C cer Research Clinical 9
J Cancer Res Clin Oncol (1983)105:24-26
9 Springer-Verlag 1983
Intratracheal Instillation of Automobile Exhaust Condensate in Syrian Golden Hamsters* U. Green, H. Warnecke, P. Schneider, and U. Mohr Abt. experimentelle Pathologic, Medizinische Hochschule Hannover, Karl-Wiechert-Allee 9, D-3000 Hannover 61, Federal Republic of Germany
Summary. Male Syrian golden hamsters were intratracheally instilled with automobile exhaust condensate at dose levels of 0.306, 0.613, 1.25, 2.5, and 5.0 rag/ hamster once a fortnight for 78 weeks. While a total of six tumors were found in the trachea and lung, hamsters treated with the vehicle only showed no respiratory tract neoplasms. Bronchiogenic adenomatous proliferations and hyperplastic nodules in the lung occurred in all groups, with a significant difference in incidence being seen between the high-dose groups and the controls.
Materials and Methods
Key words: Automobile exhaust condensate - Syrian golden hamster - Intratracheal instillation - preneoplastic alterations Tumours of trachea and lung
One hundred and eighty male Syrian golden hamsters from an outbred colony (Coombehurst Breeding Establishment, Baughurst, Hants, England) were kept under standard laboratory conditions (room temperature 22 + 2 ~ relative humidity 50 + 5%; air change 20 times per hour) and housed five to a Makrolon cage (Type III, E. Becker, Castrop-Rauxel, FRG). They received a pelleted diet (RMHB, Hope Farms, Woerden. The Netherlands) and water ad libitum. Animals were checked twice daily.
Chemicals
AEC was collected as the particulate matter of an engine exhaust using the Europe drive cycle (Prof. Grimmer, Biochemisches Institut fiir Umweltcarcinogene, Hamburg, FRG). PAH contents were determined by concentration and analyzed as described by Grimmer et al. (1973). AEC doses were suspended in 0.2ml Tris-HCl-buffer (pH 7.4) and EDTA (0.001 M).
Animals
Introduction Polycyclic aromatic hydrocarbons (PAHs) occur frequently in the environment as a result of incomplete combustion; at high doses and after long-term exposure, some of these compounds may be hazardous to man. Experimentally, the carcinogenic effect of a number of PAHs is well established; their syncarcinogenic activity in automobile exhaust condensate (AEC) has also been examined (Kotin et al. 1954; Schmfihl et al. 1977; Pott et al. 1977). Whereas skin painting in mice was initially the most popular method of application of AEC for demonstrating carcinogenicity, this study uses local intratracheal administration into the Syrian golden hamster which is known to be a sensitive model in testing for respiratory-tract carcinogenesis.
Treatment and Histology
At 8 weeks of age, the hamsters were randomly distributed into five treatment groups and one control group, each consisting of 30 animals. Treated animals were intratracheally instilled at 2-week intervals for 78 weeks with either 0.306 rag, 0.613 mg, 1.25 mg, 2.5 mg or 5.0 mg AEC/hamster. Control animals received the solvent (0.2 ml) only. Moribund hamsters were killed with carbon dioxide; complete autopsies were performed on both these hamsters and those which were found dead. Organs were fixed in 10% buffered formalin. Paraplast sections were stained with hematoxylin and eosin and by the van Gieson method. Step sections were prepared from the larynx, trachea and lung.
Statistics
* This study was performed with the team "Investigations on the carcinogenic burden of air pollution in man" and supported by the Bundesministerium des Innern of the Federal Republic of Germany (F & E Vorhaben 10706004) Offprint requests to: Prof. Dr. U. Mohr (address see above)
0171-5216/83/0105/0024/$1.00
For statistical evaluation, only those animals were considered whose respiratory organs had been histologically examined (effective number of animals). Survival data are from the beginning of treatment and were statistically examined by variance analysis. The number of nonneoplastic lesions was tested by the chi-square method.
