Document zQ9BOBGbEJj5KR1xodon40je0

Murarron Reseurch. 203 (1988) 251-271 Elsevier MTR 08692 * The effect of exposure regimen and duration on benzene-induced bone-marrow damage in mice I. Sex comparison in DBA/2 mice Carol A. Luke *, Raymond R. Tice and Robert T. Drew * * Medical Deparrmenr. Brookhaivn Xarronal Lohoruron. Lpron. .V Y 11973 I L . S . A J (Received 11 August 1987) t Revision rectiked 4 December 1987) (ACCrpled6 JanuaT 1988) '51 Krtmordr Bone-marrow damage. benzene-induced. Mice. henzenc-induced bone-marrou damage. Exposure regmen. effect of. Mice. DBA/2. sex companson. Benzene. induction of bone-marrow damage In mice Summary In the mouse. the concurrent evaluation of micronuclei frequencies in peripheral blood polychromatlc erythrocytes (PCE) and normochromatic erythrocytes (NCE) permits an assessment of both recently-induced and chronicallyaccumulated bone-marrow damage. This assay system was used to evaluate on a weekly basis the effect of exposure duration (1-13 weeks. 6 h per day) and exposure regimen (Regimen 1 :5 exposure days per week: Regimen 2 :3 exposure days per week) on the ability of 300 ppm benzene to induce genotoxic damage in the bone marrow of male and female DBA/2 mice. In addition. an analysis of the percentage of PCE in peripheral blood was used to evaluate benzene-induced alterations in the rate of erythropoiesis. Exposure to benzene induced a marked increase in the frequency of micronucleated PCE (MN-PCE), an effect which was considerably greater in male mice than in female mice. In both sexes. the induction of MN-PCE was independent of exposure regiment and of exposure duration. Exposure to benzene also resulted in an exposure duration-dependent increase in the frequency of MN-NCE. The frequency of MN-NCE increased more slowly in female than in male mice and. wittun each sex. more slowly in Regimen 2 animals. Apparent steady-state conditions for MN-NCE frequencies were attained by about the fifth week of exposure in female mice exposed by either regimen and in male mice exposed by Regimen 2. Steady-state conditions for MN-NCE frequencies in male mice exposed to benzene by Regimen 1 did not occur during the duration of the study. An analysis of SPCE data revealed an initial severe depression in the rate of erythropoiesis in both sexes. with the return in the production of PCE to control levels being dependent on both sex and exposure regimen. Suppression of PCE production occurred throughout the course of the study in Regimen 2 males. while the percentage of PCE returned to control levels sporadically after 5 weeks in Regimen 1 males and wittun 5 weeks in females. regardless of Present address: New York State College of Vetennary Medicine. Cornell University. Ithaca. N Y 14853 (U.S.A.). Present address: American Petroleum Institute. 1220 L Street. NW. Washington. DC 20005 (U.S.A.). Correspondence: Dr. Raymond R. Tice. Medical Department. Brookhaven National Laboratory. Upton. N Y 11973 (U.S.A.). 0165-1 161/88/$03 50 ,( 1988 El!,rvler Science Publisher\ B \. IBiomeJicaI D l r i ~ o n ) I 252 regimen. Thus. while the sex-dependent induction of genotoxic damage by multiple exposures to benzene over a 13-week period was independent of exposure regimen and duration. the induction of cytotoxic damage was both sex- and regimen-dependent. The most severe depression of erythropoiesis occurred in maleJBA/2 mice exposed to benzene by the more intermittent resimen (Le.. 3 days/week versus 5 days/week). The results of this study underscores the complexity of the peripheral blood micronucleus assay and emphasizes the utilitv of evaluating micronucleated PCE and micronucleated NCE frequencies and the percentage of PCE in the same animals across time in multiple exposure situations. Benzene is a common ingredient in gasoline and is used extensively in industry as a solvent and as an intermediate in the synthesis of a variety of chemical products (e.%..detergents. explosives. pharmaceuticals. dyestuffs) (Sittig. 1979: IARC. 1982). Benzene also presents many risks in regard to environmental contamination and human health. Numerous studies have been conducted which associate occupational exposure to benzene with a variety of adverse health effects. including an increased risk for leukemia and aplastic anemia (reviewed in IARC. 1982; Aksoy. 1985; Snyder. 1987). In animal studies. benzene has been shown to be carcinogenic (Maltoni and Scarnato. 1979; Snyder et ai.. 1980: Cronkite et ai.. 1984: NTP. 1985: Stoner et al.. 1986). and to induce genotoxic damage as measured by the induction of sisterchromatid exchanges (SCE), chromosomal aberrations and micronuclei (reviewed in IARC. 1982: Dean. 1969. 1985). Although there is no doubt that exposure to benzene is hazardous to human health. there is much debate as to what constitutes safe occupational and environmental exposure limits (White et al.. 1980; Federal Registry, 1985: Dean. 1985). Consequently, information relating to whether different exposure situations. especially those involving inhalation (the primary route of exposure to humans), modulate the in vivo genotoxic and cytotoxic effects induced by benzene would be valuable. Several studies have suggested that the levels of genotoxic damage induced in bone marrow (the principle target of benzene-induced genotoxic and cytotoxic damage) diminish with increasing exposure duration (Anderson and hchardson, 1981; Tice et ai.. 1984: Dean and Clare. 1985: Choy et al., 1985: Barale et al.. 1985). In addition. several investigators have concluded that. for the same duration of exposure. chronic exposure conditions were more harmful than intermittent ones to bone-marrow integrity and function (Coffin et al.. 1977: Gill et al.. 1980: Toft et al.. 1982). However. Irons (1983) demonstrated that intermittent treatments of mice with metabolites of benzene (hydroquinone and phenol) induced more bone-marrow damage (i.e.. loss in cellularity) than chronic treatments at the same doses. The mouse peripheral blood micronucleus ( M N ) assay enables an evaluation of both acute and chronic bone marrow damage in the same animal (MacGregor et ai., 1980: Schlegel and MacGregor. 1982: Tice and Ivett. 1985). Since the lifetime of 3 newly formed polychromatic erythrocyte (PCE) in the mouse is about 24 h (Jenssen and Ramel. 1978: Cole et al.. 1981: Hayashi et al.. 1984). while that of a normochromatic erythrocyte (NCE) is around 30 days (Schalm et ai.. 1975; Schlegel and MacGregor. 1982). the presence of MN-PCE in the peripheral blood are indicative of damage that occurred in the bone marrow 1-2 days prior to sampling, while the frequency of MN-NCE indicates the accumulation of damage in t h s cell population throughout the course of the exposure period. Due to the lack of information on benzene-induced genotoxic damage under multiple exposure conditions. a study was designed using the peripheral blood MN assay to evaluate the effect of exposure regimen and duration on the ability of inhaled benzene to induce genotoxic damage in the bone marrow of male and female mice. An evaluation in the peripheral blood of the percentage of PCE among total erythrocytes was included to provide an assessment of the rate of erythropoiesis and thus a measure of bone marrow cytotoxicity (Heddle et al.. 1983). [n conducting !his study. it was decided to expose mice to 300 ppm benzene for 13 weeks, for either 5 exposure days per week or for 3 exposure lays per week. This concentration of benzene was ised because of published data demonstrating its ibility to induce adverse health effects (e.g. eukopenia. anemia. cancer) (Snyder et al.. 1978. .981; Green et al.. 1981; rodate et ai.. 1984) and ;enotoxic damage (Tice et al.. 1982: Cortina et al.. .984) in exposed mice. This concentration of hen:ene was also not expected to induce overt mortalty over the course of the study (Cronkite et al.. 1984). Furthermore. 