Document Ne0p2NpmzyZd4aaZGxe77r9vw

Environmental Health Perspectives Vol. 8.2. p ~2'.39-247, 1989 A Method for In Vitro Culture of Rat Zymbal Gland: Use In Mechanistic Studies of Benzene Carcinogenesis in Combination with 32P-Postlabehg by M. Vijayaraj Reddy,* Gary R. Blackburn,* Susan E. Irwin,* Choudari Kommineni,* Carl R. Mackerer,* and Myron A. Mehlman* Zymbal glands were excised bilaterally from the ear ducts of female Sprague-Dawley rats (threelgroup), minced into approximately four fragments per gland, and transferred into a microtiter plate containing 1.5 mL per well of Waymouth's tissue culture medium supplemented with fetal calf serum, hydrocortisone. in- sulin, and gentamicin. addition of a test compound or solvent vehicle, plates were incubated for 6,24, 48, or 96 h r at 37% in a humidified atmosphere of 5% C02 in air. Tissue in culture for 6 hr was histologi- cally indistinguishable from the freshly excised tissue, while that in culture for 24.48, and 96 h r showed a progressive deterioration often with necrosis and/or squamous metaplasia. More pronounced deterioration was noted in samples treated with 750 or 1500pg/mL of benzene. Using a nuclease P~enhanced'ZP-podabeling assay, aromatic DNA adducts were detected in cultured Zymbal glands exposed for 48 hr to benzene and its derivatives, as well as to 7 , 1 2 d i m e t h y l b e n z a n t e n e (DMBA) and 1-acetylaminofluorene (AAF).Benzene produced very low levels of adducts (0.5 adducts per lo9 nucleotides), whereas its congeners produced rela- tively high levels of adducts (50-2000 lesions per lo9nucleotides). which decreased in the order benzoquinone > hydroquinone > phenol > benzenetriol > catechol. Each adduct profile overall was characteristic for the compound studied, suggesting the formation of compound-specific electrophiles. AAF and DMBA ad- ducts were identical to those formed in u i w in animals. Our results show that the Zymbalglands are capa- ble of metabolizing different carcinogens.toDNA-reactive intermediates, a process that may be causally as- sociated with tumor formation in viuo in this organ. Introduction Rat Zymbal glands, located at the base of the external auditory canal, have a distinctive pink-yellow color, weigh about 7 to 8 mg, and have a diameter of 3 to 5 mm. These glands, also present in other rodents and insectivores,but not in humans, have a typical sebaceous morphology, Which is characterized by rounded sacs of plump, fatcontaining cells (1).These cells subsequently break down, celeasingfat droplets into the external ear canal (I).Malbni et al. (2)and the National Toxicology Program (3) have reported a dose-dependentformation of solid tumors in Zymbal glands of rats following administration of repeated high doses of benzene. The mechanism by which I&,$'Enwonmental and Health Science Laboratory, Mobil Oil Corporatkn,Princeton, NJ 08540. & h s s reprint requests to M.V. Reddy,Environmental and Health a e n c e Laboratory, Mobil Oil Corporation, Princeton, NJ 08540. benzene induces this cancer is unknown, but it has been suggested from in vitro and in vivo metabolism studies that benzene is enzymatically activated to reactive intermediates that can bind to DNA (4),thereby forming covalent adducts, which, in turn, may causally be associated with mutation and the initiation of carcinogenesis (5). The liver is generally considered to be the primary organ for benzene metabolism, but the incidence of liver tumors (hepatomas) has been reported to be three times lower than that of Zymbal gland carcinomas in rats after daily administration of 500 mgkg benzene for 104weeks (2).This difference in the susceptibility to carcinogenesis may, in part, derive from the unique metabolic capabilities of Zymbal gland tissue. In order to facilitate the study of Zymbal gland metab- olism, we have devised a culture system to maintain fragments of Zymbal glands for several hours after excision from rats. The capability of t h e cultured glands to metabolize benzene and its derivatives [catechol, -. ,'b 2.40 