Document GOdn1K632RGZKnRV603a8Xdx

Toxicology Program, Departments of Biology' and Animal. Dairy & Veterinary Sciences," Ulah State University, Logirn. Utuh 84372 Abstract. Benzene is a known contaminant found in trace amounts in ground water. I t has long been associated with myelotoxicity and associated immunologic effects. The present study concerned the immunotoxic potential of benzene following four weeks of continuous oral administration via drinking water at concentrations of 0 , 31, 166 and 790 mg/L. Benzene-treated water produced a doserelated decrease in spleen weight and increase in kidney weight; both were significantly different at the highest level. Benzene exposure caused a significant dose-response reduction of peripheral blood leukocytes, lymphocytes, erythrocytes and resulted in a severe macrocytic anemia. Splenic lymphocyte proliferation to both B cell and T cell mitogens [lipopolysaccharide (LPS), pokeweed milogen (PWM), concanavalin A (Con A) and phytohemagglutinin (PHA)] was followed by a dose-related biphasic responsiveness, enhanced at the lowest dose (31 mdL) and depressed in the higher dosage groups (166 and 790 mg/L). Cellmediated immunity as measured by mixed-lymphocyte culture (MLC) response to allogeneic cells and cytotoxic T lymphocyte (CTL) activity to YAC-I tumor cells exhibited similar biphasic phenomenon. Antibody production as assessed by enumeration of the sheep red blood cell (SRBC)-specific plaqueforming cells (PFC) indicated a significant suppression of PFC in animals exposed to 166 and 790 mdL benzene. A decrease in the a-SRBC-antibody titer corresponded to the numbers of PFC. The findings suggest that oral ingestion of benzene, at I Address correspondence to R. P. Sharma, Utah State Univerbity, Logan, Utah 84322-5600. the concentrations utilized, produced a biologically significant immunotoxic effect on both the humoral and cellular immune responses. Concern for the hazards of pollutants present in ground water is attracting attention, since ground water is a major source of drinking water supplies. The major concern is the possible subclinical effects after long-term exposure. The immune system as a target organ for detecting toxicity is being studied, since its sensitive parameters are easily evaluated (Bick rt ul. 1985; Dean et ctl. 1982). A number of industrial and environmental chemicals induce immunosuppression (Faith et d 1980). Benzene, a ubiquitous environmental pollutant, exerts its greatest toxicity on hematopoietic cells, including erythroid, myeloid and lymphoid lineages, both in the peripheral blood and in the hematopoietic organs (Fishbein 1984; Bolcsak and Nerland 1983). Repeated benzene exposure in humans and laboratory animals induces various forms of cytopenia, aplastic anemia, leukemia and the dcvelopmen1 of chromosomal abnormalities (Dean 1985; Green et ul. 1981; USEPA 1980; Brief rf (11. 1980). The mechanism for benzene-induced hematopoietoxic effects has not been well elucidated; however, most researchers believe oxidative benzene metabolites, rather than benzene itself, are required to mediate these hematopoietic effects of benzene (Sawahah et rrl. 1985; Irons 1985; Bolcsak and Nerland 1983; Greenlee et al. 1981). Lymphocytes in animals are sensitive to benzene toxicity (Rozen and Snyder 1985; Denipster et ul. 1984; Snyder et uf. 1982; Green et af. 1981;Aoyama 1986), as are those from humans who are inadver- * 152 Ci. C. Hsieh cf d. tently exposed to this chemical (Moszczynsky and Lisiewicz 1984; Brief ct al. 1980). Benzene-induced depressions in lymphocyte counts may impair immunocompetence, since lymphocytes play a principal role in immune functions. The short-term administration of benzene by inhalation or intraperitoneal injection to experimental animals has caused abnormalities of immune-associated parameters, including a suppression in mitogenic response of Band T-lymphocyte proliferations (Rozen and Snyder 1985; Rozen et (11. 1984; Wierda et rrl. 1981; Aoyama 1986), impaired humoral antibody rcsponse as mcasurcd by plaqiic-forming cells to sheep erythrocytes (Wicrda et (11. 