Genetic association of increases in naphthalene, acetanilide, and biphenyl hydroxylations with inducible aryl hydrocarbon hydroxylase in mice

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

175, 495-506 (1976)

Genetic Association of Increases in Naphthalene, Acetanilide, Biphenyl Hydroxylations with Inducible Aryl Hydrocarbon Hydroxylase in Mice’ STEVEN Developmental

A. ATLAS

AND

DANIEL

W. NEBERT

Pharmacology Branch, National Znstitute of Child Health and Human National Institutes of Health, Bethesda, Maryland 20014 Received

December

and

Development,

22, 1975

The induction of four hepatic monooxygenase activities, naphthalene trans-1,2-dihydrodiol formation, acetanilide 4-hydroxylase, biphenyl 4-hydroxylase, and biphenyl 2hydroxylase, by polycyclic aromatic compounds is genetically associated with the induction of aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity and cytochrome PI-450 in C57BL/6N and DBA/SN inbred mice and among progeny from the appropriate genetic crosses involving these two progenitor strains. These enzyme activities were studied with respect to (i) preferential inhibition by metyrapone, a-naphthoflavone, or Tween 80 in vitro; (ii) use of the microsomal inducers 3-methylcholanthrene, P-naphthoflavone, 2,3,7,X-tetrachlorodibenzo-p-dioxin, or phenobarbital; (iii) apparent K, values; and (iv) heat inactivation. Several lines of evidence suggest that aromatic hydrocarbon-induced naphthalene monooxygenase, acetanilide I-hydroxylase, biphenyl 4-hydroxylase, and biphenyl 2-hydroxylase activities are, like the induced aryl hydrocarbon hydroxylase activit.y, associated with cytochrome(s) PI-450 and that the basal activities of the first three of these enzymes are, like basal aryl hydrocarbon hydroxylase activity, associated with one (or more) forms of cytochrome P-450 other than cytochrome(s) P,-450. On the other hand, the basal biphenyl 2-hydroxylase activity in mouse liver appears to be associated solely with cytochrome(s) PI-450; this finding differs from all other basal monooxygenase activities associated with the Ah locus and studied thus far in a similar manner.

The inducibility of aryl hydrocarbon (benzo[alpyrene) hydroxylase by polycyclic aromatic compounds exhibits genetic variation in the liver (l-3) and other organs of inbred strains of mice (l-5). The genetic regulation of such induction in mice probably involves several alleles at more than one genetic locus (3-5); however, the difference between two prototype strains, C57BL/6 being “responsive” to AHIP induction by the polycyclic aromatic 1 Portions of this work were presented at the annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.J., April, 1975 [(1975) Fed. Proc. 34, 7551, and at the Fourth European Workshop on Drug Metabolism, Mainz, Germany, September, 1974. 2 The abbreviations used are: AHH, aryl hydrocarbon hydroxylase; MC, 3-methylcholanthrene;

compounds MC or BNF and DBA/2 being “nonresponsive,” is explained almost completely by a single gene difference obeying classical Mendelian segregation, the responsive allele being dominant (l-5). This induction process is genetically associated (1, 3, 5) with the formation of cytochrome P,-450, or P-448, which is distinguished from the cytochrome P-450 in untreated control animals by certain spectral characteristics (1, 6-8) and genetically controlled changes in the spin-state of the heme iron (9, 10). A variety of other inducible hepatic monooxygenase activities are closely assoBNF, /3-naphthoflavone; and TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; B6, the responsive inbred C57BL/6N strain; and D2, the nonresponsive DBA/ 2N inbred strain. 495

Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

496

ATLAS

AND

ciated with induced AHH at the so-called aromatic hydrocarbon responsiveness or Ah locus (5) in the mouse: p-nitroanisole 0-demethylase (ll), 7-ethoxycoumarin Odeethylase (ll), 3-methyl-4-methylaminoazobenzene N-demethylase (ll), zoxazolamine hydroxylase (12), 2-acetylaminofluorene N-hydroxylase (13) and, based on preliminary information, phenacetin Odeethylase (14). Despite the heterogeneity of these reactions and the structural dissimilarity of the substrates involved, their association with the Ah locus is, in some manner, specific, since there are other MC-inducible, cytochrome P-450-mediated monooxygenase activities which are not similarly associated in mice (11, 12). The administration of TCDD, an inducer more potent than MC or BNF, results in full expression of cytochrome P,450 formation and induction of each associated activity examined in nonresponsive as well as responsive strains of mice (4, 5, 13, 15-17). This suggests that nonresponsive mice possess the requisite regulatory and structural genes but fail to respond to MC or BNF at any dose (4,15) because of a faulty “receptor” mechanism (5, 17). Associated enzymatic activities are also induced in both responsive and nonresponsive strains by phenobarbital treatment (1, 5, ll-13), but the mechanism of induction is different (16). Whereas induction by phenobarbital is not principally associated with the formation of cytochrome PI-450 by spectral criteria (l), there is evidence by in vitro inhibitor studies (18) and by electrophoresis (16) that phenobarbital does induce a small amount of new cytochrome P,-450 in the mouse. In this report, we show that increases in four additional drug-metabolizing enzyme activities, all involving ring hydroxylations, are regulated at or near this same Ah locus. We also present evidence which suggests that one of these activities, biphenyl 2-hydroxylase, may be associated solely with the MC-inducible “form” of AHH activity and, therefore, presumably cytochrome PI-450. MATERIALS

AND

METHODS

Biphenyl, 4-hydroxybiphenyl (p-phenylphenol), 2-hydroxybiphenyl (o-phenylphenol), and MC were

