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Evaluation of environmental carcinogens by carcinogen-specific test systems
Exp. Pathol. 29, 65-76 (1986) VEB Gustav Fischer Verlag Jena 1) 2) 3) 4) 5) 6) 7)
Beratungsforum fur Praventivmedizin und Umwelttoxikologie, Hamburg; Biochemisches Institut fur Umweltcareinogene, Ahrensburg; Fraunhofer-Institut fur Toxikologie und Aerosolforschung, Hannover; Institut fur Mathematik der Universitat, Hannover; Medizinische Hochsehule, Institut fur Experimentelle Pathologie, Hannover; Medizinisches Institut fiir Umwelthygiene der Universitat Dusseldorf; Universitatsklinikum der Gesamthochschule Essen, Institut fiir Hygiene und Arbeitsmedizin, Essen
Evaluation of environmental carcinogens by carcinogen-specific test systems By G. GRIMMER2 ), U. ABEL4), H. BRUNEI), R. DEUTSCH-WENZELI ), M. EMURA5), U. HEINRICH 3 ), ,J. JACOB2), A. KEMENA 7), J. lVIrsFELD 4 ), u. MOHR5 ), IL NORPOTH 7), F. POTT 6) and H.-B. RrcHTER-REICHHELM5 ) (Received August 8, 1985) Address for correspondence: Prof. Dr. G. GR!1'II:;!ER, Biochemisches Institut fur Umweltcarcinogene, Sieker LandstraBe 19, D - 2070 Ahrensburg, F.R.G.
Key w 0 r d s: environmental carcinogens; test systems, carcinogen-specific; polycyclic aromatic hydrocarbons (P AH); air pollution; emissions from pyrolysis; solid fuels; liquid fuels
Introduction Carcinogenic compounds are formed by pyrolytic processes during incomplete combustion of solid or liquid organic fuels. This raises the question whether these compounds occur in concentrations which may represent a general health hazard or a specific occupational risk. Epidemiological investigations have indicated a significant inerease of lung eaneer for speeifie working places e.g. in the case of coke plant workers. For a quantitative assessment of the earcinogenic effect of emissions from coal-fired furnaces, coke plants or vehicles, and for the identification of the carcinogenic constituents of emissions, a simulation of the carcinogenic effect in suitable animal models or other biological test systems such as cell or tissue cultures is required. Presently, the only way to confirm the correlations between air pollution and disease incidence which have been revealed by epidemiological studies is by use of animal experiments to establish a clear-cut relationship between dosage and cancer incidenee. Possibly due to the lack of a well-established proof of this nature, lung cancer of workers inhaling polycyclic aromatic hydrocarbons (P AH) had not been added to the list of occupational diseases at the beginning of this project. The careinogenic effect of an emission on man be estimated best by a combination of a suitable animal test system with ceIl or tissue culture systems. The selection of the appropriate animal species, mode of application and target organ is critical for meaningful extrapolation of the results to the human situation. Further support for the transferability of results from experimental animals to man can be obtained from the comparison of metabolites formed in analogous tissues or cell cultures as well as from the test of eell-transforming potencies of emission condensates and fractions thereof in human or animal cell cultures. This has been described for automotive exhaust and its fractions in subproject V with hamster lung cell cultures as a test system. The carcinogenic effect of eoke oven exhaust ean certainly be simulated better by inhalation experiments with mammals than with mutation rates of Salmonella. Concerning the mode of application, it may be assumed that the inhalation of emissions simulates the human exposure better than does the implantation of emission condensate into the lung or its instiIla5
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tion into the trachea of experimental animals. The presently available results of subproject IV demonstrate that inhalation of aerosols from pyrolized hard-coal tar pitch results in lung carcinomas in 10 % of the exposed rats after 20 months. A disadvantage of the inhalation teehnique, however, is that although gases and aerosols sueh as atmospherie particles can be t ested, this is not feasible for subfractions of aerosol eondensates because of technical difficulties. It is very laborious to verify a causal relationship between an emission and an observed carcinogenic effect by means of animal experiments or other carcinogen-specific test systems. The more careinogen-specific th e method which is used, the mGre expensive it is. Less complicated are chemical-analytical methods, providing that the carcinogenic constituents of the emission are at least partially known. The evaluation of the carcinogenic potency of an emission from chemical analytical data becomes more precise as more individual carcinogenic compounds of the emission can be identified. One should at least know the dass of compounds mainly responsible for the carcinogenic effect. In the case of emissions from pyrolysis (combustion) of liquid or solid fuel s, the eareinogenie effed is almost exelusively caused by PAR. This can be proven after fractionation by comparing the ea,r cinogenie effect of the PAR-free and the P AR-eontaining parts with that of the original condensate. For the evaluation, suitable animal test systems and other biological careinogen-specific test systems can be used as has been done already for the evaluation of the carcinogenic risk caused by various emissions. Of course, statements on the contribution of distinct earcinogenic constituents are the more reliable if corroborated in several animal species, animal test models, and ee!! eulture systems. Renee, animal test systems such as mouse skin-painting or implantion into rat lung as well as cell and tissue cultures were applied as detectors for the carcinogenic activity of constituents in eomplex mixtures. It can be demonstrated by topical application onto the mouse skin or rat lung-implantation that more than 80 % of the carcinogenic potency of vehicle exhaust condensate from gasoline-driven engines is caused by the content of PAR eontaining more than 3 rings. The same holds true for used lubricating oil of this engine type. Similarly, using mouse s~in for evaluation it could be shown that it is the PAR-fraction containing more than 3 rings which is almost exelusively responsible for the eareinogenic potency of flue gas condensate from hard coal-fired furnaces. These results have been e011firmed by rat lung-implantation experiments used in this project (subproject II). Carcinogen-specific test systems have to meet two requirements: they must respond to all sorts of earcinogenic classes sueh as polycyclic aromatic hydrocarbons, oxygen- or nitrogen-eo ntaining polycyelic aromatie compounds, aromatic amines, nitrosamines, mycotoxins, etc. they ought to provide as far as possible for the diversity of morphologieal and biochemical reactions of human cells which finally lead to malignant transformation. The first requirement can readily be cheeked .• The lo eal applieation in mice can be ta,k en to be a detector system of universal applicability if not only PAR but also non-volatile nitroso compounds provoke skin tumors. In the frame of this project, dose-dependent earcinogellic effect of nitroso carbaryl and nitrosomethyl urea in mouse skin was verified (subproject III). According to our present knowledge the second requirement seems to be best met by comparing the morphological transformation effect and the metabolites in human and animal cell or tissue cultures. The question of transferability to humans of results obtained from the various 1:n vivo syst ems in animals can be investigat ed experimentally in cell or tissue cultures. To what extent analogous biochemi cal r eactions take place in human and animal cells of identical tissues ean be determined from both the transformation rates and the eomparison of the metabolite profiles of various eareinogens. In vitro systems such as the microsomal oxidation of PAR are very satisfactory, since the formation of metabolites (e.g. the ultimate carcinogens) can be detected analytically (subproject VIII) as well as by means of the Ames test (subproject VII). The aims of subprojects V and VI were to develop cell and tissue cultures from hamster lungs, rat livers and human lung's in order to compare the metabolism of various PAR and
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their cell transforming potency. It is still an open question whether cell cultures can also be used for the detection of carcinogenic constituents from emissions. First results comparing the cell transforming properties of automobile exhaust condensate, and four fractions thereof, with fetal hamster lung cells are encouraging. Only the fraction containing P AH with more than 3 rings results in the same dose response as the original vehicle exhaust condensate (subproject V). With regard to minimizing future animal experiments this test system should be systematically studied. In the following, the various sUbprojects are summarized.
Subproject I: Epidemiology of lung cancer MISFELD, U. ABEL)
Conclusions of an appraisal of the literature*
(J.
Key W 0 r d s: lung cancer, epidemiology; smoking; passive smoking; air pollution; emissions, environment; polycyclic aromatic hydroearbons (P AH); occupational exposure
A survey of important results of epidemiological research on associations between exogenous factors and lung cancer ri8k is given. The studies are reviewed as to the strength of their findings. The available evidence is summarized in a synoptic appraisal. After a longlasting worldwide rise of rates, lung cancer is now the most important type of cancer among males of western indnstrial countries and is growing rapidly among females. Four main causes are being discussed in the etiology of lung cancer: Smoking (above all, cigarette smoking), passive smoking, environmental air pollution, and occupational exposure. In all cases polycyclic aromatic hydrocarbons presumably are contributory causes. Smoking: As a consequence of the thorough epidemiologic investigations of the past 30 years it is no longer seriously contested today that cigarette smoking is the main risk factor for lung cancer, particularly squamous cell carcinoma. Most estimations are that smokers have 8 or more times the risk of developing lung cancer than nonsmokers (not taking into account the individual smoking habits), and the proportion of lung cancer attributable to smoking is estimated at 80 %. Recent studies show that smokers of filter cigarettes have a 10 to 50 % lower risk than smokers of non-filter cigarettes, despite a certain compensation by deeper inhalation.
Passive smoking: The opinions on the hazards of passive smoking are still contradictory today. At pres.ent, a critical examination of the existing literature justifies three conclusions.
1. Research has not succeeded in freeing passive smoking from the suspicion of being ca,rcinogenic. 2. Long-term passive smoking appears to impair lung function and thus has measurable effects. There is, however, no evidence that these effects are strong enough to produce respiratory symptoms which are comparable to those from active smoking. 3. So far, there is no conclusive epidemiological evidence that passive smoking causes an increased risk of lung cancer.
Environmental a1:r pollution: A critical survey of the published material, especially of the more recent articles, on lung cancer risk due to general air pollution permits the following conclusions: The thesis that, beside cigarette smoking and occupational exposures, general air pollution is an important factor in the etiology of lung cancer is not proven. A great number of studies have been performed on this question and there seems to be an agreement on the existence of an urban-rural gradient of rates. Many of these studies, however, were designed inappropriately and they mostly contained methodological shortcomings which might have produced erroneous results. While in none of the studies all relevant confounders were adequately controlled, the more carefully the bias was controlled the lower was the estimation of the environmental influence.
* 5*
A comprehensive monograph on the subject is forthcoming. Exp. Pathol. 29 (1986) 2
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According to the authors of the best planned studies, one must doubt the existence of a measurable lung cancer risk due to general air pollution. At most, the effects are extremely small and detectable only in large-scale, specially-designed studies. There are also indications of synergistic actions of air pollution and smoking. The findings are not conclusive, however, and can be explained by imperfections of study designs. Further research on this subject is needed. Occupational exposure: Occupational exposure concerns only a limited part of the population, but because of the possibility (at least in principle) of prevention and because of the fact that these risks are taken involuntarily, they are of great importance. This holds, in particular, for the P AH which are set free in numerous industrial processes. Among the many epidemiological studies of the association between exposure at the work place (including exposures to P AH) and lung cancer risk, there are only a few which are not disputable. Often, smoking habits were not controlled, histologic types of the lung cancer cases were not distinguished, and exposures were not quantified. On the whole, the studies (particularly the carefully planned ones) give evidence for cancer risk at places where workers are exposed to mixtures of noxae containing PAH. This is strongly corroborated by dose-response relationships. The lung cancer risk, however, becomes evident only with high concentrations of P AH such as can be found in coke plants. At present, it is impossible to reliably quantify lung cancer risk due to P AH alone.
