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337 -- REPEATED EXPOSURE OF C57BI MICE TO INHALED BENZENE AT IO ppm MARKEDLY DEPRESSED ERYTHROPOIETIC COLONY FORMATION (Benzene; depressed erythropoiesis) K.A. BAARSON.C.A. SNYDER. and R.E. ALBERT hkw York University Medical Center, Insfitute of Envimnmental Medicine. JIM F ~ KAZvenue, New York. NY 10016 W.S.A.) (Received November 2nd. 1983) (Accepted Nwankr 4th. 1983) NMMARY Exposure of CJ7B1 mice to 10 pprn k n r c n e (the current oc~~lpuionerxlposure limn) for 6 h/dav. 5 days/wcek causes P progressive dcprauon in the in vitro cuiony forming ability of one of the erythroid prolenitor cells, the colony-lomini unucryrhroid(CN-E). Cdoay uwh of from apored mrcc vu oaly 5% of concrol colony growth after I78 days of aporure. Bum-formm8-Crtl u m h was dcpreucd to 5JIR of control growth after 66 days but returned to m t r o l umh vdua at 178 days. in addition. kntcnc-exposaf mice ahibited depressions in the numbers of splenic nucleated red 4 1 s and in the numbers of drcul.ung red cdlr and lymphocyla. Thee tcnrlu suggest that low-led aposurc xo knrcne may be hemuotouc. lNfRODUCTlON Over 60 years of study have demonstrated that benzene is a potent hematotoxicant [I]. The most severe blood dyscrasias associated with benzene exposure are aplastic anemia and myeioblastic leukemia 12). Several good reviews have been written daaiiing the hematotoxic responses of both human and animals exposed to benzene 12-41. The current occupational exposure limit for benzene is 10 ppm or a time-weighed average IS].Several years ago. the OSHA attempted to lower the exposure limit to 'To whom all correspondence should be sent. Abbreviations: EFU-E.burst-forming unixiwrhrold C N - E . colonv-foming unii-er?ihroid; OSHA. Occupational Safexv and Health Administration. 0 3 3 4 2 7 4 i 8 4 / 1 03.00 E . Elsevier Science Publishers E.V. . 339 counts were determined by resuspending aliquots of the respective single-cellswpensions into a hypotonic solution of 3:1 heat-inactivated fetal calf serum and water, incubating the suspensions for 10 min and preparing smears stained with 1% dimerhoxybenzidine countered with Wright-Giemsa. loo0 cells per organ per animal were scored from these slide preparaG6ns. The BFU-E and CFU-E were quantified by the methyl cellulose method as modified by Urabe and Murphy [ 171. 6 aliquots of cells were cultured from each animal. Cells from age-matched, air-exposed animals were concurrently cultured each time the assays were performed on benzene-treated animals. RESULTS The mean daily benzene concentration t SD during the course of the exposures was 10.1 ppm i 0.3 ppm. Fig. I . Efftcx of repeated exposure IO 10 ppm benzene on femoral committed eryhroid progenitor cells. Closed circles and error bars arc mean 2 S.E.of determinations from 5 benzene-exposed animals. Dash- =ed lines are S.E.of control culture growths. Mean C N - E culture growths of cells from benzene- treated mice were sratlsticallv signtficant (P<O.OI) at all time points. Mean BFU-E culture growths of cells from benzene-treated mlcc were atatrsuallv significant (PCO.011 onlv for aswvs performed on the 66th d a y of the stud). Fig. 2 . Effect of repeated exposure io IO ppm benzene on the numbers of circulating red cells (RBC)and lymphocytes. Bars denote = S.E. .Asiaisks denote statistically different from controls (PcO.OS). 3a TABLE I SPLENIC ERYTHROID CELLS Treated Control *C F U - E / S P I ~ S.E. . 10'' 32 Days -66 Days- 4 3 z 2.1 9.3 2 2.3 -8 -b 178 Dovr 2.S 2 0.3h 23.6 z 7.3 BFU-Eisplecn z S.E. Treaicd Control 281 z 150 70 : 32 235 5 35 1s2 2 46 438 2 I64 166 = 9 Cclluluityisplnn e S.E. . IO-' Treated Control 108 z 62 93 2 3.4 123 z 5.9 110 i 7.9 76 2 1.9' 90 f 4.4 Nuclutcd red ccUs/lpiem z S.E. . IO-' Treated Control 1.8 2 0.7 0.8 2 0.3 7.9 i 1.2 3.6 i 1.6 0.6 r 0.2' 4.0 2 1.0 %ells from both treated and control mice exhibited a ZOfold in- in growth with large varianm of growh from culture to culture. Thae growths were likely artifacts. 