Document Yv18JmyvBD69xbV5qzBMavME

Enwinmmenial Health Perspectives Vol. 82, p ~21. 5-222, 1989 Pharmacokinetics and Metabolism of Benzene in Zymbal Gland and Other Key Target Tissues after Oral Administration in Rats by Lawrence K. Low,* J. Ralph Meeks,* Kenneth J. Norris,* Myron A. Mehlman,* and Carl R. Mackerer" Solid tumors have been reported in the Zymbal gland. oral and nasal cavities, and mammary gland of Sprague-Dawleyrats followingchronic oral administration of benzene. The cause for the specificity of such lesions remains unclear, but it is possible that tissue-specificmetabolism or pharmacokinetics of benzene 1 is responsible. Metabolism and pharmacokineticstudies were carried out in our laboratory with "C-benzene at oral doses of 0.15 to 500 mgkg to ascertain tissue retention, metabolite profile, and elimination kinetics in target and nontarget organsand in blood. Findings fromtheses studiesindicate thefollowing: a)theZymbal i gland is not a sink or a site of accumulation for benzene or its metabolites even after a single high dose (500 mgkg) or after repeated oral administration; b) the metabolite profile is quantitatively different in target tissues (e.g., Zymbal gland, nasal cavity), nontarget tissues and blood; and c) pharmacokinetic studies show that the elimination of radioactivity from the Zymbal gland is biphasic. lntroduction 'bo-year bioassay studies by Maltoni et al. (1,2)and by the National Toxicology Program (NTP)(3)have demon*ted that chronic oral administration of benzene in rats Produces solid tumors in a number of organs, including ZYmbal gland, nasal and oral cavities, and mammary h d . It is generally believed that the toxic effects of benzene result from the metabolism of benzene to electrophilic intermediates capable of interacting covalently mwthecariltincaaltumreacorfotmheoluelctiumleaste(4to-8x)ic.Aorltchaorucginhogtheeniecxsapcet- (3%remains unknown, several metabolic pathways proposed for benzene lead to the formation of reactive interw t e s . Of importance are those pathways giving rise t to *no- and polyhydroxylatedmetabolites (e.g., phenol, b-uinone, catechol, 1,2,4-benzenetriol),ring-opened i -bolites (e.g,, muconaldehyde, muconic acid) and bhenolic metabolites (e.g., 4,4'-biphenol)(9-15).The me%Gsm of benzene has been extensively studied in the and bone marrow (4,19-21)b,ut little effort has been I~ il Oil Corporation, Environmental and Health Science LaboraBox 1029, Princeton, N J 08540. reprint requests to L. K. Low,Mobil Oil Corporation, En- and Health Science Laboratory, P.O. Box 1029, Princeton, directed toward investigating the metabolism of benzene in other tissues (22,ZS).Target organ susceptibility to the carcinogenic or toxic effects of xenobiotics is, however, thought to be governed by many factors with tissuespec& metabolism being of key importance (24,25);such metabolism might lead to the formation and persistence, at critical levels, of genotoxic metabolites. The studies reported in this paper were designed to investigate the metabolism and pharmacokinetics of benzene in the Zymbal gland and other solid tumor target organs in Sprague-Dawley rats after oral administration. Results were evaluated to delineate differences in the metabolite profile, elimination kinetics, and bioaccumulation of benzene and/or its metabolites in Zymbal gland, nasal and oral cavities, and mammary gland, which are considered here to be target tissues, and liver and kidney, which are considered to be nontarget tissues. Materials and Methods Materials [UL-"C]Benzene (80-100 mCi/mmole) was purchased from Chemsyn Science Laboratories (Lenexa, KS); radi- ochemicalpurity of this material was determined by RPHPLC analysis(70% MeOH-30% water, vlv) to be greater I16 LOW ET AL. than 99%. Benzene was obtained from American