Document JkVVnL1z7xz8rbOZo8qRyRVv

Environmental Mutagenesis 8:29-40 (1986) Induction of Cytogenetic Damage in Rodents After Short-Term Inhalation of Benzene Gregory L. Erexson, James L. Wilmer, William H. Steinhagen, and Andrew D. Kligerman Chemical Industry lnstitute of Toxicology, Department of Genetic Toxicology,Research Triangle park, North Carolina Experiments were designed to investigate both the induction of sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs) and micronuclei (MN) in bone marrow polychromatic erythrocytes (PCEs) of mice and rats after inhalation of benzene (BZ). Male DBA/2 mice (17-19 weeks old) were exposed to target concentrations of either 0, 10, 100, or 1,OOO ppm BZ for 6 hr. Male SpragueDawley rats (11-14 weeks old) were exposed to target concentrations of either 0, 0.1,0.3, 1, 3, 10, or 30 ppm BZ for 6 hr. Blood was obtained by cardiac puncture 18hr after exposure, and PBLs were cultured in the presence of lipopolysaccharide Y (mouse B cells, 60 pg/ml) or concanavalin A (rat T cells, 30 pg/ml) to stimulate blastogenesis for SCE analysis. Femoral bone marrow smears from both species were analyzed for MN in PCEs 18 hr after BZ exposure. Mouse PBLs revealed a significant concentration-related increase in the SCE frequency over controls at 10, 100, or 1,OOO ppm BZ. Mouse bone marrow showed a significant concentration-dependent increase in MN over controls after exposure to 10, 100, or 1,OOO ppm BZ. Rat PBLs showed a significant increase in the SCE frequency after exposure to 3, 10, or 30 ppm BZ. The statistical significance of the 1 ppm BZ result was borderline and dependent on the statistical test chosen. Rat cells revealed a significant concentration-related increase in MN after inhalation of either 1, 3, 10, or 30 ppm BZ. PBLs from treated mice showed significant concentration-dependentdecreases in mitotic indices; however, cell cycle kinetics and leucocyte counts remained unaffected. Rat PBLs showed significant decreases in mitotic activity only after exposure to 3 and 30 ppm BZ,whereas cell cycle kinetics and leucocyte counts were unaffected. These results show that BZ can induce statistically significant cytogenetic effects in PBLs and PCEs of both mice and rats after a 6-hr inhalation of BZ at low concentrations. Received June 7, 1985; revised and accepted August 30, 1985 Gregory L. Erexson and Andrew D. Kligermanare now at Environmental Health Research and Testing, Inc., P.O. Box 12199, Research Triangle Park, NC 27709. James L.Wilrner is now at the Pathology and Toxicology Department, Bristol Laboratories, P.O. Box 4755, Syracuse, NY 13221. Address reprint requests to Gregory L. Erexson, Environmental Health Research and Testing, Inc., P.O. Box 12199, Research Triangle Park, NC 27709. 0 Dl36 Alan R. Lk,lnc. 30 Erexsonetal Key words: benzene, rodents, sister chromatid excbange, micronucleus, bone marrow, peripheral blood lymphocytes INTRODUCTION Benzene 032) has been implicated as a human leukemogen [Goldstein, 19771. Long-term bioassays using rats or mice exposed to BZ either by inhalation or gavage have resulted in the induction of tumors. Snyder et al [19801saw a significant increase in the frequency of hematopoietic neoplasms including thymic lymphoma in C57B1/6 mice inhaling 300 ppm BZ. Goldstein et a1 [1982] found an increased incidence of leukemias in both Sprague-Dawley rats and CD-1 mice inhaling 100 or 300 ppm BZ. The finding of myelogenous leukemia in the strains of rodents tested suggests that BZ might have played a role in the induction of leukemia, although the increase was not statistically significant [Goldstein et al, 19821. Maltoni et a1 [19831reported significant increases of Zymbal's gland carcinoma, carcinoma of the