Document Z4Qp1rMjJ30J9RGxejnEGmQ87

TOXICOLOGYA N D APPLIED PHARMACOLOGY 88,3543 ( 1 987) PLAINTIFF'S EXHIBIT Inhaled Benzene Reduces Aspects of Cell-Mediated Tumor Surveillance in Mice \ GARY J. ROSENTHAALN' D CARROLL A. SNYDER~ New York UniversityMedical Center,Institute of EnvironmentalMedicine. 550 First Avenue,New York,New York 10016 Received July 21, 1986;accepted October 10, 1986 Inhaled Benzene Reduces Aspects of Cell-Mediated Tumor Surveillance in Mice. ROSENTHAL, G. J., AND SNYDER, C. A. (1987). Toxicol. Appl. Pharmacol. 88, 35-43. Benzene is a potent bone marrow toxicant with particular activity against lymphocytes. Despite the recognized effects of benzene on lymphocyte populations, few data exist concerning the effects of benzene on in vivo immune responses. We have been conducting a series of studies concerning the effects of inhaled benzene on murine cell-mediated immune responses. The studies in this report involve the interaction of inhaled benzene with some of those cell-mediated immune responses associated with tumor surveillance. Exposures to IO0 ppm benzene (5 days/week X 20 weeks) induced lethal tumor growth in 9/10 C57B1/6 mice inoculated with IO4 viable PYB6 tumor cells. Lethal tumor incidences in air controls and mice exposed to lower benzene concentrations were 3/10 or less. Exposures to 100ppm benzene (5 days/week X 4 weeks) also reduced the tumor lytic abilities of cytotoxic T lymphocytes as determined by "Cr-release assays. In addition, splenocytes taken from mice exposed to 10 or 100 ppm benzene (5 days/week X 4 weeks) exhibited delays in peak mixed leukocyte responses. Coculture experiments demonstrated that these delays were not due to an induction of suppressor cell activity by benzene. There were no alterations in the relative percentages of B cells, T cells, or T-cell subsets among splenocytes from animals exposed to any concentration of benzene tested. These results demonstrate that inhaled benzene can inhibit some of the processesassociated with tumor surveillance, and that this inhibition is due, at least in part, to impairments of the functional abilities of some of the cells responsible for tumor surveillance. Q 1987Academic Press. Inc. Benzene is a well-known hematotoxicant (Goldstein, 1977; Ahoy, 1981). In humans, the predominant hemopathy associated with benzene exposure is pancytopenia or one of its variants(Goldstein, 1977).In animal studies, especially those employing rodents, pancytopenia is also a consistent finding with circulating lymphocytesshowingparticular sensitivity (Leong, 1977; Snyder et al., 1978, 1980, 1982). The sensitivity of lymphocytes to benzene indicates an immunotoxic potential for this ' Current address: Immunotoxicology Section, NIEHS/NTP, Research Triangle Park, NC 27709. * To whom all correspondence should be addressed. compound. Indeed, the results of a number of recent studieshave implicated the immune system as a target for benzene. For example, benzene or known metabolites of benzene have been shown to suppress in vitro lymphoproliferative responses to mitogens (Wierda et al., 1981; Pfeifer and Irons, 1981, 1982; Wierda and Irons, 1982). Although these studies further demonstrate lymphocyte sensitivity to benzene, they only bear inferentially on the possible interactions of benzene with the immune response. We have been conducting a series of studies investigating the interactions of benzene with the murine immune response. In these studies, we have employed whole-body inha- 35 0041-008X/87 $3.00 Copyright0 1987 by Academic Press, Inc. All rightsof reproduction in any form reserved 36 ROSENTHAL AND SNYDER lation exposuresin order to addressquestions lowing the 10th exposure, animals were inoculated with pertinent to occupational health. Earlier work from this laboratory showed that shortterm inhalation exposures to low concentrations of benzene suppressed