Document b5XqQ11d5Z8NGjgNDMv0w2rzk

tudies in phocytes !xposllre, !ad [19]. -A0 and d UCLA. tits, h e r . iatrics, 18 losure and activity to lannacol., ;h lead, J. , Test for n strain A *roma te, nation of ening for synthesis , glial cell cell lines chromo7. Effect ent with -spolizismage in cing von Res., 1 6 esischen - PLAINTIFF'S EXHIBIT Chem.-Biol.Interactions, 30 (1980) 241-245 a Elsevier/North-Holland Scientific Publishers Ltd. 241 BENZENE IS METABOLIZED AND COVALENTLY BOUND IN BONE MARROW IN SITU R.D. IRONS, J.G.DENT, T.S. BAKERand D.E. RlCKERT Chemical Industry Institute of Toxicology, P.0.Box I213 7, Research Triangle Park, NC 27709 (U.S.A.) (Received July 2nd, 1979) (Revision received December 10th. 1979) (Accepted December 14th. 1979) Introduction Benzene is a well-known myelotoxic agent which has recently been impli- cated as a leukemogen_[l]. The mechanism by which benzene induces myelotoxicity is not understood. Myelotoxicity could result from the action of the parent compound or any of the well-known metabolites of benzene such as phenol, catechol, hydroquinone, 1,2,4-benzenetriol or trans,transmuconic acid [2-41. Quantitatively, the liver is the major site of benzene metabolism in the body [ 51;however, the relative importance of metabolism in the liver with respect of myelotoxic action is unknown. The intermediate(s) responsible for bone marrow toxicity may be synthesized in the liver and transported t o marrow where they act directly; they may require additional metabolism in bone marrow to form the ultimate toxic intermediate; or, as an alternative, bone marrow metabolism of benzene may be the only prerequisite for benzene induced myelotoxicity. The possibdity that benzene can be metabolized directly in bone marrow, the principal target organ, has not been explored [6].Phenol, catechol and hydroquinone are found in rat blood and bone marrow following inhalation of benzene [71, but the sites of formation of these metabolites have not been established, nor has the metabolic contribution of the bone marrow been ascertained. The principal objective of this work w& t o determine if bone marrow is capable of metabolizing benzene independent of metabolism in the liver. - Materials and methods Male Fischer-344 rats (Charles River, Wilmington, MA), weighing 250-350 g, were subjected to a hind limb perfusion procedure. The left common iliac vein and artery were cannulated in rats maintained under anileridine-ketamine anesthesia. The isolated limb was then perfused a t a flow rate of 1 mi min-' with citrated oxygenated whole rat blood -maintained at 39C. A single dose (0.5-2 mCi) of [14C]benzene (Midwest Resemh Institute, Kansas City, MO) (39 mCi mmol-') was introduced directly into the bone marrow space through a 0.75 mm hole drilled in 242 the distal head of the femur. The bone was immediately sealed With bone 1 Wax. The t o w volume of benzene administered ranged between 1 and 3 pl. In control preparations the bone m m o w tissue was removed bY aspiration prior t o the introduction of radiolabelled benzene. Blood W ~ Scollected from the iliac vein in 10-min fractions for 1 h- Each 10-min fraction averaged 6.5 ml blood. Following the perfusion, collected blood samples and bone marrow were spiked with Carrier corn- POunds (50 p g each 04 phenol, catechol, hydroquinone, 1,2,4-benzene triol and tmns,tmns-muconic acid) and acidified t o a pH of 1.0 with 1M HC1Samples were then extracted with ethylacetate, dried over magnesium sulfate and filtered using Millipore Clarification Kits (Millipore CO.,h d f o d MA). Extracts were concentrated under nitrogen and passed through silica gel. Final samples were concentrated to 20 pl and subjected t o high pressure liquid chromatographic analysis (HPLC). HPLC analysis was carried out using a Waters system (Milford, MA) -equipped with two