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Cancer Lettera, 9 (1980) 277-284 @ Elsevier/North-Holland Scientific Publishers Ltd. CORRELATION OF INDUCIBILITY OF ARYL HYDROCARBON HYDROXYLASE WITH SUSCEPTIBILITY TO 3-METHYLCHOLANTHRENE-INDUCED LUNG CANCERS RICHARD E. KOURI, LEONARD H. BILLUPS*, THOMAS H. RUDE**, CARRIE E. WHITMIRE***, BERNARD SASSt and CAROL J. HENRY Department of Biochemical Oncology and Department of Experimental Oncology. Yficrobiological Associates, 5221 River Road, Bethesda, MD 20016 (U.S.A.) (Received 5 February 1980) (Accepted 12 March 1980) SUMMARY C57BL/6Cum, DBA/2Cum, first filia (Ft), and backcross progeny from these 2 parental strains of mice were evaluated for their susceptibility to 3-methylcholanthrene-induced lung cancers. In the crosses among these mice, aryl hydrocarbon hydroxylase (AHH) responsiveness segregated as a single autosomal dominant gene (the Ah locus). AHH responsive mice (Ahb allele) expressed 40--60 units AHH activity/g wet wt liver following intra- peritoneal treatment with 3-methylcholanthrene (MCA) compared to AHH non-responsive mice (Ahd allele) which expressed 7- 11 units AHH activity/ g wet wt liver after MCA treatment. Intratracheal administration of 500 µg MCA for a total of 4 times at weekly intervals yielded a variety of pulmonary cancers, including squamous cell carcinomas, alveolar adenocarci- nomas, and adeno-squamous cell carcinomas among mice that survived 1 year after the carcinogen treatment. The AHH responsive C57BL/6Cum, F1, and C57BL/6Cum X F1 animals were much more susceptible to MCA: induced lung cancers than the AHH non-responsive DBA/2Cum mice. The lung cancers were also not randomly distributed in DBA/2Cum X F, back- cross progeny since significantly more lung cancers were found in AHH- *Present address: Environmental Pathology Services, 809 Viers Mill Road, Rockville, Maryland 20851, U.S.A. **Present address: Department of Pathology, School of Medicine, The University of North Carolina at Chapel Hill, Preclinical Educational Building 228H, Chapel Hill, North Carolina 27514, U.S.A. ***Present address: National Cancer Institute/National Toxicology Program, Bethesda, Maryland 20205, U.S.A. tPresent address: Carcinogenesis Testing, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Maryland 20205, U.S.A.

278 responsive progeny than in AHH non-responsive mice. Data support genetic linkage between susceptibility to MCA-induced lung carcinomas and the Ahb,allele. INTRODUCTION Aryl hydrocarbon hydroxylase (AHH) is one of the major.multicom- ponent, microsomally bound enzyme systems functioning in the biotrans- formation of many drugs, hormones, or chemical pollutants [1,2,13]. In the inbred strains of mice, conditions can be obtained so that AHH respon- siveness* segregates as a single autosomal dominant gene [15,181, a single autosomal codominant gene [19], or one in which non-inducibility is dominant [17]. In each of these genetic systems, there is a correlation between the capacity of hepatic tissue to respond to and metabolize poly- cyclic aromatic hydrocarbons and, susceptibility to subcutaneous fibro- sarcomas induced by 3-methyicholanthrene (MCA) [6,7,9,11,12]. Conditions which preferentially alter pulmonary AHH activity have been established [8,10]. These studies suggest that, as with hepatic tissue, there is specific genetic regulation of AHH activity following intratracheal treatment with polycyclic aromatic hydrocarbons. Thus, pulmonary AHH responsive- ness can be easily inferred from determinations of AHH responsiveness of hepatic tissue [121. The methods used in these reports, coupled with those of Nettesheim and Hammons [16] for producing pulmonary carcinomas in inbred strains of mice, suggest the possibility of an animal model system in which the susceptibility to lung carcinomas may be specifically linked to the capacity of that organ to metabolize chemical carcinogens. This paper describes such a model system: ibIATERIALS AND METHODS C57BL/6Cum (B6), DBA/2Cum (D2), and B6D2F1/Cum (F1) mice of both sexes were purchased from Cumberland View Farms, Clinton, TN. Backcross animals were produced in our own laboratory. At 8--10 weeks of age, mice were inoculated intratracheally with 500 µg MCA in 0.02 ml sterile 0.2% gelatin-saline according to the procedures of Ho and Furst [4j as modified by Kouri et al. [8,10]. Immediately prior to use, the solu- tion was sonicated for 30 s at setting No. 6 using a Branson Sonicator. For intratracheal treatment, mice were anesthetized with the inhalation anesthetic, Metophane (Pitman-Moore, Co., Trenton, NJ). Preliminary studies have shown that Metophane has negligible effect on pulmonary *The term 'responsiveness', as used in this paper, denotes a relative increase in rates of de novo synthesis or of enzyme activity from pre-existing moieties, or in rate of both when compared to rate of breakdown. No particular mechanism is implied.

