THE Ah LOCUS: BIOCHEMICAL BASIS FOR GENETIC DIFFERENCES IN BRAIN TUMOR FORMATION IN MICE

Size: px

Start display at page:

Download "THE Ah LOCUS: BIOCHEMICAL BASIS FOR GENETIC DIFFERENCES IN BRAIN TUMOR FORMATION IN MICE"

Marilynn Lester
6 years ago
Views:

THE Ah LOCUS: BIOCHEMICAL BASIS FOR GENETIC DIFFERENCES IN BRAIN TUMOR FORMATION IN MICE ROY C. LEVITT, JACQUELENE M. FYSH, NANCY M. JENSEN AND DANIEL W.

1 THE Ah LOCUS: BIOCHEMICAL BASIS FOR GENETIC DIFFERENCES IN BRAIN TUMOR FORMATION IN MICE ROY C. LEVITT, JACQUELENE M. FYSH, NANCY M. JENSEN AND DANIEL W. NEBERT Developmental Pharmacology Brunch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Marylnnd 225 Manuscript received January 3, 1979 Revised copy received May 16, 1979 ABSTRACT Allelic differences at the Ah locus are shown to exist in the mouse brain. This fiding probably explains inbred mouse strain differences in polycyclic hydrocarbon tumorigenesis of the brain described more than 35 years ago and may be important in understanding the etiology of genetic differences in certain human intracranial neoplasms. ALMOST 4 years ago, the intracranial subcortical implantation of 3-methylcholanthrene (MC) pellets (1.O-1.5 mm in diameter) was shown ( SELIG- MAN and SHEAR 1939; ZIMMERMAN and ARNOLD 1941) to produce various gliomas and fibrosarcomas in C3H mice. Besides lmc ( ZIMMERMAN and ARNOLD 1941 ), subcortically implanted pellets of other polycyclic hydrocarbons, such as benzo [a] pyrene ( ZIMMERMAN and ARNOLD 1943a) and dibenz [a,c] anthracene (ARNOLD and ZIMMERMAN 1943), cause various gliomas and intracranial and extracranial sarcomas varying widely in histological classification. The importance of an underlying genetic predisposition became apparent in a survey of six inbred mouse strains: the tumor incidence with MC ranged from as IOW as 3% in DBA to as high as 6% in C57BL and 71 % in C3H (ZIMMERMAN and ARNOLD 1943b; ZIMMERMAN and ARNOLD 1944). Until recently, however, there was no biochemical understanding to explain why genetic differences predispose certain inbred strains of mice to a much higher incidence of polycyclic hydrocarbon-initiated cancers than other strains. We found these old reports to be of great interest because the strains used-and the incidence of tumors found-correlate very well with allelic differences recently shown to exist at the Ah locus (reviewed in KOURI and NEBERT 1977; NEBERT et al. 1978). The Ah locus in the mouse controls the induction of drugmetabolizing enzymes that utilize polycyclic hydrocarbons as substrates; these and other mono-oxygenase activities are associated with induced microsoma1 cytochrome PI-45. Regulation of responsiveness appears to involve several alleles at more than one locus, but differences between C57BL6 (B6, responsive: Ahb) and DBA2 (D2, nonresponsive: Ahd) inbred strains can be almost completely explained by the difference at the Ah locus. Heterozygotes (AhbAhd) Genetics 92: August, 1979.