oral cavity, hepatocarcinoma, and lymphoreticular neuplasias in Sprague-Dawley rats after inhalation of BZ. Cronkite et al [1984] observed a significant increase in the frequency of lymphoma in female C57B1/6 mice inhaling 300 ppm BZ for 16 weeks. The National Toxicology Program [1984] has reported that B6C3fl mice exposed to BZ by gavage showed significant increases of Zymbal's gland carcinoma, lymphoma, alveolarhronchiolar carcinoma, and alveolarhronchiolar adenomas. In addition, the same study reported that Fischer-344N rats exhibited increased frequencies of Zymbal's gland carcinoma, squamous cell papillomas and squamous cell carcinomas of the skin, and squamous cell papillomas and squamous cell carcinomas of the oral cavity. Epidemiological studies have demonstrated that BZ is clastogenic in peripheral blood lymphocytes of occupationally exposed workers [eg, Tough et al, 1970; Forni et al, 1971; Rinsky et al, 19811. In addition, there is limited information regarding the immunotoxic paarson et al, 1984; Rozen et al, 19841and chromosome-damaging [Cortina et al, 1982; Tice et al, 1982; Styles and Richardson, 19841 effects of inhaled BZ on the rodent hematopoietic system at concentrations relevant to human exposure. DBA/2 male mice were used in the present study because this strain and sex are reported to be sensitive to BZ-induced genotoxicity and cytotoxicity [Tice et al, 1982; Snyder et al, 19831. B lymphocytes were chosen because they are sensitive indicators of BZ-induced immunotoxicity prons and Moore, 1980; Rozen et al, 19841, and polychromatic erythrocytes (PCEs) were selectedbecause they are sensitive indicators of BZ-induced genotoxicity [Hite et al, 1980; Meyne and Legator, 1980; Siou et al, 1982; Toft et al, 19821. To investigate species and cell-type susceptibility to the effects of BZ, similar studies were done with peripheral blood T lymphocytes and bone marrow PCEs of male Sprague-Dawley rats. Preliminary experimental data showed increased cytogenetic damage after exposure to nominal concentrations of 1 and 3 ppm BZ. Therefore, a second and more definitive experiment was conducted in rats in which concentrations of 0.1, 0.3, 1, and 3 ppm BZ were monitored analytically to confirm the previous findings and accurately define the shape of the dose-response curve at low doses of BZ. h - Cytogenetic Damage in Rodents Inhaling Benzene 31 MATERIALS AND METHODS Animals Eight- to ten-week-old male DBA/2 mice and Sprague-Dawley rats were obtained from Charles River Breeding Laboratories (Kingston, NY) and placed in quarantine for 2 weeks. Sprague-Dawley rats are commonly used for bioassays [Goldstein et al, 1982; Maltoni et al, 19831and are desirable for the rat micronucleus (MN) test because of the relatively low numbers of mast cells present in their bone marrow [Gollapudi et al, 19831. The housing conditions and diet were as previously described [Erexson et al, 1983; Kligerman et al, 19831. In the first experiment, male DBAf mice (17-19 weeks old) were exposed to atmospheres of 10, 100, or 1,OOO ppm BZ for 6 hr in groups of five mice per chamber. In a second experiment, male Sprague-Dawley rats (11-14 weeks old) were exposed to atmospheres of 1, 3, 10, or 30 ppm BZ for 6 hr in groups of five rats per chamber. &cause of initial problems in measuring the 1 and 3 ppm exposures analytically, a third experiment was done to confirm the positive cytogenetic effects seen at these concentrations. In addition, 0.1 and 0.3 ppm BZ concentrations were tested. Mice (three or four per cage) and rats (five per cage) were exposed concurrently with each benzene exposure concentration to chamber room air to serve as controls. Temperature (23.5 f 0.3"C) and humidity (52.5 f 5.6%) were