1981 Aoyama 19861, and increased susceptibility to the pathogenic microorganism Listerici motzocyiogenes (Rosenthal and Snyder 1985; Aranyi (JIal. 1986). Similarly, treatment with benzene metabolites has also impaired the immune responses (Pfeifer and Irons 1982; Wierda and Irons 1982). The inbred C-S7BL mice used in experiments described above may harbor an endogeneous lymphoma virus (Kaplan 1967);activation of this latent virus after benzene treatment may influence the toxicity of benzene (Snyder et al. 1980; Longacre et til. 1981). The outbred CD-1 mice were used in the current study. There are relatively few reports on effects of benzene after oral ingestion. Because some water supplies may be contaminated with benzene (USEPA 1980). oral administration was employed via drinking water for a 4-week period. ceived untreated tap water. To minimize decomposition and lo maintain the concentration of benzene, drinking water was provided in glass water bottles, shaken frequently during trealment and was changed every 3 days. Feed and water consumption WRI monitored continuously. and animals were weighed once each week. Henzene concentration in drinking water was confirmed o n different days by gas chromatography (USEPA 1982). Gross Ohservcitions and Hematology Twenty-eight days after exposure to benzene, mice were killed hy dccapilation. iind gross piilhological examinations were performed o n ;ill mice. Major organs, ;.e., liver, spleen, thymu5. and kidney were removed. trimmed and weighed. Blood sarnplec were collected in siliconized test tubes coated with potassiiim ethylene-diaminetetraacetate. Leukocytes and erythrocyte5 were counted with an automated electronic cell counter (Model ZBI. Coulter Electronics, Inc, Hialeah, FL). Hematocrits were performed with microhematocrit equipment. Differential leukocyte counts were evaluated by Wright's-Giemsa stained smears. I.solntioti atid Ciiltiire of Splenic Lymphocytes After sacrifice, the spleen was aseptically removed from each animal. rinsed in icecold sterile isotonic saline and single cell suspensions were prepared and cultured according to Sharrnit and Gehring (1979). Cells were suspended in RPMI 1640 media (Gibco Laboratories, Grand Irland, NY) supplemented with 5% heat-inactivated fetal calf serum (Hyclone Sterile Systems. Inc. Logan. UT) and 100 units penicillin and 100 CL streptomycin per mL. Cell counts were made with a Coulter counter. Materials and Methods A tI imciIs Male, and adult CD-I mice (Charles River Breeding Laboratory Inc.. Wilmington. MA) were procured at 5 to 6 weeks of age (approximately 18 g) and were acclimatized to the animal care facility for one week before use. Mice were randomly assigned to control and treatment groups and housed five per group in plastic cages with hardwood-chip bedding. They were maintained on IiiboGitory rodent chow and tap water ctd / i h i f u m . Room conditions were kept at ;in ambient temperature or21 2 IT and ;I relative hiiinidity or 50 2 IO%. The light/d;irk cycle W ; I ~m;iint;iincd ;it 12-hr intervnls. Analytical reagent grade benzene (YY.976 purity. JT Baker Chemical Co.. t'hillipsburg. NJ), was dissolved in normal tap walcr to mikc the intended lcvcls of 40. 200. and 1,000 mgil,. IknLene has a colubility of 1.780 mg/L in water at 25C (USEPA 1980). The benzene-treated water was administered to mice continuously for 2X days via drinking water; the control group re- Lymphocyte Prol$era tiorz to Mitogens Splenic lymphocytes were plated in triplicate cultures ( 5 X 10' cells/culture well) in 96-well flat-bottom microtiter plates (Microtest 11, Falcon Plastics, Oxnard, CA) to assay their responses to E. coli lipopolysaccharide (LPS; Sigma Chemical Co. S I . Louis, MO), pokeweed mitogen (PWM; Gibco Laboratories. Grand Island, NJ), concanavalin A (Con A; Sigma Chemical. St. Louis. MO), o r phytohemagglutinin (PHA: Wellcome Kcagents Ltd. Ijeckenham, England). Culture media with or without mitogen was added, total well volume was adjustcd lo 0. IS rnl,. The optimized conccntrations of mitogens were 20 pg/mL, 30 pg/mL, 2.5 pg/mL, and 6.0 p g h L for LPS. PWM. Con A and PHA, respectively. Cultures were incubated at 37C in a humidified atmosphere with 3.5-4.0% CO, for 48 hr and harvested after a 6-hr pulse with 0.5 pCi of [methyl--'H]-thymidine OH-TdR; New England Nuclear, Boston. MA) per well. l'he cells were collected on a glass fiber filter using an aulomated multiple sample cell harvester (Model M 12. Biomedical Hcwarch and Ilevelopment Laboratory, Rockville, MD). The amount of radioactivity (dpm) incorporated was measured with a scintillation counter (Model 2660. Packard Instrument CO. Downers Grove, ILL r L: It A T If IO tH c1 fS PI $m Rw L