U. Green et al. : Automobile Exhaust Condensate in Syrian Golden Hamsters
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Table 1. Treatment of Syrian golden hamsters with automobile exhaust condensate Dose rag/ hamster
Effective no. of hamsters
Survival (weeks)
Total Dose mg/kg
Larynx/ Lung trachea hyperplasia Ad. Prolif.
No. of tumors Hyp. Nodule
(%) 0 0.306 0.612 1.25 2.50 5.00
15 17 19 27 25 24
76.2_+14.9 75.4_+14.7 73.8_+11.9 81.6+_17.3 69.4_+25.6 69.1 _+23.0
0 65.2 144.1 420.2 642.8 1,322.7
15 7 5 30 20 17
%
Week a
%
Week"
-11 27 59 36 54
40 59 54 42 29
7 7 5 7 44 17
67 88 94 98 61 69
Trachea
2 1
Lung
2 1
Other sites b
la, lb la, lb, lc ld -
Ad. Prolif. = bronchiogenic adenomatous proliferation; Hyp. Nodule = hyperplastic nodule a Initial macroscopic observation b a = pheochromocytoma of adrenal gland; b = papilloma of forestomach; c = fibrosarcoma of thorax; d = malignant melanoma
Results
Discussion
Due to traumtic injuries during intratracheal instillation and/or anesthesia, a number of animals were lost in the early part of the experiment and were not considered for histological examation and statistical analysis. The remaining hamsters tolerated both treatment and substance without signs of disease, decrease in average life expectancy or marked reduction in weight development in comparison with the vehicle control animals. The latter did not show neoplasms originating in the respiratory epithelium nor did the hamsters of the treatment group reveal neoplastic growth in the nasal cavities or larynx. However, a few animals exposed to AEC developed papillomas of the trachea (1.25 mg= 94 and 85 weeks; 2.5 rng = 61 weeks) (Table 1). A total of three tumors were also found in the lungs: a bronchiogenic adenoma (83 weeks), an adenocarcinoma (88 weeks) in the 1.25 mg group, and an epidermoid carcinoma (89 weeks) in the 5 mg group. The tumor spectrum in other organs including frequency, location, latency, and histological type corresponded to that seen in controls. So-called neoplastic precursor lesions orginating in the respiratory epithelium of the larynx, trachea and bronchi were found frequently. The incidence of severe hyperplasia and metaplasia ranged from 15%-30%, the latter rate (1.25 mg group) being significantly increased compared with that of the controls (p < 0.05). Bronchiogenic adenomatous proliferation occured as early as 29 weeks after the beginning of treatment. The frequency of these lesions was between 11% and 59% and the rates in the treatment groups were significantly higher than in controls (p < 0.001). Bronchiogenic hyperplastic nodules developed in all groups, but incidence was only significantly enhanced (p < 0.001) in the 2.5 mg group. These lesions were not observed before week 61 of treatment. A dose response relationship could not be established for the incidence of epithelial alterations or for their latency.
