Document qkzjjJQNkE6L1k26kjqVEe8DG

yyT 1PLAlNTIFF'S EXHIBIT 11 Chem.-EioL Intemctions 33 (1981) 345-360 0 Elsevier/North-Holland Scientific Publishers Ltd. 345 MODULATION OF BENZENE-INDUCEDLYMPHOCYTOPENIA IN THE RAT BY 2,4,5,2',4`,5'-HEXACHLOROBIPIlENAYNL3 3,4,3',4'-TETRACHLOROBIPHENYL W.F.GREENLEE* and R.D. IRONS Chemical Industry Institute of Toxicology. Department of Pathology, 6 Davis Drive, Research Triangle Park, NC 27709 (U.S.A.) (Received December l l t h , 1979) (Revision received June loth, 198.0) (Accepted September 6th, 1980) SUMMARY Repeated administration of benzene (440 mg/kg/day, s.c.) to 6-week-old male Fischer-344 rats resulted in a progressive decline in the number of circulating lymphocytes. fietreatment of these animals with 2,4,5,2`,4',5`hexachlorobiphenyl (HCB) or 3,4,3`,4'-tetrachlorobiphenyl(TCB) protected against benzene toxicity for as long as 7 days, but not after 10 days of repeated dosing. Representative phase I (mixed-function oxidase) and phase XI (conjugating) enzyme activities were measured to determine whether the altered susceptibility to benzene toxicity in TCB- and XB-pretreated rats could be correlated with changes in the profile of hepatic oxidative and detoxification pathways. Tleasurement of 7-ethoxycoumarin 0-deethylase and benzphetamine N-demethylase activities indicated that the loss of protection by 3CB or TCB against benzene toxicity after 7 jays was not associated with changes in the activities of hepatic mixed-function oxidases in3ucible by 3-methylcholanthrene or phenobarbital. The time course for the stimulation by TCB and return to control values, of UDP-glucuronosyl transferase activity, a potential route for the elimination of benzene metabolites, mirrored the time course for the protection against toxicity. Epoxide hydratase activity was induced 2- to %fold by I:CB. Although stimulation of this pathway could result in a decreased concentration of phenol, this activity did not decline with the loss of protection. 3epatic 10 000 X g supernatant fractions, prepared from livers of rats given TCB, were incubated with a non-saturating concentration of [ I4C] benzene (equivalent to 19 nmol/ mg wet wt. tissue). Under these conditions the metabolism of benzene was depressed (40% of control) 2 days after pretreatment; after 1 4 days, the *CIITPostdoctoral Fellow. Abbreviations: HCB, 2,4,5,2',4',5`-hexachlorobiphenyl;%IC, 3-methylcholanthrene; PB,phenobarbital; TCB,3,4,3',4'-tetrachlorobiphenyl. 346 metabolism of benzene returned to control values. This pattern correlate: temporarily with the protection against lymphocytopenia. The data indicate that the protection against benzene toxicity in rats pretreated with HCB or TCB is not necessarily related to the capacity of these compounds to induce phase I activities. In rats pretreated with TCB, the data suggest that decreasing tlie concentration of primary benzene metabolites, either by inhibiting the hepatic metabolism of benzene or increasing hepatic conjugation activity is an important factor modulating toxicity. , INTRODUCTION Chronic exposure to benzene results in a progressive degeneration of bone marrow structure and function [1,2]. In the peripheral blood benzene toxicity is expressed as leucopenia, thrombocytopenia, or anemia [3]. The metabolism of benzene has been extensively studied an6 the finciings inGcate that the toxicity of benzene t o the hematopoietic system is associated with aetabolism of the parent compound to one or more toxic species [4-6]. In tile liver, benzene is a substrate for the cytochrome P-450-dependen: rnonooxygenases [71. Several primary metabolites including phenol, catechol, hydroquinone, 1,2,4-benzenetrioi and benzene dihydrociiol, an5 sulfate or glucuronic acid conjugates have been detected in the urine of rats given [I4C]benzene [8-10]. Evidence supporting the role of metabolism in the expression of benzene toxicity has resulted from the use of various inhibitors of inducers of mixedfunction oxidase activities. Pretreatment of rats with 3-amino-l,2,4-triazole [ll] or piperonyl butoxide 1121, inhibitors of the hepatic microsomd monooxygenases, protected against benzene toxicity. Benzene-associatej leucopenia was also alleviated by pretreating animals with phenobarbital (PB), an inducer [13-14];however, pretreatment with 3-methylcholanthrene (MC), a prototype polycyclic aromatic hydrocarbon inducing a profile of hepatic microsomal monooxygenase activities different from that induced by PB 151, did not prevent the leucopenia resulting from a single large dose (equivalent to 5 ml/kg) of benzene [16]. Both PB and Y C stimulated benzene metabolism as measured by the urinary excretion of phenol [16]. Attempting to resolve the paradox of the protective effect of both inhibitors an2 inducers of benzene metabolism, Snyder e t al. [3] postulated that these agents elicit different responses in the liver (the major site of detoxification) and the bone marrow (the major site of toxicity): inducers stimulate hepatic detoxification, whereas inhibitors prevent the formation of reactive metabolites in the bone marrow. Benzene associated radioactivity binds covalently to DNA in liver [171 and to macromolecules in both the liver and bone marrow [18].It has been suggested