SI b) 3. sc C C! SE SP CC mc cu in efl tar ba let' , we PiU me "C mc tar PI( N II the nin gro she the x It splc 10% PA 1 imn calc tola Et 1 I>Oll Wac Cll. the Inim~~notoxicitoyf Benzene in Drinking Writer Mixed Lytnphocyte Cirltiire (MLC) Responses The proliferation of splenic lymphocytes (responders) from benrene-treated or control animals in response to challenge with allogeneic YAC-I tumor cells (stimulators) was evaluated. The exponential growing phase YAC-I cells previously maintained in culture medium were treated with 50 p,g/mL mitomycin C (Sigma)for I hr at 3 7 T , then washed 3 times immediately before putting into culture. Cultures were set up in triplicate in 96-well microtiter plates. Responder cells from the spleen of CD-I mice were cocultured alone or with stimulator cells at a responder,timulalor ratio of 2: I . The proliferative response was ossessed by Imethyl-JHI-thymidine incorporation during the last 24 hr ol'u 3day culture. The cultures were harvested and counted as dewibed for the mitogenesis assays above. Cytotoxic T-Lymphocyte (CTL)Responses Cylotoxic T-lymphocyte (CTL) assay was performed as described by Grabstein (19x0). The splenic lymphocytes (responder cells) were incubated with mitomycin C-treated YAC- I cclls (stimulator cells) for 5 days at 37C in a 3.5-4.0% CO, 211niusphere. These incubated splenic cells (effector cells) were cocultured in triplicate with JiCr-labeled YAC-I cells (target cells) in a total volume of 200 pL in the microtiter plater. The desired ctlkctor-to-target cell ratios were 50: I and 25: I . Effector and target mixtures were spun for 1 min at 1000 g, and then incubated for another 4 hr at 37C. After incubation, cells were pelIeted by centrifugation for 10 min at I(W0 g, and the supernatants were decanted and counted in a gamma counter (Model 3320, Rckard). The labeled target cells in the presence of complete medium or 0.5% sterilized saponin served as the spontaneous W r released and total "Cr released, respectively. The triplicate mean was used to calculate the percentage of specific lysis of target cells as described earlier (Crabstein 1980). I'lriyire Fortnitig Assuy Numbers of IgM plaque-forming cells (PFC)were determined by the method of Jerne and Nordin (1963) as modified by Cunningham and Szenberg (1968). Each mouse from control and test groups was sensitized by i.p. injection with 0.25 mL of a 20% sheep red blood cell (SRBC) suspension 4 days before the end of the benzene exposure. Splenic cell suspensions were prepared ( I x IO7 cells/mL) as described above. A mixture containing 45% splenic cells suspension, 45% of a 10% SRBC suspension, and 10%guinea pig complement (Cappel LdbordtorieS. Cochranville, PA) was delivered to counting chambers, three replicates per aninial. Cultures were incubated at 37C for I hr and PFC were calculated and expressed as plaques/106 splenic cells or plaques/ total splenic cells. Etirytne-linked Itnrniriiosorbent Asscry (ELISA) Double sandwich enzyme-linked immunosorbent assay (ELISA) was used to analyze anti-SRBC (u-SRBC) antibodies (Voller C I ul. 1979). Serum samples were collected from mice utilized in the plaque-forming assay. Plates (Gilford polystyrene EIA cu- 15.3 vettes, Medical Instruments Laboratories, Inc, Oberlin, OH) were coated with 200 p L goat red blood cell ghosts (prepared :is described by Hannhan and Ekholm 1974) containing 3.6 pg/mL protein solubilized in 0.1% sodium dodecyl sulF;ite, and kept at 4C for 24 hr. After several rinses, 200 p L aliquots of sera (dilution 1:150) were added to the plate wells and incubated for 2 hr at 37C. and rinsed. Subsequently, 150 p L peroxidase-conjugated goat anti-mouse immunoglobulins (Hyclone Sterile Systems, Inc, Logan, UT) diluted 1:2000 were pipetted to each well to determine a-SRBC antibody levels. and incubated another 3 hr at 37C. The enzyme reaction was performed by adding the phosphate-citrate buffered substrate solution containing 0.4 mg/mL o-phenytenedixnine (Sigma) and 0.4 pWmL H,Oz. prepared immediately belbre use. The results were expressed as change in absorbance (AOI)) 15 niin Lifter the addition of stibstr;ile solution. Non-5ensitiLed mice pooled ser;i were used as ii negative control. The optical density was determined by ;in automated spectrophotometer (Gilford. Model EIA 50, Oberlin, OH); readings were made at 490 nm. The results are expressed as the means and their standard