Chem.-Biol. Interactions, 13 (1976) 317-331 @ Elsevier Scientific Publishing Company; .Amsterdam- Printed in The Netherlands
STUDIES ON PULMONARY ARYL HYDROCARBON HYDROXYLASE ACTIVITY IN INBRED STRAINS OF MICE
RICHARD E. KOURI, THOMAS RUDE, PAUL E. THOMAS + and CARRIE E. WHITMIRE Department of Biochemical-Oncology, Microbiological Associates, 4733 Bethesda Avenue, Bethesda, Md. 20014, and l Department of Biochemistry and Drug Metabolism, Hoffman-La Roche Inc., Nutley, NJ. 07110 (U.S.A.) (Received July 14th, 1975) (Revision received and accepted December Sth, 1975)
SUM&IARY
Pulrt~onary and hepatic levels of aryl hydrocarbon hydroxylase (AHH) were studied in inbred strains of mice following intratracheal (i.t.) instillation of 3-methylcholanthrene QMCA). 1.t. instillation of 188 Erg MCA in sterile 0.2% @atin in saline resulted in preferential induction of pulmonary AHH. After treatment with this dose of MCA, the pulmonary AHH levels of strains C57EL/6Cum, C57BL/6J, BALB/cM&i, CSH/fMai, and C57L/J were observcjd to be induced within 24 b after treatment. Strains DBA/2Cum, AKR/J, SJL/J, DBA/BJ and RF/J expressed no such increase. At a dose of 500 pg MCA, the pulmonary tissue of DBA/S mice did express a 4-fold increase. This increase in AHH was determined to be quite different from the increase observed in C57BL/6 mice by : (1) specific activity of the enzymes, (2) genetic regulation, (3) susceptibility to inhibition by 7&benzoflavone, and (4) spectral properties of the associated cytochromes. It was of major importance that induction of pulmonary AHH was observed to be regulated by a single dominant gene in crosses involving the C57BL/6Cum and DBA/2Cum strains of mice. Results were discussed with the view in mind that these genetically regulated levels of AHH may play a role in susceptibility to cancers induced by polycyclic aromatic hydrocarbon carcinogens.
Abbreviations: AHH, aryl hydrocarbon hydroxylsse; BP, benzo(a)pyrene; DMSO, dimethylsulfoxide; MCA, 3-methylcholanthrene; TCDD, 2,3,7,Wetrachlorodibenzo@)dioxin.
317
AHH is the name given to one of the multicomponent mixed-function oxidases which convert a variety of lipid-soluble compounds to water-soluble forms, usua:lly for subsequent elimination from the body (see refs. 1,2). The presence or inducibility of AHH is associated with the cytotoxic action of certain chemical carcinogens in vivo [ 31 and in vitro [ 4,5] and the detoxification [G-81 or activation [ 9--131 of polycyclic aromatic hydrocarbons in chemical carcinogenesis. Thus, one is confronted with the paradoxical situation that the same enzyme system may both detoxify and activate the same chemical carcinogens. The AHH enzyme in inbred strains of mice possesses two properties which make it particulaly amenable for studying its role in chemically induced cancers, that is, the system is inducible l [14,15], and this inducibility is genetically controlled. Depending on the strains used, hepatic AHH inducibility can be regulated by a single autosomal dominant gene [14,16], a single autosomal co-dominant gene [17,18] or non-responsiveness can be the result of a dominant allele [ 181. Using these genetic systems, recent information suggests that this enzyme system plays a major role in the susceptibility to MCA [ 19-21] - and BP [ 221 -induced carcinogenesis. The lungs and skin of mice seem to be under a different kind of control because. these organs seem to be slightly inducible in those mouse strains in which the liver is completely non-inducible [23-261. This apparent dichotomy is very important to understand because it suggests that the genetics of AHH inducibility is organ-dependent. rather than strain-dependent as originally concleived [14,16] . In this paper, we show that there is both a qualitative and quantitative difference between the increased levels of AHH observed in the AHH “non-inducible” mice and the increase observed in the AHH “inducible” mice. Moreover, in crosses involving the C57BL/6Cum and DBA/2Cum strains of mice, pulmonary AHH responsiveness to intratracheally administered MCA is regulated by a single autosomal dominant gene. EXPERJMEN’I’ALPROCEDURE
The polycyclic hydrocarbons BP and MCA were purchased from Sigma Chem&ls (St. Louis, MO.) and purified by recrystallization from benzene. 7,8-Benzoflavonle was purchased from Aldrich Chemicals (Cedar Knolls, N.J.). Metaphane was purchased from Pitman-Moore (Washington Crossing, N. J.). The sources of mice were either Clmberland View Farms (Clinton, Tenn.), The Jackson Laboratory (Bar Harbor, Maine), or Microbiological Associates (Bethesda, MCI.), For intratreachecl instillations, a Bausch and Lomb stereo-
* The term “inducibility” as used in this paper, denotes a relative increase in rates of de novo synthesis or of activation df enzyme activity from pre-existing moieties, or in rate of both, when compared to rate of breakdown. No particular mechanism is implied.
318
microscope equipped with fiber optic illumination and Hamilton syringes with 22 ga by 38 mm (with I.5 mm balls) feeder needles were used. Methods Care and feeding of mice were as stated previously [27] . Animals were always treated between the times of 9:00 and 10:00 a.m. to avoid diurnal variations. The i.t. instillation technique was similar to that described recently by Ho and Furst [ 28 1. Animals were anesthesized by inhalation of metaphane and were placed on special boards designed to hold their mouths open and at the correct angle for instillation. After positioning the animals, 0.02 ml of solution was given i.t. directly into the lung. The solution consisd;ed of MCA suspended in 0.2% gelatin in sterile saline 1291 or MCA dissolved in trioctanoin. At various times post-treatment, the lungs and livers were excised and frozen at -7O’C until assayed. Microsomes were prepared from lung or liver tissues according to the methods of Nebert and Gielen [30] and were stored at -7O’C for up to 72 h before being assayed. Samples were diluted with 0.1 M Tris-HCl buffer (pH 7.4) to a final ratio of 1.0 ml of microsomal suspension per gram wet weight tissue. The assay for AHH was basically that of Nebert and Gelboin [15] as modified by Nebert and Gielen [30] and Thomas et al. [14]. Tissues were carefully weighed and homogenized in 0.05 M Tris: 0.25 M sucrose buffer (pH 7.4) at a 1 : 10 (w/v) dilution for lungs and 1 : 20 (w/v) dilution for livers. 