279 and/or hepatic AHH activity (unpublished observations). Mice were originally scheduled to receive 6 weekly intratracheal instillations; the same schedule shown by Nettesheim and Hammons [16] to produce bronchogenic squamous cell carcinomas (SCC) in inbred strains of mice. However, because off the severe toxicity observed, only 4 inoculations were done. Controls consisted of B6, D2 and F, mice which received 0.2% gelatin in sterile saline only. At necropsy, lungs were fixed in situ by intratracheal injection of 1.0 ml 2% gluteraldehyde using an 18 gauge needle. The organ was ligated at the trachea before removal from the animal. The lung was sectioned through a frontal plane, with serial 6 µm sections taken at 3 levels in the lung. Sections were stained with hematoxylin and eosin and examined for lung pathology. Hepatic AHH activity was determined in all animals which were termi- nally sacrificed. Mice were treated intraperitoneally (i.p) with 80 µg MCA/g body wt 24 h before sacrifice. At sacrifice, lungs were removed for treatment described above and livers excised and stored at - 70°C until assayed. The assay for hepatic AHH activity [7,18] was performed on all liver samples on the same day. Activity was calculated in terms of units (U)/g wet wt liver. A unit is defined as that amount of enzyme causing the fluorescent equivalent of 1 nmol 3-hydroxybenzo[a]pyrene (3-OH-BP) per min at 37°C. The 3-OH-BP was determined in an Aminco-Bowman spectrophotofluorometer with activation at 398 nM and emission at 520 nM. As determined previously [6,9], there is an almost 10-fold diffe- rence between non-responsive and responsive levels of AHH in these strains. MCA-treated, non-responsive animals expressed AHH levels of 7--11 U/g wet wt liver, while activity in MCA-treated responsive animals was 40--60 U/g wet weight liver. It should be pointed out that in this genetic system, detection of high levels of 3-OH-BP is indicative of the formation of high levels of virtually all known metabolites of many polycyclic aromatic hydrocarbons, particularly metabolites formed via enzymatic activity at non-K-region positions [12,14]. RESULTS Intratracheal instillation of 500 µg MCA to these strains of mice was very toxic. Ten weeks after treatment, only 20---40% of the treated animals survived. D2 mice appeared to be the most sensitive to these toxic effects. The major cause of death was determined to be acute bronchopneumonia. At monthly intervals, 5--10 of the survivir.g B6 animals were randomly killed and examined histopathologically for evidence of lung lesions. An acute inflammatory reaction was observed in the MCA-treated animals, these lesions were initially located primarily around the terminal bron- chiole, often progressed to bronchitis and, in some animals, to broncho- pneumonia. Of the 40 animals randomly sacrificed during this time, only 1 was observed to have evidence of pulmonary pathological changes (i.e.,

TABLE 1 E LUNG LESIONS OBSERVED AFTER INTRATRACHEAL INSTILLATION OF MCA IN PARENTS AND OFFSPRING FROM APPROPRIATE CROSSES INVOLVING THE B6 AND D2 STRAINS OF MICE Parent Expres- Treat- No. No. Lung histopathology° Numbers b f or off- sion ment of of - lung o spring at Ah mice mice Normal SM SN SCC AH ANCN ACN AAC ASC cancers locusa on on per test test animals at riskd B6 ++ Control 46 46 44(96) 2 (4) 1 (2) 0/46 (0) B6 ++ MCA 90r 22 6(27) 2 (9) 7(32) 3(14) 3(14) 8(36) 1 (5) 8/22(36) D2 0 MCA 50 13 11(85) 1 (8) 1 (8) 0113 (0) F, ++ MCA 50 28 4(14) 1 (4) 20(71) 19(68) 5(18) 8(29) 8/28(29) F, X B6 ++ MCA 41 24 1 (4) 3(13) 3(13) 4(17) 18(75) 10(42) 4(17) 3(12) 1(4) 8/24(33) F, X D2 0 MCA 42 12 5(42) 1 (8) 6(50) 4(33) 1 (8) 2/12(17) F, x D2 ++ MCA 10 1(10) 4(40) 6(60) 4(40) 1(10) 2(20) 6/10(60) • The phenotype expressed at the Ah locus is ranked as: ++, fully responsive or inducible. 40- -60 U/g wet wt liver; 0, non-responsive, 7- -11 U/g wet wt liver. b Mice were treated intratracheal with either 0.02 ml 0.2% gelatin-saline (Control) or 500 µg MCA in gelatin-saline once per week for 4 consecutive weeks. ° Data given in terms of numbers of mice with the observed lung histopathology at 12 months after chemical treatment. More than 1 lesion was often observed per animal so that the percent incidence (given parenthetically) may be greater than 100%. (See text for abbreviations). d Summary of incidence of lesions of the type SCC, AAC and ASC per the number of animals at risk for the 12 month observation period. The percent is given parenthetically. e A total of 40 animals were randomly sacrificed from this group during the first 8 months after MCA treatment. One SN was observed in these animals during this time interval, (i.e. at 7 months). (

