168, 609621,


Metabolism of Monohydroxybenzo(a)pyrenes by Rat Liver Microsomes and Mammalian Cells in Culture FRIEDRICH Chemistry











9, 1974

The 3-, 6-, and 9-monohydroxybenzo(a)pyrenes are metabolized by microsomes from rat liver in uitro. The metabolism of 3-hydroxybenzo(a)pyrene requires the presence of NADPH and is inhibited by carbon monoxide, suggesting that the reaction is mediated by a microsomal mixed-function oxygenase. The metabolic activity can be induced by in uiuo treatment with 3-methylcholanthrene. 7&Benzoflavone strongly inhibits the induced activity but has little effect on the constitutive enzyme. The inducibility and inhibition characteristics, as well as the metabolic rate of the conversion of 3-hydroxybenzo(a)pyrene, closely resemble those of the oxidative metabolism of benzo(a)pyrene. The microsomal NADPHdependent metabolism of [aH]3-hydroxybenzo(a)pyrene leads to the formation of a number of products of which a major fraction cochromatographs with the 3,6-quinone of benzo(a)pyrene. In mammalian cell cultures 3-hydroxybenzo(a)pyrene is converted by a mechanism different from that in hepatic microsomes. The disappearance of the phenol in cultures of hamster embryo cells is independent of the action of inducers or inhibitors of the aryl hydrocarbon hydroxylases and also occurs in the mouse L-cell line, A9, which lacks detectable aryl hydrocarbon hydroxylase activity. In A9 cells, [3H]3-hydroxybenzo(a)pyrene is largely converted to water soluble derivatives.

Microsomal mixed-function oxygenase convert benzo(a)pyrene (BP)’ to a number of more polar derivatives of which the monohydroxy-metabolites form a major fraction (l-3). The high degree of fluorescence and ease of differential extractability of the phenolic products provide the basis for a highly sensitive assay of BP-oxygenation (4-6). Benzo(a)pyrene-hydroxylation is widely used as a measure for microsomal enzymes oxygenating polycyclic hydrocarbons, i.e., the “aryl hydrocarbon hydroxylases” (6), although it is recognized that hydroxylation may proceed through an epoxidation step and subsequent nonenzymatic rearrangement to the phenol (7-9) and may compete with other reactions such as epoxide hydration (7-10) or conjugation

with low molecular weight compounds (11-13). Hydroxy-derivatives of polycyclic hydrocarbons may be subject to metabolic conversion, e.g., conjugation reactions or further oxygenation (13, 14). The metabolism of monohydroxy-benzo( a)pyrenes (OHBP) is of particular interest in a number of aspects: (1) The measurement of the “aryl hydrocarbon (BP) hydroxylase” (6) is influenced by the further microsomal metabolism of the phenolic products and by their possible interference with the primary hydroxylation reaction; (2) OH-BP’s may be carcinogenic (15, 16), and may require metabolic activation to be carcinogenic as postulated for some of the polycyclic hydrocarbons (17-19); (3) S-OH-BP has been shown to be more toxic to cells in culture than BP (20, 21); the metabolism may either activate the phenol to its toxic form or convert it to less harmful water soluble derivatives.

’ Abbreviations used: AHH, aryl hydrocarbon (benzo(a)pyrene) hydroxylase; 7,8-BF, 7,8-benzoflavone (o-naphthoflavone); BP, benzo(a)pyrene; MC, 3-methylcholanthrene; OH-BP, hydroxy-benzo(a)pyrene; and Me,SO, dimethylsulfoxide. 609 Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.



This paper describes studies on the nature of the enzymes that mediate the microsomal metabolism of hydroxybenzo(a)pyrenes. The results indicate that the enzyme is one of the mixed-function oxygenases and is closely related if not identical to the oxygenases involved in BP metabolism, the aryl hydrocarbon (BP) hydroxylases. Observations in cells in monolayer culture indicated that the disappearance of OH-BP in cultured cells is due to a mechanism unrelated to the aryl hydrocarbon hydroxylases. MATERIALS



Materials 3-Methylcholanthrene (MC) and benz(a)anthracene were purchased from Eastman Organic Chemicals,, NY, 7,8-benzoflavone (7,8-BF) from Aldrich Chemical Co., Inc., Cedar Knolls, NJ, BP from Baker Chemical Co., Phillipsburg, NJ. BP, 7,8BF, and benz(a)anthracene were purified by recrystallization from ethanol. [‘H]BP (3 Ci/mmol) was purchased from Amersham/Searle, Arlington Heights, IL and was purified by thin layer chromatography with silica gel using benzene:hexane (1:15). [SH]3-OH-BP was prepared as described below. 3OH-BP, B-OH-BP and BP-3,6-quinone were synthesized according to NCI-contract NOl-CP-3387. 6-OHBP was a generous gift of Dr. P.O.P. Ts’o, Johns Hopkins University, Baltimore, MD. 9-Chloro-7H-dibenzo(a,g)carbazole was obtained from Dr. N.P. BuuHoi, Institut de Radium, Paris, France. 4’-Benzyloxy3’-methoxy-7,8-benzoflavone was supplied by Drs. M.G. Stout and W.S. Burnham, ICN, Irvine, CA. NADPH was purchased from Calbiochem, San Diego, CA, carbon monoxide from Matheson Gas Products, East Rutherford, NY. All culture media and sera were purchased from Grand Island Biological Company, Grand Island, NY.


