The Effects of Benzene Inhalation on Murine Hematopoietic Precursor Cells (CFU-e, BFU-e and CFU-gm)

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1 International Journal of Cell Cloning 1: (1983) The Effects of Benzene Inhalation on Murine Hematopoietic Precursor Cells (CFU-e, BFU-e and CFU-gm) Richard L. Hilderbran&, Martin J. Murphy, Jr.b Waval Medical Research Institute/Toxicology Detachment, Wright- Patterson Air Force Base; bthe Bob Hipple Laboratory for Cancer Research, Wright State University School of Medicine, Dayton, OH, USA Key Words. Benzene. Hematopoiesis * Toxicity CFU-e BFU-e - Inhalation Abstract. Male B6D2F1 mice were exposed by inhalation to 4OOO ppm of benzene for 8 h/day for 1, 2, 3, 5, 7 or 14 days and then sacrificed at 18 h following the last exposure. The cellularities of both femur and spleen were depressed for the duration of benzene exposure. BFU-e/femur declined for 3 days, rebounded to control levels by day 5, and were again depressed by day 7. CFU-e/femur were initially depressed, but by day 7, the concentrations of CFU-e were so elevated that the total number had actually rebounded to slightly higher than control Values. CFU-gm/femur remained depressed for the duration of the exposure periods. CFU-e, BFU-e and CFU-gm/spleen were depressed following all exposures. The toxic effects of benzene on hematopoiesis that are immediate are not ascribable to migration of stem cells between femur and spleen. The depressive effects of benzene inhalation on erythropoiesis may be compensated by the rapid proliferation of CFU-e. Introduction Benzene is an aromatic solvent that is widely used in chemical and industrial processes. While the hematotoxicity of benzene has been intensively investigated for many years, the mechanism producing the toxicity has not been identified. The predominant effect of chronic exposure to benzene is the production of pancytopenia, although shorter-lived peripheral blood cells, e.g., granulocytic leukocytes, reflect the hematopoietic "1983 AlphaMed Press, Inc /83/$2.00/0

2 Effects of Benzene on Hematopoiesis 24 1 disturbances more rapidly than do longer-lived peripheral blood cells, e.g., erythrocytes [ 11. Recent advances in clonal cell culture techniques allow the assessment of the functional integrity of murine and human hematopoietic precursor cells. The two major erythropoietic precursors recognizable by the cell culture technique are the colony forming units-erythroid (CFU-e) and the burst forming units-erythroid (BFU-e) [2, 31. The colony forming units-granulocytic/macrophagic (CFU-gm) represent committed precursors to the granulocytic and macrophagic cell lines and are also readily identified by cell culture techniques [4]. The CFU-e are late-stage precursors to erythrocytes and require the hormone erythropoietin (Ep) for both in vivo and in vitro development [5]. The BFU-e are early-stage erythroid precursors and are relatively insensitive to Ep [6]. Lee et al. [7] studied the appearance of 59Fe-labeled circulating erythrocytes and concluded that single doses of benzene selectively damaged pronormoblasts and normoblasts without affecting stem cells. In contrast, Uyeki et al. [8], investigating hematopoiesis in female BDFl mice exposed to benzene vapors (4680 ppm), found that following a single 8-hour exposure, CFU-gm/femur were significantly depressed without a decline in femoral cellularity. After 3 exposures of 8 h duration and 1 exposure of 4 h, CFU-gm/femur, marrow multipotential hematopoietic stem cells (CFU-s) and cellularity were all reduced. Earlier, Speck and Kissling had reported that repeated subcutaneous injections of benzene had no effect on marrow CFU-s [9]. Gill et al. [lo] found that exposure of male C57B1/6 mice to 4000 ppm of benzene on an intermittent schedule produced a rapid decrease in peripheral blood granulocytes and lymphocytes and in marrow CFU-s despite the presence of normal marrow cellularity. Following continuous exposure at lower levels of benzene, both peripheral cell counts and marrow cellularity were reduced. Exposure of nondividing CFU-s to benzene for 24 h produced no detectable effect.on the subsequent development of spleen colonies, indicating that peripheral cells must be depleted before CFU-s are affected. They concluded that benzene affects both undifferentiated and differentiated stem cells and that benzene toxicity to CFU-s is significant but is not the exclusive mechanism of action. Green et al. [ 1 I] exposed male CDI mice to a series of concentrations of benzene vapors (from 1.1 to 4862 ppm) for 6 h/day for 5 days. They found splenic and femoral cellularities and CFU-s/organ to be reduced at