281 squamous metaplasia). Serial sacrifice was discontinued 8 months after the last treatment. Twelve months after the last MCA treatment, all remaining animals were injected i.p. with MCA, 24 h later were sacrificed, hepatic AHH levels were determined, and lung tissues were fixed and analyzed histopatho- logically for presence of lung lesions. Results are shown in Table'l. A variety of lung lesions was observed in these animals and these lesions are divided inot categories of: (a) essentially normal; (b) those of squamous cell origin; (c) those of alveologenic origin; and (d) those of mixed-cell origin. Lesions of squamous cell origin are described as squamous metaplasia (SM), squamous neoplasm (SN) and squamous cell carcinoma (SCC). Lesions of alveologenic origin were alveolar hyperplasia (AH), alveolar non-compressing nodules (ANCN), alveolar compressing nodules (ACN) and alveolar adenocarcinoma (AAC). The only mixed-cell lesion observed was an adeno-squamous carcinoma (ASC). A detailed description of these pulmonary lesions will be published elsewhere [Billups, L.H. et al., unpublished]. Recent studies in our laboratory suggest a progression of alveologenic lesions from AH - ANCN -• ACN - AAC. Although less well defined, the squamous lesions SN and SCC appear closely related [9]. Pulmonary lesions SN, SCC, ACN and AAC all have been shown to transplant into newborn syngeneic animals (data not shown) and the lesions termed AAC and SCC are capable of metastasis and invasion, with the heart, bronchial lymph nodes, kidneys, pleura, or brain the most usual sites. Data in the study were given in terms of the number and percentage of animals from a particular strain which was observed to have a particular lung lesion. Most animals were observed to have more than one type of lesion, thus the total percent of incidences is usually greater than 100%. Gelatin- saline treated control animals and MCA treated D2 mice were observed to be essentially normal with only 5---10% incidence of early alveologenic lesions (AH and ANCN) at 12 months after treatment. No malignant lung lesions were observed in these animals. MCA-treated B6, B6D2F1, and F, X B6 animals (all AHH responsive) expressed a variety of MCA-induced lung lesions, including SCC, AAC and ASC. A total of 19 carcinomas out of 74 MCA-treated animals from these three AHH-responsive populations were observed 12 months after chemical treatment. In the F, X D2 population, AHH-responsiveness segregated into 2 populations: 10 mice were observed to be responsive and 12 mice non-responsive (see Table 1). Of the AHH non- responsive mice, 5 had essentially normal lungs and only 2 expressed evidence of MCA-induced lung carcinomas (1 SCC and 1 AAC). Of the 10 AHH responsive mice, all expressed some evidence of MCA-induced lung histopathology with 6 observed to have carcinomas (4 SCC and 2 AAC). DISCUSSION Intratracheal instillation of MCA was observed to cause many pulmonary changes in these inbred strains of mice. The changes observed in those

