THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 249, No. 18, Issue of Sept.ember 25, pp. 5851-6859, 1974

Printed in U.S.A.

Genetic Expression of Aryl Hydrocarbon Hydroxylase Induction . EVIDENCE FOR THE INVOLVEMENT OF OTHER GENETIC LOCI*

(Received for publication, January 10, 1974)

JOSEPH R. ROBINSON, NOREEN CONSIDINE, AND DANIET, W. NEBERT

From the Section on Developmental Pharmacology, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014

SUMMARY

The intraperitoneal administration of aromatic hydrocar- bons such as 3-methylcholanthrene or /3-naphthoflavone stimulates aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity in C57BL/6N, C57BL/6J, C3H/HeN, BALB/cAnN, CBA/HN, and PL/J inbred mice but not in the DBA/ZN, 129/J, RF/J, AKR/N, AKR/J, or AU/SsJ inbred strains. By means of genetic crosses between a “responsive” and a “nonresponsive” strain, the hydroxylase induction appears to segregate as an autosomal dominant trait in the C57BL/6N x RF/J cross, the C57BL/6N x 129/J cross, the C3H/HeN x AKR/N cross, the BALB/cAnN x AKR/N cross, the CBA/HN x AKR/N cross, the BALB/cAnN x DBA/ZN cross, the C57BL/6N x AU/SsJ cross, the PL/J x AU/SsJ cross, the C57BL/6 J x AKR/ J cross, the C57BL/6N x AKR/ J cross, and the C57BL/6J x AKR/N cross. The absence of hydroxylase induction appears to segregate in an autosomal dominant fashion in the C57BL/6N x AKR/N cross. The induction process in offspring from the C3H/HeN x DBA/ZN cross is inherited additively. In mice from many of the above men- tioned crosses, there exists a positive, significant correlation between the induced hepatic hydroxylase activity and (a) new formation of hepatic microsomal cytochrome PI-450; (b) higher inducible hydroxylase activities in lung, kidney, bowel, and skin; and (c) induction of hepatic microsomal 7-ethoxy- coumarin 0-deethylase, p-nitroanisole-0-demethylase, and 3-methyl-4-methylaminoazobenzene N-demethylase activi- ties. We have found no exceptions to this general observa- tion. The basal hydroxylase activity appears to be inherited additively, within the limits of individual variation. From data involving more than 2200 individual mice of these 12 inbred strains, the induction of the monooxygenase activities and the stimulation of cytochrome PI-450 formation by aromatic hydrocarbons appear to be regulated by at least three alleles at each of two nonlinked genetic loci.

* Previous papers in this series have been published (1-8). Por- tions of this work were presented at the Second International Symposium on Microsomes and Drug Oxidations, Stanford, California, July, 1972; at the Meeting of the Federation of the American Society for Experimental Biology, Atlantic City, New Jersey, April, 1973; and at the International Symposium on Drug Interactions, Milan, Italy, September, 1973.

In our initial reports (2, 9, lo), the expression of aryl hydro- carbon hydroxylase’ induction by aromatic hydrocarbon treat- ment in viva was shown to segregate in several tissues as a single autosomal dominant trait in the mouse. This conclusion wm based on appropriate genetic crosses between the “responsive” 2 C57BL/6N inbred strain and the “nonresponsive” DBA/2N, NZW/BLN, or NZB/BLN inbred strain (2). This conclusion has been confirmed and extended in this laboratory (3-8, 11-15) and by others (16-22). The autosomal dominant expression was also reported with the use of appropriate crosses between the responsive C57BL/6J and the nonresponsive DBA/2J strain (16). More recently, additive inheritance was demonstrated for the hydroxylase induction by aromatic hydrocarbons in genetic crosses between the responsive inbred C3H/HeJ and the nonre- sponsive DBA/BJ strain (22). In the present study, involving more than 2200 individual enzyme determinations in liver and in nonhepatic tissues during a 20.month period and appropriate genetic crosses between 12 inbred strains of mice, we present evidence that aryl hydrocarbon hydroxylase induction by aro- matic hydrocarbons is controlled by at least three alleles at each of two nonlinked loci.

EXPERIMENTAL PROCEDURE

Materials

Benzo[a]pyrene, NADPH, NADH, and p-nitrophenol were pur- chased from Sigma (St. Louis, MO.) ; MC? from J. T. Baker Chemi- cal Co. (Milwaukee, Wis.) ; umbelliferone and p-nitroanisole from Aldrich Chemical Co. (Milwaukee, Wis.) ; instrumental grade carbon monoxide gas from Matheson Co., Inc. (East Rutherford, N.J.); liquid helium from Gardener Cryogenic Division (Phila-

1 With benzo[a]pyrene as the substrate in vitro, the “aryl hydro- carbon hydroxylase” activity is equated nrith the rate of forma- tion of 3-hydroxybenzo[a]pyrene; this phenol may be formed either by a direct hydroxylation or in a two-step process via an arene oxide.