REDDY ET AL. 1,2,4-benzenetriol(BT),phenol, hydroquinone (HQ), and lowed by a solvent extraction as described previously benzoquinone (BQ)], as well as 7,12-dimethylbenzan- (11),except that the volumes of reagents were scaled thracene (DMBA)and 2-acetylaminofluorene(AAF)w,as down by 3-fold. An average yield of DNA from Pooled then assessed by measuring covalent binding of these compounds to DNA using a highly sensitive "P-postlabeling assay (6,7'). DNA adducts were readily detecta- Zymbal glands in several extractions corresponded to 17 (k6) pg DNA per rat. The presence of adducts in DNA was analyzed using a nuclease Pi-enhanced post la be^^ ble with all compounds tested, except benzene, which assay ( 7 8 . Briefly, DNA (10-15 pg) was enzymatically produced relatively low levels of adducts. DMBA- and digested to 3'-deoxyribomonucleotides (121,which were AAF-DNA adduct profiles were similar to those formed then treated with nuclease PI, which dephosphorylates in vivo in the epidermis and Zymbal gland, respectively, normal nucleotidesbut not aromatic adducted nucleotides of treated animals. *-(8).The latter were "P-labeled using y3*P-ATPand poly- nucleotide kinase and resolved by PEI-cellulose TLC cording to the following two methods. Materials and Methods In method A, which utilizes the techniques previously described (18-15), the adducted 3ZP-nucleotidesof U F , Benzene was purchased from American Burdick and benzene, and its derivatives were purified on a PEI- Jackson, a subsidiary of American Hospital Supply Corporation (Muskegon, MI). Catechol, BT, HQ, and BQ were obtained from Aldrich Chemical Company. Phenol (Mallinckrodt, Cat. No. 0025-2.5) was distilled prior to use. DMBA, AAF, and calf thymus DNA were from cellulose thin-layer plate (Fig. 1)in direction D1 in 2.3 M sodium phosphate, pH 5.77 (151,in situ magnet transferred to a second 20 x 12.5-cmsize PEI-cellulose sheet (15), and resolved by a three-directional development (15) of the sheet in the followingsolvents: D3,2.5 M lithium Sigma Chemical Company. Waymouth's culture medium formate, 4.7 M urea, pH 3.5, to 20 cm;D4,0.4 M sodium and calf serum were from Gibco Laboratories. Carrier- phosphate, 0.25 M Tris-HC1,4.25M urea, pH 8.0, to 12.5 free y - 32P-ATF'(about 4000 Cilmmole) was synthesized from "Pi (8)or purchased from ICN Radiochemicals(Cat. No. 35020). Polyethyleneimine (PEI>celluloseplates were prepared (9) in the laboratory. The sources of all other materials needed for 32P-postlabelinganalysis of adducts cm; D5, 1.7 M sodium phosphate, pH 6.0, to 12.5 cm. DMBA adducts were chromatographed the same way as above except the composition of D3 and D4 solvents were D3,3.2 M lithium formate, 6.0 M urea, pH 3.5; D4,6.4M sodium phosphate, 0.4 M Tris-HC1, 6.8 M urea, pH 8.0. have been documented previously (6-8). In method B, which was specially designed to permit For the organ culture experiments, Zymbal glands the recovery of additional adducts more polar than those were asepticallyexcised from both the ear ducts of female isolated in method A, the labeled solution was applied to Sprague-Dawleyrats (4 months of age,weighing approx- imately 280 g), minced into four to six fragments per gland, and placed into Waymouth's MB75211 tissue cul- ture medium supplemented with 10% fetal calf serum (heat inactivated at 57OC for 30 min), 350 pglmL gluta- mine, 5pg/mL hydrocortisone, 5pg/mL insulin, and 0.1% T gentamicin (10). The gland fragments from three rats were transferred into a %-well microtiter plate (1.SmL .r( of the above medium per well), to which was added a test z chemical or solvent vehicle (1%, v/v). DMBA and AAF were dissolved in DMSO, BT in ethanol, and others in wa- ter. Final concentrations in the incubation media were: DMBA, 5 pglmL; AAF, 40 pg/mL; benzene, 7500pg/mL (solubility in water is 1800pg/mL); others 750 and/or 1500 pglmL. The plates were incubated for 6,24,48, or 96 hr at 37OC in a humidified atmosphere at 5% CO2 in air. For the histopathologicalexamination, freshly excised Zym- -20- bal glands, as well