that the chronic production of small amounts of benzene oxide in the bone marrow may result in reactions with critical cellular macromolecules, ultimately leading t o bone marrow depression or leukemia [3];how- ever, studies o have indicated responsible for We have ob: 1254 (250 rng associated wit1 protected agall ture of chlorin of microsomal by the combii present findin nated bipheny penia. Potenti: studied using \ bolism of [ I 4 C tected against ated with the dase activity. METHODS Animals Male Fische (Wilmington, bedding, expo: Lab Blox; Allit Treatment Rats pretrez 2 consecutive each divided : (s.c.); the seco zene in corn c dosing regimer tific Co. (Pitt: Corporation (E Preparation of At various t group were a: cardiac punch NJ) containing in cold isotoni 5 mM EDTA; *HCB and TCB a em correlated .z - D data indicate I i with HCB or - ' l n d s to induce t that ilecreas- . by inhibiting . . gation activity . ation of bone benzene toxi3 1 . The rnetalings inclicate sociated with ,ecies [4-61 . 50-dependent nol, catechol, n~ sulfate or of rats given In of benzene ers of mixed1,2,4-triazole ' microsomal ne-associated bh enobarbital cholanthrene a profile of that induced :le large dose lted benzene 31. Attempthibitors and d that these toxification) date hepatic active meta- n liver [171 - It has been .eneoxide in rnacromolela [3] ; how- 347 ever, studies on the covalent binding of benzene to liver microsomal protein have indicated that a metabolite of phenol and not benzene oxide was responsible for the observed binding [191. We have observed that pretreatment of rats with a single dose of AAroclor 1254 (250 mg/kg) resulted in a decreased concentration of radioactivity associated with polyphenolic benzene metabolites in the bone marrow and protect& against benzene toxicity [201 . Aroclor 1254 is a commercial mix- ture of chlorinated biphenyl isomers eliciting a complex pattern of induction of microsomal mixed-function oxidase activity comparable to that produced by the combined administration of PB and MC [21,22]. In this report we present findings on the effect of pretreatment with individual polychlorinated biphenyl isomers, HCB* and TCB, o n benzene-mediated lymphocytopenia. Potential toxification and detoxification pathways in the liver were studied using various model substrates and compared with the in vitro rnetabolism of [14C]benzene. The data indicated that both HCB and TCB protected against benzene toxicity and that protection is not necessarily associated with the capacity of these compounds to induce mixed-function osidase activity. METHODS Animals Male Fischer-344 rats were obtained from Charles River Breeding Labs (Wilmington, MA). The rats were housed in plastic cages with hardwood bedding: exposed -to a day-night light cycle of 1 2 h, andgiven food (Wayne Lab Blox; Allied Mills, Chicago, IL) and water ad libitum. Treatment Rats pretreated with HCB (75 mg/kg, i.p.) or TCB (50 mg/kg, i.p.) for 2 consecutive days before the treatment period and naive animals were each divided into 2 groups: one group received 0.5 ml/kg/day corn oil (s.c.); the second group was given 1.0 ml/kg/day of a 50% solution of benzene in corn oil (s.c.). All rats weighed approx. 150 g at the beginning of dosing regimen. Benzene (thiophene-free) was obtained from Fisher Scientific Co. (Pittsburgh, PA) and HCB and TCB were purchased from RFR Corporation (Hope, RI). Preparation of tissues A t various times during a 2-week treatment period, 5 animals from each group were anesthetized using methoxyflurane. Blood was collected by cardiac puncture into Vacutainer' tubes (Becton-Dickinson, Rutherford, NJ) containing potassium EDTA. Livers were dissected out, minced, washed in cold isotonic KCl, and homogenized in 4 vols. of SET (250 mM sucrose; 5 mM EDTA; 20 mM Tris, pH 7.4) buffer. The homogenate was centrifuged *HCB and TCB are prototype inducers or PB- and MC-inducible mixed-function oxidase. 348 at 10 000 X g for 20 min and the supernatant fraction was carefully removed. Three 1-ml portions of this fraction were taken and stored at -80C. The remainder was centrifuged at 1 0 5 000 X g for 60 min. The resulting supernatant fraction was decanted and the microsomal pellet was resuspended in SET buffer (concentration equivalent to 2 g wet wt. liver/ml) and stored a t -80C. Hematology All determinations were made on blood samples within 1h of collection. ' Peripheral blood counts, hemoglobin, mean corpuscular volume and hematocrit were measured using a Coulter Counter (Model ZF, Coulter Electronics, Inc., Hialeah, FL). For the determination of differential white blood cell counts, blood smears were prepared and stained using a modified Wright stain containing added eosin. Enzyme assays 7-Ethoxycoumarin 0-deethylase [231, benzphetamine N-demethylase [241, epoxide hydratase [251 and UDP-glucuronosyl transferase [261 activities were assayed on the stored hepatic microsomal samples essentially as described in the references cited. 