0.2 ml of homogenate was added to 0.79 ml buffer containing 100 pmoles Tris, 0.80 pmoles NADPH, 0.78 pmoles NADH, 6 pmoles MgC& and 200 Erg bovine serum albumin (Cohn Fraction V). 80 nmoles BP were added in 0.01 ml acetone to initiate the reaction, and tubes were incubated with shaking at 37°C for 20 min. The assay was stopped by the addition of cold acetone-hexane (1 : 3.3) and the polar metabolites in the hexane phase were back-extracted with 2.0 ml of 1 N NaOH. The amount of fluorescence associated with the presence of 3-OH BP was determined with excitation at 398 nm and emission at 522 nm in an Aminco-Bowman spectrophotofluorometer. Microsomal preparations were assayed for AHH activity in a similar manner, however, the amount of microsomal protein was determined by the method of Oyama and Eagle [31]. A unit of AHH activity is defined to be that amount of enzyme producing the fluorescent equivalent of 1.0 nmoles 3-OH BP per min at 37°C. For hepatic and pulmonary tissues, this is given in terms of units/g wet weight tissues, and for microsomal preparations in terms of units/pg protein. 7,8-Benzoflavone inhibition of AHH in microsomal preparations was determined according to the procedures of Goujon et al. [ 321 and Wiebel et al. [24,33]. Concentrations of 7,8-benzoflavone were dissolved in DMSO and 0.01 ml was added to tubes containing all factors for the AHH assay, excluding BP. The tubes were incubated at 37°C for 1 min, and then BP was added. For the spectral studies, microsomal pellets were resuspended by homo319
genization in 0.1 M K-P04, pl-i 7.4. Spectral studies were performed on an Aminco-Chance DW-2 W/vis spectrophotometer in the split beam mode. Cytochrome P-450 was determined after bubbling CO, adding 0.5 n&M NADH, dividing the sample between both cuvettes, correcting the baseline and then recording the: spectra after adding sodium dithionite to the sample cuvette [ 34 1. This procedure cancels out the contribution from cytochrome bs and hemoglobin. RESULTS
AHH levels following i. t. administration of MCA Effect of vehicle. Tables I and II demonstrate the pulmonary and hepatic responses of BGDZF,Cum mice 24 h after treatment with various doses of MCA given in either 0.2% gelatin or trioctanoin. MCA suspended in the gelatin solution seemed preferentially to induce pulmonary AHH at dose levels < 188 pg. MCA in trioctanoin induced both pulmonary and hepatic AHH at every dose level tested. All subsequent studies were done with MCA suspended in 0.2% gelatin so that pulmonary AHH could be specifically studiled. Effect ofparental strains. The hepatic and pulmonary responses of C57BL/ 6Cum (B6) and DBA/2Cum (D2) are shown in Table III. The B6 strain responded to MCA much like the B6D2F1 hybrid (Table I); however, at doses > 138 pg, MCA induced both pulmonary andhepatic AHH of B6 and B6D2F1 mice. Pulmonary AHH levels in D2 mice were induced slightly by treatment
TABLE I EFFECTS OF INTRATRACHE%L INSTILLATION OF VARIOUS MCA DOSES IN TRIOCTANOIN ON PULMONARY AND HEPATIC AHH s IN B6D2F1Cum MICE ~.cg MCA
0
25 56 100 200 400 500
Liver AHH
Lung AHH Untreated
Trioc
MCATrioc
Ind. b
Untreated
Trioc
MCATrioc
Ind.
0.32
0.20
1.0 1.7 1.6 1.9 1.6 1.2
5.0 8.5 8.0
12.6
9.8
19.6 19.6 38.2 39.2 62.7 44.1
2.0 2.0 3.9 4.0 6.4 4.5
K 6.0
* AHH activity given in terms of units per g wet weight tissue. A unit is that amount of enzyme causing the fluorescent equivalent of 1 nmole of 3.CH BP per min at 37’C. Mean of at least three animals assayed in duplicate ; values for individ .ral mice were usually within 10% of each other. b Ind., Inducibility : the relative increase of MCA-treated tissue over vehicle-fretited control tissue.
320
TABLE II EFFECTS OF INTRATRACHEAL INSTILLATION OF VARIOUS MCA DOSES IN 0.2% GELATIN ON PULMONARY AND HEPATIC AHH a IN B6D2F1Cum MICE
/a MCA
0 5 11 23 47 94 188 375 500
Lung AHH
Liver AHH
Untreated
Gel
0.36
0.26
MCA: Gel
0.56 0.76 1.40 1.30 1.80 2.00 2.40 2.50
Ind.
Untreated
Gel
MCA: Gel
12.6
10.0
-
-
7.81 10.30 10.80 14.40 11.40 18.90 24.00 25.50
0.78 1.03 1.10 1.40 1.10 1.90 2.40 2.70
2.20 2.90 5.40 5.00 6.90 7.70 9.20 9.60
Ind.
See Table I for footnotes.
TABLE III EFFECTS OF INTRATRACHEAL INSTILLATION OF VARIOUS MCA DOSES IN 0.2% GELATIN ON PULMONARY AND HEPATIC AHH a IN C57BL/GCum AND DBA/2Cum MICE pg MCA
A. C57BLj 6Cum 0 5 11 23 47 94 188 375 500 B. DBA/ 2Cum 0 188 500
Liver AHH
Lung AHH Untreated
Gel
0.40
0.32
MCA Gel
0.73 0.89 1.50 1.40 1.70 2.40 2.50 2.40
0.20
Ind. b
Untreated
Gel
17.5
14.5
2.30 2.80 4.70 4.40 5.50 7.50 7.80 7.50
9.80
0.18 0.17 0.56
1.00 3.10
9.50
MCA Gel
Ind.
-
-
8.3 13.3 13.1 14.5 24.7 32.3 35.0 33.2
0.57 0.92 0.90 1.00 1.70 2.20 2.40 2.30
10.1. 10.2
1.10 1.10
See Table I for footnotes.
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with 500 c(g MCA but not by 188 pg MCA, whereas hepatic AHH was unaffected at either dose. The average inducibility for pulmonary tissue of ten D2 mice following MCA treatment was 3.1 k 0.6 (s.e.) In terms of specific activity, the D2 strain has very little pulmonary AHH activity; the highest induced activity was about the same as the constitutive (or control) activity of the other two strains. Both sexes of the two in.bred strains and their
L -
I.0
02 B6 B6DZFl
30
0 0
1
2
3
4
s
6
7
8
9
16
II
I2
13 11
DAYS
Fig. 1. Kinetics of pulmonary and hepatic AHH following i.t. administration of MCA. (a) Time-dependent increase in pulmonary AHH following i.t. treatment with 188 pg MCA }. (b) Time-dependent increase into D2 (A-A), B6 (w) and B6D2FI (in pulmonary AHH following i.t. treatment with 600 &g MCA into D2 (A---A), B6 -o) mice. (c) Time-dependent increase in hepatic AHH (V) and B6D2F1 (following i.t. treatment with 500 pg MCA into D2 (A-), B6 (w) and B6D2Fl (M) mice.
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19.2 22.2( 1.2)
0.71 3.1(4.4)
BALB/cMai
7.9 7.3(0.9)
0.33 2.5(7.2)
CSH/fMai
13.3 27.4(2.1)
0.64 3.4( 5.3)
C57L/J
16.1 32.6(2.0)
0.28 2.4(&O)
C57BL/6J
17.5 15.6(0.9)
0.28 0.45( 1.6)
AKR/J
10.8 11.7(1.1)
0.19 0.29(1.5)
SJLlJ
8.9 8.5(0.9)
0.20 0.28(X.4)
DBAlZJ
13.5 13.8(1.0)
0.41 0.54( 1.3)
RF/J
a AHH activity given in terms of units per g wet weight tissue. A unit is that amount of enzyme causing the Ruoreseent equivalent of 1.0 nmole of 3-OH EP per min at 37*C. The relative inducibiiity is given in parentheses. b Tissues were removed 24 h after i.t. treatment with either gelatin-saline or 188 &g MCA.
