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Pergamon Environment International, Vol. 22, Suppl. 1, pp. $917-$925, 1996 Copyright O1996 Elsevier Science Lid Printed in the USA. All rights reserved 0160-4120/96 $15.00+.00 PII S0160-4120(96)00202-4 CO-CARCINOGENIC EFFECTS OF VARIOUS AGENTS INRATS FOLLOWING EXPOSURE TO RADON AND RADON DAUGHTERS G. Monchaux, J.P. Mortier, P. Fdtsch, P. Rochefort, E. Douriez, M. Morin, and R. Maximilien Commissariat ~ l'Energie Atorriique, Direction des Sciences du Vivant, D6partement de Radiobiologie et Radiopathologie, Laboratoire de Canc~rologie Exp6dmentale, 92265, Fontenay aux Roses Cedex, France E19512-403 M (Received 5 December 1995; accepted 23 June 1996) Combined exposure to radon and to various occupational or environmental airborne pollutants may lead to synergistic effects for lung cancer induction. Experimentally, a co-carcinogenic effect results in increased tumour incidence after combined administration of the potential carcinogens. This paper is a review based on a standardized l~rotocol developed to identify potential co-carcinogenic agents, using an in vivo model in rats. Rats were exposed to 3.6 J h m"3 (1000 WLM) of radon, followed by exposure to the agent to be studied. Different types of compounds were studied, inotuding chemicals, mineral particles and fibres, and diesel exhaust particulates. The greatest synergistic effects were observed after administration of chemical compounds known to be cytochrome P-450 1A1 inducers which are metabolized to mutagenic or non-mutagenic forms. The results observed after treatment by cytochrome P-450 1A 1 inducers indicated that radon exposure seems to specifically increase early proliferation of target cells during the co-carcinogenic process. Combined exposure to radon and tobacco smoke resulted in a multipticative synergistic effect. For the same cumulative radon exposure, the incidence of lung carcinomas increased with the cumulative exposure to tobacco smoke, lntrapleural injection of various mineral fibres following exposure to radon resulted only in an additive co-carcinogenic effect, whereas intratracheal instillation of different minerals associated with metallic mine ores did not result in significant synergistic effects. Under the experimental conditions used, no synergistic effect was observed after combined exposure to radon and diesel exhaust. Copyright 1~1996 Elsevier Science Ltd INTRODUCTION Combined exposure to radon and its progeny and various occupational or environmental airborne pollu- tants may lead to synergistic effects for lung cancer induction. In humans, an increased incidence ofpulmo- nary neoplasia has been observed in different groups exposed to radon and its daughters. These include uranium miners (Archer et al. 1973; Sevc et al. 1976; Howe et al. 1986; Samet et al. 1989, 1991), iron miners (Pham et al. 1983; Jorgensen 1984; Radford and Renard St. Clair 1984), and other miners (Fox et al. 1981; Solli et al. 1985), especially cigarette smokers ( Edling 1982; Saccomanno et al. 1988), suggesting that co-carcino- genic mechanisms may be involved in the Pathogenesis of lung cancer. In laboratory animals, a co-carcinogenic effect results in increased tumour rates after combined administration of the potential carcinogens (Berenblum 1969). A standardized protocol was developed in Sprague-Dawley rats to identify potential co-carcino- genic agents. In dais, rats are exposed to 3.6 J h m-3 (I000 WLM) of radon followed by exposure to the agent to be studied (Monchaux et al. 1994a). All the experiments reported here were lifespan studies in which, after exposure, rats were allowed to live until they died or were moribund and then killdd. In these $917

$918 experiments, rats were exposed at a potential alpha energy concentration (PAEC) of 25 mJ m"3 (1202 WL), 5 h/d, 4 d/week for a total exposure of 142 h over 7 weeks, resulting in a cumulative exposure of about 3.61 J h m"3 (1004 WLM) rbunded to. 3.6 .J h m"3 (1000 WLM). Exposure to 3.6 J h m"3 .