the mitogenicresponses of B and T lymphocytes (Rozen ef viable PYB6 tumor cells. ARer inoculation, the animals continued their respective air or benzene exposures until a total of 100 exposure days had been completed (5 days/ week). For the lymphocyte functional assays, groups of five animals were exposed to either air, or 10 pprn, 30 ppm, a/.,1984).More recently,we reported that in- or 100pprn benzene for 20 days (5 days/week). halation of benzene altered the murine cellmediated immune response to sublethalchallenges by Listeria monocytugenes (Rosenthal and Snyder, 1985).We now report on further Tumorchallenge.A polyoma virus-induced tumor developed in C57B1/6 mice (PYB6) was kindly provided by Dr. Michael I. Luster (NIEHS, Research Triangle Park, NC) and maintained by passage through syngeneic male C57B1/6 mice. Two fresh tumors were taken from pas- interactions of inhaled benzene with murine sage mice and minced in suspensions of complete me- cell-mediated immune responses, specifically some of those associated with tumor surveillance. dium (RPMI 1640 medium containing 25 mM Hepes buffer plus 10%fetal calf serum (GIBCO, Grand Island, NY)).Following preparation of a single-cell suspension and viability testing (trypan blue exclusion), mice were inoculated subcutaneously in the right rear flank with ei- METHODS ther 5 X IO' or 1 X IO' viable tumor cells suspended in RPMI 1640 medium. In our hands, these inocula caused a lethal tumor incidence of IO-30% in normal C57B1/6 Animals. Male, 6-week-old C57B1/6J mice were received from Jackson Laboratories (Bar Harbor, ME). Animals were quarantined for 2 weeksduring which time they were acclimatized to a 12-hr light/dark cycle and observed for anomalous behavior and disease. Animals were randomly distributed into groups of 10 mice each for the tumor cell challenge study and 5 mice per group for the lymphocyte functional assays. Inhalation exposure regimens. Animals were exposed in whole-body, 1.O-m' dynamic inhalation chambers. During exposure periods, animals were housed in wire exposure cages. Benzene atmospheres were generated as mice. Animals were palpated weekly and tumor frequency as well as latency (time to tumor development) were recorded. Tumor incidences were evaluated for differences using the log rank x 2 method (Peto and Pike, 1973) which determines tumor incidence while correcting for mortality. Splenic cell digerential. Spleens were disrupted by forcing them through wire-mesh sieves into 10 ml of RPMI 1640 supplemented with gentamycin (50 pplml), 10%fetal bovine serum, and 0.1% sodium azide. A single-cell suspension was achieved by gentle aspiration with a 3-cc syringe fitted with a 25-gauge needle. The cell previously described (Snyder et al., 1978). Briefly, a suspensionswere pooled from five mice in each exposure stream of conditioned air was passed over a reservoir of group and centrifuged at 200g for 10 min. The cell pellet benzene (chromatoquality grade, Alltech Associates, was then exposed to I ml of 0.84% ammonium chloride Ann Arbor, MI). The benzene-laden air was then deliv- solution for 10 min to lyse red blood cells. Cold medium ered to the chamber supply air. Benzene concentrations ( 10ml) was then added to the tubes and cellswere washed were controlled by metering the flow rate of air that twice. Cell concentrations were then determined by he- passed over the liquid benzene. Benzene concentrations mocytometer counting. Viability, measured by trypan of 30 and 100 ppm were monitored at 0.5-hr intervals blue exclusion, was between 86 and 93% in these prepa- during the exposures by means of a Miran IA infrared rations. spectrophotometer (Foxboro Analytical, South Nor- T and B lymphocytes were enumerated by a direct im- walk, CT) at a wavelength of 9.8 bm. Concentrations of munofluorescent technique described in the Monoclonal 10 ppm were monitored at 