Hematopoietic Effects of Benzene InhalationAssessed by Long-term Bone Marrow CultureNadar G. AbrahamThe Rockefeller University, New York, New York

The strong and long-lasting hematotoxic effect after benzene exposure in vivo (300 ppm, 6hr/day, 5 days/week for 2 weeks) was assessed in mice with bone marrow cells grown in long-term bone marrow culture (LTBMC). Bone marrow cultures initiated 1 day after the last benzeneexposure did not produce adequate numbers of hematopoietic cells over 3 weeks and, in mostcases, no erythroid or myeloid clonogenic cells could be recovered. The adherent cell layer ofthese cultures had a lower capacity for supporting in vitro hematopoiesis after the second seedingwith normal bone marrow cells compared with control cultures. Two weeks after the last benzeneexposure, body weight, hematocrit, bone marrow cellularity, and committed hematopoieticprogenitor content (BFU-E and CFU-GM) were regenerated to normal or subnormal values,whereas hematopoiesis in LTBMC was very poor. Over 8 weeks, little or no significant committedprogenitor production was observed. Treatment of mice exposed to benzene with hemin (threedoses of 3 pg/g bw iv over 2 weeks for a total dose of 9 pg/g) partially overcame the toxic effectof benzene on the hematopoietic system as measured by the LTBMC method. Cultures frommice treated with hemin had a modest recovery of BFU-E and CFU-GM clonogenic potential after5 to 6 weeks in LTBMC. In contrast, little or no recovery was obtained for the adherent cell layerclonogenic capacity, even after hemin treatment. These results clearly indicate a strong, long-lasting toxic effect on the bone marrow stroma and a limited recovery of hematopoietic potentialby clonogenic cells of the nonadherent population after in vivo hemin treatment. Environ HealthPerspect 104(Suppl 6):1277-1282 (1996)

Key words: benzene, long-term bone marrow culture, hematopoiesis, bone marrow stroma

IntroductionBenzene is a dangerous environmentaltoxin produced in nature and in largeamounts by industry. Chronic exposure ofanimals to benzene is known to lead to pro-gressive degeneration of the bone marrowand the drug metabolic system, leukopenia,aplastic anemia, and eventual leukemia(1-4). The clinical evidence indicatingbenzene as a primary inducer of diseasessuch as aplastic anemia and leukemia iswell documented (5). Benzene can pro-duce such toxic metabolites as phenol, cat-echol, and hydroquinone; apparently theseagents can accumulate in the bone marrow(6-8). We have shown that hematotoxictargets for benzene and its metabolites maybe related to depletion of heme and heme

This paper was presented at Benzene '95:An International Conference on the Toxicity,Carcinogenesis, and Epidemiology of Benzene held17-20 June 1995 in Piscataway, New Jersey.Manuscript received 16 January 1996; manuscriptaccepted 14 June 1996.

Address correspondence to Dr. N. G. Abraham,The Rockefeller University, New York, NY 10021.Telephone: (212) 327-8762. Fax: (212) 327-8510.

Abbreviations used: LTBMC, long-term bone mar-row culture; CFU, colony-forming unit.

protein and induction of heme oxygenase(9). The direct effect of benzene on pluripo-tent stem cells (10,11), erythroid (12-14)and myeloid progenitors (15), lymphocytes(16), macrophages (17,18), and stromalelements (19) has been documented. Clonalcultures of bone marrow erythroid (BFU-E,CFU-E) and myeloid (CFU-GM) progeni-tors and spleen colony assays for pluripotentstem cells (CFU-S) have also verified thehematotoxic effect of benzene (11,12).

