Combined oral benzo[a]pyrene and inhalatory ozone exposure have no effect on lung tumor development in DNA repair-deficient Xpa mice

Esther M. Hoogervorst1,3, Annemieke de Vries1, Rudolf B. Beems1, Conny Th.M. van Oostrom1, Piet W. Wester1, Joseph G. Vos1,3, Wendy Bruins1, Marianne Roodbergen1, Flemming R. Cassee2, Jan Vijg4, Frederik-Jan van Schooten5 and Harry van Steeg1,6

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is considerable concern about an enhanced risk of lung tumor development upon exposure of humans to polycyclic aromatic hydrocarbons (PAHs), like benzo[a] pyrene (B[a]P), in combination with induced lung cell proliferation by toxic agents like ozone. We studied this issue in wild-type (WT) C57BL/6 mice, the cancer prone nucleotide excision repair-deficient Xeroderma pigmentosum complementation group A mice (Xpa–/–) and the even more sensitive Xpa–/–/p53+/– mice. The mice were treated with B[a]P through the diet at a dose of 75 p.p.m., in combination with intermittent ozone exposures (0.8 p.p.m.). First, a dose-range finding study with WT and Xpa–/– mice was conducted to determine the optimal ozone concentration giving high cell proliferation and low toxic side effects. We show by BrdU incorporation that cell proliferation in the lung was induced by ozone, with an optimal concentration of 0.8 p.p.m., which was subsequently used in the (sub)chronic studies. In the subchronic study, in which lacZ mutant frequency and BPDE–DNA adduct formation were measured, the mice were treated for 13 weeks with B[a]P and/or ozone, whereas in the chronic study this treatment protocol was followed by a 6 month period on control feed and filtered air. As expected, oral B[a]P exposure appeared to be highly carcinogenic to Xpa–/– and Xpa–/–/p53+/– mice and to a lesser extent to WT mice. A high incidence of forestomach tumors and some tumors of the esophagus were found. In the lung, a clear genotoxic effect of B[a]P was found as shown by the presence of BPDE–DNA adducts. However, these DNA adducts in combination with induction of cell proliferation did not result in increased lacZ mutations, nor in lung tumor formation not even in the highly sensitive Xpa–/– and Xpa–/–/p53+/– mice. The implication of these findings for tumor risk assessment will be discussed.

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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAHs), like benzo[a]pyrene (B[a]P), are components of oils, asphalt and coal tars and are formed as by-products during the incomplete combustion of organic material. They have been identified as environmental contaminants and human exposure occurs both occupationally, inhalatory and through dietary intake. Upon exposure, B[a]P is metabolically converted into several genotoxic compounds, a process that is mediated by cytochrome P450-dependent monooxygenase and epoxide hydrolase. It has been shown that benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) is the major mutagenic metabolite of B[a]P (1,2). Its carcinogenic action is achieved through its covalent binding with mainly deoxyguanosine-N2 in the DNA, leading to dG-N2–BPDE DNA adduct formation. The actual tumor site observed in mice after B[a]P administration depends on the strain, sex and administration regimen employed. Topical application induces tumors of the skin (3,4), whereas intraperitoneal injection as well as oral treatment results in lung tumor formation in susceptible mouse strains (57). In our previous studies, using C57BL/6 mice, we showed the induction of generalized T-cell lymphomas mainly residing in the spleen after oral B[a]P treatment (by gavage) (8). In the lung we found high BPDE–DNA adduct levels; however, no tumors were observed in this tissue, not even in DNA repair-deficient Xpa–/– mice (Xeroderma pigmentosum complementation group A mice; also in a C57BL/6 background) (9). Based on the results obtained in these studies we proposed that in the lung, B[a]P-related DNA adducts do not result in mutation fixation and tumor induction due to the low proliferative status of this organ. If true, one could envision there could be a cancer risk when cell proliferation in the lung is induced by e.g. air pollutants.