errors. Statistical significances for differences between benzeneexposure and corresponding normal water control values were determined by a one-way analysis of variance (ANOVA) design followed by multiple comparison procedures of Fisher's least significant differences (LSD) test (Dowdy and Wearden 1983). when F ratios indicated significant difference. A level of p < 0.05 was considered statistically signiticant. Results The average concentration of benzene in drinking water as determined by gas chromatographic analysis on several days is indicated in Table I . There was a 10% loss of benzene on the first day, an additional 10% loss per day followed on second and third day, when the water was changed. Based on observed average intake of benzene-treated water, the daily dose of benzene in each animal was estimated to be 0, 8, 40 a n d 180 mdkg, respectively. Body rind Orgtrti Weig1it.v Continuous exposure of adult, CD-I male mice to various levels of benzene via drinking water over a 4-week period did not produce overt clinical symptoms of toxicity. Overall, there was no change in food and water consumption. No gross lesions were observed on any of the organs of mice in all treatment groups. There was no apparent alteration in body weight gain at termination of the study. Although the two groups receiving higher doses (40 and 180 mg/kg/day) had a slight increase in growth ` 154 G . C. tisieh til. Tahle I . 0rg;tn and body weights of mice following 4 weeks of benzene exposure" Concentration in water. mgiL Nominal Observed` 00 40 31 200 166 Io00 790 Dose nig/kg/day Control 8 40 I80 Body weight (gib Day 0 Day 28 23.20 t 0.37 22.68 t 0.33 23.74 t 0.22 23.14 f 0.29 31.22 2 0.62 29.94 t 0.98 32.98 t 0.65 32.26 +- 0.86 Organ weight (g/IM g body weight)b Spleen 0.33 2 0.02 0.30 f 0.01 0.28 t 0.01 0.26 t 0.01* Liver 5.57 t 0.13 5.46 t 0.29 5.86 2 0.07 5.98 t 0.20 Kidney ~~~ ~~~ 1.55 f 0.06 1.58 f 0.08 1.65 t 0.10 1.87 t 0.05* - Thymus ~~ 0.16 lr o.o! 0.12 2 0.01 0.12 2 0.01 0.11 t 0.01 a Benzene was administered continuously via drinking water for 4 weeks Values are given as mean t SE (n = 5) The observed overall ranges of benzene concentration were 28 l o 36. 147 to 178. and 734 to 855. i n the three treatment groupc. respectively * Significantly (p < 0.05) different from control values Tahle 2. Effects o f benzene exposure o n selected blood paramelen Leukocyte absolute differentials Control 8 40 I 80 ~ 7.01 t 0 . 6 ~ c o x t 0.58 4.57 t 0.2X* 48.80 t 0.46 3 . 6 ~ o . ~46.20 c 0.40* 3 42 -c 0.39* 45.00 t l.22* 74.01 5 6.03 IOX.30 2 6 4Y* I ? ~ . I cX IS.^ 136.76 2 12.57* 5.96 t 0.60 4.23 -c 0.07* 4.22 2 0.57* 2.X7 t O.l6* Benzene was administered continuously via drinking water for 4 weeks Mean corpuscular volume Including monocytes. eosinophils and basophils Values are given as mean t SE (n = 5 ) * Significantly (p < 0.05) different from control values 4.38 t 0.52 2.Y9 t 0.07* 2.35 2 o.3o* 1.61 t O . l O * ~ 1.10 t 0 . 1 3 0.85 f 0.10 1.46 t 0.24 1.02 2 0.07 0.45 t 0.m 0.38 2 O(1C 0.40 t o w 0.25 t OOY rate, growth was not significantly different than the controls (Table 1). The weights of selected organs are also presented in Table I . Weights of spleen, a secondary lymphatic tissue, decreased in a dose-related manner. Kidney weights correspondingly increased. Thymus weight was reduced in all treated groups but not significantly. after oral ingestion of benzene. Lymphocyte population decreased significantly when leukocyte tlifferentials were expressed on an absolute basis. Thc number of neutrophils and other white blood cclls (monocytes, eosinophils and basophils) were not altered by the benzene exposure. Hematological Paratneters The effects of benzene exposure on erythrocyte counts, hematocrits, mean corpuscular volumes (MCV), leukocyte counts, and leukocyte differential counts are presented in Table 2. Erythrocytes decreased significantly as dose increased. Hematocrits were depressed significantly at the 40 and 180 mg/kg/day of bcnzcne. However, all of the MCV values of treatcd animals, whcn calculated from the hematocrits and erythrocyte counts, exhibited a significant increase. There was a significant doserelated reduction in total circulating white cells Mi[ogeri-indiiced Lymphocytic Proliferations Following four weeks of benzene treatment, the proliferative response of either mitogen-stimulated or nonstimulated splenic lymphocytes were elcvated