300 ppm benzene was beieved to be withn the linear portion of the dose 'ange for the absorption of inhaled benzene in nice (Wells and Nerland. 1984). DBA/Z mice were used to enable direct comparison with previius genotoxicity studies on benzene (Tice et ai.. 1980. 1982). An exposure duration of 13 weeks was used to provide data over approximately 2 - 3 VCE lifetimes (Schalm et al.. 1975: Schlegel and LlacGregor. 1982). The selection of the regimens xas based on studies by Irons (19831, in which nore cytotoxic damage occurred when mice were :rested with various metabolites of benzene on 3 d a y per week as compared to daily treatments. Finally, because the genotoxlc and carcinogenic mivity of benzene is highly sex-dependent (reviewed by Dean, 1985). both male and female mice were included in the study. Materials and methods Chemicals. Benzene (CAS No. 71-43-2). " reagent grade". "thiophene free", was obtained from Mallinckrodt Chemical Co. (Paris. KY), wlule acridine orange (AO) was obtained from Sigma Chemical Co. (St. Louis, MO). Animals. Male and female DBA/2 mice were obtained from Jackson Laboratories (Bar Harbor. ME) at 9 weeks of age. After receipt. the mice were acclimated for a period of 2 weeks in standard laboratory stainless steel cages with corn cob bedding (Bed 0 Cobs, J.R. Nielson and Sons. Inc.). The population density was kept at no greater than one animal per 90 cm'. under controlled laboratory conditions [Le., 22 & 2 C . 50 k 15% relative humidity, 20 air exchanges per hour and a 12-h photoperiod (light: 0700-1900)]. Food 253 (Purina Lab Rodent Chow 5001) and water were provided ad libitum. The initial weight range at the onset of exposure was: male. 26-30 g; female. 20-22 g. Exposure protocol. Groups of male and female DBA/2 mice ( n = 6) were exposed to either 300 ppm benzene or to ambient air for a period of 13 weeks. using one of two exposure regimens (Regimen 1: live consecutive days exposed. two consecutive days unexposed: Regimen 2: three consecutive days exposed. four consecutive days unexposed). Regimen 1 animal5 were exposed Mondays through Fridays and Regimen 2 animals were exposed Tuesdays through Thursdays. Exposure was for 6 ti a day. generally from 0900 to 1500 hours. The mice were housed in stainless steel mesh cage packs measuring 44 cm x 51 cm that were divided into 8 equal cubicles ( 2 rows of 4 each). 3 mice of the same sex were housed in each cubicle. Each cage pack housed all animals of a particular exposure group. Animals were housed continuously in these cage packs for the 13 weeks of exposure. with clean cages provided on a weekly basis. Temperature. humidity. air exchange. lighting, food and water were maintained as was described for their acclimation period. except during the actual hours of exposure. Coded toe clipping enabled identification of individual animals throughout the experiment. Exposure to benzene was carried out in stainless steel and lucite chambers. Filtered compressed air was bubbled through liquid benzene and diluted to produce the desired concentration of benzene vapor. Benzene concentration was measured every 30 min by a gas chromatograph (Packard. model 417, equipped with a column of 10% silicone SE-30 on chromosorb W-HP). with an automatic sampling valve. Temperature in the chambers ranged between 21 and 25OC. with the benzene chamber averaging about 1"C higher than * -the control chamber. Relative humidity was main- tained at 50 15% with 15 air exchanges per hour. Food was not provided during the 6-h exposure period. althou& water was provided ad libitum. Control animals were treated in a manner identical to the exposed animals. excluding the presence of benzene. The time weighted average (TWA) concentration of benzene for Regimen 1 254 and Regmen 2 was 299.8 ppm (63 exposure days) and 300.4 (39 exposure days), respectively. a. Sampling, slide preparation, staining and analysis. Peripheral blood smears were made from all animals prior to the initiation of the first exposure and weekly thereafter. Both regmcn groups were sampled on the morning after the third day of each weekly exposure period (Le.. Regmen 1 on Thursdays. Regimen 2 on Fridays), to allow for equal expression of damage in the peripheral blood. The smears (two per mouse) were prepared using blood obtained by snipping off approximately 1 mm from the end of the tail. Slides were fixed in absolute methanol for about 10 min, air dried. and stored in slide boxes until stained for analysis. Slides were stained for 8 min with A 0 (0.02 mg/ml in pH 7.3 phosphate buffer) following the protocol of Kato et al. (1974) for SCE analysis. and rinsed in buffer for 10 min. Coverslips were floated onto the wet slides, and blotted to remove excess buffer. The slides were analyzed at 800 x magnification using epi-illuminated fluorescence microscopy (450-490 nm excitation. 520nm emission) for the number of micronucleated cells per lo00 NCE. the number of micronucleated cells per 1000 PCE. and the number of PCE per lo00 erythrocytes. Although PCE with as many as six MN were observed. all data are presented as the number of micronucleated cells. not as the number of micronuclei. Scoring was restricted to areas of the slide with no overlapping cells. In some instances. PCE formation in the bone marrow of benzene exposed mice was suppressed to such an extent that scoring 1000 PCE was impossible. In some cases, 500 PCE were counted for the presence of micronuclei. and the number adjusted to equal the frequency of micronucleated PCE per 1OOO. In cases were i t was impossible to score MN-NCE or the percentage of PCE and the statistical analysis required a complete data set, the missing data was estimated by averagmg the two nearest data points for that animal. Using temporal averages. a two-way Brown-Forsythe analysis of variance (ANOVA), based on separate group variances (BMDP, 1985). with dose and regimen as factors was conducted for each sex. Where a significant sex by dose interaction term was found (at an alpha level of 0.05). group mean data were compared using Student's t test based on separate variances after the alpha level had been Bonferroni corrected for the appropriate number of multiple comparisons. To evaluate sex differences. a two-way Brown-Forsythe ANOVA with sex and dose as factors, was conducted for each endpoint defined by regimen. unless differences between regimens were found to be nonsignifi\.ant, in wtuch case the appropriate regmen data were pooled. In the event of a significant sex by dose interaction term, group mean data were compared using Student's t test based on separate variances after the alpha level had been Bonferroni corrected for the appropriate number of multiple comparisons. Student's t tests were also used to determine at whch sample times the percentage of peripheral blood PCE in the benzene-exposed mice were significantly different from control values. To evaluate for an exposure duration-dependent alteration in the induction of genotoxic damage in the bone marrow of benzene-exposed mice. weekly frequencies of MN-PCE in each animal were analyzed by multiple regression analysis. In this analysis. MNPCE data were evaluated for a nonsignificant trend. a significant positive trend, or a sigmficant negative trend across time. As with the calculation of temporal averages, pre-exposure MN-PCE data were omitted from each analysis of benzene-exposed mouse data. Statistical analysis. Temporal