Although the mechanisms that inducehematotoxicity remain unclear, the appar-ent selectivity of benzene toxicity (ormetabolites) for hematopoietic tissue maybe connected with its capacity to be accu-mulated by bone marrow several timesgreater than that for other tissues (8).Depression of drug metabolism, growthfactor production, and ribonucleic acidsynthesis by cells of the hematopoieticmicroenvironment may also result fromexposure to benzene or its metabolites(12,18,20). Furthermore, these metabolitescan inhibit heme synthesis and induce theheme-degrading enzyme, heme oxygenase(12). Inadequate levels of heme may leadto a decrease in cytochrome P450 levels,

disturbance of the drug-metabolizingsystem (12) and a variety of cellular distur-bances throughout the hemopoietic sys-tem; these effects could be attributed tobenzene. We have found that exogenoushemin can counteract some toxic effectsbrought about by inhibition of heme syn-thesis by heavy metals and drugs (21-23),and more recently we have studied theeffect of hemin on hematopoiesis in long-term bone marrow culture (LTBMC)(24). Heme is an essential component ofcytochrome P450 and the drug-metaboliz-ing system and plays a central role in theregulatory network of hematopoiesis underconditions of stress (25).LTBMC represents a near-physiologic

system for assessing growth characteristicsof bone marrow cells in vitro. The prolifera-tion and differentiation of stem cells aredependent on the intimate contact betweenthe hematopoietic cells and a confluentstroma (26). The importance of studyingthe long-term stromal effects of benzene areobvious, but effects on bone marrow growthin LTBMC have not yet been well character-ized. Only a few reports have described ben-zene hematotoxicity in LTBMC (27,28).These studies demonstrated a reduction inthe CFU-S compartment in LTBMCsderived from mice treated with benzene; thiswas thought to be due to the action of ben-zene on both hematopoietic and stromalcells. This conclusion was reached after co-culture of normal hematopoietic cells onstromal-adherent cells derived from miceintoxicated with benzene and vice versa.Unfortunately, for the second seeding, ahigh dose of bone marrow cells (l07) had tobe used. It remains possible that a highinoculum of cells contributes to the regener-ation of stromal progenitors for productionof stromal adherent cells and overcomes theprevious benzene toxicity.

Hemin probably has several beneficialeffects on the hemopoietic system in nor-mal and abnormal states. The aims of thisproject were to evaluate benzene toxicity tomurine hematopoietic bone marrowstroma with LTBMCs and to study theeffectiveness of hemin administration onhemopoietic regeneration in mice after ben-zene inhalation. A strong residual effect ofbenzene exposure on the capacity of hema-topoietic cells to generate a favorable hema-topoietic microenvironment in LTBMCwas observed. Because hemin has beenshown to enhance hematopoietic progenitorproliferation and differentiation, as well as

Environmental Health Perspectives * Vol 104, Supplement 6 * December 1996 277

N.G. ABRAHAM

total cellularity, we examined the beneficialeffect of hemin on clonogenic and stromalhematopoietic recovery. Results indicatepartial recovery of erythroid and myeloidelements after hemin treatment, whereasdamage to the adherent stromal cells wasmore permanent.

MethodsMale, specific pathogen-free DBA/2 mice,10 to 14 weeks old (Charles RiverLaboratory, Charles River, Massachusetts),were used for benzene and control groups.For second seedings of bone marrow cul-ture, female DBA/2 mice weighing 20 gand 8 to 12 weeks old were used. Miceused in each experiment were maintainedunder specific pathogen-free conditions.

Exposure to benzene was carried out inan isolation chamber system provided byC. Snyder of the New York UniversityDepartment of Environmental Medicine.Chromatography-grade benzene was usedfor inhalation. Groups of 20 mice wereexposed to conditioned air or to 300 ppmbenzene for 6 hr/day, 5 days/week, 2 weeks.

Starting 2 days after the last benzeneexposure, some of the mice intoxicatedwith benzene were given intravenoushemin injections (three doses of pg/g bw)over 2 weeks.

All bone marrow cultures were preparedunder sterile conditions as described earlier(24,26,29). Methylcellulose techniques forerythroid (BFU-E) and myeloid (CFU-GM)colony assays have been described in detail(23). The nonadherent cells were countedand appropriate dilutions were made.