7-Ethoxycoumarin and 7-hydroxycoumarin were purchased from Aldrich Chemical Co. (Milwaukee, WI), benzphetamine from Applied Science Labs, Inc. (State College, PA) and benzo[a]pyrene-4,5-oxide from Midwest Research Institute (Kansas City, MO). Assay of in vitro benzene metabolism The assay was performed in 10-ml conical flasks fitted with screw cap Mininext? valves (Supelco, Inc, Bellefonte, PA). The reaction mixture, in a total volume of 0.9 ml, contained 100 pmol Tris-buffer (pH 7.5), 10 pmol MgC12, 0.5 pmol NADPH, 0.5 pmol NADP, 1 5 pmol glucose 6-phosphate, 0.5 U glucose-6-phosphate dehydrogenase and 0.10 ml 10 000 X g hepatic supernatant. Each flask was purged with O2 before adding the substrate. The reaction was started by the addition of 560 nmol (0.5 pCi) of [14C]benzene.The substrate was added in a total volume of 1pl and the diluted benzene was prepared by adding the appropriate volume of methanol. The reaction mixture was incubated for 30 min a t 37C with vigorous shaking and the reaction was stopped by the addition of 0.125 ml of 15% (w/v) trichloroacetic acid. The samples were transferred to 15 ml conical centrifuge tubes and centrifuged at 2000 X g for 5 min. Portions (100-p1 or 200-p1) of the supernatants were analyzed for benzene metabolites using reverse phase high pressure liquid chromatography. Metabolites were separated on a Radial Pak A column containing octadecylsilane using a 12-min linear gradient (flow rate: 2.4 ml/min) from water to methanol. Both solvents were acidified with 100 pl/l formic acid. The metabolites eluted from the column within 10 min. Fractions (0.5-min) were collected into 20 ml glass scintillation vials and 10 ml of ACS scintillation cocktail (Amersham CC quantified in a by automatic samples rangec metabolites wc and catechol. Chemical Co. Chemical Co. ( For the syswere divided i 0.1 ml 0.2 M .nl sulfatase - saccharolacton from Sigma (=h Statistical anal: Statistical 5 ANOVA in COI RESULTS Effect of preti penia As shown i resulted in a s4 days, the ly: depressed ( P . with TCB (Fig 7 days of ben. treated with values for no benzene toxic (Figs. 1 and TCB pretreate cytes as indica count at days receive benzer hemoglobin, m of the treatme Depressed le intake [27]. I body weights Table I, initial than in contrc we observed 2 carefully re-edat -80C. f i e resulting !t was remswt. liver/ml) >f collection. volume and ZF, Coulter rential white g a modified demethylase f e m e [26] ?sessentially 7-hydroxyaukee, WI), ge, PA) and (ansas City, I screw cap mixture, in 3H 7.5), 10 :ose B-phos- 10 000 x g adding the 01 (0.5 pCi) 1p1 and the if methanol. t h vigorous ml of 15% ml conical or benzene iphy. Metaidecylsilane er t o methmetabolites re collected on cocktail 349 (Amersham Corp., Arlington Heights, IL) were added. Radioactivity was quantified in a Searle Mark I11 scintillation counter. quenching was correctel by automatic external standardization and I4C efficiency in the various samples ranged from 90% t o 95%. The retention volumes of the separated metabolites were compared with authentic samples of phenol, hydroquinone and catechol. Phenol and hydroquinone were purchased from Aldrich Chemical Co. (Plilwaukee, WI) and catechol was obtained from Sigma Chemical Co. (St. Louis, MO). For the systematic hydrolysis of conjugates, various incubation mixtures were divided into 0.3-rnlportions and incubated for 1 2 h at 37C with (a) 0.1 d 0.2 M sodium acetate (p1-I 4.5). (b) 0.1 ml buffer containing 3000 U/ in1 sulfatase - 100 000 U/ml p-glucuronidase or (c) same as (b) with 15 m?l saccharolactone. Both p-glucuronidase and saccharolactone were purchased from Sigma Chemical Co. (St. Louis, KO). Statistical analysis Statistical significance was assessed at the 5% level using a one-way ANOVA in combination with a least significant difference test. RESULTS Effect of pretreatment with HCB or TCB on benzene-induced lymphocytopenia As shown in Figs. 1 and 2, daily benzene administration (440 mg/kg) resulted in a steady decline in the number of circulating lymphocytes. After 4 days, the lymphocyte count in the benzene-treated group was significantly depressed (P < 0.05) compared to control animals (Fig. 1).Pretreatment with TCB (Fig. 1)or HCB (Fig. 2) protected against benzene toxicity. After 7 days of benzene dosing, the values for the lymphocyte count in rats pre- treated with TCB or HCB were significantly greater ( P < 0.05) than the values for non-pretreated animals, but after 1 0 days, protection against benzene toxicity was not observed in rats in either pretreatment group (Figs. 1 and 2). One factor contributing to the loss of protection in the TCB pretreated rats may have been a delayed toxicity of TCB t o lympho- cytes as indicated by a significant (P < 0.05) depression in the lymphocyte count at days 10 and 14 in the TCB-pretreatment group which did not receive benzene (Fig. 1).N o significant changes in the total red cell count, hemoglobin, mean corpuscular volume and hematocrit were observed in any of the treatment groups (data not shown). Depressed leucocyte counts have been reported in rats on a restricted food intake [27].In order to assess this factor in our studies, we monitored the body weights of the rats in the various treatment groups. As shown in Table I, initially, the body weight gains in the benzene-treated rats were less than in control animals. However, toward the end of the treatment period, we observed a recovery in the body weights of the benzeneexposed rats, 350 4.0 9 bc 3.0 -X E \ v) u 2.0 U 0z z 5 1.0 DAY 