Burdick and Jackson (Muskegon,MI) (99.7% pure by GC analysis). Metabolite standards (phenol, hydroquinone, catechol, 1,2,4-benzenetriol, 2,2`-biphenol, 4,4'-biphenol, Erans,tmns-muconic acid, phenyl-/3-D-glucuronidew) ere purchased from Aldrich Chemical Co. (Milwaukee, WI) or Sigma Chemical Co. (St. Louis, MO) and were greater than99% pure. Phenylsulfate was isolated a s a metabolite from the urine of rats treated with 14C-benzene, characterized by selective enzymatic hydrolysis with sulfatase,and purified by HPLC. Glucuronideconjugates of hydroquinone, catechol, and 1,2,4-benzenetriolwere synthesized enzymatically using uridine diphosphate [`4Clglucuronic acid (UDPGA)(304 mCilmole, New E n g land Nuclear) or unlabeled UDPGA and rat liver microsomes (22)and puritied by HPLC. Tetrabutylammonium dihydrogen phosphate, ascorbicacid, and ammonium acetate were obtained from Aldrich Chemical Co. and olive oil from Sigma Chemical Co. HPLC grade solvents (acetonitrile, methanol and tetrahydrofuran) were purchased from J. T. Baker (Phillipsburg, NJ). Animals Female Sprague-Dawley[Crl:CD(SD)BR]rats were obtained from Charles River Laboratory (Kingston, NY). Rooms were maintained at 20 to 22OC with a relative humidity of 40 to 60% and a 12-hrlightldark cycle. Animals received food (rodent chow, (15002 pellets, Ralston purina, St.Louis, MO)and tap water (automatic water feeder) ad libitum. At study initiation, rats were 12 to 18weeks of age and weighed 225 to 375 g. Methods oral Gavage Studies and Tissue Collection.SpraqyeDawley rats received single doses by oral gavage of Cbenzene, at 0.15, 1.5, 15, 150, and 500 mgkg, in olive oil (* 0.8-1.0 mL per animal). The specific activity of the oral gavage doses was adjusted such that each animal received 30 to 60pCi. Generally, groups of three animals were sacrificed at 1,3,6,9, 12, and 24 hr after oral administration and blood (cardiac puncture), Zymbal gland, nasaland oral cavity tissues, mammary gland tissue, bone marrow (femur), liver, and kidney were collected. Samples were frozen immediately after necropsy and stored at -7OOC until analyzed. One experiment was carried out in which three rats received "C-benzene for 2 weeks (500 mg/kg daily, 5 days a week for 2 weeks) and were sacrificed on day 14one hr after the last radioactive dose. The amount of radioactivity in the Zymbal gland was determined. Radioactivity Analyses. Determination of total radioactivity, in target and nontarget tissues, and blood was carried out by measuring the amount of 14C02produced from combustion of the samples o r a homogenate of the samples. When homogenization of the sample was performed, one equivalent volume (mL)of cold water or cold 0.1 M ascorbic acid solution was added for each gram of wet tissue. Combustion was carried out for 3 min on a Harvey Model OX-300 instrument. Radioactivity meab urements were quantitated on a Beckman Model liquid scintillation spectrometer using appropdate quench corrections. Tissue concentrations we& lated using specific activities of the radiolabeled b- oral doses and are expressed in units of l4C-k- equivalents (ng or pg) per gram or per milliliter. Metabolite Isohtion and HPLC Analysis. Isohtj,,,, aa metabolites from blood and the tissues was out according to the following procedure: samples were homogenized or minced in cold 100mM ascorbic a lution [blood, 0.5 mL per 1mL; liver, kidney, nasal a d oral cavity tissues, mammary gland, 250-500 mgm; Zymbal gland (pooled), 50 mg/500 pL;bone marrow (pooled), 100-200 mg/500 pL1. Homogenates were ex. tracted with two volumes of ethyl acetate to isolate unconjugated metabolites, and ethyl acetate and a~ueous layers were separated by centrifugation. The a~ueaus alayer was extracted a second time with ethyl acetab the organic fractions combined. Ethyl