ResultsEffet of in Vivo Benzene Exposureon Hematopoietic IndicesWe examined the effect of benzene inhala-tion on hematopoietic compartmentsimmediately after exposure or 12 days afterthe last exposure. As seen in Table 1, the

body weights of DBA/2 mice exposed to300 ppm benzene for 6 hr/day, 5days/week for 2 weeks were decreased by15% compared to body weights of the con-trol group. Peripheral blood anemia andleukopenia were also observed, and hemat-ocrits decreased to 74 to 76% of normal.Bone marrow cellularity was suppressed byas much as 93% in mice exposed to ben-zene. Furthermore, hemopoietic clonal effi-ciency was decreased and was seen as areduction in the ability to generate BFU-Eand CFU-GM. In particular, mice thatinhaled benzene had diminished clonalcapacity for BFU-E and CFU-GM contentper tibia. This was most striking for BFU-E/tibia and was decreased by as much as90% compared with control mice.

As shown in Table 1, significant regen-eration of hematopoietic elements occurredafter 2 weeks, and recovery of BFU-E to96% of control levels was observed. In addi-tion, bone marrow cellularity improved(68% of control) and CFU-GM (66% ofcontrol) regenerated after discontinuationof benzene exposure. Because hemin hasbeen used to overcome bone marrow toxic-ity by exogenous chemicals and drugs(21-23), hemin was injected intravenouslyinto mice during the recovery time at adose of 3 pg/g bw, (over 2 weeks for atotal dose of 9 )ig/g bw), and hematopoi-etic indices were compared. This dose ofhemin has been shown to be effective inmyelodysplastic patients (30). Better recov-ery of all hemopoietic indices occurred inmice exposed to benzene and treated withhemin compared with mice subjected tobenzene exposure alone (Table 1).

Effect of in Vivo Benzene Exposureon Stromal Adherent Celi Capacityto Support LTBMCControl and benzene-exposed mice werekilled 1 day after the last benzene exposureand bone marrow was used for LTBMC.Bone marrow cells from mice exposed to

benzene were able to form an adherent celllayer in LTBMC, but production of thenonadherent cell population was almostnegligible. Consequently, studies were con-ducted to evaluate the ability of the stromaladherent cells to support the proliferationand differentiation of normal hemopoieticcells. LTBMCs from control mice and ani-mals exposed to benzene were allowed togrow for 3 to 4 weeks, after which time thecultures were reseeded with normal bonemarrow cells. The cultures were secondary-seeded with a single-cell suspension of 1.5 x106 normal bone marrow cells [the stromalcell content in the number of cells is notsufficient to provide an adequate hemopoi-etic microenvironment (31)]. Thus, culturescontained stromal adherent cells derivedfrom cells of mice exposed to benzene andfresh cells from unexposed animals. Theresults are presented in Figure 1. In all caseswe saw depression in cellularity and clono-genic potential in reseeded LTBMCs grownin the presence of stromal adherent cellsfrom mice that had previously beenexposed to benzene.

Effect of in Vivo Benzene Exposureon Nonreseeded LTBMC CapacityLTBMCs were set up 12 days after the lastbenzene exposure. Cumulative cell produc-tion during the next 4 to 8 weeks was sup-pressed in cultures obtained from bonemarrow of benzene-exposed mice com-pared with controls (Figure 1). Figure 2Ashows that the cellularity of control cul-tures for weeks 7 and 8 ranged between12.5 and 16 x 106 cells/culture for thesame period.

Erythroid (BFU-E) and myeloid (CFU-GM) colony-forming progenitor cells werequantitated from LTBMCs derived fromcontrols and mice exposed to benzene.Control cultures exhibited significantcloning potential during weeks 4 through8, whereas cultures derived from animalsthat inhaled benzene had negligible

Table 1. Effects of benzene exposure and hemin treatment on body weight and hematopoietic indices in mice (means ± SEM).

