Abbreviations: B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-diol-9,10-epoxide; BrdU, 5'-bromo-2'-deoxyuridine; NER, nucleotide excision repair; PAH, polycyclic aromatic hydrocarbon; WT, wild-type; Xpa, Xeroderma pigmentosum complementation group A.
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Introduction |
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One of the major air pollutants known to induce cell proliferation in the lung is ozone (10). It is a component of photochemical smog and its biochemical effect is caused by oxidative destruction of biomolecules directly and/or via the formation of free radicals and reactive intermediates. Exposure to ozone causes lung injury involving the ciliated cells in the airways and type I epithelial cells in the alveolar region (centriacinar region). To repair the acquired damage, several repair processes will occur, including proliferation of metabolically active cells like alveolar type II cells and bronchiolar clara cells (11).
Here we report the effects of cell proliferation in the lung, induced by ozone, on mutation fixation and lung tumorigenesis in mice orally exposed to B[a]P. For this we used wild-type (WT) mice and the more sensitive nucleotide excision repair (NER)-deficient Xpa/ mice (further referred to as Xpa mice), which have lost the ability to repair the acquired BPDEDNA adducts. These mice are known to respond with higher tumor incidences and shorter latency times upon exposure to several dermal and orally administrated genotoxic agents as compared with WT mice (12,13). In order to further enhance the cancer susceptibility, we crossed Xpa mice with p53+/ mice (annotated as Xpa/p53 mice), as these mice are known to be even more sensitive to genotoxic agents as compared with single transgenic Xpa and p53+/ mice (14).
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Materials and methods |
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The mice were housed (2 mice/cage) in a stainless steel and glass inhalation chamber in which an airflow of 6 m3/h was maintained (17). The animal rooms were kept on a 12-h lightdark cycle with a relative humidity of 50 ± 5% and a temperature of 22 ± 1°C.
Treatment protocol
Ozone dose-range finding study.
A pilot study to determine the optimal ozone concentration resulting in high cell proliferation in the lung, was performed with 69-week-old WT and Xpa mice. One male and one female mouse per genotype were exposed once a week to 0, 0.4, 0.8, 1.2 or 1.6 p.p.m. ozone (8 h, 11 p.m. to 7 a.m.), for 1, 2 or 4 weeks. Ozone was generated by irradiation of oxygen with UV light. This ozoneoxygen mixture was metered into the inlet air streams with stainless steel mass-flow controllers at a rate of 30 ml/min. The exposures were performed automatically using a control program running on an Altos 1086 microcomputer interfaced to the exposure equipment. Ozone concentrations in the chambers were measured at 2-min. intervals with Monitor Labs 8810 O3 analyzers and adjustments of the flow controllers were made to maintain concentrations at the desired levels. The analyzers were checked several times per day against a reference air mixture and zero air generated by a Monitor Labs 8550 calibrator, which was calibrated weekly by means of gas-phase titration.
One day after the final ozone treatment, the mice were anaesthetized (0.05 ml KRA) and minipumps (Alzet, model 1003D with a nominal pumping rate of 1 µl/h, from Alza Corp., Palo Alto, CA) filled with 10 mg 5'-bromo-2'-deoxyuridine (BrdU, Sigma, St Louis, MO) in 100 µl PBS were implanted subcutaneously in the dorsal intercapsular region. After 4 days the mice were killed and necropsied.
(Sub)chronic exposure to ozone and B[a]P.
To study tumor formation, a chronic exposure regimen was performed with WT, Xpa and Xpa/p53 mice, 69 weeks of age, which were treated for 13 weeks with B[a]P (75 p.p.m. in feed), ozone (inhalation, 0.8 p.p.m.), or a combination of these two treatments. All treatment groups consisted of 10 males and 10 females of each genotype. After treatment the mice were put on control feed and filtered air for an additional 6 months as was done in our previous studies (8,14). The control groups consisted of five males and five females per genotype, which were fed normal feed and inhalated filtered air for the experimental period (9 months).