acetate was re moved under a stream of nitrogen gas and the residue reconstituted in t h e HPLC mobile phase (6% acetonitrile-95% 0.1 M ammonium acetate, pH 4.0, vhk separation of the unconjugated metabolites was out by HPLC using the conditions described below. remaining aqueous fraction was treated with an equivolume of methanol to precipitate proteins, follow by centrifugation. The supernatant fraction was isolaa and methanol removed under a stream of nitrogengrs; separation of the water-soluble, conjugated metabolik present in this aqueous sample was carried out by ionpair HPLC as describedbelow. HPLC analyseswere per. formed on a Beckman model 330 binary gradient high performanceliquid chromatographic system consisthg d two model llOA pumps, a model 420 controller and a model 210 injector (Beckman Instruments, Fullerton, CA) or on a Varian 5020 binary gradient HPLC system with two single-piston solvent delivery pumps, an inter- nal microprocessor controller and a Varian 9090 iqjee- tor/autosampler (Varian Instruments, Walnut Creek, CA). Reverse-phase (RP)columns (Altex OD$, 4.6 mm x 15 cm or 25 cm) with Brownlee C8 (4.6 mm x 30 mm) guard columns were used in most of the HPLC analyses. Identification of radiolabeled benzene metabolites was based on comparison of retention times of radiolabeled HPLC peaks with those of standards. Detection of these standard metabolites was carried out by monitoring the UV absorbance (254 nm) of the HPLC effluent with a Beckman model 153fixed wavelength instrument. Radi- olabeled glucuronide and sulfate standards were moni- tored with a Berthold flow-through model LB506C radi- oactivity detector. -Radiometn'oHPLCA n d p i s ofMetabolites. UWn- jugated metabolites present in the evaporated ethyl acetate sample were separated on an Altex ODS H P X column (4.6 mm x 15an)using the chromatographk con- ditions reported by Lunte and Kissinger (ad); elution carried out with 5% acetonitrile-95% 0.1 M axnrnonim acetate (pH 4.0) (vh) at a flow rate of 1ml/min. water-sgluble metabolites were separated using an ion- -., BENZENE PHARMACOKINETICS A N D METABOLISM IN ZYMBAL GLAND 21 7 procedure slightly modified after the one Sabourin et al. (22).Briefly, an Altex ODS mm x 25 cm) was initially equilibrated with methanolic tetrabutylammonium dihydrogen phosphate(TBAP, 30mM)I 90% aqueous TBAP (50mM). FolBowing injection, the HPLC column was eluted using a 1Omin linear gradient from 10% methanolic TBAP to 45% methanolic TBAP at a flow rate 1 mllmin. At 10 min, the methanolic TBAP solvent was replaced with a mlvent mixture containing 90% methanolic TBAP (30 ! mM)and 10% tetrahydrofuran (THF) (v/v)and the HPLC column eluted isocratically with 45% methanolic TBAPPTHF (9O:lO) /%% aqueous TBAP. The column was allowed to reequilibriate with 106 methanolic TBAP M o aqueous TBAP before another injection was made. 1 The HPLC eMuent was monitored with a Berthold Model LB506C flow-through radioactivity detector. AlternaI tively, fractions of the eluate (0.5 mL) were collected in scintillation vials and 15mL of cocktail fluid (Ready Sovl CP, Beckman, Fullerton, CAI added to each vial. Radivity in the fractions was determined with a Beckman 1 LS9OOO liquid scintillation instrument. Results Absorption and Distribution The time course of disposition and elimination of radioactivity in Zymbal gland and other organs after oral administration of 0.15 and 1.5m g k g 14C-benzeneis shown in Figures 1and 2.The highest levels of radioactivity for all tissues and organs were seen at the earliest sampling period (1hr), suggesting that peak levels could have occurred somewhat earlier. However, levels differed among certain tissues and organs over the entire sampling period. One hour after single oral doses of 0.15 and 1.5 mgikg 14C-benzene(Table