Chem.-Biol. Interactions, 59 (1986) 265-280 Elsevier Scientific Publishers Ireland Ltd.

265

EFFECTS OF INDUCERS ON THE REGIO- AND STEREOSELECTIVE METABOLISM OF BENZ0 [a] PYRENE BY MOUSE TISSUE MICROSOMES

MICHAEL HALL and PHILIP L. GROVER

Chester Beatty Laboratories, Instituteof CancerResearch: Royal Cancer Hospital. Fulham Road, London SW3 6JB (U.K.)

(Received March 19th, 1986) (Revision received June 30th. 1986) (Accepted June 30th, 1986)

SUMMARY

The metabolism of benzo[a] pyrene (BP) by microsomal fractions of the skin, lungs and liver of the mouse, and the effects on this process of pretreat- ment with the xenobiotics phenobarbital (PB) and 3-methylcholanthrene (3-MC) were examined. Differences between the untreated tissues were found both in terms of the total amounts of diol recovered and in the relative proportions of the individual diols extracted following incubation. Induction with PB or 3-MC significantly altered the profiles of metabolic dials obtained with epidermal and hepatic microsomes compared with their respective controls. Pulmonary microsomes showed similar trends to those ob- tained with liver microsomes but these were not statistically significant. The optical purity of the BP-7,8-diol that was formed by each microsomal type was examined by direct resolution of the enantiomers on HPLC using a chiral stationary phase. In each case the (-)-7R,BR-enantiomer predominated. Pretreatment with 3-MC significantly decreased the optical purity of BP-7,8- diol recovered from incubations with skin microsomes, but significantly increased the optical purity of the diol extracted from incubations with lung and liver microsomes. In addition to the diols, an unidentified BP metabolite was found that eluted between BP-9,10- and 4,5-diol on a reverse-phase high- performance liquid chromatography (HPLC) system and which represented a

Abbreviations: BP, benzo[a]pyrene; 3-OH-BP, 3-hydroxybenzo[a]pyrene; BP-1,5-dial, trans-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene; BP-7,8-dial, trans-7,8-dihydro-7,8-dihy- droxybenzo[a]pyrene; BP-9,10-diol, trans-9,10-dihydro-9,10-dihydroxybenzo[a]pyrene; BP-11,12-diol, trans-l1,12-dihydro-ll,lP-dihydroxybenzo[a]pyrene; 3-OH-BP-7,8-dial, 3-hydroxy-trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene; BP-7,8-diol 9,10-epoxide, 7,8-dihydroxy-9,10-oxy-7,8,9,1O-tetrahydrobenzo[a]pyrene; chrysene-1,2-diol, trans-1,2- dihydro-1,2-dihydroxychrysene; HPLC, high-performance liquid chromatography; 3-MC, 3-methylcholanthrene; PAH, polycyclic aromatic hydrocarbons; PB, phenobarbital; TLC, thin-layer chromatography.

0009-2797/86/$03.50 o 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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major product in extracts of incubations of BP with both induced and un- induced skin and lung microsomal fractions.

Key words: Benzo[a] pyrene - Diols - Stereoselective metabolism - Mouse - Induction

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAH), of which BP is an example, are widespread environmental pollutants produced by partial combustion of fossil fuels and other materials of organic origin and which are known to be potential carcinogens in animal systems [ 11. Although of themselves chemi- cally and biologically inert, their metabolism within tissues yields a form, the ‘ultimate carcinogen’, which is capable of binding to cellular macromolecules including DNA. This is most commonly a bay-region diol-epoxide [2], a type of metabolite first identified in the case of BP and characterised as the BP-7&diol 9,10-epoxide [3] . Although four stereoisomers of this metabolite may be formed, one, the (+)-anti-BP-7,%diol 9,10-epoxide, derived from (-)-BP-7R,SR-diol, has been found to possess greater biological activity than the other three [ 2,4].

