Document MMmQVQvGe4Bvbw73vmmD22pdz

%~, X K O L O G Y AND APPLIED PHARMACOLOGY 49,417-423 (1979) I Benzene Disposition in the Rat after Exposure by Inhalation' DOUGLAS E. RICSERTT. ERRIsE. BAKER. JAMES S. Bus. CRAIG S. BARROW, AND RICHARD D. IRONS Clirmical Industry Insrirule of Toxicology. Research Triangle Park, ,Vorth Carolina -77709 Received .411gustIS, 1978: accepted March 7, 1979 Benzene Disposition in the Rat after Exposure by Inhalation. RICKERTD, . E., BAKER, T. S., Bus, J. S., BARROW, c. s. AND IRONS, R. D. (1979). Toxicol. Appl. Pliarmacol. 49, 417423. Little information is available on benzene disposition after exposure by inhalation despite the importance of this route in man. Benzene metabolites as a group have been measured in bone marrow, but quantitation of individual metabolites in this target tissue has not been reported. Male Fischer-344 rats were exposed to 500 ppm benzene in air and the uptake and elimination was followed in several tissues. Concentrations of free phenol. catechol, and hydroquinone in blood and bone marrow were also measured. Steady-state concentrations of benzene (1 1.5, 37.0, and 164.0 pg/g in blood, bone marrow. and fat, respectively) were achieved within 6 hr in all tissues studied. Benzene half-lives during the first 9 hr were similar in all tissues (0.8 hr). A plot of amount of benzene remaining to be excreted in the expired air was biphasic with tf values for the a and /9 phases of 0.7 and 13.1 hr. respectively. Phenol was the main metabolite in bone marrow at early times (peak concentration, 19.4 pgjg). Catechol and hydroquinone predominated later (peak concentrations. 13.0 and 70.4 pg/g, respectively). Concentrations of these two metabolites declined very slowly during the first 9 hr. These data indicate that free catechol and hydroquinone persist in bone marrow longer than benzene or free phenol. The disposition of benzene in mammals has been studied for a number of years. The most ,- complete set of data concerning benzene distribution and elimination after exposure by inhalation is that of Schrenk et ai. (1941). These workers exposed dogs to benzene a t '-c.oncentrations ranging from 500 to 1300 ppm. Steady-state concentrations of benzene in the body were achieved rapidly ( <2 hr); hnzene concentrations were highest in bone marrow and adipose tissue, and benzene disappeared from the blood in a biphasic manner. Determination of the half-life of s+CULC r-A preliminary report of this work was presented I t the joint meeting of the American Society for %rmacology and Therapeutics and the Society of ~ ~ c o l o gAyu,gust 1978, Houston, Tex. benzene in blood was precluded by a n insufficient number of data points, and quantitation of benzene metabolites in tissues was not attempted. Benzene is converted to a number of meta- bolites in mammals. By 1949, the following compounds had been identified or suggested as urinary metabolites of benzene: phenol, catechol, hydroquinone, 1,2,4-trihydroxybenzene, trans,trans-muconic acid, phenylmercapturic acid, and dihydrocyclohexadiene (Porteus and Williams, 1949). Certain of these metabolites (phenol, catechol, hydroquinone) are much more toxic to bone marrow cultures than is benzene (Harrison and Randoll, 1948). Attempts to correlate benzene metabolism with benzene-induced a z, 415 RICKERT ET AL. myelotoxicity have produced some con- Wisc.). Deuterated benzene (de) was flicting data (Ikeda, 1964; Ikeda and Ohtsuji, 1971 ; Snyder and Kocsis, 1975; Ikeda el ui., 1972), but the bulk of evidence suggests that a benzene metabolite is responsible for the myelotoxicity. This can be illustrated by the Merck and Company, Inc. (Pointe