Aryl hydrocarbon (benzo(a)pyrene) hydroxylase induction in cells in culture

Pharmac. Ther. Vol.4. pp. 587-599.

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ARYL H Y D R O C A R B O N (BENZO(A)PYRENE) H Y D R O X Y L A S E I N D U C T I O N IN C E L L S IN C U L T U R E JAMES P. WHITLOCK, JR.* AND HARRY V. GELBOINt

INTRODUCTION Although modern technology undoubtedly provides many benefits for modern society to enjoy, it simultaneously creates an increasingly wide variety of potentially toxic compounds with which the population must live. The epithelial cells of man and other mammalian organisms are continuously exposed to industrial pollutants, food additives, dyes, drugs, carcinogens and other foreign compounds. The microsomal mixed-function oxidases, cytochrome P-450 containing enzyme systems, coupled with other metabolically linked enzymes, provide an important pathway whereby the cell can metabolize and eliminate such xenobiotics. Many of the mixed-function oxidase drug-metabolizing enzyme systems are inducible; i.e. after exposure to certain cliemicals the amount of enzyme activity increases. This process may thus provide an important mechanism for increasing the rate of metabolism of foreign compounds (Conney, 1967; Gelboin, 1967; Gelboin et al., 1972; Conney and Burns, 1972). An understanding of the factors and mechanisms involved in regulating drugmetabolizing enzyme activity, and, in particular, the biochemical events involved in enzyme induction, is relevant to several important problems. First, such enzyme systems play a central role in the metabolism of a wide variety of both endogenous and exogenous compounds. Thus, knowledge of the factors involved in enzyme induction may lead to a better understanding of the regulation of activation and detoxification of chemical carcinogens, drug synergism and antagonism, environmental and occupational medicine, genetic diseases and developmental malformations, toxicology, and ecology. Second, enzyme induction is a fundamental problem in molecular biology. Knowledge of the mechanism of induction of drug-metabolizing enzymes may lead to a more complete understanding of the regulation of gene expression in eukaryotic cells. The use of cells in culture offers several advantages for studying the mechanism of induction of drug-metabolizing enzymes. The extracellular environment can be rigorously controlled, and effects due to environmental, hormonal or nutritional factors can either be minimized or studied under controlled conditions. In addition, cell culture offers the potential for developing mutant cell lines with specific defects in enzyme induction and for constructing somatic cell hybrids with specific enzyme induction properties. However, the cell culture technique also has several disadvantages. First, there is no guaranteethat a cell will maintain its stable, differentiated functions in culture. Second, experiments must be performed under less than physiologic conditions, since the normal intercellular interactions have been disrupted. Third, the quantities of available tissue are usually quite small; therefore, sensitiv.e enzyme assays are required, and the isolation of specific macromolecules, such as receptors, regulatory proteins, or specific messenger RNAs, is often not feasible. Several laboratories have used cell culture techniques to study the induction of the microsomal enzyme complex, aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH); this substrate-inducible enzyme system is responsible for the metabolism of polycyclic aromatic hydrocarbons (PAH), which are major environmental contaminants and known carcinogens. The hydroxylase can both detoxify polycyclic *Developmental Biochemistry Section, Laboratory of Nutrition and Endocrinology, NIAMDD (Present address: Dept. of Pharmacology, Stanford University School of Medicine, Stanford California, 94305). tChemistry Branch, NCI, National Institutes of Health, Bethesda, Md. 20014, U.S.A. 587

588

J. P. WHITLOCK, JR. and H. V. GELBOIN

hydrocarbons as well as activate them to more carcinogenic forms. Thus, the AHH system plays a central role in the production of cancer by PAH (Gelboin, 1%7, 1969; Gelboin et al., 1972; Jerina and Daly, 1974; Huberman et al., 1976; Yang et al., 1976). It is likely that the A H H system is representative of an entire class of cytochrome P-450 containing mixed-function oxidases. Thus, the results of many studies of A H H induction may also apply to mixed-function oxidases in general. The study of this enzyme system in cell culture was greatly facilitated by the development of a sensitive spectrofluorometric assay for the phenolic metabolic byproducts of benzo(a)pyrene (Alfred and Gelboin, 1967; Nebert and Gelboin, 1968a). In general, there are two major classes of compounds which induce hepatic A H H in vivo. The barbiturates and certain insecticides (e.g. p,p'-DDT) represent one class, while P A H represent the other (Conney, 1%7). However, in vitro (in primary cultures of fetal rodent liver) hydroxylase induction by phenobarbital requires very high, non-physiologic concentrations of the inducer; furthermore, the response to this compound lessens with increasing age of the culture (Gielen and Nebert, 1971a,b). These phenomena may reflect the failure of these hepatocytes to maintain this liver-specific function in culture. For this reason, there have been relatively few studies using cells in culture to examine the mechanism of hydroxylase induction by phenobarbital-type compounds. In contrast, cells derived from either hepatic or non-hepatic tissue respond in vitro to concentrations of PAH which are similar to those found in the environment; this response is consistent and reproducible even after long-term culture. This corresponds to in vivo studies which indicate that A H H is inducible in most extrahepatic tissues by PAH-type compounds but is generally not inducible by phenobarbital-type compounds. Therefore, most studies of A H H induction in cells in culture have employed P A H as inducers. It remains to be determined whether the phenobarbital-inducible hepatic hydroxylase is induced by similar, or different mechanisms. A H H INDUCTION BY P A H - - K I N E T I C S The first studies of A H H induction by PAH in cell culture employed secondary cultures of fetal hamster cells (Alfred and Gelboin, 1%7; Nebert and Gelboin,