NEBERT obtained from Eastman Kodak Company (Rochester, N.Y.). Naphthalene, acetanilide, p-hydroxyacetanilide, a-naphthoflavone, and BNF were purchased from Aldrich Chemical Company (Milwaukee, Wis.); benzolalpyrene, bovine serum albumin, and NADH from Calbiochem (LaJolla, Calif.); and sodium phenobarbital from Merck and Company, Inc. (Rahway, N.J.). [l-*4C]naphthalene (5.1 mCi/ mmol), from Amersham-Searle Corporation (Arlington Heights, Ill.) and uniformly labeled (ring) [14C]acetanilide (2.13 mCi/mmol), from Mallinckrodt (St. Louis, MO.), were diluted with nonradioactive substrates, resulting in specific activities of 2.0 and 0.5 mCi/mmol, respectively. Liquifluor was purchased from New England Nuclear (Boston, Mass.). We deeply appreciate the following generous gifts: metyrapone (2-methyl-1,2,3,3-pyridyl-I-propanone) from J. J. Chart, Ciba Pharmaceutical Company, Summit, N.J.; TCDD from Dr. Alan P. Poland, University of Rochester, Rochester, N.Y.; and naphthalene-truns-1,2-dihydrodiol from Dr. Donald M. Jerina, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Md. All other chemicals and solvents were of the highest grade available. Inbred strains of mice were obtained from the National Institutes of Health Veterinary Resources Branch. Treatment of animals. Animal room conditions and the preparation of liver microsomes have been described previously (1). MC- or BNF-treated mice received an intraperitoneal injection of 80 mg/kg of the inducer in corn oil 40 h prior to sacrifice. TCDD (100 pg/kg) was administered intraperitoneally inpdioxane 72 h before sacrifice, and phenobarbital was given intraperitoneally (60 mg/kg) in 0.85% NaCl for 4 successive days, the first day’s dose being divided. Enzyme assays. Biphenyl hydroxylation was measured by a modification of the method of Creaven et al. (19). The l-ml reaction mixture contained 50 pm01 of Tris-chloride buffer, 3 pmol of MgCl,, 1.2 pmol of NADPH, 0.9 pmol of NADH, 0.7 mg of bovine serum albumin, and between 0.75 and 1.25 mg of microsomal protein, final pH 8.5. Biphenyl was dissolved in a minimal volume of benzene3 (0.5 mg/pl) and diluted with methanol to a 3 Benzene, present in the final reaction mixture at a concentration of 6 PM, is probably a substrate for a microsomal monooxygenase (20). The theoretical product, phenol, although having a fluorescent spectrum similar to that of 4-hydroxybiphenyl (282nm excitation, 328-nm emission) at pH 5.5, is approximately 1000 times less fluorescent than the 4hydroxybiphenyl, and there is no detectable contribution of benzene hydroxylation to the fluorescence measured in this assay. Furthermore, results obtained are no different when biphenyl is first dissolved in dimethylsulfoxide (final concentration 0.05 M) instead of benzene.

GENETICS

OF MOUSE

LIVER

concentration of 50 mM; the reaction was started by the addition of 40 ~1 of this solution containing 2 pmol of biphenyl. Incubations were carried out in a shaker bath at 37°C for 15 min and the reaction was stopped by the addition of 2 N HCl (0.5 ml) on ice. After n-heptane (4 ml) had been added, the incubation flasks were returned to the 37°C water bath for 10 min and were then further extracted manually (30 s) in conical tubes. Aliquots (3 ml) of the organic phase were extracted for 30 s with 0.1 N NaOH (4 ml), and 2 ml of the alkali extracts were buffered with 0.5 ml of 9.25 M succinic acid (final pH 5.5) and were allowed to equilibrate in the dark for 20 min. Samples were read in an Aminco-Bowman spectrophotofluorimeter, first at 278~nm excitation and 338nm emission, then at 290-nm excitation and 415-nm emission. After background fluorescence (microsomes added after stopping the reaction), was subtracted, the amounts of 4-hydroxybiphenyl and 2hydroxybiphenyl formed were calculated according to Creaven et al (191, using standard curves obtained by carrying product standards through the entire assay procedure. Under these optimal conditions, the assay for both activities was linear for 20 min and with up to 1.5 mg of microsomal protein. Acetanilide 4-hydroxylase was assayed according to the method of Daly (211, and naphthalene-trans1,2-dihydiodiol formation according to that of Oesch and Daly (22), with slight modifications. Incubation mixtures for both assays contained 40 pmol of Trischloride buffer, 0.6 Fmol of NADP, 7 pmol of glucose 6-phosphate, 0.1 unit of glucose-6-phosphate dehydrogenase and 2 to 4 mg of microsomal protein in a volume of 0.8 ml, pH 9.0. Incubations were carried out at 37°C for 20 min following the addition of substrate (2.0 p.mol of P4Clacetanilide or 0.5 pmol of [Wlnaphthalene) in 50 ~1 of acetonitrile. Reactions were stopped by adding 0.1 ml of 5 N HZSOa and placing the samples on ice. The final pH of the terminated rea.ction mixtures was 4.2. Although some instability of the naphthalene dihydrodiol product is possible under these conditions, calculated activities were identical with those when the reaction was stopped simply by placing the samples on ice without the addition of acid. Following the addition of 1 /bmol of nonradioactive product (4hydroxyacetanilide or naphthalene-truns-1,2-dihydrodiol), the reaction mixtures were saturated with NaCl and extracted with ether (5 ml). The extracts were dried over Na&SO, and evaporated. Residues were dissolved in a small volume of methanol and chromatographed on silica gel plates (Quantachloroform:benzene:ethyl aceGram) using tate: methanol (60:10:10:5). R, values were 0.49 and 0.18 for acetanilide and its 4-hydroxylated metabolite, respectivel,y; and 0.95 and 0.29 for naphthalene and the dihydrodiol product, respectively. Product bands were visualized under ultraviolet light, scraped, and extracted with 2 ml of methanol. Ali-