monitored each hour during exposures. Benzene Exposures Food and water were not given to any of the animals during the 6-hr exposures. I Animals were exposed in a 102-liter glass and Teflon chamber parrow and Stein- hagen, 19821 with a 20-liter/min air flow. Generation of all concentrations except 1,OOO ppm was done by metering a BZ-nitrogen mixture (Matheson Compressed Gas, Morrow, GA) from a cylinder to the chamber air supply inlet. Generation of the 1,OOO ppm concentration was done by metering liquid BZ with an FMI pump (Fluid Metering Inc., Oyster Bay, NY) at 0.072 ml/min into a heated flask. Chambers were analyzed hourly by continuous monitoring with a Miran 1A infrared gas analyzer (Foxboro Analytical, S. Norwalk, CT) for the 10, 30, 100, and 1,OOO ppm BZ concentrations. The 0.1,0.3, 1, and 3 ppm BZ exposures were analyzed two or three times per hour with a model GC-8APF gas chromatograph (Shimadzu Scientific Instruments, Columbia, MD) equipped with a flame ionization detector. All BZ mixtures were analyzed and certified by Matheson. Lymphocyte Culture and Slide Preparation Lymphocyte culture methodology and slide preparation for sister chromatid exchange (SCE) analysis were as previously described [Kligerman et al, 1982; Erexson et al, 1983; Wilmer et al, 19831. Briefly, blood was removed by cardiac puncture from anesthetized animals 18 hr after exposure to BZ, and lymphocytes were cultured in the presence of either 60 pg lipopolysaccharide/ml (mouse B cells) or 30 pg concanavalin A/ml (rat T cells) to stimulate blastogenesis. Peripheral blood leucocyte counts were determined from a 20-4 aliquot of washed blood with a Coulter counter as previously described [Kligerman et al, 19821. 5-Bromo-2'-deoxyuridine (mice, 2 pM;rats, 4 pM) was added at 24 hr after culture initiation, and the cultures were harvested at either 52 hr (rats) or 60 hr (mice) following a 4-hr demecolcine (1.35 pM) treatment. 32 Erexsonetal Preparation and staining of slides of femoral bone marrow were a modification of the technique described by Schmid [1976]. The same animals (18 hr after BZ exposure) that were used for the SCE studies were used for the MN analysis. MayGrunwald and Giemsa stains (Bio/medical Specialties, Santa Monica, CA) were used .for mouse bone marrow smears. Acridine orange (Polysciences Inc , Warrington, PA) was used to stain rat bone marrow because there is a potential problem of artifacts caused by scoring mast cell granule inclusions as MN when using the classical Giemsa staining technique [Hayashi et al, 19831. Slide Analysis Two or three slides were prepared per animal for the SCE experiments. Slides from five treated and three to five concurrent control animals were coded,combined, and randomized prior to cytogenetic analysis. Each exposure concentration and its concurrent control were analyzed separately. For the MN experiments, four slides were prepared per animal. Slides were coded, combined, and randomized as described for the SCE experiments. One to four bone marrow smears were analyzed per animal for MN. The same scorer quantitated the frequencies of SCE and MN to alleviate possible scorer-to-scorer variability. Only second-division metaphases containing the diploid number of chromosomes were used for the SCE analyses. Twentyfive seconddivision metaphases and 1,000-2,000 PCEs were analyzed from each animal for SCE and MN frequencies, respectively, unless noted otherwise. One thousand nuclei and 100 metaphases were scored consecutively for mitotic index and cell cycle kinetics, respectively. Statistical Analysis Concurrent control values for the MN frequencies were compared to those of the BZ-exposed groups using Student's t test (one-tailed) [Snedecor and Cochran, 