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FIG. 1. The effect of the polycyclic hydrocarbon inducer, benz(a)anthracene on aryl hydrocarbon hydroxylase activity in Buffalo rat liver cells. Cells were grown in control medium alone (C)~O) or in medium containing benz(a)anthracene (O--~); the medium was changed every 24~r. Inset, the effect of actinomycin D ( l l - - I ) and cycloheximide (&--&) on anjl hydrocarbon hydroxylase induction by benz(a)anthracene; antibiotics were given simultaneously with the inducer. Each point represents the average of duplicate determinationsof both aryl hydrocarbon hydroxylase and protein concentration on a single plate of cells. The concentrations of inducer and inhibitors used were: benz(a)anthracene, 10p.g/nd; cycloheximide, 10/~g/ml; actinomycin D, 1/~gJml. One unit of AHH activity is equivalent to I pmole 3-OH benzo(a)pyrene/30 mJn/mg protein.

Aryl hydrocarbonCoenzo(a)pyrene)hydroxylaseinduction in cells in culture

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TABLEI. Cells in Culture which contain PAH-lnducible AHH* l. Embryonic(primary or secondary cultures) A. Wholeembryo Hamster, rat mouse, chick, rabbit B. Individualtissues Liver, lung, gut, limbs 11. Animal cell lines A. Hamster BHK (kidney) OBP (embryo) B. Mouse 3T3 (fibroblast) JLSV-5 (thumus-spleen) C. Rat H-35 (hepatoma)

D. Human HeLa (cervicalcarcinoma) JEG (chor~ocarcinoma) VA-2 (fibroblast) 1II. Somaticcell hybrids JEGNA-2 (human/human) OBP/BHK (hamster/hamster) PO/3T3-4C2(mouse/mouse) HTC/3T3 (rat/mouse) RAG/hone marrow (mouse/human) IV. Normal human cells Lymphocytes Monocytes Macrophages Skin Bone marrow

*The list is not intended to be all-inclusive. 1968a,b). these investigations showed that a wide variety of polycyclic aromatic compounds, both carcinogenic and non-carcinogenic, induced hydroxylase activity to varying extents. Benz(a)anthracene was the most potent inducer studied, producing a thirty-fold increase in AHH activity over a period of 16 hr. Other studies indicate that AHH activity may continue to increase for 48 hr following exposure to PAH (Fig. 1). AHH has been shown to be present and inducible in a variety of ceil types (Table 1). It is unclear whether the effectiveness of a particular compound as an inducer is related primarily to its three-dimensional geometry (which might affect its interaction with hypothetical 'receptor' macromolecules) or to a more general property, such as its hydrophobic character (which might affect its general interaction with membrane lipoprotein, for example). Glucocorticoids, known inducers of several soluble enzymes in cell in culture, were inactive as inducers of AHH. There is a lag period of about 30 min between the exposure of the ceil to an inducer and the onset of increased hydroxylase activity. During this time, a fraction of the inducer molecules reaches the cell nucleus, apparently by a passive process (Nebert and Bausserman, 1970a,b). The concentration of inducer in the nucleus reaches equilibrium after about thirty minutes. Thus, there is a temporal correlation between the arrival of the inducer within the nucleus and the increase in AHH activity, suggesting that a nuclear event(s) is required for hydroxylase induction. We do not yet know whether there are specific nuclear acceptor molecules or specific nuclear recognition sites with which the inducer interacts. AHH INDUCTION BY P A H ~ R E Q U I R E M E N T FOR RNA SYNTHESIS Evidence that AHH induction requires RNA synthesis is that actinomycin D, when administered simultaneously w i t h the inducer, prevents the rise in hydroxylase activity; this suggests that transcription is required for enzyme induction, but does not reveal the type of RNA which is required (Nebert and Gelboin, 1970; Gielen and Nebert, 1971c; Whitlock and Gelboin, 1974). Studies using the mouse thymus-spleen cell line JLSV-5 and low concentrations of actinomycin D, showed that A H H induction occurs in the absence of detectable ribosomal RNA synthesis (Wiebel et al., 1972b). This finding was confirmed in another study which did not employ inhibitors of macromolecular synthesis. In this study hydroxylase induction was examined in a mutant line of B H K ceils which are temperature-sensitive for the synthesis of 28S ribosomal RNA. Benz(a)anthracene induced the hydroxylase seven- to eight-fold even at the non-permissive temperature, demonstrating that in these cells the synthesis of 28S rRNA is not required for A H H induction (Wiebel et al., 1976). Studies using actinomycin D suggest that the RNA species whose synthesis is required for A H H induction is 'heterogeneous' in size (i.e. is not rRNA or 4-5S RNA).