3 Hilderbrandl Murphy 242 all concentrations above 103 ppm. CFU-gm/ 105 femoral nucleated cells tended to increase while the CFU-gm/ 105 splenic nucleated cells, CFU-gm/ spleen, and CFU-gmlfemur decreased in animals exposed to benzene above 103 ppm. Although the CFU-gm/l05 marrow cells were increased, the decrease in cellularity accounted for a reduction in CFU-gm/femur. Thus, the pluripotent stem cell and CFU-gm are affected by short-term exposure to benzene at concentrations of 103 ppm and above [ This work was initiated to further investigate the responses of hematopoietic tissues to short-term benzene exposure. CFU-e, BFU-e and CFU-gm cultures were selected as assays indicative of the function of two hematopoietic cell populations that were sensitive to benzene, thus demonstrating several rapidly occurring myeloid responses to this benzene exposure. Materials and Methods Animals Male B~D~FI mice (C57B1/6J X DBA, Jackson Laboratories, Bar Harbor, ME) weighing from g were used in all studies. Mice were kept in plastic cages and given Purina Chow (5008, Ralston Purina, St. Louis, MO) and water ad libitum. During inhalation exposures neither food nor water was available. In halation Exposure Mice were exposed to benzene (ACS, analytical reagent, MAL, American Scientific Products, Obetz, OH) by inhalation for 1, 2, 3, 5, 7 and 14 days. The time-weighted average concentration for all the 8 h exposures, based on hourly samplings for all experiments, was 4062 ppm (SD = 203 PPm)* The exposures were started at 7:30 a.m. and terminated at 3:30 p.m.; tissue for assays was collected the morning following termination of the exposure for the appropriate number of days. Sham-treated control animals were held in a chamber to which air but no benzene was introduced. The dynamic exposures were carried out in a 31-liter inhalation chamber as described by Leach [12]. A reciprocating piston pump provided an air flow of about 6 literdmin. which was divided into two flow controlled streams, One stream at a low flow was bubbled through benzene in a gas-washing bottle. Droplets were filtered out by glass wool and

4 Effects of Benzene on Hematopoiesis 243 the air saturated with benzene vapor was diluted with the second air stream and introduced to the chamber. The ratio of flow was controlled to give the desired benzene concentration in the air entering the chamber. A second reciprocating pump with a flow control was used to adjust outlet air flow to maintain a negative pressure of 2-3 in of H20 in the chamber. The concentration of benzene in the outlet air was monitored by a Hewlett Packard 5830A Gas Chromatograph equipped with a flame ionization detector at 300 C and a 6 X % stainless steel column packed with 10% OV-17 on mesh Chromasorb W-HP. The column was maintained at 100 C and N:! at 32 ml/min was used as a carrier gas. Injections of standards or test samples were made by a manually operated external gas sample loop into which the sample was pulled by a vacuum pump. The instrument was standardized by external methods using benzene vaporized in an air volume measured by a wet test meter into a 10 liter teflon gas sampling bag. A sample of the outlet air from the exposure chamber was collected every hour in a teflon gas sampling bag, the concentration determined, and necessary chamber airflow adjustments made. Cell Culture A treated or control mouse was killed by cervical dislocation and the body and hind limbs were rinsed with 70% ethanol. The femurs were excised using aseptic technique and gently scraped free of muscle while in 1 x a medium (GIBCO Laboratories, Grand Island, NY). The ends of the femur were clipped off and the marrow was carefully flushed out with a 22-gauge needle on a 1 cc sterile syringe using 1 ml of 1 X CY medium. The cells were collected in a Petri dish and transferred to a capped 17 x 100 mm sterile polyethylene culture tube (Fisher Scientific, Cincinnati, OH). The femurs from two more mice were treated sequentially in the same manner and the cells were combined in a total volume of approximately 6 ml. The final volume of this single cell suspension was measured and cells were counted to derive the total number of cells/femur and a dilution was made to provide 2 x 105 cells/ml of medium per culture plate. Cell counts were made using an automated hematology analyzer (Ultra Logic 800, Clay-Adams, Parsippany, NJ) calibrated for mouse cells. Spleens These mice were treated as above except that the spleens were excised aseptically, trimmed of fat, cut into halves and placed in 2 ml of 1 x a