To study lacZ mutant frequencies and DNA adduct formation a subchronic experiment was performed with WT/lacZ, Xpa/lacZ and Xpa/p53/lacZ mice (aged 69 weeks, three males and three females of each genotype). The dose regimen used was the same as in the chronic study, and mice were killed 13 weeks after start of the treatment. The control, unexposed mice were fed normal diet for 5 weeks.
B[a]P-containing or normal feed and water were available ad libitum throughout the study. B[a]P diet was prepared by Altromin (Lage, Germany). Ozone exposure took place once a week for 8 h (11 p.m. to 7 a.m.) starting 1 week after beginning of the B[a]P administration.
Necropsy and (histo)pathology
Throughout the studies, all mice were monitored daily for clinical abnormalities, and body weights were measured every 2 weeks. Mice who became moribund or lost >20% of their body weight were killed intercurrently. All surviving mice were killed by cervical dislocation at the end of the study. After gross examination, the tongue, esophagus, stomach, intestines, mesenteric lymph node, spleen, kidneys, liver, lung, reproductive organs, thymus and organs showing abnormalities were collected. For the pilot study, the lung and small intestine were isolated. All tissues were fixed in 3.8% buffered formaldehyde for 24 h. Subsequently, the samples were embedded in paraffin wax and cut into 5 µm sections, and stained with hematoxylin and eosin (H&E, chronic study) or BrdU (ozone dose-range finding study).
lacZ gene mutant frequency analyses
Lung, spleen and liver tissue were isolated from the WT/lacZ, Xpa/lacZ and Xpa/p53/lacZ mice, and snap frozen in liquid N2. Total genomic DNA was isolated using a procedure described earlier by de Vries et al. (9). LacZ mutant frequencies were determined using a procedure described by Dollé et al. (18). Briefly, pUR288 plasmids were rescued from the total DNA (2050 µg) with magnetic beads coated with the lacZ/lacI fusion protein. These plasmids were subsequently transfected into electrocompetent Escherichia coli strain C. A sample (2 µl of the 2 ml total) of the bacterial sample was plated on non-selective X-gal plates in order to determine the rescue efficiency. The remainder was plated onto selective P-gal plates to select for mutants. The lacZ mutant frequencies were calculated by dividing the number of mutants by the total number of rescued colonies x1000.
DNA adduct analysis
B[a]P induced DNA adduct formation in the lung was determined by 32P-postlabeling studies with the same total genomic DNA samples (10 µg) as used for the lacZ rescue studies. These assays were performed by using the nuclease P1 enrichment procedure (19). Thin-layer chromatography was done on polyethyleneimine (PEI)-cellulose sheets (Macherey Nagel, Düren, Germany) utilizing traditional urea containing solvents systems. For standardization, control samples of [3H]BPDE modified DNA with known modification levels (1 adduct/107 and 108 unmodified nucleotides) were run in each experiment. Quantification was done using a Phosphor Imager (Molecular Dynamics, Sunnyvale, CA) with a lower detection limit of 1 adduct/109 nucleotides.
Immunohistochemistry
Cell proliferation in the lung and small intestine (positive control for BrdU incorporation) was determined by BrdU staining. The staining was performed with the TSA kit (NENTM life Science, Boston, MA) in order to obtain a high-density labeling. In brief, the AAS-precoated slides were deparaffinized in xylene and graded alcohol. Endogenous peroxidase was blocked with 0.3% hydrogen peroxidase (Merck, Darmstadt, Germany) in 50% methanol (Merck), followed by an incubation with 250 U pepsin (Sigma)/1 ml 0.1 N HCl for 30 min at 37°C. After washing with TNT buffer (0.1 M Tris, 0.15 M NaCl, 5x104% Tween-20), the sections were incubated with 1 N HCl for 30 min at 56°C, washed with 0.1 M di-sodium tetraborate decahydrate buffer pH 8.5 (borax, Merck) and blocked for 15 min with TNB medium (NENTM Life Science). Incorporated BrdU was detected with an -BrdU antibody (Boehringer, Mannheim, Germany), diluted in TNB and incubated overnight at 4°C. Subsequently, the slides were incubated with goat anti-mouse IgG1/HRP (Southern Biotechnology Associates, Birmingham, AL). After washing, the HRP signal was amplified by Biotinyl Tyramide (NENTM Life Science) and additionally streptavidine-HRP (NENTM Life Science) was added. The HRP substrate was, after washing with PBS, visualized by 3,3'-diaminobenzidine (Sigma) with ammonium-nickel sulfate hexahydrate (Fluka, Buchs, Germany). Counterstaining was performed with hematoxylin and eosin.