590

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Furthermore, hydroxylase induction is inhibited by 3-deoxyadenosine (cordycepin), suggesting that the synthesis of polyadenylic acid sequences is required for hydroxylase induction (Whitlock and Gelboin, 1974). Thus, the evidence is consistent with the idea that the synthesis of poly(A)-containing messenger RNA is required for hydroxylase induction. It remains to be determined whether the regulating event(s) occur primarily at the level of transcription itself (i.e. the increased synthesis of AHH-specific RNA sequences) or also at a post-transcriptional, pre-translational level (i.e. the polyadenylation of RNA, the nucleolytic processing of nuclear RNA, or the capping of RNA with methylguanylate). The induction process is increasingly less sensitive and becomes entirely insensitive to actinomycin D when the inhibitor is administered at times subsequent to the addition of the inducer. This suggests that the requirement of AHH induction for transcription occurs relatively early in the induction process, and indicates that after exposure to the inducer, AHH activity may continue to increase in the absence of further RNA synthesis (Fig. 2). AHH INDUCTION BY PAH--REQUIREMENT FOR PROTEIN SYNTHESIS Hydroxylase induction does not occur in the presence of inhibitors of protein synthesis, suggesting that protein synthesis is required for AHH induction. In contrast to the case for inhibitors of RNA synthesis, the rise in enzyme activity is sensitive to inhibitors of protein synthesis throughout the induction process, suggesting that continuous protein synthesis is required for induction (Nebert and Gelboin, 1970; Gielen and Nebert, 1971c; Whitiock and Gelboin, 1974). Such studies do not reveal to what extent either stimulation of de n o v o protein synthesis or inhibition of protein degradation or both contribute to the increase in hydroxylase activity. Also, these studies do not specify whether AHH induction requires the synthesis of cytochrome P-450, or whether the synthesis of a different protein is required, perhaps one involved in AHH activation. If an inducer is co-administered with an inhibitor of protein synthesis (e.g. cycloheximide) and allowed to interact with the cell for several hours, and the inhibitor is then removed, an increase in AHH activity subsequently occurs, even in the absence of further exposure to inducer; this rise in activity is insensitive to actinomycin D. 6OO OA 400

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cultures. ActinomycinD (Act D) (1 wg/ml)was added simultaneouslywith, or at various times after, the inducerbenz(a)anthracene(B.A.)(3 wg/ml).The baseline represents AHH activityin cultures incubated in medium free from BA or Act D (CM). The preparation of monolayer cultures from hamster embryosand the assay of AHH activitywere carried out as descn'bed by Nebert and Gelboin (1968a). The specific activity of AHH is expressed as pmole alkali-extractableproduct/mgof protein/30rain.

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Such experiments outline the temporal sequence of events required for hydroxylase induction; there is an initial phase requiring RNA synthesis, but not protein synthesis; this is followed by a phase requiring protein synthesis, but not RNA synthesis (Fig. 3). Thus, the events of transcription and translation can be separated experimentally (Nebert and Gelboin, 1970; Gielen and Nebert, 1971c; Whitlock and Gelboin, 1974). DECAY OF AHH ACTIVITY Removal of the inducer from induced cells (by washing with inducer-free medium) leads to a relatively rapid decrease in AHH activity; similarly, inhibition of protein synthesis in induced cells, even when the inducer is present, is followed by a fall in hydroxylase activity. Thus, continuous protein synthesis is needed apparently to maintain AHH activity at induced levels. In the presence of an inhibitor of protein synthesis, AHH activity decays with a half-life of 4-8 hr, depending upon the cell type. This relatively short half-life, combined with the high inducibility of the hydroxylase, provides a mechanism whereby AHH can respond to changes in the concentration of PAI-I in the environment. Differences in the rates of both enzyme synthesis and degradation may be important factors both in the metabolism of xenobiotics and in chemical carcinogenesis. AHH INDUCTION BY TEMPORARY INHIBITION OF PROTEIN SYNTHESIS During the course of experiments using inhibitors of macromolecular synthesis to study hydroxylase induction in a cloned line of rat liver ceils, Whitlock and Gelboin discovered that AHH activity could be increased fifteen- to twenty-fold, without the addition of a polycyclic hydrocarbon inducer, by causing a temporary (1-6hr) inhibition of protein synthesis (Whitlock and Gelboin, 1973). Following release of the block in protein synthesis, AHH activity increased rapidly to high levels. This increase in enzyme activity was insensitive to high concentrations of actinomycin D; however, if actinomycin D was present during the period of protein synthesis inhibition, there was no subsequent rise in AHH activity; this suggested that RNA synthesis was required during the period of inhibition of protein synthesis, in order to