ARYL

497

HYDROXYLATIONS

quots (1 ml) were counted in 10 ml of Liquifluor using a Packard Tri-Carb scintillation spectrometer; the remainder of the methanol extracts were diluted, and recoveries were determined by ultraviolet spectroscopy at 260 nm. Counts were corrected for recoveries (27 + 3% for 4-hydroxyacetanilide and 36 -C 8% for the naphthalene dihydrodiol) and were converted to nanomoles of product formed. Aryl hydrocarbon (benzolalpyrene) hydroxylase activities were measured as previously described (1, 51, and microsomal protein was determined by slight modification of the method of Lowry et al. (23); bovine serum albumin was used as the standard. All enzyme specific activities are the mean values of duplicate determinations from at least two experiments and are expressed in this report as picomoles of product formed per minute per milligram of microsomal protein. RESULTS

Genetic Expression Hydroxylases

of Induced

Biphenyl

Both biphenyl hydroxylase activities were inducible by MC in responsive B6 mice (Fig. 1). The 4-hydroxylase was induced two- to threefold, whereas the 2hydroxylase, having lower basal levels than the 4-hydroxylase, was induced fourto fivefold. Activities in the MC-treated nonresponsive D2 mice were, on the other hand, not significantly different from those in control B6 or D2 mice. The distribution of biphenyl 4-hydroxylase and AHH activities in MC-treated progeny of various crosses between B6 and D2 parents (Fig. 2A) reveals similar patterns for both

I

I 0

loccl

2wo

3cal

I 0

-

I loo0

2wo

3m

SPECIFIC AHH ACTIVITY

FIG. 1. Relationship of AHH and biphenyl hydroxylase activities in individual inbred mice. Circles represent B6 mice and triangles D2 mice; closed symbols indicate MC-treated animals, and open symbols indicate controls (corn oil-treated). Each point represents the mean of duplicate determinations of each enzymatic activity for a single animal. In this and subsequent figures, specific activities are expressed as picomoles of product formed per minute per milligram of microsomal protein.

ATLAS

498

P 0 e $ MKX)186D21D2 2 5 . v; .: g Km1 7 Y = zcm-? ?i + 0

Too0

wm2,F,

AND

,. ,p

a* -$

3wo 0 Km zcol 3mo SPEClFlC At+ACTIVITY

zcmo

NEBERT

z 2 0 ? :, Gc)(yJ ,Bem,D2 _ IBmw, ,; . 5 ,4** ... -. . g 4cco** . : p M 2ma l F 0 Iwo mea 3ow0 ICCQ*cc0 3ml SPECIFIC At+ACTl”lTY

FIG. 2. (A), Relationship of AHH and biphenyl4-hydroxylase activities among offspring of appropriate genetic crosses involving the B6 and D2 progenitor strains. All animals were pretreated with MC, and each symbol represents the mean of duplicate determinations on a single animal of the indicated cross. (B), Relationship of AHH and biphenyl 2-hydroxylase activities in these same offspring.

enzymatic activities. All F, progeny were induced, as were the backcross of the F, to the responsive B6 parent. The offspring from the backcross to the nonresponsive D2 parent and the F, generation both showed a bimodal distribution, with 42% induced in the former and 80% induced in the latter. These distributions are quite close to those expected (50% responsive for the backcross and 75% responsive for the F, generation) for a single autosomal dominant allelic difference between the B6 and D2 strains, as has been shown (1, 2) for AHH alone on a larger series of animals. Control animals of each cross (not shown) all have activities not significantly different from the control parent strains or MCtreated nonresponsive animals. Figure 2B shows similar genetic linkage for biphenyl 2-hydroxylase and AHH activities in the same animals as shown in Fig. 2A. The distributions were identical, and an even better correlation between the two enzymatic activities was apparent. Genetic Expression of Induced lene Dihydrodiol Formation anilide Hydroxylase

Naphthaand Acet-

Naphthalene trans-1,2-dihydrodiol formation is the combination of a monooxygenation by one (or more) forms of cytochrome P-450 and hydration of the arene

oxide intermediate by microsomal epoxide hydratase (24). Because MC treatment of B6 and D2 mice does not induce the hydratase (25), however, we feel that any increases in naphthalene trans-1,2-dihydrodiol formation found in this report represent increases in the P-450-mediated monooxygenation reaction.4 Figure 3 illustrates that both naphthalene trans-1,Z dihydrodiol formation and acetanilide 4hydroxylase activity were similarly inducible in MC-treated B6 mice but not in MCtreated D2 mice. All F, progeny were inducible by MC. The relationships between either of these activities and AHH activity in MC-treated progeny from the (B6D2)F, x D2 backcross and in MC-treated F, offspring are demonstrated in Figs. 4A and B, respectively. Although the number of 4 This study (25) used styrene oxide as substrate for the hydratase. Although not specifically studied in mice, both naphthalene oxide and styrene oxide appear to be substrates for the same hydratase activity in both guinea pigs and rats (24). Naphthalene dihydrodiol was the major metabolite of naphthalene from B6 and D2 microsomes; 1-naphthol, the spontaneous rearrangement product, represented only 12 and 9% of total metabolites from control and MC-induced microsomes, respectively. Although the dihydrodiol may arise from a tightly coupled monooxygenase-hydra&e system following MC treatment (22, 241, arene oxide formation is probably the rate-limiting step in this sequence of reactions.