5 Hilderbrand/Murphy 244 medium. Forceps were used to squeeze cells from the halves, leaving behind a translucent splenic capsule. The spleen cells were passed through a 22-gauge needle to give a single cell suspension and were transferred to a 17 X 100 mm sterile capped polyethylene tube. Again the cells from three animals were combined, the total volume measured and nucleated cells counted. A dilution to 1 X lo7 cells/ml of 1 X a medium was made for cell culture, yielding a final concentration of 106 cells/plate in a total volume of 1 ml. CFU-e and BFU-e Assay Mouse erythroid progenitor cell assays were carried out in methylcellulose [5, 131. To 0.1 ml of a single cell suspension was added 0.8% methylcellulose (Method A4M, Dow Chemical Co., Midland, MI), 1% deionized bovine serum albumin (Calbiochem, San Diego, CA), 30% heat-inactivated fetal calf serum (Lot # , Flow Laboratories, Inc., Rockville, MD), 1.0 unit of step I11 preparation of sheep plasma erythropoietin (Lot #3042-1, Connaught Laboratories, Ltd., Willowdale, Ontario, Canada), supplemented with 1 x a! medium to 5 ml total volume. To the culture medium was added 1 x 10-4 M 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO). To assay murine erythroid precursor cells, 1 ml of this mixture was plated in each of quadruplicate 35 mm Lux standard nontissue culture dishes (Lux Scientific, Newbury Park, CA) and cultured for 2 days for the CFU-e assay and 9 days for the BFU-e assay at 37 C in a humid atmosphere of 5% COz in air. CFU-e colonies were scored at x 100 magnification with an inverted microscope (Wild, M40, Heerbrugg, Switzerland), and BFU-e colonies were enumerated at X 40 magnification. The results were expressed as the number of clones/:! x 105 cells which were then converted to the total number of committed progenitors on a per organ basis. CFU-gm Assay Mouse CFU-gm colonies were assayed in methylcellulose by a modification of the technique described by Worton et al. [4]. For the assay, 0.4 ml of a single cell suspension was mixed with 0.8% methylcellulose, 2% deionized bovine serum albumin, 2 X 10-4 M 2-mercaptoethanol, 10% fetal calf serum and 20% GCT conditioned medium (GIBCO) supplemented with 1 X a medium to 5.0 ml total volume. One ml of the mouse bone marrow cell 'mixture was plated in 35 mm Lux standard nontissue culture dishes and cultured for 9 days at 37 C in a humid atmosphere

6 Effects of Benzene on Hematopoiesis 245 flushed with 5% COz in air. Colonies were counted using an inverted microscope (Olympus Optical Co., Tokyo, Japan) at x 40 magnification. Colonies containing more than 50 cells were scored and these data were displayed graphically as colonies/ 1 X 106 cells plated. Controls and Statistics Control animals held in a chamber for daily matched treatments were found to maintain normal proliferation of the hematopoietic precursor cells being investigated. Therefore, the control values represent the combined data from untreated animals taken at various times throughout the experimental period and are designated as 0 day values. The raw data were derived for each clonal assay by counting quadruplicate plates of pooled cells obtained from marrow or spleen cells from three animals. Repetitions included another set of four plates of pooled cells from three animals. Statistical analysis was performed using analysis of variance procedures with a nested design with groups (i.e., replication) nested within each day to examine the day s effect. When this effect was significant, individual days were compared to the control day using Dunnett s t statistic [ 141. The mean differences were tested using the error term for the group within each day. Significance of the t test was determined using conservative criteria of the Dunn-Bonferroni procedure based upon 4 df. Results Weights Over a 6-day period, the average weight of 10 matched control animals increased from g (SE = 0.50) to g (SE = 0.62) while the average weight of 10 animals inhaling benzene vapor decreased from g (SE = 0.18) to g (SE = 0.52). The weight changes progressed evenly over the 6-day time period. Average splenic weights from 5 matched control animals remained constant at 87 mg over a 7-day period. The splenic weights of treated mice declined rapidly (but steadily) to an average of 22 mg following 7 days of exposure. In a 7-day recovery period following the exposure, those spleens increased in weight over five-fold to 125 mg/spleen and then returned to approximately that of controls by 14 days following termination of exposure.

7 Hilderbrand/Murphy 246 Fig. 1. The number of nucleated cells/femur. Each point represents the number of cells in the pooled cell preparation from six femurs and is the average of the repetitions done at each time period. Fig. 2. Erythrocytic burst forming units (BFU-e)/femur. The values marked by an asterisk are significantly different from controls (p ); mean f SE.