0.5-hr intervals by bubbling Anlibody Source Book (Becton-Dickinson, 198I ) using a known volume of chamber air through ethanol and fluorescein isothiocyanate (FITC)-conjugated antibody. comparing the uv absorbance at 254.5 nm of the resul- For T lymphocytes, the antibody used was monoclonally tant benzene-ethanol solution with a previously pre- derived anti-Thy-1.2 (Becton-Dickinson, Mountain pared calibration curve. Chamber temperature and hu- View, CA). For B lymphocytes, the antibody used was +midity conditions were also recorded at 0.5-hr intervals. monoclonally derived anti-mouse IgM IgG (Tago, Matched air control mice received the same exposure Mountain View, CA). Working dilutions of 150 and +regimens but to conditioned air only. Exposures were 1:70 were used for Thy-1.2 and IgM IgG antibodies, conducted for 6 hrlday to either benzene or condi- respectively, after preliminary studies with these dilu- tioned air. tions gave results comparable to the published percent- For the tumor challenge experiments, groups of mice ages for these cell types in normal C57B1/6J mice. One were exposed to either air, 10ppm benzene, 30 ppm ben- million cells in 50 pl of medium were combined with 50 zene, or 100ppm benzene for 10days ( 5 days/week). Fol- pl of antibody in a flat-bottomed microtiter plate. Cells BENZENE REDUCES TUMOR SURVEILLANCE 37 with nals inti1 w 0.9 0 2aysl 0.e 'five f 0.7 Pm, 5 0.6 I2 0.5 ' de- aE 0.4 3 by 0 ark, 0.3 :de a 0.2 Pas- -me- >c_ 0.1 -I gQQBBBBBB 2:pes 0.0 -cFmq-o I I I I I I I I I I -I ind, ion rere I ei- RG. 1. Mortalitycorrected plot oftumor incidence vs time; IO4 PYB6 tumor cell inoculum. Data evalu1 in ated are from 10animals per exposure concentration. sed 11/6 fre- i: mixed with antibody were incubated on ice for 30 rnin Labs) or both antibodies were added in final concentra- :nt) : and then washed twice with medium. The washed pellet tions of 1:30 and 1:lOOO. respectively, and incubated for for i was then resuspended in 25 pl ofFluoromount-G (South- 60 rnin at 4C. These antibody concentrations were +ike, f em Biotechnology, Atlanta, GA) and the smears enu- shown in preliminary studies to yield results comparable 2ct- merated for Thy-1.2-positive or IgM IgG-positivecells to the published percentages for these cell types in nor- by immunofluorescent microscopy using a Zeiss micro- mal C57B1/6 mice. Following incubation, cellswere cen- by scope with an attached uv source and appropriate filters. trifuged once at 200g and the supernate was discarded. of Between 200 and 400 cells on each of three slides were Cells were resuspended to original volume in Low-Tox- nl), counted within 24 hr of smear preparation. M rabbit complement (Cedarlane Labs) and this suspen- ;in- Monocytejmacrophages, polymorphonuclear leuko- sion was incubated for 60 rnin at 37'C. Cells were then ion cytes, lymphocytes, and nucleated red blood cells were placed on ice and trypan blue was added 3-5 rnin before 3211 'scored from thin smears of nucleated spleen cells sus- scoring for dead cells in a hemocytometer. Controls in- ure pended in fetal calf serum. The smears were stained for cluded (1) cells alone, (2) cells with antibody alone, and llet nonspecific esterase (Koski ef al., 1976) and counter- (3) cells with Low-Tox rabbit complement alone. The ide stained with Giemsa. numbers of lymphocytes in each subset were determined im T-cell subset enumeration. T-cell subsets were enu- in three replicates from the spleen cells pooled from five led merated using a cytotoxicity technique. Spleen cell sus- mice in each exposure group. The percentages of Lyt suble- pensions pooled from five mice in each group were pre- sets in the splenic cell population were calculated as C +Ian pared as previously described with two modifications: all = total Lyt population; C - B = Lyt-1 set; C - A +la- cellular handling and incubations were carried out in = Lyt-2,3 set;A B - C = Lyt-1,2,3 set (Reske-Kunz et RPMI 1640 with 25 mM Hepes buffer and 0.3% bovine al., 1979). m- tal , serum albumin (BSA, Cedarlane Cytotoxicity Medium, Antisera Cedarlane Labs, Ontario, Canada); and it was necessary Lyt set lysed 46 Lysed ng lY. IlY +.in 'as to remove red cells and dead cells by purification of viable lymphocytes on Lymphocyte-M cell separation medium. Purification was carried out by layering 5 ml of the splenic cell suspension over 5 ml of Lymphocyte-M (Ficoll400 and sodium diatrizoate; d = 1.0875 k 0.0005 Lyt- 1.2 Lyt-2.2 Lyt- I .2 Lyt-2.2 Lyt-1 Lyt-1,2,3 Lyt-2,3 Lyt-1,2,3 Lyt-1 Lyt-2,3 Lyt-1,2,3 A B C ;0, at 25'C). The suspension was centrifuged for 20 rnin at Mixed lymphocyte culture (MLC). Spleen cell suspen- Id SOOg at room temperature, a well-defined lymphocyte sions from five individual mice were prepared as de- :S, layer was removed at the interface, and these cells were scribed above. Following one wash in RPMI 1640, total U- washed two or three times in medium before further pro- numbers of nucleated cells were determined with a It- cessing. Cell concentrations were adjusted to 1 X 106/ml Coulter counter (Model ZBI, Coulter Electronics, Hia- ie in medium and five I -ml aliquots were placed into five 12 leah, FL) and viability (always >go%) was assessed by SO X 1 W m m polystyrene culture tubes. Either monoclonal trypan blue exclusion. Viable cells were resuspended at a 'Is anti-Lyt-1.2 or monoclonal anti-Lyt-2.2 (Cedarlane concentration of 2 X 106/mlofculture medium in sterile, 38 ROSENTHAL AND SNYDER conical 15-ml polystyrene centrifuge tubes. The culture medium was RPMI 1640 supplemented with Hepes buffer (20 mM), L-glutamine (2 mM), 5% fetal calf serum (GIBCO), and 50 pglml gentamycin (Microbiological Associates, Walkersville, MD). One-way mixed-leukocyte cultures were performed employing 5 X IO5 mitomycin C (MMC; Sigma, St. Louis, M0)-treated &Is from DBA/2 mice (stimulator cells) per well. MMC treatment was performed by incubating stimulator cells with MMC (50 &IO7 cells/ml) for 40 min at 37'C. Following three washes in RPMI 1640, the stimulator cells were resuspended at 4 X lo6 cells/ml in fresh RPMI 1640 medium with supplements to which was added 5 X lo-' M 2-mercaptoethanol (Sigma). The stimulator cells and cells from either benzene-treated or air-exposed mice were added in 0. I -ml aliquots such that an effector to stimulator cell ratio of 1:2 was established (2 X 10':4 X lo5)in each well. MLCs were harvested daily from the second through the eighth day of incubation following an 18-hr pulse with ['Hlthymidine (1.0 pCi/well; sp act 5.0 Ci/mmol, Amersham, Northridge, IL). Cells were harvested using an automated cell harvester (PHD cell harvester, Cambridge Technology, Cambridge, MA) onto glass-fiber filters (grade 9H4 AHA). The filters containing radioactive matenal were placed in 7-ml glass scintillation vials containing 5 ml Aquasol scintillation fluid (Amersharn), and the vials shaken vigorously. Following dark adaptation, the vials were counted for j3H]thymidine content using a LKB 1215 Rackbeta liquid scintillation counter (Wallac Oy Co., Turku, Finland). Suppressor cell studies. Suppressor cell activity was examined by coculturing pooled MMC-blocked spleen cells (1 X 105/well)from air or benzene-treated C57BI/ 65 mice with spleen cells (1 X 105/well)from untreated C57B1/6J mice along with DBA/2 stimulator cells (4 X IO'/well, prepared as previously described) followed by examination of the mixed-leukocyte responsiveness at Days 2,3, and 4 ofculture. Counts were made on three replicates of pooled spleen cells from each group of five mice. Cell-mediated cytolysis. Cytotoxic T-lymphocyte-mediated cytolysis was examined in vitro using splenocytes from benzene-exposed and air control animals as described by Brunner et al. (1968). Mice were exposed for 10days to