19671. Customarily, the same procedure would be applied to the animal-to-animal mean SCE frequencies. However, analysis of the SCE data revealed that the animalto-animal variation was consistently less than expected if cell-to-cell variation within animals were the only source of variability. At the present time there is no clear explanation for this unexpected result. Similar observations, however, have also been seen in other data sets from our laboratory. Accordingly, both cell-to-celland animalto-animal variability are reported, but the larger cell-to-cell variation was used in the statistical analyses at the individual metaphase level. In addition to the parametric t test, the nonparametric Mann-Whitney U test [Lehmann, 19751 was used, because the SCE distribution was not normal in some instances. The chosen level of signifi- cance was 0.05. The raw data are given in Tables I3 and N to present a complete view of the experimental results and to facilitate independent assessments of the data. RESULTS Mice BZ induced significant, concentration-related increases in the SCE frequencies at all exposure concentrations examined (Tables I and 11).The concentration-related SCE increase is particularly apparent in Table 11as a shift towards cells exhibiting higher SCE frequencies and a concomitant decrease in metaphases showing low SCE frequencies. In addition, BZ caused a significant, concentration-dependent decrease *I-*L Cytogenetic Damage in Rodents Inhaling Benzene 33 TABLE I. The Induction of Cytogenetic Damage in Male DBAl2 Mice and Sprape-Dawley Rats Benzene (ppm/hr) Number of animals SCEs/metaphase" Number of MN/l ,OOO PCEsa DBAR mice 0 10 (SCE), 1 1 (MN) 5.9 f 0.2 (0.14)b *2.1 0.3 10.3 f 0.9 *100.1 1.6 *991.2 5.9 5 5 5 7.6 f 0.2 (0.17)*.** 9.5 f 0.2 (0.23)'*" 13.8 f 0.9 (0.37)'." 9.0 f 0.6' 20.3 Ifr 0.7' 28.1 It 0.8' Sprague-Dawley rats Experiment 1 0 10.0 f 0.2 10 5 *8.6 f 0.1 (0.17) 10.4 0.2 (0.27)*,** *2.0 0.1 6.2 f 0.4' 30.0 f 0.3 5 1 1 . 1 f 0.1 (0.23)*." 7.6 f 0.4* Experiment 2 0 0.1 f 0.01 0.3 f 0.02 *1.0 0.02 *3.0 0.05 20 5 5 5 5 8.2 f 0.1 (0.13) *8.2 0.1 (0.24) *8.2 0.1 (0.19) *9.1 0.2 (0.28)' *10.5 0.2 (0.25)'*'* *aMean standard error among animals within a group. 1.7 rt 0.2 1.6 f 0.3 2.0 f 0.3 4.4 f 0.3" 4.8 f 0.4' bThe numbers in parentheses denote the standard error among cells within a group. 'Significantly different from the concurrent controls (P < 0.05) using Student's t test (one-sided). *ISignificantly different from the concurrent controls (P < 0.05) using Mann-Whitney U test (one- sided). in the mitotic activity but did not significantly affect the leucocyte counts (Table In). No significant effect was seen on the cell cycle kinetics at any exposure concentration examined (data not shown). Femoral bone marrow smears made from the same mice showed that BZ caused significant, concentration-related increases in the number of PCEs containing MN of up to 13.4 times the control value at 1,OOO ppm (Tables I and II). The concentration-response curves for SCE and MN are similar in shape. Rats BZ induced significant, concentration-dependentincreases in the SCE frequency after a 6-hr exposure to 3, 10, or 30 ppm (Tables I and IV).The concentration-related SCE increase is manifested primarily as an increase in the number of cells exhibiting higher SCE frequencies, which appear as "outliers". The significance of the effect seen on SCE induction at 1 ppm is borderline and dependent on the statistical test chosen. The SCE-inducing effect of 1 pprn BZ is statistically significant using the t test (one-sided; P = 0.036), but not statistically significant using the Mann-Whitney U test (one-sided; P = 0.055). The SCE frequency for the 1-ppm BZ exposure does fall in line between the