2 DAY 4 DAY 7 DAY 10 DAY 14 Fig. 1. Effect of repeated benzene administration o n peripheral lymphocytes in rats pretreated with TCB. 0, corn oil controls; a, benzene treated; B, TCB pretreated; m, pretreatment with TCB before receiving benzene. All values represent the mean 2 S.E.M. of duplicate determinations on 5 animals. Asterisks indicate values significantly different (P < 0.05) from corn oil controls for a given group. indicating that the hematologic changes in these animals were associated with the benzene treatment and were not the result of general debility. Characterization of hepatic xenobiotic metabolism Since benzene has been shown t o be metabolized by cytochrome P-450- dependent monooxygenases in the liver [71, we measured representative phase I (mixed-function oxidase) and phase I1 (conjugating)enzyme activities t o determine if the altered susceptibility to benzene toxicity in pretreated rats c o d d be correlated with changes in the profile of hepatic oxidative and detoxification pathways. As shown in Fig. 3, pretreatment with 9CB resulted in a sustained induction (3-fold) of benzph+unine Ndemethylase activity. 7-Ethoxycoumarin Odeethylase activity was induced (4- to 5-foli) by either TCB or iICB throughout the treatment period. These data indicate that the loss of protection against benzene toxicity in HCB- or TCB-pretreated animals (Figs. 1and 2) cannot be associated with changes in representative XC- or PB-inducible mixed-function oxidase pathways. Epoxide hydratase and UDP-glucuronosyl transferase activities were measured in livers from the same rats using benzo[a]pyrene-4,5-oxide and 'ir-hydroxycoumarin, respectively, as model substrates. Epoxide hydratase activity w a s induced 2- to 3-fold by HCB. The activity of this enzyme did not decline after 2 weeks (Fig. 4). UDPglucuronosyl transferase activity r3.5 DC ~ Fig. 2. Effect of pretreated with H treatment with H of duplicate deter ( P< 0.05) from CI TABLE I EFFECT O F CH RATS PRETREAT. Male Fischer-344 tive days before t ment. Each value ranged from 5 t o : Time (days) Treatn - Corn o (0.5 m Group 0 159 4 173 : 7 192 2 : 10 197 5 1 14 215 * : .Benzene was admi bNumben in paren . T1 1 351 DAY 14 iocytes in rats pretreated; 0, mean t S.E.M. antly different .e associated !ral debility. rome P-450!presentative me activities n pretreated xidative and ICB resulted ase activity. ki) by either zte that the B-pretreates< presentative ivities were 5-oxide and e hydratase enzyme did raSe activity DAY 2 DAY 7 DAY 14 Fig. 2. Effect of repeated benzene administration o n peripheral lymphocytes in rats pretreated with HCB. g , corn oil controls; E, benzene treated; q, HCB pretreated; a,pretreatment with HCB before receiving benzene. All values represent the mean i S.E.M. of duplicate determinations on 5 animals. Asterisks indicate valuesSignificantly different (P < 0.05)from corn oil controls for a given group. T-IBLE I EFFECT OF CHRONIC BENZENE ADMINISTRXTION ON BODY WEIGHTS OF RATS PRETREATED WITH HCB OR TCB Male Fischer-344 rats were given HCB (Group 111) or TCB (Group IV) for two consecutive days before trpating with benzene (Methods). Groups I and I1 received no pretreatment. Each value represents the mean t S.E.M. The number of animals in each group ranged from 5 to 25. Time (days) Treatment Corn oil (0.5 ml/kg/day) Group I Benzene' (0.5 ml/kg/day) Group I1 Group I11 Group IV 0 159 5 8 156 f 7 (100) 155 5 10 (100) 1 4 2 2 ll(100) 4 173 5 13 (109) 158 5 10 (101) 150 f 13 (97) 147 5 1 8 ( 1 0 4 ) " 1 192 * 15 (121) 162 + 12 (104) 158 f 1 4 (102) 155 f 12(109) 10 197 * 16 (124) 172 5 9 (110) 1 6 8 5 22 (108) 160 * 12(113) 14 215 * 18 (135) 191 t 11 (122) 185 t 22 (119) 181 f lO(127) 'Benzene was administered as a 30% solution in corn oil. bNumben in parentheses represent percent of body weight at day 0 for each group. 352 I 7-ETHOX Y COU MA R I N 0- DEET H Y LASE - 12F .CE - II 1 -zr 01 0 I 2 I 4 I 6 I I I+ 8 IO 14 00 I6 .cEl BENZPHETAMINE N-DEMETHYLASE t- 4 - 16 .c- E I-x I 4500 2 4 6 8 IO 14 4j+- TIME (doys) Fig. 3. Time course for the induction of 7-ethoxycoumarin 0-deethylase and benzpheta- mine N-demethylase activities by TCB or HCB. A, TCB pretreated; =, HCB pretreated; 0 , no pretreatment. Each point represents the mean +- S.E.M. of duplicate determinations on 4 animals. was induced by TCB. The time course for the stimulation (4-fold increase in activity) and return to control values of glucuronosyl transferase activity precisely paralleled the time course for the protection against benzene toxicity (Figs.1and 4), suggesting a possible relationship between glucuronidation and the modulation of benzene toxicity. Hepatic metabolism of benzene The metabolism of benzene by hepatic 10 000 X g supernatant fractions Fig. 4. Effect of UDP-glucuronosyi the mean 2 S.E.M. was measured u results are shol authentic sampj (Fig. 5A). Unc quinone and c (Table 111). Inc saccharolactone formed in vitrc detected after h: In measuring i .i .....?._.... .. i a ?'- ; I ~~~~~~ .C- E EPOXIDE HYDRATASE 353 n "0 20 I 2 I 4 I 6 I I .. 