Metabolism of Polycyclic Aromatic Hydrocarbons …...sembles the liver microsomal cytochrome P-448 system. This metabolism generates reactive metabolites that bind covalently and,

[CANCER RESEARCH 42, 1479-1486, April 1982)0008-5472/82/0042-OOOOS02.00

Metabolism of Polycyclic Aromatic Hydrocarbons and Covalent Binding ofMetabolites to Protein in Rat Adrenal Gland1

Johan Montelius, Dimitrios Papadopoulos, Margot Bengtsson, and Jan RydstrÃ¶m2

Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden

ABSTRACT

The metabolism of the carcinogenic and adrenocorticolyticpolycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)an-thracene in rat adrenals was investigated. Both 7,12-dimeth-ylbenz(a)anthracene and benzo(a)pyrene, which also is a well-known carcinogen but has no short-term effects on rat adrenals, appear to be metabolized by one common type of cyto-chrome P-450-dependent monooxygenase localized in the en-

doplasmic reticulum. Studies of the kinetic properties of thiscytochrome P-450 reveal that the Km values for 7,12-dimeth-

ylbenz(a)anthracene and benzo(a)pyrene are lower than 3 Â¡IM.Identification of metabolites indicates that, with both 7,12-di-

methylbenz(a)anthracene and benzo(a)pyrene, phenols anddiols were formed the relative rates of formation of which weremarkedly influenced by the expoxide hydrase inhibitor cyclo-hexane oxide, suggesting that epoxides are intermediate metabolites. Added or endogenous microsomal glutathione S-transferase B had little or no effect on the distribution ofmetabolites. A rather selective binding of metabolites of 7,12-

dimethylbenz(a)anthracene to soluble and microsomal proteinswas demonstrated. The adrenal cytochrome P-450 involved inthe conversion of these polycyclic aromatic hydrocarbons appears to be unrelated to those responsible for the synthesis ofmineralocorticoids and glucocorticoids from cholesterol.Among androgens and estrogens, estradici proved to be themost inhibitory steroid, suggesting a role of the hydrocarbon-metabolizing cytochrome P-450 in estrogen biosynthesis. How

ever, no such function could be demonstrated conclusively.The metabolite patterns and the effects of nonsteroid inhibitorsof liver monooxygenases, e.g., a-naphthoflavone, SU 9055,

and ellipticine, suggest that the properties of this cytochromeP-450 resemble those of the 3-methyl-cholanthrene-induciblecytochrome P-488 from rat liver.

INTRODUCTION

Certain aromatic hydrocarbons are well-known and potent

carcinogens which, for unknown reasons, may have a relativelyhigh specificity with respect to the target organ, e.g., liver,lung, skin, and mammary glands (for reviews, see Refs. 10,16, 27, and 34). The general mechanism of action of this classof compounds is believed to involve the formation of an epoxidecatalyzed by one or several cytochrome P-450-dependent

monooxygenases localized in the endoplasmic reticulum. Reactive epoxides as well as radical derivatives and peroxidesare believed to be important intermediates in the generation ofcancer cells. Other products are diols, phenols, and conju

gates, the relative rates of formation of which are determinedby the activities of the monooxygenases, epoxide hydrase,conjugation systems (e.g., glutathione S-transferases), and

nonenzymatic reactions, respectively (7, 14, 15, 23). In someorgans, metabolism of chemical carcinogens results in destruction of mitochondria and necrosis rather than cancer, possiblybecause these organs have a relatively large amount of itsmonooxygenase systems localized in the mitochondria. However, it is generally agreed that the basic mechanisms responsible for the generation of cancer and necrosis following activation of xenobiotics are similar (4, 30, 47, 53).

An important and thus far unsolved problem in chemicalcarcinogenesis, particularly in steroid-metabolizing organs,

concerns the identity and biological function of the cytochromeP-450 systems responsible for the activation of polycyclichydrocarbons to reactive intermediates. In adrenals, DMBA3

and 2,2-bis(4-chlorophenyl,2-chlorophenyl)-1,1-dichloroeth-

ane but not BP or other known polycyclic hydrocarbons causenecrosis of the 2 inner zones of the cortex, the zona fasciculataand the zona reticularis, possibly mediated by steroid-metabo

lizing hydroxylases (2, 5, 27, 28, 36). In the rat, the adrenalxenobiotic-metabolizing system(s) is not inducible by polycyclic

aromatic hydrocarbons (12, 13, 18, 22). Specific effects ofDMBA have also been observed in testes and ovaries (1, 27,35), and it appears that the polycyclic hydrocarbon-metabolizing cytochrome P-450 systems in all these organs are regulated

by pituitary peptide hormones (22, 32). The active adrenocorticolytic metabolite of DMBA has been proposed to be 7-OHM-12-MBA and/or 3,4-dihydro-3,4-dihydroxy-7-hydroxymethyl-12-methylbenzo(a)anthracene or reactive products of these

substances formed after further metabolism (8, 27, 56). Thepresent study forms part of an investigation that aims at elucidating the mechanisms of metabolism and action of carcinogenic polycyclic hydrocarbons in steroidogenic organs, in particular, adrenals and gonads. Evidence is presented to indicatethat in rat adrenals DMBA and BP are metabolized by a monooxygenase localized in the endoplasmic reticulum which resembles the liver microsomal cytochrome P-448 system. This

metabolism generates reactive metabolites that bind covalentlyand, in the case of DMBA, rather selectively to soluble andmicrosomal proteins. A preliminary account of these resultshas been published elsewhere (36, 44).

MATERIALS AND METHODS

Adrenal glands were removed from male Sprague-Dawley rats (>180

g) after decapitation and kept frozen until used. Fractionation of theglands was carried out essentially as described previously (41). Sub-

' This work was supported by the Swedish Cancer Society (Grant 1269-B80-

01X) and the Swedish Council for Planning and Coordination of Research (GrantA1-5/241).

2 To whom requests for reprints should be addressed.

Received August 17, 1981 ; accepted December 15, 1981.

3 The abbreviations used are: DMBA, 7,12-dimethylbenz(a)anthracene, BP,benzo(a)pyrene; 7-OHM-12-MBA, 7-hydroxymethyl-12-methylbenz(a)anthra-cene; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitroben-zene.