8 Effects of Benzene on Hematopoiesis 247 Cell Culture The number of clones of each hematopoietic type cultured was calculated on an organ basis which reflected two parameters: the cellularity of either the spleen or marrow and the cloning efficiency (number of clones produced/number of cells plated) of the particular hematopoietic precursor. Femur. Upon initiation of exposure the total number of nucleated cells in bone marrow (Fig. 1) dropped rapidly in comparison to the controls. Following 3 days of exposure, recovery occurred to about 65% of control levels, followed by a plateau in the number of cells. Values for BFU-e/femur are shown in Figure 2. An initial depression in proliferative capability is observed, which is followed by a transient high proliferative capability at day 5, and then again by depressed levels of BFU-e/femur. In contrast, the proliferative capability of femoral CFU-e is immediately depressed (Figs. 3 and 4). By day 7 the CFU-e/femur are back to control levels and remain there even following 14 days of exposure. The rebound in the number of CFU-e/femur is accounted for by an actual increase in cloning efficiency (CFU-e/2 x 105 cells), as shown in Figure 3, that compensates for the depression in marrow cellularity (Fig. 1). Both BFU-e/ femur and CFU-e/femur from treated animals are significantly different from the controls (Figures 2 and 4). The CFU-gmlfemur decreased as shown in Figure 5. Spleen. The results of the spleen cultures are shown in Figure 6. On a per spleen basis, the cellularity and cloning efficiency of all hematopoietic progenitor cells examined dropped precipitously over 7 days of exposure (spleens were not taken at 14 days of exposure). This corresponds well with the observed weights of the spleens, since following the 7-day exposure the spleen consisted of a translucent capsule and relatively few cells. Recovery. In 7-, 14- and 21-day recovery periods following a 7-day exposure, CFU-e, BFU-e and CFU-gm and marrow and splenic cellularity returned to untreated control values (data not shown) based on one group of three animals each taken on day 14,21 and 28 of the experiment. Discussion In adult mice, the spleen continues to be an active hematopoietic organ; however, in the adult human, marrow is primarily responsible for hematopoiesis. In mice a physiological perturbation such as hypoxia or preg-

9 Hilderbrandl Murphy 248 Fig. 3. Erythrocytic colony forming units (CFU-e)/2 x 105 femoral cells. The values marked by an asterisk are significantly different from controls (p ); mean f SE. Fig. 4. Erythrocytic colony forming units (CFU-e)/femur. The values marked by an asterisk are significantly different from controls (p ); mean f SE.

10 Effects of Benzene on Hematopoiesis 249 Fig. 5. Granulocytic macrophagic colony forming units (CFU-gm/femur. The values marked by an asterisk are significantly different from controls (p 50.05); mean f SE. Fig. 6. The splenic response to benzene inhalation. The ordinate refers to: o = nucleated cells (X 1O7)/spleen; 0 = CFU-gm ( X 103)/spleen; + = CFU-e (x 103)/spleen; x = BFU-e ( x 103)/spleen. Mean * SE.

11 Hilderbrand/Murphy 250 nancy results in a depression of myeloid erythropoiesis while active splenic erythropoiesis continues or increases [ 151. The inhalation of benzene at high levels produces a nonphysiological perturbation of erythropoiesis and results in an essential loss of function in the spleen and a very depressed marrow function. The depressed marrow function following inhalation of benzene may result from a decrease in proliferative capability and/or a decrease in organ cellularity. BFU-e and CFU-e were initially depressed and had increased proliferative capability at 5 days of exposure. At that time, the BFU-e apparently failed to maintain increased proliferation, although the CFU-e continued to demonstrate increased proliferative capability even after a 14-day inhalation regimen. The ability of CFU-e to proliferate independently of earlier precursors in the erythroid series and to provide precursors for maturation has been demonstrated earlier [6, 161. In this case, the CFU-e compartment was apparently capable of maintaining a fairly normal output of erythroid precursors despite the hematopoietic toxicity of benzene. Whether this is a true compensation remains to be proven, since the susceptibility of more mature erythroid cell populations to benzene is not known. The possibility exists that pronormoblasts or normoblasts are sensitive to benzene. These data are in basic accord with the previous data of Uyeki et al. [8] and Green et al. [ 111. Although we did not investigate relative toxicity of benzene to pluripotent hematopoietic stem cells, the committed precursors studied were clearly sensitive. The differential sensitivity of particular precursors is evident, especially in comparing femoral CFU-e and BFU-e proliferative capability at 7 and 14 days. In the case of CFU-gm following exposure for 5 days to 4860 ppm, Green et al. [l 11 reported a normal or increased cloning efficiency with a decrease of CFU-gm/femur due to reduction in organ cellularity. Uyeki et al. [8] reported both a decrease in femoral cellularity and cloning efficiency of CFU-gm for an overall decrease of 87% in CFU-gm/femur following 3.5 x 8 h of benzene inhalation. Our data fell between these results with a moderate decrease in cloning efficiency and a marked decline in femoral cellularity, which results in a significant decrease in CFU-gm/femur. Migratory streaming is the facile movement of proliferative cells from the marrow to the spleen, or vice versa, and has been found to occur under a variety of conditions which place stress on hematopoietic tissue [ 171. In this work, the decrease in the proliferative capability of the femur alone is not necessarily indicative of a toxic effect of benzene on the hematopoietic system. However, the concomitant decrease in proliferative capability of