of Microsomes

Male Sprague Dawley rats weighing 120-140 g were fed ad libitum on a Wayne Lab Blox diet. Animals were fasted for 18 h before sacrifice. 3-Methylcholanthrene (40 mg/kg) was injected intraperitoneally in 0.5 ml corn oil 18 h prior to sacrifice. Control rats were given 0.5 ml corn oil. Livers were homogenized (1:5, w/v) in 50 mM Tris-HCl, pH 7.5; 250 mM sucrose, using a Potter-Elvehjem glass-glass homogenizer (10 strokes). The homogenates were centrifuged at 750g for 10 min and the supernatants at 12,000g for another 10 min period. Microsomal pellets were obtained by centrifugation of the supernatant at 100,OOOg for 60 min. The pellets were gently homogenized in sucrose-Tris buffer (1 ml/pellet/2 g liver wet weight)

J. WIEBEL and aliquots were immediately assayed for enzyme activities or frozen at -70°C for later use. Protein concentrations were determined by the method of Lowry et al. (22) using ribonuclease A as standard.

Assays of BP- and OH-BP Metabolism by Microsomes General conditions. The metabolism of BP and its hydroxy-derivatives was determined by modifications of the method of Wattenberg et al. (4) and of Nebert and Gelboin (6). Incubation mixtures contained in 1 ml: 50 pmol Tris-HCl, pH 7.6; 3 pmol MgCl,; 0.5 rmol NADPH; microsomal preparations as stated in Results and substrate added in 0.02-0.04 ml methanol. The mixtures were incubated at 37°C in a water bath under mild shaking. The reaction was stopped by chilling and addition of 1 ml of cold acetone followed by 3 ml of hexane. The mixture was shaken at 37°C for 10 min, except for the determination of 6-OH-BP (see below). Fluorescence was determined in an Aminco Bowman spectrophotofluorometer (model 4-8106 SFP). Metabolism of3- and g-OH-BP. A stock solution of 3-OH-BP was prepared in methanol that was stable in the dark during several weeks. The amount of 3-OHBP was measured by its fluorescence in the acetone: hexane extract of incubation mixtures at 380 nm excitation and 440 nm emission, the fluorescence of g-OH-BP at 378 nm excitation and 430 nm emission. In preliminary experiments 1 ml aliquots of the organic phase were extracted with 3 ml 1 N NaOH and the fluorescence of the alkaline phase was determined at 395 nm excitation and 522 nm emission for both phenols. The fluorescence in the alkaline phase paralleled that in the organic layer. Metabolism of 6-OH-BP 6-Monohydroxybenzo(a)pyrene was dissolved in methanol and used on the same day. Four nanomoles of the compound were added in 0.04 ml methanol to the incubation mixture. After completion of the reaction and addition of 4 ml acetone:hexane (1:3) the mixtures were transferred to tubes and shaken by hand for 30 s. Fluorescence in the organic phase was measured immediately at 398 nm excitation and 455 nm emission. Monohydroxybenzo(a)pyrene-specific fluorescence in the organic phase was stable for at least 10 min. An emission wavelength 15 nm longer than that of the emission peak of 6-OH-BP (440 nm) was chosen to allow a better separation of excitation and emission wavelengths. The loss of fluorescence by this procedure was less than 10% of the emission peak intensity. 6-Monohydroxybenzo(a)pyrene could be quantitatively extracted from the organic phase into 1 N NaOH and the fluorescence be measured at 430 nm excitation and 522 emission. The short half life of the compound in alkali (about 5 min) renders this second extraction step impractical for larger sample numbers. Metabolism of BP. Incubation mixtures were ex-




