Immunotoxicity of aminocarb

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

36, 35-45

(1990)

lmmunotoxicity II. Evaluation

of Aminocarb

of the Effect of Sublethal Exposure to Aminocarb Cells by Flow Cytometry’

on Bone Marrow

JACQUES BERNIER,* MAREK ROLA@LESZCZYNSKI,* DENIS FLIPO, KIUYSZTOF KRZYSTYNIAK, AND MICHEL FOURNIER* Mont&al,

Ddpartement des Sciences Biologiques, Universit6 du QuCbec ci Montrial, Quebec, Canada H3C 3P8, and *Fact&C de Mbdecine, UniversitC de Sherbrooke, Sherbrooke, Qudbec, Canada JlK 2Rl Received May

30, 1989;

accepted September 6, 1989

The potential immunotoxic effect of the carbamate pesticide aminocarb on murine bone marrow cell subpopulations was evaluated by flow cytometry, C57BY6 mice were exposed by gavage to sublethal doses of the pesticide and lymphocyte precursors from bone marrow population were stained with PNA lectin and a panel of monoclonal antibodies against cell surface antigens. In regard to the microenvironment-dependent maturation of B lymphocytes, the pesticide effect on the lymphoproliferative potential of bone marrow was assessed by marrow transplantation from aminocarb-exposed donor mice to normal, syngeneic, X-irradiated recipient mice. The sublethal exposure of 0.08-5.0 n@kg body wt aminocarb to donor animals did not affect regenerating bone marrow in the recipient mice. No marked effect on bone marrow cell number was noted in pesticide-exposed animals. However, a marked shii in surface IgM density on marrow B cells was noted at 0.08 and 0.31 mg/kg body wt aminocarb. This was correlated with decreased cell frequency in GJG, phase and increased frequency of cells in the S phase of the cell cycle. Thus, altered maturation of B lymphocytes, expressed as a shift in the density of surface IgM on mature B cells and not the lymphocyte count, was related to the direct effect of aminocarb and/or to the pesticiderelated changes in bone marrow microenvironment. Overall, exposure to the carbamate pesticide aminocarb activated the cell maturation process in bone marrow. Q 1990AC&.~~C press.inc. INTRODUCTION

with human intestinal microflora showed no major destabilizing effect of aminocarb (4). This pesticide, however, has been demonstrated to produce, at sublethal doses, route-dependent, transient, and reversible effects (with a maximum peak at 10 days after exposure) on humoral immune response in mice, without affecting cellular immune response (5-7). In general, dermal and gavage exposures stimulated humoral response in mice, whereas an intraperitoneal (ip) injection of aminocarb suppressed this response (S-7). Immunomodulatory functions of carbamates can probably be related to the effect of the OC(O)NHCH, group, which is responsible for the acetylcholinesterase effect of the carbamate (1, 8). Different carbamate esters are known to act either as antileukemic drugs (9) or as immunostimu-

Aminocarb, a phenylsubstituted methylcarbamate (4-dimethylamino-3-methylN-carbamate, Matacil), is an important environmental pollutant that can be absorbed mostly through inhalation, oral, and dermal routes (l-3). Acute toxic doses of aminocarb were shown in animal studies to result in severe but transient inhibition of tissue esterases (3). Inhalation studies of oilformulated, aerosolized Matacil showed minor alterations in pulmonary biochemical parameters after acute exposure to the insecticide (2). Previous interaction studies ’ Supported by Natural Sciences and Engineering Research Council of Canada and TOXEN, Universite du Quebec a Montreal. ’ To whom all correspondence should be addressed.

35 0048-3575/90 Copyright All rights

$3.00

Q 1990 by Academic F’ms, Inc. of reproduction in any form reserved.

36

BERNIER

latory agents (10). The carbamate pesticide, methyl carbamate, however, was found to have little or no effect on selected immune responses in mice (5, 11). Nonetheless, the carbamate pesticide aldicarb was shown to reduce PFC response at the 1 ppb level in drinking water (12). Hematologic changes and/or changes in bone marrow cells and the subsequent shift in lymphocyte subpopulations were proposed as possible targets for another carbamate pesticide, carbofuran (13). In this study, we examined the possible interaction of aminocarb with bone marrow cells in inbred C57BW6 mice by flow cytometry. In addition, we assessed the proliferative potential of bone marrow from aminocarb-exposed mice, subsequently transplanted to normal, syngeneic, X-irradiated mice. The data confirm our previous observations of the activating potential of aminocarb on the immune system. MATERIAL

AND METHODS

Animals Female C57BL/6 mice, 8-12 weeks old, were obtained from Charles River Company (St. Constant, Quebec). Upon arrival, all mice were quarantined for 2 weeks prior to use. Animals received by gavage a single sublethal dose of g5.0 mg/kg body wt aminocarb (98.9% purity, Chemagro Agricultural Division, Mobay Chemical Corp., Kansas City, MO) dissolved in 0.1 ml of corn oil as vehicle. Control mice were treated with the vehicle only. Lethal ip dose of aminocarb, determined previously, was 20 mg/kg body wt (6). Depending on the pesticide formulation and animal strain, the ip LD50 was reported to be from 4.7 to 10 mg/kg body wt in mice (1). The oral LDSO of aminocarb was estimated as about twice less toxic than the ip LDSO dose (1). Cells Ten days after the pesticide exposure, mice were killed by ether anesthesia, and femur and tibia bones were collected in

ET AL.