benzene or air and then sensitized by inoculation of P8 I5 mastocytoma cells. Exposuresto benzene or air were then continued for an additional 10days. Spleen cells from five individual mice per group were tested for cytolytic activity using a %-release assay employing P8 15cellsas targets. P8 15 mastocytoma cells (H-2d h a p lotype) were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in vitro. Tumor cells were collected from tissue cultures, washed three times in RPMI 1640, counted, and used to immunize mice at a concentration of 1 x 10' per mouse. Following a total of 20 exposures and IO days of alloge- neic cell sensitization, animals were sacrificed and spleens removed. Splenic single cell suspensions were made as previously described in the MLC section. Target cells were labeled with 250 pCi of sodium chromate-5 I (Amersham) by incubation at 37'C for 90 min under gentle agitation and then washed, counted, and resuspended at 2 X lo' cells/ml. A constant number of "Cr- labeled target cells( 2 X IO' in I 0 0 pl) were added to each well of a plastic 96-well microtiter plate (Flow Laboratories, Bethesda, MD) followed by the addition of 10O-pl aliquots of effector cells (splenic cells from air control or benzene-treated mice).Effector to target cell ratios were 100:1, 33:1, and 11:l. The 1OO:l effector to target cell ratio was optimal for target cell lysis. Microtiter plates were incubated for 4 hr at 37%. The plates were then centrifuged at 200g for IO min at room temperature and 100 pl of supernatant fluid from each well was removed and counted in a gamma spectrophotometer. Counts were corrected for volume and compared to counts present in control wells containing target cells but no effector cells (spontaneous release). Maximum release of "Cr from target cells was determined by addition of 50 pl of a 0.1% solution of Triton-X 100. Specific cytolysis was calculated as test release - spontaneous release x loo. maximum release Statistics.Analyses of variance were performed on the lymphocyte functional assay data. When F ratios indi- cated significant differencesamong the exposure groups, individual Student t tests were performed at a level of confidence such that the overall probability of a Type I error occurring was ~ 0 . 0 5A. two-tailed test with a,combined p s 0.05 level was considered significant. RESULTS Eflects of Benzene Inhalation on Resistance to PYB6 Tumor Challenge The mean daily benzene concentrations +SD for the exposureswere 101.4 k 7.1,29.3 k 2.1, and 10.8 k 1.5 ppm. Following a total of 100 exposure days (5 days/week), the lethal tumor incidences for those mice receiving the lo4 cell inoculum were 3/ 10, 1/ 10, 2/ 10, and 9/ 10 for air control, and 10, 30, and 100 ppm benzene, respectively. The lethal tumor incidences for those mice receiving the 5 X lo3 cell inoculum were 2/ 10, 2/ 10, 3/ 10, and 3/ 10 for air control, and 10, 30, and 100 ppm benzene, respectively. A significantdifference, asdeter- BENZENE REDUCES TUMOK SURVEILLANCE 39 TABLE 1 COMPARISON OF SPLENIC CELL POPULATIONS BETWEEN MICE EXPOSED TO 100 ppm BENZENE AND AIR CONTROLS Air control loo ppm benzene Since the proportions of the various lymphocyte subpopulations were undisturbed, the lymphocyte hnctional assays described below could be normalized for particular lymphocytepopulationsby using equal numbers of splenic cells. Lymphocytes Thy-1.2' cells 1g+cells Granulocytes Esterase+ ceUs Splenicdifferential (YO)" 90.7 f 1.5 30.3 k 3.1 56.3 k 4.0 4.7 f 2. I 2.3 f 1.5 +88.7 f 3.5 33.0 2.6 . 