Labeling indices were determined in eight different centriacinar regions of the lung of each mouse. The labeling index was calculated by dividing the number of labeled cells by the total number of cells countedx100. A total number of 7501000 cells were counted per lung.
Statistical analysis
The one-tailed Students t-test with a two-sample unequal variance was used to analyze the difference between the various treatments compared with the control group. The level of statistical significance was taken as P < 0.05.
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Results |
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Tumor development in WT, Xpa and Xpa/p53 mice after B[a]P, ozone or combined treatment
WT, Xpa and Xpa/p53 mice were treated for 13 weeks with B[a]P [75 p.p.m. in feed ad libitum, comparable with 39 mg/kg body weight used in our previous studies (8,14)], ozone (by inhalation, 0.8 p.p.m., 8 h/day), or the combination of these two treatments. After the treatment the mice were put on normal feed for an additional 6 months, again comparable with the treatment regimen followed in our previous studies. The control groups were fed normal feed for the entire experimental period (9 months). Potential target organs for tumor development upon B[a]P and/or ozone administration were isolated from all mice (killed intercurrently or at the end of the study) and were analyzed for histopathological changes.
Within 3 months after start of the treatment, one out of 70 WT mice died of unknown cause. These numbers were five out of 70 for the Xpa and three out of 70 for the Xpa/p53 double knockout mice. The death of these animals was considered as being incidental.
A summary of tumor incidences and hyperplasia occurring in mice that were killed at the end of the study or within the last 6 months of the study is given in Table I. Six Xpa/p53 mice could not be evaluated due to autolysis. B[a]P-related changes were observed in both the forestomach and the esophagus. Changes in the forestomach included epithelial hyperplasia, occasionally accompanied by focal atypia or hyperkeratosis, and squamous cell papillomas or carcinomas (for histopathological features: see Figure 3A and B
). The squamous cell tumors of the forestomach after B[a]P exposure, had a distinctly higher incidence in Xpa (16 out of 20) and in Xpa/p53 (12 out of 15) mice, compared with WT mice (two out of 20), reflecting the DNA repair-deficiency of Xpa mice. The tumors included predominantly squamous cell papillomas, but some squamous cell carcinomas were also observed. There were no consistent differences in incidence and severity of stomach lesions between B[a]P-treated mice and B[a]P in combination with ozone treated mice in the three genotypes tested, indicating that ozone had no additional effect on tumor development in this tissue.
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In the tongue, epithelial hyperplasia was found in a few animals distributed over all treatment groups (including a single animal treated with ozone alone), but incidences were very low.
No ozone-related pathological changes could be observed in any of the treatment groups. In addition, treatment of the mice with a combination of B[a]P and ozone had no effect at all on tumorigenesis in the lung, in none of the genotypes tested.
A variety of histopathological changes were observed that could be assigned to the common age-related background pathology of C57BL/6 mice. These included for WT and Xpa mice lymphomas and intestinal polypoid or sessile adenomas. Besides these C57BL/6-related background tumors, several spontaneous tumors, possibly caused by a p53+/ haplo-insufficiency, were encountered in Xpa/p53 mice. These included osteosarcoma, sarcoma, mammary adenocarcinoma, splenic hemangioma, skin basosquamous tumor and hemangiosarcoma and a poorly differentiated epithelial tumor, possibly an islet cell carcinoma. These tumors occurred randomly without an obvious treatment relationship, and are considered to be spontaneous (16,20,21).