averages for MN-PCE, MN-NCE and the percentage of PCE were calculated for each animal by summing the values across time (except for the MN-NCE data. week 0 was omitted from the exposed mouse calculations) and dividing by the number of samples evaluated. Animals that died prior to the 12th week of exposure were omitted from this analysis. Results Frequency of MN-PCE Males. There was a dramatic increase (between 50- and 100-fold) in the number of MN-PCE observed in the peripheral blood of benzene-exposed male mice sampled at the end of the first A 1501 255 024 6 8 IO 12 EXPOSURE DUAATION (weeks) Fig. 1 Regimen companson of the group mean penpherd blood \ l N - P C E frequency across time for male DBA/2 mice. ( A ) D s r i on benzene-exposed mice. (B)Data on control nuce. 0 . Cor ReGmen 1: 3.for Regmen 2. Error bars indicate 95T confidence limits. The dashed line in Panel A indicates the upper 954 confidence limit for \.IN-PCE frequency in control mice. The kalues in parenthesi, indicate the upper bound cf [he 95% confidence Iinuts for that weekly mean frequency. Vote the difference in scale for the Y axis between Panels A and B. See .4ppendix 1A for indiv~dualand group mean data. week of exposure (Fig. 1; see Appendix 1A for individual animal data and weekly group means). A two-way ANOVA of temporal averages with regmen and dose as factors revealed a significant effect due to exposure to benzene without a significant regimen difference (Table 1). Regression analvses to evaluate for an exposure durationdependent difference in MN-PCE levels among the control or exposed animals revealed one control male in Regmen 2 with a significant negative slope and one exposed male in Regimen 1 with a TABLE 1 TWO-WAY ANOVA OF MN-PCE TEMPORAL AVERAGES FOR MALE MICE. WITH REGIMEN AND DOSE AS FACTORS Regmen Opprn 1 2.05f0.131 2 1.5320.150 Brown-Forsythe ANOVA Regmen Dose [nteracrion .V 6 6 300pprn 107.85k5.529 121.23~1.700 P values 0.0768 O.OoO1' 0.0615 N 6 6 Mean number of MN-PCE per IO00 PCE? the standard error of the mean among .V animals. Significant at u = 0.05. EXPOSURE DURATION (weeks) Fig. 2. Regimen comparison of the group mean penpheral hlood XIN-PCE frequency across time for female DBA/2 mice. (.4) Dat.i on benzene-exposed mice. ( B )Data on control mice. 0 . for Regmen I : 5. for Regmen 2. Error bars indicate 95% confidence limits. The dashed line In Panel .A indicates the upper 95% confidznce limit for MN-PCE frequency in control mce. Note the difference in scale for the Y A Sh e t w e n Panels .4m d 8. See .Appendix 1 B for individual and group mean data. significant positive slope (Appendix 14). The lack of a significant slope (negative or positive) among the majority of the exposed mice indicates that the frequency of benzene-induced MN-PCE remained relatively constant throughout the 13-week exposure period. Females. Both regimens produced a significant increase in MN-PCE frequency, with the MN-PCE levels increasing approximately 15-fold above control levels (Fig. 2; see Appendix 1B for individual animal data and weekly group means). A two-way ANOVA comparison of temporal aver- TABLE 2 TWO-WAY ANOVA OF MN-PCE TEMPORAL AVERAGES FOR FEMALE MICE. WITH REGIMEN AND DOSE AS FACTORS ReGmen Oppm 1 1.5520.211 2 1.9OkO.188 Brown- Forsvthe ANOVA Regmen Dose Interaction N 6 6 300ppm 20.93k3.173 24.40k4.454 P dues 05450 o.Ooo1 0.6286 N 6 5 Mean number of MN-PCE per lo00 PCE +_ the standard error of the mean among N animals. Significant at u = 0.05. 256 TABLE 3 TWO-WAY .ANOVA OF MN-PCE TEMPORAL AVERAGES FOR DBA/2 MICE. WITH SEX A N D DOSE AS FRCTORS `A c75 w Sex Zt ale Female 0 PPm 1.79k0.123 1.72k0.145 .V 12 12 Brown- Fc,rsL the 4 N O V 4 sex Dose Interaction 300ppm 114.54+3.417 22.51 k2.579 P Lalues oO001* oO001 oO001* .v I2 11 ~~ Mean number of \IN-PCE per IO00 PCE? the btandard error of the mcin Among m m ; \ l s Significmt at a = 0 05 ages (Table 2 ) indicated a significant benzene effect which was independent of the regimen used. A regression analysis of individual female MNPCE frequencies against exposure duration revealed the absence of a significant slope among all control and exposed animals (Appendix 1B). Maies L`S. females. A visual comparison of Figs. 1 and 2 illustrates the sex-dependent difference in the frequency of MN-PCE in the peripheral blood of male and female mice exposure to benzene. Since regimen differences in the temporal averages of MN-PCE were not significant within each sex. MN-PCE data were pooled between regimens before analyzing by two-way ANOVA for sex differences in the frequency of MN-PCE in unexposed and exposed mice (Table 3). Because of the significant sex by dose-interaction term. pairwise comparisons were conducted using Student's I test. with the alpha level Bonferroni corrected for 4 pairwise comparisons, to evaluate for sex differences among control and exposed groups. A significant difference in MN-PCE levels between male and female control mice was not present ( P= 0.7287). However, in the benzene-exposed groups, male mice e h b i t e d about a 5-fold greater level of damage than female mice ( P = 0.0001). Frequency of M N - N C E Males. There was an exposure duration-dependent increase in the frequency of MN-NCE in the male mice exposed to benzene (Fig. 3; see /3-Ae -+- .& 0 ` 0 2 4 6 8 IO 12 EXPOSURE DURATICN (weeks) Fig. 3. Repment comparison of the group mean penpheral blood MN-NCE frequency across time for male DBA/2 mice. ( A ) Data on benzene-exposed mice. ( B ) Data on control mice. 0. for Repmen 1 : 3. for Regmen 2. Error bars indicate 95T confidence limits. The dashed line in Panel A indicates the upper 95'? confidence limit for MN-NCE frequencv in control rmce. Note the difference in scale for the Y axis between Panels A and 8. See Appendix ?A for individual and group mean data. Appendix 2A for individual animal data and weekly group means). At about week 7, a significant difference between regimens in the accumulation of MN-NCE in exposed mice is apparent. with the Regimen 1 animals exhibiting the greater -frequency of MN-NCE (week 13 group average MN-NCE levels being - 4 7 and 17 MNNCE/1000 NCE for Regimen 1 and Regimen 2 mice. respectively). Examination of the results obtained from a two-way ANOVA of temporal averages (Table 4) with benzene exposure and regimen as factors revealed a hghly significant interaction TABLE 4 TWO-WAY ANOVA OF MN-NCE TEMPORAL AVERAGES FOR MALE MICE. WITH REGIMEN AND DOSE AS FACTORS Regimen Oppm 1 1.70+0.157 2 1.35+0.034 Brown- Forsythe ANOVA Regtmen Dose lnteraciion N 6 6 300ppm 23.47+1.040 10.55k0.633 P values O.OOol* o.OOo1 O.o001* .V 6 6 Mean number of MN-NCE per 1000 NCEI. the standard error of the mean among N animals. Significant at u = 0.05. term ( P = 0.00011. Conducting pairwise compari- sons using Student's 1 test, with the alpha level Bonferroni corrected for 4 painvise comparisons. indicated that while There was not a significant difference between regimens In control data ( P = 0.0771). a significant difference due to benzene within ;1 regmen ( P < 0.0001) and for exposed groups between regmens ( P < 0.0001) existed. Also. while there was no evidence in male mice exposed bv Regimen 1 of a plateauing o \ e r time in the frequencv of MN-YCE. steady-stae conditions in the level of peripheral blood MN-NCE appears to have txcurred in the Regmen 2 mice by about the iourth week of exposure (see Fig. 3). Fenruler. The kmetics of the exposure duration-dependent increase in the frequency of MNNCE in the penpheral blood of female mice was similar to that observed in males. but not nearly as dramatic (Fig. 3 ) . Although there was not a great difference in MN-NCE frequency between