in low-dose groups and depressed in highcrdose groups (Table 3). At the optimal concentration of LPS (20 pg/mL), 3H-TdR uptake increased significantly in the group receiving 8 mg/kg/day of benzene. In contrast. the benzene-treated groups receiving 40 and I80 mg/kg/day manifested a significant suppression of 3H-TdR incorporation of splenocytes upon LPS Stimulation. Similarly, thcrc were significant biphasic alterations of lymphopro- .'K lmrnunotoxicity of Benzene in Drinking Water I55 Table 3. Effect of benzene exposure on splenic lymphocyte proliferiitive response to mitogem DoseP nig/kg/day Spleen cellularityb ( x IO-') Mitogen responbes' None L1s' I'WM __- Cori A ~ ~~~~~~~~ l'tl A Control 8 40 I80 7.51 t 0.4W 7.91 2 0.62 7.50 2 0.41 5.17 t 0.64* 1.68 t- 0.31 3.55 2 O.XY* 1.13 2 0.13 0.91 '' 0.19 102.04 f 23.66 261.23 -t 25.62* 22.43 2 3.48' 25.44 -t 6.20' 7.45 Z 1.90 12.02 2 2.193.25 -t 0.71 3.65 -c 0.72 154.69 5 49.33 383.90 z IOX.61* 2Y.30 5 7 . W 14.61 c 2.66* 68.02 z 1x37 285.81 z XX.OI* 16.43 2 3.94' 8 3 7 t 1.83* ' Benzene was administered continuously via drinking water Ibr 4 weeks Recovered number of splenocytes per spleen x IO-' dpm x 10-3/106 splenic cells: response evaluated by incorporation of lmrthyl-'tl I-thymidine into day 2 hplenocyte cultures for 6 hi. klsing Values are given as mean 2 SE (n = 5 ) Significantly (p < 0.05) different from control viilues 'ruble 4. Effect of benzene exposure on mixed lymphocyte culture response' Doseb nig/kg/day Responders Responders + Stimulators Control 8 40 I80 2.11 2 0.3OC 3.59 t 0.87 1.71 2 0.42 1.58 2 0.18 31.49 rf: 2.77 56.24 2 1.59* IX.77 f 2.M* 9.30 rf: 0.88* ' Kesponder cells were the syngeneic splenic cells of CD-I mice and stimulator cells were the allogeneic YAC-I cells. A ratio of I:?stimulators to responder w a b used, and the stimulator cell$ were treated with mitomycin-C before adding to the culture. hlLC was evaluated by incorporation of Imerhyl-'Hl-~hyniidine into day 3 MLC cultures after 6 hr pulsing Uenzene was administered continuously via drinking w;ilrr for 1weeks Values are given as mean 2 SE ( n = 5) * Significantly (p < 0.05) different from control viilucs I liferation in T cell mitogen-treated cells; both the Con A and PHA-induced response were enhanced in the lowest treatment group and inhibited at the [wo high levels. The PWM or nonmitogen-stimulated splenocytes increased significantly in the aninials treated with the lowest dose of benzene but were insignificantly depressed in groups receiving higher amounts. Total spleen cellularity dccrcased signiticantly at the highest dose of benzene treatment. MLC and CTL Responses to Allogi~nricCells The results of MLC presented in Table 4 demonstrate a dose-related biphasic response, i.e, )H-TdR uplake was inhibited at the higher benzene doses but increased in the lowest dose group. The effect 'lnhle 5. Effect of benLene exposure on cytotoxic 'f-lymphocyte response I)L)\ea Ing/kg/d/day VICytotoxicity 50: I 25: I Control X 40 I xo 15.72 2 1.16' 17.94 1?: 1.61 8.77 z I.X7* 5.82 z 0.94' 15.98 2 2.98 23.32 2 ?.OS* 11.09 -c 1.71 Y.17 c 1.53* a Benzene W;IS administered continuously via drinking water for 4 week5 Effector (day 5 MLC lymphocytes)-to-target (%r labelled YAC-I cells) cell ratio c Viilues itre given as mean 5 S E ( n = 5 ) * Significantly ( p < 0.05) different from control values of benzene exposure on CTL activity is shown in 'hblc 5 . The results of 51Cr-rele:~seassay revealed that benzene signit?cantly impaired the capacity of cytotoxic T-lymphocytes to lyse alloantigenic cells (farget cells) at 25: I and SO: I effector (E)-to-target (T) cell ratios after 4 weeks of oral ingestion. The lowest dose (8 rndkdday) of benzene obviously enhanced the CTL responsiveness in 25: 1 E:T ratio. The primary antibody response to SRBC was assessed four days later after sensitization with SRBC; the SRBC-specific plaque-forming cells (PFC) were counted, and the a-SRBC antibody titer determined. As shown in Tdble 6 , ability of treated animals to produce SRBC-specific antibody changed. The number of PFC in the animals receiving 40 and 180 mg benzene/kg/day were significantly reduced, when expressed on either specific activity (PFC/106 splecn cells) or whole spleen .I56 G . C. 1-lsichc v nl. 