Subproject II: Comparison of the carcinogenic activity of airborne particulate matter extract with that of flue gas emitted from hard coal-fired furnaces allil fractions thereof by implantation into rat lungs (R. DEUTSCH-WENZEL, H. BRUNE) Key W 0 r ds: airborne particulate matter extract; flue gas eondensate; hard coal-fired furnaces; lung; carcinogenicity
The objective of the study was to compare the carcinogenic activity of airborne particulate matter of a large town with that of flue gas condensate from hard coal-fired furnaces as well as to identify such substances predominantly responsible for the carcinogenic effect of the emission of the latter. To determine how much P AH components contribute to the total carcinogenic potency of the flue gas condensate from coal-fired furnaces, the emission condensate was separated into individual fractions consisting of a P AH-free fraction and several P AH containing fractions (P AH containing 2 and 3 rings; P AH containing 4 to 6 rings; a combined fraction consisting of 6 and more rings and basic nitrogen containing P AH (Azaarenes). The carcinogenic effect of these fractions was eompared with that of the total condensate as well as with that of a total condensate recombined from all individual fractions. To examine the carcinogenic action, different concentrations of the condensate and fractions thereof (dosed proportionally) were implanted into the lungs of 3-month-old female Osborne "Mendel rats. Airborne particulate matter extract as well as flue gas emitted from coal-fired residential furnaces regularly induced epidermoid carcinomas and sporadically induced pleomorphic sarcomas at the implantation site. With respect to the materials administered, flue gas condensate proved to be more potent. The results of the examination of the fractions indicate that neither the PAH-free fraction participating with 45 (by weight) in the total weight of the condensate, nor the fraction containing 2 and 3 rings (12 % by weight) and the soot (about 7 % by weight) contribute to the carcinogenic effect of the condensate. The fraction of P AH consisting of 4 to 6 rings and the combined fraction containing 6 and more rings and azaarenes reveal a comparable carcinogenic potency. The effect of the latter has, however, to be deduced mainly from the P AH containing 6 and more rings, since it was shown in previous experiments with flue gas condensate and fractions thereof using the epicutaneous test with mice that the fraction containing azaarenes alone contributed only 4 to 7 % of the total activity.
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With the condensate combined from all individual fraetions (reeombinate) a carcinogenic effect eomparable with that of the original sample was observed. Aeeording to a provisionally raw estimation, about 2 to 3 % of the total carcinogenic activity is expected to be explained by the benzo(a)pyrene (BaP) content of the condensate (1.14 mg BaPjg eondensate). Similar to previous investigations with gasoline exhaust condensate using the lung implantation model in rats and the skin painting model in mice it was seen that the total carcinogenie activity of the flue gas emitted from hard coal-fired residential furnaces is predominantly attributable to P AH containing more than 3 rings. Thus, similar results were obtained using the skin and lung of two different species as carcinogen-specifie test systems.
Subproject III: Loeal application to mouse skin as a carcinogen-specific test system for nonvolatile nitroso compounds (H. BRUNE, R. DEUTSCH-WENZEL) Key w 0 r d s: carcinogen test; non-volatile nitroso compounds; emission condensates; skin
The objeetive of these studies was the demonstration of local carcinogenie activities of the nitroso eompounds methylnitrosourea, nitrosonornieotine and nitrosocarbaryl using the skin painting model with CFLP mice. This earcinogen-specific detection system is particularly well suited for eomparing the careinogenic poteney of emission condensates with that of fraetions prepared therefrom, sinee the action of carcinogenie substanees is barely influenced even by considerable amounts of aecompanying materials. The loeal application to the skin of miee is well established as a careinogen-speeifie animal model for both pure P AH and P AH in eomplex mixtures sueh as emission condensates from automobile exhaust, coal furnaees, domestic heating and used engine oil. The question of whether local carcinogenic effects can be provoked by nitroso compounds on the mouse skin is the more important, since aromatic nitro so compounds (e.g. nitrosocarbaryl) appear in P AH-containing fraetions, whereas other nitroso eompounds such as llitrosomethylurea and nitrosonornicotine are found in the hydrophilic fraetions. In the latter, however, no carcinogenic poteney has been found to date using the mouse skin model. Therefore, even if hydrophilic nitroso compounds have a slight carcinogenie aetivity, the contribution of this elass of compounds to the total carcinogenic potential of the emission condensates of automobiles, coal-fired residential furnaces or used engine oil is expected to be very small. In a chronic epicutaneous experiment, 65 female CFLP mice were treated twiee weekly over a period of 104 weeks by dropping three dosages of nitrosomethylurea, nitrosonornicotine or nitrosoearbaryl to the dipped interscapular region. Benzo(a)pyrene was used as the reference substance. When compared with the remaining groups, survival times were lower in the groups with higher dosages of nitrosomethylurea and nitrosocarbaryl. Following treatment with nitrosonornicotine, a weak careinogenic effect was observed but the tumor incidence showed no dependence on dose. With regard to induced earcinomas and sarcomas, elear dose-response relationships were obtained for nitrosomethylurea and nitrosocarbaryl as well as for the referenee substance benzo(a)pyrene. According to the results of pro bit analysis, the carcinogenic potencies of the nitroso compounds investigated in the mouse skin painting model rank as follows: nitrosocarbaryl, 0.18; nitrosomethylurea, 0.04 and nitrosonornicotine 0.008; (benzo(a)pyrene, 1.00).
Subproject IV: Squamous cell carcinoma of rat lungs after inhalation of PAH-rich emissions (U. HEINRICH, F. POTT, U. MOHR, L. PETERS, R. FUHST, J. Ki)NIG) Key W 0 r ds: polycyelic aromatic hydrocarbons (PAR); carcinogenic effect; co-carcinogenic effect; PAR-rich exhaust; lung carcinoma; occupational diseases
Aims and motivation: The aim of the investip;ation was to examine the carcinogenic and cocarcinogenic effeet of P AH -containing emissions. The motivation to earry out this study Exp. Pathol. 29 (1986) 2
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came from the fact that although numerous P AH and exhaust emission condensates have been shown to be carcinogenic in several animal species by various methods, and although there is increased lung cancer incidence in coke oven workers, there had been no attempt to apply these findings to occupational health guidelines. In the Federal Republic of Germany up to the beginning of our investigation, lung cancer in workers who were exposed to P AHcontaining exhaust for many years was not included in the register of occupational diseases. At present, no threshold limit value for benzo(a)pyrene or any other cancer producing P AH exists either for the workroom air or for the outdoor ambient atmosphere. Since 1983, a maximum allowable emission concentration for benzo(a)pyrene and dibenz(a, h)-anthracene is included in the Technical Instructions for Ensuring Clean Air (TA Luft). From the environmental hygiene and occupational medicine point of view, therefore, there was a need for additional animal experiments giving unequivocal evidence that P AHcontaining exhaust is carcinogenic. A further aim of our investigation was the development of a test model in inhalation carcinogenesis for the examination of the strongly suspected cumulative and inhibitive effects of definite particulate matter or gaseous substances. Methods: For the exhaust generation a domestic coal oven was used. A particular disadvantage of the coal oven is that, during the 2 to 3 h initial burning phase extremely high and varying concentrations of pollutants are emitted. During the following "glowing" burning phase, the levels of P AH and other components of emission (with the exception of CO) fall almost to ambient air levels. The emission of the initial burning phase could not be used for long-term animal inhalation experiments, however, because of the considerable variations in toxic components despite standard dilution. Therefore, the P AH-poor exhaust of the "glowing" coal used for the animal exposure had to be enriched with P All without significantly increasing simultaneously acute toxic substances, such as carbon monoxide. Initially this was achieved by continuously adding droplets of a tar/pitch solution to the glowing coal and later, more effectively, by mixing the coal oven exhaust with the effluent of pitch pyrolized at about 750 DC under nitrogen atmosphere. The proportion of this exhaust mixture used for inhalation exposure is given as follows: 1 part pyrolized pitch effluent, 400 parts coal oven exhaust, 4,000 parts clean air. (The PAH enrichment of the coal oven exhaust by adding small amounts of pyrolized pitch exhaust is very effective; in view of the conditions about 10 years ago this procedure is not too unrealistic because 7 pitch by mass was used as binding medium in the production of hard coal briquettes at that time.) The experimental animals (Wistar rats) were exposed for 19 h a day, 5 d a week, for about 20 months. The average exposure with P AH-enriched coal oven exhaust did not amount to more than 80 h/week, however, because of the time needed for maintenance of the pitch pumping and pyrolizing device. The particle bound benzo(a)pyrene concentration in the exposure chamber during the last 12 months of the experiment was 90,ug/m3 on average. Some other P AH have been determined in correspondingly high concentrations. Among the exhaust components like CO 2, CO, S02' NO, N0 2, total hydrocarbons and particulate matter measured continuously in the exposure chamber, only CO and S02 exceeded the maximum allowable concentration at workplaces (MAK) in the Federal Republic of Germany. The concentrations of the gaseous components of the coal oven exhaust did not change very much when the pyrolized pitch effluent was added. Some of the rats were additionally treated with intratracheal instillations of dibenz(a, h) anthracene or crocidolite to examine the influence of the exhaust on the tumor rate induced by these known carcinogens (2-phase model). Preliminary results: Approximately half of the 108 animals which were exposed to the exhaust without an additional treatment were still alive after two years. Up to now, 10 lung carcinoma bearing animals have been detected in the exhaust-exposed group and none in the clean air control group. The tumors were identified as squamous cell carcinomas and one small cell carcinoma. The histological examination of the other experimental groups has not yet been started.
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After one year of exposure, extensive clinical-chemical and hematological investigations with satellite groups hardly showed any differences between exposed animals and controls. The evaluation of the differential leukocyte count of the lung lavage fluid resulted in a significantly higher proportion of macro phages from the exhaust exposed groups and the phagocytic activity was also strongly increa,sed. Furthermore, the lung function tests using a whole body plethysmograph showed an exposure-related reduction of the compliance, but the alveolar lung clearance measured by a radioactive test aerosol had not deteriorated.