(1000 WLM) of radon alone results in a lung cancer incidence of 20%. About 30% of the tumours are squamous cell carcinoma, 50% adeno- carcinoma, and 20% bronchioloalveolar carcinoma. The latency period is about 700 d. An increase in lung cancer incidence has been reported in rats exposed first to radon and its daughters and then to tobacco smoke (Chameaud et al. 1974; Cross et al. 1991). The effects of combined exposure to radon and tobacco smoke, and the effects of combined exposure to radon and various cytochrome P- 450 inducers are reviewed, based on the hypothesis that polycyelie hydrocarbons are involved.in the carcinogenic activity of several compounds. Moreover, as combined exposure to various carcinogens is common in some mining or industrial environments, the potential co- carcinogenic effects of environmental or industrial air- borne pollutants such as mineral fibres, diesel exhausts, or minerals associated with metallic mine ores in rats in combination with radon exposure are reviewed. COMBINED EFFECTS OF RADON AND OTHER AIRBORNE POLLUTANTS Combined exposure to radon and tobacco smoke The first experiments were carried out to investigate the effects of inhalation of radon and its daughters at various cumulative doses, before or after various passive exposures to tobacco smoke (Chameaud et al. 1982), using cigarettes with and without filters. For tobacco smoke exposures, the CO concentration within the inhalation chambers was about 40-50 ~tL L"I. For a 3.6 J h m-3 (I000 WLM) radon exposure, the incidence of lung carcinomas was slightly lower in rats exposed to tobacco smoke before radon exposure than in rats exposed to radon alone (Table 1), but the distribution of the different histological types of tumours were similar in the two groups. In contrast, a highly significant excess of lung carcinomas (P = 0.0014, Fisher's exact test), mainly of the squamous cell type, was observed in the group exposed to tobacco smoke after radon exposure. In this group, the incidence of lung carcinomas was almost four times greater than in the group exposed to radon alone. The results of further studies in which rats were exposed to cigarette smoke following exposure to radon are summarized in Table 2. For a 350-h exposure to passive tobacco smoke, the incidence of lung carcino- mas increased with the cumulative dose of radon. TI~ incidence of lung carcinomas was twice as high in the" group exposed to 5.76 J h m"3 (1600 WLM) of radon and tobacco smoke for 350 h than in the exposed to radon at 5.76 J h m"3 (1600 WLM) and was statistically significant (P = 0.0003, exact test). For a cumulative dose of radon and its daughters corresponding to 5.76 J h m3 (1600 WLM), the inci- dence of lung carcinomas increased with the cumulative exposure to tobacco smoke. The synergistic effect of combined exposure to radon and tobacco smoke decreased when the cumulative exposure to tobacco smoke decreased. Differences between rats exposed to 1000 WLM only and the groups exposed to radon at 5.76 J h m3 (1600 WLM) and to tobacco smoke for • 60 h or 100 h were not statistically significant, butthere Was a marginally significant difference between the group e:~posed to 5.76 J h m"3 (1600 WLM) of radon and tobacco smoke for 30 h compared with the group exposed to radon at 5.76 J h m"3 ( 1600 WLM) only, (P = 0.0557, Fisher's exact test). ,.~ The induction of lung carcinomas ~vas less efficient in rats exposed to tobacco smoke produced by filter cigarettes than in those exposed to cigarettes without filters. The incidence of lung carcinomas was higher (Fig. I), but not statistically significant in the groups exposed to radon and tobacco smoke combined than in the group exposed to radon alone. The proportion of o~ lung carcinomas was lower in the group exposed to filter cigarettes than in the group exposed to unfiltered cigarettes. In the group exposed to radon and filter cigarettes, adenocarcinomas were the commonest type oftumour and the incidence of this type of tumour was similar to that observed in the group exposed to radon only. The increased incidence of lung carcinomas in the group exposed to radon and non-filter cigarettes was ' mainly accounted for by an increased incidence of squamous cell carcinomas. Thes.e findings suggested a stronger synergistic effect of radon and non-filter cigar- ettes compared to that of radon and filter cigarettes. In rats exposed to radon and tobacco sffioke combined, for the same radon exposure, the incidence of lung carcinomas was greatly increased in the group exposed to radon and tobacco smoke compared ~vith the group exposed to radon only. Tumours observed in the groups exposed to radon and tobacco smoke were larger and more invasive than in the groups exposed to radon alone. These tumours also spread more to the pleura and the presence of intrapulmonary metastases or of multiple tumours in the lung was observed. For the same radon :~/..