54.0k2.6 6.3 f 2.5 3.0 & 1.0 Lyt-I+ Lyt-2,3+ Lyt-1,2,3+ Splenic T-cellsubsets(%)" 44 k 7 9 +2 48 k 6 40 + 6 9 +.4 50 k 9 a Mean rt SD of triplicate determinationsmade from pooled (n = 5) spleen cell suspensionsper treatment. mined by log rank x2analysis (Peto and Pike, 1973),was observed in the mice receiving the lo4 cell inoculum and exposed to 100 ppm benzene. A plot of the data corrected for mor- tality shows this difference (Fig. 1). No differences in tumor incidences were observed among any groups inoculated with 5 x io3tumor cells. Kinetics of Mixed-Lymphocyte Culture following Benzene Inhalation Assessments were made of the proliferative responses of spleen cells from air- or benzeneexposed mice in one-way mixed-lymphocyte cultures. Following 20 days of exposure of mice to either air, 10 ppm benzene, or 100 ppm benzene spleen cells were cultured and the amount of [3H]thymidine incorporated was assessed on Days 2 through 5 of culture. On Days 2, 3, and 4 of culture, the amounts of [3H]thymidineincorporated by cells from benzene-exposed mice were depressed relative to air control values (Fig. 2). On Day 5 of culture, however, the amounts of [3H]thymidine incorporated by spleen cells from benzene-exposed mice were increased over control values (Fig. 2). Thus, benzene exposure, even at 10 ppm induced a delay in the peak response of splenocytes to MMC-treated stimulator cells. Lymphocyte Functional Assays The mean daily benzene concentrations kSD for the exposures used for the functional assays were 99.7 rt 7.0,29.5 14.4, and 11.1 k 1.5 ppm. 6.0 0" 5.0 x: 4.0 '$E 3.0 -a v) 2.0 E 0" 1.0 0 - Air W' - 1Oppm - 100ppm .-17-5 p c 0.05 CharacteristicsofSpleen CellPopulationsfollowing Benzene Inhalation 0.0 23 4 Day of MLC 5 AS shown in Table 1, twenty exposures to 100ppm benzene induced no changes in the relative p r o p o ~ o n sofsplenic leukocytes, in the percentages Of subsets, Or in the ratio of splenic helper/suppressorcells (Lyt-1+/Lyt-2,3+). FIG. 2. Kinetic analysis of mixed-lymphocyte responses at Days 2 through 5 of culture following 20 days exposure to air or 10 or 100 ppm benzene. Individual functional tests were performed on cells isolated from each of five animds per exposure concentration. Error bars equal k 1 SE. *Significantly differentfrom air control. 40 ROSENTHAL AND SNYDER TABLE 2 EFFECTOF 20-DAY EXPOSURE TO 100 ppm BENZENE ON POTENTIAL INDUCTION OF SUPPRESSOR CELL ACIIVll" IN MIXED LEUKOCYTCEULTURE [3H]Tdrincorporated (cpm)" Coculture 1. Normal + stimulators +( M M C ) ~ 2. Air (MMC) stimulators +(MMC) 3. Benzene (MMC) stimulators +(MMC) 4. Normal stimulators(MMC) ++benzene (MMC) 5. Normal stimulators(MMC) + air (MMC) Day 2 3020 f 345 1748 f 40 1301 2 308 5570 2 310 4125 f 499 Day 3 7,9202 873 1,822f 210 1,3102 140 11,988 f 1323 11,832f 752 Day 4 15,685 f 1278 1,808 f 208 1,3762 121 18,756k 756 17,249 f 2279 " Mean f SD of triplicate determinationsmade from wold (n= 5) spleen cell suspensionsper treatment. MMC = mitomycin C treated. -16 14 -- 12 -- .-x$ lo-- 0 --.i$=- 8 =0aI . 7*v) ae -- 4 -- i 2 -- rl J 0- Suppressor Cell Studies exposed to air or 10 or 100 ppm benzene for 20 days. Spontaneous release k SD under ex- To determine whether benzene exposure perimental conditions employed was 723 could induce suppressor cell activity and 2 29 cpm. Total release k SD of the 5 X lo4 thereby cause the depressed [3H]thymidine "Cr-labeled target cells was 3974 k 196 cpm. incorporation observed in the MLC at Days The results of this experiment, presented in 2 through 4, a series of coculture experiments Fig. 3, demonstrate that exposureto 100ppm were performed. These coculture experi- benzene for 20 days significantly ( p < 0.05) ' ments were performed on Days 2,3, and 4 of altered the ability of splenic lymphocytes to culture, when depressed [3H]thymidine in- lyse the sensitizing alloantigen-bearingtarget corporation was observed. Table 2 showsthat cells at the effector to target (Em) ratio of coculturing of spleen cells from untreated 100:1. No significantdifference was observed C57B1/6 mice with MMC-blocked spleen between control and 10-ppm-benzene expo- cells from either benzene-treated or air con- sure groups at any of the E/T ratios tested trol mice induced no