D1:scnss'ion: The lung carcinoma incidence rate of about 10 %, according to the present state of evaluation, can be regarded as clear evidence of the cancer-inducing effect of the exhaust exposure. An incidence rate of more than 25 % is expected at the end of the experiment. These positive findings could be established under exposure conditions which, up to now, did not shorten the survival time of the non-tumor-bearing animals. It is important to note that the PAR-content of the diluted exhaust in the inhalation chambers did not exceed by an unrealistic amount the PAR concentration to which the coke oven workers on the oven platform may be exposed (rats: exposed to 90,ug BaP/m3 for 80 hi week for about 1 year; coke oven workers on the oven platform: exposed to about 30,ug BaP 1m3 for 40 hjweek). From the present data no conclusions can be drawn as to what extent the lung tumor induction in the exhaust exposed rats was caused by P AH, the measured irritant gases, or the many other organic compounds the bulk of which are still unknown. This question will be the subject for further investigations. Snbprojecf V: Identification and assessment of environmental carcinogens by cell and tissue culture (U. MOHR, 1\1. E1vIURA, H.-B. RICHTER-REICHHELM) Key W 0 r ds: cell and tissue culture; carcinogen test; motor vehicle exhaust condensates; respiratory epithelial cells
Organ and tissue culture systems were developed from hamster and human epithelial tissue and mixed tissue of the respiratory tract with the aim of identifying and assessing the biological activity of environmental carcinogens. A motor vehicle exhaust condensate (KAK) showed a clear transforming effect on mixed cultures of hamster lung cells. From the exhaust condensate the following five fractions were produced in the usual way: KAK-1: total motor vehicle exhaust condensate; KAK-2: methanol (70%) + cyclohexane; KAK-3: PAR (2 and 3 rings); KAK-4: PAH (4 to 7 rings); KAK-5: polar PAC and N-P AC. The fraction KAK-4 induced a specific dose response, in which it showed a considerable weaker transforming activity than the total condensate. It was further established that the condensate fractions KAK-1 to KAK-3 in the dilution rate of 1: 500 already showed signs of a toxic effect. The fractions KAK-4 and KAK-5, however, revealed a slight growth stimulation at this degree of dilution. An established clonal ccllline from hamster lung epithelium (M3E3/C3) showed bi-potential differentiation abilities dependent on the culture conditions. With increased Ca++content and reduced serum concentration in the medium, the cells produced keratin-like filaments. The presence of EGF, insulin, hydrocortisone, estradiol and vitamin A in the medium induced a marked mucus production in the cells. The hormones, including EGF, particularly stimulated the development of the endoplasmatic reticulum, whereas vitamin A caused the increased development of Golgi apparatus and mucus granules. The cell line M3E3/C3 grew undifferentiatedly under conventional culture conditions and was not transformed by benzo(a)pyrene. After the addition of hormones and EGF to the medium, the cells of the M3E3/C3 were transformed by benzo(a)pyrene. Vitamin A reduced the transformation rate considerably. The characterisation (tumor type) of the induced growths from transformed cells is the subject of further investigations at the moment. By optimizing the culture conditions it was possible to cultivate tracheal epithelial cells of a human foetus in the 16th week of gestation for up to 10 passages. For this Ca++-reduced RPMI 1640 was used. The cells of this human cell line were treated with different doses of Exp. Pathol. 29 (1986) 2
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diethylnitrosamine (DEN). The colony formation test for t oxicity showed a clear decrease in the number of surviving colonies with increasing DE N doses. In transformation exp erim ents in which soft agar colonies with a size great er than O.lnll11 diameter were evaluated, a dosedependent transformati on rate resulted. Samples of the transformed eells were injeeted sub eu ta neously into ath ymi e nude mice t o cheek the tumor form ation ability of the cells. These investigations are not yet finalized. At the moment it is still also being checked whether the eells grow when infiltrated in mesenehymal tissue. For this transformed cells were transpl anted in v'itro onto chiek embryo skin. In another study using organ cultures from fetal hamster tracheae, the inducti on of histopathological ehanges such as metaplasia, dysplasia and exophytie growth was investigated after treatment with benz(a)anthracene (BaA), benz(a)aeridine (BaAc), benzo(b)fluoranthrene (BbF), pyrene (PYR) , benzo(k)fluor anthene (BkF ), and benzo( e)pyrene (BeP). The eompounds BaA, BaAe, Bb F, and BkF prove d to be eff ectiv e here. The findings showed a high correspondenee both with the results of in m:vo tests as well as with th ose of in vitro cell transformation t ests. The scanning eleetron microscopie interpr"etation used here offers the advantage that small dysplasti c ehanges, in particular exophytieally proliferating epithelial areas (neoplasia,s), r eliably recorded and eompared with in vivo situations ean be rated as clear indications of an earl y change.
Subproject V I: Developm ent of a cell culture system to (letermine carcinogenic activities of polycyclic hy(lrocarbons (D. PAUL) K ey word s: c.ell culture system; eal'cinogen test ; polycyclic a,ro mati c hydrocarbons (PAR); hepatocyte cultu res, metabolicall y compet ent
The aim of the pres ent r esearch proj eet is to establi sh metabolically co mpetent hepat ocyte eultures in which the cell s continue to expr ess various liver specific funetions and t o utilize them for short-term in vitro t est syst ems fo r the determination of eeotoxie activities. Such hepato cyt e cultures r epresent a novel, highly differentiat ed syst em in contrast to convent ional fibroblastic cells. They would therefore be particularl y useful as a model system for the study of human careinoma. Therefore, th e specific aim of t his projeet is t o utilize hepat ocyte culture syst ems that actively express enzymes capable of metabolizing xenobiotie compo unds in order t o provide useful systems to det ermine carcinogenic and potentially carcinoge nic compounds. By optimizing complex culture media it has been possible t o maintain proliferating hepat ocytes of fet al, newborn and adult r at livers in primar y cultures. The cells ar e differentiat ed hepato cyt es expressing specific liver fun etions which are expected t o be expressed in such cell types. F etal rat hepat oeytes in primary cultures multiply in a newl y developed medium lVIX-83 in the presence of Epidermal Gro wth Factor (EGF), insulin and transferrin. Hepatoe.ytes derived from newb orn rat s also r equire hydro cor tiso ne, and adult rat hepato cyt es gro w in the presence of EGF, insulin, transferrin, glucagon, hydro cortisone and serum. The mitogenic activity in serum ean be partiall y r eplaeed by pl atel et peptide(s) that are distinct frolll Platelet Derived Growth Factor (PDGF). Cells express a,]pha-fetopro t ein, albumin, t yrosine amino transferase, and various other liver-specifie markers. lVIorris hepatoma 7777 D NA has been shown to transform normal mouse 3T3 fibr oblas ts t o malignant cells after transfe ction. It is eoncluded fr om t hese studies that hepatoma DNA eontains transforming DNA sequences. Preliminary studies in which newborn rat hepat ocyt es have been used as r ecipient cells in transfection experiments involving various cloned oneo genes suggest that the oneogene of Moloney sarcoma virus (mos) cloned in expressio n vect or flanked by LTR's (plVISV-U1) is able t o transform hepatocytes pro vided that additional treatment of transfected cultures with a car cinogen (benzo(a)pyrene (BaP) or NlVIU) oecurs 3 d post transfeetion. Mos alone or either BaP or NlVI U treatments alone do not lead t o transformation. From these studies we eonelude that t he malignant transform ation of hep at oeyt es in monolayer eultures oceurs by at least two independent eoo perating events. We will attempt
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to isolate rnos-transfected hepatocytes (which could perhaps be immortalized cells) and to establish permanent cell lines which could be useful for studies in which the mechanism of the action of chemica.! carcinogens can be investigated.
Su.bproject V II: Influence of various inducers on the metabolic activation by "S9-1Uix" in the Ames-tcst (A.
KElIIENA,
C.
SCHUMANN, J. JACOB,
K.
NORPOTH)
Key wor ds: Ames-test; S9-Mix; polycyclic aromatic hydro carbons (PAR); metabolic activities; mutagenicity
'iVith the aid of the baeterial mutagenieity test-system (AMES), the indueing effeet of several environmentally relevant PAR on liver microsomal monooxygenases was investigated. After optimization of the test-system, 18 different PAR of high purity grade were injeeted into rodents and the resulting inducing effeets monitored by the mutagenic activity of benz(a)anthracene, a PAR known as a weak mutagen. Benzo(j)fluoranthene, benzo(b )fluoranthene, indeno(1,2,3-ed)pyrene, and dibenz(a,h)allthracene turned out to be the most potent of the substances screened, with revertant colony numbers as high as 15 times the va.!ue of the uninduced control. The potencies of these indueers are comparable to that of Clophen A50, a known strong indueer. Employing the same inducers (benzo(k)f1uoranthene or Clop hen A50), revertant colony numbers in the test with benz(a)anthraeene as substrate reached 50 ';;, of those obtained with the strong mutagen benzo(a)pyrene. It is thereby evident that the interaction between inducer and mutagen plays a vital role in the mutagenic risk. There are some good corrclations between the inducing effect of the PAR in the Ames-test (with benz(a)anthracene as mutagen) and the eontent of hydroxylation products in the metabolite pool of benz(a)anthracene in comparable induetion experiments. The best eorrelation eo efficient was found between the mutagenicity and the K-region derivatives (r = 0.776, P < 0.01) , whieh led us to cone/ude that the mutagenie aetivity observed is mainly due to K-region epoxides. For further characterization of the biochcmic.al mechanisms indueed by PAR, the mutagenieity was compared with two isozyme-selective dealkylating mono oxygenase activities and also with the total cytochrome P-450f448 eoncentration. We found statistically hig'hly significant eorrelations between mutagenieity and the activity of ethoxyeou marin deethylase (mainly selective for cytoc.hrome P-448 isozymes) (r = 0.77, P < 0.01) and between the aminopyrine demethylase ac.tivity (speeifie for cytoehrome P-450 isozymes) and total eonc.entration of cytoehrome P-450f448 (r = 0.82, P < 0.001). The two enzymic activities did not eorrelate with eaeh other, and only a poor eorrelation (1' = 0.52, P < 0.05) was found between mutagenie aetivity and total eonc.entration of eytoehrome P-450f448. An explanation of these data is that the mlltagenieity of benz(a)anthraeene in the Ames test after induetiOll with P AlI is mainly due to eytoehrome P-448 isozymes. Furthermore, total conc.entration of eytochrome P-450f448 is no reliable parameter to estim ate the mutagenic. risk. The prineiple of seleetive induction of mierosomal lllonooxygenase isozymes also seems to be applieable for some but not all of the P AlI. Eaeh of these substanees seems to possess a speeific induction pattern, whereas the importance of the interaction between indueer and mutagen is underlined.