* Aromatic hydrocarbon “responsiveness” appears to represent a “threshold effect,” because exposure of fetal cell cultures derived from so-called genetically nonresponsive mice to aromatic hydro- carbons dissolved in the culture medium (1) and in vivo treatment of genetically nonresponsive mice with certain halogenated aro- matic hydrocarbons (11) produce rises in hydroxglase activity that are as high as those found in genetically responsive mice.

8 The abbreviations used are: MC. 3-methvlcholanthrene; hv- droxylase, aryl hydrocarbon hydroxyiase; 0-deethylase, 7-e&0x$- coumarin 0-deethylase; 0-demethylase, p-nitroanisole O-demeth- ylase; N-demethylase, 3-methyl-4.methylaminoazobenzene N-de- methylase.

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delphia, Pa.); and sodium dithionite from Fisher Scientific CO. (Fair Lawn, N.J.). The 3-methyl-4-methylaminoazobenzene was

a generous gift from Drs. James A. and Elizabeth C. Miller, Uni- versity of Wisconsin; the 7-ethoxycoumarin was generously given to us by Professor Volker Ullrich, Physiologisch-Chemisches Institut der Universitiit des Saarlandes, Homburg/Saar, Germany. In this report the strains of mice from the National Institutes of Health Animal Supply are denoted with the ending “N.” those purchased from The Jackson Laboratory, Bar Harbor, MAine, are denoted with the ending “J.” Unless otherwise indicated, sexually immature (3 to 6 weeks of age) mice of either sex were used.

Methods Treatment of Animals-The environment in the animal room,

feeding of the mice, the intraperitoneal administration of MC (80 mg per kg body weight), and the preparations of liver microsomes or tissue homogenates from lung, kidney, bowel, and skin were exactly as previously described (2, 5). Throughout this report, the “MC-treated” mice received an intraperitoneal dose of MC 44 to 48 hours before killing. In each cross examined and reported here, o-naphthoflavone (80 mg per kg body weight) substitut,ed for MC gave no obvious differences between these two inducers.

Enzyme Assays-The hydroxylase (2, 9), 7-ethoxycoumarin 0-deethvlase. D-nitroanisole 0-demethvlase. and 3-methyl-4- methyl~min~az~benzene N-demethylase (6) activities and thepro- tein determinations (2,9) were determined as described previously in the references cited. One unit of hvdroxvlase activitv is defined (2,9) as that amount of enzyme catalyzing,“per min at 37”, the for- mation of hydroxylated product causing fluorescence equivalent to that of 1 pmole of the recrystallized 3-hydroxybenzolalpyrene standard. For the 0-deethylase, 0-demethylase, and N-demeth- ylase activities, 1 unit is arbitrarily defined (6) as 1 nmole of 7-hydroxycoumarin (umbelliferone), p-nitrophenol, and formalde- hyde, respectively, formed per min. The enzyme activities were determined at 37”, except for the 0-deethylase which was examined at room temperature. Specific activity denotes units per mg of liver microsomal protein or units per mg of whole tissue homGge- nate protein for lung, kidney, bowel, and skin. All liver micro- somal samples were freshly prepared and assayed on the day of killing; the nonhepatic tissues were always frozen at -20” and then examined for hydroxylase activity the following day. We have found no significant differences in the enzyme activity be- tween fresh and once-frozen nonhepatic tissues. The assays were performed in exactly the same way each day, and one to three MC- treated inbred parents were always included to serve as “controls” for testing the assay conditions (pH, temperature, and extraction procedure) each day.

Specttophotometr&Concentrations of the CO-binding hemopro- teins were determined as described recentlv in detail (2, 5, 7). Wavelength measurements were standardize& by the use oi a.hol- mium oxide crystal (Fisher Scientific Co.). We have demon- strated (7) that the room temperature procedure for measuring the reduced minus oxidized difference in absorption between 645 and 700 nm correlates well4 with the g = 8.01 signal height measured with EPR spectroscopy below 10 K.

EPR Spectroscopy-EPR measurements below 10 K were carried out exactly as recently described (5, 7). The g = 8.01 signal

* The membrane-bound CO-binding hemoproteins may exist in three or more distinct types, or species, detectable by spectral or electrophoretic differences (24-31). The iron associated with each of these cytochromes may exist in either of two forms (oxidized or reduced) and in either of two spin states (high or low spin). The spin state is predominantly influenced by an endogenous or exoge- nous substrate binding (32) in the vicinity of the active site, but we suggest (5, 7) that lipoprotein or other membrane configura- tional changes also affect the spin state of the hemoprotein iron. Hence, the formation of cytochrome PI-450 (25), also called “P- 448” (26), in freshly prepared oxidized microsomes is highly corre- lated with increases in the g = 8.01 EPR signal height (5) and with increases in the ~~~~~~~~~ of the oxidized-minus-reduced difference spectrum (7). The extent to which the high spin iron is converted to low spin iron, by the binding of type II substrates, the treat- ment with organic solvents or detergents, or with the changes in temperature or pH, probably differs among the mouse, rat, and rabbit (cf. Refs. 5 and 7 for further discussion).

height, representative of high spin cytochrome P-450 iron, is arbitrarily described (5,7) in chart units per gain per mg of micro- somal protein. Since the widths of these EPR signals at half- height were never found to decrease when the signal height in- creased, we feel that changes in the g = 8.01 signal height accu- rately reflect relative changes in the high spin ferric iron of the CO-binding cytochromes (7).