2 126 R. c. LEVITT et al. are responsive because the trait is expressed as an autosomal dominant between B6 and D2 strains. The genetic trait appears to be systemic, occurring not only in liver, but also in numerous nonhepatic tissues such as lung, kidney, bowel, skin, lymph nodes, bone marrow, retinal pigmented epithelium of the eye, ovary, uterus, mammary gland and testis (KOURI and NEBERT 1977; NEBERT et al. 1978). Studies with B6, D2 and C3H inbred strains, and offspring from the appropriate genetic crosses thereof, have shown that allelic differences at the Ah locus are associated with MC- and benzo [a] pyrene-initiated subcutaeous fibrosarcomas and MC-initiated pulmonary tumors (reviewed in KOURI and NEBERT 1977). Among individuals in the same family, therefore, the genetically responsive mouse has an increased risk for these types of chemically induced cancers, most likely because tissues at the site of carcinogen exposure have higher induced aryl hydrocarbon hydroxylase (AHH) activity (and its associated cytochrome P1-45), when compared with the same tissues in the genetically nonresponsive mouse that has received the same dose of carcinogen. MC and benzo [a] pyrene are thus believed to be metabolized to ultimate carcinogenic metabolites in the mouse via quantitative increases andor qualitative changes in the forms of polycyclic hydrocarbon-induced P,-45 ( KOURT and NEBERT 1977). Other tumorigenic and toxicologic effects associated with the Ah locus have been recently reviewed (NEBERT et al. 1978). The purpose of this report is to show that allelic differences at the Ah locus are also expressed in the mouse brain. MATERIALS AND METHODS Male mice, four to six weeks of age, were purchased from Charles River Breeding Laboratory (Wilmington, MA) and held for at least one week before experimentation in our animal room under rigidly controlled environmental and nutitional conditions, as described by NEBERT (1978). MC treatment consisted of 2 mg per kg body weight given intraperitoneally 48 hr prior to killing; controls received the vehicle corn oil alone (5 mlkg). Following cervical dislocation, the brain tissue was dissected free of meninges and placed in cold isotonic potassium phosphate buffer, ph 7.4. In the experiment involving inbred strains of mice, the brain tissue from six mice was combined. The tissue was homogenized in ten volumes of the buffer with a Teflon-glass tissue grinder, and the homogenate was centrifuged at 15, x g for 15 min. The supernatant fraction was then centrifuged at 15, x g for 6 min to obtain the microsomal pellet, which was resuspeilded in 3 p1 3% glycerol-potassium phosphate buffer, ph 7.6 All procedures were performed at 4. Protein content was determined by the method of LOWRY et al. (1951) with bovine serum albumin as the reference standard. The brain enzyme was assayed by the micro-assay described in detail by NEBERT (1978). Incubation times were 45 min. The fluorescence of the blank was four or more times greater than that of control brain microsomal samples. Liver microsomal AHH was assayed by the usual method (NFBERT 1978). One unit is defined as that amount of enzyme catalyzing per min at 37 the formation of hydroxylated product causing fluorescence equivalent to that of 1 pmol of 3-hydroxybenzo [alpyrene. Specific activity denotes units per mg microsomal protein. Statistical analysis included use of Student s t test. RESULTS AND DISCUSSIORT Brain AHH activity was induced about six-fold and 2.5-fold in the genetically

GENETICS AND BRAIN TUMORS 127 TABLE 1 Comparison in three inbred strains of MC-inducible brain AHH specific activity with previous data on intracranial tumors caused by subcortically implanted MC

3 GENETICS AND BRAIN TUMORS 127 TABLE 1 Comparison in three inbred strains of MC-inducible brain AHH specific activity with previous data on intracranial tumors caused by subcortically implanted MC pellets Specific AHH activity' Number of mice with tumors Inbred strain Control MC per total number of mice: B (6%) C3H.OM (71'%) D (1%) Bagg albino 1421 (67%) A albino 622 (27%) * Values represent determinations on microsomal pellets obtained from the combined brain tissue from six inbred mice. Two other identical experiments yielded similar results. Coefficient of variance was less than.24 for all groups. + As described by ZIMMERMAN and ARNOLD (1944.). responsive B6 and C3H inbred strains, respectively, and less than 2% in the nonresponsive D2 strain (Table 1). These differences in AHH inducibility between the two responsive and the one nonresponsive strain are highly statistically significant (P <.1). AHH inducibility by MC, moreover, is highly correlated with the brain tumor study (ZIMMERMAN and ARNOLD 1944). Of interest, the (C3H) (D2)F, hybrid showed an intermediate tumor incidence of about 43% (ZIMMERMAN and ARNOLD 1944). The Ah locus and MC- or benzo- [a] pyrene-initiated tumorigenesis are expressed in this particular F, hybrid as additive (midparent) traits (reviewed in KOURI and NEBERT 1977). It is believed that there has evolved-during the past 6 or 7 years of developing numerous inbred mouse strains-a stable mutation whereby certain nonresponsive strains (such as D2) lack a normally functioning cytosolic receptor that binds avidly to polycyclic aromatic inducers (reviewed in NEBERT et al. 1978). We thus conclude from Table 1 that, among these particular inbred mouse strains developed more than 6 years ago (STAATS 1972), allelic differences at the Ah locus already existed by 194. Also included in the study of ZIMMERMAN and ARNOLD (1944) were Bagg albino (present-day BALBc) and A albino (present-day AJ) inbred strains, having brain tumor incidences of 67% and 27%, respectively. Both of these inbred strains are known to be responsive at the Ah locus (KOURI and NEBERT 1977). Unfortunately, other nonresponsive strains were not included in these original brain tumor studies. It thus appears that the A strain was intermediate in MC-initiated brain tumorigenesis between the other responsive strains and the one nonresponsive strain, D2. Tn-o possible explanations exist for this apparent intermediate value: (1) the Ah locus of the A strain of 1944 was not the same as that of the present-day A strain; or (2) other genes besides the Ah locus are important in modifying individual susceptibility to polycyclic hydrocarboninitiated cancer. Consistent with ths latter possibility is the fact reviewed in KOURI and NEBERT 1977) that the carcinogenesis index for subcutaneous benzo[a]pyrene is 56 in the C3H, ten in the B6, and 3.7 in the D2 inbred strain, i.e., the mwe than five-fold greater carcinogenesis index in C3H than in B6 can-

