Identification of metabolites of benzo[j]fluoranthene formed in vitro in rat liver homogenate

227

Chem-BwL Interactwns, 63 (1987) 227-237 Elsevier Scientific Pubhshers Ireland Ltd

I D E N T I F I C A T I O N OF M E T A B O L I T E S O F BENZO[/]FLUORANTHENE FORMED IN VITRO IN R A T L I V E R H O M O G E N A T E

JOSEPH E RICE, NORA G GEDDIE and EDMOND J LAVOIE Naylor Dana Institute for Dzsease Preventwn, Amemcan Health Foundatmn, Valhalla, N Y 10595 (USA) (Received January 5th, 1987) (Revision rece,ved April 17th, 1987) (Accepted June 16th, 1987)

SUMMARY

The metabohtes of benzoD]fluoranthene (BjF) as formed In vitro using the 9000 × g s upe r nat ant from Aroclor-pretreated rats have been Identified Two dlhydrodlols, trans-4,5-dlhydro-4,5-dihydroxyBjF and trans-9,10-dlhydro9,10-dlhydroxyBjF have been ldentlhed as major metabohtes by comparison of their spectral and chromatographic properties with those of pure synthetic standards. T her e was no evidence that any of the isomerm 2,3dlhydrodlol was formed as a metabohte of BjF under these incubahon conditions Neither of the metabolic dIhydrodlols of BjF were formed with a high degree of stereoselectIvlty The enanhomerIc purity of the 4,5dlhydrodlol was 20% while that of the 9,10-dlhydrodlol was 46% At least four phenols were detected among the metabohtes of BjF. These were identified as 3-, 4-, 6- and 10-hydroxyBjF based upon comparison of their UV spectra and HPLC retention times with those of synthetic reference standards. BjF-4,5-dlone was also identified as a metabohte under these mcubatmn conditions

Key words

BenzoD]fluoranthene Polycyclic aromatic hydrocarbon

-

Metabolism

--

Non-alternant

--

INTRODUCTION

BenzoD]fluoranthene (BjF, Fig 1) is a non-alternant polycycllc aromatic hydrocarbon (PAH) which has been Identified In cigarette smoke condensate, 0009-2797/87/$03 50 © 1987 Elsevier Scmntlflc Publishers Ireland Ltd Printed and Published In Ireland

228

10~'~12 1~'~3 11

2

7

6

Fig 1 Structure and numbering of BIF

air particulates, ground and tap water, soil, smoked foods, and automobile and dmsel engine exhaust [1-7]. This compound is mutagenlc in S typh~mumum TA100 when assayed in the presence of rat liver homogenate [8]. BjF when applied topmally to mouse skin IS active as a tumor initiator and a complete carcinogen [1,9,10]. BenzoD]fluoranthene is also carcinogemc when implanted into the lungs of rats [11] and has recently been shown to be tumorigemc in newborn mine [12]. The metabohtes of B]F as formed m vitro employing a slmdar mlcrosomal activatmn system as used in assaying its mutagemc activity was investigated Using liver homogenate from Aroclor-lnduced Fisher rats, we identified metabohtes by comparison with synthetic reference standards. The identity and the enantmmerlc purity of those metabolic dihydrodmls which are proximate mutagens of BjF were also determined MATERIALS AND METHODS

Instrumentation Analytmal high performance liquid chromatographic (HPLC) analyses were performed using a Hewlett-Packard Model 1090 liquid chromatograph equipped with a HP Model 1040A high speed spectrophotometrlc detector (diode-array) Preparative HPLC was performed using a Waters Associates, Inc. Model ALC/GPC-204 high speed hquld chromatograph eqmpped with a Model 6000A solvent dehvery system, an automated gradient controller, a Model 440 UV-vislble detector momtoring at 254 nm and a Model U6K septumless injector. A Waters Associates, Inc. Model 712 WISP was used for automatic rejections. Fractions were collected using an ISCO, Inc Model 2150 peak separator and a FOXY Fraction Collector (ISCO, Inc.)