RESULTS

Hydroxylase Induction and CO-binding Cytochrome Content as Function of Age, Sex, Time, Course, Dose Dependency, and Seasonal Variation-The magnitude of hydroxylase induction in MC-treated mice of the inbred strains studied varied considerably with age, the maximal response occurring in weanling mice and a decline found after about 8 or 10 weeks of age. Mice of 12 weeks of age generally possessed MC-inducible hydroxylase activities which were about one-half to two-thirds of those found in mice aged 3 to 6 weeks. The basal enzyme activity was found not to vary as much. In mice over 10 weeks of age and even more striking in mice of 20 weeks of age, the hepatic hydroxylase ac- tivity in females was generally higher than that in males. The inducible and basal enzyme activities were both depressed in the pregnant mouse. The time course of hydroxylase induction varied slightly among the strains; however, the level at 48 hours was always as high as, or higher than, the specific activity after 24 hours of MC treatment. A dose of 80 mg of MC per kg body weight induced maximally the hepatic hydroxylase activity; the dose-response curve for the inducible enzyme activity in extra- hepatic tissues is the topic of a separate report (33).

The microsomal CO-binding cytochrome concentration also was highest in the 3- to g-week-old mouse and decreased with age. During the course of 1 year, we found that the CO-binding cyto- chrome content in MC-treated and in control mice of the same age, sex, and strain varied by as much as about 400 pmoles per mg of microsomal protein; whether these variations reflect seasonal influences or other factors is uncertain. On the other hand, the basal or MC-induced hydroxylase activity in these same mice appeared to vary by no more than 15% throughout the year. No statistically significant circadian rhythmicity in the MC-in- ducible or basal hydroxylase activities or in the CO-binding cytochrome content was found5 in the liver of C57BL/6N mice. Individual variation in the enzyme activity is worth emphasizing. On one day we examined in the same assay 30 MC-treated AKR/N 4-week-old females with the same birth date, and the mean =t standard deviation for the hydroxylase activity was 613 f 184, with a range of 160 to 880 units per mg of liver microsomal protein. Therefore, to calculate an index of induci- bility (16,22) based on only two or four individual mice per group may be quite unreliable.

The data in Fig. 1, for example, illustrate the importance of studying mice of similar age when one is looking for genetic char- acteristics such as additive, completely dominant, or completely recessive inheritance. Throughout this study, sexually immature mice, 3 to 6 weeks of age and of either sex, were therefore used.

Examples of Autosomal Dominance in Various Crosses between Two Z&red Strains-Fig. 2 demonstrates the basal and MC-in- ducible hydroxylase activity in seven inbred strains and in MC- treated progeny resulting from the possible backcrosses and inter- cross. The enzyme in all MC-treated offspring of the AKR/N x DBA/2N cross was noninducible. The dominant expression of hydroxylase induction segregated very clearly in the C57BL/6N X RF/J cross and the BALB/cAnN x DBA/BN cross. We

6 G. F. Kahl and D. W. Nebert, unpublished data.

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C3H/HeN

8 8 300

C57BL/6N ia

C3H/HeN

0 40 a0 120 0 40 a0 120 DAYS OF AGE

FIG. 1. Aryl hydrocarbon hydroxylase (AHH) specific activity and CO-binding cytochrome content in liver microsomes from MC- treated or control C57BL/6N and C3H/HeN inbred mice, as a function of age. Each point represents the combined livers of 4 to 8 mice of a given age. For mice 6 weeks or older, only males were used. RUTS represent the standard deviation.

have also found a clearly dominant genetic segregation in the C57BL/6N x DBA/BN cross, the C57BL/6N X NZW/BLN cross, the C57BL/6N x NZB/BLN cross (2,9, lo), the C57BL/ 6N x 129/J cross, the C57BL/6N x AU/&J cross, and the PL/J x AU/SsJ cross (data not shown). In the C3H/HeN X AKR/N cross, the BALB/cAnN x AKR/N cross, and the CBA/ HN x AKR/N cross (Fig. 2), the segregation is not as clear-cut, presumably because of the relatively high enzyme activity in the AKR/N inbred mouse. We therefore conclude that, in the above mentioned 10 crosses between a responsive and a nonresponsive strain, the expression of hydroxylase induction segregates funda- mentally as an autosomal dominant trait, but other genetic or environmental modifying factors might be inufluential. These same conclusions are probably valid for the C57BL/6J X AKR/J cross, the C57BL/6J X SJL/J cross, the C57BL/6J X AU/SsJ cross, and the C57BL/J X DBA/lJ cross recently re- ported (22), in which only 2 to 7 mice in each group were studied.