regimens in the exposed animals at any one sampling time. the animals exposed by Regmen 1 (i.e., for 5 days a week) consistently e h b i t e d higher frequencies of MN-NCE than mice exposed by Regimen 2 (i.e.. for 3 days a week). An analysis of MN-NCE temporal averages supported this inference (Table 5 ) . revealing both a sigmficant reg- A -75 W 0 2 0 50 UB 1 I I I 0 2 4 68012 EXPOSURE WRATION (wseks) Fig. 4. Regimen cornpanson of the group mean peripheral blood MN-NCE frequencv across time for female DBA/2 mice. ( A ) Data on benzene-exposed rmce. (B)Data on control mice. a. for Regimen 1: 3 . lor Regmen 2. Error bars indicate 958 confidence limits. The dashed line in Panel A indicates the upper 95% confidence limit for MN-NCE frequency in control mice. Note the difference in scale for the Y axis between Panels A and B. See Appendlx ?B for individual and group mean data. 257 TABLE 5 TWO-WAY ANOVA OF MN-NCE TEMPOML AVERAGES FOR FEMALE MICE. WITH REGIMEN AND DOSE AS FACTORS Regmen Oppm 1 1.35+0.102 2 1.1510.177 Broun- Forsvthe ANOVA Regimen Dose Interaction .V 6 6 300ppm 7.72k0.995 4.98k0.497 P values 0.0325 * o.ooo1 0.0548 .Y 6 5 Mean number of M S - N C E per IO00 N C E k the standard error of the mean among .V animals. Significant at a = 0.05. imen and a significant benzene effect. An svaluation of pairwise differences among the MN-NCE data using Student's f test. with the alpha level Bonferroni corrected for 4 pairwise comparisons. indicated the lack of a significant regimen difference between MN-NCE frequencies in control mice ( P= 0.3558). Among exposed female mice. a saturation of MN-NCE levels appears to have occurred in both exposure regimens by about the fifth week of exposure. Males us. females. A visual comparison of Figs. 3 and 4 illustrates the sex-dependent difference in the peripheral blood levels of MN-NCE in response to the exposure to benzene. The frequency of MN-NCE in females exposed to benzene for 5 days a week ( R e g m e n 1) was much less than that which occurred in similarly-exposed males. The difference in MN-NCE frequency between male and female mice was not as striking for the animals exposed for 3 days a week (Regimen 2). However, the peripheral blood MN-NCE frequencies in females were consistently lower than MN-NCE levels in males. A two-way ANOVA of temporal averages for each regimen using pooled control animal data. with sex and exposure as factors, revealed a significant sex by exposure interaction term (Table 6). Temporal averages of MN-NCE frequencies in control mice pooled between regimens were not significantly different between the sexes ( P = 0.8289) whde. for both regmens. the MN-NCE temporal averages among exposed mice 258 TABLE 6 TWO-WAY ANOVA OF MN-NCE TEMPORAL AVERAGES FOR DBA/2 MICE. WITH SEX A N D DOSE AS FACTORS ( A ) Regimen I Sex 0 QPm Male Female 1.52+_0.093 1.25tO.102 Brown-Forsvthe ANOVA Sex Dose lnteraction .v 12 12 300ppm 13.4751.040 7.72+0.995 P values o.ooo1* O.o001* o.ooo1 .v 6 6 Mean number of MN-NCE per IO00 9 C E k the standard error of the mean among .V animals. Control data are po<ileddue to lack of regmen difference. Significant at a = 0.05. ( E ) Regimen 2 Sex 0 PPm Male Female 1.52-tO.093 1.25k0.102 Brown-Forsvthe ANOVA Sex Dose Interaction ,V 12 12 300ppm 10.55+0.633 4.9850.497 P values o.ooo1 o.oO01 o.oO01 `1, 6 5 Mean number of MN-NCE per 1oO0 N C E k t h e standard error of the mean among .\' animals. Sigruficant at a = 0.05. while the percentage of PCE in the peripheral blood of Repmen 1 exposed males returned to control levels at week 6 and exceeded control levels at week 9 after the start of the study. A two-way ANOVA of temporal averages using dose and regmen as factors resulted in a significant dose by treatment interaction term (Table 7). From a pairwise comparison analysis, based on Student's test with the alpha level Bonferroni corrected for 4 pairwise comparisons. a significant benzene ef- fect occurred in both regimens ( P< 0.0002). with the depression in Regimen 2 being significantly greater than that in Regrnen 1 (P=O.O016). AIthough the group S P C E temporal averages in the peripheral blood of the control mice were not jignificantly different between reiimens ( P = 0.8223) and remained relatively comtant throughout the course of the study. there was a surprising amount of intersample variability among individual mice in the percentage of PCE in their peripheral blood. Tlus was particularly evident near the final weeks of the study. This within animal variability was even more pronounced among the ben- were significantly higher in male mice ( P< 0.000 1). PCE frequency Males. Exposure to 300 ppm benzene resulted in a marked depression of PCE levels in the peripheral blood of male mice (Fig. 5B: see Appendix 3A for individual animal data and weekly group means), with the magmtude and the duration of the depression being strikingly dependent upon the exposure regmen. In both regmens, the -frequency of PCE in the exposed male mice de- clined from 3.0% to almost 0% by the completion of the first week of exposure. The percentage of PCE in male mice exposed to benzene by Regimen 2 remained significantly depressed below control levels throughout the duration of the study, EXPOSURE OURATION (wodcr) Fig. 5. Group mean frequency of peripheral blood PCE in DBA/2 mice across time. presented as the percentage of the corresponding control group daia. (A) Female mice. (B) Male mice. 0. for Regimen 1: 0. for Regmen 2. Solid symbols indicate statistically significant differences between control and benzene-exposed mice as determined by Student's r test at a=0.05. See Appendix 3A for individual and group mean data. 259 `4HI F 7 I W O - W A Y ANOVA OF %PCETEMPORAL AVER4GES F O R \taLE t 4CTORS MICE. W.+ITH REGIMEN AND DOSE 4s within animal variability in the percentage of PCE in the peripheral blood that was observed in the male mice during the study was also observed in the female mice (Appendix 3B). Regimen Oppm V 300pm .V I 3.49k0.152 6 1.4820.264 6~ ~- Males cs. feniales. ,Although animals of both 3.56f0.236 6 0.48k0.072 6 sexes experienced a marked suppression of PCE Uroun -Fors)the ANOVA P values - production by the end of the first week of exposure, the effect was more pronounced and was Regimen Do,e In[irJction 0.0324 0.o0010.0173 much more persistent in male mice (Fig. 5). A two-way A N O V A of temporal averages for each regimen with sex and exposure as factors (with Lfcr.in percentage of PCE per lo00 erythrocytes fthe standard m o r L>t the mein among .V m m a l s . Sieniiicant at a = 0.05. control data pooled between regimens) resulted in both a significant sex and and a significant dose effect for Regimen 1 (Table 9A). and ;1 significant interaction term for Regimen 2 (Tabl: 9B). Pair- lene-exposed rmce. with the percentage of PCE Laning as much as 20-fold (from 0.6% to 11.6%) wise comparisons of the various groups revealed no difference between the percentage of PCE in uithin a single animal from one meek to the next (Appendix 3A). TABLE 9 Females. Although the percentage of PCE in the penpheral blood of exposed female mice were significantly depressed by the completion of the first week of exposure to benzene, they returned to control levels withn the next few weeks (Fig. 5A: see Appendix 3B for individual animal data and weekly group means). A two-way ANOVA of the temporal averages with exposure and regimen as factors indicated that while there was a significant benzene effect. there was not a significant difference between regimens (Table 8). The same TWO-WAY ANOVA OF IPCE TEMPORAL AVERAGES FOR DBA/2 .MICE. WITH SEX A N D DOSE AS FACTORS ( A ) Regimen I Sex 0 ppm Male Female 3.53k0.134 3.67k0.115 Brown-Forsvthe ANOVA Sex Dose Interaction .V 12 12 300ppm 1.48k0.264 2.40k0.316 P values 0.033' o.ooo1 0.1167 N 6 6 TABLE 8 TR'I )-WAY ANOVA OF TPCE TEMPOWL AVERAGES FOR FEMALE MICE. WITH REGIMEN A N D DOSE AS FACTORS Mean percentage of PCE per IO00 erythrocyres k the standard error of the mean among ,V animals. Control data are pooled due 10 lack of regmen difference. Significant at (I= 0.05. ( E ) Regimen 2 Sex 0 ppm .V 300oom :V Regmen Oppm N 300ppm N Male 3.53k0.134 12 0.48It0.072 6 -1 3.51k0.129 6 2.40t0.316 6 1 3.83k0.178 6 3.09+0.520 5 Female 3.67k0.115 Brown-Forsythe ANOVA 12 3.09k0.520 P values 5 Brown-Forsythe ANOVA P values Sex 0.0040 * Regimen Dose 0.1523 0.0201 ' Dose Interaction 0.0011 0.0067 Interaction 0.5733 ~ ~Me-an percentage of PCE per lo00 erythrocytes+_the standard Mean percentage of PCE per 1000 erythrocytes 2 the \t.indard error of the mean among N animals. Control data are pooled error of the mean among N animals. due to lack of regrnen difference. Significant at a = 0.05. Significant at a = 0.05. 