Chemwals BenzoD]fluoranthene was purchased from the Community Bureau of Reference (BCR), Brussels, Belgium with a purity of > 99 5%. The syntheses of trans-2,3-dIhydro-2,3-dIhydroxyBjF, trans-4,5-dlhydro-4,5-dihydroxyBjF, trans-9,10-dlhydro-9,10-dihydroxyBjF, BjF-2,3-dlone, BjF-4,5-dlone, BjF-9,10dlone, and 4- and 10-hydroxyBjF have been previously described [13]. 1HydroxyBjF was prepared from 1,2,3,12c-tetrahydroBjF by Triton B

229 oxidation [14] at the 12c-position followed by acid-catalyzed dehydration. The resultmg dihydro compound was epoxldlzed with m-chloroperoxybenzoic acid and the epoxlde rearranged to the 1-keto demvatlve with boron trifloumde. The phenol was prepared from the ketone by heating with 10% palladmmon-charcoal. 3-HydroxyBjF was prepared m a similar manner from 1,12cdihydrobenzoD]fluoranthen-3(2H)-one [15]. Treatment of the trans-4,5dlhydrodml of BjF with warm tetrabutlyammomum hydroxide afforded hydroxyBjF The regmselectlwty of this dehydration was verified by HPLC which demonstrated that the isomerm 4-hydroxyBjF (whmh elutes 5.6 min later than 5-hydroxyBjF) was not formed as a by-product. The addltmn of phenethylmagnesmm bromide to 8-methoxyacenaphthen-l(2H)-one yielded 1hydroxy-8-methoxy-l-(2-phenylethyl)acenaphthene. Heating this compound with phosphorous pentoxide afforded 6-methoxy-BjF. Treatment with boron tmbromlde gave the corresponding phenol. Acid-catalyzed dehydration of the 9,10-dihydrodlol of BjF yielded 9-hydroxyBjF. Analysis of the dehydratmn mixture by HPLC demonstrated the absence of 10-hydroxyBjF whmh elutes 3.0 mm after 9-hydroxyBjF. Details of these syntheses will be pubhshed elsewhere. M e t a b o h s m zn w t r o w~th rat l~ver homogenate

For analytmal metabohsm studms, 400 gg of BjF was dissolved m 200 ~1 of dlmethylsulfoxlde (DMSO). This solution was added to 4 ml of S-9 mix m a 25 ml Erlenmeyer flask The mixture was shaken for 20 mm at 37°C m a Dubnoff metabohc shakmg incubator. These mcubatmn conditions have been commonly employed m our laboratorms for the metabohsm of alternant and non-alternant PAH Incubatmns were terminated by the addition of 2 ml of me-cold acetone. The mixture was extracted five times with 10-ml portions of ethyl acetate. The extracts were combined, drmd over sodmm sulfate, and evaporated to dryness under reduced pressure below 40°C. The residue was dissolved m MeOH and rejected onto a Vydac 10 ~m reverse-phase C18 column, 4.6 mm (1 d.) × 25 cm (the SEP/A/ RA/TIONS Group, Hespema, CA) Chromatograms were momtored simultaneously at 230, 254, 315 and 450 nm The solvent program employed was 50% MeOH/H20 for 20 min and then a hnear gradmnt of 50--100% MeOH over 50 ram. Preparative scale metabohsm was performed by incubation of 2 mg of BjF dissolved m 1 ml of DMSO for 20 mm at 37°C with 20 ml of the same S-9 mix employed for the analytmal scale metabohsm. Incubations were terminated by the addltmn of 20 ml of me-cold acetone Metabohtes were extracted from the incubation mixture as descmbed above and rejected onto a Hlbar Prep 10 LIChrosorb 10 ~m RP-18 column, 10 mm h.d.) × 25 cm (EM Reagents, Cincinnati, OH). The elution program employed for these separatmns was 60% MeOH for 20 mm, a hnear gradmnt of 6 0 - 8 0 % MeOH over 20 mm and then 80--100% MeOH over 10 mm. The flow rate was 4 ml/mm The S-9 mix used for these metabohsm studms was prepared from the hvers of male Fischer 344 rats The rats weighed between 250 and 300 g and were given Lp rejections of Aroclor 1254 (500 mg/kg body weight) five days