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observe the subsequent increase in AHH activity. This phenomenon was explained in terms of a putative labile protein which acted to limit the translation of hydroxylasespecific RNA. During the period of inhibition of protein synthesis, this labile protein decayed, while, at the same time, there was ongoing accumulation of hydroxylasespecific RNA, being synthesized at basal rates, but not translated. FoLlowing removal of the block in protein synthesis, this RNA was rapidly translated for several hours, prior to the resynthesis of the hypothetical regulatory protein, which subsequently caused the increase in AHH activity to cease. Thus, these experiments suggested that, in addition to an induction mechanism involving control at the level of transcription, there was another important mechanism which regulated AHH induction at a posttranscriptional level (Whitlock and Gelboin, 1973). These induction mechanisms seem to be distinct, since the simultaneous induction of AHH by both mechanisms (that is, by the administration of a polycyclic hydrocarbon together with the production of a block in protein synthesis) leads to a fifty- to sixty-fold increase in enzyme activity, a much higher increase than can be obtained using either process alone (Fig. 4). AHH INDUCTION BY CYCLIC AMP A possible third mechanism of AHH induction was described by Yamasaki et al., who found that compounds which presumably increase the intracellular concentration of cyclic AMP also lead to an increase in hydroxylase activity in BHK cells. Administration of dibutyryl cyclic AMP and/or aminophylline led to increases in AHH activity which were up to 100-fold higher than basal levels (Yamasaki et al., 1975). The increase in AHH activity was continuously sensitive to cycloheximide, and was initially sensitive, but subsequentially resistant to inhibition by actinomycin D. Thus transcription, followed by translation, was apparently required for AHH induction by these compounds. These compounds may influence a distinct regulatory step in hydroxylase induction, since combined AHH induction by aminophyLline plus

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I~G. 4. Aryl hydrocarbon hydroxylase activity in Buffalo rat liver cells exposed to: control medium alone (A--A); control medium followed by medium containing benz(a)anthracene ( 0 - - 0 ) ; medium containing cycloheximide, followed by medium containing acfinomycin D (A--A); medium cootainln~ benz(a)anthracene plus cycloheximide, followed by medium containing benz(a) anthracene plus actinomycin D (C)----C)). After the initial treatment, each plate received two 15-rain washes with the second medium. Concentrations used were those described in the legend to Fig. 1.

Aryl hydrocarbon (benzo(a)pyrene)hydroxylaseinduction in cells in culture

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benz(a)anthracene led to greater increases in hydroxylase activity than were produced by either compound alone. A H H INDUCTION AND TYROSINE AMINOTRANSFERASE INDUCTION The A H H induction process bears certain similarities to that for tyrosine aminotransferase (TAT) a soluble, steroid-inducible hepatic enzyme. Many of the induction studies with the latter have utilized TAT-specific antibodies to detect enzyme protein. Studies of the induction of TAT suggest that the rise in aminotransferase requires the synthesis of mRNA, although the amount of mRNA has not been quantitated. These studies indicate that increased enzyme activity is accompanied by a rise in antibodyprecipitable TAT activity. Furthermore, there are no enzymatically inactive precursor TAT molecules detectable immunologically (Granner et al., 1970). Thus, such studies indicate that TAT induction requires de novo enzyme synthesis, rather than activation of pre-existing enzyme molecules. Whether this is also true for A H H induction is unknown. The induction of both A H H and TAT was studied simultaneously in the Reuber H-35 hepatoma cell line (Whitlock et al., 1974). These studies revealed that (1) both enzymes require similar inducer concentrations for induction: (2) both induction processes have a similar lag period preceding a detectable increase in enzyme activity; (3) both induction processes require RNA and protein synthesis, and that the RNA synthesis and protein synthesis steps can be dissociated; and (4) the initial rates of increase in enzyme activities are similar. Such experiments emphasize the similarities between the induction processes for the two enzymes and suggest that hydroxylase induction, like TAT induction, requires the de novo synthesis of mRNA and protein; to learn whether the induction-specific protein is actually the A H H enzyme requires in vitro systems capable of translating this hypothetical mRNA and identifying by immunological or other means the proteins which are synthesized. The studies of A H H induction in H-35 cells also revealed an unexpected phenomenon which suggests some interaction between the gene-action systems of the TAT and A H H enzymes. Dexamethasone, which induces TAT up to six-fold, has no effect on hydroxylase activity when administered alone; likewise, benz(a)anthracene, which induces A H H up to twenty-five-fold, has only a minimal effect on TAT activity. However, the simultaneous administration of both inducers has a synergistic effect on the induction of both enzymes (Table 2); the activities of each are increased substantiaily more than would be predicted from the known effect of the inducers administered separately. This synergistic effect is inhibited by actinomycin D, but not by cycloheximide (Table 2); thus, the interlocking effects of the A H H and TAT systems occur at the level of transcription rather than translation. A H H INDUCTION IN SOMATIC C E L L HYBRIDS---AHH EXPRESSION RELATED TO H U M A N CHROMOSOME 2 In vivo, genetic studies of A H H induction in inbred strains of mice indicate that the regulation of this enzyme system is complex (Nebert et al., 1975). In vitro, the technique of somatic cell hybridization has been a powerful tool for the genetic mapping of eukaryotic cells. Following the isolation of the appropriate interspecific hybrids formed by cell fusion, the ios~ of specific chromosomes can be correlated with changes in the expression of the gene(s) being studied. Using this technique to analyze hybrids formed between non-inducible mouse cells and normal, inducible human bone marrow cells, Brown et al. (1976) examined the linkage between A H H and twenty-two human isozymes; they found that the expression of A H H activity was linked with the expression of human malate dehydrogenase and isocitrate dehydrogenase, both of which had previously been mapped on human chromosome 2. The results summarized in Table 3 show that in each of thirty-one clones, the presence (or absence) of A H H activity was correlated with the presence (or absence) of malate