260 the peripheral blood of male and female control mice ( P= 0.4292). and greater suppression of erythropoiesis in exposed male mice than in expo'sed female mice in Regimen 2 ( P = 0.0072). Discussion Interpretation of peripheral blood MN-NCE data requires the integration of the results of analysis on MN-PCE frequencies with an understanding of the lunetics of the hematopoietic system. The frequency of MN-NCE is a Function of: (i) the daily production of micronucleated cells in the differentiating cell population which leads to the formation of VS-PCE. (ii) the daily production of PCE. (iii) the transit time between enucleation and identification as an NCE. and (iv) the lifespan of both the normal and micronucleated NCE under different exposure conditions (see Hayashi et al.. 2984). Theoretically. under multiple exposure conditions. MN-NCE frequencies should attain steady-state conditions after the duration of the exposure has exceeded the average NCE lifetime. In the mouse. damaged erythroid precursors in the bone marrow take about 6-12 h to give rise to MN-PCE. and approximately one day to be detected in the peripheral blood as MN-NCE (Jensen and Ramel, 1978; Cole et ai., 1981; Salamone and Heddle, 1983; Hart and Hartley-Asp, 1983: Hayashi et al.. 1984). The life- -time of NCE in the mouse is 30 days under normal conditions (Schiegel and MacGregor, 1982). Therefore. in following an extended exposure protocol. steady-state conditions for MNNCE frequencies should be attained by about 5 weeks after the first exposure to benzene. Barale et al. (1985) observed in Swiss CD-1 mice steady-state conditions for peripheral blood MN-NCE frequencies after about 4 weeks of exposure to benzene by gavage in male mice administered low doses and in female mice administered low or high doses. but not in male mice administered benzene at a tu& dose. Barale and his colleagues suggested that the lack of steady-state conditions and the apparently diminished MN-NCE response at extended exposure times in male mice administered high doses of benzene by gavage could be due to the selection of resistant cells, an increased detoxification of benzene. and/or a decreased abil- ity of the animals to metabolize benzene. Similarly, Tice et al. (1984) and Choy et al. (1985) failed to observe steady-state conditions for MNNCE frequencies in the peripheral blood of C57B1/6 and B6C3F1 mice exposed to benzene for 16 weeks by inhalation or for 2 years by gavage, respectively. In all cases. a diminished level of MN-NCE as a function of expo3ure duration appeared to support an interpretation that the level of genotoxic damage induced by benzene diminished with increasing exposure duration. In agreement with this interpretation. metabolic studies on benzene have demonstrated an increased rate of benzene clearance/ metabolism in rodents under repeated exposure conditions (Snyder et al.. 1967: Gonasun et al.. 1973; Snvder et al.. 1981: Post and Snvder. 1983: Driscoll and Snyder. 1984: Pathiratne et al.. 1986). In the present study, apparent steady-state conditions for MN-NCE levels were attained after about 5 weeks in female mice exposed to benzene by both regimens and in male mice exposed to benzene for 3 days per week (Figs. 3 and 4). However, in male mice exposed to benzene 5 days per week. the frequency of MN-NCE continued to increase over the duration of the study. As opposed to the other peripheral blood MN studies discussed earlier. this temporal pattern of increasing frequencies of MN-NCE in male DBA/2 mice exposed to 300 ppm benzene for 5 days per week suggests that the induction of genotoxic damage by benzene increases with increasing exposure duration. By examining the effect of exposure duration on benzene-induced MN-PCE frequencies. this question of an exposure durationdependent alteration in the induction of genotoxic damage in bone marrow can be examined more critically. Inhalation of benzene produced a markegipcrease in the frequency of MN-PCE in the periphe?al blood of exposed mice. particularly in males. n e level of damage was independent of the exTosure regimen u d of the exnosure duration. The constant frequency of MN-PCE in the peripheral blood of these mice over a 13-week period, regardless of the kinetics of MN-NCE accumulation. strongly suggests that modulation of sensitivity and/or metabolism did not occur or. at least, did not affect the induction of micronucleated 26 1 erythrocytes by benzene and/or its metabolites. This lack of an exposure duration-dependent alteration in benzene-induced levels of genotoxic damage in the bone"marrow of DBA/2 mice appears to conflict with the chromosomal aberration data of Anderson and kchardson (1981) and of Dean and Clare (1985). and the peripheral blood micronucleated erythrocyte data of Tice et ai. (1984), Barale et ai. (1985). and Choy et 31. (1985). However. the chromosomal aberration studies were conducted over an exposure period of less than a week in rats. suggesting the possibility for species and/or endpoint differences in response. The previous peripheral blood micronucleated erythrocyte studies conducted in mice were limited to an analysis of NCE populations only. involved different strains of mice and. in two studies. a different route of exposure. Either the effect is mouse strain/ route of exposure-specific or benzene-induced. exposure duration-dependent differences in the rate of erythropoiesis may be responsible for the apparent time-dependent differences in MN-NCE levels observed in these peripheral blood studies. In this context. i t should also be noted that the age of the animal can have a profound effect on the induction of genotoxic damage induced by single acute exposures to ben- zene (Tice et ai.. 1982). suggesting that age-related differences in sensitivity and/or metabolism may confound the interpretation of exposure duration-related data. The suppression of PCE production by exposure to benzene comes as no surprise in view of the large number of studies demonstrating the toxic effects of benzene on the hematopoietic system (reviewed in IARC. 1982; Dean. 1985: Snyder. 