Subproject V III: lUicrosomal oxidation of phenanthrene, pyrene, chrysene, benzo( e)pyrelle and the induction of mixed-functional mOllooxygenases by polycyelic aromatic hy(lrocarbons and related compounds (J. JACOB, G. RAAB, G. GRIMMER) Key w 0 [(ls: mic.rosomal oxi{lation; polyc.ycl ic. aromatic hydrocarbons (PAR); phenanthrene ; pyrene ; chrysene; benzo( e)pyrene ; metabolite profiles ; carcinogenicity; liver; lung
The aim of this investigation was to reeord the metabolite profil es formed in rat liver with various environmentally relevant PAR (phenanthrene, pyrene, ehrysenc and benzo(e)pyrcne). It was further intended to study the dependenee of these profiles on various pretreatments of the animals (Wistar rats with differently potent induc.ers of the cytochromedependent monooxygenases). In this eOllneetion, it was of particular interest to know Exp. Pathol. 29 (1986) 2
73
whether the formation of ultimate carcinogens can be stimulated by P-448-monooxygenase induction. Metabolite profiles were recorded by means of capillary gas chromatography after a selective dean-up of the incubations and after conversions of the metabolites into their trim ethylsilylethers. The various components were characterized by their gas chromatographic retention times and their mass spectra which were compared with those of authentic standard:;. Phenanthrene is exclusively metabolized to the trans-9,10-dihydrodiol (K-region) by microsomes of untreated rats. Additional oxidation at the 1,2- and 3,4-position is observed with liver microsomes of P-448-induced rats. Pyrene is predominantly oxidized to 1-hydroxypyrene and 4,5-dihydroxy-4,5-dihydropyrene by liver microsomes of untreated rats. Pretreatment with P-488-inducers (various P AH) increases the oxidation at the I-position and induces the formation of two diphenols (1,6- and 1,8-dihydroxypyrene). Chrysene is a poor substrate for liver microsomes of untreated rats; it is oxidized predominantly to both the 1,2- and 3,4-dihydrodiol, and both oxidations are stimulated by P-448-inducer treatment. In the case of chrysene, the formation of the ultimate carcinogen can be enhaneed significantly by P AlI-pretreatment. In eontrast, the formation of an ultimate carcinogen from benzo( e)pyrene cannot be stimulated. Main metabolites from liver microsomes of non-induced rats are the 4,5-dihydrodiol (K-region) and the 1- and 3-hydroxybenzo(e)pyrene. Pretreatment with P-448-inducers gives rise to both the K-regioll dihydrodiol and the I-phenol. In all cases, however, inereased activities of monooxygenases resulted in the formation of secondary metabolites (triols and tetrols). In the case of benz(a)anthracene and pyrene, comparative studies on the metabolism with liver and lung microsomes were carried out which indicated a qualitatively different enzyme pattern in these tissues. K-region oxidation is only a minor pathway in the lung. Predominant pathways in the lung are oxidation at the 8,9-position in the ease of benz(a)anthracene and at the I-position in the case of pyrene. With liver microsomes of untreated rats, benz(a)anthracene is predominantly oxidized at the 10,Il-position, whereas pyrene is oxidized at the 1- and 4,5-position. Pretreatment with P-448-inducers shifts the metabolism of benz(a)anthracene to both the 5,6- and the 8,9-dihydrodiol and supresses the formation of the 10,Il-dihydrodiol. The same treatment favours the oxidation at the I-position in the case of pyrene. The liver microsomal metabolism of benz(a)anthracene was investigated with various species (rat, mouse, dog and man) and was found qualitatively similar in all of them. The metabolite profiles found in the rat and mouse resemble that found in human liver. The formation of the ultimate carcinogen was not observed either in human or in liver microsomes of untreated rats and mice. This metabolite, however, was detected with liver microsomes of P-448-induced rats. These results appear meaningful for the extrapolation of animal experiments to the human situation, since the presence or absence of proximate and ultimate carcinogens may decide on the fate of the cell. The metabolism of benz(a)anthracene was also studied in hamster lung cells. In prineiple, similar metabolite profiles as in lung microsomal incubations were observed. No hints for a K-region oxidation in these cells were obtained, and the main metabolites were the trans8,9- and the trans-lO,Il-dihydrodiol. There were minor amounts of phenols and secondary metabolites. Since this model - as a complete system - also contains those enzymes which catalyse the conjugation of hydroxy derivatives to glucuronic and sulphuric acid and to macromolecules, additional water-soluble metabolites were detected. Indications for the presenee of a methyltransferase system converting P AH base structures to their methyl derivatives were observed. The latter often possess more pronounced carcinogenic potencies. In the case of benz(a)anthracene, for example, the formation of I2-methylbenz(a)anthracene was detected.
Acknowledgement The present studies were carried od in areordance with the Cll',-irol1lreatal plan of the Federal Environment Agency by the order of the Federal Ministry of the Interior. 74
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Literature List of publications in the frame of the project "Evaluation of Environmenta I Carcinogens by Carcinogen-Specific Test-Systems". ABEL, D., and J. MISFELD, KrebsgeHihrdung durch polyzyklisehe aromatische Kohlenwasserstoffe am Arbeitsplatz - eine kritische Wiirdigung neuerer Ver6ffentlichungen. Zbl. Arbeitsmed. 34, 226-233 (1983). DEUTSCH- WENZEL, R., H. BRUNE, G. GRIMMER, G. DETTBARN and J. MISFELD, Experimental studies in rat lungs on the carcinogenicity and dose-respome relationships of eight frequently occurring environmental polycyclic aromatic hydrocarbons. JNCl 71, 539- 544 (1983). - - - - and J. MISFELD, Local application to mouse skin as a carcinogen specific test system for non-volatile nitroso compounds. Cancer Lett. (1985) in press. E~lURA, M., I-I.-B. RICHTER-REICHHELM, W. BONING, R. EICHINGEI{, C. SCIIOCH, J. ALTHOFF and D. MOHR, A fetal respiratory epithelial cell line for studying some problems of transplacental carcinogenesis in Syrian golden hamsters. J. Cancer Res. Clin. Oncol. 104, 133-144 (1982). - P. SCHNEIDER, C. SCHOCH and D. MOHR, Comparison of toxic and transforming effects of ten environment-related polycyclic hydrocarbons, including benz(a)pyrene, on fetal hamster lung cell cultures. J. App!. Toxico!. 2, 167-171 (1982). D. MOHR, T. KAKUNAGA and J. HILFRICH, Growth inhibition and transformation by long term exposure to diethylnitrosamine (DEN) in a human fetal tracheal cell line. Proc. Am. Assoc. Cancer Res. 25, 135 (1984). GmMMER, G., F. DETTBARK, H. BRUNE, R. DEUTSCH-WENZEL and.T. MISFELD, Quantification of the carcinogenic effect of polycyclic aromatic hydrocarbons in used engine oil by topical application onto the skin of mice. Int. Arch. Occup. Environ. Health 50, 95-100 (1982). H. BRUNE, R. DEUTSCH- WENZEL, K.- W. NAUJACK, J. MISl!'ELD and J. TIl\!l\l, On the contribution of polycyclic aromatic hydrocarbons to the carcinogenic impact of automobile exhaust condensate evaluated by local application onto mouse skin. Cancer Lett. 21, 105-113 (1983). - - G. DETTRARN and .T. MISFELD, Contribution of polycyclic aromatic hydrocarbons to the carcinogenic impact of gasoline engine exhaust condensate evaluated by implantation into the lungs of rats. J. Nat!. Cancer lnst. 72, 733-739 (1984). - J. JACOB, K.- W. NAUJACK, G. DETTBARN, H. BRUNE, R. DEUTSCH- WENZ~~L, J. JIIIrsFELD and J. TIMM, Contribution of polycyclic aromatic compounds to the carcinogenicity of flue gas from hard-coal-fired residential furnaces and characterization of arcnes (P AHl, azaarenes, oxaarenes and thiaarenes. 9th International Symposium on Polynuclear Aromatie Hydrocarbons, Columbus, Ohio 1984. H. BRUNE, R. DEUTSCII- WENZEL, G. DETTBARN, J. iYhSFELD, D. ABEL and J. TunI, The contribution of polycyclic aromatic hydrocarbons to the carcinogenic impact of emission eondensate from coal-fired residential furnaces evaluated by topical application to the skin of mice. Cancer Lett. 23, 167-176 (1984a). - J. JACOB, A. SCHMOLDT, G. RUB, D. MOHR and JIlL E~IURA, Metabolism of benz(a)anthracene in hamster lung cells in culture in eomparison to rat liver microsomes. In: ]\1. COOKE and A. J. DENNIS (eds.): Polynuclear Aromatic Hydrocarbons. Battelle Press, Columbus, Ohio 1985, pp. 521532. HEINRICH, D., W. STOBER and F. POTT, Long-term diesel exhaust inhalation studies with hamsters. In: W. E. PEPELKO, R. JIll. DANNER and N. A. CLARKE (eds.): Health Effects of Diesel Engine Emissions: Proceedings of an International Symposium, Vo!. 2. 1980, pp. 1026-1047. L. PETERS, W. FUNCKE, F. POTT, D. MmIR and W. STOBER, Investigation of toxic and carcinogenic effects of diesel exhaust in long-term inhalation exposure of rodents. In: J. LEWTAS (ed.): Toxicological Effects of Emissions from Diesel Engines. Elsevier Biomedical, New York, Amsterdam, Oxford 1982, pp. 225-242. and F. POTT, Possibilities and limitations of toxicity and carcinogenicity studies on emissions in inhalation. experiments. In: R. BASS, P. GIWSDANOFF, U. HACKENBERG, D. HENSCHLER, D. MULLER and H.-J. KLIMISCH (eds.): BGA-Schriften 5/84. Problems of Inhalation Toxicity Studies. MMV-VerJag Mfrnchen 1985, pp. 439-45l. - D. MOHR and W. STOBER, Experimental methods for the detection of the carcinogenicity and/or cocarcinogenieity of inhaled polyeyclic-aromatic-hydrocarbon-containing emissions. In: JIlL J. MASS, D. G. KAUFMAN, .T. lVI. SIEGFRIED, V. E. STEELE and ST. NESNOW (eds.): Carcinogenesis - A Comprehensive Survey. Vol. 8, Canem' of the Respiratory Tract: Predisposing Factors. Raven Press, N.Y. 1985, pp. 131-146. - - Lung tumors in rats and mice after inhalation of PAH-rich emissions. Exp. Pathol. 29, 29-34 (1986). JACOB, J., G. GRIMMER and A. ScnCiIOLDT, The influence of polycyclic aromatic hydrocarbons as inducers of monooxygenases on the metabolite profile of benz(a)anthracene in rat liver microsomes. Cancer Lett. 14, 175-185 (1981). - - G. RAAB and A. SCHl'IIOLDT, The metabolism of pyrene by rat liver microsomes and the influence of various monooxygenase inducers. Xenobiotica 12, 45-53 (1982). Exp. Pathol. 29 (1986) 2
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- and A. SCHMOLDT, Comparison of the metabolic profiles of pyrene and benz(a)anthracene in rat liver and lung by glass capillary gas chromatography/mass spectrometry. In: M. COOKE, A. J. DENNIS and G. L. FISHER (eds.): Polynuelear Aromatic Hydrocarbons. Battelle Press, Columbus, Ohio 1982, pp. 383-388. - A. SCH~IOLDT and G. GRIMMER, Formation of carcinogenic and inactive chrysene metabolites by rat liver microsomes of variolls monooxygenase activities. Arch. ToxicoI. 51, 255-2G5 (1982). - - Influcnce of monooxygcnase inducers on the metabolic profile of phenanthrene in rat liver mierosomes. ToxieoI. 25, 333-343 (1982). - - Benzo(e)pyrene metabolism in rat liver microsomes: dependence of the metabolite profile on the pretreatment of rats with various monooxygenase inducers. Carcinogenesis 4, 905-910 (1983). - G. RAAB, M. HAMANN and G. GRIMMER, Induction of specific monooxygenases by isoteric heterocyeJic compounds of benz(a)anthracene, benzo(c)phenanthrene and chrysene. Cancer Lett. 20,341-348 (1983). W. KARCHER, G. GRIMlIIER and A. SCHilIOLDT, The influcnce of various mono oxygenase inducers on rat liver microsomal chrysene oxidation. 9th International Symposium on Polynuclear Aromatic Hydrocarbons, Columbus, Ohio 1984. A. SCHMOLDT, G. GRIMMER, M. HAMANN and G. RAAB, Benzo(e)pyrene metabolism: Identification and quantitative analysis of metabolites formed by liver microsomes of variously pretreated rats by means of capillary gas chromatography/mass spectrometry. In: M. COOKE and A . .T. DENNIS (cds.): Polynuclear Aromatic Hydrocarbons. Columbus, Ohio 1985, pp. G27-G41. KEMENA, A., C. SCHih!ANN, .J. JACOB and K. NORPOTH, Induction of dealkylating enzymatic activities in mouse liver by 14 environmentally relevant polycyclic aromatic hydrocarbons and Clophcn A50. Carcinogenesis 1984, in press. MOHR, D., and M. El'IIUHA, The use of human and hamster tissue cells for the testing of carcinogens. In: F. HOMBURGER (eel.): Safety Evaluation and Regulation of Chemicals (2nd International Conference). Kager, Basel 1985, in press. - - Fetal human respiratory eells for£n vitro testing for careinogcnesis. In: F. HOMBURGER (cd.): Safety Evaluation and Regulation of Chemicals (3rd International Conference). Kager, Basel 1985, in press. NORPOTH, K., A. KEMENA, J. JACOB and C. SCHUMANN, The influence of 18 environmentally relevant polycyclic a,romatie hydrocarbons and Clophen A50, as liver monooxygenase inducers, on the mutage.J.lic activity of benz(a)anthraccne in the Ames test. Careinogenesis 5, 747-752 (1984). POTT, F., Dbcrsicht liber die Ergebnisse mit dem Subkutantest. Teil des AbschluBberichts der Arbeitsgruppe "Dntersuchung liber die kanzerogenc Belastung des Mensehen dureh Luftverunreinigung" fiir das Dmweltbundesamt 1983. 15 gez. 131. (iYIaschincnschriftl. Manuskript). - und U. HEINRICH, Versuehsmodell zur tierexperimentellen Kanzerogenitiitspriifung von Gesamtttbgasen. Umwelthygiene, Jahresbericht 1982, Band Hi, 8G-97 (1983). RICHTER-REICHHELM, H.-B., M. EIVIURA and J. ALTHOFF, Sc
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Beratungsforum fur Praventivmedizin und Umwelttoxikologie, Hamburg; Biochemisches Institut fur Umweltcareinogene, Ahrensburg; Fraunhofer-Institut fur Toxikologie und Aerosolforschung, Hannover; Institut fur Mathematik der Universitat, Hannover; Medizinische Hochsehule, Institut fur Experimentelle Pathologie, Hannover; Medizinisches Institut fiir Umwelthygiene der Universitat Dusseldorf; Universitatsklinikum der Gesamthochschule Essen, Institut fiir Hygiene und Arbeitsmedizin, Essen
Evaluation of environmental carcinogens by carcinogen-specific test systems By G. GRIMMER2 ), U. ABEL4), H. BRUNEI), R. DEUTSCH-WENZELI ), M. EMURA5), U. HEINRICH 3 ), ,J. JACOB2), A. KEMENA 7), J. lVIrsFELD 4 ), u. MOHR5 ), IL NORPOTH 7), F. POTT 6) and H.-B. RrcHTER-REICHHELM5 ) (Received August 8, 1985) Address for correspondence: Prof. Dr. G. GR!1'II:;!ER, Biochemisches Institut fur Umweltcarcinogene, Sieker LandstraBe 19, D - 2070 Ahrensburg, F.R.G.