This metabolic activation of PAH via the diol-epoxide involves a sequence of three enzyme-catalysed steps: mono-oxygenation by cytochrome P-450, hydration by epoxide hydrolase and further mono-oxygenation again in- volving cytochrome P-450. This is both a regio- and stereoselective process [2,4,5], the proportion of parent hydrocarbon finally appearing in the form of its ultimate carcinogen being a reflection of the specificities and relative levels with the tissue of the enzymes involved. In this respect the cytochrome P-450 profile rather than epoxide hydrolase activity seems to be of greater importance [6,7]. A number of distinct P-450 activities have been identified in the livers of rats [B-lo], rabbits [ 111 and mice [12-141, and also in extrahepatic tissues including mouse skin [ 151. Some of these activities are constitutive while others may be induced by treatment of the animal with xenobiotics such as PB or 3-MC. Since the isozymes of P-450 within a given tissue differ with respect to overall substrate specificity and also regio- and stereoselectivity for the metabolism of different substrates [16,17], then induction would be expected to effect changes in the metabolic fate of PAH within different tissues. Such variations have indeed been found, for instance in the stereoselective metabolism of PAH on incubation with liver micro- somes from differently-induced rats [ 5,18-211.

Rat liver, however, is not thought to be a target tissue for PAH-induced carcinogenesis. This is in contrast to mouse skin which has long been known to be sensitive to tumour induction by BP [22] and mouse lung which is also susceptible [ 11. In the present work a comparison has been made of the regio- and stereoselective metabolism of BP to its diol derivatives in the skin, lung and liver of the mouse; the effects of enzyme induction on this process

267

has also been investigated. It is believed that this is the first such compre- hensive study to be conducted on a species known to be sensitive to the carcinogenic action of PAH, previous work having either concentrated on a particular tissue or inducer [23-251 or been conducted using rat liver microsomes as the metabolising system [26-281.

MATERIALS AND METHODS

3H-Labelled BP was prep ared from the unlabelled hydrocarbon by catalytic exchange (Amersham International plc, Amersham, Bucks, U.K.) and was diluted by addition of unlabelled BP. This was purified immediately prior to use by chromatography on a silica gel column.

Unlabelled BP-4,5-, -7,8-, -9,10- and -11,12-diols were obtained from NC1 Chemical Carcinogen Standard Repository, NIH, Bethesda, MD, U.S.A. Other chemicals were obtained as described [ 291.

Induction of animals Three groups of 10 adult male Parkes mice were employed for each

experiment. Group A (controls) were untreated; group B received PB (5 mg) in phosphate-buffered saline (0.1 ml) a day for 3 days by intraperitoneal injection, the final injection being given 24 h before killing; group C received a single intraperitoneal injection of 3-MC (1 mg) in arachis oil (0.1 ml) 48 h before killing.

Preparation of microsomes Animals were killed by cervical dislocation and their livers, lungs and an

area of shaved dorsal skin excised. Livers and lungs were retained on ice prior to homogenisation; skin samples were frozen in liquid nitrogen, the dermal surfaces scraped to remove excess fat and then powdered in liquid nitrogen by consecutive use of an Atomix blender (MSE Ltd., Crawley, Sussex, U.K.) and a Ultraturrax (Janke and Kunkel, Staufen, F.R.G.) [30]. All tissues were then homogenised in 25 ml 0.1 M phosphate buffer (pH 7.4), using a Potter-Elvehjem homogeniser fitted with a teflon pestle, and microsomal fractions prepared by differential centrifugation as described [31]. Microsomal pellets were finally resuspended in either 5 ml (epidermis and lung) or 15 ml (liver) 0.1 M phosphate buffer (pH 7.4) and retained at -70°C for 4-12 days prior to incubation with BP.

Metabolism of BP by microsomal fractions Incubations were conducted essentially as described previously [ 291.

Reaction mixtures consisted of 0.1 M phosphate buffer (pH 7.4), NADP’ (0.4 mM), glucose 6-phosphate (8.2 mM), magnesium chloride (2.5 mM), glucose-6-phosphate dehydrogenase (0.28 U/ml) and microsomal protein (0.25 mg/ml) in a final volume of 5 ml. The reaction was initiated by addition of 20 ~1 [3H] BP (4.0 mM, 0.55-0.61 mCi/pmol) in acetone and the mixture incubated with shaking at 37°C for 30 min. The mixture was then extracted

268

with ethyl acetate (2 X 1 vol.), the combined extracts dried (Na,SO,) and evaporated. Metabolic diols were separated from other oxidation products by preparative TLC on silica gel plates in benzene/ethanol (9 : 1, v/v). The appropriate bands of silica (Rf = 0.3-0.5) were eluted with ethanol (25 ml) and the extracts filtered prior to HPLC.