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TABLE 2. E~ect of lnhibitors of Macromolecular Synthesis on the Simultaneous Induction of Aryl Hydrocarbon Hydroxylase and Tyrosine Aminotransferase* Treatmentt Initial Control medium Benz(a)anthracene Dexamethasone Benz(a)anthracene + dexamethasone Benz(a)anthracene + cycloheximide Dexamethasone + cycloheximide Benz(a)anthracene + dexamethasone + cyclohexim/de Benz(a)anthracene + cycloheximide Benz(a)anthracene + cycloheximide Benz(a)anthracene + dexamethasone + cycloheximide Benz(a)anthracene + dexamethasone + cycloheximide Dexamethasone + cycloheximide Dexamethasone + cycloheximide Dexamethasone + benz(a)anthracene + cycloheximide Dexamethasone + benz(a)anthracene + cycioheximide

Secondary None None None None None None None Benz(a)anthracene + actinomycin D Benz(a)anthracene + dexamethasone + actinomycin D Benz(a)anthracene + actinomycin D Benz(a)anthracene + dexamethasone + actinomycin D Dexamethasone + actinomycin D* Dexamethasone + benz(a)anthracene + actinomycin D~ Dexamethasone + actinomycin D*

Hydroxylase Transaminase activity** Activity 3.6 12.9 23.6 !.4 1.2

8.5 17.2 34.0 5.8 8.1

18.5 17.2 27.3 26.8

Dexamethasone + benz(a)anthracene + actinomycin D~

16.9 14.9 33.6 27.6

*Values are the averages from dupficate plates of H-35 cells. Results from identically treated plates of cells varied less than 10% (--) indicates that no measurement was made. Concentrations used: benz(a)anthracene, I gg/ml; dexamethasone 0.4 ~g/ml; actinomycin D, 1 ~g/ml; cycloheximide, i t~g/ml. tTreatments were for 4 hr except as noted. • **Picomole 3-OH benzo(a)pyrene/min/mg protein. *2 hr treatment.

dehydrogenase (MDH-1); likewise, in thirty of thirty-one clones, the presence (or absence) of A H H activity was correlated with the p r e s e n c e (or absence) of isocitrate dehydrogenase ( I D H - I ) . T h e s e results suggest that the structural genes for a protein related to constitutive and induced A H H activity are located on c h r o m o s o m e 2 (Brown et al., 1976). Other studies employing the cell fusion technique suggest that the gene(s) for A H H induction are dominantly e x p r e s s e d in hybrid cells, whereas the gene(s) for T A T induction are suppressed (Benedict et al., 1972; Wiebel e t a / . , 1972a). Thus, despite similarities in their induction mechanisms, certain regulatory processes apparently differ for these two e n z y m e s . The cell fusion technique offers the potential for finding as yet u n k n o w n biochemical events in A H H induction. Such studies would involve isolation of mutants in A H H induction, their classification into c o m p l e m e n ration groups using cell fusion, and the subsequent determination of the biochemical lesion.

VARIATION IN AHH INDUCTION IN DIPLOID CLONES Studies on the variability of A H H inducibility a m o n g diploid clones of rat liver suggest that a variety of factors are involved in the regulation of h y d r o x y l a s e activity. Whitlock et ai. isolated eight near diploid clones f r o m a cloned line of rat liver cells and found that their A H H inducibility ranged f r o m near zero to ten-fold (Table 4). Thus, new diploid cells derived f r o m the s a m e parental population cell exhibit marked differences in A H H activity suggesting that non-mutational events can influence A H H activity. The f r e q u e n c y with which variants in A H H inducibility can be isolated suggests that epigenetic events are important in the regulation of h y d r o x y l a s e activity (Whitlock et al., 1976).