Example of Add&e inheritance-Fig. 3 (top) demonstrates an additive effect in the expression of hydroxylase induction among offspring from the C3H/HeN x DBA/2N cross. Thus, MC treatment resulted in a bimodal distribution among progeny of the two backcrosses: in the F1 X C3H/HeN backcross approxi- mately one-half “high responsive” and one-half “intermediate responsive”; in the Fi X DBA@N backcross about one-half “intermediate” and one-half “nonresponsive.” The MC-treated Fz generation displays three populations; about one-fourth non- responsive, one-half intermediate and not statistically different from the Fi heterozygote, and about one-fourth “high respon- sive.” Similar data were recently illustrated (22) for the C3H/ HeJ X DBA/2J cross. In this regard, we conclude that the

C3H/He inbred strains from The Jackson Laboratory and from the National Institutes of Health have retained this gene, or genes, for the more than 20 years that they have been separated (34) ; the same holds true for the DBA/2 strains.

In the (C3H/HeN x DBA/2N)Fi hybrid, is the gene-dose effect exactly additive, or does the hydroxylase induction more closely resemble one or the other of the inbred parents? Table I shows that the genetic expression is derived very closely, if not exactly, 50% from each parent. Previous work from this lab- oratory has shown that the genetic response to aromatic hydro- carbons is associated in mice with new microsomal cytochrome Pi-450 formation, which can be detected by the blue shift in the Soret peak of the reduced hemoproteinC0 complex (2, 9), by the increase in absorption of the reduced minus oxidized difference spectrum (7), and by the increase in the g = 8.01 EPR signal (5,7). The genetic response is also associated with the induction of three other monooxygenase activities (6). Table I shows that. all of these parameters are intermediate in the (C3H/HeN X DBA/2N)Fr hybrid but are expressed dominantly in the (C3H/ HeN X C57BL/6N)F1, the (C3H/HeN X AKR/N)Fi, and the (BALB/cAnN x DBA/2N)F1 hybrids.

Evidence for Involvement of Two Genetic Loci-The enzyme ac- tivity in C57BL/6N liver microsomes was generally more induci- ble than that in C3H/HeN microsomes (Fig. 3, bottom). In the C57BL/6N X C3H/HeN cross and in either the Fi X C57BL/6N or the F1 X C3H/HeN backcross, all individuals were genetically responsive to MC, having hydroxylase specific activities greater than 1000. Of interest among the 49 offspring of the Fz genera- tion, however, was that the enzyme was not inducible in five mice.

Example of Dominant Expression of Absence of Hydroxylase Induction-Fig. 4 shows the expression of MC-inducible hydrox- ylase activity among several possible crosses between C57BL/6N, AKR/N, C57BL/6J, and AKR/J inbred strains. We found the hydroxylase induction among offspring from the C57BL/6N X AKR/N genetic cross (Fig. 4A) to be expressed just the opposite as that from the C57BL/6N X AKR/J cross (Fig. 4B); the MC-inducible enzyme activity in the C57BL/6J X AKR/N cross and the C57BL/6J x AKR/J cross (data not shown) segre- gated in a fashion similar to that shown for the C57BL/6N X AKR/J cross. Hence, the (C57BL/6N X AKR/N)Fi hybrid was completely nonresponsive, the (C57BL/6N X AKR/N)Fz progeny displayed a bimodal distribution with three-fourths of the individuals nonresponsive, and so forth. This dominantly expressed lack of hgdroxylase induction has been found in more than 150 offspring from more than 20 C5713L/6N X AKR/N and AKR/N X C57BL/BN individual matings. We have taken the C57BL/6N male, whose progeny from a mating with an AKR/N female were nonresponsive, and subsequently mated him with an AKR/J female, and the resulting offspring were responsive. By means of reciprocal matings, we have found no evidence for a factor which is maternally inherited in any of the C57BL/6 x AKR crosses. Previously (2) we had found no evidence for a maternally inherited factor in several other genetic crosses dis- playing autosomal dominant inheritance.

Fig. 4C illustrates that the hydroxylase in C57BL/6N mice is more highly inducible than the enzyme activity in C57BL/6J mice and that the enzyme in AKR/N mice is higher than the hydroxylase activity in AKR/J mice. We have observed that the basal hydroxylase activity in C57BL/6N mice was also sig- nificantly higher than that in C5713L/6J mice (data not illus- trated). In virtually all offspring from the C57BL/6N X (AKR/N)(AKR/J) cross, the C57BL/6J x (AKR/N)(AKR/J)

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(C3AK)F2 , a N=24

A 0 2'Bl 4CW 6C'X SPECIFIC ACTIVITY

B O 2000 4m 6000 SPECIFIC ACTIVITY

lc0tdTR0~ Ic3 1 J+ N: 15

c3 N: 15

MC- 66 N:3tl TREATED

c3 N=ZS

0 2000 4000 6000 SPECIFIC ACTIVITY

FIG. 3. Histograms of hepatic microsomal hydroxylase specific activity in two pairs of control and MC-treated inbred mice and in MC-treated offspring from genetic crosses involving these two pairs. Abbreviations are the same as those in Fig. 2.