Time after last Bone marrowGroup exposure, days Body weight, g Hematocrit, % Leukocytes cellularity, x 106 BFU-E, per femur CFU-GM, per femur

Experiment 1Control 1 28 ± 1 44.1 3.0 11.0 ± 0.5 3250 ± 34 13,640 ± 104Benzene 1 24 ± 1 32.5 0.6 0.7 ± 0.3 280 ± 15 7,140 ± 97

Experiment 2Control 12 29 ± 1 44.2 4.7 11.3 ± 0.6 791 ± 21 23,746 ± 111Benzene 12 30 ± 1 39.1 1.9 7.7 ± 0.7 760 ± 36 15,708 ± 87Benzene + hemin 12 30 ± 2 44.6 2.7 9.5 ± 1.0 809± 42 23,370 ± 118

Indices were examined 1 and 12 days after benzene exposure (330 ppm, 6 hr/day, 5 days/week for 2 weeks). Hemin was given during recovery at 3 pg/g, iv, 3 times in 2 weeks.

Environmental Health Perspectives * Vol 104, Supplement 6 * December 19961 278

HEMATOPOIETIC EFFECTS OF BENZENE INHALATION

B15 -

CD

10 -=

CD,x

LLmM 5-

4 5 6 7 8 4 5 6 7 8 4 5 6 7 8

Weeks after second seeding

Figure 1. Capacity of the adherent cell layer and the effect of reseeding on hematopoietic recovery in long-term bone marrow culture (LTBMC). LTBMCs of cells from controlsand animals exposed to benzene (taken 1 day after last exposure) were allowed to grow for 3 to 4 weeks, after which time the cultures were reseeded with normal bone marrowcells. (A) Cumulative nonadherent cellularity; (B) cumulative erythroid progenitor (BFU-E) during weeks 5 to 7; (C) cumulative myeloid progenitor (CFU-GM) during weeks 5 to 7.

A Control- S - Benzene--4-- Benzene + hemin

B

Cu

4=

x

m

2 3 4 5 6 7 8

C12

10

= 8C.,

6

LL 4C-

4 5 6 7 8

Weeks

Figure 2. Effect of in vivo benzene exposure and hemin on long-term bone marrow culture (LTBMC) capacity. Cellularity and clonogenic potential were determined in LTBMCswith bone marrow from controls and animals exposed to benzene and then given hemin 12 days after the last benzene exposure. (A) Cumulative nonadherent cellularity during8 weeks of culture; (B) cumulative erythroid progenitor production (BFU-E) during weeks 4 to 8 of culture; (C) cumulative myeloid progenitor production (CFU-GM) duringweeks 4 to 8 of culture.

colony-forming potential (Figure 2B,C).During weeks 4 to 8, control BFU-Ecolonies/culture ranged from 1.3 to 6 x

102/culture and CFU-GMs ranged from2.2 to 9 x 103/culture (Figure 2B,C).Almost no colony growth could be quanti-tated from cultures derived from animalsexposed to benzene during the same cul-ture period. Hence, the regeneration ofstromal progenitors capable of producingfunctionally active stromal adherent cells

was not complete 2 weeks after the lastbenzene exposure.

Effect ofHemin on HematopoieticRecovery ofMice Intoxicatedwith BenzeneCellularity and clonogenic potential were

determined in LTBMCs with bone marrow

from mice exposed to benzene and treatedwith hemin (Figure 2). In all cases, heminwas found to have significant beneficial

effects on the capacity of LTBMCs to

support bone marrow cell growth after micehad been exposed to benzene. Duringweeks 1 to 8 of culture, cultures derivedfrom mice given hemin had increases incellularity form 1.5 x 106 cells/culture(hemin) (Figure 2A). In addition, BFU-Eand CFU-GM growth potential duringweeks 4 to 8 increased from negligible valuesto 1.2 x 102 BFU-E/culture and 2.1 x 103CFU-GM/culture (Figure 2B,C). Thus,