significantdepression in (Fig. 3). [3H]thymidine incorporation. These data therefore suggest that the depressedresponses observed at Days 2,3, and 4 of the MLC were DISCUSSION not due to benzene-induced suppressor cell activity. The results presented here demonstrate \ that inhaled benzene can cause immunodeCell-Mediated Cytolysis following Benzene pressive effects asmanifestedby reduced host Inhalation resistance to a transplantable syngeneic tu- mor and by altered in vitro lymphocyte func- The ability of sensitized cytotoxic T lym- tions. To our knowledge, this is the first study phocytes to recognize and destroy a sensitiz- to investigate the effects of inhaled benzene ing target cell was tested in a "Cr-release cy- exposure on host resistance to a tumor chal- totoxicity assay using spleen cells from mice lenge and the results may bear on the known BENZENE REDUCES TUMOR SURVEILLANCE 41 Tl6 14 -12 -- .-xf lo- - -- 1.gY 8 -- lp 4 -- 2 -- 0 - Air control -V 10 ppm benzene -0 100 ppm benzene T 01 I 11:1 i 33:l 1003 Effector to target cell ratio FIG. 3. Cytolytic activity of spleen cell pop.ulation obtained from mice exposed for 20 days to air or 10 or 100 ppm benzene. Individual functional tests were performed on cells isolated from each of five animals per exposure concentration. Error bars equal + I SE. *Significantly different from air control. link between human exposure to benzene and hematopoietic neoplasms. . The tumor used in our host resistance study was a polyoma virus-induced tumor from C57B1/6 mice (PYB6). There is a strong evidence that host resistance to this tumor and other DNA virus-induced tumors is mediated primarily via T lymphocytes (Dean et al., 1975;Howell et al., 1974; Law and Ting, 1965).Thus, these data suggest a benzene-induced impairment of T-cell-mediatedimmunity. However, in an in vivo situation, alterations of other effector cells (e.g., natural killer cells or macrophages) cannot be ruled out. It is conceivable that a benzene-induced decrement in the number of immunocompetent cells caused the increased tumor suscep tibility; however, using a "Cr-release assay we also demonstrated a benzene-induced impairment in the lytic ability of those T cells responsible for tumor cell lysis. In the "Crrelease assay we employed P815 mastocy- toma cells which induce the production of cytotoxic T lymphocytes(CTL) (Brunner et al., 1968).While an in vitro"Cr-release assay utilizing PYB6 cells as targets would correlate better with the tumor challenge data, generally syngeneic cells (as PYB6 are to C57B1) do not elicit a marked CTL response in vitro. Although in vitro lysis of allogeneicP815cells was employedin our studies, the mechanisms by which CTL destroy these cells in vitro is believed to be identical to the mechanisms by which CTL destroy virally infected or transformed cells in vivo (Wells, 1982). In our studiesthe samebenzene concentration (100 ppm) which altered host resistance to the PYB6 tumor challenge also impaired CTL cytotoxicity toward P8 15 mastocytoma cells. This further strengthensthe association between a benzene-induced impairment of T-cell-mediated immune surveillanceand increased susceptibility to the PYB6 tumor challenge. The MLC was employed as a sensitive assay for the functional capacity of lymphocytes derived from animals exposed to benzene vapor. This assay is dependent upon cellular communication and T-cell recognition of antigen (F. H. Bach et al., 1976,B. A. Bach et al., 1978). The murine cell type widely thought to respond in the MLC is the Lyt1+23- lymphocyte which is believed to lie within the same population of cells responsible for helper and delayed-typehypersensitivity reactions (Cantor and Boyse, 1975; Cantor et al., 1976;Huber et al., 1976).Cellsfrom mice exposed to 100 and 10 ppm benzene showed increased [3H]thymidineincorporation at Day 5 of the MLC, preceded by 3 days of