C1 Erposrire procediire. Rats were exr two dynamic air flow chambers. i chamber, operated at an air Row rate was used to determine approach concentrations of benzene. The r,, ( work of Andrens er a/. (1972) who used depression of red cell s " up~ take as an esti- mate of benzene-induced myelotoxicity. Their data sugzest that [3H]benzene metabolism and toxicity can be altered by coadministra- 99:; of nominal benzene concentr chamber was 23.9 min as calculated b. Silver (1946). Exposure for eliininati was carried o u t in a I100-litcr glas steel chamber operated at an air f l ~ literslmin (Ipo = 20.3 min). Duration I tion of toluene, which resulted in less depression of red cell 59Fe uptake than did administration of benzene alone. This protective effect of toluene was associated with a decrease in non-toluene-extractable radio- measured from the time that benze was begun. Benzene concentration. chamber were monitored by gas c (Cow-Mac 750 Series FID) using a glass column (Tcnax GC 60/90 niesi large chamber, benzene concentratic activity (presumably benzene metabolites) in bone marrow. It is clear from the foregoing that the rational design of experiments to assess hazards of benzene exposure in man and to tored by infrared spectrometry (Wilk a wavelength of 3.214 jtm. Concentra in the small chamber ranged from i The mean timeweighted average (T. periments was 484 ppm hith a S D oi large chamber, the benzene concer: determ i ne the mech ani sin of benzene-i nd uced myelotoxicity requires a more complete knonledge of benzene disposirion after inhalation exposure. I t is especially important that concentrations of benzene and its metabolites be measured individuallv in bone from 474 to 499 ppin (mean TWA = 10 ppm). Exposure of animals to 57 for 6 hr in each chamber produced si: centrations of benzene. Sarnp/i/i: procehrre. Animals \\e: decapitation. In the experiments, approach to steady state, animals marrow. This report describes benzene distribution during and following exposure of Fischer-344 rats to 500 ppm benzene in air. In addition, three major benzene metabolites, phenol, catechol, and hydroquinone. were after 0.5, I . 1.5, 2, 4, 6. o r 5 hr o f t elimination experiments. animals w, 0.5, 1 , 1.5, 2, 2.5, 3. 4, 6, or 9 hr af exposure. Blood was collected in t heparin. Tissue samples were remove the following order: liver. lunz. quantitated in the blood and bone marrow adipose tissue rrom the peritoneal c: of the exposed animals. Bone marrow was removed from ' Samples were kept on ice at all tinx cessed (see below) immediately after !VI ETHODS Snmpie rreofnwir. One hundrec blood conraining heparin was mi Animals nnd c-hemicnls. Male Fischer-344 rats (Charles River Co.. Wilmington. Mass.) neighing 200-30 g were used (three:group). For 3t lc3jt I week prior to benzene exposure, the animals \\ere housed in a humidity and temperature-sontrolled room and given fond (Wavne Lab Blou. .Allied 54ills. Chicago. 111.) 3nd ivater ad libifitm. Benzene v.3~ ohtained from Fisher Scientific Company (cenitied A.C.S.. lot No. 770535). Methanol \vas ohtsincd I'rom Burdick and Jackson (Muskegon. Xlich.). Standard phenol, catechol, hydroquinone, and de-phenol and trifluoroacetic anhydride were ob- internal standard solution ( 1 rr?' methanc>l).Weighed samples oi 50 mixed \rirh 100 111 internal .;t;lnd;lrd samples \\ere homogenized u\ing 3 mizer in 3 nil Inrrrnal i r a n h r d Particul~teninrter u a s remobed by the cold 3t JOOO rpm for 15 min. supernate \&ereanalyzed by selectec (see below). The practice of chilli mixing as soon as possible with i minimized bcnzene loss, yielding \ 95 % of those expcctcd in preliminar tained from Aldrich Chemical Company (Milwaukee, The benzene metabolites, phenc ... I; . - .