Environmental Health Perspectives * Vol 104, Supplement 6 * December 1996

A24 -

A Control- v - Benzene

CD

' 20--UCD

x 16-

.r7, 12-Cu)C.)a)

8-CID

EC-) 4-

C

10 -

C.)

x

U-

8 -

6 -

4 -

2 -

A

Cu)=

x 10-

Cu

Cu

Cu

.2- 5.Cu

EC-,

4 5 6 7 8

l1279

N.G. ABRAHAM

there was modest sparing of clonogenicpotential in LTBMC by cells from animalsexposed to benzene and treated with hemin.

Effect ofBenzene and Hemin onHematopoiesis by Adherent CellsHemin was given to mice treated with ben-zene 12 days after the last exposure to ben-zene in a manner similar to the proceduredescribed in Table 1. Animals were thenkilled and LTBMCs were established forcontrols, mice exposed to benzene and miceexposed to benzene but treated with hemin.Cultures were grown for 8 weeks, afterwhich time all of the adherent cells wereremoved from cultures, and cellularity andclonogenic potential were quantitated withonly adherent cells. Results of these studiesare depicted in Figure 3. The data demon-strate that some population of cells in theadherent layer ofLTBMCs has the capacityto give rise to erythroid and myeloid clono-genic growth. Control cultures gave rise toapproximately 7.0 ± 1 x 102 BFU-E/cultureand 6.3 ± 1.1 x 103 CFU-GM/culture(Figure 3B,C). It is dear that cultures fromanimals exposed to benzene had markedreductions in the total number of adherentcells/culture. Note that the cellularity ofcontrol cultures was 15.6 ± 1 x 106 adher-ent cells/culture, whereas cellularity of cul-tures from mice exposed to benzene was 3.1± 1 x 106 adherent cells/culture (Figure 3A).In addition, Figure 3A shows that in vivohemin treatment improved the cellularity ofLTBMCs previously exposed to benzene.Results demonstrate that adherent cellnumbers in cultures from animals exposedto benzene and treated with hemin were

,, A, 15

10-oX

L- 5 -E

a_

=

a)

0 L

6.9 ± 1.2 x 106 adherent cells/culturecompared with 3.1 ± 1 x 106 adherentcells/culture for benzene exposure alone.No significant improvement was seen inBFU-E/CFU-GM clonogenic potential byadherent cells (slight improvement inCFU-GM clonogenic capacity was seen),but this did not appear to be significant(Figure 3B,C). Thus, in vivo hemin treat-ment was not as effective for hematopoieticrecovery by adherent cells when contrastedwith the nonadherent cell populationshown in Figure 2.

DiscussionExcessive benzene exposure in humans andother mammals often results in disorderssuch as leukemia and aplastic anemia(3,5). Disturbed or impaired stroma maycontribute to abnormal hematopoiesis andthus to the pathogenesis of these disorders(32,33). Results from our studies clearlydemonstrate the long-term toxic effects ofbenzene on the hematopoietic stromalprogenitors and the capacity to generate aviable adherent cell layer that can supportsustained hematopoiesis. Hemin had somebeneficial effects on the recovery of stromalcapacity to generate an adherent cell layerable to support cellularity and clonogenicityby cells from LTBMC. However, adherentcell layers from mice exposed to benzeneand treated with hemin recovered only par-tially both quantitatively and qualitatively;in these cultures, the relative content ofclonogenic progenitors in stromal adherentcells with cells in suspension was sharplydecreased. These toxic effects of benzeneand the sparing influence of hemin are not

CB15-I

= 10

xLa~

5-CD

15

a)