High performance liquid chromatography (a) Metabolic dials. UV-absorbing quantities of non-radioactive BP diols

were mixed with the extracts containing radioactive metabolites and the mixtures evaporated to dryness under reduced pressure. The residues were redissolved in methanol/water (1: 1, v/v) and subjected in their entirety to HPLC using a Waters liquid chromatography system (Waters Associates, Harrow, Middlesex, U.K.). The column (250 X 4.6 mm) of Zorbax ODS (HPLC Technology Ltd., Macclesfield, Cheshire, U.K.) was eluted at room temperature with a convex gradient (Waters 660 solvent programmer, gradient 3) of 50-70s (v/v) methanol in water over 60 min at a flow rate of 1.5 ml/min. Eluates were monitored at 254 nm (Waters 440 UV detector). 1 min fractions of the eluates were collected and aliquots (0.1 ml) removed for determination of radioactive content by liquid scintillation counting.

(b) Stereochemical analysis of BP-7,8-diol formation. Fractions of eluates from the reverse-phase HPLC separation of metabolic BP dials which were identified as containing BP-7,8-diol by their UV spectra were pooled and evaporated to dryness, then further dried in a vacuum dessicator for at least 30 min. These samples were then redissolved in hexane/ethanol/acetonitrile (17 : 2: 1, by vol.) and subjected to normal phase HPLC using a stationary phase consisting of chiral N-(3,5-dinitrobenzoyl)phenylglycine ionically bonded to y-aminopropyl silanised silica [32], otherwise referred to as a Pirkle 1-A column (Phase Separations Ltd., Queensferry, Clwyd, U.K.). The column was eluted at room temperature with a mobile phase of hexane/ ethanol/acetonitrile (17 :2: 1, by vol.) at a flow rate of 2.0 ml/mm [33]. Eluates were monitored at 254 nm, and 1 min fractions collected. The entire volume of each was used for the determination of radioactive content by liquid scintillation counting.

Protein determinations The protein content of microsomal fractions was determined by the

method of Lowry et al. [34] with bovine serum albumin as standard.

Statistical analysis The statistical significance of results was tested on a paired basis using

a one-sided Student’s t-test, with n - 1 d.f. Values throughout are presented as means + S.E.M.

RESULTS

HPLC elution profiles for radioactive metabolic diols extracted from incu-

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bations of [3H] BP with epidermal, pulmonary and hepatic microsomes ob- tained in turn from PB-treated, 3-MC-treated or control mice, respectively, are presented in Fig. 1. These results show that BP was metabolised to its 4,5-, 7,8- and 9,10-diol derivatives by all three tissue types under the incubation conditions employed. In certain samples two further peaks of radioactivity were noted. One (Figs. 1A and 1C) was found to chromatograph in the region of authentic BP-11,12-diol, and was most consistently seen in extracts of liver microsomal incubations. The second (Fig. 1A) eluted prior to BP-4,5- diol and apparently represents a major radioactive product. A similar peak was obtained as a major metabolite on HPLC profiles from incubations with both induced and uninduced skin microsomes, uninduced and PB-induced lung microsomes, and generally as a minor metabolite on profiles from incu- bations with PB- and 3-MC-induced liver microsomes (data not shown). However, only in one HPLC profile from incubations of BP with uninduced liver microsomes was a radioactive metabolite detected in this region (cf. Fig. 1C). Of the three HPLC profiles from separate incubations with 3-MC- induced lung microsomes, two showed a major peak with similar elution time (data not shown) whilst in the third (Fig. 1B) this was not detected. Neither of these metabolites have been positively identified so far.

The results of triplicate experiments designed to determine the relative amounts of BP diols present following a 30-min incubation of [3H]BP with microsomal fractions from different tissues of variously-induced animals al-e presented in Table I. Diols were quantitated from the amounts of radio- activity found to cochromatograph with authentic diols (Fig. 1) and from the specific activity of the parent hydrocarbon. These data indicate that both in the uninduced and induced systems, liver microsomal fractions contained higher BP hydroxylating activities than did skin or lung micro- somes, which both showed similar activities. On induction the total amounts of diols detected in extracts of liver microsomal incubations were significantly altered, being decreased by PB but increased by 3-MC. A similar, but non- significant trend was observed with lung but not with skin microsomes.