GENETICS

OF MOUSE

LIVER

Fz animals studied was too small to confirm the expected ratio, genetic linkage is supported in both cases by the absence of recombinants; that is, there were no aniA. lax k

r. 8-

I -B6

D2

tB6D2)F,

0B6

D2 (B6D2)F,

FIG. 3. Distribution of naphthalene truns-1,2dihydrodiol formation (A) and acetanilide 4-hydroxylase (B) activities in B6, D2, and (B6D2)F, mice. As previously, open symbols represent corn oil-treated controls and closed symbols MC-treated animals.

ARYL

499

HYDROXYLATIONS

mals which were induced for one activity but not for the other. Induction of These Four Monoowgenases -by Other inducers The effects of various inducers on these monooxygenase activities are compared in Table I. It was previously shown (26) that BNF is an inducer equivalent to MC in terms of eliciting genetic specificity of aromatic hydrocarbon responsiveness in inbred mice. This was found also to be the case for the biphenyl hydroxylases, because they were induced by BNF (to levels comparable to those induced by MC) in B6 but not in D2 mice. TCDD is capable of fully expressing induction of cytochrome P,-450-related enzymatic activities in nonresponsive as well as responsive mice (15). This was also true for the four enzymatic

FIG. 4. Relationship between AHH activity and (Al rate of naphthalene truns-1,2-dihydrodiol formation or (B) acetanilide 4-hydroxylase activities in MC-treated (B6D2)D2 and (B6D2)F, progeny. TABLE I EFFECT OF VARIOUS INDUCERS ON HEPATIC MONOOXYGENASE ACTIVITIES IN B6 AND D2 MICE Strain

Inducer”

B6

None MC BNF TCDD PB

520b 1270 k 93

78b 580 k 45

13906 940 '- 74

670b 240 -r- 44

None MC BNF TCDD PB

600b 580 + 48

1040 98 t 9

1510b 1050 r 81

620b 280 L 22

D2

-

Naphthalene truns-l,t-dihydrodiol formation

Acetanilide 4-hydroxylase

Biphenyl

4-hydroxylase

Biphenyl2-hydroxylase

2580 4930 4860 5240 5310

? 4 -e + k

120 190 220 160 300

1050 4580 5050 4990 1160

k f + L r

70 140 170 210 1Off

2330 2210 2410 5070 5100

4 2 ? 2 2

130 60 170 240 380

1060 940 1120 5120 1200

k + ? + +

110 80 80 190 8W

a Treatment of animals with inducers is described under Materials and Methods. All activities are expressed as picomoles of product formed per minute per milligram of microsomal protein. IJnless otherwise indicated, values represent the means of three to eight determinations and are expressed as means t SE. b Means of duplicate determinations on pooled livers from four animals. ’ Not significantly different from corresponding controls (P > 0.05).

500

ATLAS

AND

NEBERT

previously shown for AHH (18). Since metyrapone is known to enhance epoxide hydratase activity in vitro (24), the observed inhibition of naphthalene dihydrodiol formation by metyrapone (Fig. 5A) supports our contention that arene oxide formation, and not epoxide hydration, is the ratelimiting step in this pathway. The data with a-naphthoflavone (Fig. 5B) are opposite from the metyrapone results: The activities from MC-treated B6 mice were more sensitive to inhibition by o-naphthoflavone in vitro than the activities from Preferential Inhibition by Metyrapone, (Y- control B6 or D2 or MC-treated D2 mice. Naphthoflavone, or Tween 80 in Vitro The response of the basal and induced biphenyl 2-hydroxylase activities to either The increased sensitivity of induced AHH activity in vitro to inhibition by CY- test compound in vitro appears to represent a special case. Both control and innaphthoflavone and the increased resistance of induced AHH activity in uitro to duced forms were resistant to metyrapone inhibition, and both were sensitive to LYinhibition by metyrapone occur in MCinhibition. Also, this actreated B6 (18) and TCDD-treated B6 or naphthoflavone tivity was far more sensitive to cw-naphD2 (15) but not in MC-treated D2 mice thoflavone inhibition than were the other (18). This same sensitivity or resistance three enzymes studied in this report.6 was seen for three other inducible and Such preferential inhibition of monooxybasal monooxygenase activities associated with the Ah locus (11). Accordingly, it is genase activities is not limited to hydrophobic substrates known to interact with felt (18) that a-naphthoflavone interacts cytochromes P-450. Figure 6 shows that with greater affinity toward cytochrome the nonionic detergent Tween 80, which PI-450, whereas metyrapone interacts has been used (19) to solubilize substrate with greater affinity toward another form in the assay for biphenyl hydroxylase, of cytochrome P-450. enhanced AHH and biFigure 5A demonstrates the in vitro ef- preferentially phenyl 4-hydroxylase activities from MCfect of metyrapone on the four monooxytreated B6 mice, as compared with those in genase activities studied in this report. either control mice or MC-treated D2 mice. The three activities other than biphenyl2hydroxylase all showed varying degrees of AHH activity was inhibited in the control samples, whereas biphenyl 4-hydroxylase inhibition by metyrapone; in each of these activity was not affected. There was, on three cases, there was relative resistance of the activity from MC-treated B6 mice to the other hand, considerable enhancement 2-hydroxylase activity from inhibition by metyrapone, as had been of biphenyl both control and MC-treated mice. Thus, the use of Tween 80 to solubilize substrates 5 Using rat microsomes, Oesch and Daly found for monooxygenase activities may lead to (22) that the rate of naphthalene dihydrodiol formaspurious results. tion from the 1,Zoxide is induced threefold by pheThe extreme sensitivity of the biphenyl nobarbital, whereas that from naphthalene is inhydroxylases to inhibition by less than 10 duced only twofold; the rate of dihydrodiol formation activities studied here, which were maximally induced in D2 as well as B6 mice. Phenobarbital is also capable of inducing monooxygenase activities in all strains of mice, but the mechanism of induction differs from that by MC, BNF, or TCDD (1, 5). All enzymatic activities studied in this report were induced to variable degrees by phenobarbita15, except for biphenyl 2-hydroxylase, which was not significantly increased above control levels in either strain.