12 Effects of Benzene on Hematopoiesis 25 1 both the spleen and the marrow eliminates migratory streaming as the cause of the decline in proliferative capability. This indicates that toxicity accounts for the observed decline in proliferative capability. Previous work has documented that a metabolite of benzene is responsible for the toxicity, but the actual metabolite or mechanism has not been unequivocally identified [ 181. Recent interest has focused on a potential metabolite of benzene, trans, trans-muconaldehyde [ 191, which, along with polyphenols, has been identified as a primary product of the opening of a benzene ring following irradiation of an aqueous solution of benzene in the presence of oxygen [20]. This ring opening is not enzymatically catalyzed. Recent work has shown that benzene treatment of rabbits induces CFU-e and BFU-e inhibiting activity in adherent mononuclear peripheral blood cells [21]. Thus, it is apparent that new directions are being taken in addressing the mechanisms of benzene toxicity. The method of dosing animals with a volatile hydrocarbon such as benzene is recognized as having significant effects on toxicity, especially when the toxic effect is due to a metabolite of the material administered. In this work, the inhalation of a high concentration of benzene probably does not produce significantly greater toxicity than inhalation of a much lower dose, as was observed by Green et al. [ 111. Recent kinetic work has shown that physiological factors may limit metabolism of inhaled vapors at low concentrations [22]; however, at high concentrations, metabolism will be saturated [23]. An increase in concentrations, beyond certain levels, of inhaled vapor will not necessarily produce increased quantities of toxic metabolite. Thus, if enzymatic metabolism is responsible for producing a toxic metabolite of benzene, future studies of the toxicity of inhaled benzene can focus on much lower ambient concentrations of vapor. Doses administered subcutaneously are difficult to evaluate because of first-pass metabolism, partition coefficients and exhalation of vapors. In summary, the effects of inhaled benzene on hematopoietic precursor cells in male B~D~FI mice are rapid. The BFU-e and CFU-gm are severely affected and show little ability to compensate for the toxic effects of benzene. CFU-e have an apparent ability to compensate, but the physiological significance of this proliferative capability is not known, The spleen is more susceptibie than the marrow to the toxic effects, which discounts the possibility that changes in proliferative capability can be attributed to migration of stem cells between femur and spleen.

13 Hilderbrand / M urphy 252 Acknowledgments This work was supported by the Naval Medical Research and Development Command, Research Task No. MR and the Bob Hipple Memorial Committee for Cancer Research. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the Naval Service at large. The experiments reported herein were conducted according to principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Resources, National Research Council, DHEW Publication No Portions of this work were presented at the Twenty-first Annual Meeting of the Society of Toxicology, Boston, MA, February, We would like to thank Doctors Kohsaki, Sloman and Pitts for helpful discussions and assistance in this work. Thanks also are due to HM3 Walt, HM3 Kisner, and Mrs. Jenny Kwak for their excellent technical assistance. Dr. Ross Vickers, Jr. and Mrs. Linda Hervig from the Naval Health Research Center, San Diego, generously donated time and assistance in performing the statistical analyses. References Leong, B. : Experimental benzene intoxication; in Laskin, Goldstein, Benzene Toxicity, pp (McGraw-Hill, New York 1977). Stephenson, J.R.; Axelrad, A.A.; McLeod, D.L.; Shreeve, M.M.: Induction of hemoglobin synthesizing cells by erythropoietin in vitro. Proc Natl Acad Sci 68: (1971). Axelrad, A.A.; McLeod, D.L.; Shreeve, M.M.; Heath, D.: Properties of cells that produce erythrocytic colonies in vitro, in Robinson, Hemopoiesis in Culture, pp (U. S. Government Printing Office, Washington 1974). Worton, R.G.; McCulloch, E.A.; Till, J.E.: Physical separation of hemopoietic stem cells from cells forming colonies in culture. J Cell Physiol 74: (1969). Iscove, N.N.; Sieber, F.; Winterhalter, K.H.: Erythroid colony formation in cultures of mouse and human bone marrow: Analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agaroseconcanavalin A. J Cell Physio18.3: (1974). Iscove, N.N.: The role of erythropoietin in regulation of population size and cell cycling at early and late erythroid precursors in mouse bone marrow. Cell Tissue Kinet 10: (1977). Lee, E.W.; Kocsis, J.J.; Snyder, R.: Acute effect of benzene on 59Fe incorporation into circulating erythrocytes. Toxicol Appl Pharmacol 27: (1974). Uyeki, E.M.; Ashkar, A.E.; Shoeman, D.W.; Bisel, T.U.: Acute toxicity of benzene inhalation to hemopoietic precursor cells. Toxicol Appl Pharmacol 40: ( 1977).