Control MCA
Liver
MCA
control
Lung
Tissue b
EFFECTS OF INTRATRACHEAL INSTILLAmON OF 188 c(g MCA IN 0.2% GELATIN ON PULMONARY AND HEPATIC AHN a IN VARIOUS STRAINS OF MICE
TABLE IV
hybrid were assayed for their pulmonary and hepatic AHH responses following MCA and no differences were observed (data not shown). Changes in pulmonary AHH with time following i.t. instillation in gelatin of 188 pg MCA into B6, D2 and B6D2Fl mice are shown in Fig, la. The induction time course was similar for both B6’ and B6D2F, mice; maximum induction was observed by 24 h and remained constant for up to 96 h. The maximum observed increase for each strain and hybrid was similar; about 6-fold. The responses of pulmonary and hepatic AHH following i.t. administration of 500 c(g MCA are shown in Fig. lb and Fig. lc. Pulmonary AHH levels remained maximally~ induced for at lea& 7 days. Hepatic levels decreased by day two and were almost at background level by the third day. A subsequent treatment on the seventh day resulted in levels of AHH activity similar to that observed for the first treatment. Effects on various strains of mice. Table IV demonstrates the hepatic and pulmonalry AHH levels following i.t. instillation of 188 pg MCA. The AHH levels in pulmonary tissues of strains BALB/c Mai, C3H/f Mai, C57L/J and C57BL/6J were induced by MCA while strains AKR/J, SJL/J, DBA/BJ and RF/J showed httle or no increase. Hepatic responses were low for all strains except perhaps for the C57BL/6J and C57L/J, which did show a 100% increase. Genetic regulation of pulmonary AHH induction. Induction of pulmonary AHH by i.t. instilled MCA segregated as a single autosomal dominant gene in crosses between the B6 and D2 strains of mice (Table V). Backcross and F2 animals were classified as inducible or non-inducible if, after i.t. treatment with 188 pg MCA, pulmonary AHH levels were 2.5 (kO.5) units/g tissue (inducible) or 0.3 (kO.05) units/g tissue (non-inducible). Among 90 backcross animals ,tested, 47 were inducible (52%) and among 50 F1 animals tested, 36 were inducible (72%). These numbers were not statistically differTABLE V GENETIC SEGREGATION OF PULMONARY AHH IN CROSSES INVOLVING THE C57BL/6Cum AND DBA/2Cum STRAINS OF MICE I’ ----___-----Number induced Strain Number treated % Induction __-L__.--_-I-_B6 50 50 100 D2 50 0 B6D2F 1 100 1; 100 B6D2F, X B6 52 62 100 B6D2F, X D2 92 47 62 B6D2Fz 60 36 72 a Mice were treated with 163 l.(g MCA/O.OB ml 0.2% gelatin solution it. and 24 h later, the pulmonary AHH was assayed. A mouse was considered inducible if, after MCA treatment, pulmonary AHH was 2.5 (t0.6) units/g tissue and considered noninducible if pulmonary AHH was 0.3 (fO.05) units/g tissue. The sex of the progeny played no role in this segregation pattern.
324
I
*
5
50
500
w 7,6-benzoflovone/ml Fig. 2. In vitro effects; of 7,8-henzoflavone on AHH activity of pulmonary microsomal preparations from control (M ) and MCA-treated (w) B6 mice, and control a) and MCA-treated (_ A) D2 mice. Specific activities of the microsomal (Apreparations were 5.0, 89.0, 1.7 and 3.8 units AHH activity per&g protein, respectivt!y.
ent (P < 0.01, using chi square analysis) from the expected 50% and 75% ratios that would be expected if a single autosomal dominant gene was regulating this inducib~i~, AH. levels in microsomal preparations. The effect of 7&benzoflavone, a known competitive inhibitor of the induced, or P-446mediated enzyme, on the in vitro metabolism of BP by microsomes derived from pulmonary tissues of the B6 and D2 mice is shown in Fig. 2. Results with the untreated (no 7,8benzoflavone) preparations generally agreed with the results shown in Tables III and IV; that is, D2 pulmonary tissue expressed much less enzyme activity than B6 tissue, MCA treatment of D2 mice resulted in a small increase in pulmonary AHH levels to about that observed in control B6 mice, and MCAtreatment of B6 mice resulted in a much higher level of pulmonary AHH activity (about 14-fold in these microsomes). 7,&Benzoflavone inhibited AHH activity from control and induced D2 tissue and control B6 tissue in a similar manner, while the AHH activity from induced B6 tissue was much more ~nsitive. For example, at concen~ations of 7,8-benzo~avone equimolar to the BP concentration, the enzymes from the D2 tissue and from control B6 tissue were inhibit by about QO%,while the enzymes from induced B6 tissues were inhibited by greater than 80%. ~~ect~~ studies in mic~sama~ preparations. Spectral destinations of P450 in lung microsomes from both control and MCA induced mice were attempted (Table VI). These studies were complicated by a large absorbance 325
TABLE VI SPECTRAL STUDIES ON PULMONARY MICROSOMES DERIVED FROM D2 AND B6 MICE Strains
Treatment a
Spectral peak (nm)
nmoles cyrochrome P-450/ mg protein
D2 D2 B6 B6
Gel MCA Gel MCA
450 449-449.5 450 446
0.198 0.093 0.101 0.095
a Twenty mice of each strain were treated i.t. with either 500 pg MCA of 0.2% gelatinsaline solution and sacrified 48 h later. Lung microsomes were prepared as given in MATERIALS AND METHODS.
in the 420-424 nm region of the CO difference spectra which equalled or exceeded the absorbance in the region of 450 nm. This “P-420-like” material was present even though the CO difference spectra were recorded under conditons designed to blank out cytochrome b5 and hemoglobin, and regardless of whether the microsomes were fresh or frozen. We do not know to what extent this “P-420-like” material might interfere with the P-450 determinations. Even under these conditions, a shift in the CO difference spectra from 450 nm to 448 nm in C57BL/6J lung 2 days after administration of MCA was observed. DBA/BJ mice analyzed at the same time showed at most a 1 nm shift from 450 nm to 449 nm after MCA induction. These results are very similar to those recently described for microsomal preparations of rat lung [35]. Following treatment with MCA, the content of P-450 in pulmonary tissue of D2 mice actually decreased {see Table VI). Total P-450 content of pulmonary tissue from B6 mice was not altered by MCA treatment. The nature of this decrease is inexplicable at this time. DISCUSSION
In this report we describe some of the major parameters regulating the constitutive and MCA-induced levels of pulmonary AHH activity in inbred strains of mice. The intratracheal route was used so as to attempt to limit the enzymatic response to pulmonary tissue. Utilizing the inbred strains C67BL/6 and DBA/B, whose hepatic AHH responses have been extensively studied [ 14-18 1, we show in this report that: (a) MCA given in either trioctanoin or a 0.2% gelatin solu$ion induces pulmonary AHH, and this induction is dosedependent (Tables I, II, 111, IV); (b) a dose of 188 pg MCA given in 0.2% gelatin induces pulmonary AHH, but has very limited effect on hepatic AHH levels (Tables II and III); (c) pulmonary AHH can be induced in D2 mice by it. admimstration of MCA, but hepatic AHH levels are never induced (Table III, Fig. 1); (d) although pulmonary AHH is induced in D2 mice, the levels 326
are very low, with about a 5-lo-fold difference between D2 and B6 puhnonary tisues; and (e) this strain difference is under the sa,ne genetic control as that of hepatic tissue [14,16], that is, the highly responsive B6 strain differs from the D2 strain by a single autosomal dominant gene controlling this heightened responsiveness (Table V). Results with other inbred strains of mice agree with this contention, that is, strains that are non-responsive to MCA in their hepatic tissues are low responders to MCA when given Lt. and vice versa (Table IV). In agreement with the observations of Wiebel et al. [24], Burki et al. [23], and more recently Abramson and Hutton [25 ] and Van Canfort and Gielen [26], pulmonary AHH seems to be inducible in every strain of mouse regardless of hepatic AHH responsiveness. However, the mean basal AHH activities in lungs from strains of mice classified as hepatic AHH inducible are significantly higher than the activities for the same pulmonary enzymes in hepatic non-inducible strains (ref. 25; see Tables II, III and Fig. 1). In fact, Abramson and Hutton [25] suggest that the same genes may be affecting both liver AHH inducibilities and levels of basal AHH activity in pulmonary tissue. Of interest is the fact that the