1987). Barale et al. (1985) also reported a decrease in the percentage of peripheral blood PCE due to ,benzene. when administered chronically by gavage, but did not present data on temporal or sex-dependent variability. Particularly striking, and a finding unique to this study, was the regmen-dependent persistence of PCE suppression in male mice. Male mice exposed to benzene for only 3 days per week exhibited suppressed erythropoiesis throughout most of the 13-week exposure period. while male mice exposed 5 days per week exhibited an apparent recovery in hematopoietic activity to control levels by about the sixth week of exposure. These data are in agreement with the benzene metabolite studies of Irons (1983) and provide the tirst expenmental evidence that the extent of benzene-induced toxicity can be inFerseIy related to the number of exposure days per week. t ~ t u stincling is in contrast to the studies of .m n et al. (1977). Gill et al. (1980). and Toft et al. (1982). for which it was concluded that chronic exposures were more harmful to bone marrow f a o n and integrity than intermittent ones? Rowever, these investigators used different end- ' points (bone-marrow cellularity. the onset of leucopenia). other species (rats) o r other strains of mice (C57Bli'6. NMRI). and different exposure regimens. I t is not obvious why greater suppression of rvthropoiesis occurred in male mice exposed to 300 ppm benzene for 3 days per week. However, since proliferating cells appear to be much more susceptible to the cytotoxic actions of benzene than are the normallyquiescent stem cells and since benzene appears to inhbit stem cell proliferation. the 3-day exposure regmen may provide a greater opportunity for stem cell mobilization in response to a benzene-induced depletion of differentiated cells and. thus. a greater opportunity for toxicity (R.D. Irons. 1983; Snyder. 1987). The increased variability of PCE frequency encountered among individual control and exposed animals during the later weeks of exposure is of concern, particularly in terms of extended exposure studies. Infections among the animals. due to the repeated blood samplings, and fighting which occurred among some of the mice was probably responsible for such variability. These are factors that need to be addressed in future experiments of this kind. perhaps necessitating increased care in obtaining the blood samples and by caging the animals individually. The observation that the ability of benzene to suppress erythropoiesis and to induce micronucleated erythrocytes was less pronounced in female than in male mice was not unexpected. There are numerous reports indicating that male mice are more susceptible than are female mice to the genotoxic and carcinogenic effects of benzene (reviewed in Dean. 1985). Siou and Conan (1980) . implicated the influence of testosterone in male mice on the metabolism of benzene as the basis for the sex difference in sensitivity. 'The induction of genotoxic and cytotoxic damage in the hematopoietic system of the mouse f o i b w i n g multiple exposures to benzene has proven to be a complex interaction of many variables. An analysis of MN-PCE frequencies throughout the 13 weeks of exposure revealed that the level of genotoxlc damage induced by benzene remained fairly constant during the course of the extended exposure period. These data indicate that interpretations of bone marrow sensitivity based simplv on an evaluation of peripheral blood MNNCE frequencies may be inadequate. Since t h s observation may in part be mouse strain and route of exposure specific. studies to compare the timedependent induction of MN-PCE/MN-NCE in other strains of mice (see Luke et al.. 1987) and by other routes of exposure appear warranted. It was of interest to find that whle the induction of genotoxic damage was independent of exposure regimen. the induction of cytotoxic damage was not. The fact that exposure to benzene for 3 d a y p e r week as opposed to 5 davs per week.resulted, in greater bone-marrow cvtotoxicitv has implica:, ' tions in the extrapolation of animal data to human exposure situations. However, the concentration of benzene ( 3 0 0 ppm) used in ths study greatly exceeds the current and recent occupational exposure limits (a time-weighted average of 1 and 10 ppm. respectively). Furthermore, although 300 ppm benzene was believed to be within the linear portion of the dose range for the absorption of inhaled benzene in mice (Wells and Nerland. 1984). more recent data indicate that this dose lies above the linear portion of the dose-response curve for inhaled benzene metabolism (Sabourin et al., 1987). These factors suggest the need to evaluate t h s observation at lower concentrations of benzene. Regardless. the effects of benzene exposure on PCE production has a number of implications in regard to the interpretation of MN-PCE and MN-NCE data resulting from such exposure situations. The results of this study underscore the need for an analysis of PCE frequency in periph- eral blood studies of MN induction and indicate some future directions for research on the chronic effects of benzene and other agents. Acknowledgements The authors greatfully acknowledge the assistance of V. Miller. .4. Giovanelli and C. Brooks for their assistance in collecting the peripheral blood samples. The authors also wish to greatly thank Dr. E. Margolin for many helpful discussions on statistical analyses, Dr. R. Irons for discussions about benzene. Dr. R. Kutzrnan for helpful comments. This research was supported at Brookhaven National Laboratory by the Drpartment of Energy under prime contract DE-ACOZ76CH00016. Accordingly, the US. Government retains a nonexclusive. royalty-free licence to publish or reproduce the published form of tfus con- tribution. or allow others to do so. for US. Government purposes. References ..\ksov. M. (1985)Benzene as a leukemogenic and carcinogenic agent. Am. J. Indust. Mtd.. 8.9-20. Anderson. D.. and C.R. Richardson (1981)Issues relevant to the assessment of chemically induced chromosome damage in vivo and their relationship to chemical mutagenesis. Mutation Res.. 90.261-272. Barale. R.. F. Giorgelli. L. Migliore. R. Ciranni. D. Casini. D. Zucconi and N. Loprieno (1985)Benzene induces rmcronuclei in circulating erythrocytes of chromcallv treated mice. Mutation Res.. 144. 193-196. BMDP Statistical Software (1985)W .J. Dixon (Ed.). University Press. Berkeley. California. Choy. W.N.. J.T. MacGregor. M.D. Shelby and R.R. Maronpot (1985)Induction of micronuclei by benzene in B6C3F1 mice: Retrospective analysis of peripheral blood smears from the NTP carcinogenesis bioassay. Mutation Res.. 143. 55-59. Coffin. D.L.. D.E. Gardner. G.I. Sidorenko and .M.A. Pinigin (1977)Role of time as a factor in the toxicity of chermcal compounds in intermittent and continuous exposures. 11. Effects of intermittent exposure. J. Toxicol. Environ. Health. 3. 821-828. Cole. R.J.. N. Taylor. J. Cole and C.P. Arlett (1981)Short-term tests for transplacentally active carcinogens. 1. Micronucleus formation in fetal and maternal mouse erythroblasts. Mutation Res.. 89,141-157. Cortina. T.A.. E.W.Sica. N.E. McCarroll. W. Coate. A. Thakur and M.G. Farrow (1984)Inhalation cytogenetics in mlce and rats exposed to benzene. in: H.N. MacFarland. C.E. Holdsworth. J...\. MacGregor, R.W. Call and M.L. Lane ( u s . ) . Applied Toxicology of Petroleum Products. Adv. Modern Environ. Tooxicol.. Vol.. 6, Princeton Scientific Publishers. Princeton. NJ. pp. 81-88. Cronlute. E.P.. J.E. Bullis. T. Inoue and R.T. Drew (1984) Benzene inhalation produces leukerma in truce. Toxicol. Appl. Pharmacol.. 75. 358-361. Dean. B.J. (1969)Chemical-induced chromosome damage. Lab. Anim.. 3. 157-174. Dean. B.H.(1985)Recent findings on the genetic toxicology of benzene. toluene. xylenes and phenols. Mutation Res.. 154. 153-181. 263 Dean. B.J.. and M.J. Clare (1985) p. 168. cited in Dean. B.J. (1985) Recent findings on the genetic toxicology of be- nzene. toluene. xylenes and phenols. Mutation Res.. 154. 153-181. d. Dnscoll. K.E.. and C.A. Snyder (1984) The effects of ethanol injection and repeated benzene exposures on benzene pharmacokinetics. r (icol. Appl. Pharmacol.. 