APRIL 1982 1479

Research. on February 28, 2020. © 1982 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

J. Montelius et al.

cellular fractions were used immediately after preparation or frozen,even though slightly decreased monooxygenase activities were normally observed after freezing. Incubations for estimating DMBA and BPmetabolism were carried out in a medium composed of 2 mM isocitrate,6 mM MgCI2, 30 /ig isocitrate dehydrogenase, 0.4 ^g rotenone, 1 to 50JIM [dimef/7y/-''1C]DMBA or [14C]BP, 2 mw EDTA, 15 mw potassium

chloride, 5 mM potassium phosphate, and 50 mM Tris-HCI (pH 7.0), ina final volume of 0.2, 0.5, or 1 ml. After preincubation for 5 min at 30Â°,

the reaction was started by the addition of 1.5 mM NADPH and stoppedafter 60 min with 4 ml ethyl acetate. Analysis of the extent of conversionwas achieved by 2 extractions of metabolites (and unreacted DMBA orBP) with 4 ml ethyl acetate containing 0.04% (w/v) butylated hydrox-ytoluene, followed by thin-layer chromatography on silica gel with

toluene as eluant. Alternatively, the conversion was assayed by thedistribution method described by van Cantfort ef al. (54), which wasfound to be applicable also for DMBA. The thin-layer and the distribution assays differed by less than Â±15%. High-pressure liquid Chromatographie analysis of ethyl acetate-extractable DMBA metabolites

was carried out by a Waters Associates instrument using a NucleosideCis column equilibrated with 35% methanol and eluted with a 35 to95% methanol gradient during a period of 70 min, including a hold at50% methanol for 10 min and a second hold at 95% methanol for 10min. Covalent binding of DMBA metabolites was measured by precipitation of proteins on filter paper discs according to the method of Wallinef al. (55) or after precipitation with 10% (final concentration) trichlo-

roacetic acid, 4 extractions with 6 ml ethyl acetate and 2 extractionswith acetone. In the latter case, the residue was dissolved in 10%sodium dodecyl sulfate (pH 9.0) containing 10 mM mercaptoethanoland applied to a 5 to 15% sodium dodecyl sulfate "slab" polyacryl-

amide gel. After autoradiography, the gel was sliced, and the radioactivity was estimated by scintillation counting. All handling of DMBA, BP,and their metabolites during preparations, incubations, and assays wascarried out in the dark or in diffuse light. Radioimmunoassay of estradiciwas carried out after exchange of DMBA or BP for 10 JUMandrostene-

dione, quenching of the reaction with 4 ml diethyl ether, and separationand evaporation of the organic phase. 21-Hydroxylation of progesterone was assayed with 100 /JM [""C]progesterone (57.2 mCi/mmol).

Other conditions were as in the assay of DMBA and BP metabolism,except that the reaction was stopped by addition of 4 ml chloro-

form:methanol (4:1); the water phase was extracted once more withchlorofornrmethanol, and the thin-layer chromatography was devel

oped twice in chloroform:ethyl acetate (4:1).The effects of long-term treatments of male Sprague-Dawley rats

were followed by injecting 25 mg DMBA per kg i.p., using a solution of9 mg DMBA per ml of corn oil, for 6 consecutive days. A control groupreceived the corresponding amount of corn oil only at these occasions.Two rats from each group were sacrificed after 0, 3, 6, and 10 days,respectively. The 4 adrenals were homogenized and sonicated gently(4 times for 10 sec each) in 2 ml of 50 mM Tris-HCI (pH 7.0) containing0.25 M sucrose. Glutathione S-transferase was measured as glutathi-

one conjugation with CDNB or DCNB as substrates according to themethod of Habig ef al. (24). Cytochrome oxidase was measured withreduced cytochrome c (52), lactate dehydrogenase according to themethod of Pesce ef al. (43), and cytochrome P-450 as the dithionite-

reduced minus oxidized spectrum in the presence of carbon monoxide(42). The content or reduced glutathione in the adrenals was estimatedaccording to the method of Moron ef al. (38).

Ciba SU 9055 was a gift from Ciba-Geigy (Basel, Switzerland), and

ellipticin was a gift from National Cancer Institute (NIH, Bethesda, Md.).BP and DMBA metabolite references were gifts from Dr. J. W. DePierre(University of Stockholm, Stockholm, Sweden) and Dr. P. L. Grover(Chester Beatty Research Institute, London, England), respectively.Glutathione S-transferase B isolated from rat liver was a gift from Dr.Claes Guthenberg (University of Stockholm). [14C]DMBA (100 mCi/mmol) and ['4C]progesterone (57.2 mCi/mmol) were obtained fromMEN Chemicals (Dreeich, West Germany). [MC]BP (30 Ci/mmol) was

purchased from Amersham (Amersham, Buckinghamshire, England).

Other biochemicals were purchased from Sigma Chemical Co. (St.Louis, Mo.).

RESULTS

Subcellular Distribution of Activity. The mitochondrial andmicrosomal fractions of rat adrenal cortex were found to convert DMBA at a rate of 44.0 and 138.8 pmol/min/mg protein,respectively. Extensive washing of the mitochondrial fractiondecreased the metabolism in this fraction to 27.2 pmol/min/mg protein, which corresponds to about 19% of the microsomalactivity. A 100,000 x g supernatant fraction was inactive(Table 1). Since mitochondria under these conditions are contaminated with 10 to 15% microsomes (51 ), pure mitochondriawere assumed to be inactive. For a comparison, the adrenalmicrosomes also catalyzed conversion of BP at a rate of 120.71pmol/min/mg protein (cf. RÃ©f.22) with a similar relative conversion by the mitochondrial fraction as in the case with DMBA.

The metabolism of DMBA was linear for at least 120 min(Chart 1>\) and with protein in the range of 0.5 to 1.5 mgmicrosomal protein per ml (Chart 1B). An accurate Km proved

Table 1

Subcellular distribution of DMBA metabolism activity

Rat adrenals were fractionated, and activities were estimated as described in"Materials and Methods."

Specific activityNo. of ex- {pmol/min/mg pro-

Fraction periments tein)

Mitochondria (washedonce)Mitochondria(washed 4times)MicrosomesSoluble444444.0

Â±4.6a27.2

Â±2.0138.8Â±6.0~0

Mean Â±S.D.

30 60 90

time (min)

120

90

e so

CL

30

1

(mg>protein

Chart 1. Time W and protein (ÃŸ)dependence of DMBA metabolism in ratadrenal microsomes. The conditions were as described in "Materials and Methods." Each point represents the mean of 2 experiments, prot, protein.

1480 CANCER RESEARCH VOL. 42



Metabolism of Polycyclic Aromatic Hydrocarbons in Rat Adrenals

difficult to determine but was estimated to be lower than 3 JUM(not shown).