In all three tissues, pretreatment of animals with inducers led to a shift in the relative proportions of the diols recovered, regardless of whether total levels were altered. Thus the major diol product obtained from incubations of BP with uninduced liver and lung microsomes was BP-4,5-diol, whereas similar incubations with uninduced skin microsomes yielded mainly BP-7,8- diol (Table I). Following induction with both PB and 3-MC, the proportion of BP-9,10-diol recovered from incubations with epidermal microsomes was increased, with a concomitant decrease in BP-7,8-diol. BP-9,10-diol was also the major diol metabolite detected following incubations with PB-induced hepatic microsomes, whereas BP-7,Sdiol predominated in extracts of 3-MC- induced hepatic microsomal incubations. As with total amounts of metabolic diols, changes caused by induction in the proportions of individual diols detected followed a similar trend in incubations with lung as with liver microsomes. However, these differences were not significant when tested at the 0.1 level of significance.

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BP-g, BP-4, IO-diol 5-diol

1 1

2.0 - BP-11,12-diol BP-T, a-d101

A 4 4

1.5-

25

50 - C

40 -

30 -

20 -

10 -

0 10 20 30 40 50 Time (min)

Fig. 1. Separation by reverse-phase HPLC of metabolic diols of BP formed on incubation of microsomes with [SH]BP. Columns (250 x 4.6 mm) of Zorbax ODS were eluted with convex gradients of 50-702 methanol in water as described in the text. Eluates were examined for the presence of UV-absorbing materials and radioactivty. A: microsomes from the skin of animals pretreated with PB; B: microsomes from the lungs of animals pretreated with 3-MC; C: microsomes from the livers of animals left untreated. The positions of elution of the 4,5-, 7,8-, 9,10- and 11,12-diols are indicated.

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TABLE I

INFLUENCE OF INDUCERS ON THE FORMATION OF BP-4,5-, -78 AND -9,10-DIOLS FROM [‘HI BP BY MOUSE TISSUE MICROSOMAL FRACTIONS

Microsomes were isolated from the skin, lungs and livers of groups of 10 mice either pretreated with PB or 3-MC or left untreated, and incubated with [‘H]BP for 30 min as described in the text. Diol metabolites were isolated by TLC and reverse-phase HPLC and quantitated by determination of radioactivity. Data are means * S.E.M. of 3 separate experiments; figures in parentheses indicate the quantity of individual dials as a percentage of the total. *P < 0.1; **P < 0.05; ***P < 0.005 for changes caused by inducing agent when compared with control.

Tissue Inducing Amount of metabolic BP diols detected agent (pmol/min/mg microsomai protein)

-- 4,5-diol 7,8diol 9,10-diol Total

Skin

Lung

Liver

None (control) PB

3-MC

None (control) PB

3-MC

None (control) PB

3-MC

0.30 f 0.12 (21 * 10%)

0.69 +_ 0.56 (24 t 8%)

0.20 f 0.11 (18 f 5%)

0.99 f 0.59 (62 f 19%)

0.23 t 0.10 (26 + 16%)*

0.21 r 0.01 (38 t 17%)*

1.63 f 0.97 0.27 t 0.09 (67 +_ 7%) (18 + 6%)

0.44 * 0.16 0.11 t 0.08 (53 i 18%) (13 + 8%)

3.04 t 0.84 6.29 f 3.86 (44 t 20%) (41 r 14%)

17.08 i 4.48 (50 * 6%)

4.31 * 0.44 (23 t l%)**

10.51 i 2.40 (23 i 4%)**

3.87 t 1.20 (11 f 1%)

3.52 i 0.52 (19 f 2%)**

32.16 + 11.42 (60 * 5%)***

0.15 f 0.11 (16 f 10%) 1.44 r 0.59

5.34 + 5.19 (50 i 20%)* 6.26 f 5.85

0.60 t 0.46 (44 t 14%)** 1.01 t 0.57

0.37 * 0.18 (15 5 5%) 2.27 t 1.16

0.25 f 0.15 (35 t 23%) 0.80 i 0.07

2.28 t 1.14 (15 + 8%) 11.62 f 5.52

12.99 + 4.51 (39 r 7%) 33.94 f 8.74

11.42 t 1.94 (59 iT 2%)* 19.24 f 2.70*

8.63 * 2.96 (17 r 2%)* 51.30 +_ 16.13*

Figure 2 shows the HPLC profiles obtained when mixtures of purified metabolic and synthetic BP-7,Sdiols were subjected to normal-phase HPLC on Pirkle 1-A columns as described above. The percentage of each enantio- mer of the BP-7,8-diol that was recovered from incubations with microsomes was then determined from the amounts of radioactivity that coeluted with the authentic reference materials. These values are presented in Table II. Each tissue type shows a high degree of metabolic stereoselectivity in the conversion of BP to BP-7,8-diol, with the R,R enantiomer predominating in each case. Pretreatment of the animals with PB did not appear to signifi- cantly affect the proportions of the two enantiomers obtained with micro- somes from any of the three tissues. Induction with 3-MC, however, signifi- cantly increased the optical purity of the BP-7,8-diol obtained from incu- bations with lung and liver microsomes, but decreased the optical purity of the product from skin microsomal incubations. Taken together, the data