230 prior to sacrlhce The S-9 fraction was obtained as descmbed prewously [16] Each ml of S-9 m~x contained 50 mol of potassium phosphate buffer (pH 7 4), 8.0 ~mol of MgC12, 33 ~mol of KCI, 5.0 ~mol of glucose 6-phosphate, 4 0 ~mol NADP ÷ and 0.5 ml of the 9000 × g supernatant of the rat hver homogenate. The protein content of the S-9 mix used for these studms was 28 mg/ml as determined using the method of Lowry [17].

Enantwmemc pumty of the metabohc dzhydrodwls of B)F The enanhomers of the metabolic 4,5-dihydrodlol of BjF were directly resolved by HPLC using an ionic D-phenylglycine modified Spherlsorb 5 m Pirkle column, 4.6 mm (i.d.) × 25 cm (Regis Chemical Co, Morton Grove, IL) with an lsocratm solvent system of 7% (2:1 ethanol/acetonitrlle) in hexane at a flow rate of 2 ml/min Similar conditions have been employed by others for the resolution of dIhydrodiol metabohtes of several PAH [18] Suitable conditions could not, however, be found to directly resolve the enantiomers of BjF-9,10-dlol. BjF-9,10-dlol was converted into its bls(menthoxyacetate) derivative by reaction with (--)-menthoxyacetyl chloride [19] in pyridlne as described previously for related compounds [20,21]. The dlastereomerm bls(menthoxyacetates) of BjF-9,10-dlol were resolved by normalphase HPLC on a 10 ~m SI-60 LIChrosorb column, 4 mm (i.d) × 25 cm (EM Reagents, Darmstadt, F R.G.) eluting with 15% (10% THF/hexanes containing 0.2% MeOH) in hexanes at a flow rate of 2 ml/mm. RESULTS

A HPLC prohle of the ethyl acetate extractable metabohtes of BjF is presented in Fig 2 HPLC retention times and UV spectral maxima for each

c'n

250-

200-

D

,< E

150-

10050O-

10

2'0

30

4b

s'o

6o

70

Time (rain) Fig 2 HPLC profile of the ethyl acetate extractable m e t a b o h t e s of BjF as formed m vitro m r a t h v e r homogenate The lower trace was m o m t o r e d at 254 nm The trace m the insert was m o m t o r e d at 450 nm The a r r o w m d m a t e s the r e l a h v e r e t e n h o n time of s y n t h e t m BjF-2,3-dlol