cross, the AKR/N X (C57BL/6N)(C57BL/6J) cross, the AKR/J X (C57BL/6N)(C57BL/6J) cross, and the (C57BL/6N)- (C57BL/6J) X (AKR/N)(AKR/J) cross, inducible hydroxylase activity was observed. In progeny from the (C57BL/6N)

FIG. 2. Histograms of hepatic mi- crosomal hydroxylase specific activity in six pairs of control and MC-treated inbred mice and in MC-treated off- spring from genetic crosses involving these six pairs. In this figure and in subsequent figures and tables, IV de- notes the number of individual mice examined for each group. Abbrevia- tions of the inbred strains include: AK, AKR/N; 02, DBA/PN; B6, C57BL/6N; RF, RF/J; CS, C3H/HeN; BA, BALB/cAnN; and CB, CBA/HN. Although the F1 hybrid is denoted (AKDP)Fl, for example, (DPAK)Fi hybrids were also studied. The same is true for most of the other genetic crosses between two inbred strains shown by histograms throughout this report, i.e. the results in the progeny were not dependent on the sex of either parent.

(AKR/N) x AKR/J cross, 33 out of 34 were nonresponsive; what this one individual having a very high MC-induced hy- droxylase activity represents is unknown. Fig. 5 shows the genetic expression of hydroxylase induction in MC-treated off- spring originating from two, three, or four inbred strains.

Hydroxylase Induction in Nonhepatic Tissues and Hepalic 0-Deethylase, 0-Demethylase, and N-Demethylase Induction among Various Crosses-Table II demonstrates that a positive highly significant correlation exists between the hepatic hydroqlase induction in MC-treated progeny derived from these various genetic crosses and the induction of hydroxylase activity in lung, kidney, bowel, or skin,6 or the induction of three other hepatic monooxygenase activities. In no cross-including the C57BL/ 6N X AKR/N cross in which the absence of hydroxylase induc- tion is a dominant trait-have we found an exception to this general observation.

Genetic Expression of Basal Hydrozylase Activity in Liver-

6 In the genetically nonresponsive strains of mice administered MC in Go, we found severalfold increases in the hydroxylase ac- tivity from lung, kidney, bowel (2), and skin (12), compared with the low basal enzyme activities in these tissues. Likewise, in benz[a]anthracene-treated fetal cell cultures from these nonre- sponsive strains (1,35), severalfold increases in the enzyme activ- ity were found. Over a wide range of doses of aromatic hydrocar- bon inducer in uivo or in cell culture, we have found (33) that the inducible hydroxylase activity in nonhepatic tissues of responsive mice is always higher than that of nonresponsive mice. That the hydroxylase is slightly inducible in nonhepatic tissues (17, 18) or fetal liver cell cultures (19) from nonresponsive inbred strains but is much more highly inducible in these tissues or cultures of respon- sive strains by aromatic hydrocarbons has also been shown re- cently in the references cited.

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5855

TABLE I Liver microsomal hydroxylaze activity, optical and paramagnelic characteristics of the CO-binding cytochromes, and other

monooxygenase activities in MC-treated inbred strains and FI hybrids

Abbreviations for the mouse strains are the same w those described in legends t.o Figs. 2 and 3. All four monooxygenase specific activities are expressed in units per mg of microsomal protein. All parameters were studied on the same day with preparations of liver microsomes combined from eight inbred or hybrid mice for each group.

Inbred or hybrid mice

c3 1610 D2 360 (C3D2)Fl 1020

B6 2210 (C3B6)Fl 1780

AK 690 (C3A0Fl 1920

BA 1960 (Bm2)Fl 1810

Iydroxylase specific activity

Cyto- chrome P450 01 p1450a

Soret maxi- mum

1040 4g.8 710 450.2 960 449.5

1360 447.8 1520 448.2

1000 450.0 1060 448.7

1050 448.4 910 448.8

1

I I I F

22 28 8 5

15 17

28 32 23 31

g=8.01 o- 3- J- EPR de- 3e- le- signal heightC

ethyl- nethyl- nethyl- ase ase me

17 5.0

11.4

24 14

3.8 18

13 12

2.5 0.8 1.9

3.6 3.2

1.1 2.5

3.0 2.2

24 6.1 8.8

25 21

9.9 22

16 14

aExpressed in pmoles per mg of microsomal protein. b Expressed as the difference in absorbance units per mg of microsomal protein x 10 -4 ,

when dithionite-reduced microsomes are compared with oxidized microsomes (7).

'Expressed in chart units per gain per mg of m:icrosomal protein x 10 -4 (5, 7).