Aryl hydrocarbon (benzo(a)pyrene) hydroxylase induction in cells in culture

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TABLE 3. Correlation of Induced Aryl Hydrocarbon Hydroxylase* and Human lsozyme Activities in Hybrid

Clones Isozyme/AHH +/+ Chromosome 1 1 2 2 4 5 6 7 9 10 II 12 13 14 15 16 17 18 19 20 21 X

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AK-2 PEP--C MDH-! IDH-I§ PGM=2 HEX-B MF_~I /3-GLU AK-I GOT-I LDH A PEP B ES-D NP MPI-I APRT GALK PEP A GPI ADA SOD-I G6PD

3 3 3 3 1 1 3 0 0 3 2 3 3 3 2 2 0 2 1 0 3 4

10 10 10 10 2 1 6 0 0 5 7 6 9 9 1 7 0 7 0 0 10 11

5 3 0 1 I 2 I 0 0 7 4 3 9 5 3 4 2 2 3 0 4 8

4 4 0 0 3 0 5 0 0 5 3 1 8 4 0 2 0 3 0 0 9 8

1 1 0 0 2 2 0 3 3 0 2 1 0 0 2 1 3 2 2 3 I 0

0 0 0 0 8 9 4 0 0 5 3 4 1 3 9 3 2 3 10 0 0 7

3 5 9 8 5 4 8 6 9 2 4 5 0 4 5 2 3 6 6 9 4 0

5 5 9 9 5 2 4 0 0 4 5 7 I 3 8 6 0 5 8 0 0 0

9 7 0 1 4 2 6 0 0 12 7 4 17 9 3 6 2 5 3 0 13 9

1 1 0 0 10 iI 4 3 3 5 5 5 1 3 11 4 5 5 12 3 1 7

Total clones tested 31 31 31 31 27 21 31 9 12 31 30 30 31 31 30 27 10 30 30 12 31 38

*Cells were exposed to inducers in fresh growth medium for 18hr. AHH activities were assayed in homogenates of cells from duplicate cultures. Incubation time was 3 hr. AHH activity is expressed as + or - as described in Brown et al. (1976). tHuman isozymes were determined by starch gel analysis. Mouse isozymes were always expressed. Isozyme symbols are as follows: G6PD, glucose 6-phosphate dehydrogenase (E.C. 1.1.1.49); P E P A, PEP B, P E P C, peptidase (E.C. 3.4.11.x); A K - I , AK-2, adenylate kinase (E.C. 2.7.4.3); MDH-1, malate dehydrogenase (E.C. 1.1.1.37); IDH-I, isocitrate dehydrogenase (E.C. 1.1.1.42); PGM-2, phosphoglucomutase (E.C. 2.7.5.1); HEX-B, hexosaminidase (E.C. 3.2.1.30); M E - l , malic enzyme (E.C. 1.1.1.38, i.1.1.39, 1.1.1.40); /3--GLU, /~-glucuronidase (E.C. 3.2.1.31); GOT, glutamate oxaloacetate transaminase (E.C. 2.6.1.1.); LDH A, lactate dehydrogenase (E.C. 1.1.1.27); ES-D, esterase (E.C. 3.1.1.1); N-P, nucleoside phosphorylase (E.S. 2.4.2.1); MPl, mannose phosphate isomerase (E.C. 5.3.1.8); APRT, adenine phosphoribosyltransferase (E.C. 2.4.2.7); GALK, galactokinase (E.C. 2.7.1.6); GPI, glucosephosphate isomerase (E.C. 5.3.1.9); ADA, adenosine deaminase (E.C. 3.5.4.4); SOD--I, superoxide dismutase (E.C. 1.15.1.1). ~10 represents primary clones; 2° represents subclones. §IDH-I (E.C. 1.1.1.42) is the NADP oxodoreductase form of this enzyme.

TABLE 4. Aryl Hydrocarbon Hydroxylase Activity in Predominantly Diploid Rat Liver Cell Subclones* Treatment (U/mg protein) Subclone Parents (BRL-3C4) A B

C H J L N P

Total chromosome no.t 42 42 42 42 42 43 42 43 42

(40-42) 42 (41--43) (40-43) (41-43) (42--44) (40-43) (41-43) (41-43)

14/20 20/20 14/20 13/20 15/20 15120 12/20 12/20 6/20

Medium change§

Benz(a)anthracene~

57 5 22 45 27 4 34 17 47

285 58 115 336 172 6 197 63 263

*Each value represents the average from duplicate plates of cells. Hydroxylase activity of identically treated plates of cells varied less than 15 per cent. One unit of AHH activity is equivalent to 1 picomole 3-OH benzo(a)pyrene/30 min/mg protein. *Values indicate means and ranges and the fraction with a chromosome number equal to the mean. §Cells were exposed for 8 hr to fresh medium. $Cells were exposed for 8 hr to medium containing benz(a)anthracene (1 ttg/ml).