8 10 14 UDP-GLUCURONOSYL TRANSFERASE T and benzphetaCB pretreated; determinations ' Id increase in :rase activity ienzene toxiglucuronida- ant fractions I I I I 1 14- 0 2 4 6 8 I O 14 TIME (days) Fig. 4. Effect of pretreatment with TCB or HCB on hepatic epoxide hydntase and UDP-glucuronosyl transferase. Symbols are the same as in Fig. 4. Each point represents the mean t S.E.M. of duplicate determinations on 4 animals. was measured using reverse-phase high pressure liquid chromatography. The results are shown in Fig. 5 and Tables I1 and 111. Peaks coeluting with authentic samples of hydroquinone and phenol were consistently detected (Fig. 5A). Under these incubation conditions we did not detect hydroquinone and only a small peak coeluting with phenol was observed (Table 111). Incubation of this sample with sulfatase in the presence of saccharolactone (a 0-glucuronidase inhibitor), indicated that the conjugate formed in vitro was the sulfate ester of phenol. Hydroquinone was not detected after hydrolysis of the conjugate fraction. In measuring the in vitro metabolism of benzene, a concentration equiva- 354 2500 2000 1500 1000 500 z1 V 50 3m B 1500 w t N t 500 - 011 1 P EA L U 1 1 I I 11.1 I I 1 1 1 3 5 7 9 11 1 3 1 5 RETENTION TIME, MIN. Fig. 5. HPLC profile of benzene metabolites after incubation of ["Clbenzene with hepatic 10 000 x g supernatants from naive rats. Metabolites were analyzed by reverse phase high pressure liquid chromatography using a Radial Pak A column containing octadecylsilane. A: incubation buffer containing NADPH and NADPH regenerating system. B: same as (A) plus ATP,sodium sulfate, and UIIP-glucuronic acid. lent t o 19 nmol/mg wet wt. tissue was used. This is a nonsaturating substrate concentration, but more accurately reflects the hepatic concentrations of benzene reported in vivo [28,29]. In the presence of conjugating cofactors, the metabolism of benzene (assessed by total metabolite formation) was significantly depressed (P < 0.05) 2 days after pretreatment with TCB (day 0 of the start of the treatment period) and then returned to control values within 14 days (Table 111). Under these same conditions a moderate, but statistically insignificant, increase in benzene metabolism was observed in livers from HCB-pretreated animals. In the absence of conjugating cofactors IICB-pretreatment stimulated the formation of hydroquinone at all time points examined (Table 11); however, in the presence of these cofactors (Table 111), hydroquinone was not detected in either free or conjugated form TABLE I1 METABOLISM OF TCB OR IICB Male Fischer-344 r; a t the times indica1 benzene (0.89 pCi, (Methods). All valuf Day Pretreatn 0 Control HCB TCB 7 Control HCB TCB 14 Control HCB TCB dNumbers in parenr TABLE I11 METABOLISM O TCB OR HCB All conditions wen ATP, sodium sulfa for 3 animals. Day Pretreatr 0 Control HCB TCB L' I 7 i Control HCB TCB 14 Control HCB TCB t =Values shown incl, bNurnbers in paren cSignificantly diffe and least significa! dAverage value for -i 4 : ` 355 TABLE11 METABOLISM O F [`TIBENZENE IN LIVERS FROM RATS PRETREATED WITH TCBORXCB Male Fischer-344 rats were pretreated with TCB or HCB (Methods). Animals were killed at the times indicated and 560 nmol (equilvalent t o 9 nmol/mg wet wt. tissue) of [ " C l benzene (0.89 pCi/pmol) was incubated with hepatic 10 000 x g supernatant fractions (Methods). All values represent the mean t S.E.M. for 3 animals. Day Pretreatment Hydroquinone Phenol Total metabolitesa (nmol/assay) (nmollassay) (nmol/assay) 0 Control HCB TCB 7 Control HCB TCB 0.114 t 0.003 0.240 f 0.014 0.094 f 0.002 0.126 f 0.002 0.313 f 0.025 0.154 i 0.005 2.76 i 0.23 4.18 k 0.43 1.60 f 0.11 3.55 f 0.19 4.98 * 0.33 3.03 f 0.15 2.93 f 0.32 (100) 4.45 * 0.49 (151) 1.70 * 0.24 (58) 3.68 * 0.23 (100) 5.36 f 0.57 (146) 3.19 f 0.21 (87) 1 4 Control HCB TCB 0.170 f 0.007 0.285 r 0.025 0.197 f 0.010 3.12 f 0.08 5.01 f 0.13 3.58 2 0.44 3.31 f 0.12 (100) 5.31 f 0.24 (160) 3.78 r 0.54 (114) `Numbers in parentheses represent percent of control value for each group. benzene with ed by reverse in containing regenerating irating subcentrations g cofactors, iation) was with TCB to control i moderate, i s observed king cofaclone at all e cofactors gateJ form TABLE I11 METABOLISM OF [ ` T I BENZENE IN LIVERS FROM RATS PRETREATED WITH TCB OR HCB All conditions were the same as in Table I1 except that the incubation buffer contained ATP, sodium sulfate, and UDP-glucuronic acid. All values represent the mean t S.E.M. for 3 animals. Day Pretreatment Phenol (nmollassay) Conjugate (nmol/assay) Total Metabolitesab (nmol/assay) 0 Control HCB TCB \ 7 Control HCB TCB 0.466 f 0.022 1.03 f 0.08 0.103 f 0.001 0.607 f 0.132 0.741d 0.434 r 0.020 3.32 2 0.59 5.16 2 0.17 1.76 * 0.45 5.16 * 0.18 4.5.1d 3.58 f 0.39 4.34 * 1.31 (100) 6.21 f 0.37 (143) 1.91 t O.4lc (44) 5.76 f 0.61 (100) 5.28d (92) 4.01 f 0.41 (70) 14 Control HCB TCB 0.437 f 0.001 0.06 f 0.001 0.293 f 0.001 4.69 f 0.48 7.56 f 6.1 5.46 f 3.8 5.12 * 0.51 (100) 8.62 6.0 (170) 5.76 f 3.8 (112) aValues shown includes contribution from a third metabolite (presumably hydroquinone). bNumbers in parentheses represent percent of control value for each group. CSignificantlydifferent (P < 0.05) from the control value as assessed by one-way ANOVA and least significant difference test. dAverage value for two animals. 