Analysis of Metabolites. High-pressure liquid Chromatographie analysis of reaction products indicated that, with[14C]DMBA as substrate, 3 major labeled metabolites were

formed (Table 2), the R( of which did not coincide with any ofthe available reference compounds (cf. RÃ©f.9); in addition,some minor unlabeled and unknown metabolites were formed.The more polar of the labeled metabolites (R( 0.53) was tentatively identified as a diol or a derivative of a diol. The majorunknown hydrophobic metabolite (R( 0.89) was slightly morehydrophobic than was 12-hydroxymethyl-7-methylbenzo(a)-

anthracene and may be a phenol; little or no hydroxymethylderivatives were formed. The minor hydrophobic metabolite (Rf0.91) is presumably not a phenol but may be an aldehyde or adione (quinone). This identification is supported by the factthat, in the presence of cyclohexane oxide or cyclohexaneoxide plus reduced glutathione, the formation of the hydrophilicmetabolite was decreased whereas the formation of the majorand minor hydrophobic metabolite was increased and unchanged, respectively. This is consistent with the possibilitythat the hydrophilic metabolite is primarily formed by the actionof an epoxide hydrase on an intermediate epoxide metabolite;in the presence of the epoxide hydrase inhibitor cyclohexaneoxide (40), the epoxide will undergo a nonenzymatic conversion to a phenol. However, the inhibitor also caused an overallinhibition of the conversion of DMBA which may reflect the

Table 2

Metabolism of DMBA by rat adrenal microsomes: effect of cyclohexane oxide,cyclohexane oxide plus reduced glutathione, and reduced glutathione plus

glutathione S-transferase B

Incubations, activity measurements, and analysis of metabolites were carriedout as described in "Materials and Methods." Additions were 1 HIMcyclohexane

oxide, 10 RIM reduced glutathione, and 2 units of glutathione S-transferase B.

Table 3

Metabolism of BP by rat adrenal microsomes: effect of cyclohexane oxide,cyclohexane oxide plus reduced glutathione, and reduced glutathione plus

glutathione S-transferase B

Incubations, activity measurements and analysis of metabolites were carriedout as described in "Materials and Methods." Additions were the same as those

described in the legend of Table 2.

Specific activity (pmol/min/mgprotein)Metabolite3Unknown7-OHM-1

2-MBA-5.6-diol07,12-diOHMBAUnknown7,12-diOH-DMBA7-OHM-1

2-MBA-5.6-oxide7.12-DMBA-7.12-epidioxideUnknown7-OHM-1

2-MBA17-OH-DMBA}12-OHM-7-MBAUnknownUnknown7,12-DMBA-5,6-dioneDMBATotal

activityRi0.530.600.650.680.700.730.770.790.840.850.890.910.921.00Control32.920.201.702.740.280.841.012.231.992.6959.8016.900.60123.90+

Cyclohexane oxide7.400.141.423.200.611.070.932.762.102.3671.7615.690.86110.30+

Cyclohexane oxideandglutathi

one5.190.121.601.840.300.600.351.172.001.7574.5816.760.40106.66+

Glutathione

and glutathioneS-trans

feraseB34.950.211.941.750.250.180.261.851.481.5451.667.910.32104.30

a Listed metabolites were identified with reference compounds.7-OHM-12-MBA-5,6-diol, 5,6-dihydro-frans-5,6-dihydroxy-7-hydroxymeth-

yl-12-methylbenz(a)-anthracene; 7,12-diOHMBA, 7,12-dihydroxymethylbenz-(a)anthracene; 7,12-diOH-DMBA, 7,12-dihydroxy-7,12-dimethylbenz(a)anthra-cene; 7-OHM-12-MBA-5,6-oxide, 7-hydroxymethyl-5,6-epoxy-12-methylbenz-(aÂ¡anthracene;7,12-DMBA-7.12-epidioxide, 7,12-dimethylbenz(a)anthracene-7,12-epoxide; 7-OH-DMBA, 7-hydroxy-7,12-dimethylbenz(a)anthracene; 12-OHM-7-MBA, 12-hydroxymethyl-7-methylbenz(a)anthracene; 7,12-DMBA-5.6-dione. 7,12-dimethylbenz(a)anthracene-5,6-dione.

Specific activity (pmol/min/mgprotein)Metabolite8EpoxidesBP-gjO-diol"UnknownBP-4,5-diolBP-7,8-diolQuiÃ±ones9-OH-BP3-OH-BPBPTotal

activityR.0.07-0.110.21

10.23J0.320.430.51-0.680.730.761.00ControlNDC8.06NO13.147.8143.4948.21120.71+

Cyclohexane oxideND4.28ND2.1912.8642.1051.53112.96+

Cyclohexane oxideandglutathi

oneND1.31ND1.236.9834.2543.9387.70+

Glutathione

and glutathioneS-trans

feraseBND6.42ND13.599.2329.6637.8596.75

Listed metabolites were identified with reference compounds.0BP-9,10-diol. 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene; BP-4,5-diol,

4,5-dihydro-4,5-dihydroxybenzo(a)pyrene; BP-7,8-diol. 7,8-dihydro-7,8-dihy-droxybenzo(a)pyrene; 9-OH-BP, 9-hydroxybenzo(a)-pyrene; 3-OH-BP, 3-hy-

droxybenzo(a)pyrene.0 ND, not detected.

interaction of this compound with cytochrome P-450 (ci. RÃ©f.

40). No or few changes were seen in the absence of cyclohexane oxide but in the presence of conjugation systems, e.g.,transferase B (Table 2), except for a 50% decrease in theformation of the minor hydrophobic metabolite. Exchange ofmicrosomes for mitochondria in the incubation gave a metabolite pattern which was qualitatively identical to that obtainedwith microsomes (not shown). This supports the assumptionthat the mitochondrial activity represents a microsomal contamination and suggests, in addition, that mitochondria do notcatalyze a secondary conversion of DMBA metabolites. With[14C]BP as substrate, all metabolites were labeled; these were

mainly 3-hydroxybenzo(a)pyrene, 9-hydroxybenzo(a)pyrene,and an unknown metabolite with a Rf close to that of 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene, tentatively identifiedas a diol (Table 3). QuiÃ±ones and 7,8-dihydro-7,8-dihydroxy-

benzo(a)pyrene were also formed. Also in this case cyclohexane caused an overall inhibition and a diol-phenol shift of boththe 9,10- and 7,8-diols, indicating 2 possible intermediate

epoxide metabolites, but the extent of the increased phenolformation was less pronounced than with DMBA as substrate.