') 10

CD,.5LJC-,

~s Control Benzene Benzene+ hemin

Figure 3. Effect of benzene and hemin on hematopoiesis by cells from the adherent cell layer. LTBMCs of cellsfrom animals exposed to benzene and treated with hemin 12 days after the last exposure were allowed to growfor 8 weeks, after which time all of the adherent cells were removed from cultures and cellularity and clonogenic-ity potential were determined. (A) Hematopoietic cellularity of adherent cell layer at terminations of cultures; (B)erythroid progenitor (BFU-E) content of adherent cell layer at termination of cultures; (C) myeloid progenitor(CFU-GM) content of adherent cell layer at termination of cultures.

obvious if one examines only the hema-topoietic indices directly from mice afterbenzene exposure.

Inadequate production of specificgrowth factors or extracellular matrix sub-stances by the stroma may be one of thedamaging effects of benzene. It is notclear whether benzene itself is the primarytoxin or whether more toxic metabolitessuch as phenol are responsible. We havedemonstrated that phenol is more toxic tobone marrow CFU-E and stromal growththan benzene (12). Furthermore, benzeneis known to promote a marked increase inbone marrow heme oxygenase activity, andthis could contribute to our results (12).Hemin has been found to reverse some ofthe bone marrow toxicity caused by heavymetals (21) and drugs such as azidothymi-dine (AZT) (23). It is thought that thetoxicity of these agents, in part, is causedby a disturbance in the heme biosyntheticpathway and a decrease in cellular hemo-protein, including the cytochrome P450system. Thus, exogenous hemin can bypassthe defective steps and restore adequate lev-els of heme (21) needed for cell function,including drug biotransformation.

Macrophages are a population of adher-ent cells with the capacity to metabolizebenzene; cells of this type are included inthe stromal adherent cell layer of LTBMCs(17,18). Macrophages and other adherentcells may also have the capacity to store oraccumulate exogenous or endogenous meta-bolic products for extended periods of time(34). The lack of significant in vivo heminresponse may, in part, be related to theformation of more toxic benzene metabo-lites in vivo. Damage to cells of the adher-ent cell population may thus be morepermanent, as evidenced by a poor recoveryin vitro. In this regard, benzene exposurein vivo produced cell populations thatremained depressed in cellularity and clono-genic potential over the 8-week period ofLTBMC. This is in marked contrast toresults obtained with short-term CFUcultures performed 12 days after benzeneexposure. As seen in Table 1, significantrecovery ofCFU potential/femur cellularity,hematocrit and body weight occurred; theseresults do not bear out the more permanentlong-term effects of benzene. The moreimmediate recovery of CFUs as seen heremay be derived mainly from cells of thenonadherent population in the bone mar-row. Our results from LTBMCs supportthis inference (Figure 2).A direct permanent cytotoxic effect of

benzene on the hemopoietic stroma is

Environmental Health Perspectives * Vol 104, Supplement 6 * December 19961 280

HEMATOPOIETIC EFFECTS OF BENZENE INHALATION

further evidenced by the reduced numberof adherent hematopoietic cells obtained inLTBMCs from animals exposed to benzene.Previous studies suggest that some cells ofthe adherent hematopoietic cell layer ofLTBMCs are actually a subpopulation ofCFU-S with regard to pluripotence andproliferation capacity (35). Thus, a depres-sion in adherent cell numbers by benzeneor its metabolites would result in a perma-nent depression in stem cell numbers, lim-ited capacity to regenerate progenitor cellcompartments on demand, or both. Thiswas seen as a marked depression of CFU

potential by LTBMC for up to 8 weeks inculture. Nevertheless, this extreme toxiceffect does not express itself so severely inthe more immediate population of nonad-herent cells, which are probably in a moreactive phase of the cell cycle.

