Isosafrole-induced Cytochrome P2-450 in DBA/2N Mouse Liver CHARACTERIZATION AND GENETIC CONTROL OF INDUCTION*

Mouse “cytochrome Pz-450” is defined as that form of isosafrole-induced P-450 in DBA/ZN liver most specifically correlated with isosafrole metabolism. Isosaf- role pretreatment does not induce aryl hydrocarbon hydroxylase activity (“cytochrome PI-450”) in C57BL/ 6N or DBA/2N mice, induces acetanilide 4-hydroxyl-ase activity (“cytochrome P3-450”) more than %fold in C57BL/6N but not in DBAIZN mice, and induces isosafrole metabolite formation more than %fold in both C57BL/6N and DBA/ZN mice. P2-450 was, therefore, purified from isosafrole-treated DBA/2N liver microsomes having negligible amounts of contaminating PI- 450 and P3-450. The apparent molecular weight of P2-450 is 55,000, and the protein appears homogeneous on sodium dodecyl sulfate-polyacrylamide gels. The Soret peak of the reduced purified cytochrome*CO complex is 448 nm. Purified P2-450, reconstituted in vitro, metabolizes acetanilide’poorly and benzo[a]py-rene hardly at all. Anti-(P2-450) inhibits (90 to 100%) liver microsomal isosafrole metabolite formation, yet has no effect on aryl hydrocarbon hydroxylase, acetan- the previously published techniques. Ah Receptor Assay-The postmicrosomal supernatant of individual livers from untreated mice was exposed to 10 nM ['HITCDD (in the absence or presence of nonlabeled 1 p~ TCDD) for 1 h at 4 "C. Following dextran-charcoal adsorption, the material was centrifuged on a linear (5 to 20%) sucrose density gradient, as detailed (29). The saturable radioactivity in the Ah receptor peak was equated with femtomoles of receptor per mg of cytosolic protein and with the number of TCDD-binding sites per cell (29).

proteins are controlled by the Ah locus, either a P-450 protein polymorphism exists between C57BL/6N and DBA/ZN mice or subtle differences may exist in the interaction of various inducers with Ah receptor.
The biochemical purification and characterization of a growing number of distinct cytochrome P-450 proteins' has received increasing attention in recent years. More than 300 foreign chemicals, steroids, and peptide hormones are known to enhance P-450-mediated monooxygenase activities (reviewed in Refs. 4 and 5), suggesting the induction of one or more P-450 proteins in each instance, and the list grows larger each year.
T o understand the genetic mechanisms by which a particular chemical is able to induce a particular P-450 protein has been the central goal of this laboratory. Most of our work has involved the expression of mouse PI-450, controlled by the Ah receptor (6)(7)(8). These studies have always taken advantage of the fact that the B6 mouse (Ahb/Ahb) has a large amount of the high affinity receptor and the D2 mouse (Ahd/Ahd) has a poor affinity receptor. Following 3-methylcholanthrene treatment, PI-450 is thus highly induced in B6 mouse liver and negligibly induced in D2 mouse liver. PI-450 induction by 3-methylcholanthrene is inherited in the B6D2F1 heterozygote (Ahb/Ahd) as an autosomal dominant trait (9).