Effect of Inhibitors. Microsomal DMBA metabolism wasNADPH dependent and sensitive to carbon monoxide, indicating that the enzyme system(s) involved is cytochrome P-450

dependent (Table 4). Other inhibitors included BP, estradiol,and SU 9055, an inhibitor of 17a-hydroxylase (6). Aminopyrine

and metyrapone, a substrate (17) and inhibitor (33), respectively, of cytochrome P-450 from liver microsomes and adrenal

mitochondria, were without effect at 500 /Â¿M(Table 4). On theother hand, a- and /S-naphthoflavone (58) and ellipticine (21),inhibitors of methylcholanthrene-inducible cytochrome P-448

in rat liver, were strongly inhibitory, the most potent beingellipticine (Table 4). The inhibition of DMBA metabolism by BPindeed suggests that these 2 polycyclic hydrocarbons weremetabolized by the same cytochrome P-450 system. To further

explore this possibility, the inhibition of DMBA metabolism by

APRIL 1982 1481



J. Montelius et al.

Table 4Effects of various cytochrome P-450 inhibitors on DMBA metabolism in rat adrenal microsomes

The concentration of DMBA was 50 JIM.

ConditionsMicrosomes-*-

Carbonmonoxide0+

BP+Metyrapone+Aminopyrine+a-Naphthoflavone+/Â¡-NaphthoflavonetEllipticine+

SU9055+SKF525-A-I-Ethyl morfinConcen

trationofinhibitor<|iM)50050050050050010500500500No.ofexperi

ments144444444444Specific

activity"(pmol/min/mg

protein)77.7

Â±9.763.8

Â±1.917.3Â±4.271

.6 Â±8.673.1Â±10.117.5Â±

2.865.0Â±10.76.0Â±1.212.7Â±

2.077.4Â±5.866.4Â±14.0Inhibition(%)95.177.77.85.973.416.292.383.60.314.4P<0.001<0.001<0.01<0.05<0.001<0.01<0.001<0.001MS"<0.05

All values have been corrected for the activity obtained (3.3 Â±0.4 pmol/min/mg protein; nwhere NADPH was omitted.

0 Mean Â±S.D.0 Bubbling for 3 min.'' NS, no statistically significant difference.

13)

100

500

[DMBA]or [BP]Chart 2. Inhibition of adrenal DMBA metabolism by BP and wee versa. The

concentration of DMBA (D) and BP (O) was 50 /IM.

100

50

100 200 300 Â¿00

[SU 9055] (HM)

500

Chart 3. Inhibition of adrenal DMBA and BP metabolism by SU 9055. Theconcentration of DMBA O and BP (O) was 50 Â¡iu.

BP and vice versa was investigated more carefully. As may beseen in Chart 2, increasing concentration of the inhibiting, I.e.,unlabeled, hydrocarbon led to a maximal inhibition of about75%. With 50 fiM DMBA as substrate and BP as inhibitor (Chart2), half-maximal inhibition was obtained at a BP concentrationof about 20 juM. In the reverse situation, with 50 fiM BP assubstrate, 60 /Â¿MDMBA was required for half-maximal inhibition(Chart 2). Inhibition of DMBA and BP metabolism by SU 9055showed a similar discrepancy; i.e., as compared to DMBAmetabolism, BP metabolism required more than 2 times theconcentration of SU 9055 to reach 50% inhibition (Chart 3).

A variety of steroids and steroid analogs were also tested aspossible inhibitors of DMBA metabolism (Table 5). Althoughnone was particularly potent, progesterone and estradiol werethe most efficient among the steroids whereas spironolactonewas the most efficient steroid analog. Attempts to characterizethe cytochrome P-450 responsible for the adrenal metabolismof polycyclic hydrocarbons by measuring the effects of DMBAon various steroid hydroxylases (not shown) have confirmedthe results shown in Table 5, i.e., that the substrates of theside-chain cleavage systems for cholesterol and 17a-hydrox-ypregnenolone and the 11/3-, 17a-, 18-, and 19-hydroxylasesystems had no effect on DMBA metabolism, indicating thatnone of these hydroxylases are involved in DMBA metabolism.The 21-hydroxylase activity assayed with 100 Â¡J.Mprogesterone

as substrate was inhibited to 36% by 500 /tw DMBA (notshown). However, as judged from the inhibition of DMBA metabolism by progesterone (cf. Table 5), the DMBA-metabolizingcytochrome P-450 has an approximately 20-fold higher affinityfor DMBA than for progesterone. If 21-hydroxylase was responsible for DMBA metabolism, one would therefore expectan almost total inhibition of progesterone conversion at aDMBA:progesterone ratio of 5. The fact that this was not seensuggests that 21-hydroxylase is unrelated to DMBA metabolismand that the effects of steroids on DMBA metabolism and viceversa are unspecific. The activity of the aromatase system withandrostenedione as substrate was not measurable by radioim-munoassay (less than 0.5 pmol per mg microsomal protein perhr), and the possible effect of DMBA on this system couldtherefore not be tested (not shown).

Covalent Bindingof DMBA Metabolites to Protein. Chart 4shows 4 different peptides in a microsomal plus soluble adrenalfraction (25,000 x g supernatant) that were demonstrated tobe labeled after incubation with [14C]DMBA, electrophoretic

separation on a slab polyacrylamide gel, autoradiography, andscanning. The molecular weights of these peptides were about47,000, 28,000, 25,000, and 13,000. The corresponding experiment with only microsomes gave 5 major labeled peptideswith molecular weights of 59,000, 50,000, 47,000, 25,000,and 13,000. These peptides may tentatively be identified as





cytochrome P-450 (M, 59,000 and 50,000; cf. Refs. 20, 22,and 25) and glutathione S-transferases (M, 25,000 and 28,-

000; cf. Ref. 24). The peptide with a molecular weight of13,000 comigrates with cytochrome c from beef. For unknownreasons, peptides labeled by covalent binding of BP metabolites could not be separated properly. Omission of NADPH oraddition of SU 9055 or carbon monoxide markedly inhibitedlabeling of protein (Table 6), indicating that generation ofprotein-bound radioactivity was related to the cytochrome P-450-dependent conversion of [14C]DMBA or [14C]BP to reactive

metabolites. Quantitatively, there was no significant differencebetween DMBA and BP in the generation of protein-bound

metabolites (Table 6). Addition of mitochondria to the incubation of microsomes with DMBA resulted in a decreased specificbinding of metabolites to protein (Table 6).

Table 5

Effects of various steroids and steroid analogs on DMBA metabolism in ratadrenal microsomes

All steroids and steroid analogs were added at a final concentration of 500pM. The concentration of DMBA was 50 /IM.