'lndiutes significinily depressed relative to control values. P < 0.05. .) Progenitor cells from benzene-exposed mice showed markedly reduced abilities to form colonies compared to cells from control mice. The'numbers of marrow C N - E colonies from benzene-urposed mice showed a progressive decline during the exposure period reaching W o of control values after 178 days (Fig. I). Colony numbers declined exponentially during the exposures with a half-life of about 2 months (Fig. 1). Marrow B N - E colonies from benzcne-exposed mice were significantly depressed to 55% of control values at 66 days but returned to control values at 178 days (Fig. 1). Splenic C N - E colony formation was depressed to IOmo of control values in benzene-exposed mice at 178 days (Table I). The exposures had no effect on splenlc BFU-E colony growth at any monitoring period (Table I). Bcnzene-exposed mice also exhibited changes in the numbers of differentiated hematopoieric ceils. Significant depressions in the numbers of circulating red cells and lymphocytes were observed in exposed mice. Red cells were depressed at 66 and 178 days while lymphocytes were depressed at all three monitoring periods (Fig.2). Levels of circulating neutrophils were unaffected by the exposures (nor shown). Ai 178 days. benzene-exposed mice exhibited depressions in splenic nucleated cellularity and in splenic nucleated red cell numbers. The latter were depressed to about 15% of control values (Table I). Marrow celluiarity and marrou-nucleared red cell numbers were unaffected by the exposures (not shown). 341 DlSCUSSlON Exposure to 10 ppm benzene cieariy inhibited the ability of the mouse C N -E to form colonies in .itro. By the end of the exposures, 95% of the C N -E were unable to form colonies. It is unlikely that 95% of the C N -E were destroyed since a loss of that magnitude could not maintain the nucleated red cell numbers found in the marrow. A more likely explanation would be a progressive inability of the CFU-E to replicate and differentiate in culture under the stimulus of erythropoie~in.The inability of CFU-E to differentiate in culture may not reflect an inability of these cells to differentiate in vivo. However, an inability to differentiate in culture indicates that at least one functional characteristic of these cells has been changed. Alternatively, damage to the ability of the preceding ccfl types to replace C N - E could have a role in this effect. C N -E have limited proliferative potential and are progeny of BFU-E which have a large proliferation potential (18, 19). Even a minor amount of benzene-induced damage to B N -E could result in significant depressions in C N -E numbers. Evidence for damage to the B N - E population was observed at 66days when the in vitro colony forming ability of these cells was rcduced to 55?0 of control values. Under conditions of erythropoietic stress, the spleen can become a site of ex- tradmedullary erythropoiesis in the rodent 120, ti].SplenicC N - E and the numbers of more differentiated nucleated red cells in the splem were reduced at 178 days in mice exposed to benzene. Thus, erythropoiesis was ais0 disrupted in this compensatory erythropoietic organ. The failure of the erythrons (marrow and spiem erythropoiesis) of benzeneexposed mice to s u p p o ~normal red cell mass is illustrated by the significant reduction in peripherai red cell numbers in these animals during the last two monitoring periods. It is clear from t h w data that exposures to levels of benzcne considered occupationally safe markedly disrupt normal erythropoiesis in t h e e mice. Since peripheral lymphocyte numbers were also depressed, 10 ppm benzene exposure may also be affecting other cell types. We have acquired evidence for this io preliminary work in which ue found that femoral pluripotent stem cells (CFU-S) were reduced to 50% of control values after 178 days exposure to 10 k m (not shown). Further work concerning the hematotoxic effects of inhaled benzene at or below 10 ppm seems to be warranted. ACKNOWLEDGEMENTS Work completed in partial fulfillment of requirements for degree of Doctor of Philosophy (senior author) under the sponsorship of Dr. Snyder. This research was supported in part by center grant number ES00260 from the National Instituies of Environmental Health Sciences and by center grant number CAI3343 from the National Cancer Institute. The senior author is a pre-doctoral inhalation toxicology .. 342 trainee.on grant number ESO7065 from the National Institutes of Environmental Health Sciences. Erythropoicrin was generously supplied by the National lnstitutes of Health Red Blood Cell Program, Blood Diseases Branch. DBDR.The authors wish to acknowledge the efforts of Kmnah Magar for operating the inhalation chambers and Maureen Freitag for typing the manuscript. REFERENCES I L. W i n g . Benzol as a leucotoxin. Studies on the degeneration of the blood and hmatopoiaic organs. Johns Hopkins Hospital Repon. I7 (1916) 83-142. 2 s. Laskin and B. Goldsic@. Benzene toxicity, a critical d u a r i o n . J. Toxicol. Environ. Health. Suppl. 2 (1977). 3 R. Snyder and 1. Koair. Current concepts of chronic benzene toxicity, C.R. 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