as the treated tissues, from each of the time points were fixed in neutral formalin, embedded in FIGUR1E. Scheme of PEI-celluloseTLC for pdication and separation paraffh, cut at 6 pm thick, and*stained with hematoxylin and eosin. For the adduct study, tissues were collected at 12 hr (DMBA) or 48 hr (others) after treatment. In vivo-modifiedU F - D N A was prepared from the Zymbal glands of rats a t 24 hr after daily oral gavage doses of 40 of adducted '*P-nucleotides(method A). This scheme is essentially the same as the one previously described by Lu et al. (IS)with a few minor modifications. "P-Labeled DNA digests were applied to the origins (a to g) of a PEI-cellulosesheet that had been attached to a Whatman paper No. 1 wick by stapling (A).The sheet was then developedin direction D1 to remove leftover j-"P-ATP and '*P-labeled m g k g AAF in ethanol/DMSO/olive oil (1:2:14, v/v, 3 m a g ) for 5 days; control animals received solvent vehi- cle alone. DNA was isolated from the Zymbal glands by treat- ment with ribonucleases (A, TI)and proteinase K, fol- contaminants.Each origin area(shadedarea)containingpurified a m matic adducts was cut out and attached to a second PEIcellulose sheet (B) with the aid of buttontype magnets for c o n t a c t - t d e r Of adducts(15).The sheet was then developedalong D3,D4,and D5with solventsgiven in the text to transfer and resolveadducts. Measure- ments are in centimeters. i i .-e --@- "P-POSTLABELING OF DNA ADDUCTS IN CULTURED ZYMBAL GLANDS 24I theorigin (OR)of a PEI-cellulose sheet (20 x 20 cm)(Fig. 2). It was then developed overnight (16-17 hr) in direction D1 in 1.7 M sodium phosphate, pH 6.0, onto a Whatman No. 1paper wick stapled to the top of the sheet. The wet sheet was cut below the paper wick, which was discarded and soaked twice in 1L of deionized water for 5 mineach. After drymg, it was developed in D3, the same direction as the preceding development, with water to 1 cm from the bottom edge, then with 1.9 M lithium formate, 3.8 M urea, pH 3.5, to the top of the sheet. The sheet was soaked in water as before, dried, and developed in D4, perpendicular to the preceding development, in water to 1cm, followed by 0.36 M sodium phosphate, 0.23 M Tris-HC1, pH 8.0, to the top, and dried. 32P-adducts were detected by screen-enhanced autoradiography (6,7). Adduct radioactivity was determined by excising the spots from the chromatograms and Cerenkov counting ( 6 3 . An estimation of adduct levels, expressed as relative adduct labeling (RAL),was made from the values of adduct count rates, adjusted for background radioac- tivity, and the specific activity of Y-~'P-ATPdetermined by measuring the incorporation of 32Pinto a known mount of dAp (7,s). Results Histopathological Examination of Tissues M i ~ s c o p i cexmination of the Zymbal glands main- e e d in tissue culture for time periods without I - - - - - - - - - - PAPER WICK i I D 1,D3 c1 1 20 PE'-CELLULoSE I I -D4,D5 -- 20 2. Scheme of PEIellulose TLC for purification and separation Ofpresumabl less hydrophobicadductsderivedfrom phenol and HQ (method B). 'P-Labeled digests were applied at the origins(OR) of %' a~Icellulosethin-layerplate, which was developedalong D1 to re- move7-'*P-ATPand partially resolve adducts.Further resolutionof dductswas obtained by developmentalong D3,D4,and D5 with sol- vents of composition given in the text. Measurements are in cen- timeters. any test substance revealed that the glands a t 6 hr (not shown) were histologically indistinguishable from those at 0 hr, Le., the freshly removed tissue (Fig. 3A), while the ones at 24 (not shown), 48 (Fig. 3B), and 96 hr (Fig. 3 0 exhibited a prog?essive deterioration often with necrosis and/or squamous metaplasia. Analysis of DMBA and AAF Adducts Using the nuclease PI-version of the "P-postlabeling assay (7,8),we detected DNA adducts in the Zymbal glands treated in culture with DMBA (5pg/mL)and AAF (40 pg/gL), both being used in the present study as positive controls (Fig. 4). DMBA produced two major (spots 1and 8)and five