In conclusion, the strong hematopoieticresidual effects of benzene were demon-strated in LTBMC. Hematopoiesis in cul-ture over 8 weeks was on the threshold level,and little or no progenitor production wasobserved by cells from animals exposed tobenzene. Perhaps this concealed and long-lasting damage of hematopoietic and stromal

progenitors is an important part of the leuke-mogenic activity of benzene. Furthermore,hemin may augment benzene detoxificationand alleviate its cytotoxic effect on thehematopoietic microenvironment.

Irrespective of the mechanisms involvedin the accelerated hematopoietic regenera-tion induced by hemin in mice exposed tobenzene, the use of hemin along or with acombination of growth factors may be ofclinical importance (25). In fact, heminhas a beneficial effect on improvement ofthe anemia seen in patients with myelodys-plasia (30).

REFERENCES

1. Snyder R, Lee E, Kocsis J, Witmer C. Bone marrow depressantand leukemogenic actions of benzene. Life Sci 21:1709-1722(1977).

2. Cronkite EP, Bullis J, Inoue T, Drew R. Benzene inhalationproduces leukemia in mice. Toxicol Appl Pharmacol75:358-361 (1984).

3. Cronkite EP. Chemical leukemogenesis. Benzene as a model.Semin Hematol 24:2-11 (1987).

4. Longacre SL, Kocsis J, Witmer CM, Lee EM, Sammett S,Snyder R. Toxicological and biochemical effects of repeatedadministration of benzene in mice. J Toxicol Environ Health7:223-231 (1981).

5. Rinsky RA. Benzene and leukemia. N Engl J Med 316:1044-1051 (1987).

6. Snyder R, Longacre SL, Witmer CM, Kocsis JJ. Metabolic cor-relates of benzene toxicity. In: Biological ReactiveIntermediates (Snyder R, Parke D, Kocsis J, Jollow J, GibsonC, Witmer C, eds). New York:Plenum Press, 1988;245-256.

7. Rickert D, Baker T, Bus J, Barrow C, Irons R. Benzene disposi-tion in the rat after exposure by inhalation. Toxicol ApplPharmacol 49:417-22 (1979).

8. Andrews LS, Sasame AA, Gillette JR. [3H]-Benzene metabo-lism in rabbit bone marrow. Life Sci 25:567-572 (1979).

9. Abraham NG, Friedland ML, Levere RD. Heme metabolism inhepatic and erythroid cells. Prog Hematol 13:75-130 (1983).

10. Cronkite EP, Inoue T, Carsten AL, Miller ME, Bullis JE, DrewRT. Effects of benzene inhalation on murine pluripotent stemcells. J Toxicol Environ Health 9:411-421 (1981).

11. Green JD, Snyder CA, LoBue J, Goldstein BD, Albert RE.Acute and chronic dose/response effects of inhaled benzene onmultipotential hematopoietic stem (CFU-S) and granulocyte/macrophage progenitor (GM-CFU-c) cells in CD-1 mice.Toxicol Appl Pharmacol 58:492-503 (1981).

12. Abraham NG, Lutton JD, Levere RD. Benzene modulation ofbone marrow hematopoietic and drug metabolizing systems.Biochem Arch 58:492-496 (1985).

13. Seidel HJ, Barthel E, Zinser D. The hematopoietic stem cellcompartments in mice during and after long-term inhalation ofthree doses of benzene. Exp Hematol 17:300-303 (1989).

14. Seidel HJ, Beyvers G, Pape M, Barthel E. The influence ofbenzene on the erythroid cell system in mice. Exp Hematol17:760-764 (1989).

15. Irons RD, Heck Hd'A, Moore BJ, Muirhead KA. Effects ofshort-term benzene administration on bone marrow cell cyclekinetics in the rat. Toxicol Appl Pharmacol 51:399-409(1979).

16. Rosen MG, Snyder CA, Albert RE. Depressions in B and Tlymphocyte mitogen-induced blastogenesis in mice exposed tolow concentrations of benzene. Toxicol Lett 1984;20:343-349.