Key w 0 r d s: environmental carcinogens; test systems, carcinogen-specific; polycyclic aromatic hydrocarbons (P AH); air pollution; emissions from pyrolysis; solid fuels; liquid fuels
Introduction Carcinogenic compounds are formed by pyrolytic processes during incomplete combustion of solid or liquid organic fuels. This raises the question whether these compounds occur in concentrations which may represent a general health hazard or a specific occupational risk. Epidemiological investigations have indicated a significant inerease of lung eaneer for speeifie working places e.g. in the case of coke plant workers. For a quantitative assessment of the earcinogenic effect of emissions from coal-fired furnaces, coke plants or vehicles, and for the identification of the carcinogenic constituents of emissions, a simulation of the carcinogenic effect in suitable animal models or other biological test systems such as cell or tissue cultures is required. Presently, the only way to confirm the correlations between air pollution and disease incidence which have been revealed by epidemiological studies is by use of animal experiments to establish a clear-cut relationship between dosage and cancer incidenee. Possibly due to the lack of a well-established proof of this nature, lung cancer of workers inhaling polycyclic aromatic hydrocarbons (P AH) had not been added to the list of occupational diseases at the beginning of this project. The careinogenic effect of an emission on man be estimated best by a combination of a suitable animal test system with ceIl or tissue culture systems. The selection of the appropriate animal species, mode of application and target organ is critical for meaningful extrapolation of the results to the human situation. Further support for the transferability of results from experimental animals to man can be obtained from the comparison of metabolites formed in analogous tissues or cell cultures as well as from the test of eell-transforming potencies of emission condensates and fractions thereof in human or animal cell cultures. This has been described for automotive exhaust and its fractions in subproject V with hamster lung cell cultures as a test system. The carcinogenic effect of eoke oven exhaust ean certainly be simulated better by inhalation experiments with mammals than with mutation rates of Salmonella. Concerning the mode of application, it may be assumed that the inhalation of emissions simulates the human exposure better than does the implantation of emission condensate into the lung or its instiIla5
65
tion into the trachea of experimental animals. The presently available results of subproject IV demonstrate that inhalation of aerosols from pyrolized hard-coal tar pitch results in lung carcinomas in 10 % of the exposed rats after 20 months. A disadvantage of the inhalation teehnique, however, is that although gases and aerosols sueh as atmospherie particles can be t ested, this is not feasible for subfractions of aerosol eondensates because of technical difficulties. It is very laborious to verify a causal relationship between an emission and an observed carcinogenic effect by means of animal experiments or other carcinogen-specific test systems. The more careinogen-specific th e method which is used, the mGre expensive it is. Less complicated are chemical-analytical methods, providing that the carcinogenic constituents of the emission are at least partially known. The evaluation of the carcinogenic potency of an emission from chemical analytical data becomes more precise as more individual carcinogenic compounds of the emission can be identified. One should at least know the dass of compounds mainly responsible for the carcinogenic effect. In the case of emissions from pyrolysis (combustion) of liquid or solid fuel s, the eareinogenie effed is almost exelusively caused by PAR. This can be proven after fractionation by comparing the ea,r cinogenie effect of the PAR-free and the P AR-eontaining parts with that of the original condensate. For the evaluation, suitable animal test systems and other biological careinogen-specific test systems can be used as has been done already for the evaluation of the carcinogenic risk caused by various emissions. Of course, statements on the contribution of distinct earcinogenic constituents are the more reliable if corroborated in several animal species, animal test models, and ee!! eulture systems. Renee, animal test systems such as mouse skin-painting or implantion into rat lung as well as cell and tissue cultures were applied as detectors for the carcinogenic activity of constituents in eomplex mixtures. It can be demonstrated by topical application onto the mouse skin or rat lung-implantation that more than 80 % of the carcinogenic potency of vehicle exhaust condensate from gasoline-driven engines is caused by the content of PAR eontaining more than 3 rings. The same holds true for used lubricating oil of this engine type. Similarly, using mouse s~in for evaluation it could be shown that it is the PAR-fraction containing more than 3 rings which is almost exelusively responsible for the eareinogenic potency of flue gas condensate from hard coal-fired furnaces. These results have been e011firmed by rat lung-implantation experiments used in this project (subproject II). Carcinogen-specific test systems have to meet two requirements: they must respond to all sorts of earcinogenic classes sueh as polycyclic aromatic hydrocarbons, oxygen- or nitrogen-eo ntaining polycyelic aromatie compounds, aromatic amines, nitrosamines, mycotoxins, etc. they ought to provide as far as possible for the diversity of morphologieal and biochemical reactions of human cells which finally lead to malignant transformation. The first requirement can readily be cheeked .• The lo eal applieation in mice can be ta,k en to be a detector system of universal applicability if not only PAR but also non-volatile nitroso compounds provoke skin tumors. In the frame of this project, dose-dependent earcinogellic effect of nitroso carbaryl and nitrosomethyl urea in mouse skin was verified (subproject III). According to our present knowledge the second requirement seems to be best met by comparing the morphological transformation effect and the metabolites in human and animal cell or tissue cultures. The question of transferability to humans of results obtained from the various 1:n vivo syst ems in animals can be investigat ed experimentally in cell or tissue cultures. To what extent analogous biochemi cal r eactions take place in human and animal cells of identical tissues ean be determined from both the transformation rates and the eomparison of the metabolite profiles of various eareinogens. In vitro systems such as the microsomal oxidation of PAR are very satisfactory, since the formation of metabolites (e.g. the ultimate carcinogens) can be detected analytically (subproject VIII) as well as by means of the Ames test (subproject VII). The aims of subprojects V and VI were to develop cell and tissue cultures from hamster lungs, rat livers and human lung's in order to compare the metabolism of various PAR and
66
Exp. Pathol. 29 (1986) 2
their cell transforming potency. It is still an open question whether cell cultures can also be used for the detection of carcinogenic constituents from emissions. First results comparing the cell transforming properties of automobile exhaust condensate, and four fractions thereof, with fetal hamster lung cells are encouraging. Only the fraction containing P AH with more than 3 rings results in the same dose response as the original vehicle exhaust condensate (subproject V). With regard to minimizing future animal experiments this test system should be systematically studied. In the following, the various sUbprojects are summarized.
Subproject I: Epidemiology of lung cancer MISFELD, U. ABEL)
Conclusions of an appraisal of the literature*
(J.
Key W 0 r d s: lung cancer, epidemiology; smoking; passive smoking; air pollution; emissions, environment; polycyclic aromatic hydroearbons (P AH); occupational exposure
A survey of important results of epidemiological research on associations between exogenous factors and lung cancer ri8k is given. The studies are reviewed as to the strength of their findings. The available evidence is summarized in a synoptic appraisal. After a longlasting worldwide rise of rates, lung cancer is now the most important type of cancer among males of western indnstrial countries and is growing rapidly among females. Four main causes are being discussed in the etiology of lung cancer: Smoking (above all, cigarette smoking), passive smoking, environmental air pollution, and occupational exposure. In all cases polycyclic aromatic hydrocarbons presumably are contributory causes. Smoking: As a consequence of the thorough epidemiologic investigations of the past 30 years it is no longer seriously contested today that cigarette smoking is the main risk factor for lung cancer, particularly squamous cell carcinoma. Most estimations are that smokers have 8 or more times the risk of developing lung cancer than nonsmokers (not taking into account the individual smoking habits), and the proportion of lung cancer attributable to smoking is estimated at 80 %. Recent studies show that smokers of filter cigarettes have a 10 to 50 % lower risk than smokers of non-filter cigarettes, despite a certain compensation by deeper inhalation.
Passive smoking: The opinions on the hazards of passive smoking are still contradictory today. At pres.ent, a critical examination of the existing literature justifies three conclusions.
1. Research has not succeeded in freeing passive smoking from the suspicion of being ca,rcinogenic. 2. Long-term passive smoking appears to impair lung function and thus has measurable effects. There is, however, no evidence that these effects are strong enough to produce respiratory symptoms which are comparable to those from active smoking. 3. So far, there is no conclusive epidemiological evidence that passive smoking causes an increased risk of lung cancer.