Isosafrole is a naturally occurring plant constituent (10). Studying Ahb/Ahb and Add/Ahd mice, Fennel1 et al. (11) concluded that isosafrole induces a form of P-450 specific for The term "P-450" is used to designate any or all forms of microsomal cytochrome P-450. Mouse "P~-450" is defined as that form of isosafrole-induced P-450 in DBA/2N liver most specifically associated with isosafrole metabolism. Mouse "PI-450" and "P3-450 are defined as those froms of 3-methycholanthrene-induced P-450 most specifically associated with induced aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase activities, respectively. The concentration of P3-450 protein in control mouse microsomes is at least five times greater than that of P1-450; after 3-methylcholanthrene induction of C57BL/6N liver, P3-450 is the major induced form, being about five times greater in concentration than P1-450 (2). The size of all three proteins is 55,000 daltons, as judged by NaDodS04-polyacrylamide gel electrophoresis. The Soret peaks of the reduced hemeprotein. CO complex for PI-450, P2-450, and P3-450 are 449.3, 448, and 448 nm, respectively. Because we have now characterized two proteins with a spectral maximum of 448 nm, we have changed the name of "P-448" studied 5 years ago (3) to "P3-450." Other abbreviations used are: isosafrole, I-propenyl-l,2-methylenedioxybenzene; B6, the C57BL/6N inbred mouse strain; 0 2 , the DBA/2N inbred 2676 Isosafrole-induced P2-450 in 0 2 Mouse isosafrole metabolism and that this form is not identical to the major phenobarbital-or 3-methylcholanthrene-inducible forms of P-450. Subsequently, isosafrole-induced P-450 was purified from rat liver microsomes (12,131. From these studies it was apparent that this isosafrole-induced P-450 is similar in many ways to the major rat 3-methylcholanthrene-inducible P-450 yet did not metabolize benzo[a]pyrene. These valuable studies (3, 6-8, 12, 13) were each performed with an antibody that was not claimed to be monospecific; despite this shortcoming many important conclusions have been made.
Whereas 3-methylcholanthrene induces P1-450 in B6 but not D2 liver, isosafrole induces a form of P-450 almost equally well in both B6 and D2 liver (1,11). In this report we show the isolation of the isosafrole-induced form (P2-450) from isosafrole-treated D2 mouse liver microsomes, in which there is virtually no contaminating P1-450. Further, we demonstrate that the P2-450 induction by isosafrole is controlled by the Ah receptor but that the inheritance of this genetic trait is somewhat different from inheritance of the trait of PI-450 induction by 2-methylcholanthrene.
Purification of Cytochrome &-&io from Isosafrole-treated 0 2 Mouse Liver Microsomes-Isosafrole induces PI-450 in B6 but not D2 mouse liver (1,17). We, therefore, chose to isolate P2-450 from D2 liver microsomes, in which there would be no contaminating problems with PI-450. The method developed by Imai and Sat0 (18) was applied to the purification of P2-450. NaDodS04-polyacrylamide gel electrophoresis was performed by the method of Laemmli (19), with slight modifications in this laboratory (16).
Development of Anti-(P2-450)-A mixture of purified P2-450 (500 pg for each dose, with complete Freund's adjuvant the first time, and incomplete Freund's adjuvant the next two times) was injected three times, a t 2-week intervals, into a goat. Preimmune and postimmune immunoglobulin G fractions were prepared by ammonium sulfate fractionation. After dialysis the fractions were dissolved in 100 mM potassium phosphate buffer (pH 7.5). These fractions were used for inhibition of microsomal monooxygenase activities in vitro (3) and determination of tritiated anti-(P2-450)-precipitable protein from cholate-solubilized microsomal membranes that had been previously labeled with NaB[3H]4 in the presence of 1 mM pyridoxal phosphate (3,20), by the methods cited. The values of anti-(P2-450)-precipitable material are expressed as: (disintegrations min" of immunoprecipitate per mg of microsomal protein)/(disintegrations min-I per mg of total microsomal protein).
Spectrophotometry-Total P-450 content was determined by the method of Omura and Sat0 (21); an extinction coefficient of 91 mM" cm-I was used for the difference in absorption between the Soret peak around 450 nm and the base-line at 490 nm for the dithionitereduced hemoprotein. CO complex. The isosafrole metabolite. cytochrome P2-450 complex was assayed (13,22) with an extinction coefficient of 75 mM" cm-' for the difference in absorption between 455 nm and the base-line a t 490 nm for the dithionite-reduced form (Fig. 2). Protein concentrations were determined by the method of Lowry et al. (23). as modified by Dulley and Grieve (24) to eliminate interference by detergents.
Step 2, incubation of the microsomes with 17 mM 2methylbenzimidazole was carried out (15 min a t 37 "C), and the reaction mixture was applied to a Sephadex G-50 column (1.7 X 20 cm) that had been equilibrated with 0.1 M potassium phosphate (pH 7.4), 5 mM MgCIZ, and 0.1 mM ethylenediaminetetraacetic acid. This procedure displaces the isosafrole metabolite.