tracted with acetone:hexane (see above) and aliquots of the organic phase were extracted with 3 vol of 1 N NaOH. The total metabolism of BP was measured by the disappearance of BP-specific fluorescence from the organic phase (360 or 380 nm excitation and 415 nm emission). Neither BP-dihydrodiols nor the BP-quinones, two major organic soluble solvent-extractable metabolites of BP (2, 3) fluoresce sufficiently to interfere with the assay of the highly fluorescent parent compound. The interference by phenolic products, the third major group of metabolites (2, 3), was eliminated from the organic solvent by the alkali. Benzo(a)pyrene-hydroxylation. The conversion of BP to phenolic products was measured by the appearance of fluorescence at 398 nm excitation and 522 nm emission in the alkali extract (4, 6). Since the fluorescence spectra of the products from rat liver microsomes resemble closely those of 3-OH-BP, this compound was used as standard. Column and high pressure liquid chromatography suggest that the bulk of the phenol derivatives consists of 3-OH-BP with a minor contribution of g-OH-BP (3, 23). Preparation of BH-lnbeled Of&BP. Incubation mixtures containing 50 nmol of [“H]BP (3 Ci/mmol) and 0.5 mg of microsomal protein from livers of MCtreated rats were incubated in the presence of NADPH for 10 min and extracted with 4 vol of acetone:hexane (1:3). Phenolic products were extracted from the organic solvent by 0.02 N NaOH (1:l v/v); the alkaline solution was then extracted with two volumes of ethyl acetate. The organic solvent fraction was dried over anhydrous MgSO, and evaporated under vacuum. The residue was taken up in methanol and the labeled [3H]BP-products were separated by high pressure liquid chromatography (23). The water:methanol fractions containing [3H]3-OHBP were pooled and the solvent evaporated under vacuum. The residue was then dissolved in methanol and unlabeled 3-OH-BP was added to give the desired specific radioactivity (cf. Results). The labeled 3-OHBP was extractable from aqueous solution into acetone:ethyl acetate (1:5) by 99.7%. Liquid chromatographic analysis of the organic extract showed only one major peak of labeled material which cochromatographed with authentic 3-OH-BP (Fig. 4) and gave the same fluorescence spectra as 3-OH-BP. Counting procedure. Radioactivity was measured in Aquasol (New England Nuclear Corp.) in a Packard Liquid Scintillation Spectrometer (model 3380). Mixtures containing proteins or membrane fractions were solubilized with 1 ml of NCS (Amersham/ Searle) and counted in a toluene:PPO:POPOP solution (4 g PPO, 50 mg POPOP/liter). Counts were corrected for quenching by internal standards. Tissue culture conditions. Hamster embryo cell cultures were prepared as previously described (6) and grown in Eagles Minimum Essential medium

with Earle’s salt solution containing 10% calf serum, 100 units of penicillin, and 100 pg of streptomycin. Experiments were carried out in secondary cultures approaching confluency, i.e., containing approx. 1.5 x lo6 cells per 100 mm culture dish (Falcon). A9 cells which are derived from mouse L cells (24) were generously supplied by Dr. H. G. Coon (Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD). They were grown in Ham’s nutrient mixture F-12 (25) with 10% fetal calf serum, 106 units of penicillin, and 100 pg of streptomycin. Confluent monolayers were used for experiments. The growth medium was renewed 16 h prior to the start of experiments. Benzo(a)pyrene-hydroxylation and 3-OH-BP disappearance in cell culture. Benzo(a)pyrene or 3-OHBP was added in acetone to fresh growth medium to give a solvent concentration of 0.1%. After various incubation times with cells 0.3-ml aliquots were removed sterilely from the growth medium and extracted into 1.5 ml of acetone:hexane (1:3) by mild shaking for 1 min. A 0.75 ml portion of the organic phase was then extracted into 2 ml of 1 N NaOH. Phenol-specific fluorescence was measured in organic and alkaline solutions as described above. The phenolic products from hamster cells in culture consist mainly of 9-OH-BP.Z 3- and g-OH-BP have very similar fluorescence-coefficients at 395 nm excitation and 520 nm emission. Thus the amounts of phenols recovered from the medium are recorded on the same scale in Fig. 5. Fluorescence in organic solvent extracts from medium without addition of polycyclic hydrocarbon was negligible. Treatment of cultures with benz(a)anthracene or the presence of 9chloro-7H-dibenz(a,g)carbazole in the medium (cf. Results) does not interfere with the fluorescence assay. The amount of fluorescence in aliquots of the growth medium was taken as a measurement of the amount of phenol per plate since it was found that the cellular layer contained less than 15% of the total amount of phenol and its conversion products (cf. Results). RESULTS


of Hydrorybenzo(a)pyrenes

in Vitro NADPH-dependency. Incubation of 3OH-BP with microsomes in the presence of NADPH at 37°C results in a decrease of fluorescence specific for 3-OH-BP. This decrease is not found in the absence of NADPH (Table I). The disappearance of a small amount of 3-OH-BP during the 10 min incubation period in the absence of added NADPH might be due either to a 2 Unpublished


cf. Refs.

3 and







Complete” at 0°C Complete” at 37°C Minus NADPH Plus acetoned Heated’ (SO’C, 45 min)

3-OH-BP Fluorescenceb (units)

270 188 266 263 270

3-OH-BP Metabolism’ (nmol/mg proteimmin) 0.474 0.024 0.040 0

(I Complete incubation mixtures contained in 1 ml: 50 pmol Tris-HCl, pH 7.6, 3 pmol MgCl,; 0.50 pmol NADPH; 0.125 mg microsomal protein from liver of MC-treated rats and 2.0 nmol 3-OH-BP in 0.020 ml methanol. Incubation time was 10 min. b Incubation mixtures were extracted with 4 vol of acetone: hexane (1:3, v/v) and fluorescence of the organic extract was measured as described in Materials and Methods. Values represent units of fluorescence at 380 nm excitation/440 nm emission. c Numbers give the amount of 3-OH-BP (nmobmg proteimmin) which disappeared during incubation at 37°C. They are calculated from the decrease in fluorescence at 380 nm excitation/440 nm emission using 3-OH-BP as standard. d One milliliter of acetone was added prior to incubation. Following incubation, the mixture was extracted with 3 ml hexane. e Microsomes suspended in buffer (50 mM Tris-HCl, pH 7.5; 250 mM sucrose) were heated at 80°C for 45 min prior to the addition to incubation mixtures.