231 of the designated peaks m Fig 2 are presented in Table I. Peak A has an ldentmal UV spectrum and coelutes with a synthetic reference standard of trans-4,5-dlhydro-4,5-dihydroxyBjF (BjF-4,5-dlol) Warming an acetic acid solution of this collected metabohte with a drop of concentrated HC1 results m formation of a phenol which has identical HPLC retention and UV spectral characteristics as 4-hydroxyBjF. Catalytic hydrogenation of collected Peak A (10% trmthylamme in THF, platinum oxide (Adam's) catalyst, 15 lbhn 2 H 2, 20 min) yields a compound which has a UV spectrum closely resembling benzo[c]fluorene On the basis of these data metabohte A was identified as BjF-4,5-dlol Metabohte B has been identified previously as trans-9,10dlhydro-9,10-dihydroxyBjF (BjF-9,10-dlol) by comparison with a synthetic standard The UV spectra of the metabolic dlhydrodlols of BjF are presented in Fig 3. Peak purity checks (comparison of the UV spectrum at 5 points on the peaks) of the metabolic 4,5- and 9,10-dlhydrodlols of BjF show that these peaks are free from any coelutlng metabohtes BjF-2,3-dlol was not observed as a metabohte under these incubation conditions (the arrow in Fig 2 marks the relative retention time of synthetic BjF-2,3-dlol) Synthetic reference standards of other potential metabolic dlhydrodlols of BjF such as the 7,8- or 11,12-dlhydrodiols are not presently available These metabohtes, if present, would be minor by comparison with the 4,5- and 9,10-dlhydrodlols Metabohte C was tentatively identified as BjF-4,5-dlone by comparison of Its HPLC retention time and UV spectrum with a synthetic standard Synthetic standards of BjF-2,3-dlone and BjF-9,10-dlone eluted in the same region of the chromatogram as BjF-4,5-dlone. The UV spectra of these qumones, however, did not correspond to those of any of the minor metabohte peaks which eluted between 35 and 40 mln. Seven out of the twelve possible isomers of hydroxyBjF were synthesized to serve as reference standards for this metabolism study. Peaks F and G were tentatively identified as 10- and 6-hydroxyBjF, respectively, by comparison of their UV spectra and HPLC retention times with synthetic reference standards (Figs. 4C and 4D) When monitored at 254 nm Peak H was observed as a poorly resolved shoulder This metabohte peak was more clearly detected when the chromatogram was monitored at 450 nm (see the insert in Fig 2) On the basis of its UV spectrum, and by colnjectlon with a synthetic standard, Peak H was tentatively identified as 3-hydroxyBjF (Fig 4A) 4-HydroxyBjF (peak I) had the longest retention time (50 6 min) among the metabolic phenols of BjF (Fig 4B) There was no indication that 1-, 5- or 9-hydroxyBjF were present among the ethyl acetate extractable metabohtes of BjF formed under these incubation conditions The use of a diode-array detector, which allowed for the collection and storage of UV spectra every 3 5 S greatly facilitated this analysis of BjF metabohtes. It was possible, using this detector, to obtain high-quality UV spectra of Peaks F, G and H whmh had extensive overlap. Such an analysis would have been much more difficult using the conventional technique of peak collection followed by UV spectral determination The enanhomeric purity of BjF-4,5-diol was determined directly by chlral stationary-phase HPLC as described above in Methods The metabolic dlhydrodlol was isolated by preparative HPLC and then Injected onto a

B]F-4,5-dlol BjF-9,10
A B C D E F G H I J

26 9 28 7 37 9 45 0 45 7 47 7 48 4 48 8 50 6 60 0

Ret time (mm)~ 344, 393, 410, 354, 380, 389, 394, 386, 394, 383,

331,320, 289, 374, 356, 328, 360, 328, 290, 350, 336, 324, 362, 345, 328, 369, 352, 324 377, 340, 324, 367, 332, 318, 374, 356, 330, 375, 365, 357,

278, 258 max, 250, 229 307, 297, 285, 275 sh, 241 max, 237 255, 236 max 310, 287, 277, 268, 243 max 317, 303 sh, 288 sh, 241 max max, 311, 282, 272, 257, 242, 219 310, 299, 285, 247 max 297, 286, 278 sh, 246 max, 224 317, 303 sh, 277 sh, 257, 242 max, 223 348, 333, 319, 308, 294, 282, 241,225 max

UV Spectrum (nm)d

a

Peak refers to the lettered peaks m Fig 2 b Identlhcatmn of peaks ~s based upon their UV spectra and comjectmn with synthetic reference standards c Retention times were recorded using the HPLC conditions hsted m Methods d UV spectral data for BjF derivatives not detected among the metabohtes formed in vitro BjF-2,3~ilol, 353, 335, 335, 320, 299, 285 sh, 268, 258 max, 238, 233 sh, 1-HydroxyBjF, 338, 324, 296, 287, 268 max, 260, 5-HydroxyBjF, 390, 370, 342, 325, 317, 292, 280, 273, 253 sh, 244 max, 9HydroxyBjF, 384, 366, 320 max, 310 sh, 267, 236, BjF-2,3-dlone, 347, 330, 270 max, 233, BjF-9,10-dlone, 352 max, 342, 260,238

Identlhcatlon b

Peak a

IDENTIFICATION OF METABOLITES OF BjF FORMED IN VITRO IN RAT LIVER HOMOGENATE

TABLE I

t'o

233

OH

D

E

)