Environmental a1:r pollution: A critical survey of the published material, especially of the more recent articles, on lung cancer risk due to general air pollution permits the following conclusions: The thesis that, beside cigarette smoking and occupational exposures, general air pollution is an important factor in the etiology of lung cancer is not proven. A great number of studies have been performed on this question and there seems to be an agreement on the existence of an urban-rural gradient of rates. Many of these studies, however, were designed inappropriately and they mostly contained methodological shortcomings which might have produced erroneous results. While in none of the studies all relevant confounders were adequately controlled, the more carefully the bias was controlled the lower was the estimation of the environmental influence.
* 5*
A comprehensive monograph on the subject is forthcoming. Exp. Pathol. 29 (1986) 2
67
According to the authors of the best planned studies, one must doubt the existence of a measurable lung cancer risk due to general air pollution. At most, the effects are extremely small and detectable only in large-scale, specially-designed studies. There are also indications of synergistic actions of air pollution and smoking. The findings are not conclusive, however, and can be explained by imperfections of study designs. Further research on this subject is needed. Occupational exposure: Occupational exposure concerns only a limited part of the population, but because of the possibility (at least in principle) of prevention and because of the fact that these risks are taken involuntarily, they are of great importance. This holds, in particular, for the P AH which are set free in numerous industrial processes. Among the many epidemiological studies of the association between exposure at the work place (including exposures to P AH) and lung cancer risk, there are only a few which are not disputable. Often, smoking habits were not controlled, histologic types of the lung cancer cases were not distinguished, and exposures were not quantified. On the whole, the studies (particularly the carefully planned ones) give evidence for cancer risk at places where workers are exposed to mixtures of noxae containing PAH. This is strongly corroborated by dose-response relationships. The lung cancer risk, however, becomes evident only with high concentrations of P AH such as can be found in coke plants. At present, it is impossible to reliably quantify lung cancer risk due to P AH alone.
Subproject II: Comparison of the carcinogenic activity of airborne particulate matter extract with that of flue gas emitted from hard coal-fired furnaces allil fractions thereof by implantation into rat lungs (R. DEUTSCH-WENZEL, H. BRUNE) Key W 0 r ds: airborne particulate matter extract; flue gas eondensate; hard coal-fired furnaces; lung; carcinogenicity
The objective of the study was to compare the carcinogenic activity of airborne particulate matter of a large town with that of flue gas condensate from hard coal-fired furnaces as well as to identify such substances predominantly responsible for the carcinogenic effect of the emission of the latter. To determine how much P AH components contribute to the total carcinogenic potency of the flue gas condensate from coal-fired furnaces, the emission condensate was separated into individual fractions consisting of a P AH-free fraction and several P AH containing fractions (P AH containing 2 and 3 rings; P AH containing 4 to 6 rings; a combined fraction consisting of 6 and more rings and basic nitrogen containing P AH (Azaarenes). The carcinogenic effect of these fractions was eompared with that of the total condensate as well as with that of a total condensate recombined from all individual fractions. To examine the carcinogenic action, different concentrations of the condensate and fractions thereof (dosed proportionally) were implanted into the lungs of 3-month-old female Osborne "Mendel rats. Airborne particulate matter extract as well as flue gas emitted from coal-fired residential furnaces regularly induced epidermoid carcinomas and sporadically induced pleomorphic sarcomas at the implantation site. With respect to the materials administered, flue gas condensate proved to be more potent. The results of the examination of the fractions indicate that neither the PAH-free fraction participating with 45 (by weight) in the total weight of the condensate, nor the fraction containing 2 and 3 rings (12 % by weight) and the soot (about 7 % by weight) contribute to the carcinogenic effect of the condensate. The fraction of P AH consisting of 4 to 6 rings and the combined fraction containing 6 and more rings and azaarenes reveal a comparable carcinogenic potency. The effect of the latter has, however, to be deduced mainly from the P AH containing 6 and more rings, since it was shown in previous experiments with flue gas condensate and fractions thereof using the epicutaneous test with mice that the fraction containing azaarenes alone contributed only 4 to 7 % of the total activity.
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With the condensate combined from all individual fraetions (reeombinate) a carcinogenic effect eomparable with that of the original sample was observed. Aeeording to a provisionally raw estimation, about 2 to 3 % of the total carcinogenic activity is expected to be explained by the benzo(a)pyrene (BaP) content of the condensate (1.14 mg BaPjg eondensate). Similar to previous investigations with gasoline exhaust condensate using the lung implantation model in rats and the skin painting model in mice it was seen that the total carcinogenie activity of the flue gas emitted from hard coal-fired residential furnaces is predominantly attributable to P AH containing more than 3 rings. Thus, similar results were obtained using the skin and lung of two different species as carcinogen-specifie test systems.
Subproject III: Loeal application to mouse skin as a carcinogen-specific test system for nonvolatile nitroso compounds (H. BRUNE, R. DEUTSCH-WENZEL) Key w 0 r d s: carcinogen test; non-volatile nitroso compounds; emission condensates; skin
The objeetive of these studies was the demonstration of local carcinogenie activities of the nitroso eompounds methylnitrosourea, nitrosonornieotine and nitrosocarbaryl using the skin painting model with CFLP mice. This earcinogen-specific detection system is particularly well suited for eomparing the careinogenic poteney of emission condensates with that of fraetions prepared therefrom, sinee the action of carcinogenie substanees is barely influenced even by considerable amounts of aecompanying materials. The loeal application to the skin of miee is well established as a careinogen-speeifie animal model for both pure P AH and P AH in eomplex mixtures sueh as emission condensates from automobile exhaust, coal furnaees, domestic heating and used engine oil. The question of whether local carcinogenic effects can be provoked by nitroso compounds on the mouse skin is the more important, since aromatic nitro so compounds (e.g. nitrosocarbaryl) appear in P AH-containing fraetions, whereas other nitroso eompounds such as llitrosomethylurea and nitrosonornicotine are found in the hydrophilic fraetions. In the latter, however, no carcinogenic poteney has been found to date using the mouse skin model. Therefore, even if hydrophilic nitroso compounds have a slight carcinogenie aetivity, the contribution of this elass of compounds to the total carcinogenic potential of the emission condensates of automobiles, coal-fired residential furnaces or used engine oil is expected to be very small. In a chronic epicutaneous experiment, 65 female CFLP mice were treated twiee weekly over a period of 104 weeks by dropping three dosages of nitrosomethylurea, nitrosonornicotine or nitrosoearbaryl to the dipped interscapular region. Benzo(a)pyrene was used as the reference substance. When compared with the remaining groups, survival times were lower in the groups with higher dosages of nitrosomethylurea and nitrosocarbaryl. Following treatment with nitrosonornicotine, a weak careinogenic effect was observed but the tumor incidence showed no dependence on dose. With regard to induced earcinomas and sarcomas, elear dose-response relationships were obtained for nitrosomethylurea and nitrosocarbaryl as well as for the referenee substance benzo(a)pyrene. According to the results of pro bit analysis, the carcinogenic potencies of the nitroso compounds investigated in the mouse skin painting model rank as follows: nitrosocarbaryl, 0.18; nitrosomethylurea, 0.04 and nitrosonornicotine 0.008; (benzo(a)pyrene, 1.00).
Subproject IV: Squamous cell carcinoma of rat lungs after inhalation of PAH-rich emissions (U. HEINRICH, F. POTT, U. MOHR, L. PETERS, R. FUHST, J. Ki)NIG) Key W 0 r ds: polycyelic aromatic hydrocarbons (PAR); carcinogenic effect; co-carcinogenic effect; PAR-rich exhaust; lung carcinoma; occupational diseases
Aims and motivation: The aim of the investip;ation was to examine the carcinogenic and cocarcinogenic effeet of P AH -containing emissions. The motivation to earry out this study Exp. Pathol. 29 (1986) 2
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came from the fact that although numerous P AH and exhaust emission condensates have been shown to be carcinogenic in several animal species by various methods, and although there is increased lung cancer incidence in coke oven workers, there had been no attempt to apply these findings to occupational health guidelines. In the Federal Republic of Germany up to the beginning of our investigation, lung cancer in workers who were exposed to P AHcontaining exhaust for many years was not included in the register of occupational diseases. At present, no threshold limit value for benzo(a)pyrene or any other cancer producing P AH exists either for the workroom air or for the outdoor ambient atmosphere. Since 1983, a maximum allowable emission concentration for benzo(a)pyrene and dibenz(a, h)-anthracene is included in the Technical Instructions for Ensuring Clean Air (TA Luft). From the environmental hygiene and occupational medicine point of view, therefore, there was a need for additional animal experiments giving unequivocal evidence that P AHcontaining exhaust is carcinogenic. A further aim of our investigation was the development of a test model in inhalation carcinogenesis for the examination of the strongly suspected cumulative and inhibitive effects of definite particulate matter or gaseous substances. Methods: For the exhaust generation a domestic coal oven was used. A particular disadvantage of the coal oven is that, during the 2 to 3 h initial burning phase extremely high and varying concentrations of pollutants are emitted. During the following "glowing" burning phase, the levels of P AH and other components of emission (with the exception of CO) fall almost to ambient air levels. The emission of the initial burning phase could not be used for long-term animal inhalation experiments, however, because of the considerable variations in toxic components despite standard dilution. Therefore, the P AH-poor exhaust of the "glowing" coal used for the animal exposure had to be enriched with P All without significantly increasing simultaneously acute toxic substances, such as carbon monoxide. Initially this was achieved by continuously adding droplets of a tar/pitch solution to the glowing coal and later, more effectively, by mixing the coal oven exhaust with the effluent of pitch pyrolized at about 750 DC under nitrogen atmosphere. The proportion of this exhaust mixture used for inhalation exposure is given as follows: 1 part pyrolized pitch effluent, 400 parts coal oven exhaust, 4,000 parts clean air. (The PAH enrichment of the coal oven exhaust by adding small amounts of pyrolized pitch exhaust is very effective; in view of the conditions about 10 years ago this procedure is not too unrealistic because 7 pitch by mass was used as binding medium in the production of hard coal briquettes at that time.) The experimental animals (Wistar rats) were exposed for 19 h a day, 5 d a week, for about 20 months. The average exposure with P AH-enriched coal oven exhaust did not amount to more than 80 h/week, however, because of the time needed for maintenance of the pitch pumping and pyrolizing device. The particle bound benzo(a)pyrene concentration in the exposure chamber during the last 12 months of the experiment was 90,ug/m3 on average. Some other P AH have been determined in correspondingly high concentrations. Among the exhaust components like CO 2, CO, S02' NO, N0 2, total hydrocarbons and particulate matter measured continuously in the exposure chamber, only CO and S02 exceeded the maximum allowable concentration at workplaces (MAK) in the Federal Republic of Germany. The concentrations of the gaseous components of the coal oven exhaust did not change very much when the pyrolized pitch effluent was added. Some of the rats were additionally treated with intratracheal instillations of dibenz(a, h) anthracene or crocidolite to examine the influence of the exhaust on the tumor rate induced by these known carcinogens (2-phase model). Preliminary results: Approximately half of the 108 animals which were exposed to the exhaust without an additional treatment were still alive after two years. Up to now, 10 lung carcinoma bearing animals have been detected in the exhaust-exposed group and none in the clean air control group. The tumors were identified as squamous cell carcinomas and one small cell carcinoma. The histological examination of the other experimental groups has not yet been started.