NADPH-independent mechanism or to residual amounts of NADPH contained or generated in the microsomal preparation. The difference between OH-BP disappearance in the presence and absence of NADPH was taken as a measure of its “metabolism” or “utilization.” Table I demonstrates that heat denaturation of the microsomal proteins as well as the presence of acetone largely suppresses the NADPHdependent disappearance of 3-OH-BP. Sensitivity to carbon monoxide. The presence of a carbon monoxide:oxygen gas mixture (9:l) inhibits 3-OH-BP metabolism in hepatic microsomes from both control and MC-treated animals (Table II, first and second columns). However, MC-


induced metabolism is considerably less sensitive to CO inhibition than the activity of the constitutive enzyme. Carbon monoxide also inhibits the hydroxylation of BP, at either non-saturating concentrations equimolar to the OH-BP (2 nmol) or at saturating concentrations (100 nmol) (Table II). Thus, the different CO effect appears to be independent of the substrate concentration. The differential inhibition of constitutive and MC-induced BPhydroxylation by CO has also been observed by Estabrook and co-workers (personal communication). The inhibition by CO suggests the involvement of cytochrome P450, the terminal electron acceptor of the microsomal mixed-function oxygenases (26-28). The nearly identical CO effects on OH-BP- and BP-metabolism (cf. Table II, bottom line) TABLE



J-OH-BP Metabolismb (nmobmg protein/min)

BP-Hydroxylatior+ (nmol/mg proteimmin) 2 nmol


100 nmol








Air co:o,

0.114 0.057

0.253 0.196

0.038 0.019

0.286 0.209

0.112 0.069

0.769 0.584








a Microsomes were prepared from MC-treated (= Induced) and from corn oil-treated (= Basal) rats. The amount of microsomal protein per incubation was 0.12 mg. b 3-OH-BP metabolism and the production of OHBP from BP (= BP Hydroxylation) were determined as described in Materials and Methods. Substrate amounts were 2 nmol of 3-OH-BP and 2 nmol or 100 nmol of BP. Values represent nmol of 3-OH-BP metabolized or of phenolic product formed from BP/mg protein/min. ~Gas mixtures were bubbled through incubation mixtures on ice at a flow rate of 100 ml/min 2 min prior to and 2 min after addition of the substrate. Incubation flasks were then tightly stoppered and incubated at 37°C for 10 min.




indicate that the 3-OH-BP metabolizing enzymes are closely related to the “BPhydroxylases” (“aryl hydrocarbon hydroxylases”) which have previously been characterized as mixed-function oxygenases (1, 6, 29). Rates of OH-BP and BP metabolism. Figure 1 compares the metabolism of 3and g-OH-BP to the total metabolism of BP and to phenol production from BP. The rates of all these reactions are nearly linear for 5-10 min, but fall off rapidly with longer incubation times. In fact, the amount of phenolic products decreases after a 20-min incubation period, i.e., the relative amount of phenols in total BPmetabolites becomes progressively smaller with time. The rates of 3-OH-BP and BP-metabolism are remarkably similar. In four experiments using microsomes from untreated or MC-treated rats at 2.0-2.5 PM

substrate concentrations, the metabolic rates of BP- and 3-OH-BP differed only 2-2077~from each other. Localization in microsomes. Table III demonstrates that 3-OH-BP metabolism resides in microsomes and is not due to contamination by lOO,OOOg supernatant protein. No 3-OH-BP metabolism is found in the 100,000~ supernatant. Furthermore, the activity in the “washed” microsomal preparation is reduced by only 25% when compared to microsomes prepared by a single centrifugation. It appears unlikely that the loss of activity is due to the removal of some components specific for 3OH-BP metabolism since it is matched by a nearly identical reduction in BP-hydroxylase activity (Table III). Inducibility and in vitro response to benzoflauones. The similarity of the metabolism of OH-BP to that of BP is also TABLE





of spin

Pellet Unwashed (0.14 mg) Washed (0.13 mg) Supernatantd (0.25 mg) (0.50 mg)

n “0

I 10


BP - Metabolism

&---A c -4

BPOH Production 3. BPOH - Metabohsm 9 BPOH Metabolism



20 TIME (min)


FIG. 1. Metabolic utilization of BP, 9- and 3-OHBP and production of OH-BP from BP by microsomes of rat liver. Benzo(a)pyrene, 9-, and 3-OH-BP (2.5 nmol) were added in 0.025 ml methanol. Incubation mixtures contained 0.055 mg microsomal protein from liver of MC-treated rats. The metabolism of BP or of 3- and g-OH-BP and the metabolism of BP to OH-BP were assayed as described in Materials and Methods. Enzyme activities are expressed as nmol of substrate utilized or product formed/mg protein.