'I'alile 6. Elfccts (11' liiiii. week\ (iI'hcn/.cnc cxposurc on the antibody rcsponws to thyrnic-dependen! ;intieen sheep erythrocyte (SKUCI' Doseh mg/kg/day Spleen cellularity ( x 10-7) PFC/I06 Splenic cells PFCltoIal spleen cells ( x 10-3) dRBC titer Control 8 40 Ixo 22.59 f 3.02c 16.W t 1.5Y* 15.36 f 0.65* 12.70 t 1.51* 1.254 2 171 1.576 2 65* 643 -c 4Y* 229 f 40* 295.72 f 74.51 268.80 2 36.10 Y9.76 2 10.32* 31.41 f 9.S2* 0.44 f 0.06 0.57 t 0.1 I 0.31 f 0.05 0.21 f 0.01' a Mice were sensitized with SRBC 4 days before the end of the benzene exposure. Splenic lymphocytes were analyzed for antibody forming cells and sera were detected for antibody titer (a-SRBC) Benzene was administered continuously via drinking water for 4 week5 Values are given a s mean 2 SE ( n = 5 ) * Significantly (p < 0.05) different from control valiics basis. However, there were more PFC per IOh splenic cells in animals cxposetl to the lowest level of benzenc. The total number of recovcrcd splenocytes was significantly reduced in a dose-related manner. The titers of a-SRBC antibodies corresponded to the number5 of PFC, but significant cleprcssion was only foiincl al thc highcsl coticcntr;1tion of benzene (Table 6 ) . Discussion The results demonstrated that oral administration of various dosages of benzene via drinking water crtl lihitrrm to CD-I mice markedly altered the immune function. The alteration of immunocompetence was grossly evidenced by a decrease of thymic and splenic weights, which are useful indicators of immune dysfunction, and a loss of lymphocytes from peripheral blood and spleen. Benzene appeared to specifically affect lymphocytes since neutrophil and other leukocytes did not decrcase significantly (Table 2). Various studies have revealed that lymphocytes are particularly sensitive to benzene toxicity via inhalation (Rozen and Snyder 1985; Dempster et ai. 1984: Snyder PI crl. 1982; Green et (11. 1981). Benzene increases the permeability of lyrnphocyte lysosomal membranes and releases lysosomal enzymc into the cytoplasm, resulting in ;i dccrease in the number of lymphocytes (MoSzczynsky and Lisiewicz 1984). Mice exposed to 300 ppm benzene via inhalation showed significant depressions in the numbers of B-lymphocytes (sIgM+ cells) in bone marrow and spleen and in t h e numbers of T-lymphocytes (Thy 1,2+ cells) in thymus and spleen (Rozen and Snyder 1985). Similar effects on these two lymphocyte populations in blood and spleen were also reported in mice after 7 or I4-day inhalation exposure to SO or 200 ppm bcnzcne (Aoyama 1986). Occupational exposure to benzene showed significantly decreased T-lyrnphocyte counts (Moszczynsky and Lisiewicz 1984).I n our studies, mice exposed to benzene had significantly lower peripheral erythrocyte counts and hcmatocrit. The increase in mean corpuscular volume (MCV) indicates that benzene ingcstion induced a macrocytic anemia. I n humans, chronic benzene poisoning is most easily detected by RBC reduction and increased MCV, but an accurate diagnosis also requires the other hematologic parameters (Haley 1977). Baarson et ai. (1984) reported that repeated exposure to low levels of benzene (10 ppm) via inhalation in mice depressed the in vitro colony- forming ability of one of the erythroid progenitor cells, the colony-forming unit-erythroid (CFU-El. the number of splenic nucleated red cells, and the number of circulating erythrocytes and lyrnphocytes. Generally, the size of lymphatic organs associated with immunologic function decreased. The effect of benzene on immune parameters in the present study was often a biphasic response, enhanced at the lowest dose (8 mg/kg/day) and suppressed at the higher doses (40 or 180 mg/kg/day). Several workers have reported the in vivo imrnunosuppressive effect of benzene (Aoyama 1986: Rozen and Snyder 1985; Pandya et af. 1986). However, differences in animal models, doses and duration of exposure. and routes of administration makc comparisons difficult (Longacre et ai. 1981). Inhalation of benzene depressed host resistance to the infectious agents, Listeria monocytogenes and Klehsiella pnerrmoniae (Rosenthal and Snyder 1985; Aranyi 1986). Mitogen-induced lymphoproliferation was suppressed in mice treated with bcnzene metabolites, i.e., hydroquinone and catechol (Wierda and Irons 1982). The cell renewal rate as well as the rate of DNA, RNA, and protein synthesis in cultured leukocytes and lymphoma cell lines exposed to benzene or its metabolites were inhibited (USEPA 1980; Walker el al. 1985; Post eta/. I 1 1.1 ( I / . <KBC). )C 0.06 . 