356 in control or pretreated rats, suggesting that the conjugation of phenol effectively competed with the further metabolism of this compound to hydroquinone. The decline and subsequent recovery to control values for benzene metabolism in livers from TCB-pretreated rats correlated temporally with the protection against lymphocytopenia (Fig. 1;Table 111). DISCUSSION In this report we have shown that pretreatment of rats with HCB or TCS protects against benzene toxicity. Measurement of MC- and PB-inducible enzyme activities using the well-characterized prototype substrates, 7- ethoxycoumarin and benzphetamine, indicated that the protection against benzene toxicity elicited by TCB or 9CB was not associated with changes in the profile of phase I activities. However, the data suggest that pathways purported to govern the concentration of free phenol (anA thus metabolites of phenol such as hydroquinone) in the liver mediate the expression of benzene toxicity. This is indicated by the temporal correlation between protection against lymphocytopenia and (a) depressed benzene metabolisrr: (40% of control) by hepatic 10 000 X g supernatant fractions (Fig. 1a n i Table 111) or (b) the stimulation and return of control values of UDPglucuronosyl transferase activity (Figs. 1and 4)in rats pretreat& with TCB. The depressed hepatic metabolism of benzene in vitro at a non-saturating substrate concentration (Tables I1 and 111) suggests that TCB may compete with benzene for specific enzyme sites. At a 20-fold greater substrate concentration than reported in Table 11, benzene metabolism in livers from rats pretreated with either TCB or HCB was approx. 2 times greater than in livers from control animals (W.F. Greenlee and R.D. Irons, unpublished datzl. In the broken cell preparation used in this study only the sulfate conjugate was detected (Table 111);however, in vivo both the sulfate and glucuronide conjugates of phenol have been identified [8,30] and it has been reported that at higher concentrations, the percentage of phenol excreted as the glucuronide increases in the absence of significant depletion of inorganic sulfate [31]. In a recent series of experiments characterizing the urinary metabolites of benzene (W.F. Greenlee et al., in prep.), we have found that hydroquinone is eliminated as the glucuronide conjugate, whereas phenol is excreted as both the sulfate and glucuronide conjugate. Further, the amount of hydroquinone found in the urine from control rats was greater than in urine from rats pretreated with Aroclor 1254, a regimen which protects against benzene toxicity [20]. N o obvious relationship between protection against benzene toxicity and metabolism was seen in rats pretreated with I-ICB. The stimulation of epoxije hydratase activity by HCB (Fig. 4) would result in a decreased concentration of phenol and subsequent metabolites such as hydroquinone, but this activity did not decline with the loss of protection against lymphocytopenia (Figs. 2 and 4). Since catechol is a putative product from the oxidation of benzene dihydrodiol [3],catechol may have achieved a toxic concentration after 7 days of Catechol was nc 10 000 X g sur rats after admini In reviewing studies of benze bations of benz low expression benzene metabc concentration o situation and pr in the expressior High concent the bone marro mum concentra approx. 2 h ai Rickert et al. [ 2 in the bone mz Sammett et aI. reduced both th that in rats give with hydroquinc marrow and lyrr these tissues W E et al. [34]have in vitro; howeve less than 0.24% in the rat with to 800-fold gre benzene t o mice lites formed wa: suggest; (a) that for the concentr and (b) that t h t expression of to: and catechol bei toxicity such as t Hydroquinone to semiquinone ( mediates or the factors leading t anion, the low le could be a deter that tissue. In summary, v protects against compound to r o l values for elated tempole 111). i HCB or TCB PB-inducible mbstrates, 7- xtion against with changes h a t pathways LS metabolites expression of between pro? metabolism s (Fig.1 and ues of UDPe2 with TCB. on-saturating nay compete ubstrate coni livers from eater than in Aished data). ulfate conjuand glucuroit has been 11excreted as of inorganic the urinary e founl that eas phenol is , the amount ?ater than in ich protects toxicity and n of epoxije xicentration le, but this hocytopenia Dxidation of mcen tration 357 after 7 days of repeated benzene dosing in the HCB-pretreated animals. Catechol was not detected in the in vitro metabolism of benzene by hepatic 10 000 X g supernatants; however, this species is detected in the urine of rats after administration of benzene [8-lo]. In reviewing the apparent discrepancies between in vivo and in vitro studies of benzene metabolism, Gut [32] suggested that either low concentrations of benzene in the liver or product inhibition could account for the low expression of induced hepatic rmcrosomal monooxygenase activity in benzene metabolism in vivo. Thus, it would appear that the non-saturating concenhation of benzene used in our investigation approximates the in vivo situation and provides a reasonable index of the role of hepatic metabolism in the expression of benzene toxicity. High concentrations of benzene metabolites have been demonstrated in the bone marrow of mice given 880 mg/kg ['HI benzene [29]. The maximum concentration of these metabolites in the bone marrow occurred approx. 