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 BPDE–DNA 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).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice
The mice used in these experiments were all in a C57BL/6 background. The generation of Xpa mice has been described by de Vries et al. (12,15). Xpa mice were crossbred with p53+/– mice (16) in order to obtain Xpa/p53 double knockout mice. Additionally, for mutant frequency analyses, mice of the three different genotypes (WT, Xpa and Xpa/p53) were crossed with mice containing the pUR288 lacZ reporter gene. To confirm the genotypes, PCR analyses with DNA isolated from tail tips were performed, as described previously (9).

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 light–dark 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 6–9-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 ozone–oxygen 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, 6–9 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 6–9 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 (20–50 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, 5x10–4% 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 {alpha}-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 750–1000 cells were counted per lung.

Statistical analysis
The one-tailed Student’s 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.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Ozone dose-range finding study in WT and Xpa mice
Cumulative cell proliferation in the lung was measured in two WT and two Xpa mice per dose group in order to determine the maximal tolerated ozone dose showing high cell proliferation induction. Proliferation was measured by cumulative BrdU incorporation in the centriacinar acini as this is the area of the respiratory tract that is particularly affected by ozone (11). The mice were exposed once weekly to 0, 0.4, 0.8, 1.2 or 1.6 p.p.m. ozone for 1, 2 or 4 weeks. After the first treatment with 1.6 p.p.m. ozone, severe toxicity was found in mice of both genotypes. Therefore, ozone treatment at this concentration was discontinued. There was no significant difference in ozone-induced lung cell proliferation between WT and Xpa mice, nor any sex difference was observed. Therefore, data obtained with the mice of the different genotypes and sexes were combined. As is shown in Figure 1Go the first ozone exposure resulted in high cell proliferation induction particularly at a dose of 0.8 and 1.2 p.p.m. of ozone. A typical example of BrdU incorporation in lung cells of an ozone treated mouse is shown in Figure 2AGo. In Figure 2BGo, BrdU incorporation in the lung of an unexposed mouse is depicted. Subsequent treatments resulted in some induction of cell proliferation, but they were not as effective as the first one.



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Fig. 1. Cell proliferation in lungs of ozone exposed WT and Xpa mice. Cell proliferation was determined in the centriacinar region, and calculated as the amount of BrdU positive cells divided by the total amount of cells * 100. {blacklozenge}, 0.4 p.p.m.; {blacktriangleup}, 0.8 p.p.m.; {blacksquare}, 1.2 p.p.m.

 


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Fig. 2. (A) A typical example of the BrdU immunhistochemical staining of a lung of a WT mouse exposed to 0.8 p.p.m. ozone for 1 week (LI 50) and (B) untreated control (LI 5). Only the BrdU positive cells in (B) are indicated by an arrow. Magnification: 50x.

 
Based on these results, we decided to use a dose of 0.8 p.p.m. of ozone for subsequent experiments.

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 IGo. 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 BGo). 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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Table I. Incidence of tumors and hyperplasia in WT, Xpa and Xpa/p53 mice
 


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Fig. 3. Histopathological features of a squamous cell papilloma (A) and carcinoma (B) of the forestomach. Magnification: 12.5x.

 
In the esophagus, a low incidence of epithelial hyperplasia and an occasional squamous cell papilloma was observed (Table IGo). These changes were not related to ozone treatment, as they occurred with the same frequency in animals treated with B[a]P alone compared with mice exposed to B[a]P in combination with ozone. Epithelial hyperplasia was absent in WT mice and occurred with a relatively low incidence in Xpa and Xpa/p53 mice, treated with B[a]P with or without ozone. Squamous cell papillomas were only observed in B[a]P-treated Xpa/p53 mice.

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 4Go. In the three tissues analyzed, the mean number of lacZ mutants in the untreated WT, Xpa and Xpa/p53 mice were comparable (i.e. 5.7–8.4x10–5). 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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Fig. 4. LacZ mutant frequencies in lung, liver and spleen of WT, Xpa and Xpa/p53 mice. Mice were treated for 13 weeks with B[a]P, ozone or a combination of these two as described in Materials and methods. *Significantly increased compared to untreated controls (P < 0.05). {dagger}Significantly increased compared to mice treated with B[a]P only (P < 0.05). {square}, WT mice; {blacksquare}, Xpa mice; {blacksquare}, Xpa/p53 mice.