t, Co-carcinogenic effects of various agents in rats $919 Table 1. Incidence of the different histological types of lung carcinoma in rats exposed to tobacco smoke before and after radon exposure. Number of rats Proportion Squamous Bronehiolo Adeno with lung (%) of lung cell alveolar carcinomas carcinomas carcinomas carcinomas carcinomas Tobacco smoke'(350 h) 8 16 3 1 4 before radon 3.6 J h m"3 (~ 000 WLM) " Radon 3.6 J h m"3 I 1 22 3 1 7 (1000 WLM) only Tobacco smoke (350 h) 39 78 30 3 7 after Radon 3.6 2 h m"3 (I 000 WLM) Table 2. Incidence of lung carcinomas after combined exposure to radon and tobacco smoke according to the cumulative dose of radon and progeny and to the cumulative exposure to tobacco smoke. Number Number Proportion of rats of lung (%) of lung carcinomas carcinomas Radon only (40 WLM) 21 Tobacco smoke (350 h) 27 after radon (40 WLM) 5 3.5 Radon only (200 WLM) 63 Tobacco smoke (350 h) 75 after radon (200 WLM) Radon only (1600 WLM) 208 7 I1 16 21 81 39 Tobacco smoke (350 h) after radon (1600 WLM) 138 106 77 Tobacco smoke (100 h) after radon (1600 WLM) 35 II 32 Tobacco smoke (60 h) after radon (1600 WLM) Tobacco smoke (30 h) after radon (1600 WLM) 64 19 30 35 1 3 eXposure, the mean latent period for lung carcinomas was shorter in the group exposed to radon and then to tobacco smoke compared with the group exposed to radon alone (e.g., 682 d and 748 d, respectively, at 200 WLM radon exposure). For an identical tobacco .~oke exposure of 350 h, the mean latency period was ~nVersely related to the cumulative radon dose (e.g., .600 d in the 5.76 J h m-3 (1600 WLM) group and 682 d t~ the 0.72 J h m3 (200 WLM) group). All these results sho:ved a clear co-carcinogenic effect of exposure to radon and radon daughters and tobacco smoke in rats. Combined exposure to radon and various CYP 1A1 inducers In Sprague-Dawley rats exposed to radon at 3.6 J h m"3 (I000 WLM), about 30% of radon-induced lung carol-

$920 G. Monchaux et al. 100" 80' 60' 40' I~ Squamous ccll c:~rcinomas [] Adcnocan:inomas [] Bronchioloalwolar ca~inomas [] All lung carcinomas 20' 1: radon 1600 WLM, 2: radon 1600 WLM + rig. with filters, 3: radon 1600 WLM + rig. without filters . Fig. 1. Histologic types of lung carcinomas after exposure to radon and tobacco smoke produced by filtered and unfiltered cigarettes. nomas are of the squamous cell type (Chameaud et al. 1984; Poncy et al. 1993; Morin et al. 1994). This proportion rose to 75% in rats exposed first to radon and then to tobacco smoke (Table 1). It has also been demonstrated that lung carcinomas, mostly of the squamous tell. type, can be induced in laboratory animals after either a single or repeated treatments with different chemical compounds (Hirano et al. 1974; Witschi et al, 1977; Cross et al. 1985; Gray et al. 1986; Gies et al. 1987; Archer 1989). An experimental model of co-carcinogenesis was developed, based on the hypothesis that polycyclic hydrocarbons are involved in the carcinogenic activity of several agents. Among tobacco smoke components, polycyclie aro- matic hydrocarbons (PAH) are metabolized to carcino- • genie substances by the cytochrome P-450 multigenic enzyme family (Queval et al. 1979). The metabolites generated by cytochrome P-450 1A1 (CYP 1A1) for most PAH are about 100-fold more mutagenic than those generated by the other P-450 enzymes. The aim of this study was to characterize the role of chemicals known to be cytochrome P-450 1A1 inducers and which are metabolized to mutagenic or non-mutagenic com- pounds, after exposure to radon and its decay products. The results were compared with those observed after combined exposure to radon and tobacco smoke. Rats were first exposed to radon and its daughters by inhalation at a cumulative exposure of about 3.6 J h m"3 (1000 WLM), and then treated by different CYP 450 IA1 inducers. These included methylcholanthrene (MC) which is metabolized to strong mutagenic compounds, 5,6 benzoflavone (,13NF) which is metabolized to a non-