250

300

350

400

250

300

350

400

Wavelength (nm) Fig 3 Comparison of the UV spectra of metabohtes A (left) and B (right) with synthetic BjF-4,5dlol and BjF-9,10-dml Metabohte spectra are presented as sohd hnes (--) and UV spectra for synthetic standards are presented as dotted hnes ( )

Pirkle column. BjF-4,5-diol was found to exist as a 2 : 3 m i x t u r e of e n a n t l o m e r s with r e t e n t i o n times of 58.2 and 60.3 rain and a resolution value of 0.84*. The e n a n t i o m e r i c purity** of this dihydrodlol was t h e r e f o r e 20%. Metabolic BjF-9,10-dlol was c o n v e r t e d into a pair of d i a s t e r e o m e r i c bls(menthoxyacetates) and t h e s e w e r e s e p a r a t e d by normal-phase H P L C with r e t e n t i o n times of 15.8 and 17.7 rain and a resolution value of 1.15 as described above. T h e ratio of t h e s e d i a s t e r e o m e r s was 7 3 : 2 7 with the e a r h e r eluting d t a s t e r e o m e r predominating. T h e enantiomerlc p u r i t y of the m e t a b o h c 9,10-dihydrodiol of BjF f o r m e d u n d e r t h e s e incubation conditions is t h e r e f o r e 460/0. DISCUSSION S e v e r a l studms on the blologmal activity of benzo[?]fluoranthene have been r e p o r t e d . M u t a g e n i c l t y studies using Salmonella typh~murzum T A 100 have shown t h a t BjF is active in the p r e s e n c e of a microsomal activation s y s t e m [8]. W y n d e r and Hoffman [1] r e p o r t e d t h a t BjF was a c t w e as a complete carcinogen w h e n applied to the skin of Swiss mice t h r e e times weekly for life. More r e c e n t l y , BjF has been shown to elicit squamous-cell carcinomas of the lung in Osborne-Mendel r a t s [11]. BjF also gives rise to lung adenomas and liver t u m o r s when assayed in n e w b o r n mice [12]. A m o n g the isomeric b e n z o f l u o r a n t h e n e s which have been bloassayed, BjF is second only to benzo[b]fluoranthene m tumorigenic p o t e n c y [9,12]. *Resolution Value = 2(t G - tb)/(W a + W b) w h e r e t a and t b are the retenUon times of the two peaks and W a and W b are the peak base widths -B[ **Enantiomertc p u r i t y = I AI T-~-~ × 100 w h e r e A and B r e p r e s e n t the a m o u n t s of the t w o enantlomers

234

.

B

A OH

D ,<

E D

25o

3oo

4oo

25o

36o

4oo

Wavelength (nm) Fig 4 Comparison of the UV spectra of synthetm (-----)and metabohc ( hydroxyBjF, B, 4-hydroxyBjF, C, 6-hydroxyBjF, D, 10-hydroxyBjF

) phenols of BjF A, 3-

P r e h m m a r y investigations into the m e c h a m s m of activation of BjF to a genotoxic a g e n t have shown t h a t BjF-9,10-dlol is formed as a m e t a b o h t e m vitro m h v e r h o m o g e n a t e from A r o c l o r - p r e t r e a t e d male Fischer-344 rats [8] This dlhydrodlol was found to be a more p o t e n t m u t a g e n than BjF at low doses (<20 ~g/plate) when a s s a y e d with m e t a b o h c a c t w a t l o n [8]. BjF-9,10dlol has also b e e n assayed for tumor-initiating a c t w l t y on CD-1 mouse skin relative to BjF [9] A t total m~tmtmg doses of 4.0 and 0.4 ~mol/mouse BjF9,10-dlol was clearly less t u m o r l g e m c than the BjF. This was m a m f e s t e d as both a lower incidence of t u m o r - b e a r i n g mice as well as a lower t u m o r mult~phclty A second m u t a g e m c dlhydrodlol m e t a b o h t e of BjF of unknown s t r u c t u r e was also isolated m the e a r h e r s t u d y [8] This same m e t a b o h t e was t e n t a t i v e l y ldentffmd m vlvo m mouse skin as BjF-4,5-dml, based upon a comparison of its UV s p e c t r u m with t h a t of 7-ethyhdenebenzo[c]fluorene [22]