 
Evidently, B[a]P and/or ozone treatment had hardly any influence on the amount of lacZ mutants occurring in lung or spleen. In contrast, B[a]P treatment results in an increased amount of mutations in liver tissue of NER-deficient mice, irrespective of the p53 status. Also, in this tissue, no additional effect of ozone treatment was observed.

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 BPDE–DNA standard. As shown in Figure 5Go, BPDE–DNA 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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Fig. 5. BPDE–N2-dG adduct levels in total DNA isolated from lungs of WT/lacZ, Xpa/lacZ and Xpa/p53/lacZ mice. Mice were treated with B[a]P, ozone or both for 13 weeks. For details see Materials and methods. *Significantly increased compared to untreated controls (P < 0.05). {square}, WT mice; {blacksquare}, Xpa mice; {blacksquare}, Xpa/p53 mice.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is considerable concern that oral exposure to PAHs in combination with cell proliferation in the lung (due to air pollutants) may lead to enhanced lung cancer incidences in the human population. We studied this issue in cancer prone Xpa and Xpa/p53 mice, known to be sensitive to genotoxic agents (1214) using an experimental design, which reliably mimics human exposure.

First, we showed that cell proliferation in the centriacinar area of the lung could be induced after exposure to ozone (Figure 1Go). 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 5Go). Comparing the BPDE–DNA adduct levels in WT mice with those measured in Xpa and Xpa/p53 mice, revealed 2–3-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 4Go) nor in lung tumor formation (Table IGo). 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).