73. 1 3 - 5 3 5 . Federal Register I 1985 . Occupational exposure to benzene: Proposed rule and notice of heanng. 50. 50512-50586. Gill. D.P.. V.K. Jenkins. R.R. Kempen and S. Ellis ( 1980) The importance of plunpotential stem cells in benzene toxicity. T0xicoli)gy. 16. 163-171. Gonasun. L.M.. C.M. Witmer. J.J. Kwsis And R. Snyder (1973) In vivo met.iholisni in mouse liver microsomes. Toxicol. Appl. 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World Health Organtzation International Agency for Research in Cancer. Lyon. France. pp. - -93-148. Irons. R.D. I 1983) Metabolism of benzene and mechanisms of i its toxlcitv. Collegmn R a m m i n i International Conference /I - on Benzene. November 3-4. Grand Hyatt Hotel. New : York. p* Jenssen. D.. and C. Ramel (1978) The micronucleus test as part of a short-term mutagemcity test program for the predic- tion of carcinogenicitv evaluated by 143 agents tested. Mutation Res.. 7 5 . 191-202. Kato. H. (1974) Spontaneous sister chromatid exchanges de- tected by a BudR-labeling method. Nature (London). 251. 70-72. Luke. C.A.. R.R. Tice and R.T. Drew (1988) T h e effect of exposure regimen and duration on benzene-induced bone marrow damage in mice. 11. Strain compansons involving B6C3F1. C57B1/6 and DBA/2 male mice. Mutation Res.. 203. 273-295. MacGregor. J.T.. C.M. Wehr and D.H. Could (1980) Clasto- gen-induced micronuclei in peripheral blood erythrocytes: The basis of an improved micronucleus test. Environ. Lfutagen.. 2. 509-514. Maltoni. C.. and C. Scamato (1979) First expenmental dernonbtration of the carcinogenic effects of benzene: Long-term bioassays on Sprague-Dawley rats by oral administration. .%led. Lav.. 70. 353-357. NTP Technical Report 289 (1985) NTP Technical Report on the toxlcology and carcinogenesis studies of benzene in F244/9 rats and B6C3F1 mice (gavage studies). NIH Publication No. 84-2545. US. Department of Human Services. Pathiratnr. A.. R.L. Puyear and J.D. Brammer 11986) A comparative >cud? of the effects of benzene. toluene. and xvlenes on their in vitro metabolim~and drug-metaboliLlne, enzvrnes in rat liver, Toxicol. Appl. Pharmacol.. 82. 272-280. Post. G.B.. and R. Snyder I 1983) Effects of enzyne induction on mcrosomal benzene induction. J. Toxicol. En\ iron. Health. 1 I . 8 I 1-825. Sabounn. P.J.. T.-H. Chen. G . Lucier. L.S. Birnbaum. E. Fisher and R.F. Henderson 11987) Effect of dose on the absorption and excretion of " C benzene administered orally or hv inhalation in rats and mice. Toxicol. Appl. Pharmacol.. 87. 325-336. Sdlamone. M.F.. and J.A. Heddle ( i -W? I The bone marrou micronucleus ~ s s a v : Rationale for a revised protocol. in: F.J. de Serres and A. Hollaender ( E A . ) . Chemical .Mutagens. Pnnciples and Methods for Their Detection. Vol. 8. Plenum. NCH York. pp. 111-149. Schalm. O.W.. N.C. Jam and E.J. Carrol 11975) Vetennary Hematology. 3rd edn.. Lea and Febiger. Philadelpha. p. 390. Schlegel. R.. and J.T. MacGregor (1982) The persistence of rmcronuclei in peripheral blcod erythrocytes: detection of chronic chromosome breakage in mice, Mutation Res.. 104. 367-369. Siou. G.. and L. Conan (1980) Contribution h la connaissance de I`effet mutagene du benzene. I. Influence de la Matunte sexuelle sur la sensibilite de la souris a I'action mutagene du benzene. Cahers Notes Docum. INRS. 101. 531-534. Sittig. M. (Ed.) (1979) Benzene. in: Hazardous and T o m Effects of lndustnal Chemicals. Noyes Data Corp.. Park k d g e . NY. pp. 52-59. Snyder. C.A. (1987) Benzene. in: R. Snyder (Ed.). Ethyl Browrung's Toxlcity and Metabolism of lndustnal Solvents. Elsevier, Amsterdam. pp. 3-37. Snyder. C.A.. B.D. Goldstein. A. Sellakumar. S.R. Wollman. 1. Bromberg. M.N. Erlichman and S . Laskin (1978) Hematotoxicity of inhaled benzene t t i Sprague-Dawley rats and AKR mice at 300 ppm. J. Toxicol. Environ. Hlth.. 4. 605-618. Snyder. C.A., B.D. Goldstein. A.R. Sellakumar. I. Bromberg. S. Laskm and R.E. Albert (1980) The inhalation toxicology of benzene: incidence of hematopoietic neoplasms and hematoxicity in AKR/J and C57B1/6J mice. Toxlcol. Appl. Pharmacol.. 54, 323-331. Snyder. C.A.. M.N. Erlichman. S. Laskin. B.D. Goldstein and R.E. Albert (1981) The phamacokmetics of repetitive benzene exposures at 300 and 100 ppm in AKR mice and Sprague- Dawley rats. Toxicol. Appl. Pharrnacol.. 57, 164-171. Snyder. R.. F. Uzuki. L. Gonasun. E. Bromfield m d A. Wells (1967) The metabolism of benzene in vitro. Toxicol. Appl. Pharrnacol.. 11. 346-360. Stoner. G.D.. P.B. Conran. E..4. Greisiger. J. Stoher. M. Morgan ;ti.i \i .A Pereira (1986) Comparison of two routes of cnemcsl sdrmnistration on the lung adenoma response in ,\rain .A,, J rmce. Toxicol. Appl. Pharrnacol.. 82. 19-31. ' ~ e K. R.. m d J.L. Ivett (1985) Cytogenetic ..nalysis of bone rnsrrow damage. in: R.D. Irons (Ed.). Toxicology of the B l m d .md Bone bf3rrOw. Raven. New York. pp. 119-140. 1.1ie. K R D.L. Costa and R.T. Drew (1980) Cytogenetic effects of inhaled benzene in murine bone marrow: Induct i a n of \isier chromatid exchanges. chromosomal aberra[ion\. Jnci cellular proliftration inhibition in DBA/Z mice. Proc. Yi3t1. Acad. Sci. (U.S.A.). 77. 2148-1152. Tiie. K.R.. .T.F. Vogt and D.L. Costa (1982) C>togenetlc cffttcn of inhaled benzene In murine bone marrow. in: R.R. T~ce.D.L. Costa and K . M . Schaich (Eds.). Genotoxic Effects of Airborne Agents. Plenum. Yew York. pp. 757-275. Tice. R.R.. M.J. Sawey, R.T. Drew and E.P. Cronlclte (1984) Benzene-induced micronuclei in the peripheral blood of rmce: A retrospective analysis. Enwon. Mutagen.. 6. 421. Toft. K.. T. Olofsson. A. Tunek and M. Beriin (1982) Toxic effects on mouse bone marrow caused by inhalation of benzene. Arch. Toxicol.. 51. 195-302. Wells. M..and D.E. Nerland (1984) Nonlinear relationships between benzene exposure and blood levels of benzene in mce. Fed. Proc.. 43. 759. White. M.C.. P.F. Infante and B. Walker Jr. (1980) Occupational exposure to benzene: A review of carcinogenic and related health effects idlowing the U.S. Supreme Court Decision. Am. J. Indust. .Med.. 1. 233-243. 765 nI-no-w rw- ?n05.0lPmnm*QOINr O?0mN0 000500 0000s's' " ?O ooor'*o3 3 0 .. a -Idri 3 a! -C!oe aE m OP-NON N "- ' 90 -PmNNO 3n- 0 ; PrnVLPOI- cd d -omo~n I- Yzoz9 " "0 mln 01.0 , 266 --Nm-N N "- r0! 0-c30m 0 "-? 00 -0omo- n ?-? 30 -r?NONN N ?-? -0 --wo-- Y 3; NPO-NO Y *P -c 0-0-0- CI 2: Nr-n NNN -9- ??I 300 = w N v m- 3 2 - -Q InN 3 . m n p "OXNN 91 19 0- v "? 03 2% 3.14 N 19 -0 3. 99 -0 r? Y -. -0 9 1Y -0 N '?? NO - lv, nP( 2 29 m y0! -0 - -F. 0 9 0N YY NO 3.10, " E. 2 2n Q-O C N - ~ 0 H P a99 -0 II N I- ? ? NO nu, ?Y -0 E Em 267 APPENDlX 2 I N D I V I D U A L ANIMAL DATA FOR MY-NCE FREQUENCY .9. 1 . 4 ) D B A I.?ilale .Vice 1 2 2 4 5 6 \lean SEkl 1 2 2 4 : 2 2 1 4 1 3 1 4 3 2.3k0.30 '102 2 0 1 2 2 2 0 0 4 1.5f0.33 201 2 0 1 0 1 2 0 3 I 1 3 1.2 t0.28 1 2 3 1 1 I) I 13333 1 * 1 1.8t0.28 1 0 5 1 2 0 3 2 0 2 2 1 1 1 !.Sf0.36 1121 I 4 0' 30 52 4 I 1.9t0.41 1.3 1.0 2.2 1.8 1.5 1.5 1.0 1.3 2.3 1.3 3.0 1.3 2.0 2.2 0.21 0.36 0.70 0.48 0.43 0.62 0.52 0.21 0.56 0 4 9 0.45 0.42 0.68 0.54 Reqrnwr i. Benzene concentratton = 300 ppm 13 129 6 1 7 2 0 IS 43 17 50 35 35 14 3 3 2 4 9 1 3 21 14 28 23 39 4.4 15 o 2 15 16 30 '0 --34 4.4 11 26 3' 16 1'4 4 1 2 1 2 15 16 19 33 3 1 41 17 145 819 5 17 45 41 32 3; 37 18 049 8 2 7 1 1 74 35 17 24 33 23 5 2 43 '4.8 2 4 76 19 4s 22.2 5.04 *81 59 27.6 2 6.00 50 ( 46 )a '0.4 4 49 37 47 23.9 2 4.58 53 40 22.0 2 4.01 Mean 1.0 3.3 5 . 