from the 1,2-oxide is induced only 50% by MC, yet that from naphthalene is induced sixfold. Hence, although phenobarbital does induce the epoxide hydratase (22, 251, naphthalene oxide formation is likely to be the rate-limiting step. We therefore believe that the control and induced activities of dihydrodiol formation shown in Table I are an accurate reflection of naphthalene monooxygenation.

’ Although maximally inhibited at a concentration of 50 FM a-naphthoflavone, the 2-hydroxylase activity from MC-treated B6 mice is even somewhat less sensitive than that from MC-treated D2 or control mice at lower a-naphthoflavone concentrations. This phenomenon, though unexplained, was observed consistently in three separate experiments.

GENETICS

OF MOUSE

NAPHTHALENE TRANS.I, Z-DIHYDRODIOL FORMATION

LIVER

ARYL

ACETANILIDE 4-HYDROXYLASE

1w

loo0

501

HYDROXYLATIONS

BIPHENYL 4.HYDROXYLASE

EIPHENYL Z-HYDROXYLASE

10

10

METYRAPONE.

loo

1000

100

1000

&,

B ACETANILIDE 4-HYDROXYLASE

a-NAPHTHOFLAVONE. PM

FIG. 5. In vitro effect of metyrapone (A) or a-naphthoflavone (B) on the four hepatic monooxygenase activities from control B6 (01, MC-treated B6 (O), control D2 (A), and MCtreated D2 (A) mice. The procedure was performed as described previously (18). Following the addition of metyrapone, in 10 ~1 of ethanol, or cY-naphthoflavone, in 10 ~1 of acetone, to incubation mixtures containing microsomal protein, samples were incubated for 1 min at 37°C prior to addition of substrate. Activities are expressed as percentages of control samples containing 1% ethanol or 1% acetone alone. Each point represents the mean of duplicate determinations. A difference of 20% is considered to be statistically (P < 0.05) significant. FM a-naphthoflavone, despite a substrate concentration of 2 mnq suggests either that a-naphthoflavone and biphenyl have vastly different affinities for a common site or that inhibition is not competitive. As seen in Fig. 7, the latter appears to be the case. Inhibition of both activities by crnaphthoflavone fits the pattern of noncompetitive inhibition; by plotting l/u versus the inhibitor concentration separately for each substrate concentration (data not shown), we have estimated the apparent Ki for MC-induced biphenyl4-hydroxylase and biphenyl2-hydroxylase activities with a-naphthoflavone to be approximately 25 and 12 PM, respectively. The differences between the biphenyl4hydroxylase and biphenyl 2-hydroxylase activities in response to metyrapone, (Ynaphthoflavone, or Tween 80 in vitro suggest that the two specific hydroxylations are catalyzed, at least in part, by different

enzyme active-sites. This suggestion is consistent with our observation (Table I) that phenobarbital induces the 4-hydroxylase but not the 2-hydroxylase activity. The data in Table II, showing differences in the apparent Michaelis constants for the two activities, may support this contention. In particular, there was a small change in the apparent K, value for the 4-hydroxylase in MC-treated B6 mice, whereas the apparent K, value for the 2hydroxylase remained the same. Sensitivity Heat

of Biphenyl

Hydroxylases

to

There were no significant differences between control and MC-induced samples in the heat inactivation as a function of time at 55°C of either activity. However, both control and induced biphenyl 4-hydroxylase activities were somewhat less heat sensitive than the 2-hydroxylase, and

ATLAS

502

AND

NEBERT DISCUSSION

there appeared to be a fraction (approximately 10%) of biphenyl 4-hydroxylase which was relatively heat stable. Although the thermolability profiles (from 48 to 58°C) were fairly similar for the two activities, there was a slight difference in the temperature required to produce 50% inactivation: 52.5”C for the 4-hydroxylase and 51.5”C for the 2-hydroxylase. AHH

We have shown here (Figs. l-4) that the capacity for induction of four additional monooxygenase activities by polycyclic aromatic compounds segregates as a single autosomal dominant gene in the progeny of crosses between the B6 and D2 progenitor strains. The expression of this induction is apparently controlled at the same BIPHENYL BIPHENYL locus or genetic cluster responsible for 2.HYDROXYLASE 4.HYDROXYLASE AHH induction and cvtochrome PI-450 formation in mice.