    Notes
 
6 To whom correspondence should be addressed Email: h.van.steeg{at}rivm.nl Back


    Acknowledgments
 
We would like to thank Ton van de Kuil, Jan Bos, Paul Reulen, Coen Moolenbeek, Sacco Luypen, Gerard van Leuveren, Ton de Liefde, Paul Fokkens and John Boere for their skillful (bio)technical support and Hilde van Gijssel for helpful discussions. The work presented here was in part supported by the NCI grant 1RO1 CA75653.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Sims,P. and Grover,P.L. (1974) Epoxides in polycyclic aromatic hydrocarbon metabolism and carcinogenesis. Adv. Cancer Res., 20, 165–275.[Medline]
  2. Gelboin,H.V. (1980) Benzo[a]pyrene metabolism, activation and carcinogenesis: role and regulation of mixed-function oxidases and related enzymes. Physiol. Rev., 60, 1107–1166.[Free Full Text]
  3. Levin,W., Wood,A.W., Yagi,H., Dansette,P.M., Jerina,D.M. and Conney,A.H. (1976) Carcinogenicity of benzo[a]pyrene 4,5-,7,8- and 9,10-oxides on mouse skin. Proc. Natl Acad. Sci. USA, 73, 243–247.[Abstract]
  4. Albert,R.E., Miller,M.L., Cody,T.E., Barkley,W. and Shukla,R. (1991) Cell kinetics and benzo[a]pyrene-DNA adducts in mouse skin tumorigenesis. Prog. Clin. Biol. Res., 369, 115–122.[Medline]
  5. Stoner,G.D. (1991) Lung tumor in strain A mice as a bioassay for carcinogenicity of environmental chemicals. Exp. Lung Res., 17, 405–423.[ISI][Medline]
  6. Weynand,E.H., Chen,Y.-C., Wu,Y., Koganti,A., Dunsford,H.A. and Rodriquez,L.V. (1995) Differences in the tumorigenic activity of a pure hydrocarbon and a complex mixture following ingestion: benzo[a]pyrene vs manufactured gas plant residue. Chem. Res. Toxicol., 8, 949–954.[ISI][Medline]
  7. You,M., Candrian,U., Maronpot,R.R., Stoner,G.D., Marshall,W. and Anderson,M.W. (1989) Activation of the Ki-ras protooncogene in spontaneous occurring and chemically induced lung tumors of the strain A mouse. Proc. Natl Acad. Sci. USA, 86, 3070–3074.[Abstract]
  8. De Vries,A., Van Oostrom,C.Th.M., Dortant,P.M., Beems,R.B., Van Kreijl,C.F., Capel,P.J.A. and Van Steeg,H. (1997) Spontaneous liver tumours and benzo[a]pyrene-induced lymphomas in XPA-deficient mice. Mol. Carcinogen., 19, 46–53.[CrossRef][ISI][Medline]
  9. De Vries,A., Dollé,M.E.T., Broekhof,J.L.M., Muller,J.J.A., Kroese,E.D., Van Kreijl,C.F., Capel,P.J.A., Vijg,J. and Van Steeg,H. (1997) Induction of DNA adducts and mutations in spleen, liver and lung of XPA-deficient/lacZ transgenic mice after oral treatment with benzo[a]pyrene: correlation with tumour development. Carcinogenesis, 18, 2327–2332.[Abstract]
  10. Evans,M.J. (1982) In Witschi,H.P. and Nettesheim,P. (eds) Mechanisms in Respiratory Toxicology. CRC Press, Boca Raton, FL, Vol. I, pp. 189–218.
  11. Schwartz,L.W., Dungworth,D.L, Mustafa,M.G., Tarkington,B.K. and Tyler,W.S. (1976) Pulmonary response of rats to ambient levels of ozone: effect of 7-day intermittent or continuous exposure. Lab. Invest., 34, 565–578.[ISI][Medline]
  12. De Vries,A., Van Oostrom,C.Th.M., Hofhuis,F.M.A. et al. (1995) Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA. Nature, 377, 169–173.[CrossRef][ISI][Medline]
  13. Van Steeg,H., De Vries,A., Van Oostrom,C.Th.M., Van Benthem,J., Beems,R.B. and Van Kreijl,C.F. (2001) DNA repair-deficient Xpa and Xpa/p53+/– knockout mice: nature of the models. Toxicol. Pathol., 29 (suppl.), 109–116[CrossRef][ISI][Medline]
  14. Van Oostrom,C.Th.M., Boeve,M., Van den Berg,J., De Vries,A., Dollé,M.E.T., Beems,R.B., Van Kreijl,C.F., Vijg,J. and Van Steeg,H. (1999) Effect of heterozygous loss of p53 on benzo[a]pyrene-induced mutations and tumors in DNA repair-deficient XPA mice. Environ. Mol. Mutagen., 34, 124–130.[CrossRef][ISI][Medline]