- -i - 420 RlCKERT ET AL. and mlr 84 were calculated and plotted against nanograms of benzene per microliter. Thac standard .a: I ;*:.TABLE 1 -4,+8% STEADY-~TATBEENZENE CONCEWRATIO~NS _f* p curves were used to calculated the concentrations of benzene in blood, bone marrow, and tissues. Concentrations of benzene eliminated in expired k-EXPOSURE OF RATS TO 500ppm BENZENE FOR 6 "- AND HALF-TIME FOR UPTAKE AND ELIMINATION 01 BENZENE. "..@ air were measured in three animals. Immediately after a 6-hr exposure to 500 ppm benzene, animals were removed to all-glass metabolism units (one rat/unit). . Air flow was adjusted to 400ml/min. Benzene was trapped on 1 g activated coconut charcoal (6-14 mesh, Fischer Scientific CO.) in 5-ml disposable pipets. Traps were changed at 0.5, 1.0. 1.5. 2.0, 2.5, 3.0, 4.0, 6.0. 9.0, 12.0, 24.0, 36.0. and 48.0 hr after introduction of the rat into the unit. The benzene was eluted from the traps with carbon disulfide; the charcoal was transferred to 5-ml amber reaction vials. and 3 ml CS2 (Fisher A.C.S. Reagent) was injected through a septum fitted over the vials. The CS, was then injected into a gas chromatograph with a flame ionization detector (Gow-Mac, Series 750) Sample Blood Bone marrow Fat Liver Lung Kidney Spleen Brain' Steady-state concentration (gg/g or /ml) 11.5C 0.7 37.0k 2.2 164.42 15.0 9.9k 0.7 15.12 0.9 25.32 1.3 4.92 0.5 6.5+ 0.6 -k Half-times (itin hrji-: J Approach tr. to steady- Elimim.. state' tione- J_ . 1.4 0.1 i 4--0..5 T 2.0 1.6 L 1.9 0.4 1.5 0.4 1.3 0.6 0.9 0.8 2.6 0.6 . * 1.0 0 t 4 e tz Y 0 t 0 0 Y t 2 OJO 0m c Ya under the following conditions: 6 ft x 2-mm i.d. glass column (Tenax GC 60/80 mesh) held at 135C. The carrier gas was nitrogen at a flow rate of 30 ml/ min. The temperatures of the injection port and detector were held at 165C. Standard curves of benzene in CSI were prepared ranging in concentration from 0.5 to 5 pl/ml and plotted as concentration benzene vs peak height. In a preliminary experiment. this method of trapping and eluting benzene was shown to result in a recovery of >99%. Percentage of benzene remaining to be excreted in the expired air was calculated for each time interval for each animal by dividing the amount of benzene eliminated during the time interval by the total amount eliminated in expired air. The trifluoroacetate derivatives of phenol, cate- chol and hydroquinone were analyzed by gc/ms. The compounds were separated on a 6 ft x 2-mm i.d. glass column (3:; OV1 on 60/80 mesh DMCS Chro- masorb W) held at 90T. SIM was used, monitoring the derivative of the internal standard (d,-phenol) at nrlc 195; phenol at rn/r 190; catechol and hydroquinone at rn/e 302. Standard solutions (50 pg/pf to 50 ngipl) of phenol. catechol, and hydroquinone were prepared in methanol. The concentration of the N = 3. Data are expressed as mcans+SE. 'Determined graphically from data points at 0.54 hr after beginning of exposure. Determined graphically from data points at '0.5-9 hr after end of exposure.' Steady-state approached too rapidly for determination of half-time. elimination are summarized in Table 1. This table indicates the similarity between elirnination half-lives for all tissues except fat. Phenol was present in both blood and bone 1 marrow after a 6-hr exposure to benzene (Fig. 2). but it rapidly disappeared. Catechol. and hydroquinone concentrations remained' fairly constant over the time period studied. Benzene was eliminated in the expired air in a biphasic manner (Fig.3). The two corn- . ponents of the curve \\.ere determined graphically for each of the three animals ex-. posed and the coefficients were averaged. The, inc SE iisjues ad;:?.; cc bl na: A stead!. tissues be dogs ( [his sl internal standard was held at I ng/jrl. Peak area equation found to represent the curve waJ ratios (compound!d,,-phenol) were calculated for each compound and plotted against nanograms per microliter. 