inducibility of AHH in hepatic non-responsive strains (e.g. D2) is dose-dependent and the differences in specific activity following exposure to 188 pg MCA segregate as a single autosomal dominant gene in crosses between the B6 and D2 strains; just as hepatic AHH inducibility [ 14,161. Thus, although the pulmonary tissue in D2 mice is induced by high levels of MCA (-500 gg), this increase can be discriminated genetically from that response observed in B6 mice. These results could be explained in terms of hypothetical receptor sites for AHH induction [36]. Pulmonary and hepatic tissue in B6 mice contain efficient receptor sites and hence are responsive to low-level treatment with an inducer, such as MCA. Receptor sites in pulmonary and hepatic tissue in D2 mice are much less efficient and there may even be slight differences in efficiency between the receptor sites in the pulmonary and hepatic tissue in this strain, the lung receptor site being more efficient. Thus, high levels of inducers would be required to effect AHH induction in D2 mice and responsiveness may be limited to just pulmonary tissue. This explanation isvery similar to that proposed by Poland and Glover [36] for describing the induction results using TCDD in B6 and D2 strains of mice. These authors observed that TCDD is capable of inducing AHH activity in both B6 and D2 mice but that the level required for maximal induction of D2 mice is about 100 times higher than that required for B6 mice. The fact that AHH can be induced in hepatic tissue of D2 mice (with its concomitant cytochrome, P-448) indicates that the structural genes necessary for synthesis of these induction-specific enzymes are present in D2 tissue. A mutation in a receptor site (or receptor protein) was hypothesized as the most logical explanation for the variation in responsiveness between the two strains. Our results suggest that, not only may the D2 strain possess an inefficient receptor site, but also, this site is different in pulmonary and hepatic tissue. This pulmonary AHH seems unique for it is fairly resistant to inhibition by 327
7,8-benzoflavone (see Fig. 2) and the blue spectral shift normally associated with hydrocarbon induction could not be observed in MCA-treated D2 lung tissue (see Table VI). The expected sensitivity to 7,8-benzoflavone inhibition and presence of P-448 was observed in MCA-treated B6 lung tissue. Thus, although increases in AHH are observed in MCA-treated pulmona,ry tissues in D2 mice, thfere is no indication that it is the same kind (or form) of induction that occurs in B6 mice. Direct quantitation of the cytochromes in MCAtreated D2 lung tissue may be important in understanding the mechanism of this kind of induction. The recent report of Capdevila et al. [37] may allow for unambiguous study of the nature of pulmonary inducibility in hepatic non-responsive strains. These authors report a sensitive chromatographic method for the isolation of cytochromes P-450 and P-448 from pulmonary microsomes. This approach is currently in progress in our laboratory. Results recently observed by Poland et al. [38], on maximal absorbance peaks of CO difference spectra in both C57BL/6N and DBA/BN mouse lung with and without intraperitoneal MCA administration, do not agree with our data in some respects. Namely, their spectral peaks for CO reduced cytochrome P-450 is at 455-456 nm instead of at 450 nm which most probably results from improper compensation for the presence of hemoglobin However, their results corroborate in a qualitatnre manner, for they did observe a 1 nm blue shift in the CO reduced spectra of C57BL/6N mouse lung with MCA treatment, but no shift in DBA/BN mouse lung when treated with MCA. This genetically controlled enzyme response may p!ay a major role in the ultimate susceptibility of pulmonary tissue to chemically induced cancer, just as hepatic AHH inducibility is correlated with increased susceptibility to MCA-induced subcutaneous tumors in mice [19-211. Moreover, this idea seems to have precedence in the human situation, where recent results (presently requiring confirmation) suggest a relationship between the higher levels of AHH inducibility (as measured in lymphocyte culture) and presence of bronchogenic squamous+ell carcinoma (cigarette smoke-associated) [ 391. Nettesheim and Hammans [29] have reported conditions for induction of squamous cell carcinomas in inbred strains of mice and their data suggest a relationship between AHH inducibility and lung cancer. These authors utilized the (C57BL/6 X C3H/f) F1 and the DBA/B strains of mice, and 500 c(g MCA (in 0.2% gelatin) was given i.t. at weekly intervals for 4-6 weeks. These authors observed that the AHH inducible (C57BL/6 X C3H/f) Fi hybrid was much more sensitife to MCA-induced squamous cell carcinomas than the AHH “non-inducible” DBA/2 strain. Hepatic and pulmonary AHH levels of responsive (B6) or ‘low responsive (D2) mice to this treatment schedule are shown in Fig. lb and Fig, lc. There are obvious differences between the two strains in their ability to respond to the MCA and it is very likely that the increased susceptibility to chemically induced carcinomas of AHH inducible animals reflects this heightened ability to metabolize chemical carcinogens. This theory can be tested by it. instillation of various doses of MCA into backcross of F2 animals derived from the B6 and D2 strains and observing 328
the relationship between AHH responsiveness and susceptibility to MCAinduced lung cancers. Actually, since lower doses (e.g. 188 pg) of MCA maximally induce pulmonary AHH and have xly limited effect on hepatic AHH levels, this may be the dose of choice. These procedures are currently being evaluated in our laboratories. ACKNOWLEDGEMENTS
Supported in part by contracts from the Council for Tobacco Research. The authors thank Drs. R. Imblum, P. Price and J. Kreisher for their helpful comments concerning this study. REFERENCES Mason, Mechanisms of oxygen metabolism, Adv. Enzymol., 19 (1957) 79. 2 A.H. Conney, Pharmacological implications of microsomal enzyme induction, Pharmacol. Rev., 19 (1967) 317. 3 A. Somogyi, K. Kovacs, R. Solymoss, A.H. Conney and R. Kuntzman, Suppression of 7,12_dimethylbenz(a)anthracene produced adrenal necrosis by steroids capable of inducing aryl hydrocarbon hydroxylase, Life Sci., 10 (1971) 1261. 4 H.V. Gelboin, E. Huberman and L. Sachs, Enzymatic hydroxylation of benzo(a)pyrene and its relationship to cytotoxicity, Proc. Natl. Acad. Sci. USA, 64 (1969) 1188. 5 R.A. Lubet, D.Q. Brown and R.E. Kouri, The role of 3-OH benzo(a)pyrene in mediating benzo(a)pyrene induced toxicity 7nd transformation in cell culture, Res. Commun. Chem. Path. Pharmacol., 6 (1973) 929. 6 L.W. Wattenberg and J.L. Leong, Inhibition of the carcinogenic action of benzo(a)pyrene by flavones, Cancer Res., 30 (1970) 1922. 7 T.L. Dao, Inhibition of tumor induction in chemical carcinogenesis in mammary gland, Prog. Exp. Tumor Res., 14 (1971) 59. 8 D.N. Wheatley, Enhancement and inhibition of the induction by 7,12-dimethylbenz(a)anthracene of mammary tumors in female Sprague-Dawley rats, Br. J. Cancer, 22 (1968) 787. 9 H.V. Gelboin, F.W. Wiebel and L. Diamond, Dimethyl-benzanthracene tumorigenesis and aryl hydrocarbon hydroxylase in mouse skin: inhibition of 7,8-benzoflavone, Science, 170 (1970) 169. 10 P.L. Grover, A. Hewer and P. Sims, Epoxides as microsomal metabolites of polycyclic hydrocarbons, FEBS Letters, 18 (1971) 76. 11 J.K. Selkirk, E. Huberman and C. Heidelberger, An epoxide is an intermediate in tbe microsomal metabolism of the chemical carcinogen, dibenz(a,h)anthracene, Biochem Biophys. Res. Commun., 43 (1971) 1010. 12 P.L. Grover, P. Sims, E. Huberman, H. Marquardt, T. Kuroki and C. Heidelberger, In vitro transformation of rodent cells by K-region derivatives of polycyclic hydrocarbons, Proc. Natl. Acad. Sci. USA, 68 (1971) 1098. 13 H. Marquardt, T. Kuroki, E. Huberman, J. Selkirk, C. Heidelberger, P. Grover and P. Sims, Malignant transformation of ceils derived from mouse prostate by epoxides and other derivatives of polycyclic hydrocarbons, Cancer Res., 32 (1972) 716. 14 P.E. Thomas, R.E. Kouri and J.J. Hutton, The genetics of aryl hydrocarbon hydroxy lase induction in mice: A single gene difference between C57BL/6 and DBA/SJ, Bio1 H.S.