  15. Van Oostrom,C.Th.M., De Vries,A., Verbeek,S.J., Van Kreijl,C.F. and Van Steeg,H. (1994) Cloning and characterization of the mouse XPAC gene. Nucleic Acids Res., 22, 11–14.[Abstract]
  16. Jacks,T., Remington,L., Williams,B.O., Schmitt,E.M., Halachmi,S., Bronson,R.T. and Weinberg,R.A. (1994) Tumor spectrum analysis in p53-mutant mice. Curr. Biol., 4, 1–7.[ISI][Medline]
  17. Marra,M. and Rombout,P.J.A. (1990) Design and performance of an inhalation chamber for exposing laboratory animals to oxidant air. Inhalat. Toxicol., 2, 187–204.
  18. Dollé,M.E.T., Martus,H.-J., Gossen,J.A., Boerrigter,M.E.T.I. and Vijg,J. (1996) Evaluation of a plasmid-based transgenic mouse model for detecting in vivo mutations. Mutagenesis, 11, 111–118.[Abstract]
  19. Van Schooten,F.J., Godschalk,R.W.L., Breedijk,A., Maas,L.M., Kriek,E., Sakai,H., Wigbout,G., Baas,P., Van‘t Veer,L. and Van Zandwijk,N. (1997) 32P-Postlabelling of aromatic DNA adducts in white blood cells and alveolar macrophages of smokers: saturation at high exposures. Mutat. Res., 378, 65–75.[ISI][Medline]
  20. Donehower,L.A., (1996) The p53-deficient mouse: a model for basic and applied cancer studies. Semin. Cancer Biol., 7, 269–278.[CrossRef][ISI][Medline]
  21. Van Kreijl,C.F., McAnulty,P.A., Beems,R.B., Vynckier,A., Van Steeg,H., Fransson-Steen,R., Alden,C.L., Forster,R., Van der Laan,J.-W. and Vandenberghe,J. (2001) Xpa and Xpa/p53+/– knockout mice: overview of available data. Toxicol. Path., 29 (suppl.), 117–127.
  22. Van der Wal,W.A.A., Van Bree,L., Marra,M. and Rombout,P.J.A. (1994) Attenuation of acute lung injury by ozone inhalation—the effect of low level pre-exposure. Toxicol. Lett., 72, 291–298.[CrossRef][ISI][Medline]
  23. Rajini,P. and Witschi,H. (1995) Cumulative labeling indices in epithelial cell populations of the respiratory tract after exposure to ozone at low concentrations. Toxicol. Appl. Pharmacol., 130, 32–40.[CrossRef][ISI][Medline]
  24. Culp,S.J., Gaylor,D.W., Sheldon,W.G., Goldstein,L.S. and Beland,F.A. (1998) A comparison of the tumors induced by coal tars and benzo[a]pyrene in a 2-year bioassay. Carcinogenesis, 19, 117–124.[Abstract]
  25. Neal,J. and Rigdon,R.H. (1967) Gastric tumors in mice fed benzo[a]pyrene: a quantitative study. Texas Rep. Biol. Med., 25, 553–557.[ISI]
  26. Ide,F., Iida,N., Nakatsuru,Y., Oda,H., Tanaka,K. and Ishikawa,T. (2000) Mice deficient in the nucleotide excision repair gene XPA have elevated sensitivity to benzo[a]pyrene induction of lung tumors. Carcinogenesis, 21, 1263–1265.[Abstract/Free Full Text]
  27. Waldman,J.M., Lioy,P.J., Greenberg,A. and Butler,J.P. (1991) Analysis of human exposure to benzo(a)pyrene via inhalation and food ingestion in the Total Human Environmental Exposure Study (THEES). J. Exposure Anal. Environ. Epidemiol., 1, 193–225.[Medline]
  28. Hassett,C., Mustafa,M.G., Coulson,W.F. and Elashoff,R.M. (1985) Murine lung carcinogenesis following exposure to ambient ozone concentrations. J. Natl Cancer Inst., 75, 771–777.[ISI][Medline]
  29. Last,J.A., Warren,D.L., Pecquet-Goad,E. and Witschi,H. (1985) Modification of lung tumor development in mice by ozone. J. Natl Cancer Inst., 75, 771–777[ISI][Medline]
  30. Herbert,R.A., Haily,J.R., Grumbein,S., Chou,B.J., Sills,R.C., Haseman,J.K., Goehl,T., Miller,R.A., Roycroft,J.H. and Boorman,G.A. (1996) Two-year and lifetime toxicity and carcinogenicity studies of ozone in B6C3F1 mice. Toxicol. Pathol., 24, 539–548.[ISI][Medline]
  31. Witschi,H., Espiritu,I., Pinkerton,K.E., Murphy,K. and Maronpot,R.R. (1999) Ozone carcinogenesis revisited. Toxicol. Sci., 52, 162–167.[Abstract]
  32. Amos,C.I., Xu,W. and Spitz,M.R. (1999) Is there a genetic basis for lung cancer susceptibility? Recent Results Cancer Res., 151, 3–12.[Medline]
Received September 17, 2002; revised December 23, 2002; accepted December 27, 2002.