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J.P. WHITLOCK,JR. and H. V. GELBOIN GENETIC ASPECTS OF A H H IN H U M A N LYMPHOCYTES AND MONOCYTES

Twin (Alexanderson et al., 1969; Andreasen et al., 1973; Kellermann e t a / . , 1975; Vessel and Page, 1968a,b,c) and family studies (Asberg et al., 1971; Kellermann et al., 1973a,b; Motulsky, 1964; Whittaker and Price-Evans, 1970) in normal human volunteers showed that genetic factors are primarily responsible for maintaining large interindividual variations in rates of elimination of several commonly used drugs. A recent study of A H H inducibility in cultured iymphocytes suggested that genetic factors accounted for approximately three-fourths of the total interindividual variation in A H H inducibility, which ranges from 1.5 to 4.0 (Busbee et al., 1972; Kellermann et al., 1973a,b) but that either a poor or no correlation occurred between A H H inducibility in vitro and the biological half-life of various drugs (Atlas et al., 1976). A H H induction in lymphocytes generally requires addition of a mitogen such as phytohemagglutinin, the presence of which may contribute to variability. A H H has also been identified in monocytes from human peripheral blood (Bast et al., 1976). AHH inducibility can be measured in cultured monocytes without addition of mitogen and ranges up to thirtyto forty-fold. Interindividual and intraindividual variations in the basal and benz(a)anthraceneinduced levels of A H H were investigated in cultured monocytes obtained from ten sets of monozygotic and seventeen sets of dizygotic normal adult twin volunteers (Okuda et al., 1977). The values for basal levels and, therefore, for induction ratios were more variable than those of the induced level. The mean values for the induced levels ranged from 4.26 to 17.69. Intratwin differences in the induced levels were quite small within monozygotic and most dizygotic twins. However, several sets of dizygotic twins had much greater intratwin differences than did the monozygotic twins; these discordant dizygotic twins were responsible for raising heritability indices, which were between 0.57 (uncorrected) and 0.71 (corrected). Genetic factors accounted for approximately one-half to two-thirds of the total interindividual variation in A H H inducibility that occurred in this study, the other one-half to one-third of the total variation being attributable to environmental factors. Small intratwin differences among most dizygotic twins suggest that a relatively small number of genes may be involved in regulation of the induced A H H levels. Thus, both the studies with human l~/mphocytes and monocytes suggest that individuals vary in a reproducible way in A H H inducibility. These differences may relate to individual differences in drug metabolism and carcinogen susceptibility. COMMENTS To date, studies of the mechanisms of A H H induction in cells in culture have relied heavily upon the use of inhibitors of macromolecular synthesis; such experiments can yield only indirect evidence regarding the induction process. Despite their limitations, such studies are important in that they suggest potentially fruitful lines of investigation for the future. For example, there is substantial indirect evidence that there is an important event(s) at the level of transcription in hydroxylase induction. Development of methods to isolate and translate in vitro the putative AHH-mRNA would provide direct evidence that regulation at the level of transcription is important in hydroxylase induction. Likewise, the characterization of the postulated labile regulatory protein as well as the protein specie(s) whose synthesis is required for A H H induction will yield more direct, specific information about the mechanism of enzyme induction. In addition, the effect of cAMP and aminophylline on hydroxylase activity suggests that a protein kinase(s) activity and the phosphorylation of specific proteins, perhaps chromosomal proteins, may be important in A H H induction. Finally, studies of the mechanism of action of various steroid hormones have implicated the participation of cytoplasmic receptor proteins which are involved in the transport of the hormone to the nucleus (Jensen and DeSombre, 1973; O'Malley and Means, 1974). The possibility exists that analogous receptors exist for inducers of