17. Thomas DJ, Reasor MJ, Wierda D. Macrophage regulation ofmyelopoiesis is altered by exposure to the benzenemetabolite hydroquinone. Toxicol Appl Pharmacol 97:440-53(1989).

18. Post G, Snyder R, Kalf FG. Metabolism of benzene and phenolin macrophage in vitro and the inhibition of RNA synthesis bybenzene metabolites. Cell Biol Toxicol 2:231-246 (1986).

19. Gaido K, Wierda D. In vitro modulation of stromal cell func-tion in DBA/2J and B6CF1 mice exposed to benzene or phe-nol. Toxicol Appl Pharmacol 81:469-475 (1985).

20. Brandt J, Srour EF, Van Beisien K, Briddell RA, Hoffman R.Cytokine-dependent long-term culture of highly enriched pre-cursors of hematopoietic progenitor cells from human bonemarrow. J Clin Invest 86:932-937 (1990).

21. Lutton JD, Abraham NG, Friedland M, Levere RD. The toxiceffects of heavy metals on rat bone marrow in vitro erythro-poiesis: protective role of hemin and zinc. Environ Res35:97-103 (1984).

22. Lutton JD, Levere RD, Abraham NG. Physiologic role of hemeand cytochrome P450 in hematopoietic cells. Proc Soc ExpBiol Med 196:260-269 (1991).

23. Abraham NG, Bucher D, Niranjan U. Microenvironmentaltoxicity of azidothymidine. Partial sparing with hemin. Blood4:139-144 (1989).

24. Chertkov JL, Jiang S, Levere RD, Lutton JD, Abraham NG.Hemin stimulation of hematopoiesis in murine long-term bonemarrow culture. Exp Hematol 19:905-909 (1991).

25. Abraham NG, Levere RD, Lutton JD. Eclectic mechanisms ofheme regulation of hematopoiesis. Int J Cell Cloning9:185-210 (1991).

26. Dexter TM, Allen TD, Lajtha LG. Conditions controlling theproliferation of hemopoietic stem cells in vitro. J Cell Physiol91:335-342 (1991).

27. Harigaya K, Miller ME, Cronkite EP, Drew RT. The detectionof in vivo hematotoxicity of benzene by in vitro liquid bonemarrow culture. Toxicol Appl Pharmacol 60:346-353 (1981).

28. Garnett HM, Cronkite EP, Drew RT. Effect of in vivo expo-sure to benzene on the characteristics of bone marrow adherentcells. Leuk Res 7(6):803-810 (1983).

29. Chertkov JL, Drize NJ, Gurevitch OA, Udalov G.Hemopoietic stromal precursors in long-term culture of bonemarrow. Exp Hematol 11:231-237 (1984).

30. Volin L, Ruutu T, Knuntila S. Heme arginate treatment formyelodysplastic syndromes. Leuk Res 12:423-429 (1988).

31. Chertkov JL, Gurevitch OA. Hematopoietic Stem Cell and ItsMicroenvironment. Moscow:Meditsina, 1984;1-237.

32. Marsh JCW, Chang J, Testa NG, Hows JM, Dexter TM. Thehematopoietic defect in aplastic anemia assessed by long-termmarrow culture. Blood 76:1748-5317 (1990).

Environmental Health Perspectives * Vol 104, Supplement 6 * December 1996 1281

N.G. ABRAHAM

33. Hotta T, Kato T, Maeda H, Yamao H, Yamada H, Saito H.Functional changes in marrow stromal cells in aplastic anemia.Acta Haematol 74:65-71 (1985).

34. VanFurth R, ed. Mononuclear Phagocytes. The Hague:Nijhoff.

35. Reincke U, Rosenblatt M, Hellman S. Adherent stem cells:Frequency in mouse marrow and terminal clone sizes in long-term culture. Exp Hematol 13:545-551 (1985).

1282 Environmental Health Perspectives * Vol 104, Supplement 6 * December 1996