Step 3, both absorption bands can be reproduced in oitro from the microsomes in the sample cuvette of Step 2 by the aerobic incubation with isosafrole and 1 mM NADPH. Bottom, the usual CO difference spectrum (21) of dithionitereduced P-450 from fresh isosafrole-induced D2 liver microsomes (0.5 mg/ml). O B . , optical density. coumarin 0-de-ethylase (28) activities were carried out by the procedures cited. Reconstitution experiments were performed with 30 pg of dilauroylphosphatidylcholine, 0.5 unit of NADPH P-450 reductase, and 100 pmol of P2-450 incubated a t room temperature for 10 min; then the regular buffer and assay conditions for each activity were used, according to the previously published techniques.
Ah Receptor Assay-The postmicrosomal supernatant of individual livers from untreated mice was exposed to 10 nM ['HITCDD (in the absence or presence of nonlabeled 1 p~ TCDD) for 1 h at 4 "C. Following dextran-charcoal adsorption, the material was centrifuged on a linear (5 to 20%) sucrose density gradient, as detailed (29). The saturable radioactivity in the Ah receptor peak was equated with femtomoles of receptor per mg of cytosolic protein and with the number of TCDD-binding sites per cell (29).

RESULTS
The p2-450 Protein-As detailed under "Experimental Procedures" and in Fig. 1, an electrophoretically homogeneous protein was purified from isosafrole-treated D2 liver microsomes. The Soret peak of the reduced cytochrome.CO complex was 449 nm in microsomes (Fig. 2), but the Soret peak of the purified P2-450. CO complex was 448 nm. The apparent molecular weight was 55,000 ( Fig. 3). By means of Ouchterlony double diffusion plates, fusion of a major precipitin line between neighboring wells (Fig. 4) indicates immunologic homology of the anti-(P2-450)-precipitable protein in isosafrole-treated B6 and D2 and control D2 liver microsomes. An increased intensity of the precipitin line following isosafrole treatment is consistent with relatively equal induction of anti- Comparison between intact liver microsomes and purified P2-450 on a NaDodS0,-polyacrylamide gel. Polyacrylamide concentration of 7.5% was used (16,19). Direction of migration is from top to bottom. Twenty pg of microsomal protein from control and isosafrole-treated B6 and D2 mice and 5 pg of purified P2-450 protein were applied to each well.
(P2-450)-precipitable material in both D2 and B6 mice. A possible minor precipitin line nearer to well I than the major line ( Fig. 4) suggests that the antiserum may recognize a t least a second microsomal protein; we are not claiming, however, that anti-(Pz-450) is monospecific. There are clearly no spurs seen. Between the P2-450 and P2-450 proteins (data not shown), there is no fusing of precipitin lines indicating that anti-(Pz-450) does not cross-react with P1-450.
Association of P2-450 with Isosafrole Metabolism-Whereas isosafrole did not induce aryl hydrocarbon hydroxylase activity in the intact liver microsomes of D2 and B6 mice (Table   I), isosafrole did induce acetanilide 4-hydroxylase activity about 3-fold in B6 but not a t all in D2 mice. These data indicate that isosafrole is not a good inducer of P1-450 in either D2 or B6 mice and does not induce P3-450 in D2 mice. induced both aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase activity more than 6-fold in B6 mice and not at all in D2 mice. These data have been reported previously (3,27).
The P2-450-reconstituted activities of aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase (Table I) are about one-thirtieth and one-tenth, respectively, of these activities in 3-methylcholanthrene-induced B6 liver intact microsomes. The rate of benzo[a]pyrene metabolism by purified P1-450 reconstituted in vitro (3) was 2.95 nmol/min/nmol of P-450, as compared with the P2-450 value of 0.14 in Table I Table I. These findings suggest that, whereas there exist overlapping substrate specificities, neither benzo[a]pyrene nor acetanilide is a particularly good substrate for P2-450. Further, the results in Table I show that isosafrole-induced P2-450 in D2 mice is different from 3-methylcholanthrene-induced PI-450 or Ps-450 in B6 mice.