272

A

IS, ES ‘lR, 8R

4 4

0.5

0.25

IS. ES Ill, 8R B

4 4 0

1 ,”

160-

; 120-

E

,g EO-

T, c5 0 -2 40-

!x

O-

IS, ES IR, 8R

C 4 4

60

-0.02

- 0.01

-0

-0.02

-5

s E!

:, .d -0.01 E

5i

z

-0

-0.015

- 0.010

- 0.005

0 20 30 40 50

Time (min)

Fig. 2. Resolution by chiral HPLC of the radioactive S,S and R,R enantiomers of BP- 7&diol that were formed on incubation of [)H] BP with microsomes. Pirkle 1-A columns (250 x 4.6 mm) were eluted isocratically with a mixture of hexane/ethanol/acetonitrile (17 : 2 : 1, by vol.) as described in the text and the eluates were examined for the presence of UV-absorbing materials and radioactivity. A: microsomes from the skin of animals pretreated with PB; B: microsomes from the lungs of animals pretreated with 3-MC; C: microsomes from the livers of animals left untreated. The enantiomers were assigned as S,S or R,R according to Yang et al. [33].

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TABLE II

INFLUENCE OF INDUCERS ON THE ENANTIOMERIC PURITY OF BP-7,&DIOL FORMED FROM [‘HI BP BY MOUSE TISSUE MICROSOMAL FRACTIONS

[‘H]BP-7,8diol, isolated from reverse-phase HPLC, was used for the estimation of optical purity following resolution into enantiomers on a Pirkle 1-A column as described in the text. Data are means of 3 separate experiments, except for skin PB (n = 2). Optical purity is defined as the difference between the percentage of the two enantiomers, and is given here as the means * S.E.M. of 3 separate experiments, except for skin PB (n = 2). *P < 0.1; **P < 0.01 for changes caused by inducing agent when compared with control,

Tissue Inducing Relative amount of BP- agent 7,8-diol enantiomer (%)

Optical purity (%)

Skin None (control) PB 3-MC

Lung None (control) PB 3-MC

Liver None (control) PB 3-MC

4 3

3

2 <l :

5

4 2

98 96i3

96 93 f 4 97 93 r 4*

97

98 >99

95

96 93 f 2 98 97 t 1**

94t3

95 f 1 99 t o*

90 f 1

presented in Tables I and II indicates that induction with PB and 3-MC de- creased the amount of the proximate carcinogen (-)-BP-7R,8R-diol ex- tracted from incubations of BP with skin microsomes, but increased it from incubations of BP with lung and liver microsomes.

DISCUSSION

This work has investigated the regio- and stereoselective metabolism of BP by microsomes obtained from skin, lung and liver of the mouse, and the effects of pretreatment with the xenobiotics PB and 3-MC upon this process. Specifically, this was examined in relation to the production of metabolic diols from the parent hydrocarbon. Inducer-mediated variations in both the relative proportions of the diols found and the stereoselectivity of the BP-7,8-diol recovered following incubation were observed for all three tissues. With the exception of an unknown metabolite which was found to elute between BP-9,10-diol and -4,5-diol on reverse-phase HPLC of extracts from lung microsomal incubations, the metabolic profile obtained with pulmonary microsomes in general paralleled that with hepatic microsomes, although

274

activities were smaller and variations less pronounced. Epidermal microsomes produced rather contrasting results.

Such a tissue-specific difference was observed in the case of the major diol detected following incubation of BP with uninduced microsomes (Table I). With skin microsomes this was BP-7,8diol, in agreement with a previous study [35] in which mouse skin was treated with [3H] BP in vivo. However, when skin was incubated with BP in short-term organ culture, more than half the diol metabolites present were identified as BP-9,10-dial 135,361. This difference was thought perhaps to be due [35] to a greater propensity of BP-7,8-diol for non-covalent binding with subcellular components when compared with BP-9,10-diol. In this respect, then, microsomal incubations might provide a better model for the in vivo state than short-term organ culture.