Epidemiological studies link atmospheric pollution in industrial areas with respiratory tract disease in general and with lung cancer in particular (Wynder and Hammond 1962). In this context, a number of PAHs are thought to be the active components of automobile exhaust fumes. Treatment of mouse skin with AEC resulted in the development of tumors originating in the epidermis (Kotin et al. 1954; Wynder and Hoffmann 1962; Brune 1977). After short-term intatracheal instillation (four times in four weeks) of AEC, the respiratory epithelium of Syrian golden hamsters showed minimal pathological changes in the lung, such as slightly increased interstitial cellularity, enlarged bronchial epithelial cells and squamous cell metaplasia (Stenbfick 1974). After chronic exposure to AEC, proliferative changes of the bronchial epithelium were described as being pulmonary adenomas originating from peripheral bronchioles (Reznik-Schiiller and Mohr 1977). Similar changes were found in the present study. These changes corresponded more to reactive and proliferative lesions than to true neoplasms; therefore, they were thought to represent adenomatous bronchiogenic hyperplasia and hyperplastic nodules which may convert to neoplastic growth as was found in a small number of cases after exposure to high doses over a long period. A squamous cell carcinoma could only be found after a total dose of 195 mg AEC and a treatment period of 89 weeks. Some experiments with AEC have demonstrated lower tumor-inducing potencies than were expected from the amount of B(a)P present in the condensate (Hoffmann and Wynder 1962; Hoffmann et al. 1965; Pott et al. 1977). Similarly, reconstituted mixtures of PAHs may be less effective than assumed from the proportion of one of the components (Pfeiffer 1977). Results after intratracheal instillation of AEC into Syrian golden hamsters contrast with those obtained by epicutaneous testing of mice, where dose-related effects
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U. Green et al. : Automobile Exhaust Condensate in Syrian Golden Hamsters
and a strong carcinogenicity were observed. Reasons for this observation may depend on differences in the mode of application, vehicle or solvent and clearance machanisms. Since data from two species now exsist indicating a biologic effect of AEC, further studies are needed to examine the risk of chronic respiratory disease and (co-) carcinogenesis in laboratory animals and man. Acknowledgement. The authors are grateful to Susan Hamilton for help with the manuscript.
References Brune HFK (1977) Experimental results with percutaneous applications of automobile exhaust condensate in mice. IARC Sci. Publ. No. 16:4147 Grimmer G, Hildebrandt A, B6hmke H (1973) Investigations on the carcinogenic burden by air pollution in man. IL Sampling and analytics of polycyclic aromatic hydrocarbons in automobile exhaust gas. 2. Enrichment of the PAH and separation of the mixture of all PAH. Zentralbl Bakteriol [Orig B] 158:3549 Hoffmann D, Theisz E, Wynder EL (1965) Studies on the carcinogenicity of gasoline exhaust. J Air Pollut Control Assoc 15:162165 Hoffmann D, Wynder EL (1962) Analytical and biological studies on gasoline engine exhaust. J Natl Cancer Inst 9:91-116
Kotin P, Falk H, Thomas M (1954) Aromatic hydrocarbons. II. Presence in particulate phase of gasoline engine exhaust and carcinogenicity of exhaust extracts. AMA Arch Indust Hyg 9:164177 Pfeiffer EH (1977) Oncogenic interaction of carcinogenic and noncarcinogenic polycyclic aromatic hydrocarbons in mice. IARC Scientific Pulbication 16:69-77 Pott F, Tomingas R, Misfeld J (1977) Tumours in mice after subcutaneous injection of automobile exhaust condensates. IARC Sci Publ No 16:78-87 Reznik-Schiiller H, Mohr U (1977) Pulmonary tumorigenesis in Syrian golden hamsters after intratracheal instillation with automobile exhaust condensate. Cancer 40:203-210 Schm/ihl D, Schmidt KG, Habs M (1977) Syncarcinogenic action of polycyclic hydrocarbons in automobile exhaust gas condensates. IARC Sci Publ No 16:53-59 Stenb/ick F (1974): Morphogenesis of experimental lung tumors hin hamsters: The effects of carrier dust. In: Karbe E, Park JF (eds) Experimental lung cancer. Springer, Berlin Heidelberg New York (International Symposium, pp 161 172) Wattenberg LW ( 1961): Chemoprophylaxis of carcinogenesis: A review. Cancer Res. 26:1520-1526 Wynder EL, Hammond EC (1962) A study of air pollution carcinogenesis. I. Anlaysis of epidemiological evidence. Cancer 15:7992 Wynder EL, Hoffmann D (1962) A study of air pollution carcinogenesis. III. Carcinogenic activity of gasoline engine exhaust condensate. Cancer 15:103-108 Received March 23, 1981/Accepted June 3, 1982