1' = 93e-O.W-t -4.93e-0.55." . The first corn ponent (line 4)hss J half-life of 0.7 hr. and the second component (line B) has a half-life RESULTS of 13.1 hr. ,?.t Benzene concentrations reached stead! state within 1 hr in blood. 6 hr in fat. and less than 1hr in bone marrow (Fig. I). DISCLSSION _c . 1- Data collected here essentially confirm the Steady-state benzene concentrations and information obtained in dogs with regard to ' half-times for approach to steady state and relative concentrations of benzene in various BENZENE DISPOSITION L 4 'I ( -a 0 ? L 2 c -I c L .) 3 L D u Y 5 2 0 0 4 c w 1 r L... E.' FIG. 2. Concentration of metabolites following a 6-hr inhalation exposure to benzene. Asterisks i. indicate points at which a compound was not determined or not detected due to technical difficulties. 5. SE were all less than 50%. Each point is the mean for three animals. $& $ti7ssues (Shrenk et a!., 1941). Bone marrow, cribed mathematically by a two-compart&pose tissue, and kidney contained con- ment model. The half-time for the more h r a t i o n s of benzene in excess of that in rapid phase of elimination in expired air blood. This is probably due to the lipophilic agrees well with the half-times calculated for &ure of benzene. Half-times for achieving disappearance from various tissues (0.7 vs &dy-state were of the same order for all 0.4-1.6 hr). The assay for benzene in tissues &sues except bone marrow, and they appear was not sufficiently sensitive to describe the *@+ be somewhat longer than those found in slow phase of benzene elimination from dogs (Shrenk et ul., 1941). The reasons for tissues. The half-life for the slow phase of species difference could include differ- benzene e1.imination in expired air (13.1 hr) gys in fraction absorbed or differences in suggests that some accumulation of benzene &r*aAt,es of excretion or metabolism. The data on could occur under this regimen of exposure. 52k*n+'tene approach to steady-state indicate The maximum benzene concentration in a commonly used protocol for inhalation blood which could be achieved can be ,ylosure (6 hrlday) would result (at least for approximated by: $2) in maintenance of steady-state con- .--bations of benzene for about 2 hr. most complete description of benzene where Cl is the concentration achieved during a single exposure, tf = 13.1, and At is the interval between exposures (24 hr). Substituting the actual values yields a 422 c RJCKERT ET AL. in b servc hydr agen the t (e.g.. n. - r n;.ii: Th assist. ..,.*. AhD; J. . TIME lhr) thc Ftc;. 3. Percentage o i benzene remaining to be excreted in expired air. Each point is the mean+_SE for t h r a animals w i t h the exception of the I 3 h r time point which represents only a single value. to3 19 H~R. '1 .- I blood concentration of 16 jig/ml after an in- 19481, but their ability to produce myelofinite number of exposures. One may also toxicity after administration to intact rats has predict that after four daily exposures, blood not been established. Mitchell (1971) was un- It ., I .3. . concentrations of benzene will be 15.9 pgzml. able to produce aplastic anemia in rats at This agrees very well with the experimental doses of phenol, catechol, or hydroquinone value found (15.5 i 1.6 jlgjml) in preliminary which produced significant lethality, but experiments. benzene