a.carcinogcnic ~ffects of various agents in rats $921 lutagenic compound and 2,3,7,8-tetrachlorodibenzo-p- .ioxin (TCDD) which is not metabolized and con- idered to be non-mutagenic. The same radon exposure protocol was used through- ~ut and eight groups .of experimental animals were .tistributed as follows: Group 1 was exposed to radon 8 h a day, 5 d a week for 4 ~,eeks so that the cumulative exposure was in the range of 3.6 J h m"3 (1000 WLM). Groups 2; 3, and 4 were given respectively 6 intra- muscular injections at formightly intervals of either [3NF (25 mg/kg), MC (25 mg/kg), or TCDD (1.34 ~tg/kg) dissolved in 2 mL of corn oil. Groups 5, 6, and 7 were exposed first.to radon in the same way as Group 1 and one month after the end of radon exposure were treated as Groups 2, 3, and 4, respectively. Group 8 comprised unexposed control animals. The results of lung carcinogenic and co-carcinogenic effects of the different CYP 1A1 inducers in Sprague- Dawley rats are summarized in Table 3. For the dif- ferent CYP 1 A1 inducers administered alone, there was a decreasing carcinogenic effect through MC to 13NF and TCDD. After radon exposure, each CYP 1A1 in- ducer was shown to have a clear co-carcinogenic effect. All the lesions induced were of the squamous cell type, irrespective of whether the inducer was meta- bolized into mutagenic or non-mutagenic compounds or not. The latent periods for squamous cell lesions in- duction ~vere short, i.e., less than 100 d for MC and [3NF, but longer, about 200 d, for TCDD. Biochemical studies based on the ethoxy resorufineO- de-ethylase (EROD) enzymatic activities associated with CYP 1A 1, showed that one month after the end of chemical treatment, a similar induction of CYP IA1 xvas observed in the lungs of all treated groups whether or not the rats were exposed to radon. This induction was about 30 to 40 fold greater than in controls. The EROD activity measured in rats exposed to radon alone was identical to that of controls. Thus, the role of radon in the carcinogenic process did not appear to involve specific changes in CYP IA1 ihduction. Histopathological analysis showed that lung squa- raous cell nodules and/or lung squamous cell carcino- mas occurred in the animals exposed to radon and the different CYP 1A1 inducers. The latency period for the induction ofsquamous cell lesions varied according to the inducer. In MC or I3NF treated rats, such lesions were observed systematically one month after the end of treatment whereas in the TCCD group, they were observed about three months later. In contrast, in animals not exposed to radon, such lesions were observed only in the MC-treated group. The early lesions consisted of hyperplastic areas of bronchiolar- type cells in the alveolar region which originated near the respiratory bronehioles and expressed CYP 1A1 from the beginning of the treatment. Metaplastic squa- mous cells arose from bronchiolar-type cell hyper- plasia and were gradually transformed into poorly- differentiated squamous cell nodules, squamous papil- lomas and ultimately squamous cell carcinomas (Poncy et al. 1993). The expression of CYP IA1 disappeared concomitantly with the development of squamous cell metaplasia. Thus, in this experimental model, the ex- pression of CYP 1AI in target cells appears to be restricted to the first stages of the co-carcinogenic pro- cess (Douriez et al. 1994), but neoplastic lesions ap- peared to be derived directly from the squamous cell nodules. These results could be in agreement with a specific co-carcinogenic effect in relation to the metabolism of endogenous compounds by CYP 1AI which could be initiated by metabolit~s formed during the catabolism of xdhobiotic inducers. Among these endogenous com- pounds, retinoic acid could be involved. A decreased lung concentration of retinoic acid has been reported after benzo-(a)-pyrene administration and an increased retinoic acid metabolism was observed in epidermal microsomes of MC-treated rats (Van Den Bossche et al. 1991). It has also been shown that depletion of vitamin A stimulates cell proliferation, induces squa- mous cell differentiation, and increases susceptibility to the development of chemically-induced lung cancers (Nettesheim et al. 1979; Chytil 1992). Additional studies are still in pro~ess to determine whether cyto- chrome P-450 1.A1 induction is a primary step in the aetiolog2¢ ofsquamous cell carcinoma. However, such a two-stage model of lung c~ircinoma seems to be restricted to the Sprague-Dawley strain, since it has not been possible to reproduce it in other strains of rats in which severe lung vasculitis, pleural exudate, and fatal heart failure were frequently observed after treatment by [3NF (Masse et al. 1992). Combined exposure to radon and mineral fibres The experimental protocol described above was also used to study the potential co-carcinogenic effects of radon and mineral fibres. Acid leached chrysotile fibres were shown to exhibit less carcinogenic activity in vivo than untreated fibres (Morgan et al. 1977; Monchaux et al. 1981). Since mesothelial cells are considered to be the target cells for the induction oftumours by mineral fibres, this experiment was designed to investigate the