ConditionsMicrosomes+

Cholesterol+Pregnenolone-I-Progesterone+Deoxycorticosterone+Corticosterone+

17-Hydroxypregnenolone+17-Hydroxyprogesteroneâ€¢f

Testosterone+

Androstendione+19-Hydroxytestosterone+

19-Hydroxyandrostendione+^-Estradici+Estrone+Estriol+3-Desoxyestrone"+

D-Equilenin+Stilbestrol+

SpironolactoneNo.

ofexperiments14444444444444444444Specific

activity8 (pmol/

min/mg protein)77.7

Â± 9.7b76.0

Â±8.774.7Â±11.565.3Â±11.670.4

Â±6.970.7Â±5.077.3Â±4.473.2Â±8.669.1Â±8.367.1Â±6.869.5Â±11.666.9Â±7.662.8Â±7.969.6Â±6.566.8Â±9.886.5Â±3.065.0Â±1.273.2Â±3.654.4Â± 5.3Inhibi

tion(%)2.13.716.09.39.00.55.811.013.610.413.819.210.314.016.36.029.9PNSCNS<0.05NSNSNS<0.01<0.01<0.001<0.05<0.001<0.01<0.01<0.001<0.01NS<0.001

See Footnote a in Table 4.' Mean Â±S.D.: NS, no statistically significant difference.' Resulted in 11.3% stimulation (p < 0.05).

start

t t t t

68K60K 43K 29K

Chart 4. Covalent binding of DMBA metabolites to soluble plus microsomalproteins (lower trace) and microsomal proteins (upper trace). The conditionswere as described in "Materials and Methods."

Table 6

Effect of inhibitors on covalent binding of DMBA and BP metabolites to adrenalmicrosomal, soluble, and mitochondrial protein

The conditions were as described in Table 1 except that the concentration ofDMBA and BP was 20 UM in a final volume of 200 pi. Additions were 500 /IM SU9055, 0.2 mg protein of microsomes, 0.2 mg protein of mitochondria, and 0.24mg protein of cytosol.

ConditionsMicrosomes-

NADPH+

SU9055+Carbonmonoxide+

Soluble-I-MitochondriaMicrosomes-

NADPH+

SolubleSubstrateDMBADMBADMBADMBADMBADMBABPBPBPNo.

ofexperiments424442424Covalent

binding8

(pmol/min/incuba-tion)0.830

Â±0.00960.051

Â±0.0060.062Â±0.0050.113Â±0.0131

.000 Â±0.0460.749Â±0.0020.852

Â±0.0400.131Â±0.0081.102Â±0.022

a Estimated according to Wallin ef al. (55).6 Mean Â±S.D.

I20

-100

80

120

J10

90

3 6Days

3 6Days

Chart 5. Effects of DMBA administered in vivo on adrenal enzyme activities.Sprague-Dawley rats were administered DMBA i.p. during 6 days (see "Materialsand Methods") after which various enzyme activities of the adrenals were

measured. Total glutathione S-transferase activity (A: â€¢CDNB assay; â€¢,DCNBassay) and total lÃ¡clate dehydrogenase activity (B) in adrenals from DMBA-treated rats are expressed as a percentage of those from untreated rats. Day 0denotes the start of DMBA administration.

Effect of DMBA on Adrenal Cortex Enzymes in Vivo. Attempts were made to correlate covalent binding of [14C]DMBA

metabolites to microsomal and soluble proteins with the in vivoactivities of these proteins following DMBA administration. MaleSprague-Dawley rats were fed DMBA for 6 days (cf. "Materialsand Methods") and sacrificed, and the activities of the various

proteins were measured. As shown in Chart 5A, the totalactivity or reduced glutathione S-transferase declined about20% after 10 days of exposure to DMBA. Calculated as specificactivity, the decrease was about 40% (not shown). With DCNBinstead of CDNB as substrate, the total activity remained virtually constant (Chart 5A). The decreased activity found withCDNB as substrate indicates that a glutathione S-transferaseof the B-type (24) was inhibited by the DMBA treatment through

covalent binding of reactive DMBA metabolites to the protein.In contrast, the total activity of lactate dehydrogenase increased about 25% after 10 days of exposure to DMBA (Chart56). The same patterns were shown for mitochondrial andmicrosomal cytochrome P-450 content of reduced glutathione

and cytochrome oxidase (not shown). This general increase inprotein and total activity presumably represents the normalDMBA-induced proliferation described by others (2).

DISCUSSION

The present data show that the adrenocorticolytic agent

APRIL 1982 1483



J. Montelius et al.

DMBA is metabolized by one or more P-450-dependent mono-

oxygenase systems localized in the endoplasmic reticulum.The mitochondrial fraction containing the bulk of cytochromeP-450 (23) was inactive. In the presence of BP, which is not anadrenocorticolytic agent (27) but which has been shown to bemetabolized by adrenals (1 3, 18, 22, 56), the DMBA metabolism was inhibited to a maximum of 75%. Since this interactionalso occurred in the reverse case, i.e., BP metabolism wasinhibited to 75% by DMBA, it may be concluded that the 2hydrocarbons are metabolized predominantly by one commoncytochrome P-450. The residual activity, which was inhibited

by SU 9055 close to 100% independently of the substrateused, may involve the same cytochrome P-450, in which case

the incomplete inhibition by a competing substrate may be duea lack of sufficient substrate accessibility in the presence oftoo high concentrations of hydrocarbons. Thus, contributionsof more than one cytochrome P-450 to the overall DMBA

metabolism appear unlikely but cannot be excluded at thepresent stage.

Both DMBA and BP appear to be metabolized to diols andphenols, which together with the striking diol-phenol transitions

occurring in the presence of cyclohexane oxide may be takenas strong evidence for the involvement of epoxide intermediates. This is also consistent with a previous demonstration ofthe presence of epoxide hydrase in this tissue (39). However,the detailed metabolite patterns for DMBA and BP are different.DMBA apparently generates only one type of diol. Inhibition ofepoxide hydrase leads to the conversion of this metabolite tothe second major but more hydrophobic metabolite, consequently proposed to be a phenol derivative. The third mainmetabolite is also hydrophobic but is influenced only by gluta-thione S-transferase from rat liver and 1s therefore probablynot derived from an epoxide. Interestingly enough, it has beenshown (37) that glutathione S-transferase B catalyzes the formation of glutathione conjugates with quiÃ±ones. The minorhydrophobic metabolite may therefore be tentatively identifiedas a quinone, which may be involved in the subsequent reactions to epoxide, diol, and phenol; a definite identification ofunknown metabolites will have to await a mass spectrometricanalysis. Nevertheless, the data allow the conclusion that ratadrenals, in contrast to rat liver (cf. Refs. 29, 48, and 49), donot form significant amounts of the potent adrenocorticolyticand carcinogenic agent 7-OHM-12-MBA (8, 27, 57).