LacZ mutant frequency analyses after B[a]P and ozone exposure of WT, Xpa and Xpa/p53 mice
In previous studies, oral B[a]P treatment (by gavage) resulted in increased lacZ mutant frequencies in several tissues (9). To investigate whether B[a]P mixed in feed had the same effect, and to see whether ozone had an additional effect, we measured lacZ gene mutant levels in lung, liver and spleen tissue after 13 weeks of B[a]P exposure, with or without ozone, and after exposure to ozone alone. The control mice were fed a normal diet for 5 weeks. The results of these studies are presented in Figure 4. In the three tissues analyzed, the mean number of lacZ mutants in the untreated WT, Xpa and Xpa/p53 mice were comparable (i.e. 5.78.4x105). This indicates that, at least in mice
13 weeks old, there is no clear influence of DNA repair or p53 status on the spontaneous mutations occurring in lung, liver or spleen. Ozone treatment had no detectable effect on the amount of lacZ mutants in any of the genotypes or tissues tested. However, after 13 weeks of B[a]P treatment, a significant increase of lacZ mutants as compared with background values was found in liver tissue of both Xpa and Xpa/p53 mice (1.5- and 1.6-fold, respectively). Some increase in lacZ mutant frequency was seen in the lungs of Xpa mice and the spleens of Xpa/p53 mice after B[a]P exposure; however, these effects were rather small. Comparing mutant frequencies in tissues of B[a]P-treated WT, Xpa or Xpa/p53 mice with mice of the same genotypes, treated with B[a]P in combination with ozone, in general no significant increase in mutant levels could be found in any of the tissues tested. In livers of WT mice treated with B[a]P in combination with ozone a significant, but small increase in mutant frequency was detected as compared with livers of WT mice treated with B[a]P only. This might be due, however, to the relatively low amount of mutants measured in the WT mice treated with B[a]P.
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DNA adduct levels in lungs of WT, Xpa and Xpa/p53 mice exposed to B[a]P and ozone
As no clear induction of mutations was detectable in the lungs of B[a]P exposed mice, we wanted to analyze whether B[a]P-induced DNA damage had occurred in this tissue. For this, DNA adduct levels were measured in lungs of WT/lacZ, Xpa/lacZ and Xpa/p53/lacZ mice, treated for 13 weeks with B[a]P, ozone or the combination of B[a]P and ozone. B[a]P treatment resulted in a major DNA adduct spot that co-chromatographed with the BPDEDNA standard. As shown in Figure 5, BPDEDNA adduct levels were significantly increased in the lungs of all genotypes tested, whereas no BPDE adducts could be detected in the control mice nor in mice treated with ozone only. Furthermore, a clear difference in the amount of BPDE adducts was found in the Xpa and Xpa/p53 mice as compared with the WT mice, showing the inability of these mice to repair these B[a]P-induced DNA adducts. The p53 haplo-insufficiency had no additional effect on BPDE-adduct levels in Xpa/p53 mice as compared with the Xpa single knockout mice. Furthermore, ozone treatment had no effect on the amount of BPDE adducts induced by B[a]P, as the levels found in the lungs of mice exposed to both B[a]P and ozone, were comparable with those found in mice treated with B[a]P only.
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Discussion |
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First, we showed that cell proliferation in the centriacinar area of the lung could be induced after exposure to ozone (Figure 1). This cell proliferation induction was most effective after the first ozone exposure. Subsequent ozone treatments still resulted in increased cell proliferation in the lung, but the effect of these subsequent treatments was not as overt as found after the first ozone exposure. However, cell proliferation indices were still higher than those found in untreated control animals. This observation is in line with other studies, showing that lung epithelial cells can become adapted to ozone induced cell proliferation upon repeated exposure (22,23). In this study we have chosen to use, despite the decline in ozone responsiveness, repeated exposures to ozone, to mimic the human situation in which several episodes of increased photochemical smog may occur during a summer season.
Oral B[a]P administered at the dose used in this study appeared to be highly carcinogenic to Xpa and Xpa/p53 mice, and to a lesser extent to WT mice. After 13 weeks of oral exposure to B[a]P (in feed), followed by an 6-month period on control feed, a high incidence of forestomach tumors were found. In addition, some mice developed tumors of the esophagus, an observation also demonstrated by others (6,24,25). In our previous studies, conducted with WT, Xpa and Xpa/p53 mice of the same age, B[a]P administration (by oral gavage) resulted mainly in multicentric or generalized T-cell lymphomas (8,14), and to a lesser extent tumors of the forestomach. In the study presented here, no lymphomas induced by B[a]P were found, possibly due to the different dose regimen applied.