SCE frequencies observed for the 0.3 and 3 ppm exposures, and all rats exposed to 1 ppm BZ had a higher SCE frequency than all their concurrent controls. Atmospheres of 0.1 or 0.3 ppm BZ did not significantly affect the SCE frequency. Whereas exposure of rats to 0.1, 0.3, 1 or 10 ppm BZ revealed no significant change in mitotic activity, both 3 and 30 ppm BZ caused a significant decrease in mitotic activity (Table 111). In addition, no significant effect was observed on the leucocyte counts (Table HI) or cell cycle lunetics (data not shown) at any exposure concentration studied. Femoral bone marrow smears prepared from the same rats revealed that BZ induced significant, concentration-related increases in the number of PCEs containing a- -- N -N d r n d -d m \o d vi \omNmP\o m i - m rg m i - -10 vi r c d 10 d d N -m --m- Nm0 CytogeneticDamage in Rodents Inhaling Benzene 35 TABLE III. The Effects of Inhaled Benzene on Mitotic Indices and Leucocyte Counts of Male DBAl2 Mice and Sprague-Dawley Rats Benzene (ppm/hr) Number of animals Mitotic index (%)a Leucocytes/ml of whole blood ( DBAR mice 0 10.3 f 0.9 *100.1 f 1.6 991.2 5.9 *10 4.4 f 0.3 4.13 0.5 *5 2.9 f 0.2' 3.20 0.2 *5 2.3 f 0.6' 5.57 1.0 *5 1.0 f 0.1' 4.94 0.4 Sprague-Dawley rats Experiment 1 0 *10.0 0.20 *30.0 0.30 20 4.6 f 0.1 14.80 f 0.7 5 3.7 f 0.3 14.70 f 0.9 5 3.5 f 0.2' 15.00 f 2.2 Experiment 2 0 0.1 f 0.01 *20 5.1 f 0.2 14.10 0.7 * *5 6.1 0.2 14.60 1.9 0.3 f 0.02 5 5.6 f 0.2 12.40 1.0 1.0 jl 0.02 5 5.5 f 0.1 14.00 f 2.7 3.0 jl 0.05 5 3.9 f 0.1* 15.60 f 0.8 aMean f standard error among animals within a group. *Significantly different from the concurrent controls (P < 0.05) using Student's t test (one-tailed). MN at concentrations from 1 to 30 ppm (Tables I and IV). As with the SCE frequency, there were no observed effects at 0.1 or 0.3 ppm. The concentration-response curves for the rat reveal that the SCE and MN responses are similar in shape. Observations at 10 ppm show that the responses for induced SCE frequencies are similar for both the rat and mouse, whereas mouse PCEs appear to be somewhat more sensitive to the MN-inducing effects of BZ. DISCUSSION These results confirm and extend the work of several investigators, who have also observed cytogenetic effects from lowdose BZ exposureon mouse bone marrow. Hite et a1 [1980] reported significant increases in the number of PCEs with MN in both male and female CD-1 mice given 125 p1 BZ/kg/day by gavage and sacrificed 18 hr after the second dose. Similarly, Meyne and Legator [1980] found that male CD-1 mice exhibited higher frequencies of chromosome aberrations and MN than females after exposure to 500 4 BZlkglday by gavage or intraperitoneal injections at 24-hr intervals for 3 consecutive days. Also, Siou et al [19821 observed a significant increase in PCEs containing MN from male Swiss mice after two gavages of 25.6 p1 BZ/kg/day at 24-hr intervals. Toft et al [1982] found that NMRI mice exposed to 14 ppm BZ by inhalation continuously for 1-8 weeks had a significant increase in PCEs containing MN. Tice et a1 [1982] have demonstrated significant increases of SCE after a 4-hr inhalation exposure to 28 ppm BZ in both male and female DBA/2 and C57B1/6 mice (28 ppm x 4 hr = 112 ppm x hr), which represents the previously reported lowest cumulative concentration of BZ to cause a significant induction of SCE in the bone marrow. Tice et a1 [19821 observed an induced SCE frequency in male DBA/2 mice of 3. I , 8.4, 11.9, and 17.9/metaphaseat 112, 1,960, 4,072, and 9,541 ppm x hr BZ, m0 N -- NN 38 Erexsonetal respectively. For comparison, BZ induced an increase of 1.7, 3.6, and 7.9 SCEsl metaphase at 62, 601, 5,948 ppm x hr BZ, respectively in the present study. In addition, the