III OF 3-OH-BP ACTIVITY” protein/mix


Ratio: WI

2.47 2.42 -

a Microsomes were prepared from 3-methylcholanthrene-treated rats (Materials and Methods). The microsomal pellets of the first 100,OOOg centrifugation (= Pellet, unwashed) were resuspended in sucrose-Tris buffer, pH 7.6, and subjected to a second 100,OOOg centrifugation for 60 min. The amounts of microsomal and supernatant protein used are given in parentheses. h The metabolism of S-OH-BP (2.5 nmol added in 0.025 ml methanol) and BP-hydroxylation, i.e., the production of OH-BP from BP (106 nmol added in 0.04 ml methanol) were assayed as described in Materials and Methods. c Values represent nmol/mg protein/min of 3-OHBP metabolized or of phenolic products formed from BP. d Supernatant of the first 100,OOOg centrifugation.



shown by: (1) The metabolism of OH-BP and BP is similarly increased after treatment of rats with MC (Table II and Fig. 2). The 3- to 4-fold increase corresponds to the induction of total BP-oxygenation, but is somewhat lower than the induction of phenol formation. At low substrate concentrations the phenolic products appear to comprise a relatively larger fraction of metabolites in microsomes from inducer-treated than from untreated animals.3 (2) We have previously shown that BP-hydroxylase in microsomes from untreated and MCtreated rats can be distinguished by their in vitro response to 7,8-benzoflavone (30). This is also true for the metabolism of 3-OH-BP. 3-Methylcholanthrene-induced activity is strongly inhibited by the 7,8benzoflavone, whereas constitutive activity is not or only slightly affected in the range of 7,8-benzoflavone concentrations tested (Fig. 2). (3) Similarly, the 7,8-benzoflavone derivative, 4’-benzyloxy-3’-methoxy-7,8benzoflavone, stimulates BP-hydroxylation (31) and utilization of 3-OH-BP (Table IV): In vitro addition of the flavone derivative increases OH-BP-metabolism by microsomes from untreated animals more than 2-fold.

NADPH-dependent utilization BP. 6-Monohydroxybenzo(a)pyrene

of 6-OH-

is far less stable than its isomers, 3- and g-OHBP. As shown in Fig. 3, the half life of the 6-OH-BP in incubation mixture in the absence of microsomes was less than 3 min. However, when microsomes (approx. 0.5 mg protein) were added to the incubation mixture in the absence of NADPH, the half life of the phenol was considerably prolonged. Addition of NADPH, in turn, caused a rapid utilization of the 6-OH-BP of approximately 1.5 nmol/mg protein/ min, i.e., of a similar magnitude as the NADPH-dependent utilization of 3OH-BP (Fig. 2). These results suggest that in a microsomal environment 6-OH-BP may not simply be subject to nonenzymatic conversion, but may be metabolized, possibly by the same mechanism as the other phenols or BP. B Unpublished



O/L--l 7.8~BENZOFLAVONE (Log Molartty) FIG. 2. Effect of 7$benzoflavone on the metabolism of 3-OH-BP in hepatic microsomes from untreated and MC-treated rats. Two nanomoles of 3-OH-BP were added in 0.02 ml methanol, and various amounts of 7,8-BF in 0.01 ml methanol. Incubation mixtures contained approximately 0.125 mg of microsomal protein from liver of untreated and MC-treated rats. Incubation time was 10 min. Other conditions as in Materials and Methods. Enzyme activity is given in nmol of substrate metabolized/mg protein/min.


3-OH-BP Incubation time (min) 3 6



Metabolism* Control

1.62 2.66

(nmol/mg Benzyloxy7,8-BF’ 3.77 6.16

protein) Factor of stimulation 2.3 2.3

4 Samples contained 0.125 mg of microsomal protein and 0.500 mg of 100,OOOg supernatant protein from livers of untreated rats. b 3-OH-BP metabolism was determined as described in Materials and Methods. Two nanomoles of 3-OH-BP were added in 10 ~1 of methanol. c One nanomole of 4’-benzyloxy-3’-methoxy-7,8benzoflavone (Benzyloxy-7,8-BF) was added in 6 ~1 of MelSO.



01 0



I 1

I 2 TIME (mm)

I 3

FIG. 3. Metabolism of 6-OH-BP by microsomes of rat liver. Four nanomoles of B-OH-BP added in 0.04 ml of methanol were incubated under various conditions: (a) in buffer solution containing 50 rmol of Tris-HCl, pH 7.6; 3 pmol of MgCl,, and 0.5 rmol of NADPH (0-O); (b) in the same buffer solution including 0.5 mg of microsomal protein in the presence (A-A) or absence (V- - -‘I) of NADPH. Microsomes were obtained from liver of MC-treated rats. Other conditions as described in Materials and Methods.

Separation of [3H]3-OH-BPmetabolites.