235 In the present analysis we have confirmed that BjF-4,5-dlol is a major in vitro metabohte by comparison of its UV spectrum and HPLC retention time with a synthetic reference standard, and by identification of its dehydration and reduction products The enantlomerm pumties of BjF-4,5-dlol and BjF-9,10-dlol as formed in vitro in rat hver homogenate were determined to be 20% and 46%, respectively We have observed that the metabolic 2,3-dihydrodlol of fluoranthene, which is structurally similar to BjF-4,5-diol, is formed with the same low enantiomerlc purity as BjF-4,5-dlol under similar incubation conditions (unpublished results). An emplrmal relationship has been found to exist between the relative retention times of dlhydrodlol metabohtes on chlral stationary phase (CSP) HPLC and their absolute configuration It has been noted by several investigators that the (S,S)-enantlomer of transdlhydro and tetrahydrodiols elutes more rapidly on CSP-HPLC than the (R,R)-enantlomer [23,24]. This relationship can be used to tentatively assign the more abundant (later eluting) enantlomer of BjF-4,5-diol as BjF-4R,5Rdihydrodiol A similar relationship exists between retention time on normalphase HPLC and absolute configuration of (-)-menthoxyacetate esters of trans-dihydrodlols. The less polar (earlier elutmg) diastereomer has the (R,R)configuration [25] Based upon this correlation, BjF-9R,10R-diol can be tentatively assigned as the major enantiomer formed m vitro In rat liver homogenate. Studies have shown that the enantiomeric purity of metabolic dlhydrodmls can vary significantly depending on the strain of animal used and on the selection (if any) of an enzyme inducer [26]. In view of this we are currently investigating the enantiomeric purity and absolute configuration of the metabolic dlhydrodlols of BjF as formed in vivo in mouse skin under conditions similar to those used to evaluate the tumor-initiating activity of BjF Several metabolite peaks were detected which eluted between 30 and 40 min in the HPLC profile shown in Fig 2 These peaks are more prominant when monitored at 450 nm than at 254 nm BjF-4,5-dione has been shown to elute in this same area of the HPLC profile On the basis of these observations it would not be inconsistent for these metabohtes to be BjF qutnones None of these metabohtes, however, was found to coelute with a synthetic reference standard of either BjF-2,3-dione or BjF-9,10-dione BjF has a four-sided pseudo-bay region in the area between the 1- and 12positions. X-ray crystallography of 5,10-dlmethoxyBjF has shown that the steric lnteractmns occuring between the hydrogens on the 1- and 12-carbons are similar to those occuring in the bay-regions of phenanthrene and benz[a]anthracene [27] Although it might be anticipated (based upon studies with other PAH) that enzymatic oxidation of BjF-9,10-dlol to a pseudo-bay region diol-epoxlde would be a major activation pathway for BjF this does not appear to be the case Ab initlo calculations indicate that the pseudo-bay region does not confer any special stability to the carbocatlon resulting from ring-opening of the 9,10-diol-ll,12-epoxlde of BjF [28]. This calculated result Is consistent with bioassay data which show that the 9,10-dihydrodlol IS not

236 the major proximate tumomgemc metabohte of BjF [9]. Huckel molecular orbital calculatmns have shown that the carbocatmn resulting from opening of the 4,5-dml-6,6a-epoxlde of BjF ~s substantmlly more stable than that demved from the 2,3-dml-l,12c-epoxlde of BjF (B D Sflverman, pers comm ) Studms are currently m progress to evaluate the tumor-mltmting a c h w t y of BjF-4,5-dml and BjF-9,10-dml on mouse skin relahve to BjF ACKNOWLEDGEMENTS

This study was supported by Grant No ES 02030 from the Natmnal Inshtute of Enwronmental Health Scmnces REFERENCES 1 2

3 4 5

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9

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12 13 14 15

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