With regard to BP metabolism, it is interesting to note thatadrenal microsomes produce a metabolite pattern that resembles that of 3-methylcholanthrene-induced liver microsomes(46, 48, 49), suggesting the involvement of a C-type of cytochrome P-450 (Ref. 3; see also Refs. 20, 22, and 25).

The presence of both, 9,10- and 7,8-diols suggests that theproposed ultimate carcinogen 7,8-dihydroxy-9,10-epoxy-7,-8,9,10-tetrahydrobenzo(a)pyrene is an intermediate in theoverall adrenal BP metabolism. It may also be concluded thatthe selective adrenocorticolytic effect of DMBA as comparedto BP presumably is not due to any major qualitative differences, i.e., with respect to intermediate epoxides, phenols,and diols, or quantitative differences in metabolite patternsbetween the 2 hydrocarbons.

As to the identity and biological function of the DMBA-me-

tabolizing system, the slightly inhibitory effects of high concentrations of steroids, especially progesterone and estrogens,presumably represent unspecific interactions with the DMBA-

metabolizing system. This conclusion is further substantiatedby the fact that the affinities of steroid hydroxylases for theirsteroid substrates appear to be in the fiM range (31, 45).However, it cannot be excluded at the present time that 21-hydroxylase contributes to the metabolism of DMBA. A role ofa steroid hydroxylase in DMBA metabolism is supported by thefinding that SU 9055, a potent inhibitor of 17a-hydroxylase (6)

and liver BP metabolism (44), also inhibits DMBA metabolism.Such a mechanism is in agreement with a previous report (cf.Ref. 19 but see Ref. 11 ) on the relationship between metabolism of steroids and xenobiotics in the guinea pig. It is obvious,however, that the specificity of the hydrocarbon-metabolizingsystem(s) in the guinea pig is very different from that in the rat.Direct effects of estradiol on DMBA-induced adrenal necrosis

have been demonstrated earlier (26, 50).That covalent binding of DMBA metabolites to adrenal protein

apparently occurs essentially without the formation of 7-OHM-12-MBA as a major metabolite suggests that 7-OHM-12-MBA

is not an obligatory intermediate in the generation of reactivemetabolites. However, since 7-OHM-12-MBA is generated by

the liver (49) and is metabolized by the adrenal (49), it is stillpossible that, in vivo, adrenal necrosis is caused by metabolitesof 7-OHM-12-MBA generated in the adrenal. Moreover, prevention of necrosis by induction of liver cytochrome P-448 by,e.g., BP or 3-methylcholanthrene, which causes an increased

ring hydroxylation of DMBA as well as hydroxylation of methylgroups (13, 29, 48, 49), may involve different types of protective mechanisms. Ring-hydroxylated derivatives may competewith methyl-hydroxylated derivatives for further adrenal metab

olism, and/or adrenal metabolism of liver products may beinhibited through a direct and competing binding to the cytochrome P-450 by the inducer.

The extensive binding to microsomal and soluble proteinsmay indicate that the seemingly specific and primary destruction of mitochondria that precedes necrosis (2) is a secondaryevent possibly caused by a direct effect of microsomal metabolites. Alternatively, a mitochondrial metabolism of DMBA orDMBA metabolites has been considered (cf. RÃ©f.2). However,the present results seem to exclude at least a quantitativelyimportant mitochondrial pathway for DMBA and/or its metabolites. This conclusion is substantiated by recent and preliminary results that indicate that 7-OHM-12-MBA is not metabo

lized when incubated with mitochondria. Both the in vitro andthe in vivo data may suggest that inactivation of a glutathioneS-transferase through covalent modification may contribute to

the toxic effects of DMBA on rat adrenals. Whether there areany differences between DMBA and BP in this respect whichmay provide a partial explanation for the specific adrenocorticolytic effect of DMBA is presently being investigated.

ACKNOWLEDGMENTS

The authors are indebted to Dr. Peter Eneroth, Hormone Laboratory, Department of Obstetrics and Gynecology. Karolinska Hospital. Stockholm, for carryingout the radioimmunoassay of estradiol.

REFERENCES

1. Ahlquist, K. A. Enzyme changes in rat testis produced by the administrationof busulphan and of 7,12-dimethylbenz(a)anthracene. J. Reprod. FÃ©rtil.,12:377-379, 1966.

2. Belloni, A. S., Mazzocchi, G., Robba. C., Gambino, A. M., and Nussdorfer,G. An ultrastructural, morphometric and autoradiographic study of the





effects of 7,12-dimethylbenz(a)anthracene on the rat adrenal cortex. Vir-chows Arch. B Cell. Pathol., 26: 195-214. 1978.

3. Botelho, L. H., Ryan, D. E., and Levin, W. Amino acid compositions andpartial amino acid sequences of three highly purified forms of liver micro-somal cytochrome P-450 from rats treated with polychlorinated biphenyls,phÃ©nobarbital, or 3-methylcholanthrene. J. Biol. Chem., 254; 5635-5640,

1979.4. Brodie, B. B.. Reid, W. D., Cho, A. K., Sipes, G., Krishna, G., and Guette, J.

R. Possible mechanism of liver necrosis caused by aromatic organic compounds. Proc. Nati. Acad. Sei. U. S. A., 68: 160-164, 1971.

5. Cefis, F., and Goodall, C. M. Distribution and species limitation of the adrenallesions induced by 7,12-dimethylbenz(a)anthracene. Am. J. Pathol., 46:227-243, 1965.

6. Chart, J. J., Sheppard, H.. Mowles, T., and Howie, N. Inhibitors of adrenalcorticosteroid 17a-hydroxylation. Endocrinology, 71. 479-486, 1962.

7. Chen, C., and Tu, M.-H. Transannular Dioxygenation of 9,10-dimethyl-1,2-benz(a)anthracene by cytochrome P-450 oxygenase of rat liver. Biochem.J., reo. 805-808, 1976.

8. Chou, M. W., and Yang, S. K. Identification of four frans-3,4-dihydrodiolmetabolites of 7.12-dimethylbenz(a)anthracene and their in vitro DNA-bind-ing activities upon further metabolism. Proc. Nati. Acad. Sei. U. S. A., 75.5466-5470, 1978.