2 7.5 16.7 15.2 19.2 31.2 27.7 30.7 33.5 35.3 55.3 47.4 SEhl 0.45 0.49 1.30 1.67 2.54 3.55 1.30 5.41 4.99 4.32 1.89 3.02 5.92 3.23 Reyqrn7rn 2. Benzene concenrrarion = 0 ppm -7 0 2 1 1 0 4 7 I 1 2 1 1 2 2 1.420.27 8 2 2 0 I O 4 0 1 1 I 1 1 3 2 1.4t0.31 9 0 0 0 1 3 2 2 0 1 1 (2lU 2 3 3 1.4t0.31 10 11 -12 1 1 0 2 1 2 ,' I 1 0 (0)U 0 0021 2 1 0 2 0 1 4 1 01000 3 4 0 0 2 1 3 3 -7 1 3 1.250.28 3 1.420.32 1 l.2r0.37 Mean 0.5 1.0 0.5 1.0 1.0 2.7 1.7 0.8 0.7 1.2 2.0 1.3 2.5 2.3 SEM 0.34 0.36 0.34 0.26 0.52 0.49 0.62 0.31 0.21 0.31 0.71 0.42 0.22 0.33 Reyrnen 1. Benzene concentration = 300 ppm 19 0 1 1 3 7 14 19 11 11 15 (17)u 19 13 14 10.4t1.78 20 0 4 5 8 9 8 9 6 22 11 19 20 11 13 10.4k1.69 --21 0 3 3 5 IO 9 10 14 3 7 13 21 13 26 9.8k1.95 17 0 3 9 16 11 19 21 6 13 IO 9 71 25 23 13.3k2.07 23 2 7I 3 5 19 10 10 18 18 IO 20 16 17 1 0 . 8 ~ 1 . 9 2 74 1 1 4 4 9 1 6 7 1 6 1 0 4 1 4 1 6 4 14 8.6+ 1.58 Mean 0.5 2.5 3.8 6.5 8.5 14.2 12.7 10.5 12.8 10.8 13.0 19.5 13.7 17.8 SEM 0.34 1.02 1.22 2.04 3.47 1.96 2.38 1.67 2.70 2.09 1.76 0.76 2.30 2.21 269 APPENDIX 2 (continued) ( 8 )D BA / 2 Female Mire ~ Animal Week number 0 1 2 3 4 Regimen 1 . Benzene concentration = 0 pprn 25 003 I? 26 210 22 27 021 11 28 122 10 29 003 01 30 I10 32 Mean SEM 0.7 1.0 1.5 1 . 3 1.3 0.33 0.36 0.56 0.42 0.33 56 21032 11011 10121 01030 01333 1'132 0.8 I O 0 8 2.7 1.5 0.31 0.26 0.48 0.21 0.43 Temporal average I l 12 13 ( M e m + S E M ) 1 5 3 I 1.750.38 3 0 1 I 1.2+0.24 1 1 1 2 1.1 ~ 0 . 1 8 3 0 3 0 1.1 20.33 3 2 2 I 1.5 r0.33 I 3 I 0 1.520.27 I 8 I S 1.0 0.8 0.40 0.79 0.36 0.31 Reqrmen /. Benzene concentration = 300 pprn 37 0 0 1 I4 5 14 6 3 9 4 7 9 13 6.6 c_ 1.29 38 032 9 11 14 1 1 12 17 13 15 IS 17 22 11.7k1.71 39 2 1 4 16 6 IO 4 9 7 1 3 1 0 7 5 5 7.1 k 1.12 40 0 2 5 10 16 16 11 3 7 14 4 IO 17 20 9.6k1.69 41 0 I 2 11 7 I O 2 6 4 12 9 5 7 7 5.9k 1.01 42 0 1 2 6 6 9 2 1 1 5 7 7 5 7 8 5.450.85 Mean SEM 0.3 1.3 2.7 11.0 8.5 12.2 6.0 7.3 8.2 10.5 8.7 8.7 10.3 12.5 0.33 0.42 0.62 1.46 1.73 1.17 1.69 1.61 1.90 1.65 132 2.01 L.17 2.91 Regimen 1. Benzene concentration = 0 pprn 31 0 2 0 0 2 2 2 1 0 2 4 4 I 1 1.6k0.36 32 1 1 0 2 0 0 1 1 1 1 1 0 1 2 0.9k0.18 33 101 10 Ill20 1 I 4 0 1.0k0.28 34 0000025320 2 1 0 3 1.3k0.42 35 0 0 2 2 1 l l 2 l O 2 2 3 5 1.6k0.36 36 210 IO 11000 U 0 0 1 0.5k0.18 Mean SEM 0.7 0.7 0.5 1.0 0.5 1.2 1.8 1.5 1.0 0.5 2.0 1.3 1.5 2.0 0.33 0.33 0.34 0.36 0.34 0.31 0.65 0.43 0.36 0.34 0.55 0.62 0.67 0.73 Regimen 2. Benzene concentration = 300 ppm 43 201 23 3 44 301 6 4 I2 45 1 3 2 2 9 9 8 46 a 47 332 32 5 48 110 396 6 1 1 5 3 5 4 3 4 7 1 7 3 6 3 4 3 5 6 5 3 10 2 17 86 (8)u9 3 12 3 4 3.4k0.67 4 22 6.1 k1.73 (7)a (6)a 5.9C0.91 6 9 5.1k0.64 3 6 4.4k0.89 Mean SEM 2.0 1.4 1.2 3.2 5.4 7.0 4.6 6.6 4.0 4.0 4.6 10.8 4.0 10.2 0.45 0.68 0.37 0.74 1.50 1.58 1.21 1.69 1.10 0.71 0.51 1.83 0.71 4.05 Number ofMN-NCE per lo00 NCE means and standard error ofthe means (SEM) calculated for each exposure group at each sample time. The mean responsefthe SEM across time for each mouse is presented as a temporal average. Abhorortons: a. animal died; u. slides unscorable due to techrucal reasons: ( ), estimated value by averaging two nearest data points. these values used in the calculation of the temporal averages but not in the calculatlon of the weeklymeans. number 0 1 2 3 4 5 6 7 8 9 10 11 12 13 average (Mean 5 SEM) Rtyinirrr 1 . Benzene concentration = 0 ppm 1 31 64 30 29 26 34 5 46 60 15 -3 36 5 2 67 41 17 114 8 42 50 7 3 73 14 38 62 32 18 10 62 23 37 3 33 38 40 33 26 48 14 25 51 33 5 76 43 48 32 20 10 13 46 64 34 6 36 49 37 34 22 2.5 14 30 24 14 42 21 16 41 46 49 59 35 6 33 44 12 77 36 24 34 57 5 35 33.1k4.46 23 42.4k7.20 52 33.6k5.14 34 33.Ik3.00 15 34.8t5.19 65 32.6k4.58 M e a n 33 2 4 . 3 43.3 38.5 23.8 41 5 10.7 41.8 45.3 23.3 47.7 39.X 18.7 37.3 SEXt 1.57 6.59 5.29 4.97 2.17 15.46 1.50 5.37 7.24 5.22 6.99 4.98 6.70 7.53 Reqrnien I . Benzene concenrrarion = 300 ppm 13 37 3 0 5 14 7 0 15 17 77 14 36 1 0 1 8 19 17 I 2 37 29 15 23 0 3 8 7 10 I O 12 24 74 16 2 6 0 0 4 8 2 8 3 5 48 17 15 0 0 2 10 4 18 4 16 11 18 1 1 2 2 8 5 5 7 8 16 18 27 14 10 12 15.5+S.54 23 11 28 39 9 9 13.5+ 3.10 9 110 25.7k8.92 39 35 36 (36)a 17.4k3.61 0 27 9 23 9.5t2.62 956 6 7.5k 1.31 Mean 75.5 1.0 0.8 4.7 8.7 7.8 10.0 9.3 19.2 42.8 21.0 21.8 13.2 32.0 SEM 4.36 0.52 0.54 1.20 1.26 2.50 2.14 1.78 4.35 11.53 5.79 5.65 4.60 19.71 Regrmen 2. Benzene concentration = 0 ppm 7 31 40 83 40 21 21 8 13 34 40 14 27 16 22 30.9t 5.22 8 32 44 55 28 19 11 8 45 26 40 20 39 6 116 36.1+ 7.89 9 25 35 31 57 31 25 20 19 12 20 (30)u 41 26 104 34.05 6.13 I O 23 71 77 55 7 64 10 43 22 50 (36)u 23 14 155 46.4k10.34 11 24 32 31 47 37 7 25 42 18 24 29 52 18 41 30.6+ 3.58 12 37 45 63 54 I8 4 13 27 30 47 61 27 24 73 35.55 5.57 Mean 28.7 44.5 56.7 46.8 23.2 22.0 14.0 31.5 23.7 36.8 31.0 34.8 17.3 85.2 SEM 2.26 5.68 9.07 4.56 4.37 9.03 2.86 5.61 3.28 4.98 10.46 4.52 2.95 20.22 Regimen 2. Benzene concentration = 300 ppm 19 3 7 1 1 7 1 3 4 3 3 6 (8)u 11 I 17 5.1k1.32 20 2 1 0 5 3 7 1 7 2 2 3 8 1 4 7 3.8k0.75 21 17 0 0 24 2 1 0 38 1 11 350 8 7.2k3.18 22 26 3 13 1 16 0015 9 5 0 5 1 11 6.1k1.67 23 2 8 0 0 1 1 2 5 9 0 3 1 14 1 22 4.5* 1.85 24 3 4 0 0 3 3 0 2 9 0 6 1 0 1 3 2.2k0.76 Mean 27.2 0.7 3.2 6.5 5.0 1.2 3.0 12.7 2.5 5.7 2.6 6.0 1.3 11.3 SEM 3.09 0.49 2.12 3.61 2.38 0.48 1.15 5.42 1.38 1.20 1.44 2.25 0.56 2.86 APPENDIX 3 (continued) -( A ) D BA / 2 Femaie Mice Animal Week 27 1 Ternooral Regimen I . Benzcne concentration = 0 ppm 25 18 50 38 51 24 16 15 27 18 42 26 31 77 30 41 36 46 10 34 43 30 27 23 43 40 45 24 24 26 30 4b 40 28 18 45 37 86 27 I5 24 45 14 56 29 24 64 40 49 42 I 5 18 47 39 57 30 33 55 34 48 30 19 29 31 45 28 48 21 7 68 31.624.77 33 27 17 68 37.4e4.74 27 39 5 77 34.9t4.43 19 13 12 11 33.0k5.48 32 36 18 82 40.2k5.08 25 43 37 22 3.7.5_+2.80 Mean 24.5 55.7 36.5 53.3 30.5 22.5 20.3 35.7 34.2 2.2 30.7 31.5 14.3 59.7 SEM 2.59 5.26 1.59 6.69 2.94 4.90 2.95 3.40 5.85 5.05 4.04 3.70 3.30 9 5 0 Regimen I . Benzene concentration = 300 ppm 37 32 14 46 23 8 10 5 5 14 25 38 40 0 4 4 1 7 9 1 5 16 22 14 39 22 3 41 33 9 5 25 19 28 18 40 16 2 26 6 3 11 9 34 31 6 41 30 21 36 35 15 58 38 68 16 58 42 16 5 28 63 24 9 7 32 49 7 IO 69 5 1 4 28.Xi10.62 16 '3 io 52 18.0k 4.28 10 23 3 37 19.55 3.60 30 15 12 36 17.0k 3.48 5 53 11 68 37.1 5 6.17 24 31 8 20 23.62 4.88 Mean 26.0 7.5 36.8 26.8 11.0 SEM 3.93 3.35 3.41 9.18 3.04 -Regrmen 2. Benzene concentralion 0 ppm 31 31 50 29 36 39 32 19 35 23 48 30 33 17 33 34 50 14 34 37 42 40 57 21 35 23 48 61 50 27 36 21 41 53 53 13 17.0 8.24 11 30 19 28 16 23 16.5 5.21 45 26 26 25 29 38 29.0 8.94 26 22 40 68 22 30 26.7 5.21 38 21 14 12 13 26 21.3 7.88 49 40 39 61 33 44 15.8 3.88 13 36 16 49 32 (47)u 36.5 8.32 27 7 33 37 29 50 8.2 58.8 1.45 17.54 7 100 17 182 19 91 14 134 14 124 5 137 35.8+ 6.11 38.3+11.40 32.2+ 5.82 44.6+ 8.24 37.2+ 7.66 41.5 8.38 Mean 24.7 41.5 40.0 49.0 24.0 21.2 31.5 34.7 20.7 44.3 29.2 30.5 12.7 129.0 SEM 3.16 2.77 5.93 2.90 4.08 2.96 3.33 7.20 4.11 3.98 6.64 5.76 2.26 12.63 Regmen 2. & w n c concentration = Mo ppm 43 40 3 20 48 14 2 16 I5 16 58 13 61 3 141 31.5k 10.70 44 42 14 2 13 19 1 20 54 0 13 32 21 0 118 23.6_+ 8.90 45 24 9 1 10 10 2 7 43 7 31 22 18 (20)a (19)a 15.3k 3.32 46 a 47 23 23 1 9 11 42 46 52 11 59 (54) 49 99 82 41.41 8.20 48 25 33 36 50 24 1 36 29 21 14 33 31 2 242 42.5k17.06 Mean 30.8 16.4 12.0 26.0 15.6 9.6 25.0 38.6 11.0 35.0 25.0 36.0 26.0 145.8 SEM 4.19 5.29 7.01 9.42 2.62 8.10 7.04 7.37 3.62 10.11 4.71 8.27 24.34 34.30 Number of PCE per lo00 erythrocytes with means and standard error of the means (SEM) calculated for each exposure group at each sample time. The mean response+- the SEM across time for each mouse is presented as a temporal average. Ab&euiarronr: a. animal died: u. slides unscorable due to technical reasons: ( ), estimated value by averaging two nearest data points. these values used in the calculation ofthe temporal averages but not in the calculation of the weeklv means.