Isosafrole induced total P-450 content in D2 liver (Table  IJ), whereas 3-methylcholanthrene had no effect in D2 liver. In B6 liver, 3-methylcholanthrene induced total P-450 content better than isosafrole treatment. It is known (12,13,22) that isosafrole is metabolized by isosafrole-induced P-450 and that the metabolite forms a complex with the induced protein.  The formation of this complex can be measured by following the increase of absorption at.456 nm (Fig. 2). The amount of isosafrole metabolite bound to P2-450 in uivo was about the same as that bound in vitro following incubation of microsomes with substrate and NADPH at 37 "C. Anti-(P2-450) inhibited isosafrole metabolite formation completely in isosafrole-treated D2 mice and more than 90% in isosafrole-treated B6 mice (Table 11). The antibody had little effect on isosafrole metabolite formation in 3-methylcholanthrene-treated D2 mice and blocked more than half of the activity in 3-methylcholanthrene-treated B6 mice. Anti-(Pz-450) had no appreciable effect on isosafrole metabolite formation in control or phenobarbital-treated B6 or D2 mice. Fig. 5 illustrates that anti-(Pz-450) is a good inhibitor of isosafrole activity but does not inhibit aryl hydrocarbon hydroxylase, acetanilide 4-hydroxylase, biphenyl 2-or 4-hydroxylase, or 7-ethoxycoumarin 0-de-ethylase activities in isosafrole-treated or 3-methylcholanthrene-treated B6 and D2 mice. These findings lend further support to the concept that Pz-450 is distinct from both mouse P1-450 and P3-450.

Aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase activity in intact liver microsomes and with purified Pz-450 reconstituted in vitro
Effect of Inducers-To quantitate the amount of Pz-450 in liver microsomal membranes, we treated isolated intact microsomes with pyridoxal phosphate and then reduced the microsomal mixture with NaB[3H]4. After centrifugation, the radioactive microsomes were incubated with cholate and then centrifuged to obtain the solubilized microsomes. The immunoprecipitates were formed by addition of anti-(P2-450) to the solubilized radiolabeled microsomes and then analyzed by NaDodS04-polyacrylamide gel electrophoresis. It was determined ( Fig. 6) that 5 mg of antiserum were sufficient to obtain maximal amounts of the anti-(P2-450)-precipitable protein.
In control, 3-methylcholanthrene-treated, isosafrole-treated, and phenobarbital-treated B6 and D2 mice (Fig. 7), a single sharp peak of 55,000 daltons was observed. By means of measuring the radioactivity of this 55,000-dalton peak, anti-(Pz-450)-precipitable material was present in control B6 and D2 mice and was induced about 6-fold in both strains. 3-Methylcholanthrene increased the immunoprecipitate about 12-fold in B6 mice and less than 2-fold in D2 mice. Phenobarbital did not induce the moiety in either strain. Of interest, phenobarbital induced isosafrole metabolite formation about 5-fold (Table 11). The data in Table I1 and Fig. 7, therefore, suggest that phenobarbital induces one or more forms of P-450 that metabolize isosafrole, but phenobarbital does not induce Pz-450, the form specific for isosafrole metabolism.
In addition to these inducers, TCDD at high doses (10 pg/ kg) was as effective as 3-methylcholanthrene for induction of the immunoprecipitate in B6 mice. TCDD also induced Dz

Formation of the P-450 complex with isosafrole metabolite and the effect of anti-(P2-450) on this formation
Values represent at least two determinations on liver microsomes pooled from six mice. Each determination of specific activity was also performed in duplicate.   D2 (bottom) mice. The immune serum (immunoglobulin G ) in milligrams of protein is depicted on the abscissa. The starting liver microsomes (10 mg of protein/ml) were dissolved in 100 mM potassium phosphate (pH 7.5) containing 200 mM KCl, 20% glycerol, and 2% sodium cholate. Following centrifugation at 105,000 X g for 60 min, aliquots of the supernatant (40 pg) were used for precipitation by the immune serum. The incubation was carried out overnight at 4 "C. Immunoprecipitates were washed twice with 100 mM potassium phosphate buffer (pH 7.5) containing 200 mM KCl, 1% sodium cholate, and 0.25% SBM, and then once with water to remove potassium ions.