present study demonstrates that BZ is capable of inducing SCE in lymphocytes and increasing MN in bone marrow at a substantially lower timeweighted average concentration than previously examined. BZ is metabolized in mammals to phenol, hydroquinone, catechol, 1,2,4benzenetriol, and p-benzoquinone [Teisinger et al, 1952; Dean, 19691. The decreases in mitotic indices seen after the inhalation of BZ might be attributed to the formation of hydroquinone and p-benzoquinone; these metabolites react with sulfhydryl groups of cytoskeletal proteins vital for blastogenesis [Irons and Neptun, 1980; Irons et al, 1981; Heifer and Irons, 19811. Possibly, a majority of the SCEs induced in vivo in the mouse B lymphocytes and rat T lymphocytes are caused by catechol, p-benzoquinone, and 1,2,4-benzenetriol; these compounds are relatively efficient SCE inducers in cultured human T lymphocytes exposed in vitro [Morimoto and Wolff, 1980; Erexson et al, 19851. The data obtained in the present study show parallel induction of SCE and MN in both mice and rats, and this indicates that a common BZ metabolite(s) might be involved. MN induction is indicative of either spindle disruption or chromosome breakage leading to lagging acentric fragments or entire chromosomes at anaphase [Heddle et al, 19831. The formation of MN in bone marrow might be a result of either the accumulation of phenol, hydroquinone, and catechol at this site or the minor metabolism of BZ and its metabolites by the marrow [Rickert et al, 1979; Irons et al, 1980; Greenlee et al, 19811, leading to disruption of the spindle and aneuploidy. The idea that chromosome loss is involved in MN induction by BZ is supported by an in vitro study [Irons et al, 19811that has shown that microtubule assembly of rat brain tubulin is inhibited by hydroquinone. A number of studies also support the hypothesis that the MN are a result of chromosome breakage. Evidence of a concentration-dependent increase of chromosome aberrations in bone marrow of male Wistar rats has been reported after exposure to concentrations of 1, 10, 100, or 1,OOO ppm BZ for 6 hr [Styles and Richardson, 19841. Tice et a1 [19801 found significant increases of chromatid-type aberrations in bone marrow cells of DBA/2 male and female mice exposed to 3,100 ppm BZ for 4 hr after pretreatment with phenobarbital, and other investigators have reported chromosome aberrations in rat bone marrow after exposure to BZ alone [Philip and Jensen, 1979; Lyon, 1976; Dean, 1978; Anderson and Richardson, 19811. The present study accurately defines the shape of the concentration-response curves for these cytogenetic endpoints and indicates that a 6-hr exposure to 1 ppm BZ and above can induce measurable cytogenetic effects in rodents. It should be noted that the shape of the concentration-response curves for both rats and mice for both endpoints are nonlinear, with the greatest slope at the lower concentrations. A semilogarithmic plot of the data yields a straight line. The magnitude of the SCE response to BZ, although modest, was greater than that observed for rats exposed to ethylene oxide by inhalation [Kligerman et al, 19831. The continued monitoring of human exposure to BZ with sensitive cytogenetic tests of peripheral blood lymphocytes might be warranted to determine whether or not these rodent models accurately approximate the human situation. These results suggest that further studies on the cytogenetic effects of repeated exposure to low concentrations of BZ are needed to assess the persistence and accumulation of cytogenetic lesions in rodent lymphocytes. Cytogenetic Damage in Rodents Inhaling Benzene 39 REFERENCES Anderson D, Richardson CR (1981): Issues relevant to the assessment of chemically