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330
36 37 38 39
microsomal fractions of rodent respiratory tract and their characterization, Cancer Res., 34 (1974) 2196. A. Poland and E. Glover, Genetic expression of aryl hydrocarbon hydroxylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin: evidence for a receptor mutation in genetically non-responsive mice, Mol. Pharmacol., 11 (1975) 389. 3. Capdevila, SW. Jakobsson, B. Jernstrom, 0. Helia and 8. Grrenius, Gharacte:rization of a rat lung microsomat fraction obtained by Sepharose 2B uitra~itration, Cancer Res., 35 (1975) 2820. A.P. Poland, E. Glover, J.R. Robinson and D.W. Nebert, Genetic expression of aryl hydrocarbon hydroxylase activity, J. BioI. Chem., 249: (1974) 5599. G. Kellerman, C.R. Shaw and M. Luyten-Kellernan, Aryl hydroxylase inducibility and bronchogenic carcinoma, New Engl. J. Med., 289 (1973) 934.
331
STUDIES ON PULMONARY ARYL HYDROCARBON HYDROXYLASE ACTIVITY IN INBRED STRAINS OF MICE
RICHARD E. KOURI, THOMAS RUDE, PAUL E. THOMAS + and CARRIE E. WHITMIRE Department of Biochemical-Oncology, Microbiological Associates, 4733 Bethesda Avenue, Bethesda, Md. 20014, and l Department of Biochemistry and Drug Metabolism, Hoffman-La Roche Inc., Nutley, NJ. 07110 (U.S.A.) (Received July 14th, 1975) (Revision received and accepted December Sth, 1975)
SUM&IARY
Pulrt~onary and hepatic levels of aryl hydrocarbon hydroxylase (AHH) were studied in inbred strains of mice following intratracheal (i.t.) instillation of 3-methylcholanthrene QMCA). 1.t. instillation of 188 Erg MCA in sterile 0.2% @atin in saline resulted in preferential induction of pulmonary AHH. After treatment with this dose of MCA, the pulmonary AHH levels of strains C57EL/6Cum, C57BL/6J, BALB/cM&i, CSH/fMai, and C57L/J were observcjd to be induced within 24 b after treatment. Strains DBA/2Cum, AKR/J, SJL/J, DBA/BJ and RF/J expressed no such increase. At a dose of 500 pg MCA, the pulmonary tissue of DBA/S mice did express a 4-fold increase. This increase in AHH was determined to be quite different from the increase observed in C57BL/6 mice by : (1) specific activity of the enzymes, (2) genetic regulation, (3) susceptibility to inhibition by 7&benzoflavone, and (4) spectral properties of the associated cytochromes. It was of major importance that induction of pulmonary AHH was observed to be regulated by a single dominant gene in crosses involving the C57BL/6Cum and DBA/2Cum strains of mice. Results were discussed with the view in mind that these genetically regulated levels of AHH may play a role in susceptibility to cancers induced by polycyclic aromatic hydrocarbon carcinogens.
Abbreviations: AHH, aryl hydrocarbon hydroxylsse; BP, benzo(a)pyrene; DMSO, dimethylsulfoxide; MCA, 3-methylcholanthrene; TCDD, 2,3,7,Wetrachlorodibenzo@)dioxin.
317
AHH is the name given to one of the multicomponent mixed-function oxidases which convert a variety of lipid-soluble compounds to water-soluble forms, usua:lly for subsequent elimination from the body (see refs. 1,2). The presence or inducibility of AHH is associated with the cytotoxic action of certain chemical carcinogens in vivo [ 31 and in vitro [ 4,5] and the detoxification [G-81 or activation [ 9--131 of polycyclic aromatic hydrocarbons in chemical carcinogenesis. Thus, one is confronted with the paradoxical situation that the same enzyme system may both detoxify and activate the same chemical carcinogens. The AHH enzyme in inbred strains of mice possesses two properties which make it particulaly amenable for studying its role in chemically induced cancers, that is, the system is inducible l [14,15], and this inducibility is genetically controlled. Depending on the strains used, hepatic AHH inducibility can be regulated by a single autosomal dominant gene [14,16], a single autosomal co-dominant gene [17,18] or non-responsiveness can be the result of a dominant allele [ 181. Using these genetic systems, recent information suggests that this enzyme system plays a major role in the susceptibility to MCA [ 19-21] - and BP [ 221 -induced carcinogenesis. The lungs and skin of mice seem to be under a different kind of control because. these organs seem to be slightly inducible in those mouse strains in which the liver is completely non-inducible [23-261. This apparent dichotomy is very important to understand because it suggests that the genetics of AHH inducibility is organ-dependent. rather than strain-dependent as originally concleived [14,16] . In this paper, we show that there is both a qualitative and quantitative difference between the increased levels of AHH observed in the AHH “non-inducible” mice and the increase observed in the AHH “inducible” mice. Moreover, in crosses involving the C57BL/6Cum and DBA/2Cum strains of mice, pulmonary AHH responsiveness to intratracheally administered MCA is regulated by a single autosomal dominant gene. EXPERJMEN’I’ALPROCEDURE
The polycyclic hydrocarbons BP and MCA were purchased from Sigma Chem&ls (St. Louis, MO.) and purified by recrystallization from benzene. 7,8-Benzoflavonle was purchased from Aldrich Chemicals (Cedar Knolls, N.J.). Metaphane was purchased from Pitman-Moore (Washington Crossing, N. J.). The sources of mice were either Clmberland View Farms (Clinton, Tenn.), The Jackson Laboratory (Bar Harbor, Maine), or Microbiological Associates (Bethesda, MCI.), For intratreachecl instillations, a Bausch and Lomb stereo-
* The term “inducibility” as used in this paper, denotes a relative increase in rates of de novo synthesis or of activation df enzyme activity from pre-existing moieties, or in rate of both, when compared to rate of breakdown. No particular mechanism is implied.