[3H]3-Monohydroxybenzo(a)pyrene was prepared by incubation of [3H]BP with microsomes and isolation of the labeled phenol as described in Materials and Methods. Two nanomoles of [3H]3-OH-BP were incubated with microsomal preparations in the presence or absence of NADPH and following extraction with acetoacetate (1:5 v/v) the distribution of radioactivity was determined in the aqueous and organic solvent phases. More than 93% of radioactivity was found in the organic solvent extract. The organic soluble components were then separated by high pressure liquid chromatography as shown in Fig. 4. Three major fractions are discernible: (1) the substrate with a retention time of 44-50 min; (2) a NADPHdependent metabolite with a retention time of 33-38 min which cochromato-


graphed with 3,6-quinone-BP; and (3) metabolite(s) with a very short retention time of 2-8 min which have not been identified. Table V (A) summarizes the distribution of NADPH-dependent metabolites (cf. third column) in various phases: The metabolites, tentatively identified as 3,6-quinone, form a major portion of the organic solvent extractable metabolites (cf. organic soluble, total). In the absence of NADPH little radioactive material (2% of total radioactivity) other than the substrate [3H]3-OH-BP is found in aqueous or organic solvent phases. Less than 25% of the radioactivity related to NADPH-dependent metabolism remained in the aqueous phase. As shown in Table V (B, third column), the metabolism of 3-OH-BP determined by the radioassay closely matches the NADPH-dependent disappearance of substrate assayed by spectrofluorometry. Disappearance of OH-BP in cells in culture. The initial appearance of phenolic

products in the growth medium of cells in culture upon addition of BP and their subsequent decrease accompanied by an accumulation of water-soluble derivatives (32) suggested that the phenolic products might be further metabolized analogously to the microsomal metabolism in vitro described above. As shown in Fig. 5B, 3-OH-BP added to the growth medium of hamster embryo cell cultures does indeed rapidly disappear with a half-life of about 1.5 h. However, utilization of OH-BP apparently operates through a different mechanism than that in hepatic microsomes as indicated by the following observations: (1) Prior induction of the aryl hydrocarbon hydroxylases in hamster embryo cells by benz(a)anthracene does not alter the disappearance rate of externally added OH-BP (Fig. 5B). That induction has occurred in these cells is demonstrated by the initially faster accumulation of phenolic products from BP in benz(a)anthracene-treated cultures (Fig. 5A). Since BP acts as a potent inducer (1) the gap in enzyme activities in untreated and inducer pretreated cultures will only be transient. (2) Inhibition of the cellular BP-hydroxyl-





3, 6 . Quinone I




TIME (minutes)

FIG. 4. Separation of metabolites of [*H]3-OH-BP. Triplicate samples were incubated in the absence (A-A) or presence (O----O) of NADPH (0.5 rmol). Samples contained 0.12 mg of microsomal protein from livers of MC-treated rats. Substrate, 2 nmol of [*H]3-OH-BP (spc act 0.39 Ci/mmol), was added in 0.02 ml of methanol. Other conditions as described in Materials and Methods. After a lo-min incubation period the reaction was stopped by addition of 1 ml of acetone. The mixture was extracted by 5 ml ethyl acetate. Aliquots were taken from the aqueous and the organic phases. The organic phases from triplicate samples were then pooled and dried over 2.0 g of anhydrous magnesium sulfate. The solvent was evaporated under vacuum, and the residue was redissolved in 0.15 ml of methanol. The high pressure liquid chromatography was performed as described by Selkirk et al. (23). Fractions were collected at 20 s intervals. 3,6-Quinone of BP was added to the organic phases prior to chromatography and its retention time was determined by monitoring the ultraviolet absorption at 254 nm.

ase by 9-chloro-7H dibenzo(a,g)carbazole, a strong inhibitor of the hydroxylase (31), does not affect the rate of disappearance of OH-BP from the medium (Fig. 5B). The inhibitory effect on BP-metabolism is demonstrated by the apparent, complete suppression OH-BP production (Fig. 5A) and the persistence of the substrate BP in these cultures (data not shown). Further experiments showed that 3-OHBP also disappears in cell cultures in which BP-hydroxylation is undetectable (see below). The disappearance of 3-OH-BP is dependent on the presence of intact cells: (1) OH-BP incubated in culture plates containing growth medium only decreased by 2-3%/h (see below, Table VI), i.e., 10 times slower than in the presence of intact cells. This was also observed when cells broken by homogenization were added to the growth medium. (2) The disappearance of OH-BP was largely inhibited by prior heat denaturation (lOO”C, 10 min) of the cellular proteins of monolayers cultured in glass dishes.

The disappearance of 3-OH-BP from the medium of cell cultures was further examined using [3H]3-OH-BP as substrate. Monolayers of A9 cells which do not exhibit detectable BP-hydroxylating activity (33) were incubated with 9 nmol of [3H]3-OHBP up to 3 h. Incubation of growth medium in the absence of cells served as control. Table VI shows a comparison of radioactivity and fluorescence that can be recovered in the organic and aqueous phases after extraction with either acetone:hexane (1:3, v/v) or acetone:ethyl acetate (1:5, v/v). After a 3 h incubation period in the presence of cells about 75% of the labeled compound in the medium is no longer extractable into acetone:hexane (cf. first column). Determinations of organic soluble material by radioactivity or fluorescence yielded comparable values. The fluorescent material in the organic phase was extractable into 1 N NaOH to,about 70% and showed the fluorescence spectrum of 3-OH-BP. Thus, most of the labeled and fluorescent material extractable into acetone:hexane appears to be unaltered sub-