minor adducts (Fig. 4B)that were not detected in control DNA (Fig. 4A).The adduct pattern, except for an additional product 8,was qualitatively simi- lar to that seen previously (7,8,16,17) in the epidermal DNA of mice that had been treated by topical application with DMBA. Cerenkov counting revealed that adducts 1and 8 together comprised 70% of total DNA modifkations, which corresponded to 25 adducts in lo9 DNA nucleotides. Schrneiser et al. (18)have recently characterized adduct 1as the major s y n dihydrodiol epoxide deoxyguanosine (dG)product, and adducts 3 and 4 as the major anti dihydrodiol epoxide derivatives of deoxyadenosine (dA) and dG, respectively. AAF produced four adduct spots (Fig. 4 0 ) that were chromatographidy identical to those obtained in in Zymbal gland DNA of rats dosed orally with MF (Fig. 4439. Because some aromatic amine adducts are not recovered completely by the nuclease PI-enhanced 32Pprocedure (7,8), AAF adducts were also evaluated by an alternate procedure involvingbutanol extraction of DNA digests prior to 32P-labeling(19).More radioactivity was incorporated into spot 1after butanol ex-tion (Fig. 4G, 4H) when compared with that after exposure to nuclease PI (Fig. 40,4E),suggesting the loss of this adduct by enzymic 3'-dephosphorylation. The recovery of adduct 1after nuclease Plwas 4% of that after butanol extraction; adduct 2 was recovered similarly with and without the enzyme (data not shown). Adduct 1, which has been characterized previously as dpGp-C&(NZ-AF()131, composed 89%of totalmodifications, corresponding to 31adducts per lo9 DNA nucleotides. Modification of the in vivo sample was about 540 lesions per lo9 nucleotides, with adduct 1representing 97% of the total. Adduct 2 has previously been identified as dpGp-N2<C3-AAF)(13). Analysis of Adducts from Benzene and Its Derivatives When DNA samples isolated from the Zymbal glands that had been treated in culture with various derivatives of benzene (1500CcglmL),as well as benzene itself(- 1800 pg/mL), were analyzed by the nuclease PI-amplified 32P- postlabeling assay, the autoradiograms shown in Figure 5 were obtained. DNA adducts were readily detectable 243 ". i REDDY ET AL. AI I 4 I IB 3 E t & x e FIGUR3E. Light photomicrographs of rat Zymbal gland. (A) Freshly excised and fixed tissue. Focal fresh hemorrhage ( m n )in the supporting connective tissue and the normal architecture of the gland are seen. (B)Zymbal glands maintained in the culture medium Hithout a test substance for 48 hr. Dissolution of glandular epithelium forming cystic spaces, which are partially filled with cell debris is seen. (0Zymbal glands maintained in the culture medium without a test substance for 96 hr. Large cystic spaces with cell debris and squamous metaplasia (arrow)of the glandular epithelium are present. Continued 071 next page "P-POSTLABELING OF Dh'A ADDUCTS IN CULTURED ZYMBAL GLANDS 243 FIGURE 3. Continued. C following exposure to different derivatives of benzene, with unique adduct profiles for each compound. Catechol produced one main adduct (spot a) and several minor ones (spots b to m), some of the latter migrated in areas where the DNA derivatives of BT migrate. Phenol produced one major (spot 1)and seven minor adducts; HQ induced an adduct pattern of 3 major spots (spots 5 to 7) and many minor adducts, With BQ-DNA, one major (spot 6)and a few minor alterations were detected. The major adduct was found to be a guanine derivative, as a chro- matographically identical product was formed when dGp was reacted in vitro with BQ and 32 P-labeled (data not shown). With benzene-DNA, no adducts were detected when the chromatogram was exposed to X-ray film for the same time period as that employed for the detection Of metabolite-DNA adducts; however, upon five times longer film exposure, two faint adduct spots in the area where the major products of BQ and phenol migrate were FIGURE 4. Autoradiograms of TLC maps of 32P-labeledDMBA and AAF adducts. DNA specimens isolated from the Zymbal glands exposed in culture ( a