282 animals after 12 months on test were of both alveologenic and squamous cell origin and included not only metaplastic or hyperplastic changes, but also alveologenic and squamous cell tumors which were occasionally mixed (ASC), well differentiated, invasive and/or metastatic. Such lesions were not observed in the gelatin-saline treated controls. The AHH responsive B6, F, and F, X B6 mice expressed a much higher incidence of each type of lung lesion, in contrast to the AHH non-responsive D2 strain where 85% of the mice were observed to have essentially normal pulmonary tissue. In F, X D2 progeny, the incidence and severity of MCA- induced lung lesions seemed to be associated with enhanced ability of these animals to respond. to, and metabolize, this chemical carcinogen (see Table 1). A total of 83% of the AHH non-responsive mice expressed either essentially normal lungs or were observed to have only the early alveologenic lesions of AH or ANCN. The incidence of the AH or ANCN lesions was similar in the AHH-responsive progeny suggesting a lack of genetic linkage between the presence of these lesions and AHH responsiveness (i.e., the Ahb allele). The incidence of SCC and AAC, however, was significantly higher in the AHH-responsive progeny (60% vs. 17% P = 0.038) suggesting genetic linkage between the Ahb allele and susceptibility to MCA-induced pulmonary carcinomas. Considering the progressive or interrelated nature of the lung lesions, the cummulative incidence of these lung lesions in the various parental or offspring animals makes the difference that exists between AHH-responsive and AHH-non-responsive animals even more striking. Total incidence of the lesions designated SN, SCC, ACN, AAC or ASC for the AHH-responsive progeny was 38/84 (45%), while the AHH- non-responsive progeny was 2/25 (8%). A rather long latency period for the malignant lesions was observed in this study, in contrast to the results of Nettesheim and Hammons [16]. These authors reported a high incidence of SCC's within 8 10 weeks after 6 weekly treatments with 500 µg MCA. The longer latency period (approx. 1 year) and the lower tumor incidence observed in this study could have resulted from the fact that only 4 treatments were given; how- ever, other studies in our laboratories suggest that the size of the MCA crystals in the gelatin-saline vehicle may be most important in the induction of malignant lung lesions (R.E. Kouri, unpublished observation). Other studies that are ongoing in this laboratory have also suggested that highly sonicated MCA (used in this study) is more toxic than large-particle MCA. The relative resistance of the animals reported here to SCC (compared to the results of Nettesheim and Hammons [16]) may reflect the selection of a particularly resistant subpopulation of mice due to the effects of highly toxic levels of MCA. However, the selection process must not have been closely associated with AHH activity, because the AHH non- inducible D2 strain was the most sensitive to this toxic effect and the surviving population from the F, X D2 backcross still expressed the 50% segregation pattern at the Ah locus (i.e. 10/22 or 48% were AHH respon- sive).

283 Hepatic AHH levels were used to determine the AHH responsiveness of the mice used in this study, since the pulmonary tissues were fixed in situ and were examined histologically. However, hepatic and pulmonary AHH levels are regulated by the same gene in crosses between B6 and D2 mice [10]. Thus, measurement of AHH levels in hepatic tissues is indicative of the AHH levels in pulmonary tissue. Data in this report suggest that the capacity to metabolize MCA may determine the susceptibility of that tissue to MCA-associated cancers. These results suggest that the study of the control and regulation of tumors of epithelial origin, the types of tumors observed most frequently in the human lung [3], can be initiated using model systems involving inbred animal strains. It is interesting to point out that one of the factors proposed to be a determinant in the susceptibility of man to pulmonary cancers is the level (or inducibility) of AHH [5]. Although this latter observation requires confirmation, the observations presented in this paper tend to support this conclusion: that is, the capacity to respond to polycyclic aromatic hydrocarbons through increased levels of AHH activity seems to be genetically linked to susceptibility to certain kinds of cancers. ACKNOWLEDGEMENTS This project has been supported in part by the Council for Tobacco Research, USA, Inc., and Contracts NO1-CP-43240 and NO1-CP=53519 with the Virus Cancer Program of the National Cancer Institute, NIH, TSPHS. REFERENCES 1 Conney, A.H. (1967) Pharmacological implication of microsomal enzyme induction. Pharmacol. Rev., 19, 317-366. 2 Conney, A.H. and Burns, J.J. (1972) Metabolic interactions among environmental chemicals and drugs. Science, 178, 576- 586. 3 Higginson, J. and Muir, C.S. (1973) Epidemiology. In: Cancer Medicine, pp. 241- 306. Editors: J. Holland and E. Frei. Lea and Fibiger, Philadelphia, PA. 4 Ho, W. and Furst, A. (1971) Intratracheal instillation method for lungs. Oncology, 27, 697-701. 5 Kellermann, G., Shaw, C.R. and Luyten-Kellermann, M. (1973) Aryl hydrocarbon hydroxylase inducibility and bronchogenic carcinoma. N. Engl. J. Med., 289, 934- - 938. 6 Kouri, R.E., Ratrie, H. and Whitmire, C.E. (1973) Evidence of a genetic relationship between 3-methylcholanthrene-induced subcutaneous tumors and inducibility of aryl hydrocarbon hydroxylase. J. Natl. Cancer Inst., 51, 197-200. 7 Kouri, R.E., Salerno, R.A. and Whitmire, C.E. (1973) Relationships between aryl hydrocarbon hydroxylase inducibility and sensitivity to chemically-induced sub- cutaneous sarcomas in various strains of mice. J. Natl. Cancer Inst., 50, 363-368. 8 Kouri, R.E., Demoise, C.F. and Whitmire, C.E. (1974) The significance of aryl hydrocarbon hydroxylase enzyme systems in the selection of model systems for respiratory carcinogenesis. In: Experimental Lung Cancer, pp. 48-61. Editors: E. Karbe and J. Park. Springer-Verlag, New York, N.Y.

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