Pretreatment of animals with both PB and 3-MC caused significant changes in the regioselective metabolism of BP by skin and liver microsomes, and also in the total levels of BP dials recovered from liver microsomal incubations (Table I). Similar effects of 3-MC induction on hepatic microsomal metabolism have previously been reported [23,24]. However, the results with skin microsomes are in contrast with previous work [25] where BP-7,8-diol was the major metabolite found following induction, although in this case two strains of inbred mouse were used and the inducer was applied directly to the area of skin taken as a source of microsomes.

BP dials are essentially intermediates on a pathway by which BP is sequen- tially oxidised to forms which may either be conjugated and excreted as water-soluble derivatives, or which bind to cellular macromolecules so disrupting intracellular mechanisms [4]. Thus any changes seen in the total amounts of dials found following induction may be due to alterations in either the rate of their formation from BP, the rate of their further meta- bolism or both. Such possibilities could be better resolved by time course studies rather than by the single time-point observations made in the present work. It is of interest to note that one of the enzymes involved in the con- jugation of hydroxylated metabolites of BP to form water-soluble derivatives, UDP glucuronyltransferase, is both an inducible activity and is also believed to be a structural gene product of the murine Ah gene locus which codes for the cytochromes P-450 [37].

As mentioned above, none of the apparent inducer-mediated changes in regioselective metabolism of BP by lung microsomes were statistically significant (Table I), which would seem to be due to the rather large variability in the results from the three experiments. A similar phenomenon may also be seen in the data obtained for incubations of BP with skin microsomes even though significant inducer effects were obtained with these. In contrast, the variance in the results for incubations with liver microsomes were smaller (Table I) apparently because the total amounts of diols extracted from the incubations with hepatic microsomes were con- sistently greater than with skin or lung microsomes, thus leading to greater accuracy in their measurement. Under present experimental conditions

275

the extent of variance in the results with these last two tissue types could be reduced in two ways. One method would be to increase the number of incubations performed, thereby increasing the size of the sample. Alter- natively the yield of extractable diol could be increased for each experi- ment so increasing the sensitivity of measurement. The latter might be achieved by including exogenous epoxide hydrolase in microsomal reaction mixtures since it has been reported [38] that levels of this enzyme are limiting for PAH metabolism in certain rat tissues.

Since the same amount of microsomal protein from the three tissues was used in each incubation, the larger yields of diols from liver compared with skin or lung microsomes would seem to be due either to an increased rate of their formation or to a decreased rate of their further metabolism in the former tissue compared with the latter two. Again time-course studies should help to clarify this situation. It would also be of interest to widen such investigations to include the measurement of the extent of metabolism of BP by the various types of microsomes, and also the relative amounts of metabolite, other than diols, that are formed.

The regioselective metabolism of BP by mouse tissues in the present study was evaluated by measurement of the PB-4,5-, -7,8- and -9,lOdiols. Al- though there was some evidence for the formation of BP-11,12-diol (Fig. 1A and 1C) this was neither consistent nor unequivocal. This metabolite has previously been detected in rat skin following 18-h incubations with BP [39], but not after incubation of mouse liver, lung or skin microsomes with BP [23-251 nor after incubation of BP with mouse skin either in vivo or in short-term organ culture [35] .

Figure 1A shows the presence of a further BP metabolite that was also found in the extracts of several other microsomal incubations, but which is of unknown identity. One possibility for this could be a phenol, although previous separations of BP metabolites on reverse-phase HPLC have shown that phenols elute later than diols [ 23,241. A recent study [ 401 has identified a BP triol, 3-OH-BP-7,8-diol as being present in the bile of rats injected with [3H] 3-OH-BP. When this metabolite was run on a reverse-phase HPLC system it was found to elute between the BP-9,10- and -7,8-diols. The pos- sibility that BP was being metabolised to a trio1 derivative by mouse tissue microsomes in the present set of experiments would not be entirely un- precedented since evidence has previously been obtained that a triol-epoxide of chrysene reacts with DNA following application of [ 3H] chrysene to mouse skin both in vivo [41] and in short-term organ culture [42].