l),concentrations of radioactivity fell roughly into three groups: the highest levels were found in liver and kidney; the lowest levels in Zymbal gland, nasal cavity tissue, oral cavity tissue, mammary gland, and bone marrow; and intermediate levels in blood. However, this distribution was altered at 15 mgikg where disproportionate increases were found in mammary gland and bone marrow. '- 1 \` - -+ Zymbal Gland -------t Mammary Gland Nasal Cavity Oral Cavity - - BoneMarrow 0 Blood --t Liver 1 - --0 Zymbal Gland - MammaryGland - Nasal Cavity - Oral Cavity - Bone Marrow --e- Blood -- Lnet Kidney Time (hr) . "C concentrations in the Zymbal gland, and various other d nontarget organs in Sprague-Dawley rats following a sindose of benzene (0.15mgkg). Points represent the means of 0 10 20 Time (hr) FIGUR2E. "C concentration in the Z-ynbal gland and vclrious other target and nontarget organs in Sprague-Dawley rats following a single oral dose of benzene (1.5mgkg).Points represent the means of three animals. 218 LOW ET AL. Table 1. Concentration of radioactivity in various tissues 1 hr following oral administration of 0.15, 1.5 and 15 mglkg "C-benzene. pg Benzene equivalentdg or mL, ppm' Tissue 0.15 mgkg 1.5 m g k g * *Zymbalgland * *Nasal cavity * *Oral cavity * *Mammary gland *Blood * *Bone marrow *Liver *Kidney 0.034 0.044 0.035 0.028 0.0% 0.058 0.198 0.254 0.006 0.008 0.001 0.008 0.004 0.005 0.006 0.005 0.380 0.059 0.547 0.123 0.359 0.017 0.373 0.055 0.769 5 0.073 0.490 0.077 2.043 f: 0.195 1.926 f 0.174 *'Values represent mean SEM for three animals. 15 m g k 3 *3.2 f 0.4 2.4 0.8 2.4 f 0.2 6.6 f 1.4 *6.3 f 0.9 10.1 1.3 12.8 2 1.4 12.2 f 1.2 Elimination from Target Tissues The eliminationof radioactivity in the Z y b a l gland occurred in a biphasic manner (Fig.3);the 4C elimination half-life for the rapid phase was 2.4 to 2.8 hr, while the half-life of the slow phase was 18 to 21 hr (Table 2). As evident in Figures 1and 2, elimination of 14Cfrom the nasaland oral cavities, mammary gland, blood, liver, kidney, and bone marrow also displayed biphasic kinetics. During the first 8to 10hr,eliminationof radioactivity occurred rapidly in these tissues; but af'ter this period, radioactivity disappeared a t a much slower rate. The halflives of 14Celimination (rapid and slow phases) for various target and nontarget tissues are summarized in Table 2;for the 0.15 mglkg dose, the t1/2for the rapid phase ranged from 2.2 hr for blood to 4.2 hr for kidney while the t112 for slow phase ranged from 11hr for bone marrow to 29 hr for blood. All of the radioactivity in blood and tissues 1hr after the 0.15 mglkg dose appeared as benzene metabolites, indicating very efficient first-pass metabolism of benzene by the liver after oral absorption. Accumulation in Zymbal Gland 0 10 20 T I M (hr) F I ( ; ~ K3.EComparison of I4Clevels in the Zymbal gland in ratsgiven a single oral low dose of benzene of 0.15,1.5and 15mg/kg. Points rep resent the means of three rats. For the 15mgkg dose group, tissues were not taken at the 12-hr time interval. The amount of benzene-derived material remaining in the Zymbal gland 24 hr after single gavage doses of 0.15, 1.5,15,150, and 500 mglkg 14C-benzeneconstituted less than O.OOO18 of the administered dose, indicating that ac- Table 2. Half-life of elimination of radioactivity from Zymbal dand and other tissues.' cumulation of "C does not occur in this gland. There was Rapid pharie, Slow phase, some evidence, based on tissuehlood ratios and extrac- Tissue Dose, mgikg ti,.. hr t1/21 hr tion studies, that 14C-benzenemight be sequestered in Zymbal gland 0.15 2.8 18 mammary gland, bone marrow, and adipose tissue when animals were dosed with radiolabeled benzene at or atiboonveth1a5tm'PCn<gw(aTsasbellee1c)t.ivHeolywerveetar,inthederebywathsenoZiynmdibcaal- Zymbal