$922 G. Monchaux et al. potential synergistic action of different kinds of un- leached or acid leached asbestos fib~'es and other mineral dusts injected into the pleural cavity of rats after previous inhalation of radon and its daughters (Bignon et al. 1983). In these experiments, 60 rats exposed to 10.8 J h m"3 (3000 WLM) of radon were used as controls. Ten groups of 10 rats each were exposed tO the same dose of radon and then, 2 weeks later were injected intra- pleurally with 2 mg of mineral dust, unleached or • leached asbestos fibres, glass fibres, and two varieties of quartz. No rats were exposed to mineral fibres alone. The results of this study were compared with those of previous experiments in which rats were inoculated intrapleurally with various doses of asbestos and other mineral fibres (Wagner et al. 1973; Monchaux et al. 1981). In the 157 rats examined microscopically, 83 malig- nant thoracic tumours were observed. Of the 60 control rats, 17 (28%) developed tumours, and 66 out of 97 (68%) in the group given an intrapleural inject!on of mineral dust. These tumours were classified either as lung carcinomas or pleural tumours. Lung carcinomas were differentiated into squamous cell carcinoma, bronchi01o-alveolar carcinoma, and adenocarcinoma. Some tumours displayed the characteristics of squamous cell carcinoma and adenocarcinoma and were classified as mixed pattern. Pleural tumours were differentiated into typical mesothelioma and combined pulmonary pleural tumours. The latter consisted of lung tumours, of the squam0us cell, bronchiolo-alveolar, or adenocarci- noma type which also exhibited a mesothelial pattern when they spread to the serosal surface of the pleura. Primary tumours of the pleura could not be distin- guished from an extension to the pleura of a pulmonary tumour which mimics the histological pattern of a mesothelioma. Lung carcinomas, mainly of the squamous cell and bronchiolo-alveolar types, occurred in all groups. No pleural tumours were observed in rats which inhaled radon only. Typical mesotheliomata only occurred in the group of rats injected with asbestos fibres, whereas combined pulmonary pleural tumours were observed in rats injected with the different mineral fibres (leached or unleached asbestos and glass fibres), and with the two varieties of quartz. The proportion of lung cancer increased from 28% in rats which inhaled radon only, to 68% in those given an intrapleural injection of mineral dust after radon in- halation, demonstrating the synergistic effect of this type of insult. The carcinogenicity of asbestos at the level of the pleura was amplified when intrapleural inoculations of dusts were given after the previous inhalation of radon and its daughters. In the groups exposed to radon and mineral fibres combined, espeeial'ly in the group ex- posed to radon and. chrysotile fibres, tumours were almost eyenly distributed between bronchoPuimonary carcinomas (2/6), combined pulmonary pleural tumours (2/6), and pleural mesotheliomas (2/6). Thus, combined exposure to radon and mineral fibres resulted in an additive co-carcinogenic effect, showing that roughly one third of lung carcinomas could be related to radon exposure, about one third of typical pleural meso- thelioma could be related to fibre exposure, and another third of combined pulmonary pleural tumours could be related to the combined effect of radon and mineral dusts at the level of the pleura. Combined exposure to radon and minerals from metallic.mine ores The potential carcinogenic or co-carcinogenic role of four minerals present in the ores of metallic mines was also investigated. These included nemalite (a