Method of determination



B. Fluorescence of 3-OH-BP’

Organic soluble, BP-quinone’ Unidentifiedd Aqueous soluble, Organic

+ aqueous




[3H]3-OH-BP (nmol/mg



A. Radioactivity’




totale soluble*




Metabolites proteimmin) NADPH

A plus-minus NADPH

0.018 0.016 0.002 0.005

0.399 0.280 0.119 0.106

0.381 0.264 0.117 0.101







n Experimental conditions are described under Fig. 4 and in Materials and Methods. * Incubation mixtures were extracted with 6 ml of acetone: ethyl acetate (1:5, v/v). Radioactivity in aliquots of the aqueous phase and the solvent extract was determined by liquid scintillation counting. The range of counts in triplicate samples was less than 10% of their mean. c, d Organic soluble metabolites and substrate were separated by high pressure liquid chromatography as shown in Fig. 4. Numbers represent the amount of labeled material (c) under the peak with a retention time of 33-38 min which cochromatographs with authentic 3,6-quinone of BP (= BP-quinone) and (d) under the peak with a retention time of 2.5-7.5 min (= Unidentified). p The amounts of material in various fractions were calculated from the specific activity of the substrate, [‘H]3-OH-BP (0.39 Ci/mmol). ’ The amount of 3-OH-BP was determined spectrofluorometrically in the acetone: ethyl acetate extract as described in Materials and Methods using authentic Y-OH-BP as standard. r Cf. text and footnote c.

&rate, 3-OH-BP. About 15% of the labeled material was found in the cells predominantly in an aqueous, soluble form after the 3-h incubation. Acetone:ethyl acetate extracts more of the labeled products from the aqueous incubation mixture than acetone:hexane (cf. first and third columns), however, in contrast to the microsomal metabolites of [3H]3-OH-BP more than 50% of the labeled material remains still in the aqueous phase. The material extracted into acetone:ethyl acetate showed a fluorescence spectrum which differed from that of 3OH-BP and has not yet been identified. DISCUSSION

The present observations indicate that hydroxybenzo(a)pyrenes are metabolized by microsomal mixed-function oxygenases which are similar if not identical to the “benzo(a)pyrene hydroxylases.” Benzo(a)pyrene and OH-BP metabolism re-

semble each other in (1) their requirement for NADPH and their inhibition by carbon monoxide, (2) their microsomal localization, (3) the rates of reaction, (4) the inducibility of activity by methylcholanthrene, and (5) the in vitro inhibition or stimulation by benzoflavones in microsomes from methylcholanthrene treated or untreated animals. The microsomal metabolism of hydroxyderivatives of BP may partially account for the apparent short linear kinetics of BPhydroxylation and most likely cause the decrease of the phenolic products observed at longer incubation times and in preparations with high activity (cf. Fig. 1). Furthermore the similarity of microsomal BPand OH-BP-metabolism suggests that the phenolic products may compete with the parent compound for the same enzyme site and thus contribute to the nonlinearity of the overall BP oxidation (cf. Fig. 1). The latter effect is probably insignificant in the







H w 5oz 0 t s R B


+-----------sq I

I 3 - OH




m3 0 %




Premduced AHH Inhibitor


HOURS FIG. 5. BP-hydroxylation

and 3-OH-BP disappearance in hamster embryo cell cultures. Three groups of cultures containing approximately 2 x IO8 cells were incubated with 7 ml of growth medium containing either 10 nmol/ml BP or 9 nmol/ml 3-OH-BP. (1) A-----A cultures were preincubated with medium containing 13 nmol of benz(a)anthracene for 16 b. (2)v - - - -v cultures were exposed to 10 nmol/ml of 9-chloro-7H-dibenzo (a,g)carbazole simulataneously with the benzopyrenes. (3) (GO) Control cultures were kept in growth medium without addition except for the benzopyrenes. At various times aliquots were taken from the growth medium and the amounts of phenols were determined spectrofluorometrically as described under Materials and Methods. Values represent acetone; hexane extractable nmol of phenolic BP-products ( = BP hydroxylation) or nmol of 3-OH-BP per culture which are recovered from the growth medium ( = 3-OH-BP utilization). Variation from the mean in duplicate samples was generally less than 10% of the mean.

commonly used assay of BP-hydroxylation (4, 6) in which the substrate, BP, is in excess and relatively small amounts of metabolites are formed. However, it might interfere considerably with the determination of reaction kinetics (34) that require the use of lower substrate concentrations.

The separation of OH-BP metabolites by liquid chromatography indicates that more than one derivative is formed in rat liver microsomes. This is in keeping with the observation that (a) microsomal oxygenases metabolize polycyclic hydrocarbons at more the.7 oni position (2, 3), and (b)







nmol/Culturee, Hexane’