induced chromosome damage in vivo and their relationship to chemical mutagenesis. Mutat Res 90:261-272. Baarson KA, Snyder CA, Albert RE (1984): Repeated exposure of C57B1 mice to inhaled benzene at 10 ppm markedly depressed erythropoietic colony formation. Toxicol Lett 20:337-342. Barrow CS, Steinhagen WH (1982): Design, construction and operation of a simple inhalation exposure system. Fund Appl Toxicol 2:33-37. Cortina TA, Sica EW, McCarroll NE, Coate W, Thakur A (1982): Inhalation cytogenetics in mice and rats exposed to benzene. In MacFarland HN, Holdsworth CE, MacGregor JA, Call RW, Kane ML (4s): "The Toxicology of Petroleum Hydrocarbons." Washington, DC: American Petroleum Institute, pp 122-127. Cronkite EP, Bullis JE, Inoue T, Drew RT (1984): Benzene inhalation produces leukemia in mice. Toxicol Appl Pharamacol 75:358-361. Dean BJ (1969): Chemical-induced chromosome damage. Lab Anim 3: 157-174. Dean BJ (1978): Genetic toxicology of benzene, toluene, xylenes and phenols. Mutat Res 47:75-97. Erexson GL, Wilmer JL, Kligerman AD (1983): Analyses of sister chromatid exchange and cell cycle kinetics in mouse T- and B-lymphocytes from peripheral blood cultures. Mutat Res 109:271-281. Erexson GL, Wilmer JL, Kligerman AD (1985): Sister chromatid exchange induction in human lympho- cytes exposed to benzene and its metabolites in vitro. Cancer Res 45:2471-2477. Forni A, Pacific0 E, Limonta A (1971): Chromosome studies in workers exposed to benzene or toluene or both. Arch Environ Health 22:373-378. Goldstein BD (1977): Hematotoxicity in humans. J Toxicol Environ Health 2 [Suppl]: 69-105. Goldstein BD, Snyder CA, Laskin S (1982): Myelogenous leukemia in rodents inhaling benzene. Toxicol Lett 13:169-173. Gollapudi BB, Linscombe VA, Sinha AK (1983): A note of the utility of Fischer-344 rat in the micronucleus test. Toxicol Lett 17:201-204. Greenlee WF, Gross EA, Irons RD (1981): Relationship between benzene toxicity and the disposition of ``C-labelled benzene metabolites in the rat. Chem Biol Interact 33:285-299. Hayashi M, Sofuni T, Ishidate Jr M (1983): An application of acridine orange fluorescent staining to the micronucleus test. Mutat Res 120:241-247. Heddle JA, Hite M, Kirkhart B, Mavournin K, MacGregor JT, Newell GW, Salamone MF (1983): The induction of micronuclei as a measure of genotoxicity: A report of the U S . Environmental Protection Agency GENETOX Program. Mutat Res 123:61-118. Hite M, Pecharo M, Smith 1, Thornton T (1980): The effect of benzene in the micronucleus test. Mutat Res 77: 149-155. Irons RD, Dent JG, Baker TS, Rickert DE (1980): Benzene is metabolized and covalently bound in bone marrow in situ. Chem Biol Interact 30:241-245. Irons RD, Moore BJ (1980): Effect of short term benzene administration on circulating lymphocyte subpopulations in the rabbit: Evidence of a selective B-lymphocyte sensitivity. Res Commun Chem Pathol Pharmacol27: 147-155. Irons RD, Neptun DA (1980): Effects of the principal hydroxy-metabolites of benzene on microtubule polymerization. Arch Toxicol 45:297-305. Irons RD, Neptun DA, Pfeifer RW (1981): Inhibition of lymphocyte transformation and microtubule assembly by quinone metabolites of benzene: Evidence for a common mechanism. J Reticuloendothel SOC30:359-372. Kligerman AD, Wilmer JL, Erexson GL (1982): Characterization of a rat lymphocyte culture system for assessing sister chromatid exchange. 11. Effects of 5-bromodeoxyuridine concentration, number of white blood cells in the inoculum, and inoculum volume. Environ Mutagen 4385592. Kligerman AD, Erexson GL, Phelps MC, Wilmer JL (1983): Sister chromatid exchange induction in peripheral blood lymphocytes of rats exposed to ethylene oxide by inhalation. Mutat Res 120: 37-44. Lehmann EL (1976): In "Nonparametrics." San Francisco: Holden-Day, Inc., pp 76-81. Lyon JP (1976): Mutagenicity studies with benzene. Dissert Abstr 36B:5537. Maltoni C, Conti B, Cotti G (1983): Benzene: A multipotential carcinogen. Results of long-term bioassays performed at the Bologna Institute of Oncology. Am J lndust Med 4589-630. 