depressed [3H]thymidine incorporation (with respect to aircontrols). Thus, exposures to 10ppm and exposuresto 100ppm altered the kinetics of [3H]thymidineincorporation. Similar observationswere reported by Rozen et al. (1984)in that 6 days of exposure to 10 ppm benzene wasjust as effectivein reducing femoralB-cell blastogenesis as6 days of exposure to 30, 100, or 300 ppm benzene. Appar- ently, concentrations lower than 10 ppm should be employed to ascertain concentra- 42 ROSENTHAL AND SNYDER tion-response relationships between benzene short-term exposure limit of 25 ppm (not to exposures and these parameters. exceed 15 min over an 8 hr workday). Of the It is noteworthy that 100ppm benzene de- studiesdiscussedabove, the most pertinent to pressed responses in both the MLC and CTL the threshold limit value was the delay in assays. As MLC responsiveness is generally peak MLC response in mice exposed to 10 thought to represent the initial phases in the ppm. Under certain conditions, a delay in induction of cytotoxic T cells, the benzene- immune response might be detrimental to induced loss of CTL lytic ability might be ex- the host. For example, a delay in surveillance plained by an impaired ability of T cells to of tumor cells could enable the quantity of recognize foreign tissue or tumor-associated such cellsto reach a number that would over- antigens during T-helper-cell-mediated in- whelm the normal immune response. Since duction of cytotoxic T cells. millions of individualsare occupationallyex- Alterations in immune function can be posed to benzene (U.S. Department of due, in part, to an induction of suppressorcell Health and Human Services, 1983), further activity (Gershon, 1974; Berlinger et ai., study of the effects of low-level benzeneexpo- 1976).Sincethe inhibitory effects of suppres- sure on the immune response seems war- sor cellsare exerted at the level of lymphocyte ranted. nucleotide synthesis(Kirchner et al., 1975),a straightforward method for assessing suppressor activitiesis the in vitro coculturetech- ACKNOWLEDGMENTS nique followed by [3H]thymidinelabeling. A number of studies have shown that various agentscan induce suppressorcell activity and thereby suppress in vitro lymphocyte func- This work was performed in partial fulfillment for a Doctor of Philosophy degree (G.J.R.)under the tutelage of C.A.S. This work was supported by a research grant from MEHS (1RO 1 ESO3214), by an inhalation toxicol- tion (Kirchner et al., 1975;Clark et al., 1983; ogy training grant from NIEHS (5T32 ES07065), and by Luster et al., 1980).However, in our studies, Center grants from the National Institute of Environ- excessive suppressor cell activity was not detected when coculturing experiments were performed in the context of the mixed-lymphocyte cultures (Table 2). The cultures for mental Health Sciences (ES00260), and the National Cancer Institute (CA 13343), and a Special Institutional grant (oooO9) from the American Cancer Society. The authors wish to thank Michael Barbieri for operating the inhalation chambers and Maureen Freitag for typing and these experiments were harvested at Days 2 editing the manuscript. through 4 because it was at these time points that depressions were observed in the proliferative capacities of cells from mice exposed REFERENCES to 100and 10ppm benzene for 20 days. Since the accessory functions of macro- phages (e.g., antigen presentation, interleukin-1 secretion) are known to be required for most T-cell functions, we determined the AKSOY, M. (198 1). Problems with benzene in Turkey. R e d . Toxicol.Phannacol. 1,147-155. BACH, B. A., SHERMAN,L., BENACERRAFB,., AND GREENE, M. I. ( 1978). Mechanism of regulation of cell mediated immunity. 11. Induction and suppression of effects of benzene exposure on the percentages of splenic monocytes and macrophages. 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