contami- nant of Quebec chrysotile), biotite (present in many granites and in the French uranium ore), iron pyrites (present in various iron and gold ores), and finally iron- rich chlorite (present in iron, tungsten, and gold ores). The effects of these minerals were studied in rats (Monchaux et al. 1994b), either alone or following radon exposure, in relation to the potential combined exposure for workers in some of these mines. The iron pyrite used was prepared by air ageing of the powder. Five groups of 30 rats were each given 4 intratracheal instillations of 10 mg mineral dust, suspended in phos- phate buffered saline (PBS). Five other groups of rats were given 4 intratracheal injections of the same mineral dusts, one month after the end of a 3..6 J h m-3 (I 000 WLM) radon exposure. In control rats, instilled with PBS buffer alone, 2 lung carcinomas were ob- served, a squamous cell carcinoma and an adenocarci- noma. Iri the groups treated by mineral dust alone, the only lung carcinomas observed were a squamous-cell carcinoma in the group treated with air-~iged iron pyrites and an adenocarcinoma in the group treated with chlorite. In the group exposed to radon and PBS buffer, 9 lung carcinomas were observed among 5 rats. In the groups treated by mineral dusts after previous radon inhalation exposure, lung carcinomas and one pleural mesothelioma were observed. A slight nonsignificant increase in the incidence of lung carcinomas was observed in rats exposed to both radon and minerals,

Co.carcinogenic effects ofvarious agents in rats $923 especially, nemalite, air-aged iron pyrites, and chlorite, compared to rats exposed to radon and PBS buffer. The occurrence of a pleural mesothelioma in the group exposed to biotite might be related to a specific carcinogenic effect of mineral dust at the level of the pleura. In the groups injected with mineral dusts after radon exposure, the lung carcinomas were mostly large and more invasive compared to those observed in the group treated by radon'and PBS buffer. There were also more tumors which had spread to the pleura, and more intrapulmonary metastases or multiple lung tumours in the group exposed to both radon and mineral dusts than in the group exposed to radon and PBS buffer. These results, as those previously reported with cro- cidolite asbestos administered by inhalation (Wagner et al. 1974; 1994), demonstrate neither a clear carcino- genic effec~ of the minerals from metallic mine ores insiilled intratracheally, nor a strong co-carcinogenic effect of these minerals after previou~ radon exposure. Combined exposure to radon and diesel exhausts The use of diesel-powered vehicles is steadily in- creasing worldwide. Among the numerous epidemio- logical studies on diesel-exhaust exposed populations, only two, a case control study (Garschik et al. 1987) and a retrospective cohort study in railroad workers (Garschik et al. 1988), showed a significant association between diesel exhaust inhalation and lung cancer, suggesting that occupational exposure to diesel exhausts results in a small but significant excess risk of lung cancer. Experimentally, some evidence of a carcino- genic effect has been previously reported in rats after exposure to diesel exhaust dontaining high concentra- tions of diesel soot particles for periods of up to 2 y (Heinrich et al. 1986; Mauderty et al. 1987; Brightwell et al. 1989). The potential synergistic effects of diesel exhaust were investigated in rats after previous exposure to radon and radon daughters (Monchaux et al. 1994c). Three groups of 50 rats each were used. Group 1 was exposed to radon alone; Group 2 was first exposed to radon, and one month after the end of radon inhalation, to diesel exhaust; and Group 3 was exposed to diesel exhaust only. Rats were exposed to radon and its decay products to give a cumulative dose of 3.6 J h m"3 (I000 WLM). Diesel exhaust exposure was performed at high concentrations, but for limited periods to allow com- parison with experiments in which rats were first ex- posed to radon and then to tobacco smoke. Thus, rats were exposed to the exhaust produced by a diesel- powered engine vehicle used in the Raz6s uranium mines for 5 h a day, 5 d a week, for 3 months (300 h). The CO concentration was adjusted to 20-25 gL L"1 (20-25 ppm) and the diesel particle burden, to 4-5 mg Histopathologie analysis of the 3 groups of 50 rats revealed a total of 28 malignant thoracic tumours in 25 animals. These tumours were differentiated into lung carcinomas (squamous cell carcinoma, bronchiolo- alveolar carcinoma, and adenocarcinoma) and pleural mesothelioma. The incidence of each histological type of tumour in the different groups of exposed rats showed that lung carcinomas occurred in all groups, but only one pleural mesothelioma with a fibrosarcomatous pattern was observed in the group exposed to both radon and diesel exhausts. A slight, but nonsignificant in- crease in the incidence of thoracic tumours was ob- served in rats after combined exposure to radon and diesel exhaust compared to rats exposed to radon alone. The proportion of rats with thoracic tumours rose from 20% in the group which ifihaled radon only, to 28% in the group exposed to radon and diesel exhausts combined. There was only one pleural mesothelioma in the latter group. The proportion of rats with lung carcinomas was 20% in the group exposed to radon alone, and 26% in the group exposed to radon and diesel exhausts combined. However, the number of lung carcinomas was identical in both groups: t3 lung carcinomas among 10 of the rats in the group exposed to radon alone, and 13 among 13 of the rats in the group exposed to radon and diesel exhaust. These results showed that exposure to diesel exhaust only did not increase the incidence of lung cancer in rats. Combined exposure to radon and diesel exhausts induced a nonsignificant increase in the incidence of lung carcinomas compared to exposure to radon alone. Thus, it does not appear that inhalation of diesel exhaust either alone, or after previous radon inhalation, has a clear carcinogenic or co-carcinogenic effect. CONCLUSION These results demonstrate the potential co-carcino- genic action of various environmental or industrial airborne pollutants combined with radon exposure, showing either a multiplicative, an additive, or a nul effect. The strongest co-carcinogenic effect was shown by combined exposure first to radon and then to t~bacco smoke, which resulted in an increased incidence of lung carcinomas, mainly of the squamous cell type. For the same cumulative radon exposure, the incidence of lung

$924 carcinomas increased with the cumulative exposure to tobacco smoke. The use of filter cigarettes only modi- fied the distribution of histological tumour types. In the group exposed to radon and filtered cigarettes, adeno- canzinomas prevailed but the proportion of this type of turnout was similar to that observed in rats exposed to radon alone. In rats exposed to radon and unfiltered cigarettes, the increased incidence of lung carcinomas was due mainly to an increase in the squamous type cell carcinoma. These results showed a trend toward a preferential differentiation to the squamous cell type in lung carcinomas induced in rats by combined exposure to radon and industrial or environmental airborne pol- lutants. The results observed after treatment by cyto- chrome P-450 IA1 inducers indicated that radon ex- posure increases specifically the early proliferation o,¢ target cells during the co-carcinogenio process. They suggest that expression ofCYP 1AI in some particular cell types could be an early stage ofa co-carcfnogenic process induced after local pulmonary irradiation by radon and its daughters. These also indicated the possible application of this 'radon model' to the in- vestigation of possible interactions between exposure to two occupational and/or environmental pollutants. This experimental model for risk assessment is important be- cause human industrial occupational or environmental exposures are nearly atway.s not single but multiple. Acknowledgment--This study was supported in part by the Commission of the European Communities (Contract FI3P.CT920042). REFERENCES Archer, V.E.; Wagoner, J.K.; Lundin, F.E. Lung cancer among ura- nium miners in the United States• Health Phys. 25:351-371; 1973• Archer, V.E. Comment on "A histologic study of the influence of - cigarette smoking in suppressing Rn daughters carcinogenesis in dogs". Health Phys. 56: 255; 1989. Berenblum, I. 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