gland Zymbal gland Blood Blood Blood 1.5 15.0 0.15 1.5 15.0 2.1 2.5 22 2.1 2.5 21 8b 29 23 ab F gland in comparison to mammary gland and adipose tissue. Since it has been reported that fatty tissues (e.g.,adi- pose, bone marrow) might function as a depot or sink for Mammary gland Nasal cavity Oral cavity Bone marrow 0.15 0.15 0.15 0.15 2.6 2.6 2.9 3.2 NC' 17 23 11 benzene (9,27),one experiment was carried out to specif- Liver 0.15 2.8 21 ically determine if "C-benzene residues accumulatein the Kidney 0.15 4.2 14 Zymbal gland (a sebaceous gland) after repeated oral ex- "Half-lifeof elimination of radioactivity for the rapid and slow phase: I posure. After 2 weeks of oral dosing at 500 mg/kg (sin- gle dose daily 5 days per week for two weeks), no appar- ent accumulation of I4C was observed in the Zymbal was estimated by visualizing the best line graphically on semilog plotand then best-fitting the data points for each phase using an exponer tial curve-fitting program on an HP 41CX calculator. two data points were available for determining half-life of t h t gland compared to that after a single oral dose (data not slow phase. shown). 'Not calculable. BENZENE PHARMACOKINETICS A N D METABOLISM IN ZYMBAL GLAND 219 Metabolite Profile The metabolites in Zymbal gland and other tissues were obtained by extraction, separated, and measured by HPLC and liquid scintillation spectrometry. Ethyl acetate extraction was used to isolate the unconjugated metabolites of benzene (e.g., phenol, hydroquinone, catechol, biphenol). HPLC separation of these free phenolic metabolites was achieved using an acetonitrileammonium acetate mobile phase (26). Separation of water-soluble metabolites of benzene (e.g., glucuronides, sulfates)was accomplished using an ion-pair HPLC F a dient elution method (22).Radiochromatograms, such as (A) ZYMBAL GLAND those shown in Figure 4, illustrate the excellent baseline resolution of the water-soluble metabolites using the ion- pair HPLC procedure. The retention times for various standard metabolites performed under these two HPLC schemes are listed in footnote c of Table 3. The relative percentages of unconjugated and water-soluble metabo- lites found in Zymbal gland, oral and nasal cavity tissues, bone marrow, liver, kidney, and blood 1hr after oral ad- ministration of 15 mg/kg 14C-benzeneare shown in Table 3. Zymbaf Gland. The major unconjugated metabolite identified in the Zymbal gland was hydroquinone ( n 3~0% of unconjugated metabolite fraction) but small amounts (B)BLOOD -al =m -In If r8 IU A 6000 snE -.c*- 2 4000 C 0 .-(0 0 0m -K f 2000 0 I" 1 0 -7- 1 0 20 30 40 RetentionTime (min) Ah 0 0 A I 10 20 RetentionTime (rnin) (C) NASAL CAVITY TISSUE (D) KIDNEY 2000 c A Ep 1500 .c>-. .>- c. .-0m 0 1000 Uam -* i; 500 0 1 0 20 30 4 0 C 0 10 20 30 4 0 Retention Time (min) Retentionlime (rnin) E 4. HPLC radioactivity profile of the water-soluble metabolite fraction isolated from variouh tissueh and from blood 1 hr after female S p r q u e - wley ratb \t ere orally administered 15 mglkg 'T-benzene in olive oil: ( A )Zymbal gland; (B)blood; (0nasal cant\ tissue; (D)kidney. Isolation Procedures and i m p a i r HPLC conditions are given in the materials and methods section. See Table 3 for relative percentages of each identified benzene metabolite and their HPLC retention times. 110 LOW ET .AL Table 3. Relative percentage of benzene metabolites in Zymbal gland and other tissues 1 h r after a 15 m g k g "C-benzene oral dose.8 o/c Total radioactivity in ethyl acetate or aqueous fractions in tissue Metabolite Zymbal gland Nasal cavity Oral cavity Bone marrow Mammary gland B b d Liver Kidney- C`nconjug`ated metabolites in ethyl acetate fractionb Hydroquinone Catechol Phenol Unidentified Water-soluble metabolites in aqueous fractionb Phenyl sulfate Phenyl glucuronide Muconic acid Hydroquinone glucuronide Unidentified and other metabolites 30 11 N V ND 3 29 67 60 (1major peak) (1major. 