Fig. 6 illustrates that in the (AKR/N X AU/SsJ)Fl, the (AKR/J X AU/SsJ)F1, and the (PL/J X AU/SsJ)F1 hybrid, the control hydroxylase activity is inherited additively. This finding could be most readily determined in F1 hybrids involving the AU/SsJ strain, because the basal enzyme activity in this strain is quite low. In F1 hybrids of other strains, the control enzyme appeared to be inherited additively; however, this could not be determined with a high degree of certainty due to the small differences in the mean enzyme activity between any other two strains and due to individual variation of the control hydroxylase activity among mice of the same strain.

Since the control enzyme displays additive characteristics (Fig. 6) whereas the hydroxylase induction by MC is expressed dominantly in the (PL/J X AU/SsJ)Fl, this suggests to us that there are regulatory differences between the basal and MC-in- ducible hepatic hydroxylase activities. This is perhaps not so surprising, since by spectral (2,3) and paramagnetic (5,7) studies and by studies involving preferential inhibition in vitro (4) and involving reconstitution of partially purified microsomal subfrac- tions (8), we have suggested that differences in the enzyme active site exist between the control and the aromatic hydrocarbon-in- ducible hydroxylase system.

DISCUSSION

Because aryl hydrocarbon hydroxylase “activity” reflects the integrity of electron flow between a FAD-containing reductase, membrane phospholipid, and the CO-binding hemoprotein en- zyme active site (8, 36), changes in concentration of any of these moieties, changes in configuration of any of these components, changes in the assembly of these various moieties into the mem- brane, or changes simply in the flow of reducing equivalents from NADPH to the cytochrome all could result in an increased rate of 3-hydroxybenzo[a]pyrene formation, which we equate with the “induced” hydroxylase activity. Decreases in the formation of benzo[a]pyrene dihydrodiols or glutathione conjugates or de-

creases in the amount of benzo[a]pyrene bound covalently to cellular macromolecules (cf. Ref. 14 for discussion), with a con- comitant increase in the nonenzymic conversion of benzo[a]- pyrene epoxide to the phenolic product, would similarly appear as “induction” of the hydroxylase activity. Since we have such an inadequate understanding of what aryl hydrocarbon hydrox- ylase “induction” actually signifies, this makes an understanding of its genetic regulation all the more difficult.

The Ah locus has been proposed (3, 37) to designate the.gene for aromatic hydrocarbon responsiveness. This locus is clearly associated with cytochrome PI-450 formation (2-5,7-g) and with the induction of at least four other hepatic monooxygenase ac- tivities (6, 38) which are most likely mediated by this newly formed CO-binding hemoprotein. The allele Ahb was suggested (3,37) to represent the dominant gene, and the C57BL/6N strain is arbitrarily designated the prototype inbred strain. The allele Ahd was proposed (3, 37) to represent the recessive gene, and the DBA/2N strain is arbitrarily designated as the prototype strain for this allele. Our findings in thii report with respect to ex- pression of the hydroxylase induction among progeny of the C57BL/6 and AKR strains from both National lnstitutes of Health and The Jackson Laboratory do not readily support allelism. Therefore, until we have a better understanding of these data, we feel that it would be unwise to attempt to ascribe alleles at the Ah locus for any strain other than the C57BL/6N and DBA/2N inbred mice. Because hydroxylase induction in the C3H/HeN X AKR/N cross segregates dominantly, it would appear that the AKR/N mouse may contain the Ahd allele; how- ever, the absence of hydroxylase induction in the (C57BL/6N X AKR/N)F1 hybrid indicates this cannot be the case. Likewise, further studies must be completed before any of the other inbred strains can be assigned an allele at the Ah locus.

The incidence of 5 out of 49 MC-treated (C57BL/6N X C3H/HeN)F2 mice having the noninducible hydroxylase activity may represent a genetic incidence of 1 in 16; this phenotypic

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N=20

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A 0 2000 4000 6000

SPECIFIC ACTIVITY

WNAKJ,AKJ N=l6

B 0 2000 4000 6000 SPECIFIC ACTIVITY

incidence suggests to us the homozygosity of two genes.7 This iinding may represent evidence for two loci associated with aromatic hydrocarbon responsiveness and indicates that C57BL/ 6N and C3H/HeN would differ in their alleles at both loci. Moreover, the two loci need not be linked. The most appropri- ate nomenclature for two loci is Ah-l and Ah-,?; it will be espe- cially helpful to find linkage of either or both of these loci with other markers already located on the mouse chromosomes.

We have shown in thii report that C57BL/6N is not identical to C57BL/6J and/or that AKR/N is not identical to AKR/J, with respect to basal and MC-inducible levels of hydroxylase activity and with regard to the autosomal dominant expression, or dominant lack of expression, of the inducible enzyme activity. This is perhaps not surprising, since the sublines of the National Institutes of Health and The Jackson Laboratory C57BL/6 and AKR have been separated (34) for more than 20 years. In fact, rather striking differences in lhy-1 antigen, malic enqyme, es- terase-3, principal urinary protein, expression of MuLV-gs anti- gen, and incidence of leukemias have been recently pointed out

’ At latest count, we have found 13 nonresponsive (C57BL/6N X C3H/HeN)F2 offspring out of a total of 205 individually studied.