13Hl Cells and growth medium

Growth medium Only

Growth mediumc Organic soluble Aqueous soluble

0.61 2.63

Cell 1ayeP Organic soluble Aqueous soluble

0.08 0.44







o Monolayers of A9 cells were grown in 100-mm dishes as described in Materials and Methods. Concentrations of [3H]3-OH-BP (specific activity 0.39 Ci/mmol) was 9 nmol/ml growth medium. b Five milliliters of labeled medium were incubated in dishes in the presence or absence of A9 cells at 37°C in a 5% CO,, humidified atmosphere. c At various times aliquots of the medium were extracted with a lo-fold volume of acetone:hexane (1:3 v/v) or acetone:ethyl acetate (1:5 v/v) by mild shaking at room temperature. d After 3 h of incubation the media were removed from the dishes and the cell monolayer was scraped with a rubber blade into 1 ml of buffer (50 mM Tris-HCI pH 7.5; 250 mM sucrose). The cellular material was homogenized in a Potter-Elvehjem glass-glass homogenizer and aliquots of the homogenates were extracted with organic solvents as described for the growth medium under (c). Fluorescence in the organic extracts (= Organic soluble) and radioactivity in aqueous layer (= Aqueous soluble) and organic layer were determined as described in Materials and Methods. ‘, ‘The amounts of hydrocarbon in various fractions (nmol/culture) were estimated from (e) the radioactivity based on the specific activity of the substrate 13H]3-OH-BP or (f) from the fluorescence (380 nm excitation/440 nm emission) using 3-OH-BP as standard. Ranges of fluorescence or radioactivity in triplicate cell cultures were generally less than 10% of their mean.

that the presumed primary products, arene oxides, can be further metabolized, e.g., to dihydrodiols or rearrange to hydroxy_. derivatives (7-10, 35). A major micro-


somal metabolite of 3-OH-BP appeared to be the BP-quinone, which is tentatively identified as BP-3,6-quinone. Quinones, and predominantly the 3,6-quinone, are also observed after incubation of BP with microsomes (2, 3). Since monohydroxyBP’s readily oxidize to quinones in air (2), the quinones observed as BP-“metabolites” could arise as nonenzymatic oxidation products from corresponding phenols. The present data indicates that they are products of a sequential enzymatic oxygenation of BP, first to monohydroxy-BP and then probably to monoxy-monohydroxy-BP or dihydroxy-derivatives which may readily convert to their quinones. The unidentified metabolites of 3-OH-BP which showed a very short retention time on the methanol:water gradient (Fig. 4 and Table V) are more polar; they might be dihydrodiols or dihydroxy-derivatives of another than the hydroquinone type and thus be less susceptible to further oxidation to diketones. The formation of dihydroxy-BP from 3-OH-BP by mouse liver microsomes has been suggested by Hill and Shih (14). The quantitation of labeled [3H]OH-BP metabolites from the specific radioactivity of [3H]3-OH-BP (cf. Table V) may be subject to errors introduced by a loss of tritium during metabolism that might largely depend on the extent of migration and retention of the label (“NIHShift”) (36, 37) following the oxygenation step.’ However, in view of the correspondence of total radioactive metabolites recovered ’ In accordance, radioactivity in the aqueous phase (Table V) might represent [sH]H,O. Formation of 13H]H,0 has been observed during the NADPHdependent oxygenation of [‘H]BP (41) which appears to be closely related to the metabolism of the BPphenols. About 1 out of 50 substrate molecules (BP or OH-BP) used in this study is nearly randomly labelled with a tritium atom (Amersham/Searle Radiochemical Centre, England). Assuming random labeling of the substrate, the maximum amount of tritium released during metabolism does not account for the radioactivity found in the aqueous phase (Table V). Some of the radioactivity might be due to the presence of water soluble metabolites or to the binding of metabolites to protein and RNA contained in the incubation mixture. This is currently under investigation.



and the overall 3-OH-BP-metabolism determined by spectrofluorometry, it is unlikely that the specific radioactivity of the metabolites is significantly decreased. Previous observations have suggested that cytotoxicity of BP (20,21) and of other polycyclic hydrocarbons (38) is due to the formation of phenolic derivatives. These studies estimated cytotoxicity by the effect of 3-OH-BP on the cloning efficiency of cells in sparse cultures. The rapid disappearance of 3-OH-BP from dense monolayer cultures might explain the observations (unpublished) that the phenol was not toxic in any of the crowded cultures in contrast to the strong cytotoxicity of equivalent amounts of BP in those cultures provided they contained substantial amounts of aryl hydrocarbon hydroxylase (e.g., hamster embryo cells). It is also possible that the exogenously added 3-OHBP does not reach susceptible cellular sites at effective concentrations, although histochemical studies have shown that the phenol readily enters the cytoplasm of cultured cells (39). We do not know the nature of the products to which the phenols are converted in cultured cells. The incomplete extraction of these metabolites by organic solvents from aqueous medium suggests that many or all of them are more polar than the products formed in microsomes. They might be conjugation products that have been observed for a number of polycyclic hydrocarbons in the soluble fraction of liver (11-13) and in cells in culture (40).

I thank Dr. H. V. Gelboin for his encouragement, advice and for the valuable discussions concerning the manuscript. I am indebted to Dr. J. K. Selkirk for his help with the high speed liquid chromatography and for reviewing the manuscript. The excellent technical assistance of J. C. Leutz is gratefully acknowledged. REFERENCES E. C., AND MILLER,

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Metabolism of monohydroxybenzo(a)pyrenes by rat liver microsomes and mammalian cells in culture.

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 168, 609621, (1975) Metabolism of Monohydroxybenzo(a)pyrenes by Rat Liver Microsomes and Mammalian Cells...
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