d , Jg)or in vivo (e,h)to solvent vehicle (a,DMSO, c andJ ethanol/DMSO/oliveoil)and the indicated carcinogenswere Seen when compared with the control map left in contact With the film for the same period. Adducts exhibiting Similarmobilities on the "P maps were tentatively iden- tified by overlapping the autoradiograms. Unequivocal evidence for the identity of adducts of interest would, however, require chromatography of a mixture of 32P- labeled DNA digests in additional solvents and using dif- ferent TLC systems (20).As shown in Figure 6,when 32P- labeled digests were chromatographed according to digested to n o d and adducted nucleotides. The latter were enriched, "P-labeled, contact-transferred, and resolved by TLC (15). Adducts of DMBA (a, b) and AAF (e-e) were enriched by nuclease PItreatment and resolved using solvents given in the text. AAF ad- ducts were also enriched by butanol extraction If-h) (19)and resolved according to the published conditions (14)with moditications entail- ing use of 2.3 M sodium phosphate, pH 5.77,as a D1 solvent and a magnet-transfer step after D1 development (15).Autoradiography was performed at -8OoC for 2 hr (a,b),5 hr (c, e). 15 hr (d),and 3hr If-h). Faint adduct spots requiringlonger film exposure times (15-20 hr) have been circled. REDDY ET AL. ** Lk control f catechol -A Benzenerid FICI~R5F. A: utoradiograms of TLC maps of '2P.labeled DNA adducts derived from benzene and its derivatives. DNA samples isolated from Zym- bal glands exposed in culture for 48 hr to the solvent vehicle (control) and the indicated compounds were digested and "P-labeled. Labeled adducts in DNA digests were purified, contact-transferred, and resolved by two-dimensional TLC according to method A outlined in Fig. 1. D3 development was in the vertical direction, and D4 and D5 developments were in the horizontal direction. Autoradiographic conditions were: control (top row), catechol and BT, -8OoC for 8hr: phenol and HQ, -8OoC for 2 hr; BQ, 23oC for 2 hr; control (bottom row) and benzene, -8OOC for 36 hr. Note that the sensitivity of film detection is increased 3- to 4-fold at -8OOC relative to 23OC. Chromatographically corresponding adducts on maps of catechol and BT. as well as those of phenol, HQ, BQ, and benzene, were identified with the same letters or numbers. method B, which permits recovery of derivatives more polar than those detected above (Fig. 5), one additional adduct (no. 23)with phenol-DNA and two (nos. 23 and 24) with HQ-DNA were detected. are unknown, except for adduct 6 which was found completely resistant to nuclease PI-mediated3'-dephosphorylation (21). DNA modifications with diff&rentbenzene derivatives ranged from 45 to 1980 adducts per lo9 nucleotides and decreased in the order BQ > HQ > phenol > BT > Discussion catechol (Table 1). Phenol and HQ induced dose- In this report, we show for the first time that rat Z p - I dependent adducts at two concentrations tested. More bal glands can be maintained in culture for a period long BQ adduct (no. 6)was formed when the HQ concentration enough to study early effects of genotoxicants, e.g., DNA $ 2 was increased from 750 to 1500pglmL. The adduct levels adduct formation. Employing the nuclease P1-enhanced determined represent a minimum estimate, as the effi- 32P-postlabelingassay, DNA adducts have been detected ciency of labeling of various adducts and the extent of in Zymbal glands cultured in the presence of DMBA, their resistance to 3'-dephosphorylation by nuclease PI AAF, benzene, and its derivatives. f "P-POSTLABELING OF DNA ADDUCTS IN CULTURED ZYMBAL GLANDS 245 e him 7 Control Phenol Hydroquinone 1, Benzoquinone FIGURE 6. Autoradiograms of TLC maps of "P-labeled DNA adducts derived from phenol, HQ, and BQ.DNA ~ e c i m e npsrepared as given in the legend of Fig.5 were digested and 'P-labeled. Labeled adducts in DNA digests were purified and resolved according to method B outlined in Fig. 2. D1 and D3 developmentswere in the vertical direction and D4 development in the horizontaldirection.Autoradiographic