4 128 R. c. LEVITT et al. not be explained by allelic differences at the Ah locus because both strains are responsive. Other modifier genes, such as those controlling the immune response or viral oncogene expression, are currently under consideration to explain this apparent discrepancy (reviewed in NEBERT et al. 1978). It has been recently shown (PELKONEN et al. 1979) that no significant differences exist between C3H and B6 in their rates of DNA repair, as measured by the rate at which covalently bound benzo [a] pyrene metabolites are removed from DNA from skin and subcutaneous tissue of the intact mouse. There was a strong tendency (Figure 1) for the MC-induced brain AHH activity to be correlated 2wol with the MC-induced liver AHH activity among progeny c 6 F a r a - L Y a cn CT w > : =&,. I I I BRAIN SPECIFIC AHH ACTIVITY FIGURE 1.-Relationship between AHH activity in liver microsomes and AHH activity in brain microsomes of individual MC-treated offsprng from the (B6D2)F1 x D2 backcross. The B6 (M) and D2 () reference points represent combined microsomes from three MC-treated mice each. Experimental procedures and definitions are identical to those described under MATERIALS AND METHODS. Each close circle represents a single mouse. The dashed line drawn between the closed circles was calculated with the Monro-matic computer program for leastsquares analysis; correlation coefficient r =.32 (P =.1). In other words, there is a one-inten chance that the dashed line is not significantly different from a horizontal line.

5 GENETICS AND BRAIN TUMORS 129 of the (B6D2)F1 X D2 genetic cross. In tissues such as lymph nodes and skin (BENEDICT, CONSIDINE and NEBERT 1973), pigmented epithelium of the retina (SHICHI, ATLAS and NEBERT 1975) and, in this report, the brain, this tendency of higher MC-induced AHH activity to be correlated with MC-induced AHH in liver among individual backcross animals is not nearly so striking as the correlation in such nonhepatic tissues as lung. kidney, bowel or bone marrow (NEBERT et al. 1978). This less striking correlation may reflect the much lower AHH activities detected in the lymph nodes, skin, retina or brain, or may be related to the pharmacokinetic distribution of the inducer or to the number of cytosolic receptor sites in these various tissues. The brain is, therefore, yet another tissue in which allelic differences at the Ah locus are demonstrable. It should be pointed out that only very recently were detectable cytochrome P-45 (COHEN, ALVARES and KAPPAS 1977; SASAME, AMES and NELSON 1977) and inducible AHH activity (COHEN, ALVARES and KAPPAS 1977) found to exist in the rat brain. Evidence exists for heritability of aromatic hydrocarbon responsiveness in man (reviewed in ATLAS and NEBERT 1978). Cigarette smoking (WELCH et al. 1968; NEBERT, WINKER and GELBOIN 1969), ingestion of charcoal-cooked foods (PANTUCK et al. 1976) and exposure to potent environmental pollutants such as chlorinated dibenzo-p-dioxins, chlorinated dibenzofurans and both polychlorinated and polybrominated biphenyls (BICKERS, KAPPAS and ALVARES 1974; Po- LAND and GLOVER 1974,1977; HOOK, HASEMAN and LUCIER 1975) are known to induce AHH activity. Several laboratories have claimed that there is an association between responsiveness at the Ah locus and an increased risk for bronchogenic carcinoma and several other types of cancer (reviewed in ATLAS and NE- BERT 1978). In light of the data shown in this report, it will be of interest to see if such a correlation between responsiveness at the Ah locus and an increased risk for brain tumor formation can be found in the mouse or the human. We thank MICHAEL SALCMAN for bringing to our attention the early publications about polycyclic hydrocarbon tumorigenesis in mouse brain. LITERATURE CITED ARNOLD, H. and H. M. ZIMMERMAN, 1943 Experimental brain tumors Tumors produced with dibenzanthracene. Cancer Res. 3 : ATLAS, S. A. and D. W. NEBFRT, 1978 Pharmacogenetics: A possible pragmatic perspective in neoplasm predictability. Sem Oncol. 5: BENEDICT, W. F., N. CONSIDINE, and D. W. NEBERT, 1973 Genetic differences in aryl hydrocarbon hydroxylase induction and benza[a]pyrene-produced tumorigenesis in the mouse. Mol. Pharmacol. 9: BICKERS, D. R., A. KAPPAS and A. P. ALVARES, 1974 Differences in inducibility of cutaneous and hepatic drug metabolizing enzymes and cytochrome P-45 by polychlorinated biphenyls and l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). J. Pharmacol. Exp. Ther. 188: COHEN, J. A., A. P. ALVARES and A. KAPPAS, 1977 On the occurrence of cytochrome P-46 and aryl hydrocarbon hydroxylase activity in rat brain. J. Exp. Med. 145:

6 121 R. c. LEVITT et al. HOOK, G. E. R., J. D. HASEMAN and G. W. LUCIER, 1973 Induction and suppression of hepatic and extrahepatic microsomal foreign-compound-metabolizing enzyme systems by 2,3,7,8- tetrachlorodi-benzo-p-dioxin. Chem.-Biol. Interact. 1: KOURI, R. E. and D. W. NEBERT, 1977 Genetic regulation of susceptibility to polycyclic hydrocarbon-inducecl tumors in the mouse. pp In: Origins of Human Cancer. Edited by H. H. HIATT, J. D. WATSON and J. A. WINSTEN. Cold Spring Harbor Laboratory, New York. LOWRY,. H., N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, 1951 Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: NEBERT, D. W., 1978 Genetic differences affecting microsomal electron transport: The Ah locus. Methods Enzymol. 52 : NEBERT, D. W., S. A. ATLAS, T. M. GUENTHNER and R. E. KOURI, 1978 The Ah locus: Genetic regulation of the enzymes which metabolize polycyclic hydrocarbons and the risk for cancer. pp In: Polycyclic Hydrocarbons and Cancer: Chemistry, Molecular Biology and Enuironment. Edited by P.. P. Ts'o and H. V. GELBOIN. Academic Press, New York. NEBERT, D. W., J. WINKER and H. V. GELBOIN, 1969 Aryl hydrocarbon hydroxylase activity in human placenta from cigarette smoking and nonsmoking women. Cancer Res. 29: PANTUCK, E. J., K.-C. HSIAO, A. H. CONNEY, W. A. GARLAND. A. KAPPAS, K. E. ANDERSON and A. P. ALVARES, 1976 Effect of charcoal-broiled beef on phenacetin metabolism in man. Science 194: PELKONEN, O., A. R. BOOBIS, R, C. LEVITT, R. E. KOURI and D. W. NEBERT, 1979 Genetic differences in the metabolic activation of benzo[a]pyrene in mice. Attempts to correlate tumorigenesis with binding of reactive intermediates to DNA and with mutagenesis in vitro. Pharmacology, in press. POLAND, A. and E. GLOVER, 1974 Comparison of 2,3,7,8-tetrachlorodibenzo-p-diosin, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcholanthrene. Mol. Pharmacol. 1: , 1977 Chlorinated biphenyl induction of aryl hydrocarbon hydroxylase activity: a study of the structure-activity relationship. Mol. Pharmacol. 13: SASAME, H. A., M. M. AMES and S. D. NELSON, 1977 Cytochrome P-45 and NADPH cyto. chrome c reductase in rat brain: Formation of catechols and reactive catechol metabolites. Biochem. Biophys. Res. Commun. 78: SELIGMAN, A. M. and M. J. SHEAR, 1939 Studies in carcinogenesis. VIII. Experimental produc. tion of brain tumors in mice with methylcholanthrene. Am. J. Cancer 37: SHICHI, H., S. A. ATLAS and D. W. NEBERT, 1975 Genetically regulated aryl hydrocarbon hydroxylase induction in the eye: Possible significance of the drug-metabolizing enzyme system for the retinal pigmented epithelium-choroid. Exp. Eye Res. 21: STAATS, J., 1972 Standardized nomenclature for inbred strains of mice: Fifth listing. Cancer Res. 32: WELCH, R. M., Y. E. HARRISON, A. H. CONNEY, P. P. POPPERS and M. FINSTER, 1968 Cigarette smoking: stimulatory effect on metabolism of 3,4-benzpyrene by enzymes in human placenta. Science 16: ZIMMERMAN, H. M. and H. ARNOLD, Experimental brain tumors. I. Tumors produced with methylcholanthrene. Cancer Res. 1 : , 1943a Chemical carcinogens and animal species as factors in experimental brain tumors. J. Neuropathol. Exper. Neurol. 2: , , Experimental brain tumors. 11. Tumors produced with benzpprene. Am. J. Pathol. 19: , 1944 Experimental brain tumors. IV. The ir.c dence in different strains of mice. Cancer Res. 4: Corresponding editor: R. E. GANSCHOW

Polychlorinated Biphenyls: A New Type of Inducer of Cytochrome

Proc. Nat. Acad. Sci. USA Vol. 7, No. 5, pp. 1321-1325, May 1973 Polychlorinated Biphenyls: A New Type of nducer of Cytochrome P-448 in the Liver (cytochrome P-45/rats aryl hydrocarbon hydroxylase/enzyme