318
microscope equipped with fiber optic illumination and Hamilton syringes with 22 ga by 38 mm (with I.5 mm balls) feeder needles were used. Methods Care and feeding of mice were as stated previously [27] . Animals were always treated between the times of 9:00 and 10:00 a.m. to avoid diurnal variations. The i.t. instillation technique was similar to that described recently by Ho and Furst [ 28 1. Animals were anesthesized by inhalation of metaphane and were placed on special boards designed to hold their mouths open and at the correct angle for instillation. After positioning the animals, 0.02 ml of solution was given i.t. directly into the lung. The solution consisd;ed of MCA suspended in 0.2% gelatin in sterile saline 1291 or MCA dissolved in trioctanoin. At various times post-treatment, the lungs and livers were excised and frozen at -7O’C until assayed. Microsomes were prepared from lung or liver tissues according to the methods of Nebert and Gielen [30] and were stored at -7O’C for up to 72 h before being assayed. Samples were diluted with 0.1 M Tris-HCl buffer (pH 7.4) to a final ratio of 1.0 ml of microsomal suspension per gram wet weight tissue. The assay for AHH was basically that of Nebert and Gelboin [15] as modified by Nebert and Gielen [30] and Thomas et al. [14]. Tissues were carefully weighed and homogenized in 0.05 M Tris: 0.25 M sucrose buffer (pH 7.4) at a 1 : 10 (w/v) dilution for lungs and 1 : 20 (w/v) dilution for livers. 0.2 ml of homogenate was added to 0.79 ml buffer containing 100 pmoles Tris, 0.80 pmoles NADPH, 0.78 pmoles NADH, 6 pmoles MgC& and 200 Erg bovine serum albumin (Cohn Fraction V). 80 nmoles BP were added in 0.01 ml acetone to initiate the reaction, and tubes were incubated with shaking at 37°C for 20 min. The assay was stopped by the addition of cold acetone-hexane (1 : 3.3) and the polar metabolites in the hexane phase were back-extracted with 2.0 ml of 1 N NaOH. The amount of fluorescence associated with the presence of 3-OH BP was determined with excitation at 398 nm and emission at 522 nm in an Aminco-Bowman spectrophotofluorometer. Microsomal preparations were assayed for AHH activity in a similar manner, however, the amount of microsomal protein was determined by the method of Oyama and Eagle [31]. A unit of AHH activity is defined to be that amount of enzyme producing the fluorescent equivalent of 1.0 nmoles 3-OH BP per min at 37°C. For hepatic and pulmonary tissues, this is given in terms of units/g wet weight tissues, and for microsomal preparations in terms of units/pg protein. 7,8-Benzoflavone inhibition of AHH in microsomal preparations was determined according to the procedures of Goujon et al. [ 321 and Wiebel et al. [24,33]. Concentrations of 7,8-benzoflavone were dissolved in DMSO and 0.01 ml was added to tubes containing all factors for the AHH assay, excluding BP. The tubes were incubated at 37°C for 1 min, and then BP was added. For the spectral studies, microsomal pellets were resuspended by homo319
genization in 0.1 M K-P04, pl-i 7.4. Spectral studies were performed on an Aminco-Chance DW-2 W/vis spectrophotometer in the split beam mode. Cytochrome P-450 was determined after bubbling CO, adding 0.5 n&M NADH, dividing the sample between both cuvettes, correcting the baseline and then recording the: spectra after adding sodium dithionite to the sample cuvette [ 34 1. This procedure cancels out the contribution from cytochrome bs and hemoglobin. RESULTS
AHH levels following i. t. administration of MCA Effect of vehicle. Tables I and II demonstrate the pulmonary and hepatic responses of BGDZF,Cum mice 24 h after treatment with various doses of MCA given in either 0.2% gelatin or trioctanoin. MCA suspended in the gelatin solution seemed preferentially to induce pulmonary AHH at dose levels < 188 pg. MCA in trioctanoin induced both pulmonary and hepatic AHH at every dose level tested. All subsequent studies were done with MCA suspended in 0.2% gelatin so that pulmonary AHH could be specifically studiled. Effect ofparental strains. The hepatic and pulmonary responses of C57BL/ 6Cum (B6) and DBA/2Cum (D2) are shown in Table III. The B6 strain responded to MCA much like the B6D2F1 hybrid (Table I); however, at doses > 138 pg, MCA induced both pulmonary andhepatic AHH of B6 and B6D2F1 mice. Pulmonary AHH levels in D2 mice were induced slightly by treatment
TABLE I EFFECTS OF INTRATRACHE%L INSTILLATION OF VARIOUS MCA DOSES IN TRIOCTANOIN ON PULMONARY AND HEPATIC AHH s IN B6D2F1Cum MICE ~.cg MCA
0
25 56 100 200 400 500
Liver AHH
Lung AHH Untreated
Trioc
MCATrioc
Ind. b
Untreated
Trioc
MCATrioc
Ind.
0.32
0.20
1.0 1.7 1.6 1.9 1.6 1.2
5.0 8.5 8.0
12.6
9.8
19.6 19.6 38.2 39.2 62.7 44.1
2.0 2.0 3.9 4.0 6.4 4.5
K 6.0
* AHH activity given in terms of units per g wet weight tissue. A unit is that amount of enzyme causing the fluorescent equivalent of 1 nmole of 3.CH BP per min at 37’C. Mean of at least three animals assayed in duplicate ; values for individ .ral mice were usually within 10% of each other. b Ind., Inducibility : the relative increase of MCA-treated tissue over vehicle-fretited control tissue.
320
TABLE II EFFECTS OF INTRATRACHEAL INSTILLATION OF VARIOUS MCA DOSES IN 0.2% GELATIN ON PULMONARY AND HEPATIC AHH a IN B6D2F1Cum MICE
/a MCA
0 5 11 23 47 94 188 375 500
Lung AHH
Liver AHH
Untreated
Gel
0.36
0.26
MCA: Gel
0.56 0.76 1.40 1.30 1.80 2.00 2.40 2.50
Ind.
Untreated
Gel
MCA: Gel
12.6
10.0
-
-
7.81 10.30 10.80 14.40 11.40 18.90 24.00 25.50
0.78 1.03 1.10 1.40 1.10 1.90 2.40 2.70
2.20 2.90 5.40 5.00 6.90 7.70 9.20 9.60
Ind.
See Table I for footnotes.
TABLE III EFFECTS OF INTRATRACHEAL INSTILLATION OF VARIOUS MCA DOSES IN 0.2% GELATIN ON PULMONARY AND HEPATIC AHH a IN C57BL/GCum AND DBA/2Cum MICE pg MCA
A. C57BLj 6Cum 0 5 11 23 47 94 188 375 500 B. DBA/ 2Cum 0 188 500
Liver AHH
Lung AHH Untreated
Gel
0.40
0.32
MCA Gel
0.73 0.89 1.50 1.40 1.70 2.40 2.50 2.40
0.20
Ind. b
Untreated
Gel
17.5
14.5
2.30 2.80 4.70 4.40 5.50 7.50 7.80 7.50
9.80
0.18 0.17 0.56
1.00 3.10
9.50
MCA Gel
Ind.
-
-
8.3 13.3 13.1 14.5 24.7 32.3 35.0 33.2
0.57 0.92 0.90 1.00 1.70 2.20 2.40 2.30
10.1. 10.2
1.10 1.10
See Table I for footnotes.
321
with 500 c(g MCA but not by 188 pg MCA, whereas hepatic AHH was unaffected at either dose. The average inducibility for pulmonary tissue of ten D2 mice following MCA treatment was 3.1 k 0.6 (s.e.) In terms of specific activity, the D2 strain has very little pulmonary AHH activity; the highest induced activity was about the same as the constitutive (or control) activity of the other two strains. Both sexes of the two in.bred strains and their
L -
I.0
02 B6 B6DZFl
30
0 0
1
2
3
4
s
6
7
8
9
16
II
I2
13 11
DAYS
Fig. 1. Kinetics of pulmonary and hepatic AHH following i.t. administration of MCA. (a) Time-dependent increase in pulmonary AHH following i.t. treatment with 188 pg MCA }. (b) Time-dependent increase into D2 (A-A), B6 (w) and B6D2FI (in pulmonary AHH following i.t. treatment with 600 &g MCA into D2 (A---A), B6 -o) mice. (c) Time-dependent increase in hepatic AHH (V) and B6D2F1 (following i.t. treatment with 500 pg MCA into D2 (A-), B6 (w) and B6D2Fl (M) mice.