0 02 04 06 0 02 04 06 0 02 04 06 TWEEN-8c ,%, FIG. 6. Effect of Tween 80 on AHH and biphenyl hydroxylase activities in hepatic microsomes from control B6 (O), MC-treated B6 (O), and MC-treated D2 (A) mice. Appropriate concentrations of Tween 80 were obtained by dilutions of 0.2 M Tris-chloride buffer containing 4% Tween 80 (pH 7.2 for AHH, pH 8.5 for biphenyl hydroxylases) which were then used to make up the incubation mixtures. All samples were preincubated for 1 min at 37°C prior to adding substrate, and activities are expressed as percentages of control (i.e., no Tween 80). The effect of Tween 80 on these activities from control D2 mice (data not illustrated) was the same as that from control B6 or MC-treated D2 mice. TABLE

FIG. 7. Lineweaver-Burk plots of biphenyl4-hydroxylase (A) and biphenyl2-hydroxylase (B) activities in the absence (0) or presence of a-naphthoflavone: (A), 1 mM; (m), 20 mM; (+), 100 m&Q. Microsomes were pooled from the livers of three MCtreated B6 mice and all samples were preincubated for 1 min at 37°C as previously. Points represent the means of duplicate determinations from a typical experiment. II

EFFECT OF MC OR PHENOBARBITAL PRETREATMENT ON THE KINETICS OF HEPATIC BIPHENYL HYDROX~ASE ACTIVITIES IN B6 AND D2 MICE Biphenyl 2-hydroxylase Strain Pretreatment? Biphenyl 4-hydroxylase K, (mM)

V (pm01 min-’

mn-‘1

K,

(mM)

V (pm01 min-’

mg-1)

B6

None MC PhenobarbitaP

0.19 2 0.02 0.41 s 0.03 0.19

2820 k 110 5780 k 260 4870

0.14 ? 0.01 0.17 + 0.02 0.16

1150 + 55 5240 ? 180 1200

D2

Noneb MC Phenobarbital*

0.18 0.20 ” 0.02 0.21

2580 2540 k 67 5080

0.15 0.15 L 0.01 0.18

1140 1230 + 92 1310

” Treatment of animals with inducers is described under Materials and Methods. Maximal velocities fV) and apparent Michaelis constants (K,) were derived from double reciprocal plots of velocity (picomoles per minute per milligram of microsomal protein1 for at least five substrate concentrations (0.1-4.0 mM biphenyl). Each determination was performed on pooled microsomes from livers of three appropriately treated animals. Unless otherwise indicated, values represent the means 2 SE of three or four determinations. b Values for these determinations are the means of two experiments.

GENETICS

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It was previously reported (27) that one of these hepatic activities, biphenyl 4-hydroxylase, is not inducible by MC7 in rats. A more recent report (28) indicated that TCDD does induce hepatic biphenyl 4-hydroxylase in the rat. These separate findings led to the conclusion (28) that MC and TCDD, both of which induce new cytochrome PI-450 formation (15-171, are not identical as inducers of microsomal monooxygenase activities. However, we have shown (Table I) that this biphenyl 4-hydroxylase activity is induced approximately twofold by either MC or TCDD in genetically responsive mice. We finds a similar magnitude of induction of biphenyl 4-hydroxylase by MC and TCDD in Sprague-Dawley rats. Moreover, there is no evidence that TCDD and MC differ qualitatively as inducers of other cytochrome P,-450-associated monooxygenase activities in responsive inbred mice (5, 13, 15-17). The concept has emerged that treatment of animals with MC, BNF, or TCDD causes the “formation” of cytochrome PI450. Electrophoresis of solubilized microsomes from several species (16, 29-31) suggests instead that such treatment causes increases in a polypeptide (P-450 apoprotein) band which is already present in control microsomes. Phenobarbital treatment principally causes increases in a different preexisting apoprotein in the mouse (16) and rat (31). It therefore seems likely that basal monooxygenase activities may be associated with any one (or more) of the several existing apoprotein bands and that induced monooxygenase activities may be associated with any one (or more) of the bands which increase in size following treatment with a particular inducer. Several lines of indirect evidence (5, 9, 16, 18) indicate that this is the case for hepatic AHH activities. Metyrapone, ’ These authors (27) found that, whereas 20methylcholanthrene, i.e., 3-methylcholanthrene by the commonly accepted nomenclature, did not induce the 4-hydroxylase activity, 22-methylcholanthrene, i.e., 5-methylcholanthrene, was a good inducer. * S. A. Atlas, S. S. Thorgeirsson, and D. W. Nebert, unpublished data.

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which preferentially inhibits AHH activity in microsomes from control or phenobarbital-treated mice (181, appears to inhibit AHH activity associated with the phenobarbital-inducible form(s) of cytochrome P-450, which predominate(s) (16) in control microsomes. a-Naphthoflavone, on the other hand, preferentially inhibits MC-induced AHH activity (18); it therefore seems likely that this compound selectively inhibits the MC-inducible form(s)s of cytochrome. Similar patterns of preferential inhibition have been demonstrated in this report for basal and MC-induced naphthalene trans-1,2-dihydrodiol formation, acetanilide 4-hydroxylase, and biphenyl 4-hydroxylase activities, suggesting that these basal and MC-inducible “activities” similarly reflect product formation at two (or more) distinct enzyme active-sites. Differences in the degree of deuterium retention when [4-2H]acetanilide was metabolized by liver microsomes from control and MC- or phenobarbital-treated rats indicated several years ago (32) that basal and induced acetanilide hydroxylase activities may represent product formation at two (or more) active sites. On the other hand, biphenyl2-hydroxylase activity appears to be unique. Neither the basal nor the MC-induced activity is inhibited by metyrapone, and both the basal and MC-induced activities are inhibited by a-naphthoflavone (Fig. 5). These findings suggest that hepatic biphenyl2-hydroxylase activity is associated solely with an MC-inducible, cr-naphthoflavone-sensitive form of cytochrome P-450 (presumably P,-450) in both control and MC-treated mice. The absence of signiiicant induction of this activity in the mouse by phenobarbital (Table I), which has been previously reported for the rat (271, also supports this hypothesis.‘O We cannot be 9 Recent evidence (J. S. Felton and D. W. Nebert, manuscript in preparation) indicates that the “band” increased by MC is itself heterogeneous, perhaps representing multiple MC-inducible forms of cytochrome P-450. lo Phenobarbital treatment actually causes slight, but detectable, increases in “band 4” (felt to be associated with P,-450) in the mouse (16). Although the data (Table I) may suggest slight (i.e.,