Mouse skin has previously been found to metabolise chrysene to chrysene- 1,2-diol in a highly stereoselective manner’ [42] , and a similar stereoselective formation of BP-7,8-diol from BP was found in the present work (see Table II). This diol was chosen in order to examine the changes in the stereo- selectivity of BP metabolism caused by inducers and between tissues in the mouse partly because it is the precursor of the ultimate carcinogenic form of BP and partly because, of the three diols here investigated, it is the 7,8- diol whose enantiomers are resolved most readily by HPLC on Pirkle 1-A

276

columns [33]. Under the present incubation conditions, it was found that the (-)-7R,8R enantiomer [33,43] of BP-7,8-diol was formed preferentially, the degree of optical purity of the product being either significantly increased or decreased on induction, depending on the tissue type (Table II).

In the stereoselective metabolism of BP by rat liver microsomal systems, formation of (-)-BP-7R,8R-diol leads to the formation of anti-BP 7,8-diol 9,10-epoxide, the ultimate carcinogenic form of BP [2]. Nebert and Jensen [37] have suggested that those tissues which have a greater capacity for the conversion of PAH into ultimate and proximate carcinogenic forms tend to be more sensitive to their carcinogenic action, and more specifically that Ah responsiveness in the mouse is related to sensitivity to skin tumouri- genesis. However, these authors also speculated that other gene products might be involved in determining susceptibility of certain mouse strains to PAH-induced carcinogenesis. Bickers et al. [25] in a study of BP metabolism by rodent skin microsomes found that neither the patterns of metabolism nor the quantities of proximate carcinogens that were produced were reliable indicators of tissue sensitivity to the carcinogenic action of PAH. The results of the present work (Tables I and II) would tend to confirm this latter view. Both skin and lung are target organs for PAH-induced carcinogenesis, yet showed different patterns of BP metabolism. Induction, particularly with 3-MC, decreased the amount of (-)-BP-7R,8R-diol recovered following incubation of BP with skin microsomes, but increased it after incubation with lung and also liver microsomal fractions, the latter being a non-target organ. It has previously been reported [44] that induction of mouse skin with PAH led to increased microsome-mediated mutagenesis of BP -7,8-diol and it thus seems probable that the further metabolism of proximate carcino- gens, as well as other factors such as DNA repair, must be considered along- side the regio- and stereoselective metabolism of PAH when assessing the susceptibility of a particular tissue to PAH-induced carcinogenesis.

Any change in the relative proportions or optical purities of metabolic diols produced from BP on incubation must be a reflection of a change in enzyme levels within a given tissue. In the liver of rats pretreated with 3-MC, greater than 70% of the total cytochrome P-450 levels is represented by isozyme P-450 c, whilst in the livers of rats induced with PB, enzymes P-450 b and P-450 e predominate. The levels of other isozymes, including a constitutive form, P-450 a, also vary between differently-treated animals [9]. These isozymes display different activities towards different substrates and thus the metabolic products of a given tissue will reflect the range of cytochrome P-450 isozymes present and will be altered on induction [9,16, 171. Thus whilst the formation of BP-7,8-diol by rat liver microsomes is highly stereoselective, the optical purity varies with the type of inducer used to pretreat the animal, and also between different strains of the same animal [ 51.

Both PB-and 3-MC-inducible forms of cytochrome P-450 have been ident- ified in murine hepatic [13] and epidermal [15] microsomes. They are structural gene products of the Ah locus of responsive mice [13,37] in

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which category are included random-bred mice such as the Parkes strain used in the present experiments. It has been suggested [37] that there exist between three and five forms of 3-MC-inducible cytochrome P-450 in mouse liver. Neither the substrate specificities nor the regio- and stereo- selectivities of metabolism for these forms are known. In addition, it is not known whether similar isozymes in different tissues display comparable activities or selectivities. The differences in BP diol formation between skin and liver microsomes found here (Tables I and II) might therefore be ex- plained on the basis of distinct constitutive and induced cytochrome P-450 profiles within these tissues and/or the presence of tissue-specific forms of the same isozyme possessing distinct activities. Only the isolation and characterisation of these enzymes can resolve these possibilities.

ACKNOWLEDGEMENTS

The authors wish to thank Drs. P.P. Fu and A. Weston for helpful dis- cussions and Mrs. D. Parker for skilful technical assistance. This work was supported in part by PHS grant No. CA21959 awarded by the National Cancer Institute, DHSS and, in part, by grants to the Institute of Cancer Research from the Medical Research Council and the Cancer Research Campaign.

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