9. Chou, M. W., and Yang, S. K. Combined reversed-phase and normal-phasehigh-performance liquid chromatography in the purification and identificationof 7,12-dimethylbenz(a)anthracene metabolites. J. Chromatog., 785. 635-

654, 1979.10. Clayson, D. B. The aromatic hydrocarbons and related compounds, /n: D. B,

Clayson (ed.), Chemical Carcinogenesis, pp. 135-175. London' J. & A.

Churchill, Ltd., 1962.11. Colby, H. D.. Marquess, M. L., Johnson, P. B., and Pope, M. R. Effects of

steroid hormones in vitro on adrenal xenobiotic metabolism in the guineapig. Biochem. Pharmacol., 29. 2373-2377, 1980.

12. Dao, T. L., and VÃ¡rela, R. M. On the mechanism of inducing protection ofthe adrenal cortex against injury from 7,12-dimethylbenz(a)anthracene.Cancer Res., 26. 1015-1021, 1966.

13. Dao, T. L., and Yogo, H. Effects of polynuclear aromatic hydrocarbons onbenzpyrene hydroxylase activity in rats. Proc. Soc. Exp. Biol. Med., 116:1048-1050, 1964.

14. DePierre, J. W., and Ernster, L. Enzyme topology of intracellular membranes.Annu. Rev. Biochem., 46: 201-262, 1977.

15. DePierre, J. W., and Ernster, L. The metabolism of polycyclic hydrocarbonsand its relationship to cancer. Biochim. Biophys. Acta, 4 73:149-186,1978.

16. DiGiovanni, J., and Juchau, M. R. Biotransformation and Bioactivation of7,12-dimethylbenz(a)anthracene (7,12-DMBA). Drug Metab. Rev., 11: 61-

101, 1980.17. Ernster, L., and Orrenius, S. Substrate-induced synthesis of the hydroxyl-

ating enzyme system of liver microsomes. Fed. Proc., 24: 1190-1199,1965.

18. Gelboin, H. V., and Blackburn, N. R. The stimulatory effect of 3-methylcholanthrene on benzypyrene hydroxylase activity in several rat tissues: inhibition by actinomycin D and puromycin. Cancer Res., 24: 356-360, 1964.

19. Greiner, J. W., Kramer, R. E.. Rumbaugh, R. C., and Colby, H. D. Differentialcontrol of adrenal drug and steroid metabolism in the guinea pig. Life Sci.,20. 1017-1026. 1977.

20. Guengerich, F. P. Separation and purification of multiple forms of microsomalcytochrome P-450. J. Biol. Chem., 253. 7931-7939, 1978.

21. Guenthner, T. M.. Kahl, G. F., and Nebert, D. W. NADPH-cytochrome P-450ReducÃase: preferential inhibition by ellipticine and other type II compoundshaving little effect on NADPH-cytochrome c reducÃase. Biochem. Pharmacol., 29: 89-95, 1980.

22. Guenthner, T. M., Nebert, D. W., and Menard, R. H. Microsomal aryl hydrocarbon hydroxylase in rat adrenal regulation by ACTH but not by polycyclichydrocarbons. Mol. Pharmacol., 15: 719-728. 1979.

23. Gunsalus, I. C., Pedersen, T. C., and Sugar, S. G. Oxygenase-catalyzedbiological hydroxylations. Annu. Rev. Biochem., 44: 377-407, 1975.

24. Habig, W. H., Pabst. M. J., and Jacoby, W. B. Glutathione S-transferases.The first enzymatic step in mercapturic acid formation. J. Biol. Chem.. 249:7130-7139, 1974.

25. Haugen, D. A., and Coon, M. J. Properties of electrophoretically homogenous phenobarbital-inducible and /J-naphthoflavone inducible forms of livermicrosomal cytochrome P-450. J. Biol. Chem., 25). 7929-7939, 1976.

26. Horvath, E., Somogyi, A., and Kovacs, K. Effect of estradiol on 7,12-di-methylbenz(a)anthracene-induced adrenocortical necrosis. A histochemicalstudy. Arch. Geschwultstforsch.. 37: 203-209, 1971.

27. Huggins. C. B. Selective cell destruction in the adrenal cortex and testisproduced by 7,12-dimethylbenz(a)anthracene. In: C. B. Huggins (eds.),Experimental Leukemia and Mammary Cancer. Induction, Prevention andCure, pp. 130-213. Chicago: The University of Chicago Press, 1979.

28. Huggins, C., and Fukunishi, R. J. Induced protection of adrenal cortexagainst 7,12-dimethylbenz(a)anthracene. Exp. Med., 7/9: 923-942, 1964.

29. Jellinck, P. A., and Goudy. B. Effect of pretreatment with polycyclic hydrocarbons on the metabolism of dimethylbenz(a)anthracene-12-'4C by rat liverand other tissues. Biochem. Pharmacol.. 16: 131-141. 1967.

30. Jollow, D. J., Mitchell, J. R., Potter, W. 2., Davis, D. C.. Guette. J. R.. andBrodie, B. B. Acetamineophen-induced hepatic necrosis. II. Role of covalentbinding in vivo. J. Pharmacol. Exp. Ther., 787. 195-202. 1973.

31. Kominami, S., Ochi, H., Kobayashi, Y., and Takemori, S. Studies on thesteroid hydroxylation system in adrenal cortex microsomes. Purification andcharacterization of cytochrome P-450 specific for steroid C-21 hydroxylation. J. Biol. Chem., 255: 3386-3394, 1980.

32. Lee, J. P., Suzuki, K., Mukhtar, H., and Bend, J. R. Hormonal regulation ofcytochrome P-450-dependent monooxygenase activity and epoxide-me-tabolizing enzyme activities in testis of hypophysectomized rats. CancerRes., 40: 2486-2492. 1980.

33. Liddle, G. W., Island, D., Lance, E. M., and Harris, A. P. Alterations ofadrenal steroid patterns in man resulting from treatment with a chemicalinhibitor of 11/S-hydroxylation. J. Clin. Endocrinol. Metab., 78. 906-912,1958.

34. Little, J. B., and OToole, W. F. Respiratory tract tumors in hamsters inducedby benzo(a)pyrene and 210Po-radiation. Cancer Res., 34:3026-3039,1974.

35. Mattison, D. R., and Thorgeirsson. S. S. Genetic differences in mouseovarian metabolism of benzo(a)pyrene and oocyte toxicity. Biochem. Pharmacol., 26:909-912, 1977.

36. Montelius, J., and Rydestrom, J. Metabolism of 7,12-dimethylbenz-(a)anthracene in rat adrenals. In: M. J. Coon, A. H. Conney, R. W. Estabrook,H. V. Gelboin, J. R. Gillette, and P. J. O'Brien (eds.), Microsomes, Drug

Oxidations and Chemical Carcinogens, Vol. 2, pp. 1231-1234. New York:Academic Press, Inc., 1980.