N=34

I I I

(86WAKAKI

N:34

C 0 2000 4000 600C

SPECIFIC ACTIVITY

FIG. 4. Histograms of hepatic microsomal hydroxylase specifi act.ivity in control and MC-treated pairs of inbred mice and in MC treated offspring from genetic crosses involving these pairs. Ab breviations of the inbred strains include: BUN, C57BL/6N; AKN AKR/N; AKJ, AKR/J; B6J, C57BL/6J; AKAK, the (AKR/N > AKR/J)F1 hybrid; and B6B6, the (C57BL/6N X C57BL/6J)F hybrid.

(39) between various AKR sublmes. Of further interest is that differences in the degradation rate of phenylethanolamine N- methyltransferase in adrenal medulla between BALB/cAnJ and BALB/cA~N mice have also been recently reported (40). The expression of MuLV-gs antigen segregates as a single autosomal dominant trait in crosses between C57L/J and AKR/J mice; however, the lack of expression is dominant over expression in crosses between C57BL/lOSn and the congenic BlO.D2(58N) strain (41). Since MuLV-gs antigen synthesis involves assembly of a multicomponent system, it is possible that some similarities in genetic regulation exist between this system (41) and the dominant expression of induction versus the dominant lack of expression of MC-inducible hydroxylase activity.

Listed in Table 111 is the observed incidence of hydroxylase induction in offspring derived from three or four inbred strains. These data are compared with what one empirically expects on the basis of results obtained from crosses between two inbred strains. Three types of results were found: (a) in the upper group of four genetic crosses in Table III, the observed results were not statistically different from the experimental results, and the genetic background of the offspring included either C57BL/

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5857

6N or AKR/N, but not both; (b) in the middle group of five genetic crosses, the observed results were not significantly differ- ent from the expected findings, even though the genetic back- ground of the offspring included both C57BL/6N and AKR/N; (c) in the lower group of seven genetic crosses, the observed results were very significantly different from the expected results. It is of extreme interest that the genetic background in each of these seven crosses includes both C57BL/6N and AKR/N. We have

IC- REATED (86D2lAK

N = 53

N = 31

N=58

N=52

N=44

N=l6

lC3129)F, N=7

I I 8 0 2000 4000 6

SPECIFIC ACTIVITY ‘OOC

FIG. 5. Histograms of hepatic microsomal hydroxylase specific activity in MC-treated progeny derived from two, three, or four inbred stains. Abbreviations include those already described in Figs. 2 to 4; 199, 129/J. B6 and AK always represent C57BL/6N and AKR/N, respectively; AK, denotes AKR/J. For example, (B6D2)(BGAK) represents offspring from the cross between a (C57BL/6N X DBA/SN)Fi and a (C57BL/6N X AKR/N)Fi parent.

)NTRGi

N = 16

N=II

N=20 (PLAUIF,

I 0 500 IZOO I

SPECIFIC ACTIVITY

FIG. 6. Histograms of hepatic microsomal hydroxylase specific activity in untreated, control inbred mice or Fi hybrids involving four strains.

TABLE II Correlations between liver hydroxylase activity and the hydroxylase activity in nonhepatic tissues OT the other hepatic monooxygenase

activities in MC-treated progeny from genetic crosses involving various mouse strains

Abbreviations for the mouse strains are the same as those de- tistical confidence are shown. The mice used in these experiments scribed in legends to Figs. 2, 3, and 4. The positive correlation were either progeny from the Fi backcrossed with the recessive coefficient T, in parentheses the number of individual determina- inbred parent or progeny from the Fa generation; thus both.high tions from the individual MC-treated mice, and the level of sta- and low hydroxylase activities are included.

-I nepatic hydroxylase

lung activity in offspring from genetic crosses

t r

Nonhepatic hydroxylase ac

I kidney

00 E L ‘3) E

(32) <O.OOl (25) co.01 (38) <O.OOl (24) 0.02<p<O.O5 (17) <o .Ol (45) <O.OOl (12) 0.02yO.05

(30) <O.OOl

(36) <O.OOl

Gm <O.Ol

(24) <O.OOl

-I bowel skin

-

00

0.73 0.59 0.61 0.47 0.67 0.52 0.64

0.69

0.70

0.59

0.68

0.39 (32) 0.78 (24) 0.46 (7.1) 0.69 (24)