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certain that control and induced biphenyl 2-hydroxylase activity is associated with precisely the same mouse hepatic cytochrome as MC-induced AHH activity; however, in control and MC-treated adult rabbits, in which AHH activity is present but not induced by MC (331, no significant biphenyl2-hydroxylase activity is measurable. A recent report with the reconstituted P-450 system from rat liver (34) demonstrates a similar distinction between the biphenyl4- and 2-hydroxylase activities. It has been suggested (5, 35, 36) that cytochromes PI-450 and forms other than PI-450 differ in their lipid environments in the microsomal membrane. The results shown in Fig. 6 are consistent with the idea that Tween 80, at low concentrations, preferentially enhances cytochrome P,450-associated activities, i.e., AHH and biphenyl 4-hydroxylase from MC-treated B6 mice and biphenyl 2-hydroxylase from both control and MC-treated mice. ‘I The similarity of the apparent K, values for biphenyl2-hydroxylase in control, MCtreated, and phenobarbital-treated mice also supports the notion that the nature of the catalytic site is similar in all three cases. The small change in the apparent K, value for the 4-hydroxylase in MCtreated B6 mice, but not in phenobarbitaltreated mice (Table II), suggests instead that a change in the predominant form of cytochrome P-450 associated with the enzyme activity may occur after MC treatment but not after phenobarbital treatment. An even more striking example of this was found in TCDD-treated rats by Hook and co-workers (281, leading them to a similar conclusion. Our results differ in that the apparent K, value for the 2-hyabout 10%) induction of biphenyl 2-hydroxylase activity by phenobarbital, it is not statistically significant. More recent electrophoretic studies (see footnote 9) suggest that there are at least two bands increased by MC treatment, one of which is also increased by phenobarbital treatment and one of which is not. ‘I Other authors have reported inhibition of biphenyl 4-hydroxylase by Tween 80 in rabbit liver microsomes (37) but at higher concentrations than those used in the present study.

NEBERT

droxylase is lower than that for the 4-hydroxylase and that, even so, the K,n for the MC-induced 4-hydroxylase increases. Perhaps, at least in the control and MCtreated mouse, the cytochrome P,-450-associated active site has a steric preference for hydroxylation of biphenyl in the 2-position. It thus appears that, in control mouse liver microsomes, biphenyl is para-hydroxylated predominantly at an active site associated with a phenobarbital-inducible form of P-450 and is ortho-hydroxylated entirely at an active site associated with an MC-inducible form, i.e., PI-450. Following MC treatment of the responsive mouse, a greater proportion of para-hydroxylation occurs in association with an MC-inducible, a-naphthoflavone-sensitive form of cytochrome, which might be identical to the form associated with orthohydroxylation. The higher apparent Ki value for the MC-induced 4-hydroxylase with a-naphthoflavone might further reflect the fact that, even following induction by MC, a significant proportion of this activity occurs at an c+naphthoflavone-resistant active site. The activation of certain chemical carcinogens to cause tumors in uivo (38, 39) and mutagenesis in vitro (40) is associated with the genetically determined induction of AHH and cytochrome P,-450 in mice. These associations are not simply due to increases in AHH activity, since phenobarbital treatment cannot mimic the effect of MC treatment; instead, such associations are probably related to differences in the profiles of metabolites (41-43) and of metabolically activated intermediates covalently bound to DNA (44-46) mediated preferentially by cytochrome PI-450. In this regard, biphenyl 2-hydroxylase activity may prove useful as a marker for cytochrome PI-450 associated with MC-induced AHH activity; in our laboratory this now appears to be the case for the mouse, rat, and rabbit. The idea stated some years ago by Creaven and Parke (27), that carcinogens are inducers of biphenyl 2-hydroxylase while noncarcinogens are not, is an overstatement since the noncarcinogen /3naphthoflavone is very similar to the car-