1 minor peak) 3 ND 31 16 ND ND 62 35 18 3 ND 6 14 ND ND 4 65 76 18 (1major, 2 (major peak, (4 minor minor peaks) 23.5 min, peaks) catechol glumnide, 53%) -d - - 66 ND 11 2 22 (4 minor peaks) -d 64 a 65 -- ND ND ND 2 3 26 - 34 8 9 (2peaks) (2peaks) ---d 83 26 23 223 6 5 15 - 2 86 - 6 56 53 (2 minor (major peak (2 pe& peaks) at 5 min. at 3-5 min, 27%) 31%) `See "Materials and Methods" for metabolite isolation procedure and HPLC conditions for separation of metabolites in the ethyl acetate fraction and in the aqueous fraction. bHPLC retention times of unconjugated metabolites: 1.2,4-triol(3.0min); hydroquinone (4.5 min); catechol (9.0min); phenol (20 mid. HPLC retention times (ion-pair) for water-soluble metabolites: hydroquinone glucuronide (14.0 min); triol glucuronide (major isomer. 16.0 mink muconic acid (17.5 min); phenyl glucuronide (21 min); catechol glucuronide (2L5 mink phenylsulfate (31min). `ND.not detected. dEthyl acetate extraction was carried out on bone marrow homogenate, but evaporation of solvent yielded little if any radioactive residue for HPU: analysis. Radioactivity lost most likely parent material. Similar findings occurred with mammary gland, radioactivity was not found in the evaporated ethyl acetate fraction or aqueous fraction. of phenol (- 3% of unconjugated fraction) were detected. However, about 67% of the radioactivity in the unconjugated fraction was associated with a polar, unknown peak having a retention time of less than 3 min. Efforts are underway to isolate sufficient quantities of this polar product from pooled Zymbal glands for mass spectrometric identification and structure elucidation. Free biphen- olic metabolites were not detected in the Zymbal gland. Phenylglucuronide was identified as a water-soluble metabolite in the Zymbal gland based on retention time comparison with standards (Fig. 4A). Phenylsulfdte and muconic acid were not detected as water-soluble metabolites in the Zymbal gland a t 1hr. The absence of phenyl- sulfate in the Zymbal gland was surprising since this sulfate conjugate was found as the principal water-soluble metabolite in blood (Fig. 4B),bone marrow, oral cavity, urine, and other tissues in our studies. About 65% of the radioactivity in the aqueous metabolite fraction of the Zymbal gland remained unidentified; most of the radioactivity was associated with a polar peak that elutes from the column at about 5 min (Fig. 4A,Table 3). The un- known polar peak does not appear to be prephenylmercapturic acid or a diconjugate based on relative retention times reported in the literature (22,28). However, the chromatographic nature of this polar product might be consistent with a structure such as a metabolite of m u conic acid (e.g., shorter open-chain acid or alcohol metabolite) or a S-oxide metabolite of a mercapturic acid or premercapturic acid. Further characterization studies need to be carried out to evaluate these possibilities. Blood. The unconjugated metabolites found in blood were hydroquinone and phenol, along with two uniden- tified polar products (Table 3). The HPLC profile of the water-soluble metabolites in blood 1 hr following a 15 mgkg oral dose of "C-benzene is shown in Figure 4B. The major metabolite (31min) coeluted with phenylsulfate and comprised 83% of the radioactivity in the queous fraction. In addition, muconic acid, phenylglucuronide, and hydroquinoneglucmnide were detected as minor metabolites (2to 6%)in blood ("able 3). Nasal Caoify Tissue. Unconjugated metabolites iden- tified in the nasal cavity tissue included phenol (2!