Formation of isosaf-
P2-450 almost as well. These data are consistent with the finding (30) that D2 mice have a poor affinity Ah receptor that can be overwhelmed by sufficient amounts of TCDD when this occurs, the TCDD-receptor complex enters the nucleus and elicits its effect of PI-450 mRNA induction (30). The results with 3-methylcholanthrene and TCDD in B6 and D2 mice in Fig. 7 thus suggest that induction of the anti-(P2-450)-precipitable material might be controlled by the Ah  receptor (5,6).
Benzo  HI4 and examined on NaDodS04-polyacrylamide gels. Liver microsomes were pooled from five mice for each group. A , radioactivity measured as a single 55,000-dalton peak from control, 3-methylcholanthrene (MeChoZ)-treated, isosafrole-treated, and phenobarbital-treated I36 (left) and D2 (right) mice. B, amount of [3H]protein precipitated by anti-(P2-450) from liver microsomes of B6 and D2 mice that bad received treatment with nine various chemicals known to induce one or more forms of P-450. B6 and D2 mice were also treated with three other derivatives of thep,p'-DDT insecticide,p,p'-DDE, Dicofol, and chlorobenzylate; the results were not significantly different from that seen with p,p'-DDT, dexamethasone, pregnenolone 16a-carbonitrile, or phenobarbital treatment. The amount of immunoprecipitated P-450 protein labeled with tritium was calculated on the basis of disintegrations per min per pg of protein in the cholate-solubilized fraction. Individual samples varied less than 10%. Futher experimental details are described under "Experimental Procedures." the legend to Fig. 6, and in Refs. 3 and 20. about 2-fold in B6 and negligibly in D2 mice (Fig. 7). It is known (17) that the dose of benzo[a]anthracene used induces P1-450 mRNA very effectively in B6 but not D2 mice. Ellipticine behaved like isosafrole by inducing anti-(Pz-450)-precipitable protein equally well in B6 and D2 mice.' Pregnenolone 16a-carbonitrile, dexamethasone, p,p'-DDT, p,p'-DDE, Dicofol, and chlorobenzylate behaved much like phenobarbital by having no significant effect on induction of the immu-Isosafrole, henzo[a]anthracene, and ellipticine displace 67, 61, and 40%, respectively, of [3H]TCDD from the A h receptor (31), when these test compounds are present in 100-fold excess, compared with the radioligand concentration of 10 nM; in the same experiments, 3methylcholanthrene and nonlabeled TCDD displace the radioligand noprecipitate in either B6 or D2 mice.
Association of the Ah Locus with Induction of the Immunoprecipitate by Isosafrole-An intermediate dose of isosafrole (15 mg/kg) was used to demonstrate that the additive inheritance of P2-450 induction follows Mendelian genetics and correlates well with the Ah phenotype (Fig. 9). The B6D2F1 was intermediate between the B6 and D2 parents. From offspring of the B6D2F1 x B6 backcross, about one-half appeared to be similar to B6 and about one-half similar to the F,. From offspring of the B6D2F1 X D2 backcross, about onehalf appeared to be similar to the F1, and about one-half similar to the D2 parent. Although these individual mouse values are consistent with single-gene inheritance of an additive trait, there is sufficient scatter of points to suggest more than a single gene being involved.
In Fig. 9 (far right), eleven offspring from the B6D2F1 X D2 backcross were Ah-phenotyped by the zoxazolamine paralysis test (35) 2 weeks previously and then their liver microsomes were examined for anti-(P,"I)-precipitable protein?
Because aryl hydrocarbon hydroxylase activity is not induced by isosafrole, we cannot conveniently study aryl hydrocarbon hydroxylase induction in this experiment. Whereas five Ahb/Ahd and all five Ahd/Ahd mice behaved as expected, one Ahb/Ahd mouse appeared to be a recombinant. The data in Fig. 9 provide genetic proof that P2-450 induction by isosafrole is associated with the Ah locus or a closely segregating gene. The fact that one recombinant occurred out of eleven backcross offspring examined ( Fig. 9) suggest that there exists some possible crossover event, or less than perfect association, between P2-450 induction by isosafrole and Ahphenotyping by the zoxazolamine paralysis test.