322
19.2 22.2( 1.2)
0.71 3.1(4.4)
BALB/cMai
7.9 7.3(0.9)
0.33 2.5(7.2)
CSH/fMai
13.3 27.4(2.1)
0.64 3.4( 5.3)
C57L/J
16.1 32.6(2.0)
0.28 2.4(&O)
C57BL/6J
17.5 15.6(0.9)
0.28 0.45( 1.6)
AKR/J
10.8 11.7(1.1)
0.19 0.29(1.5)
SJLlJ
8.9 8.5(0.9)
0.20 0.28(X.4)
DBAlZJ
13.5 13.8(1.0)
0.41 0.54( 1.3)
RF/J
a AHH activity given in terms of units per g wet weight tissue. A unit is that amount of enzyme causing the Ruoreseent equivalent of 1.0 nmole of 3-OH EP per min at 37*C. The relative inducibiiity is given in parentheses. b Tissues were removed 24 h after i.t. treatment with either gelatin-saline or 188 &g MCA.
Control MCA
Liver
MCA
control
Lung
Tissue b
EFFECTS OF INTRATRACHEAL INSTILLAmON OF 188 c(g MCA IN 0.2% GELATIN ON PULMONARY AND HEPATIC AHN a IN VARIOUS STRAINS OF MICE
TABLE IV
hybrid were assayed for their pulmonary and hepatic AHH responses following MCA and no differences were observed (data not shown). Changes in pulmonary AHH with time following i.t. instillation in gelatin of 188 pg MCA into B6, D2 and B6D2Fl mice are shown in Fig, la. The induction time course was similar for both B6’ and B6D2F, mice; maximum induction was observed by 24 h and remained constant for up to 96 h. The maximum observed increase for each strain and hybrid was similar; about 6-fold. The responses of pulmonary and hepatic AHH following i.t. administration of 500 c(g MCA are shown in Fig. lb and Fig. lc. Pulmonary AHH levels remained maximally~ induced for at lea& 7 days. Hepatic levels decreased by day two and were almost at background level by the third day. A subsequent treatment on the seventh day resulted in levels of AHH activity similar to that observed for the first treatment. Effects on various strains of mice. Table IV demonstrates the hepatic and pulmonalry AHH levels following i.t. instillation of 188 pg MCA. The AHH levels in pulmonary tissues of strains BALB/c Mai, C3H/f Mai, C57L/J and C57BL/6J were induced by MCA while strains AKR/J, SJL/J, DBA/BJ and RF/J showed httle or no increase. Hepatic responses were low for all strains except perhaps for the C57BL/6J and C57L/J, which did show a 100% increase. Genetic regulation of pulmonary AHH induction. Induction of pulmonary AHH by i.t. instilled MCA segregated as a single autosomal dominant gene in crosses between the B6 and D2 strains of mice (Table V). Backcross and F2 animals were classified as inducible or non-inducible if, after i.t. treatment with 188 pg MCA, pulmonary AHH levels were 2.5 (kO.5) units/g tissue (inducible) or 0.3 (kO.05) units/g tissue (non-inducible). Among 90 backcross animals ,tested, 47 were inducible (52%) and among 50 F1 animals tested, 36 were inducible (72%). These numbers were not statistically differTABLE V GENETIC SEGREGATION OF PULMONARY AHH IN CROSSES INVOLVING THE C57BL/6Cum AND DBA/2Cum STRAINS OF MICE I’ ----___-----Number induced Strain Number treated % Induction __-L__.--_-I-_B6 50 50 100 D2 50 0 B6D2F 1 100 1; 100 B6D2F, X B6 52 62 100 B6D2F, X D2 92 47 62 B6D2Fz 60 36 72 a Mice were treated with 163 l.(g MCA/O.OB ml 0.2% gelatin solution it. and 24 h later, the pulmonary AHH was assayed. A mouse was considered inducible if, after MCA treatment, pulmonary AHH was 2.5 (t0.6) units/g tissue and considered noninducible if pulmonary AHH was 0.3 (fO.05) units/g tissue. The sex of the progeny played no role in this segregation pattern.
324
I
*
5
50
500
w 7,6-benzoflovone/ml Fig. 2. In vitro effects; of 7,8-henzoflavone on AHH activity of pulmonary microsomal preparations from control (M ) and MCA-treated (w) B6 mice, and control a) and MCA-treated (_ A) D2 mice. Specific activities of the microsomal (Apreparations were 5.0, 89.0, 1.7 and 3.8 units AHH activity per&g protein, respectivt!y.
ent (P < 0.01, using chi square analysis) from the expected 50% and 75% ratios that would be expected if a single autosomal dominant gene was regulating this inducib~i~, AH. levels in microsomal preparations. The effect of 7&benzoflavone, a known competitive inhibitor of the induced, or P-446mediated enzyme, on the in vitro metabolism of BP by microsomes derived from pulmonary tissues of the B6 and D2 mice is shown in Fig. 2. Results with the untreated (no 7,8benzoflavone) preparations generally agreed with the results shown in Tables III and IV; that is, D2 pulmonary tissue expressed much less enzyme activity than B6 tissue, MCA treatment of D2 mice resulted in a small increase in pulmonary AHH levels to about that observed in control B6 mice, and MCAtreatment of B6 mice resulted in a much higher level of pulmonary AHH activity (about 14-fold in these microsomes). 7,&Benzoflavone inhibited AHH activity from control and induced D2 tissue and control B6 tissue in a similar manner, while the AHH activity from induced B6 tissue was much more ~nsitive. For example, at concen~ations of 7,8-benzo~avone equimolar to the BP concentration, the enzymes from the D2 tissue and from control B6 tissue were inhibit by about QO%,while the enzymes from induced B6 tissues were inhibited by greater than 80%. ~~ect~~ studies in mic~sama~ preparations. Spectral destinations of P450 in lung microsomes from both control and MCA induced mice were attempted (Table VI). These studies were complicated by a large absorbance 325
TABLE VI SPECTRAL STUDIES ON PULMONARY MICROSOMES DERIVED FROM D2 AND B6 MICE Strains
Treatment a
Spectral peak (nm)
nmoles cyrochrome P-450/ mg protein
D2 D2 B6 B6
Gel MCA Gel MCA
450 449-449.5 450 446
0.198 0.093 0.101 0.095
a Twenty mice of each strain were treated i.t. with either 500 pg MCA of 0.2% gelatinsaline solution and sacrified 48 h later. Lung microsomes were prepared as given in MATERIALS AND METHODS.