% of unconjugated fraction)and hydroquinone (1181,while the major unknown metabolite (- 60%) eluted as a polar peak on HPLC. The spectrum of water-soluble metabolites found in the nasal cavity tissue was different from that found in blood and other tissues in that phenylsulfate was not detected in the nasal cavity. Phenylglucuronide(1896) and muconic acid (6%) were identified as water-soluble metabolites in nasal cavity tissues, but a majority of the radioactivity in the aqueous fraction (76%)remained unidentified (Fig.4 0 . The major radioactive HPLC peak eluted at 23.5 min and represented 53% of the aqueous fraction isolated from nasal cavity tissue. OfherTissues. The metabolite profiles in various other tissues including bone marrow, oral cavity tissue, mammary gland, liver, and kidney have also been determined and are summarized in Table 3. The radioactivity found in the bone marrow 1 hr after a 15 mgkg oral benzene dose was mainly parent and water-soluble metabolites (Table 3). Phenylsulfate, muconic acid, and hydroquinoneglucuronide were identified as the major water- BENZENE PHARMACOKINETICS A N D METABOLISM IN ZYMBAL GLAND 221 soluble metabolites in the bone marrow. In oral cavity tisme,hydroquinone and phenol were the principal unconjugated metabolites, while phenylsulfate, muconic acid, phenylglucuronide and hydroquinoneglucuronide were the primary water-soluble metabolites. Based on extraction results, the I4Cmaterial present in mammary gland appeared to be the parent material. After attempted isolation of metabolites, practically no radioactivity was foundin the aqueousfraction or the evaporated ethyl acetate fraction, indicating that the radioactivity initially associated with the ethyl acetate fraction was lost as I4Cbenzene during evaporation of ethyl acetate. Unconjugated metabolites detected in the kidney were hydroquinone and phenol. Phenylsulfate (23%)and muconic acid (15%) were the major water-soluble products while the glucuronide conjugates of phenol and hydroquinone were minor water-soluble metabolites (3to 6%). The metabolite profile in the liver appears to be similar to that found in the kidney except that the relative percentages of muoonic acid and unconjugated phenol were lower in the h r than in the kidney. A substantial portion of the Water-solublemetabolites in Zymbal gland, liver, kidney, .sdnasal cavity tissue remained poorly characterized ChMe 3); most of these products were polar in nature. Discussion Although solid tumor formation occurs in a number of b e s in rats following chronic oral benzene exposure 04, information has not been reported on the metabolic dbposition and pharmacokinetics of benzene at these tar) eta sites, which include Zymbal gland, oral and nasal ?vity tissues, and mammary gland. Of particular interest UIthe Zymbal gland since this tissue is most susceptible bneoplastic changes induced by benzene. In this pres- '1ent stud ,we report the pharmacokinetics and metabo- b of C-benzene in Zymbal gland and other solid tu- mor target tissues in the Sprague-Dawley rat. Our results indicate that after oral administration, radiolabeled benzene is rapidly absorbed and distributed to the h b a l gland, with peak levels reached within 1hr of ade t r a t i o n . Subsequent elimination of radioactivity ocCms in a biphasic manner (rapid and slow phases). The slower elimination phase suggests a reduced rate of clearance of metabolites from the Zymbal gland, possibly beQuse of covalent binding to tissue proteins. HPLC radiometric analysis demonstrated the presence of benzene metabolites in the Zymbal gland after treatment with %-benzene. Free hydroquinone and phenol were detected as unconjugated metabolites, and phenyl@Wwonideas a water-soluble metabolite, on the basis ~ ~ H P LreCtention times. Other metabolites in Zymbal h d were polar in nature and have not been identified. The metabolite