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After one year of exposure, extensive clinical-chemical and hematological investigations with satellite groups hardly showed any differences between exposed animals and controls. The evaluation of the differential leukocyte count of the lung lavage fluid resulted in a significantly higher proportion of macro phages from the exhaust exposed groups and the phagocytic activity was also strongly increa,sed. Furthermore, the lung function tests using a whole body plethysmograph showed an exposure-related reduction of the compliance, but the alveolar lung clearance measured by a radioactive test aerosol had not deteriorated.
D1:scnss'ion: The lung carcinoma incidence rate of about 10 %, according to the present state of evaluation, can be regarded as clear evidence of the cancer-inducing effect of the exhaust exposure. An incidence rate of more than 25 % is expected at the end of the experiment. These positive findings could be established under exposure conditions which, up to now, did not shorten the survival time of the non-tumor-bearing animals. It is important to note that the PAR-content of the diluted exhaust in the inhalation chambers did not exceed by an unrealistic amount the PAR concentration to which the coke oven workers on the oven platform may be exposed (rats: exposed to 90,ug BaP/m3 for 80 hi week for about 1 year; coke oven workers on the oven platform: exposed to about 30,ug BaP 1m3 for 40 hjweek). From the present data no conclusions can be drawn as to what extent the lung tumor induction in the exhaust exposed rats was caused by P AH, the measured irritant gases, or the many other organic compounds the bulk of which are still unknown. This question will be the subject for further investigations. Snbprojecf V: Identification and assessment of environmental carcinogens by cell and tissue culture (U. MOHR, 1\1. E1vIURA, H.-B. RICHTER-REICHHELM) Key W 0 r ds: cell and tissue culture; carcinogen test; motor vehicle exhaust condensates; respiratory epithelial cells
Organ and tissue culture systems were developed from hamster and human epithelial tissue and mixed tissue of the respiratory tract with the aim of identifying and assessing the biological activity of environmental carcinogens. A motor vehicle exhaust condensate (KAK) showed a clear transforming effect on mixed cultures of hamster lung cells. From the exhaust condensate the following five fractions were produced in the usual way: KAK-1: total motor vehicle exhaust condensate; KAK-2: methanol (70%) + cyclohexane; KAK-3: PAR (2 and 3 rings); KAK-4: PAH (4 to 7 rings); KAK-5: polar PAC and N-P AC. The fraction KAK-4 induced a specific dose response, in which it showed a considerable weaker transforming activity than the total condensate. It was further established that the condensate fractions KAK-1 to KAK-3 in the dilution rate of 1: 500 already showed signs of a toxic effect. The fractions KAK-4 and KAK-5, however, revealed a slight growth stimulation at this degree of dilution. An established clonal ccllline from hamster lung epithelium (M3E3/C3) showed bi-potential differentiation abilities dependent on the culture conditions. With increased Ca++content and reduced serum concentration in the medium, the cells produced keratin-like filaments. The presence of EGF, insulin, hydrocortisone, estradiol and vitamin A in the medium induced a marked mucus production in the cells. The hormones, including EGF, particularly stimulated the development of the endoplasmatic reticulum, whereas vitamin A caused the increased development of Golgi apparatus and mucus granules. The cell line M3E3/C3 grew undifferentiatedly under conventional culture conditions and was not transformed by benzo(a)pyrene. After the addition of hormones and EGF to the medium, the cells of the M3E3/C3 were transformed by benzo(a)pyrene. Vitamin A reduced the transformation rate considerably. The characterisation (tumor type) of the induced growths from transformed cells is the subject of further investigations at the moment. By optimizing the culture conditions it was possible to cultivate tracheal epithelial cells of a human foetus in the 16th week of gestation for up to 10 passages. For this Ca++-reduced RPMI 1640 was used. The cells of this human cell line were treated with different doses of Exp. Pathol. 29 (1986) 2
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diethylnitrosamine (DEN). The colony formation test for t oxicity showed a clear decrease in the number of surviving colonies with increasing DE N doses. In transformation exp erim ents in which soft agar colonies with a size great er than O.lnll11 diameter were evaluated, a dosedependent transformati on rate resulted. Samples of the transformed eells were injeeted sub eu ta neously into ath ymi e nude mice t o cheek the tumor form ation ability of the cells. These investigations are not yet finalized. At the moment it is still also being checked whether the eells grow when infiltrated in mesenehymal tissue. For this transformed cells were transpl anted in v'itro onto chiek embryo skin. In another study using organ cultures from fetal hamster tracheae, the inducti on of histopathological ehanges such as metaplasia, dysplasia and exophytie growth was investigated after treatment with benz(a)anthracene (BaA), benz(a)aeridine (BaAc), benzo(b)fluoranthrene (BbF), pyrene (PYR) , benzo(k)fluor anthene (BkF ), and benzo( e)pyrene (BeP). The eompounds BaA, BaAe, Bb F, and BkF prove d to be eff ectiv e here. The findings showed a high correspondenee both with the results of in m:vo tests as well as with th ose of in vitro cell transformation t ests. The scanning eleetron microscopie interpr"etation used here offers the advantage that small dysplasti c ehanges, in particular exophytieally proliferating epithelial areas (neoplasia,s), r eliably recorded and eompared with in vivo situations ean be rated as clear indications of an earl y change.
Subproject V I: Developm ent of a cell culture system to (letermine carcinogenic activities of polycyclic hy(lrocarbons (D. PAUL) K ey word s: c.ell culture system; eal'cinogen test ; polycyclic a,ro mati c hydrocarbons (PAR); hepatocyte cultu res, metabolicall y compet ent
The aim of the pres ent r esearch proj eet is to establi sh metabolically co mpetent hepat ocyte eultures in which the cell s continue to expr ess various liver specific funetions and t o utilize them for short-term in vitro t est syst ems fo r the determination of eeotoxie activities. Such hepato cyt e cultures r epresent a novel, highly differentiat ed syst em in contrast to convent ional fibroblastic cells. They would therefore be particularl y useful as a model system for the study of human careinoma. Therefore, th e specific aim of t his projeet is t o utilize hepat ocyte culture syst ems that actively express enzymes capable of metabolizing xenobiotie compo unds in order t o provide useful systems to det ermine carcinogenic and potentially carcinoge nic compounds. By optimizing complex culture media it has been possible t o maintain proliferating hepat ocytes of fet al, newborn and adult r at livers in primar y cultures. The cells ar e differentiat ed hepato cyt es expressing specific liver fun etions which are expected t o be expressed in such cell types. F etal rat hepat oeytes in primary cultures multiply in a newl y developed medium lVIX-83 in the presence of Epidermal Gro wth Factor (EGF), insulin and transferrin. Hepatoe.ytes derived from newb orn rat s also r equire hydro cor tiso ne, and adult rat hepato cyt es gro w in the presence of EGF, insulin, transferrin, glucagon, hydro cortisone and serum. The mitogenic activity in serum ean be partiall y r eplaeed by pl atel et peptide(s) that are distinct frolll Platelet Derived Growth Factor (PDGF). Cells express a,]pha-fetopro t ein, albumin, t yrosine amino transferase, and various other liver-specifie markers. lVIorris hepatoma 7777 D NA has been shown to transform normal mouse 3T3 fibr oblas ts t o malignant cells after transfe ction. It is eoncluded fr om t hese studies that hepatoma DNA eontains transforming DNA sequences. Preliminary studies in which newborn rat hepat ocyt es have been used as r ecipient cells in transfection experiments involving various cloned oneo genes suggest that the oneogene of Moloney sarcoma virus (mos) cloned in expressio n vect or flanked by LTR's (plVISV-U1) is able t o transform hepatocytes pro vided that additional treatment of transfected cultures with a car cinogen (benzo(a)pyrene (BaP) or NlVIU) oecurs 3 d post transfeetion. Mos alone or either BaP or NlVI U treatments alone do not lead t o transformation. From these studies we eonelude that t he malignant transform ation of hep at oeyt es in monolayer eultures oceurs by at least two independent eoo perating events. We will attempt
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to isolate rnos-transfected hepatocytes (which could perhaps be immortalized cells) and to establish permanent cell lines which could be useful for studies in which the mechanism of the action of chemica.! carcinogens can be investigated.
Su.bproject V II: Influence of various inducers on the metabolic activation by "S9-1Uix" in the Ames-tcst (A.
KElIIENA,
C.
SCHUMANN, J. JACOB,
K.
NORPOTH)
Key wor ds: Ames-test; S9-Mix; polycyclic aromatic hydro carbons (PAR); metabolic activities; mutagenicity
'iVith the aid of the baeterial mutagenieity test-system (AMES), the indueing effeet of several environmentally relevant PAR on liver microsomal monooxygenases was investigated. After optimization of the test-system, 18 different PAR of high purity grade were injeeted into rodents and the resulting inducing effeets monitored by the mutagenic activity of benz(a)anthracene, a PAR known as a weak mutagen. Benzo(j)fluoranthene, benzo(b )fluoranthene, indeno(1,2,3-ed)pyrene, and dibenz(a,h)allthracene turned out to be the most potent of the substances screened, with revertant colony numbers as high as 15 times the va.!ue of the uninduced control. The potencies of these indueers are comparable to that of Clophen A50, a known strong indueer. Employing the same inducers (benzo(k)f1uoranthene or Clop hen A50), revertant colony numbers in the test with benz(a)anthraeene as substrate reached 50 ';;, of those obtained with the strong mutagen benzo(a)pyrene. It is thereby evident that the interaction between inducer and mutagen plays a vital role in the mutagenic risk. There are some good corrclations between the inducing effect of the PAR in the Ames-test (with benz(a)anthracene as mutagen) and the eontent of hydroxylation products in the metabolite pool of benz(a)anthracene in comparable induetion experiments. The best eorrelation eo efficient was found between the mutagenicity and the K-region derivatives (r = 0.776, P < 0.01) , whieh led us to cone/ude that the mutagenie aetivity observed is mainly due to K-region epoxides. For further characterization of the biochcmic.al mechanisms indueed by PAR, the mutagenieity was compared with two isozyme-selective dealkylating mono oxygenase activities and also with the total cytochrome P-450f448 eoncentration. We found statistically hig'hly significant eorrelations between mutagenieity and the activity of ethoxyeou marin deethylase (mainly selective for cytoc.hrome P-448 isozymes) (r = 0.77, P < 0.01) and between the aminopyrine demethylase ac.tivity (speeifie for cytoehrome P-450 isozymes) and total eonc.entration of cytoehrome P-450f448 (r = 0.82, P < 0.001). The two enzymic activities did not eorrelate with eaeh other, and only a poor eorrelation (1' = 0.52, P < 0.05) was found between mutagenie aetivity and total eonc.entration of eytoehrome P-450f448. An explanation of these data is that the mlltagenieity of benz(a)anthraeene in the Ames test after induetiOll with P AlI is mainly due to eytoehrome P-448 isozymes. Furthermore, total conc.entration of eytochrome P-450f448 is no reliable parameter to estim ate the mutagenic. risk. The prineiple of seleetive induction of mierosomal lllonooxygenase isozymes also seems to be applieable for some but not all of the P AlI. Eaeh of these substanees seems to possess a speeific induction pattern, whereas the importance of the interaction between indueer and mutagen is underlined.