Zorbax silica columns (Dupont, Wilmington, DE),flow programmed from 0.8-1.6 ml min-'. The solvent system was 74.9% methylene chloride, 25% ethylacetate, 0.1% methanol with 100 ctl Of 88% formic acid added per 100 ml of solvent. Fractions were collected at 0.3 min inter~alsstarting 9 min after injection and analyzed for radioactivity by liquid scintillation. Total metabolites bound in bone marrow were determined by subjecting the ethylacetate extncted bone marrow residue to further extraction with 5% trichloroacetic acid (TAC) and increasing concentrations of aqueous methanol (40, 60, 80 and 100%). The solvent concentration was changed when no radioactivity was detected above background in the supemate% each sample subjected t o an average of 17 extractions. Find residues were solubilized in a mixture of NaOH, methanol and Triton-X-100 and analyzed for radioactivity by liquid scintillation. Results and discussion A representative profile for benzene metabolites obtained by HPLC analysis is presented in Fig. 1. Radioactivity was found to co-ehte with phenol, catechol, and hydroquinone in blood perfusate and bone marrow fractions. No radioactivity coeluted with metabolites in blood spiked with radioactive benzene and extracted immediately. Peak levels for all three metabolites in blood were found in the 1O-min fraction (Fig. 2) with total radioactive metabolites recovered averaging 6.44 nmol ( 2 1.2, S.E. n = 4) after 1-h incubation. The same radioactive metabolites were recovered from bone marrow (phenol, 64 2 23; catechol, 3.9 f 1.6; and hydroquinone, 0.31 5 0.2 pmol * mg-I). No radioadive metabolites were detected in Control preparations and n o radioactivity was detected in systemic blood, liver Or urine in any of the perfusion experiments. Thus the small amount of metabolism observed is directly attributable t o the bone marrow. Covalent binding of benzene or its metabolites was investigated using the extraction procedure described. Results are presented in.Table I. Total i f 1 I f I i I i' i I ! t Fig. 1. Radioa fnpCti1 (254 u GidiO recol and 1 follo th bone 1 and lved by )r 1 h. rfusion, r comne triol M HC1. nesium edford; h silica ressure 1, MA) )Y flow 74.9% >f 88% ted at mdio- jecting n with lueous ianged mates, s were alyzed HPLC : with arrow I with three total I = 4) from none, I con, liver nt of ig the Total 243 Phenol 1 5 IO Unknown I5 20 J 25 TIME (min) Fig. 1. Analysis of benzene metabolites by HPLC. Absorbance of 254 nm -( ). Radioactivity (--.---). injection of the sample. Fractions were Total time for collected at 0.3 min sample elution: 25 minitner. v-als star,tiUnVg 7 min. after absorbance (254 urn); 01-, radioactivity (cpm). radioactivity bound in the bone marrow represents 14.7%of the metabolites recovered from the bone marrow or 2.76% of the total metabolites in blood and bone marrow. Addition of radiolabelled benzene to bone marrow in vitro followed by immediate extraction resulted in retention of 11 pmol equi- valents versus an average of 239 pmol in perfusion experiments. Thus, metabolism of benzene appears t o be a necessary prerequisite for covalent binding in bone marrow. The results of this study clearly establish the capability of bone marrow to metabolize benzene independent of metabolism of the compound by the liver. However, the amount of metabolism appears t o be very small, recovered metabolites representing only 2 X of 1%of the administered dose. It is impossible to assess in physiologic terms the contribution of bone marrow t o overall benzene metabolism under these experimental conditions. The initial levels of benzene in bone marrow in these experiments 244 1 II IO 20 ll 30 40 ll 50 60 . TIME (monl Fig. 2. Radioactive metabolites recovered from blood following the introduction of ["Clbenzene directly into bone marrow. -(94-195 pg mg-') are higher