Both in this study, and in our studies in which B[a]P was administered by gavage, a clear genotoxic effect of B[a]P in the lung was found as shown by the formation of BPDE adducts (Figure 5). Comparing the BPDEDNA adduct levels in WT mice with those measured in Xpa and Xpa/p53 mice, revealed 23-fold higher levels in the NER-deficient mice, demonstrating the inability of the Xpa mice to remove the acquired DNA damage. In the previous study, the high levels of B[a]P-induced DNA adducts did not result in increased mutant frequencies nor in the induction of lung tumors. In the present study, we show that in this tissue the induction of DNA adducts, in combination with the induction of cell proliferation, did not result in mutations (Figure 4
) nor in lung tumor formation (Table I
). One could argue that the actively dividing lung cells are not the main target for DNA adduct formation. However, this is not likely as the actively dividing alveolar type II cells are also involved in the cytochrome P450-mediated metabolic activation of B[a]P. Another explanation for the lack of lung tumor induction might be the genetic background of the mice used in this study, and the route of B[a]P administration. Ide et al. (26) treated Xpa mice (in an undefined, but different background than the mice we used) intratracheally with B[a]P. In their study, 71% of the Xpa mice developed lung adenomas, versus 35% of the WT mice, clearly showing that the lack of a functional NER pathway can result in a high incidence of lung tumors. Another indication for the importance of route of administration on the effect of B[a]P is demonstrated by comparing our previous study with the present study. The levels of B[a]P-induced DNA adducts and lacZ mutations in the lung were respectively 4- and 3-fold higher in the gavage study than those found in this feeding study. Furthermore, the highest levels of lacZ mutants in the gavage study were found in the spleen, whereas in this study, the liver showed the highest amount of mutations. So, depending on, for example, the route of administration, some variation in the actual target site of B[a]P is found. As we were particularly interested in the human risk of developing lung cancer upon B[a]P exposure in combination with ozone, we used this dose regimen to mimic the human situation in which the range and magnitude of dietary B[a]P exposure is much greater than for inhalation (27).
Several studies have been conducted to explore the carcinogenic potential of ozone itself (2831). However, no conclusive evidence exists to link ozone exposure to lung cancer development in experimental animals. From studies with A/J strain mice and B6C3F1 mice, it was concluded that ozone only gives a weak carcinogenic response (2830). However, in another study, the carcinogenic potential of ozone could not be demonstrated in A/J strain mice (31). Furthermore, in a study with Swiss Webster mice no tumors were found in the lung upon ozone exposure (29). These results, together with our results with C57BL/6 mice (both NER proficient and deficient) in which no lung tumors could be found upon 13 weeks of ozone exposure, indicate that ozone does induce cell proliferation in the lung and, dependent on the mouse strain used, can lead to a slight increase in lung tumor incidence. In our hands, neither a defect in the repair of the major B[a]P-induced DNA adducts, nor a defect in p53-mediated cell cycle control appeared to influence the development of ozone-induced lung tumors.
Based on the results shown here we can conclude that in C57BL/6 mice oral B[a]P administration results in the formation of forestomach tumors, and to a lesser extent oesophagus tumors, a finding which was also shown by others (6,24,25). These findings point towards a potential risk for the human population to develop digestive tract cancer when exposed orally to B[a]P. However, B[a]P administration, in combination with cell proliferation in the lung (induced by ozone) does not significantly contribute to lung tumor induction, not even in the highly sensitive Xpa and Xpa/p53 mice. For risk assessment, one should keep in mind, however, that human exposure can occur orally, as well as dermally and inhalatory, and animal studies clearly show that the route of B[a]P exposure is extremely important for the tissues at risk for tumor development. Furthermore, in the human population, as for experimental animals, there will be an interindividually variation of lung cancer susceptibility due to differences in biotransformation of carcinogens as a result of, for example, polymorphisms in cytochrome P450 enzymes and glutathione-S transferase (32).
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Notes |
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Acknowledgments |
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References |
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