14 Effects of Benzene on Hematopoiesis Speck, M.; Kissling, M.: A comparative toxicological evaluation of benzene, toluene, and xylene; in the 13th International Congress of Hematology, Abstract, p. 134 (1971). Gill, D.P.; Jenkins, V.K.; Kempen, R.R.; Ellis, S.: The importance of pluripotential stem cells in benzene toxicity. Toxicology 26: (1980). Green, J.D.; Snyder, C.A.; LoBue, J.; Goldstein, B.D.; Albert, R.E.: Acute and chronic dosehesponse effects of inhaled benzene on multipotential hematopoietic stem (CFU-S) and granulocytelmacrophage progenitor (GM- CFU-C) cells in CD- 1 mice. Toxicol Appl Pharmacol58: ( 198 1). Leach, L.J.: A laboratory test chamber for studying airborne materials. AEC Research and Development Report UR-629, pp (1963). DiPersio, J.F.; Brennan, J.K.; Lichtman, M.A.; Speiser, B.L.: Human cell lines that elaborate colony-stimulating activity for the marrow cells of man and other species. Blood 51: (1978). Winer, B.J.: Statistical Principles in Experimental Design, pp (McGraw-Hill, New York 1962). Lord, B.I.; Murphy, M.J., Jr.: The response of hemopoietic stem cells to hypoxia (in press). Iscove, N.N.; Guilbert, L.J.: Erythropoietin-independence of early erythropoiesis and a two-regulator model of proliferative control in the hemopoietic system, in Murphy, In Vitro Aspects of Erythropoiesis, pp. 3-7 (Springer- Verlag, New York 1978). Murphy, M.J., Jr.; Lord, B.I.: Hematopoietic stem cell regulation I. Acute effects of hypoxic-hypoxia on CFU kinetics. Blood 42: (1973). Snyder, R.; Andrews, L.; Lee, E.W.; Witmer, C. Longacre, S.; Kocsis, J.J.: Biochemical toxicology of benzene; in Hodgson, Bend, Philpot, Reviews in Biochemical Toxicology, pp (ElseviedNorth Holland, Amsterdam 1981). Rao, G.S.; Witz, G.; Goldstein, B.D.: The reactivity of trans, trans-muconaldehyde, a possible toxic metabolite of benzene towards glutathione. The Toxicologist 2: 430 (1982). Loeff, I.; Stein, G.: Aromatic ring opening in the presence of oxygen in irradiated solutions. Nature 184: 901 (1959). Haak, H.L.; Speck, B.: Inhibition of CFU-e and BFU-e by mononuclear peripheral blood cells during chronic benzene treatment in rabbits. Acta Haemat (1982). Andersen, M.E.: A physiologically based toxicokinetic description of the metabolism of inhaled gases and vapors: Analysis at steady state. Toxicol Appl Pharmacol60: (1981). Hilderbrand, R.L.; Andersen, M.E.; Jenkins, L.J., Jr.: Prediction of in vivo kinetic constants for metabolism of inhaled vapors from kinetic constants measured in vitro. Fundam Appl Toxicol 1: (1981). Accepted: May 17, 1983 Dr. Richard L. Hilderbrand, Naval Bioscience Laboratory, NSC, Oakland, CA (USA)