0.02<~<0.05 <O.OOl

0.02yO.05 <O.OOl

0.64 (27) co.001 0.86 (23) <O.OOl

0.60 (30) <O.OOl

0.78 (23) <O.OOl 0.43 (11) 0.10<~<0.20 0.74 (23) <O.OOl

r N) E

0.59 (24) co.01 0.92 (7) co.01

0.38 (34) 0.02yO.05

0.62 (11) 0.02<p<O.O5

I E

0.38 (30) 0.02yO.05

0.36 (21) 0.10<p<0.20

0.42 (36) 0.01<p<0.02

B6 x RF c3 x AK BA X AK CB X AK BA x 02 c3 x D2

B6N ' % B6N x AKJ

B6J x 54 C3D2 X AK B6D2 X B6AX B6C3 X AKD2

Hepatic microsomal monooxygenase activities

I I

0-deethylase

L (N) p

C3 X D2 0.77 (10) co.01

B6N ' % 0.89 (7) co.01

B6D2 X B6AK 0.71 (11) 0.01<~<0.02 --

0-demethylase N-demethylase

L 00 E r 00 p

0.68 (10) O.OZ<p<O.OS 0.74 (10) 0.01<~<0.02

0.66 (11) 0.02<E'O.O5 0.76 (11) co.01

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TABLE III Observed and predicted incidences of hydroxylase induction by MC in mice derived from various genetic crosses

The abbreviations for the inbred mouse strains are the same as of normal probability functions, and a two-tailed test was used. those used in Figs. 2, 3, 4, and 5; ZW denotes the NZW/BLN in- To assure oneself a 0.95 chance of making no misstatements (i.e. bred strain. The statistical confidence level, ‘P, is estimated declaring a result significant when in fact it is not), one can use the with the use of the equation 2 = (P - ?r)/d?r(l - r)/n, where P LY* = 0.004 level of significance for each individual test. We thank is the observed value, and n represents the number of observa- Dr. James Schlesselman for his help with these statistical analyses. tions. The value for 1 - F(Z) was then determined from the table

Genetic cross 0. of mice

Observed Expecteda

2; 21 34

36% 100%

50% 100% 100% 100%

0.095 1.00 1.00 0.97

53 53% 50% 0.66 36 39% 50% 0.187 58 50% 50% 1.00 52 42% 50% 0.25 44 48% 50% 0.79

17 100% 26 88% 24 100% 79

lllb 1%

52% 18 0% 19 100%

50% 50% 75% 50%

0% 50% 50%

co. 0001 0.0002 0.005

co. 0001 <0.0001 <0.0001 co. 0001

aExpected on the basis of a one-gene model, as postulated from all the data observed in the offspring derived from two inbred parents.

b On the basis of reciprocal crosses, no evidence was found for maternally inherited influences. These data are based on at least 6 individual matings (i.e. 6 different fathers and 6 different mothers). The remainder -- of the data in this table is based only on one to three individual matings, and all possibilities for maternally ruled out.

no reasonable hypothesis as to why certain genetic crosses con- taining both C57BL/6N and AKR/N genetic material display results that are predictable on the basis of data derived from crosses between two inbred parents and why other crosses con- taining both C57BL/6N and AKR/N genetic material display such an unexpected genetic expression. It is possible* to con- struct a hypothetical model in which there are three alleles, or gene products, at each of two nonlmked loci and in which each of the alleles at one locus can be assigned a certain rank, or per- missiveness of interaction, with each of the alleles at the second locus. However, even this proposed model cannot predict all of the unexpected results shown in Fig. 5 and Table III.

The importance of aromatic hydroxylations of drugs, insecti- cides, and polycyclic hydrocarbons mediated by monooxygenases to pharmacology, toxicology, and chemical carcinogenesis has been recently reviewed (42). Genetic differences in aromatic hydrocarbon-inducible hydroxylase activity in mice (12, 13, 15, 20,21,23) are associated with an increased susceptibility to MC- produced tumorigenesis. Moreover, an increased in vitro muta- genesis rate with Salmonella bacteria is highly correlated with the genetically mediated increase in mouse hepatic microsomal aryl hydrocarbon hydroxylase activity (43). Additive inheritance has recently been suggested in man: reproducible, significant

*J. G. M. Shire, personal communication.

inherited effects have not yet been

differences in the magnitude of hydroxylase induction by MC were found in cultured lymphocytes from several hundred pa- tients (44). Susceptibility to bronchogenic carcinoma in man has recently been reported to be associated with genetically mediated higher levels of the inducible hydroxylase activity (45). It is of interest that the genetic expression of hydroxylase induction in the C3H/HeJ X DBA/BJ cross and the C3H/HeN x DBA/2N cross most closely approximates the expression of this inducible enzyme activity in human lymphocytes. Further studies of the genetic regulation of cytochrome PI-450 formation and the as- sociated increases in various monooxygenase activities may lead to a better understanding of mechanisms involving carcinogene- sis, pharmacology, and toxicology.

AcknowZe&ments-We appreciate Dr. Kideo Kon for his valua- ble time in helping us with the EPR determinations. We thank Drs. Kenneth Paigen and Verne Chapman (Roswell Park Memo- rial Institute, Buffalo, N.Y.) and John G. M. Shire (University of Glasgow, Scotland) for reviewing this manuscript and offering helpful advice.

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Joseph R. Robinson, Noreen Considine and Daniel W. NebertFOR THE INVOLVEMENT OF OTHER GENETIC LOCI

Genetic Expression of Aryl Hydrocarbon Hydroxylase Induction: EVIDENCE

1974, 249:5851-5859.J. Biol. Chem. 

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