than are likely to be encountered as a consequence of exposure of the whole animal [71. In addition, the perfusion procedure provides for an examination of only a first pass effect with no recirculation of metabolites or parent compound. It was impossible to measure accurately the recovery of radioactive benzene due t o the extraction procedure employed; however, it is likely that benzene rapidly enters the TABLE I BENZENE METABOLISM AND COVALENT BINDING M BONE MARROW * v Total metabolites Metabolites Metabolites Metabolites recovered from blood recovered from recovered from bound in bone andbonemarrow , blood bone marrow marrow .8614. t 1537b 6441 t 1242 1626 f 661 239 t 107 hetabolites are expressed as picomoles and bound metabolites as picomole equivalents of benzene. ' `Ihe data are expressed as mean t standard error from five bone marrow and four blood rr) perfusatt benzene AlthOl only 25: (mean w bolites ir marrow. rapidly E bolites ir of the I exposure concentr. declined blood aF experime bolites fc 1 S. Lask Environ 2 J.W.Pc benzene imide. ( receivin. quinol a 3 D.V. p: benzene 4 D.V. Pa Pbenylg of ["C] - 5 R.Snyd C.R.C. ( 288. 6 ELSnyc linr an( 7 DX.Ric rats afte ction of :d as a rfusion vith no ibie to raction ;ers the )lites in bone r .07 iivalents ir blood p*.- 245 perfusate and thus the marrow was only exposed to a high concentration of benzene for a short time. Although the total metabolites recovered from bone marrow represent only 25% of those in blood, the concentration of metabolites in marrow -(mean weight = 20 mg) is much higher. The concentration of total meta- bolites in blood is 0.215 nmol g-' as compared to 81.3 nmol * g-' for bone marrow. The fact that the appearance of metabolites in blood diminishes rapidly after 10 min demonstrates a high retention of water soluble metabolites in bone marrow. This finding is in keeping with the results of studies of the disposition of benzene metabolites in rats following inhalation exposure (6 h) to benzene (500 ppm) in which catechol and hydroquinone concentrations persisted in bone marrow long after blood levels had declined [7].The metabolite concentration ratio between bone marrow and blood approaches 400 in this study as compared to 7 in the inhalation experiment, supporting the conclusion that a high proportion of the metabolites formed in bone marrow is retained in that tissue. We thank B.J. Moore, C.N. Smith and D.H.Williams whose expert techni- cal assistance made this work possible. 1 S. h k i n and B.D. Goldstein, Benzene toxicity: a critical evaluation, J. Toxicol. Environ. Health, Suppl. 2 (1977). 2 J.W. Portcua and R T . Williams,Studies in detoxification 19. The metabolism of benzene. 1. (a) The determination of phenol in urine with 2:6-dichloroquinonechloroimide. @) The excretion of phenol glucuronic acid, and ethereal sulphate by rabbits receiving benzene and phenol. (c) Observations on the determination of catechol, quinoi and muconic acid in urine,Biochem. J., 44 (1949)56-61. 3 D.V. Parke and R.T. Williams, Studies in detoxification 49. The metabolism of benzene containing ['4C]beazene, Biochem. J., 54 (1953)231-238. 4 D.V. Parke and RT. Williams, The metabolism of benzene (a) I b e formation of phenylgiucuronide and phenylsulphuric acide from [ I T]benzene. (b) The metabolism of [ ' C ]phenol, Biochem. J., 55 (1953)337. 5 R. Snyder and J.J. Koesis, Current concepts of benzene toxicity,in: L. Golberg (Ed.), C.R.C. Critical Reviews in Toxicology, C.R.C. Press, Cleveland, Ohio,1975,pp. 265- 288. 6 R. Snyder, E.W. Lee and J.J. Kocia, Binding of labeled benzene metabolites to mouse liver and bone marrow,Res.Comm. Chem. Path. Pharmacol., 20 (1978)191. 7 DE. Rickert, T.S. Edgar,'C.S. Barrow,J. Bus and R.D. Irons, Benzene disposition in rats after inhalation exposure, Toxicol. Appl. Pharmacal., 49 (1979)417. \