0.I I 0.05 -0.01, i t 1body pho1. In ,nifiI heume 3d a :ene tion also iley ited L inbnyitor -E), the ho- .sorhe the enu PY). 10- 16; IW- raLke lahe nd er `0- n- 01 as n- :I1 n- 11. Iininunotoxicit y o f Iknzene in Drinking Wilier 1985; Schwartz el al. 1985). Hemopoiesis in vilro in stromal cells pretreated with low doses of oxidative metabolites of benzene, such as hydroquinone, was enhanced (Gaido and Wierda 1984). Garnett et ril. (1983) also obtained biphasic responses in vifro with CFUs (colony forming units-spleen) from niicc that inhaled benzene. These results support the hypothesis that benzene or its metabolites may promote as well as suppress the hemopoietic system (Harigaya et ai. 1981). Recently, Post et ai. (1985) indicated that exposure of T-lymphocytes to p-benzoquinone, one of the metabolites of benzene, inhibited the forniation of lymphokine interleukin 2 (1L2). 1L2, produced by T lymphocytes, is an important regulatory cytokine in lymphocyte proliferation since it is a growth factor for helper, and cytotoxic T lymphocytes (Dinarello and Mier 1986). The data described in this report show that ingestion of benzene via drinking water in CD- I mice noticeably affected all aspects of the immune system, including cellular and humoral parameters. Of particular importance was the fact that the effects were evident at the lowest dose of treatment and a no-effect level could not be established. Further studies involving lower levels of exposure are necessary to fully ascertain the safety of this chemical i n groundwater. Arknowkdgrnenrs. The help of K. Ryan Dupont. Utah Water Kesearch Laboratory, Utah State University, for chromatographic analyses is gratefully acknowledged. This work was supported in part by U.S. Geological Survey Grant No. G-125504. Published as Utah State University Agricultural Experiment Station Journal Paper No. 3380. References Aoyama K (1986) Effects of benzene inhalation on lymphocyte subpopulations and immune response in mice. Toxicol Appl Pharmacol85:92- 101 Aranyi C, O'Shea WJ, Graham JA. Miller J F (1986)The effects of inhalation of organic chemical air contaminants on murine lung host defenses. Fundam Appl Toxicol 6:713-720 Baarson KA, Snyder CA. Albert R E (1984) Kepeated exposure of CS7BI mice 10 inhaled benzene at 10 ppm markedly depressed erythropoietic colony. Toxicol Lett 20:337-342 Uick H , Holsapple MP, White K L (1985) Assessment of the effects of chemicals on the immune system. In: Li AI' (ed) New Approaches in Toxicity Testing and l h e i r Application in Human Risk Assessment. Raven Press, New York, pp 165-177 Uolcsak LE, Nerland DE (1983) Inhibition of erythropoiesis by benzene metabolites. Toxicol Appl Pharmacol 69:363-368 Brief RS, Lynch J , Bernath T, Scala RA (1980) Benzene in the workplace. .4m Ind Hyg Assoc J 43:616-623 157 Cunningham AJ, Szenberg A (1968) Further improvements in the plaque technique for detecting single antibody producing cells. Immunology I4:599-601 Dean BJ (1985) Recent findings on the genetic toxicology of benzene. toluene, xylenes, and phenols. Mutation Res 154:153-I X I 1)e;in JH, Lusler MI, Ijoorman GA. m d Lauer LD (1982) l'rocedures available to examine the immunotoxicity of chemicals and drugs. Pharmacol Kev 34: 137-151 Dempster AM, Evans 1-1L, Snyder CA ( 1981) The temporal relationship between behiivioral and hematological effects of inhaled benzene. Toxicol Appl Pharmacol 76: 195-203 Dinarello CA, Mier JW (1986) Interleukins. Ann Rev Med 37: 173-178 Dowdy S, Wearden S (1983) Skitibtics t`or research. John Wiley bt Sons. New York Faith R E , Lubter MI, Vos JG (1980) Effects on immunocompe- tence by chemicols of environmental concern. Ann Rev Uiochem Toxicol 2: 173-2I I Fishbein 1 (1981) An overview of environmental and toxicological aspects ol aromatic hydrocarbons. 1. Oenzene. Sci Total Environ 40: IX9-2IX Gaido K, Wierda I) (19x4)/ , I I.~/IYJ effects of benzene metabolites on mouse bone ni;irrow stomal cellb. Toxicol Appl Pharniilcol 76:45-S.5 Garnett HM. Cronkite El', Drew KT ( 1983) Effect of itr v i t w exposure to benzene on the chmacteristics of bone marrow adherent cells. Leukemia Res 7:803-810 Grabstein K ( 1980) Cell-mediated cytolylic responses. In: Mishell BB. Shiigi SM (eds) Selected Methods in Cellular Immunology. WH Freeman, San Francisco. pp 124-137 Green ID, Snyder CA. LoBue J, Goldstein BD, Albert RE (1981) Acute and chronic dose/response effects of benzene inhalation on the peripheral blood, bone marrow, and spleen cells of CD-l male niice. `lbxicol Appl I'harmacol 59:?01214 Greenlee WF, Sun JD, Bus JS (1981) A proposed mechanism of benzene toxicity: Formation of reactive intermediates from polyphenol metabolites. Toxicol Appl Pharmacol. 