itself caused the disorder. Nomiyama 1 ' : Free phenol, catechol. and hydroquinone (1965) used much lower doses of phenol, were all found in blood and bone marrow catechol. hydroquinone, and other benzene following 6 hr of exposure to benzene. The metabolites to treat rats. He demonstrated concentrations of free phenol in blood and that catechol. given at a dose of 30 ms,'kg/day bone marrow decreased much more rapidly for 7 days. produced a decrease in pen- ; after exposure ceased than did those of free pheral leukoqte count sirntlar to that sea catechol or hydroquinone. I t is interesting when benzene Has administered. With the that both carechol and hydroquinone per- denionstrsrion. in this study, of the PO&'- sisted in blood and bone inarrotc for at least bility for accumulation of catechol and 9 hr after exposure to benzene m p p e d . This h>droquinone in bone marrow of rats re- suggests a possibility for accumulation of ceiving benzene, it seems necessary to in- these compounds. Catechol and hydro- vestigate further the role of these compounds quinone are 1 1 6 times more toxic to bone marrow cultures than phenol or certain in benzene-induced myelotoxicity. The assay procedure developed here should allow ' . Ftrihydroxybenzenes (Harrison and Randoll, correlation of individual benzene metabolites n bone marrow with the toxic effects ( bgrved. The hypothesis that catechol or lydroquinone (or both) is the causative lgent is attractive because it requires nettt er he transport of a highly reactive intermediate +g., benzene oxide) from liver to Dolie marrow nor thedemonstration of an act1 ie mixed function oxidase system in hone marrow. ACKNOWLEDGMENTS The authors thank C. Rarnaley and D. Deyo for assistance in the quanritation of bcnzene in expired air. REFERENCES ANDREWS, L. S.. LEE,E. W..WITMERC,.M.. Kocsrs, J. J., AND SNYDER. R. (1972). Effects of toluene on the metabolism disposition and hemopoietic toxicity of ['HI benzene. Biochem. Phormacol. 26, 293-300. HARRISON, K.. A N D RANDOLL, F. W. (1948). An application of bone-marrow cultures to toxicology and therapeutics. Quurr. 1. fip. Physrol. 34, 141150. IKDA, M. (1964). Enzymatic studies on benzene up intoxication. J. Biochem. (Tokyo)55.231-243. .:2 I n i ( s t 3prewon of iien~:rli*; i ! i d 5 ! . r e r c ( <IC!+ lion cii-.idrninistered tolriere I ! rat, arc1 t l k c t s of p t c i c t ~ i r b i t a i .,\efioLtotr,u :!. 101 - I 10. M I T C I I L L , I. R. (1971) M x - i a n siii f 3eri?erii:indJced 1-13sticanemia F w . .l'rx. i ed 4 r w r 50c. E r p . Brei 30, 2aW. N O M I Y P M PK,. (1965). :tucliex, o n pc1 so~ing by benzene .i td 11shomolugues 1 7 ) roxrl:it, of hrizene rneraliolirc I t o hemOp~Jlta,i5f.nd. f I c ( i / r h 3, 53- 57. -PORTEUS, W., AND WILLIAM`, IF!. 1.- (1949). StLdies in de o\icItion 19. The inetabolts rl of berizmc. 1. (a) The tletermination 3f >heno1 in urine vith ~ : 6 - d ~ c h l c i o q u i n o n e c h l o ~ o i n[ b~ti ~T~he cxcretion o f pllenol, glucurorlic w d . ,inti erhe-ea1 sul- phate h) i.ibbits receiving brnrent. . m i phenol (c) Observaiicvis on the determination o f catechol, quinone and muconic acid in urine BiOChctJI. J . 44.46-5 5. SHRENCK, 3 H.. YANT. W . P., PEARCSE., J.. PATTY. F. A., AIWD SAYERS, R. R. (1941). 4bsorption, dis- tribution lnd elirninaricn of bewrne by body tissues and fluids of dogs rxposed to benzene vapor. J. Ind. H>g. Toxtcol. 23. XL.14. SILVER, D. D (1946). Constant flow gassing chambers: Principles influencing design ana operation. J. .Lob. Clin. !.led. 31, 1153-1 161. SNYDER. R AND Kocns, J. J. (1975) Current concepts of chronic benzene toxicity. CRC Crir. Rea. Toxrcol. 3, 265-288. _.