37. Morgenstern, R., DePierre, J. W., Lind. C.. Guthenberg, C., Mannervik, B.,and Ernster, L. Benzo(a)pyrene quiÃ±ones can be generated by lipid peroxi-dation and are conjugated with glutathione. Biochem. Biophys. Res. Commun. 99: 682-690, 1981.

38. Moron. M. S., DePierre, J. W., and Mannervik, B. Levels of glutathione,glutathione reducÃaseand glutathione S-transferase activities in rat lung andliver. Biochim. Biophys. Acta, 582: 67-78, 1979.

39. Oesch, F., Glatt, H., and Schmassmann, H. The apparent ubiquity of epoxidehydratase in rat organs. Biochem. Pharmacol., 26: 603-607, 1977.

40. Oesch, F., Kaubisch, N., Jerina, O. M., and Daly, J. W. Hepatic epoxidehydrase. Structure-activity relationships for substrates and inhibitors. Biochemistry, 70:4858-4866, 1971.

41. Ogle, T. F. Effects of pregnancy on steady-state kinetics of 21-hydroxylaseand 11/8-hydroxylase in the rat adrenal gland. J. Steroid Biochem., 8:1033-1036, 1977.

42. Omura, T., and Sato, R. The carbon monooxide-binding pigment of livermicrosomes. J. Biol. Chem., 239: 2370-2378, 1964.

43. Pesce, A.. McKay, R. H., Stolzenbach, F., Cahn, R. D., and Kaplan, N. O.The comparative enzymology of lactic dehydrogenases. J. Biol. Chem., 239:1753-1761, 1964.

44. RydstrÃ¶m, J., Montelius, J., Papadopoulos, D., Hallberg, E., Helfer, L., andBengtsson, M. Metabolism of polycyclic hydrocarbons in adrenal cortex: amodel for chemical carcinogenesis in steroidogenic organs. In: J.-A. Gus-tafsson, J. Carlstedt-Duke, A. Mode, and J. Rafter (eds.), Biochemistry,Biophysics and Regulation of Cytochrome P-450, pp. 491 -497. Amsterdam:Elsevier-North Holland Publishing Co., 1980.

45. Sato, H., Ashida, N., Suhara, K., Itagaki, E., Takemori, S., and Katagiri, M.Properties of an adrenal cytochrome P-450 (P-459i,/,) for the hydroxylationsof corticosteroids. Arch. Biochem. Biophys., 790. 307-314, 1978.

46. Selkirk, J. K., Croy, R. G., Roller, P. P., and Gelboin, H. V. High pressureliquid Chromatographie analysis of benzo(a)pyrene metabolism and covalentbinding and the mechanism of action of 7,8-benzoflavone and 1,2-epoxy-3,3,3-trichloropropane. Cancer Res.. 34: 3474-3480, 1974.

47. Shimada, T., and Sato, R. Covalent binding in vitro of polychlorinatedbiphenyls to microsomal macromolecules. Biochem. Pharmacol., 27: 585-593, 1978.

48. Sims, P. Qualitative and quantitative studies on the metabolism of a seriesof aromatic hydrocarbons by rat liver preparations. Biochem. Pharmacol.,79. 795-818, 1970.

49. Sims, P. Studies on the metabolism of 7-methylbenz(a)anthracene and 7,12-dimethylbenz(a)anthracene and its hydroxymethyl derivatives in rat liver andadrenal homogenates. Biochem. Pharmacol., 79. 2261-2275, 1970.

50. Somogyi, A., and Kovacs, K. Effect of various steroids on the adrenalnecrosis induced by 7,12-dimethylbenz(a)anthracene in rats. Rev. Can.Biol.. 29: 169-180, 1970.

51. Sottocasa, G. L. The isolation of mitochondria and their membranes. In: A.H. Maddy (ed.), Biochemical Analysis of Membranes, pp. 55-78. New York:John Wiley & Sons, Inc., 1976.

52. Sottocasa, G. L., Kuylenstierna, B., Ernster. L., and Bergstrand, A. Anelectron-transport system associated with the outer membrane of livermitochondria. J. Cell Biol., 32: 415-438. 1967.

53. Thor. H.. MoldÃ©us, P., Hermanson, R., HÃ¶gberg, J., Reed. D. D., andOrrenius, S. Metabolic activation and hepatoxicity. Toxicity of bromoben-zene in hepatocytes isolated from phÃ©nobarbital and diethylmaleate-treatedrats. Arch. Biochem. Biophys., 788. 122-129. 1978.

54. van Cantfort. J., De Grave. J., and Gielen, J. E. Radioactive assay for arylhydrocarbon hydroxylase. Improved method and biological importance.Biochem. Biophys. Res. Commun.. 79: 505-512, 1977.

APRIL 1982 1485



J. Montelius et al.

55. Wallin, H., Schelin, C., Tunek, A., and Jergil, B. A rapid and sensitive method Adrenal necrosis induced by 7-hydroxymethyl-12-methyl-benz(a)-for determination of covalent binding of benzo(a)pyrene to proteins. Chem. anthracene and its prevention. Nature (Lond.), 277: 1311 -1312. 1966.Biol. Interact., 38: 109-118. 1982. 58. Wiebel, F. J., Leute. J. C.. Diamond, L., and Qelboin. H. V. Aryl hydrocarbon

56. Wattenberg, L. W., Leong, J. L., and Stroud, P. J. Benzpyrene hydroxylase (benzo(a)pyrene) hydroxylase in microsomes from rat tissues: differentialactivity in the gastrointestinal tract. Cancer Res.. 22: 1120-1125, 1962. inhibition and stimulation by benzoflavones and organic solvents. Arch.

57. Wheatly. D. N.. Hamilton, A. G., Currie. A. R., Boyland, E., and Sims, P. Biochem. Biophys., 144: 78-86, 1971.




1982;42:1479-1486. Cancer Res Johan Montelius, Dimitrios Papadopoulos, Margot Bengtsson, et al. Binding of Metabolites to Protein in Rat Adrenal GlandMetabolism of Polycyclic Aromatic Hydrocarbons and Covalent

Updated version

http://cancerres.aacrjournals.org/content/42/4/1479

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/42/4/1479To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Documents

Metabolism of Polycyclic Aromatic Hydrocarbons …...sembles the liver microsomal cytochrome P-448 system. This metabolism generates reactive metabolites that bind covalently and,