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A H H and that the presence or absence of such proteins may play a critical role in the process of A H H induction (Poland et al., 1976; Guenthner and Nebert, 1977). A major unresolved question in the understanding of the molecular events related to A H H induction involves the properties of the protein(s) whose synthesis is required for induction. Although some studies indicate an increased cytochrome content of cells exposed to a PAH inducer, the magnitude of the increase is substantially less than the overall increase in enzynte activity. Therefore, it is unclear whether de n o v o synthesis of new enzyme occurs during A H H induction, or whether pre-existing enzyme molecules are activated. Resolution of this question would be facilitated by the development of methods (e.g. immunologic) for the specific detection of enzyme-related proteins. SUMMARY The A H H enzyme system is inducible in cells grown in culture by a wide variety of polycyclic hydrocarbons. The inducible cells include secondary cultures, cell lines, cell hybrids and lymphocytes and monocytes. Generally the enzyme level rises after a 30-60 min lag period after the addition of inducer. The increase in A H H levels can reach thirty-fold or greater at 16 hr. The induction process is sensitive to actinomycin D and is therefore dependent on RNA synthesis. This dependence occurs only during the first few hours of the induction process. The increase in A H H activity is continuously dependent on protein synthesis; the accumulation of the initial induction specific RNA is independent of protein synthesis. The induction process thus requires RNA synthesis initially, followed by protein synthesis, and these steps can be experimentally dissociated. The induction-specific RNA is not ribosomal RNA and is probably poly A-containing heterogeneous nuclear RNA. Induction can be accomplished by the addition of a P A H inducer, by temporary inhibition of protein synthesis, or by cyclic AMP. These induction processes are synergistic, suggesting that there are at least three mechanisms of A H H induction. The gene action system responsible for A H H induction interacts in some manner with the steroid system regulating tyrosine aminotransferase, since synergistic effects between the steroid and polycyclic hydrocarbons are observed. Studies with cloned cells indicated that a non-genetic regulatory mechanism may influence basal and inducible A H H levels; studies with human-mouse hybrids indicate that A H H expression is associated with human chromosome 2. Studies of A H H induction in lymphocytes and monocytes of fraternal and identical twins suggest strong genetic determinants in A H H levels in the human population. REFERENCES ALEXANDERSON,B., PRXCEEVANS,D. A. and SJOQVXgr,F. (1969) Steady-state plasma levels of nortfiptyline in twins: Influence of genetic factors and drug therapy. Br. med. J. 4, 764-768. ALFRED, L. J. and GELBOIN,H. V. 0967) Benzpyrene hydroxylase induction by polycyclic hydrocarbons in hamster embryonic cells grown/n vitro. Science, N.Y. IS'/, 75--76. ANDREASEN,P. B., FROLAND,A., SKOVSTED,L., ANDERSEN,S. A. and HAUGE,M. 0973) D/phenylhydantoin half-life in man and its inhi'bition by phenylbutazone: The role of genetic factors. Acta reed. scand. 193, 561-564. ASBERG,M., PRICEEVANS,D. A. and SJOQVIST,F. (1971) Genetic control of nortriptyline kinetics in man: A study of relatives of propositi with high plasma concentrations. J. Meal. Genet. 8, 129-135. ATLAS, S. A., VESSELL, E. S. and NEBERT,D. W. (f976) Genetic control of interindividual variations in the inducibility of aryl hydrocarbon hydroxylase in cultured human lymphocytes. Cancer Res. 36, 49194930. BAST, R. C., JR, OKUDA,T., PLOTgJN, E., TARONE,R., RAPe, H. J. and GELBOIN,H. V. (1976) Development of an assay for aryl hydrocarbon [benzo(a)pyrene] hydroxylase in human peripheral blood monocytes. Cancer Res. 36, 1967-1974. BENEDICT,W. F., NEBERT,D. W. and THOMPSON,E. B. (1972) Expression of aryl hydrocarbon'hydroxylase induction and suppression of tyrosine aminotransferase induction in somatic-cell hybrids. Proc. natn. Acad. Sci. U.S.A. 69, 2179-2183. BROWN, S., WIEBEL, F. J., GELBOIN, H. V. and MINNA, J. D. (1976) Assignment of a locus required for flavoprotein-linked monooxygenase expression to human chromosome 2. Proc. natn. Acad. Sci. U.S.A. 73, 4628--4632. JPT Vol. ,4. No,

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(1974) Female steroid hormones and target cell nuclei, science, N.Y. 183, 610--620. POLAND, A., GLOVER, E. and KENDE, A. S. (1976) Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. J. biol. Chem. 281, 4936-4946. rESELL, E. S. and PAGE, J. G. (1968a) Genetic control of drug levels in man: Phenylbutazone. Science, N.Y. 159, 1479-1480. VESELL, E. S. and PAGE, J. G. (1968b) Genetic control of drug levels in man: Anti-pyrine. Science, N.Y. 161, 72-73. VESELL, E. S. and PAGE, J. G. (1968c) Genetic control of drug levels in man. J. clin. Invest. 47, 2657-2663. WHrrLOCK, J. P., JR and GELBOIN, H. V. (1973) Induction of aryl hydrocarbon (benzo(a)pyrene) hydroxylase in liver cell culture by temporary inhibition of protein synthesis..i, biol. Chem. 248, 6114--6121. WHITLOCK, J. P., JR and GELBOIN, H. V. (1974) Aryl hydrocarbon (benzo(a)pyrene) hydroxylase induction in rat liver cells in culture. J. biol. Chem. 249, 2616-2623. WHITLOCK, J. 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WIEBEL, F. J., BADER,J. P. and GELBOrN,H. V. (1976) Enzyme induction in mammalian cells defective in 28S ribosomal RNA formation. Nature, Lond. 259, 331-333. YAMASAKI, H., HUBERMAN, E. and SACHS, L. (1975) Regulation of aryl hydrocarbon Coenzo(a)pyrene) hydroxylase activity in mammalian cells..L biol. Chem. 250, 7766--7770. YANG, S. K., McCOURT, D. W., ROLLER, P. P. and GELBOIN, H. V. (1976) Enzymatic conversion of benzo(a)pyrene leading predominantly to the diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene through a single enantiomer of r-7,t-8-dihydroxy-7,8-dihydrobenzo(a)pyrene. Proc. natn. Acad. Sci. U.S.A. 73, 2594-2598.