40 Erexson et a1 Meyne J, Legator MS (1980): Sex-related differences in cytogenetic effects of benzene in the bone marrow of Swiss mice. Environ Mutagen 243-50. Morimoto K, Wolff S (1980): Increase of sister chromatid exchanges and perturbations of cell division kinetics in human lymphocytes by benzene metablites. Cancer Res 40:1189-1193. National Toxicology Program (1984): Technical report on the toxicology and carcinogenesis studies of benzene (CAS No. 71-43-2) in F-344/N rats and B6C3FI mice (gavage studies) (Pub1 NTP-TR289). Bethesda, MD: NIH. Pfeifer RW, Irons RD (1981): Inhibition of lectin-stimulated lymphocyte agglutination and mitogenesis by hydroquinone: Reactivity with intracellular sulfhydryl groups. Exp Mol Path01 35: 189-198. Philip P, Jensen MK (1970): Benzene induced chromosome abnormalities in rat bone marrow cells. Acta Path Microbiol Scand 78A.3489-490. Rickert DE, Baker TS, Bus JS, Barrow CS, Irons RD (1979): Benzene disposition in the rat after exposure by inhalation. Toxicol Appl Pharmacol49:417-423. Rinsky RA, Young RJ, Smith AB (1981): Leukemia in benzene workers. Am J Indust Med 2:217-245. Rozen MG, Snyder CA, Albert RE (1984): Depressions in B- and T-lymphocyte mitogen-induced blastogenesis in mice exposed to low concentrations of benzene. Toxicol Lett 20:343-349. Schmid W (1976): In Hollaender A (ed): "Chemical Mutagens: Principles and Methods for their Detection, Vol4." New York: Plenum Press, pp 31-53. Siou G, Conan L, Haitem M el (1982): Comparative study of the effect of the carcinogens benzene and methylmethane sulfonate on the mouse and Chinese hamster. C o r n Eur Commun 7549:353359. Snedecor GW, Cochran WG (1967): "Statistical Methods, Ed 6." Ames, IA: Iowa State University Press. Snyder CA, Goldstein BD, Sellakumar AR, Bromberg I, Laskin S, Albert RE (1980): The inhalation toxicology of benzene: Incidence of hematopoietic neoplasms and hematoxicity in AKR/J and C57B1/6J mice. Toxicol Appl Pharmacol54:323-33 1. Snyder R, Longacre SL, Sammett D, Witmer CM, Kocsis JJ (1983): Relationship between the toxicity and metabolism of benzene. In Mehlman MA (ed):"Carcinogenicity and Toxicity of Benzene." Princeton, NJ: Princeton Scientific Publishers Inc., pp 23-27. Styles JA, Richardson CR (1984): Cytogenetic effects of benzene: Dosimetric studies on rats exposed to benzene vapour. Mutat Res 135:203-209. Teisinger J, Bergerova-Fiserova V, Kudma J (1952): The metabolism of benzene in man. Prac Lek 4: 175-188. Tice RR, Costa DL, Drew RT (1980): Cytogenetic effects of inhaled benzene in murine bone marrow: Induction of sister chromatid exchanges, chromosomal aberrations, and cellular proliferation inhibition in DBA12 mice. Proc Natl Acad Sci USA 77:2148-2152. Tice RR, Vogt TF, Costa DL (1982): Cytogenetic effects of inhaled benzene in murine bone marrow. In Tice RR, Costa DL, Schaich KM (eds): "Genotoxic Effects of Airborne Agents." New York: Plenum Publishing, pp 257-275. Toft K, Olofsson T, Tunek A, Berlin M (1982): Toxic effects on mouse bone marrow caused by inhalation of benzene. Arch Toxicol 51 :295-302. Tough IM, Smith PG, Court-Brown WM, Harnden DG (1970): Chromosome studies on workers exposed to atmospheric benzene. Eur J Cancer 6:49-55. Wilmer JL, Erexson GL, Kligerman AD (1983): Implications of an elevated sister-chromatid exchange frequency in rat lymphocytes cultured in the absence of erythrocytes. Mutat Res 109:231-248.