2 h after maximum concentrations were attained in the liver. Rickert et al. 1281 reported that hydroquinone and catechol were retained in the bone marrow of rats exposed to 500 ppm benzene for 6 h and Sammett et al. [33] reported that the removal of 7040% of the liver reduced both the metabolism and the toxicity of benzene in rats. We showed that in rats given ''C-labelled benzene metabolites, radioactivity associated with hydroquinone or catechol, but not phenol, concentrated in the bone marrow and lymphoid organs [ZO]. Further, the amount of radioactivity in these tissues was reduced in rats pretreated with Aroclor 1254. Andrews et al. [34] have demonstrated benzene metabolism by rabbit bone marrow in vitro; however, the amount of phenol formed, the major metabolite, was less than 0.24%of the benzene added. After perfusion of an isolated femur in the rat with blood containing [I4C]benzene at a concentration 200to 800-fold greater than the reported concentrations G f toxic doses of benzene to mice or rats [28,29] , it was found that the amount of metabolites formed was 3% of the administered dose of benzene [35]. These data suggest; (a) that the metabolism of benzene by bone marrow cannot account for the concentration of metabolites reported in that tissue in vivo [28,29] and (b) that the metabolism of benzene in the liver is important for the expression of toxicity with poly'phenolic metabolites such as hydroquinone and catechol being taken up and concentrated by target tissues for benzene toxicity such as the bone marrow. Hydroquinone is an inherently reactive species capable of being oxidized to semiquinone or benzoquinone [ 3 6 ] .The formation of free radical intermediates or the potential generation of superoxide anion may be causative factors leading to cytotoxicity. If toxicity is mediated by the superoxide anion, the low levels of superoxide dismutase reported in bone marrow [37] could be a determinant of the selective toxicity of benzene metabolites to that tissue. In summary, we have shown that pretreatment of rats with HCB orTCB protects against benzene toxicity. This protection is a complex process 3 58 involving metabolic events other than the induction of phase I activities. In rats pretreated with TCB, the data suggest that decreasing the concentratio:i of primary benzene metabolites, either by inhibiting the hepatic metabolism of benzene or increasing hepatic conjugating activity, is an important factor regulating the expression of toxicity. ACKNOWLEDGEMENTS We would like t o thank Drs. John G. Dent and Douglas E. Rickert for helpful discussions and assistance on various aspects of this research. We also thank John P. Chism, Beverly J. Moore, Come N. Smith, Delorise A. Williams and Elizabeth A. Gross for their ex'pert technical assistance. REFERENCES 1 C.P. Carpenter, C.B. Shaffer, C.W. Weil and H.F. Smyth Jr., Studies o n the inhalation of 1:3 butadiene; with a comparison of its narcotic effect with benzol, toluol, and styrene, and a note on the elimination of styrene by the human, J. Ind. Hyg. Toxicol., 28 (1944)69. 2 H.V. Hough and S. Freeman, Relative toxicity of commercial benzene and a mixture of benzene, toluene and xylene, Fed. Proc., 3 (1944)20. 3 R. Snyder, E.W. Lee and J.J. Kocsis, Bone marrow depressant and leukemogenic actions of benzene, Life Sci., (1978)1709. 4 R. Snyder and J.J. Kocsis, Current concepts of chronic benzene toxicity, CRC Crit. Rev. in Toxicol., 3 (1975)265. 5 S. Laskin and B.D. Goldstein, Benzene toxicity: a critical evaluation, Arch. Toxicol. Environ. Health, Suppl. 2 (1977). 6 H.S. Cohen, M.L. Freedman and B.D. Goldstein, The problem of benzene in our environment: clinical and molecular considerations, Am. J. Med. Sci., 275 (1958) 124. 7 L.M. Gonasun, C.M. Witmer, J.J. Kocsis and R. Snyder, Benzene metabolism in mouse liver microsomes, Toxicol. Appl. Pharmacol., 26 (1973)398. 8 D.V. Parke and R.T. Williams, Detoxication XLIX. Metabolism of benzene containing ["Clbenzene, Biochem. J.. 54 (1953)231. 9 D.V. Parke and R.T. Williams, Detoxication L N . T h e metabolism of benzene. (a) The formation of phenyl glucuronide and phenyl sulfuric acid from ["C] benzene. (b) The metabolism of ["C]phenol, Biochem. J.. 55 (1953)337. 10 S.A. Timbrel1 and J.R. Mitchell, Toxicity-related changes in benzene metabolism in vivo, Xenobiotica, 7 (1977)415. 11 K Nomiyama, Experimental studies o n benzene poisoning, Bull. Tokyo Med. Dent. Univ., 11 (1964)297. 12 J.R. Mitchell, Mechanism of benzene-induced aplastic anemia, Fed. Proc., 30 (1971) 561. 13 M. Ikeda and H. Ohtsuji, Phenobarbital-induced protection against toxicity of toluene and benzene in the rat, Toxicol. Appl. Pharmacol., 20 (1971)30. 14 R.T. Drew, J.R. Fouts and C. Harper, The influence of certain drugs on the meta- bolism and toxicity of benzene, in: D. Braun (Ed.), Symposium o n Toxicology of Benzene and Alkylbenzenes, Industrial Health Foundation, Pittsburgh, 1974,p. 17. 15 A Conney, Pharmacologic implications of microsomal enzyme induction, Pharmacol. Rev., 19 (1967)317. 16 D.P. Gill, R P and metabolit (1 979)1633. 17 W.K. Lutz an irreversible bir 18 R. Snyder, B mouse liver ar 191. 