Association of Ah Receptor with P2-450 Induction by Isosafrole- Fig. 10 is an illustration of the difference in reponse among B6, D2, and B6D2F1 mice between induction of anti-(Pz-450)-precipitable protein by isosafrole and PI-450 induction by 3-methylcholanthrene. The former trait is inherited additively, the latter, dominantly. The results in Figs. 8, 9, and 10 thus demonstrate the involvement of the Ah receptor but indicate some slightly different manner in which the inducer-receptor complex effects its response: P2-450 induction being like UDP glucuronosyltransferase induction and different from PI-450 or P3-450 induction.

DISCUSSION
In this report we have shown the isolation and characterization of a new mouse P-450 protein called P2-450. We have demonstrated that P2-450 is unique via studies of catalytic activity, antibody inhibition of catalytic activities, spectral data, response of the immunoprecipitated protein to various classical P-450 inducers, and genetic expression of its induction among the appropriate crosses between B6 and D2 mice. An antibody's "lack of inhibition of catalytic activity" need not always be correlated with the antibody's "lack of immu- noprecipitation." It is also possible that an antibody might precipitate the protein without blocking its catalytic activity. P2-450 seems highly specific for isosafrole metabolite formation and metabolizes acetanilide and benzo[a]pyrene very poorly. Anti-(Pz-450) blocks the isosafrole metabolite formation virtually 100% and has no effect on acetanilide 4-hydroxylation, benzo[a]pyrene hydroxylation, biphenyl 2-or 4-hydroxylation, or 7-ethoxycoumarin 0-de-ethylation.
The additive inheritance of the induction of anti-(Pz-450)precipitable protein between B6 and D2 mice might be explained on the basis of a P-450 polymorphism between the two inbred strains. Complete nucleotide sequencing of the mRNA from both strains will rule this possibility in or out.
The variable types of inheritance and "induction responses" of P1-450, P2-450, and P3-450 by several polycyclic aromatic compounds may be difficult to resolve by postulating a single species of Ah receptor. It, therefore, appears that there exist at least four groups of polycyclic aromatic compounds which interact in slightly different ways with the Ah receptor to induce P1-450, Pz-450, and P3-450 differentially. First, 3methylcholanthrene, when given at the highest experimentally possible doses, induces P1-450 in B6 but negligibly in D2 mice (36). 3-Methylcholanthrene also induces the Pz-450 immunoprecipitate and P3-450 very well in B6 mice but not D2 mice. P-Naphthoflavone (37) presumably belongs to this 3-methylcholanthrene class. Second, TCDD at low doses induces P1-450 in B6 but not D2 mice yet at high doses induces P1-450 equally well in both B6 and D2 mice (30,36). TCDD also has the same effect on P2-450 (Fig. 7B) and p3-450 (2) induction. Third, isosafrole at low doses induces the anti-(Pz-450)-precipitable protein in B6 but not D2 mice and at high doses induces the immunoprecipitate equally well in both B6 and D2 mice. However, isosafrole a t high doses does not induce P1-450 and induces acetanilide 4-hydroxylase activity in B6 but not D2 mice. Fourth, benzo[a]anthracene is an excellent inducer of PI-450 mRNA in B6 but not D2 mice (17) but induces the Pz-450 immunoprecipitate very poorly in B6 mice.
In terms of classical receptor studies, when an inducerreceptor complex forms and interacts with an intranuclear target, one specific response is elicited. If a different response is elicited with a second inducer, this suggests a distinctly different inducer-receptor complex. Therefore, it seems impossible that any two of the above-mentioned four classes of compounds are acting precisely in the same manner.
One possible explanation is that the Ah receptor population is heterogeneous (31). The other possibility is that a single type of Ah receptor molecule exists but that each of several inducers allosterically produces a somewhat different structure of the inducer-receptor complex, thereby exerting different signals during the activation of the three P-450 genes, plus one or more UDP-glucuronosyltransferase(s). Further studies with these four (or more) proteins, and especially their corresponding genes, should shed new light on this perplexing problem.