conditions were similar to those given in the legend of Fig. 5. Detection of DNA adducts with the indirect carcino- gens,DMBA and AAF, indicates that Zymbal glands in culture are capable of metabolically activating these carh o g e n s to electrophilic intermediates that can bind to DNA. The activation pathways in the cultured Zymbal glands appear to be similar to those occurring in vivo,in aS much as the adduct profiles (Fig.4) were qualitatively the same as those seen in vivo in DMBA-treated mouse epidermal DNA (8,16-18) and in AAF-treated rat Zymbal glands in the present studies. DMBA, however, induced an additional product (no. 8) in cultured Zymbal gland, which has not been detected in vivo in mouse skin (8,16,1r), suggesting the formation of some tissuespecific -the intermediates. DMBA and AAF metabolism is mediated by a mixed-functionoxidase system (22,281.Adduct detection in Zymbal glands is consistent with the ob- emation by Pohl and Fouts (24), who have shown the presence of cytochrome P-450-dependent aryl hydrocarbon hydroxylase activity in rat Zymbal gland homogenates. The comparison of adduct patterns among different benzene metabolites studied (Fig. 5)enables the predic- tion of possible electrophiles formed from the individual compounds. Metabolism studies with liver and bone mar- row (4,25,26)have shown that phenol can be oxidized to catechol, HQ, and biphenol by cytochrome P-450-depen- dent monooxygenase, peroxidase, or active oxygen released during the oxidative burst. Our findings that the major derivatives of phenol (Fig. 5, adducts 1and 6)were different from those of catechol and overlapped with a fraction of HQ adducts suggest, but do not prove, that, in cultured Zymbal glands, conversion of phenol to catechol is negligible, while its conversion to HQ represents a minor pathway. This further suggests that phenol may be predominantly metabolized to biphenols, which, following oxidation to their semi-quinoneand quinone entities, can bind to DNA. The putative reactive metabolites derived from HQ are likely to be p-bemsemiquinone andlor BQ, which can be formed either by autoxidation of HQ at physiological pH in the presence of oxygen or by enzyme-mediated oxidation of HQ (4,25,26).In addition, HQ can be converted to BT, which can autoxidize to electrophiles such as ahydroxy-p-benzosemiquinone and a-hydroxy-p-benzoquinone (4,25,26). Since only a minor fraction of HQ ad- ducts (Fig. 5, adducts 6 and 8, composing < 20%) cor- responded to BQ- and BT-derived modifications (Fig.5, adducts 6,8, and b), it appears that in Zymbal gland culture, BQ and BT are minor reactive intermediates formed from HQ, while the pbenzosemiquinone may pre- dominate. The DNA-reactive species derived from catechol may be either a mixture of o-benzosemiquinone and obenzoquinone formed by enzyme mediated-oxidation or a mixture of semiquinone and quinone species of BT (4,25,26). About 20% of total modifications produced by catechol exhibited chromatographic properties similar to those derived from BT (Table l),indicating that the conversion of catechol to BT is a minor pathway. Therefore, the alternate pathway leading to the formation of the semiquinone and quinone is probably the major one. The unique "P-fingerprints for each metabolite tested (Figs. 5 and 6) are consistent with the data of Rushmore et al. (27),who have observed different Sephadex LH-20 column profiles of dG adducts in 'H-GMP-prelabeled DNA of rabbit bone marrow mitoplasts exposed in vitro to HQ, BQ, phenol, catechol, and BT. Although phenol or other metabolites of benzene i n duced relatively high levels of adducts, benzene itself produced adducts at levels barely detectable by the "Ppostlabeling assay. This could result from a)less efficient conversion of benzene to phenol in the Zymbal gland cultures; b) the high volatility of benzene, reducing the ef- fective dose reaching Zymbal glands in the culture medium; and/or c) the metabolism of benzene predominantly to a ring-opened derivative (28),which may form polar adducts not detectable by postlabeling under the ,..' 