59: 187195 Haley, 1'(1977) Evaluation of the health effects of benzene inhalation. Clin Toxicol I I:S31-54X Il;in;ih:in I I J , Ekholni J E (1974) The preparation of rcd cell ghosts (mtxnbranes). Methods EnLymol XXXI: 168-172 Harigaya K, Miller ME. Cronkite EP, Driw K T (1981) The detection of itr viw hzmatoxicity of benzene by in vifro liquid bone marrow cultures. Toxicol Appl Pharmacol 60:346-353 Irons RD (1985) Quinones ;IS toxic metabolites of benzene. J Toxicol Environ Hlth 16:673-678 Jerne NK. Nordin AA (1963) Plaque formation in agar by single antibody-producing cells. Science 140:405-407 Kapliin H S (1967) On the niitiir;11 history o f the murine leu- kemias. Cancer Kes 27:1325- 1340 Longacre S L , Kocsis J J , Snyder R (1981J Influence of strain dif- ferences in mice on tile metabolism and toxicity of benzene. `I'oxicol Appl Pharmacol 60:398-409 Moszczynsky P, Lisiewicz J (1984) Occupational exposure 10 benzene, toluene and xylene and the T lymphocyte functions. Haematologiu 17:449-453 Pandya KP, Shanker R , Guptn A , Khan WA. Kay PK (1986) Modulation of benzene toxicity by an interferon inducer (GMFA). Toxicology 39:291-305 Heifer KW, Irons KD (1982) Effect of benzene metabolites on - l?X phytolicrn;i~pl~rtinin-stini~ileldymphopoiesis in rat bone marrow. J Reticuloendothel Soc 31:155-170 Post GB, Snyder R, Kalf G F (198.0 Inhibition of RNA synthesis and interleukin-2 production in lymphocytes in vitro by benzene and i t s metabolites. hydroquinone, and p-benzoquinone. 'li)xicol Lett 29: 161- 168 Kosenthal GJ. Snyder CA ( I Y X S ) Modulation of the immune response to Listericr ,nonr,c'v/openc's by benzene inhalation. Toxicol Appl Pharmacol 80502-5 10 Rozen MG, Snyder C A ( IY85) Protracted exposure o f CS7R 116 mice to 300 ppm benzene depresses U- and T-lymphocyte numbers and mitogen responses. Evidence for thymic and bone marrow proliferation in response to the exposures. Toxicology 37:13-26 Kozen MG. Snyder CA. Alhert KI1 119x4) Ileprcssion in II- iintl T-lymphocyte mitogen-induced blastogenesis in mice exposed to low concentrations o f benzene. Toxicol L e t t 20:343-349 Sawahata T, Kickcrt DE, Circenlces W F (19x5) Metiibolism 0 1 benzene and its metabolites in hone mnrrow. In: Irons RD (ed) Toxicology o f the lllood and Bone Marrow. Raven Press, New York, pp 141-148 Schwartz CS. Snyder K. Kalf (;I;( I Y X S ) The inhibition of mito- chondrial D N A replication in i*irro by the metabolites o f benzene, hydroquinone and p-benzoquinone. Chem Bioi Interac 53:327-350 Sharma RP, Gehring PJ (1979) Immunologic effects of vinyl chlo- ride in mice. Ann N Y Acad Sci 32031-563 Snyder CA, Goldstein BD. Sellakumar A, Bromberg 1. Laskin S, Albert R E ( 1982) Toxicity of chronic benzene inhalation: C1)-l mice exposed to 300 ppm. Bull Environ Contam Tov icol 29:385-391 ( 1980)The inhalation toxicology o f benzene: Incidenceof hematopoietic neoplasms and hematotoxicity in ARKlJ and C57BU6J mice. Toxicol Appl Pharmacol 54:323-33 I US ~ f ~ i , ; r r ~ ~ f /P~r~otee/cit/iofnr /ARency ( 1980) Ambient Water Q d i t y Criteria for Benzene. (EPA 440/5-80-018). Wath. ington. DC (1982) Test Methods for Evaluating Solid Waste. Phy. sical/Chemical Methods. 2nd Edition. Office of Solid Wa~lc and Emergency Response. SW-846. Washington, DC Voller A, Bidwell DE, Bartlett A (1979)The enzyme linked irn. munosorbent assay (ELISA). A guide with abstracts of micropliitc applications. Dyniitech Laboratories, Alexandria. VA Walker PP. Walker JK. Evans HH. Blumer J L (1985) Relation. ship between the oxidation potential of bcnzene metabaliltr iind their inhibitory effect of D N A synthesis on IJI'IRYS cells. Molec Pharmacol 28560-566 Wierda D. Irons R D (1982) Hydroquinone and catechol reduce the frequency o f progenitor B-lymphocytes i n mouse spleen and bone marrow. lrnmunopharmacology 4:41-54 Wierda D. Irons RD. Greenlee W F (1981) lmrnunotoxicity in C57BU6 mice exposed to benzene and Aroclor 1254". Toxicol Appl Pharmacol60:410-4 I7 Mnnusc'ript rereit-ed Fehrrmry 18. 1987 and in revised form Jidr I O . 1987. y: AI 2' thc in tcc bcl 'I : lo\ am * dur pa1 wh ma sar