in the 420-424 nm region of the CO difference spectra which equalled or exceeded the absorbance in the region of 450 nm. This “P-420-like” material was present even though the CO difference spectra were recorded under conditons designed to blank out cytochrome b5 and hemoglobin, and regardless of whether the microsomes were fresh or frozen. We do not know to what extent this “P-420-like” material might interfere with the P-450 determinations. Even under these conditions, a shift in the CO difference spectra from 450 nm to 448 nm in C57BL/6J lung 2 days after administration of MCA was observed. DBA/BJ mice analyzed at the same time showed at most a 1 nm shift from 450 nm to 449 nm after MCA induction. These results are very similar to those recently described for microsomal preparations of rat lung [35]. Following treatment with MCA, the content of P-450 in pulmonary tissue of D2 mice actually decreased {see Table VI). Total P-450 content of pulmonary tissue from B6 mice was not altered by MCA treatment. The nature of this decrease is inexplicable at this time. DISCUSSION
In this report we describe some of the major parameters regulating the constitutive and MCA-induced levels of pulmonary AHH activity in inbred strains of mice. The intratracheal route was used so as to attempt to limit the enzymatic response to pulmonary tissue. Utilizing the inbred strains C67BL/6 and DBA/B, whose hepatic AHH responses have been extensively studied [ 14-18 1, we show in this report that: (a) MCA given in either trioctanoin or a 0.2% gelatin solu$ion induces pulmonary AHH, and this induction is dosedependent (Tables I, II, 111, IV); (b) a dose of 188 pg MCA given in 0.2% gelatin induces pulmonary AHH, but has very limited effect on hepatic AHH levels (Tables II and III); (c) pulmonary AHH can be induced in D2 mice by it. admimstration of MCA, but hepatic AHH levels are never induced (Table III, Fig. 1); (d) although pulmonary AHH is induced in D2 mice, the levels 326
are very low, with about a 5-lo-fold difference between D2 and B6 puhnonary tisues; and (e) this strain difference is under the sa,ne genetic control as that of hepatic tissue [14,16], that is, the highly responsive B6 strain differs from the D2 strain by a single autosomal dominant gene controlling this heightened responsiveness (Table V). Results with other inbred strains of mice agree with this contention, that is, strains that are non-responsive to MCA in their hepatic tissues are low responders to MCA when given Lt. and vice versa (Table IV). In agreement with the observations of Wiebel et al. [24], Burki et al. [23], and more recently Abramson and Hutton [25 ] and Van Canfort and Gielen [26], pulmonary AHH seems to be inducible in every strain of mouse regardless of hepatic AHH responsiveness. However, the mean basal AHH activities in lungs from strains of mice classified as hepatic AHH inducible are significantly higher than the activities for the same pulmonary enzymes in hepatic non-inducible strains (ref. 25; see Tables II, III and Fig. 1). In fact, Abramson and Hutton [25] suggest that the same genes may be affecting both liver AHH inducibilities and levels of basal AHH activity in pulmonary tissue. Of interest is the fact that the inducibility of AHH in hepatic non-responsive strains (e.g. D2) is dose-dependent and the differences in specific activity following exposure to 188 pg MCA segregate as a single autosomal dominant gene in crosses between the B6 and D2 strains; just as hepatic AHH inducibility [ 14,161. Thus, although the pulmonary tissue in D2 mice is induced by high levels of MCA (-500 gg), this increase can be discriminated genetically from that response observed in B6 mice. These results could be explained in terms of hypothetical receptor sites for AHH induction [36]. Pulmonary and hepatic tissue in B6 mice contain efficient receptor sites and hence are responsive to low-level treatment with an inducer, such as MCA. Receptor sites in pulmonary and hepatic tissue in D2 mice are much less efficient and there may even be slight differences in efficiency between the receptor sites in the pulmonary and hepatic tissue in this strain, the lung receptor site being more efficient. Thus, high levels of inducers would be required to effect AHH induction in D2 mice and responsiveness may be limited to just pulmonary tissue. This explanation isvery similar to that proposed by Poland and Glover [36] for describing the induction results using TCDD in B6 and D2 strains of mice. These authors observed that TCDD is capable of inducing AHH activity in both B6 and D2 mice but that the level required for maximal induction of D2 mice is about 100 times higher than that required for B6 mice. The fact that AHH can be induced in hepatic tissue of D2 mice (with its concomitant cytochrome, P-448) indicates that the structural genes necessary for synthesis of these induction-specific enzymes are present in D2 tissue. A mutation in a receptor site (or receptor protein) was hypothesized as the most logical explanation for the variation in responsiveness between the two strains. Our results suggest that, not only may the D2 strain possess an inefficient receptor site, but also, this site is different in pulmonary and hepatic tissue. This pulmonary AHH seems unique for it is fairly resistant to inhibition by 327
7,8-benzoflavone (see Fig. 2) and the blue spectral shift normally associated with hydrocarbon induction could not be observed in MCA-treated D2 lung tissue (see Table VI). The expected sensitivity to 7,8-benzoflavone inhibition and presence of P-448 was observed in MCA-treated B6 lung tissue. Thus, although increases in AHH are observed in MCA-treated pulmona,ry tissues in D2 mice, thfere is no indication that it is the same kind (or form) of induction that occurs in B6 mice. Direct quantitation of the cytochromes in MCAtreated D2 lung tissue may be important in understanding the mechanism of this kind of induction. The recent report of Capdevila et al. [37] may allow for unambiguous study of the nature of pulmonary inducibility in hepatic non-responsive strains. These authors report a sensitive chromatographic method for the isolation of cytochromes P-450 and P-448 from pulmonary microsomes. This approach is currently in progress in our laboratory. Results recently observed by Poland et al. [38], on maximal absorbance peaks of CO difference spectra in both C57BL/6N and DBA/BN mouse lung with and without intraperitoneal MCA administration, do not agree with our data in some respects. Namely, their spectral peaks for CO reduced cytochrome P-450 is at 455-456 nm instead of at 450 nm which most probably results from improper compensation for the presence of hemoglobin However, their results corroborate in a qualitatnre manner, for they did observe a 1 nm blue shift in the CO reduced spectra of C57BL/6N mouse lung with MCA treatment, but no shift in DBA/BN mouse lung when treated with MCA. This genetically controlled enzyme response may p!ay a major role in the ultimate susceptibility of pulmonary tissue to chemically induced cancer, just as hepatic AHH inducibility is correlated with increased susceptibility to MCA-induced subcutaneous tumors in mice [19-211. Moreover, this idea seems to have precedence in the human situation, where recent results (presently requiring confirmation) suggest a relationship between the higher levels of AHH inducibility (as measured in lymphocyte culture) and presence of bronchogenic squamous+ell carcinoma (cigarette smoke-associated) [ 391. Nettesheim and Hammans [29] have reported conditions for induction of squamous cell carcinomas in inbred strains of mice and their data suggest a relationship between AHH inducibility and lung cancer. These authors utilized the (C57BL/6 X C3H/f) F1 and the DBA/B strains of mice, and 500 c(g MCA (in 0.2% gelatin) was given i.t. at weekly intervals for 4-6 weeks. These authors observed that the AHH inducible (C57BL/6 X C3H/f) Fi hybrid was much more sensitife to MCA-induced squamous cell carcinomas than the AHH “non-inducible” DBA/2 strain. Hepatic and pulmonary AHH levels of responsive (B6) or ‘low responsive (D2) mice to this treatment schedule are shown in Fig. lb and Fig, lc. There are obvious differences between the two strains in their ability to respond to the MCA and it is very likely that the increased susceptibility to chemically induced carcinomas of AHH inducible animals reflects this heightened ability to metabolize chemical carcinogens. This theory can be tested by it. instillation of various doses of MCA into backcross of F2 animals derived from the B6 and D2 strains and observing 328
the relationship between AHH responsiveness and susceptibility to MCAinduced lung cancers. Actually, since lower doses (e.g. 188 pg) of MCA maximally induce pulmonary AHH and have xly limited effect on hepatic AHH levels, this may be the dose of choice. These procedures are currently being evaluated in our laboratories. ACKNOWLEDGEMENTS
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