116 HEDDY ET AL. Table 1. Levels of DNA adducts formed in Zymbal glands in culture treated with benzene and its metabolites. as estimated by a nuelPl-enhanced poetlabeling.' Compound Concentration, PdmL a RAL x lo9 for adduct spotb(No. of adducts per lo9 DNA nucleotides) b C d e Others Total Catechol BT 1500 16.2 3.0 6.0 NC' 5.2 15.0 (7) (13) (11) (33) 1500 ND' 20.2 8.7 5.1 8.4 10.3 (38) (17) (10) (16) (20) 45.4 52.7 1 5 6 7 23 24 Others Total Phenol HQ BQ Benzene 750 1500 750 1500 1500 *1800 30.7 ND ND ND 33.5 (42) (46) 75.1 ND 3.5 ND 52.9 (52) (2) (36) 36.1 317 39.6 260' 254 (3) (29) (4) (24) (24) 53.3 269 2223 202 215 (4) (22) (18) (16) (17) ND ND 1370 ND ND (69) NC 0.5 ND ND 90.2 (8) 121 (10) ND 9.4 73.6 (13) 14.8 146 (10) 83.5 1080 (8) 162 1260 (13) 614 1984 (31) 0.5 .Adduct spots (Figs. 5 and 6)were cut from the chromatograms and counted by Cerenkov assay. RAL values were calculated from count rates using the specific activity of Y-'~P-ATP(7,8). bFor adduct assignments, see Figs. 5 and 6. 'Not countable. dNumbers in parentheses indicate percentages of each adduct relative to total. 'Not detected. 'The recovery of this adduct varied markedly. The value represents a maximum estimate. experimental conditions described. Currently, none of these possibilities can be dismissed. Reaction of BQ or HQ with dG has been shown by Jowa et al. (29)to generate (30H)benzetheno(N1,N2)dGas the major derivative on the basis of HPLC and NMR analysis. In comparisonwith this data, adduct 6, which was detected as the major product of the interaction of BQ with DNA or dGp (Fig. 5), may be (3'0H)-benzetheno- (N1,N')deoxyguanosine 3',5'-[3'Pbisphosphate. The detection of multiple "P-postlabeled adducts with HQ-DNA is in accordance with the multiple HPLC peaks obtained from the hydrolysate of calf thymus DNA modified in witro by 14C-HQ(29). Analysis of AAF adducts here (Fig. 4) and those of 4-aminobiphenyl previously (7,8) indicate that the CS-guanine derivatives are susceptible to 3'-dephosphorylation by nudease PI, while the N'-guanine derivatives are not. Adducts of benzo[alpyrene, safrole, and mitomycin C, which predominantly bind at the N2 posi- tion of guanine, have also been shown to be resistant to the enzyme activity (7,8). Taken together, the data suggest that an adducted 3'-nucleotide with a carcinogen bound to a base at the exocyclic position, but not at the endocyclic position, is resistant to nuclease PI-mediated dephosphorylation. When the,attachment is simultaneously at the exocyclicand endocyclicpositions, for example, a cyclic adduct, as in the case of the major adduct of BQ-DNA above, the adduct may not be susceptible to the enzyme activity (21). Such differential enzyme activity may be useful in adduct characterization. Our results demonstrate the utility of postlabeling tech- niques to determine the capacity of tissues or cells to metabolize a mutagenlcarcinogen of interest by measuring DNA adduct formation. Because of high sensitivity and the need for only a few micrograms of DNA, the "Ppostlabelingassay affords adduct measurement in a small number of cells, as evident from the present studies on Zymbal glands weighing only about 15mg per rat. The "P-postlabeling protocol, with further refinement to reduce background noise, may potentially be applicable to monitor the formation of DNA adducts in blood cells of humans exposed to benzene and thereby provide useful information for risk assessment and management. By combining the highly sensitive postlabeling assay with the newly devised Zymbal culture system, we have begun to elucidate the metabolic pathways and macromolecular interactions in this target tissue. Insights gained from this work will subsequently be applied to the design and interpretation of analogous in vivo studies, with the ultimate goal of better understanding the mechanisms involved in the carcinogenic process. We appreciate the skillful assistance of Lisa A. 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