profile found in the Zymbal gland differs from those found in blood and other tissues and ih h c t e r i z e d by the absence of phenylsulfate. This %g is surprising since phenylsulfate is the predomi- bolewt water-soluble metabolite of blood, liver, kidney, marrow,oral cavity tissue, and urine. These results are consistent with previous findings that sulfate conjugation may not occur readily in the Zymbal gland; Irving et al. (29) have reported that Zymbal gland lacks sulfotransferase activity toward N-hydroxy-2-acetylaminofluorene in the rat. Therefore, detoxification of phenolic xenobiotics in the Zymbal gland may be mainly proceeding by glucuronidation or possibly by other conjugative or metabolic pathways leading to polar products. The presence of phenylglucuronide and an unknown polar product as major water-soluble metabolites in the Zymbal gland of animals treated with benzene appears to s u p port this hypothesis. The significant difference between the metabolite profile in blood versus that in the Zymbal gland suggests inherent metabolic capability in the Zymbal gland to biotransform benzene and/or its metabolites. If metabolites in the blood were transported to and sequestered by the Zymbal gland rather than being formed there, one would expect the two metabolic profiles to be more similar than distinctly different as experimentally observed. Pohl and Fouts (30)have demonstrated that Zymbal gland homogenates possess cytochrome P-450-dependent xenobiotic-metabolizingactivity toward berw$a)pyrene and 'I-ethoxycoumarin. Studies in our lab oratory using an in vitro tissue culture technique have demonstrated t h a t t h e Zymbal gland is capable of metabolizing benzene, 2-acetylaminofluorene, and 7,12-dimethylbenzanthraceneto reactive intermediates that interact covalently with DNA (31). At this time, we cannot rule out the possibility that phenylsulfate might be taken up by the Zymbal gland (from surrounding blood supply); once in this organ, phenylsulfate could be rapidly hydrolyzed by sulfatase to yield phenol, which in turn, could be further oxidized andlor glucuronidated. The metabolic capacity of the Zymbal gland in regard to deconjugatingor hydrolytic activities has not been well characterized(29,52).Relatively little is known on the distribution of sulfatases in the Zymbal gland ($3).However, it is interesting to speculate that the presence of sulfatases in the Zymbal gland might provide a mechanism by which sulfate conjugates of phenolic benzene metabolites can be hydrolyzed to yield free phenolic metabolites, capable of undergoing further activation or inactivation processes. Work is underway to investigate this possibility. Since the Zymbal gland is a specialized sebaceous gland in rodents (34),it is reasonable to expect that lipophilic chemicals like benzene would partition readily into the gland. However, results of these studies showed that accumulation of benzene does not occur in the Zymbal gland following single or multiple oral doses but that Zymbal gland metabolismproduces a profile of metabolites, which is somewhat different from that of other organs and tissues examined. Perhaps this differential metabolism might contribute to the carcinogenicactivity of benzene in the Zymbal gland and other target tissues. Continued metabolism and pharmacokinetic studies after single and repeated oral doses of benzene may provide further information for determining the likely reactive species and pathways involved in causing the formation of solid tumors in the rat. LOW ET AL. We thank David C. Kossor for his excellent technical assistance in carrying out rddioactivity measurements and HPLC analyses. We are also grateful to Lisa A. Carmody for her help in preparing the manu script. 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