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cinogen MC as an effective inducer. However, it may prove true that inducers of biphenyl 2-hydroxylase activity enhance tumorigenesis because they induce cytochrome PI-450. ACKNOWLEDGMENT The advice and criticisms of Dr. John W. Daly during the course of this project are sincerely appreciated. REFERENCES 1. GIELEN, J. E., GOUJON, F. M., AND NEBERT, D. W. (1972) J. Biol. Chem. 247, 11251137. 2. THOMAS, P. E., KOURI, R. E., AND HUTTON, J. J. (1972) B&hem. Genet. 6, 157-168. 3. ROBINSON, J. R., CONSIDINE, N., AND NEBERT, D. W. (1974) J. Biol. Chem. 249, 5851-5859. 4. NIWA, A., KUMAKI, K., NEBERT, D. W., AND POLAND, A. P. (1975)Arch. Biochem. Biophys. 166, 559-564. 5. NEBERT, D. W., ROBINSON, J. R., NIWA, A., KUMAKI, K., AND POLAND, A. P. (1975) J. Cell. Physiol. 85, 393-414. 6. SLADEK, N. E., AND MANNERING, G. J. (1966) Biochem. Biophys. Res. Commun. 24,668-674. 7. ALVARES, A. P., SCHILLING, G., LEVIN, W., AND KUNTZMAN, R. (1967) Biochem. Biophys. Res. Commun. 29, 521-526. 8. JEFCOATE, D. R. E., GAYLOR, J. L., AND CAM ABRESE, R. L. (1969) Biochemistry 8, 34553463. 9. NEBERT, D. W., AND KON, H. (1973) J. Biol. Chem. 248, 169-178. 10. NEBERT, D. W., ROBINSON, J. R., AND KON, H. (1973) J. Biol. Chem. 248, 7637-7647. 11. NEBERT, D. W., CONSIDINE, N., AND OWENS, I. S. (1973) Arch. Biochem. Biophys. 157, 148159. 12. ROBINSON, J. R., AND NEBERT, D. W. (1974) Mol. Pharmacol. 10, 484-493. 13. THORGEIR.MON, S. EL, FELTON, J. S., AND NEBERT, D. W. (1975) Mol. Pharmacol. 11, 159165. 14. POPPERS, I’. J., LEVIN, W., AND CONNEY, A. H. (1974) Pharmacologist 16, 262. 15. POLAND, A. P., GLOVER, E., ROBINSON, J. R., AND NEBERT, D. W. (1974) J. Biol. Chem. 249, 5599-5606. 16. HAUGEN, D. A., COON, M. J., AND NEBERT, D. W. (1976) J. Biol. Chem. 251, 1817-1827. 17. POLAND, A., AND GU)VER, E. (1975) Mol. Pharmucol. 11, 389-398. 16. GOUJON, F. M., NEBERT, D. W., AND GIELEN, J. E. (1972) Mol. Phurmucol. 8, 667-680. 19. CREAVEN, P. J., PARKE, D. V., AND WILLIAMS, R. T. (1965) Biochem. J. 96, 879-885.

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20. WISNIEWSKA-KNYPL, J. M., JABLONSKA, J. K., AND PIOTROWSKI, J. K. (1975) Brit. J. Indust. Med. 32, 42-48. 21. DALY, J. W. (1969) Anal. Biochem. 33, 286-296. 22. OESCH, F., AND DALY, J. (1972) Biochem. Biophys. Res. Commun. 46, 1713-1720. 23. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 24. OESCH, F. (1972) Xenobioticu 3, 305-340. 25. OESCH, F., MORRIS, N., DALY, J. W., GIELEN, J. E., AND NEBERT, D. W. (1973) Mol. Phurmucol. 9, 692-696. 26. NEBERT, D. W., GIELEN, J. E., AND GOUJON, F. M. (1972) Mol. Phurmucol. 8, 651-666. 27. CREAVEN, P. J., AND PARKE, D. V. (1966) Biothem. Phurmucol. 15, 7-16. 28. HOOK, G. E. R., ORTON, R. C., MOORE, J. A., AND LUCIER, G. W. (1975)Biochem. Phurmucol. 24, 335-340. 29. WELT~N, A. F., AND AUST, S. D. (1974) Biochem. Biophys. Res. Commun. 56, 898-906. 30. RYAN, D., Lu, A. Y. H., WEST, S., AND LEVIN, W. (1975) J. Biol. Chem. 250, 2157-2163. 31. HAUGEN, D. A., VAN DER HOEVEN, T. A., AND COON, M. J. (1975) J. BioZ. Chem. 250, 35673570. 32. DALY, J., JERINA, D., FARNSWORTH, J., AND GUROFF, G. (1969) Arch. Biochem. Biophys. 131, 238-244. 33. ATLAS, S. A., THORGEIRSSON, S. S., BOOBIS, A. R., KUMAKI, K., AND NEBERT, D. W. (1975) Biochem. Phurmucol. 24, 2111-2116. 34. BURKE, M. D., AND MAYER, R. T. (1975) Drug Metub. Dispos. 3, 245-253. 35. CHAPLIN, M. D., AND MANNERING, G. J. (1970) Mol. Phurmucol. 6, 631-640. 36. IMAI, Y., AND SIEKEVITZ, P. (1971) Arch. Biothem. Biophys. 144, 143-159. 37. HOOK, G. E. R., BEND, J. R., AND FOUTS, J. R. (1973) Chem. Biol. Interact. 1, 205-222. 38. KOURI, R. E., RATRIE, H., III, AND WHITMIRE, C. E. (1974) Znt. J. Cancer, 13, 714-720. 39. NEBERT, D. W., BENEDICT, W. F., AND KOURI, R. E. (1974) in Chemical Carcinogenesis (Ts’o, P. 0. P., and DiPaolo, J. A., eds.), pp. 271-288, Marcel Dekker, New York. 40. FELT~N, J. S., AND NEBERT, D. W. (1975)5. Biol. Chem. 250, 6769-6778. 41. RASMUSSEN, R. E., AND WANG, I. Y. (1974) Cuncer Res. 34, 2290-2295. 42. SELKIRK, J. K., CROY, R. G., ROLLER, P. P., AND GELBOIN, H. V. (1974) Cancer Res. 34, 34743480. 43. HOLDER, G., YAGI, H., DANSETTE, P., JERINA, D. M., LEVIN, W., Lu, A. Y. H., AND CONNEY, A. H. (1974) PFOC. Nat. Acud. Sci. USA 71,43564360.

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44. KING, H. W. S., THOMPSON, M. H., AND BROOKES, P. (1975) Cancer Res. 34, 1263-1269. 45. SIMS, P., GROVER, P. L., SWAISLAND, A., PAL, K., AND HEWAR, A. (1974) Nature (London) 252, 326-328.

NEBERT 46. NEBERT, D. W., KOURI, R. E., YAGI, H., JERINA, D. M., AND BOOBIS, A. R. (1976) in Active Intermediates: Formation, Toxicity and Inactivation (Snyder, R., Jollow, D., and Gillette, J. R., eds.), Plenum, New York, in press.