Subproject V III: lUicrosomal oxidation of phenanthrene, pyrene, chrysene, benzo( e)pyrelle and the induction of mixed-functional mOllooxygenases by polycyelic aromatic hy(lrocarbons and related compounds (J. JACOB, G. RAAB, G. GRIMMER) Key w 0 [(ls: mic.rosomal oxi{lation; polyc.ycl ic. aromatic hydrocarbons (PAR); phenanthrene ; pyrene ; chrysene; benzo( e)pyrene ; metabolite profiles ; carcinogenicity; liver; lung
The aim of this investigation was to reeord the metabolite profil es formed in rat liver with various environmentally relevant PAR (phenanthrene, pyrene, ehrysenc and benzo(e)pyrcne). It was further intended to study the dependenee of these profiles on various pretreatments of the animals (Wistar rats with differently potent induc.ers of the cytochromedependent monooxygenases). In this eOllneetion, it was of particular interest to know Exp. Pathol. 29 (1986) 2
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whether the formation of ultimate carcinogens can be stimulated by P-448-monooxygenase induction. Metabolite profiles were recorded by means of capillary gas chromatography after a selective dean-up of the incubations and after conversions of the metabolites into their trim ethylsilylethers. The various components were characterized by their gas chromatographic retention times and their mass spectra which were compared with those of authentic standard:;. Phenanthrene is exclusively metabolized to the trans-9,10-dihydrodiol (K-region) by microsomes of untreated rats. Additional oxidation at the 1,2- and 3,4-position is observed with liver microsomes of P-448-induced rats. Pyrene is predominantly oxidized to 1-hydroxypyrene and 4,5-dihydroxy-4,5-dihydropyrene by liver microsomes of untreated rats. Pretreatment with P-488-inducers (various P AH) increases the oxidation at the I-position and induces the formation of two diphenols (1,6- and 1,8-dihydroxypyrene). Chrysene is a poor substrate for liver microsomes of untreated rats; it is oxidized predominantly to both the 1,2- and 3,4-dihydrodiol, and both oxidations are stimulated by P-448-inducer treatment. In the case of chrysene, the formation of the ultimate carcinogen can be enhaneed significantly by P AlI-pretreatment. In eontrast, the formation of an ultimate carcinogen from benzo( e)pyrene cannot be stimulated. Main metabolites from liver microsomes of non-induced rats are the 4,5-dihydrodiol (K-region) and the 1- and 3-hydroxybenzo(e)pyrene. Pretreatment with P-448-inducers gives rise to both the K-regioll dihydrodiol and the I-phenol. In all cases, however, inereased activities of monooxygenases resulted in the formation of secondary metabolites (triols and tetrols). In the case of benz(a)anthracene and pyrene, comparative studies on the metabolism with liver and lung microsomes were carried out which indicated a qualitatively different enzyme pattern in these tissues. K-region oxidation is only a minor pathway in the lung. Predominant pathways in the lung are oxidation at the 8,9-position in the ease of benz(a)anthracene and at the I-position in the case of pyrene. With liver microsomes of untreated rats, benz(a)anthracene is predominantly oxidized at the 10,Il-position, whereas pyrene is oxidized at the 1- and 4,5-position. Pretreatment with P-448-inducers shifts the metabolism of benz(a)anthracene to both the 5,6- and the 8,9-dihydrodiol and supresses the formation of the 10,Il-dihydrodiol. The same treatment favours the oxidation at the I-position in the case of pyrene. The liver microsomal metabolism of benz(a)anthracene was investigated with various species (rat, mouse, dog and man) and was found qualitatively similar in all of them. The metabolite profiles found in the rat and mouse resemble that found in human liver. The formation of the ultimate carcinogen was not observed either in human or in liver microsomes of untreated rats and mice. This metabolite, however, was detected with liver microsomes of P-448-induced rats. These results appear meaningful for the extrapolation of animal experiments to the human situation, since the presence or absence of proximate and ultimate carcinogens may decide on the fate of the cell. The metabolism of benz(a)anthracene was also studied in hamster lung cells. In prineiple, similar metabolite profiles as in lung microsomal incubations were observed. No hints for a K-region oxidation in these cells were obtained, and the main metabolites were the trans8,9- and the trans-lO,Il-dihydrodiol. There were minor amounts of phenols and secondary metabolites. Since this model - as a complete system - also contains those enzymes which catalyse the conjugation of hydroxy derivatives to glucuronic and sulphuric acid and to macromolecules, additional water-soluble metabolites were detected. Indications for the presenee of a methyltransferase system converting P AH base structures to their methyl derivatives were observed. The latter often possess more pronounced carcinogenic potencies. In the case of benz(a)anthracene, for example, the formation of I2-methylbenz(a)anthracene was detected.
Acknowledgement The present studies were carried od in areordance with the Cll',-irol1lreatal plan of the Federal Environment Agency by the order of the Federal Ministry of the Interior. 74
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Literature List of publications in the frame of the project "Evaluation of Environmenta I Carcinogens by Carcinogen-Specific Test-Systems". ABEL, D., and J. MISFELD, KrebsgeHihrdung durch polyzyklisehe aromatische Kohlenwasserstoffe am Arbeitsplatz - eine kritische Wiirdigung neuerer Ver6ffentlichungen. Zbl. Arbeitsmed. 34, 226-233 (1983). DEUTSCH- WENZEL, R., H. BRUNE, G. GRIMMER, G. DETTBARN and J. MISFELD, Experimental studies in rat lungs on the carcinogenicity and dose-respome relationships of eight frequently occurring environmental polycyclic aromatic hydrocarbons. JNCl 71, 539- 544 (1983). - - - - and J. MISFELD, Local application to mouse skin as a carcinogen specific test system for non-volatile nitroso compounds. Cancer Lett. (1985) in press. E~lURA, M., I-I.-B. RICHTER-REICHHELM, W. BONING, R. EICHINGEI{, C. SCIIOCH, J. ALTHOFF and D. MOHR, A fetal respiratory epithelial cell line for studying some problems of transplacental carcinogenesis in Syrian golden hamsters. J. Cancer Res. Clin. Oncol. 104, 133-144 (1982). - P. SCHNEIDER, C. SCHOCH and D. MOHR, Comparison of toxic and transforming effects of ten environment-related polycyclic hydrocarbons, including benz(a)pyrene, on fetal hamster lung cell cultures. J. App!. Toxico!. 2, 167-171 (1982). D. MOHR, T. KAKUNAGA and J. HILFRICH, Growth inhibition and transformation by long term exposure to diethylnitrosamine (DEN) in a human fetal tracheal cell line. Proc. Am. Assoc. Cancer Res. 25, 135 (1984). GmMMER, G., F. DETTBARK, H. BRUNE, R. DEUTSCH-WENZEL and.T. MISFELD, Quantification of the carcinogenic effect of polycyclic aromatic hydrocarbons in used engine oil by topical application onto the skin of mice. Int. Arch. Occup. Environ. Health 50, 95-100 (1982). H. BRUNE, R. DEUTSCH- WENZEL, K.- W. NAUJACK, J. MISl!'ELD and J. TIl\!l\l, On the contribution of polycyclic aromatic hydrocarbons to the carcinogenic impact of automobile exhaust condensate evaluated by local application onto mouse skin. Cancer Lett. 21, 105-113 (1983). - - G. DETTRARN and .T. MISFELD, Contribution of polycyclic aromatic hydrocarbons to the carcinogenic impact of gasoline engine exhaust condensate evaluated by implantation into the lungs of rats. J. Nat!. Cancer lnst. 72, 733-739 (1984). - J. JACOB, K.- W. NAUJACK, G. DETTBARN, H. BRUNE, R. DEUTSCH- WENZ~~L, J. JIIIrsFELD and J. TIMM, Contribution of polycyclic aromatic compounds to the carcinogenicity of flue gas from hard-coal-fired residential furnaces and characterization of arcnes (P AHl, azaarenes, oxaarenes and thiaarenes. 9th International Symposium on Polynuclear Aromatie Hydrocarbons, Columbus, Ohio 1984. H. BRUNE, R. DEUTSCII- WENZEL, G. DETTBARN, J. iYhSFELD, D. ABEL and J. TunI, The contribution of polycyclic aromatic hydrocarbons to the carcinogenic impact of emission eondensate from coal-fired residential furnaces evaluated by topical application to the skin of mice. Cancer Lett. 23, 167-176 (1984a). - J. JACOB, A. SCHMOLDT, G. RUB, D. MOHR and JIlL E~IURA, Metabolism of benz(a)anthracene in hamster lung cells in culture in eomparison to rat liver microsomes. In: ]\1. COOKE and A. J. DENNIS (eds.): Polynuclear Aromatic Hydrocarbons. Battelle Press, Columbus, Ohio 1985, pp. 521532. HEINRICH, D., W. STOBER and F. POTT, Long-term diesel exhaust inhalation studies with hamsters. In: W. E. PEPELKO, R. JIll. DANNER and N. A. CLARKE (eds.): Health Effects of Diesel Engine Emissions: Proceedings of an International Symposium, Vo!. 2. 1980, pp. 1026-1047. L. PETERS, W. FUNCKE, F. POTT, D. MmIR and W. STOBER, Investigation of toxic and carcinogenic effects of diesel exhaust in long-term inhalation exposure of rodents. In: J. LEWTAS (ed.): Toxicological Effects of Emissions from Diesel Engines. Elsevier Biomedical, New York, Amsterdam, Oxford 1982, pp. 225-242. and F. POTT, Possibilities and limitations of toxicity and carcinogenicity studies on emissions in inhalation. experiments. In: R. BASS, P. GIWSDANOFF, U. HACKENBERG, D. HENSCHLER, D. MULLER and H.-J. KLIMISCH (eds.): BGA-Schriften 5/84. Problems of Inhalation Toxicity Studies. MMV-VerJag Mfrnchen 1985, pp. 439-45l. - D. MOHR and W. STOBER, Experimental methods for the detection of the carcinogenicity and/or cocarcinogenieity of inhaled polyeyclic-aromatic-hydrocarbon-containing emissions. In: JIlL J. MASS, D. G. KAUFMAN, .T. lVI. SIEGFRIED, V. E. STEELE and ST. NESNOW (eds.): Carcinogenesis - A Comprehensive Survey. Vol. 8, Canem' of the Respiratory Tract: Predisposing Factors. Raven Press, N.Y. 1985, pp. 131-146. - - Lung tumors in rats and mice after inhalation of PAH-rich emissions. Exp. Pathol. 29, 29-34 (1986). JACOB, J., G. GRIMMER and A. ScnCiIOLDT, The influence of polycyclic aromatic hydrocarbons as inducers of monooxygenases on the metabolite profile of benz(a)anthracene in rat liver microsomes. Cancer Lett. 14, 175-185 (1981). - - G. RAAB and A. 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