19 A. Tunek, K.1 species irrever: metabolism, hl 20 W.F. Greenlee. the dispositior 33 (1981)285 21 J.A. Goldstein Toxicological benzofuran in Toxicol. Appl. 22 A. Poland and lase activity: a 924. 23 W.F. Greenlee activity: induc phenobarbital, Pharmacol., Ex 24 R.A. R o u g h a oxidase and C: phys., 180 (19` 25 H.U. Schmassr activity with (1976)94. 26 I.S. Owens, GI cyclic aromatic 27 S. Oishi, H.Oi: toxicity param1 28 D.E. Rickert, 1 the rat after c 29 L.S. Andrews, on the metabo Pharmacol., 26 30 J.W. Porteous a The isolation o fraction of the 31 J.G. Weitering, as a rate limiti Pharmacol., 28 32 I. Gut, Some 1. Gut (Edr), Biotransfonnat 33 D. Sammett, E metabolism an 34 L.S. Andrews, marrow, LifeSI jle concentratio<+?- latic metabolism '5. I . mportant factor .- u. .I 5 E. Rickert for .search. We also )rise A.Vlilliams ~ , i on the inhalation enzol, toluol, and nd. Hyg. Toxicol., ene and a mixture md leukemogenic ixicity, CRC Crit. In, Arch. Toxicol. f benzene in our Sci., 275 (1978) le metabolism in mzene containing I of benzene. (a) m ["C] benzene. i e metabolism in Dkyo Med. Dent. Proc., 30 (1971) xicity of toluene ugs on the metain Toxicology of g h , 1974. p. 17. :tion, Pharmacol. 359 1 6 D.P. Gill, R.P. Kemper, J.B. Nash and S. Ellis, Modification of benzene myelotoxicity and metabolites by phenobarbital, SKF- and 3-methylcholanthrene, Life Sci., 25 (1 979) 1633. 17 W.K. Lutz and Ch. Schlatter, Mechanism of the carcinogenic action of benzene: irreversible binding to rat liver DNA, Chem.-BioL Interact., 18 (1977) 241. 18 R. Snyder, B.W. Lee and J.J. Kocsis, Binding of labeled benzene metabolites to mouse liver and bone marrow, Res. Commun. Chem. Pathol. Pharmacol., 20 (1978) 191. 1 9 A. Tunek, K.L. Platt, P. Bentley and F. Oesch, Microsomal metabolism of benzene to species irreversibly binding t o microsomal protein and effects of modifications of this metabolism, Mol. Pharmacol., 1 4 (1978) 920. 20 W.F. Greenlee, E.A. Gross and R.D. Irons, Relationship between benzene toxicity and the disposition of "C-labelled benzene metabolities in the rat, Chem.-BioL Interact., 33 (1981) 285. 21 J.A. Goldstein, J.D. McKinney,G.W. Lucier, P. Hickman, H.Bergman and J.A. Moore, Toxicological assessment of hexachlorobiphenyl isomers and 2,3,7,8-tetrachlorodibenzofuran in chicks. 11. Effects in drug metabolism and prophyrin accumulation, Toxicol. Appl. Pharmacol., 3 6 (1976) 81. 22 A. Poland and E. Glover, Chlorinated biphenyl induction of aryl hydrocarbon hydroxylase activity: a study of the structure-activity relationship., Mol. Pharmacol., 13 (1977) 924. 23 W.F. Greenlee and A. Poland, An improved assay of 7-ethoxycoumarin 0-deethylase activity: induction of hepatic enzyme activity in C57BL/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-pdioxin,J. Pharmacol., Exp. Ther., 205 (1978) 596. 24 R.A. Rough and D.M. Ziegler, The relative participation of liver microsomal amine oxidase and cytochrome P-450 in N-demethylation reactions, Arch. Biochem. Biophys., 180 (1977) 363. 25 H.U. Schrnassrnann, H.R. Glatt and F. Oesch, A rapid assay for epoxide hydratase activity with benzo(a)pyrene-4,5-(K-region)oxide as substrate, Anal. Biochem., 74 (1976) 94. 26 I.S. Owens, Genetic regulations of UDP-glucuronosyltransferase induction by poly- cyclic aromatic compounds in mice, J. Biol. Chem., 252 (1977) 2827. 27 S. Oishi, H. Oishi and K. Hiraga, The effect of food restriction for 4 weeks on common toxicity parameters in male rats, Toxicol. Appl. Pharmacol., 47 (1979) 15. 28 D.E. Rickert, T.S. Baker, J.S. Bus,C.S. Barrow and R.D. Irons, Benzene disposition in the rat after exposure by inhalation, Toxicol. Appl. Pharmacol., 49 (1979) 417. 29 L.S. Andrews, E.W. Lee, C.M. Witmer, J.J. Kocsis and R. Snyder, Effects of toluene o n the metabolism, disposition and hemopoietic toxicity of [ 'H ] benzene, Biochem. Pharmacol.. 26 (1977) 293. 30 J.W. Porteous and R.T. Williams, Studies in detoxication. p e metabolism of benzene. The isolation of phenol. catechol, quinol and hydroxyquinol from the ethereal sulfate fraction of the urine of rabbits receiving benzene orally, Biochem. J., 44 (1949) 56. 31 J.G. Weitering, K.R. Krijgsheld and G.R. Mulder, The availability of inorganic sulfate as a rate limiting factor in the sulfate conjugation of xenobiotics in the rat, Biochem. Pharmacol., 28 (1979) 757. 32 I. Gut, Some factors affecting xenobiotic metabolism in vivo, in: J.R. Fouts and I. Gut (Eds.), Industrial and Environmental Xenobiotics: In Vitro versus In Vivo Biotransformation and Toxicity, Excerpta Medica, Amsterdam 1978, p. 74. 3 3 D. Sammett, E.W. Lee, J.J. Kocsis and R. Snyder, Partial hepatectomy reduces both metabolism and toxicity of benzene, J. Toxicol. Environ. Health, 5 (1979) 785. 34 L.S. Andrews, H.A. Sasame and J.R. Gillette, 'H-Benzene metabolism in rabbit bone marrow, Life Sci., 25 (1979) 567. Chem.-BioL Inlemct 1 o Elsevier/North-Hol 35 36 Short Communica 37 INTERACTION C ISOXAZOLEACE TRANSPEPTIDA, L.M.ALLEN, MAFLI. Department of Oncc Miami,FL 331 01 (V. (Received April 18th (Revision received AL (Accepted August 24 Introduction The 7-glutamyl linking the utiliza the major enzyme (EC 2.3.2.2). Thi: predominantly wi cells transformed catalyzes the tzar amino acid accept( for amino acid me! Several analogs serine, azaserine) -Materials and M e t ) Bovine kidney