cold HBSS3 (Flow Laboratories, Toronto, Ontario). Bone marrow cells were recolted by flushing from the bone with 2.0 ml HBSS with a 26 gauge needle and syringe (14). Bone marrow cell suspensions were prepared by several pipettings, strained through fine nylon wool to avoid cell clumps, and washed three times. Flow Cytometry Lymphocyte precursors from the bone marrow population of donor mice were studied by cytofluorometric analysis with a FACScan, equipped with an argon excitation laser, and Consort 30 software (Becton-Dickinson Immunocytometry Systems, Mountain View, CA). For analysis of fluorescein isothiocyanate (FITC) and phycoerythrin (PE)/ethidium bromide, the fluorescence was monitored through 530- and 585-nm band pass filters, respectively. The cell cycle was analyzed by the method of Taylor (15). Briefly, 2 ml of suspension 1 X lo6 bone marrow cells/ml was mixed with 0.5 ml of solution 250 pg/ml ethidium bromide (Sigma Chemical Co., St. Louis, MO) containing 1% Triton X-100 (Sigma Chemical Co.) and incubated at 4°C for 15 min. Thereafter, 50-75 U of bovine pancreas RNAse (Sigma) per milliliter of cell suspension was added and incubated at 22°C for 20 min to avoid unspecific staining. The samples were incubated at 4°C before analysis by FACS. The results were analyzed by the sum of broadened rectangles model with DNA cell-cycle analysis software from Becton-Dickinson. The frequency of cells in peaks II and III of unstained bone marrow cell population, which have been reported to correspond to lymphoid cells and nonlymphoidlmyeloid 3 Abbreviations used: HBSS, Hank’s balanced salt solution; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PNA, peanut agglutinin; BSA, bovine serum albumin; MLR, mixed lymphocyte reaction; Con A, concanavalin A; PHA, phytohemagglutinin; LPS, lipopolysaccharide; DXS, dextran sulfate; RBC, red blood cells.

IMMUNOTOXICITY

cell lineage, respectively (16), was determined by direct cytometric analysis (forward scatter versus cell frequency). Lymphocyte precursors from bone marrow were analyzed with lectin and a panel of antibodies against different antigens displayed on precursor cells by direct or indirect immunofluorescence (17). Peanut agglutinin (PNA) was reported to bind to the IgM-negative precursors of B lymphocytes (18, 19). The subsequent appearance of IgM, Ia, and IgD was reported as a method for determination of the B cell maturation (19-21). Negative isotype controls of the specificity of the labeling were performed for lymphoid cell populations and were shown to be not different from the unstained cell controls used as routine negative controls. The monoclonal antibody for indirect immunofluorescence was rat anti14.8 IgG (Sera-lab Ltd., Crawley Down, England) and was used as a first antibody. Fluorescein-labeled rabbit anti-rat Ig antibody (Dimension Science), absorbed on mouse splenocytes, was used as a second antibody. Antibody for direct immunofluorescence staining was R-phycoerythrinlabeled, affinity-purified goat anti-mouse IgM (PE-anti-IgM) (Fisher Biotech, Orangeburg, NY). Fluorescein-labeled peanut agglutinin (PNA-FITC) (Dimension Science) was used to discriminate Ig-bearing cells from immature cells (18, 22). Rabbit monoclonal anti-mouse IgD antibody (Nordic Immunological Laboratories, El Toro, CA) and monoclonal mouse anti-Iab IgM (Ia.rn3) (Cederlane, Homby Ontario) were conjugated to tluorescein isothiocyanate by the method described by Goding (23). Bone marrow cell labeling was carried out by preparing a mixture of 50 ).~lof the cell suspension (2 x lo7 cells/ml) with 50 l~,l of antibody solution (2 t&ml) or 50 p,l of PNA-FITC solution (5 )&ml) and incubating at 4°C for 3W5 min. Two milliliters of medium was added, then twice centrifuged at 1200 t-pm for 10 min, and the cell pellet was resuspended in 0.5 ml of PBS supplemented with 1% sodium azide. To minimize

OF AMINOCARB

31

(II)

cell adherence, all samples were kept at 4°C in polystyrene tubes and analyzed at the day of experiment. Expression of Ia on cells, reflecting possible heterogeneity of B cell precursors/B cells, was determined according to the method of Mond et ~1. (24). Reconstitution

of X-Zrradiated

Mice

Bone marrow from aminocarb-exposed donor animals was transplanted to untreated syngeneic C57BL/6 mice (recipient), X irradiated with a dose of 800 rads at the day of reconstitution (25). Recipient mice were injected intravenously into tail with 0.3 ml of the bone marrow cell suspension (6.67 x lo7 cells/ml) from untreated animals or mice gavaged twice with O-5 mg/ kg body wt aminocarb at 10 days prior to transplantation. Reconstituted mice were maintained in sterile conditions for 8 weeks before evaluation of the regeneration of immune functions. Mixed Lymphocyte

Reaction

(MLR)

One-way MLR assays were performed for the assessmentof the immune functions in reconstituted animals, as described previously (26). Briefly, the responder spleen cells were prepared from reconstituted mice and red blood cells were removed by a hypertonic shock method with Gey’s solution. The erythrocyte-depleted, in vitro Xirradiated splenocytes from A/J mice were used as stimulator cells. Responder and stimulator cells were cocultured at 37°C 5% CO*, for 72 hr at a concentration of 5 x lo5 cells in 0.2 ml complete RPM1 1640 (Flow) in U-shape microculture plates (Linbro, Toronto, Ontario, Canada). Thereafter, the cultures were pulsed with 0.5 &i of [3H]TdR (New England Nuclear, Boston, MA) for 18hr. Cells were harvested (Titertek Cell Harvester, Flow) and the radioactivity was determined by liquid scintillation. Mitogen

Stimulation

The mitogen assay was applied for the

BERNIER ET AL.

38

assessmentof RNA and DNA synthesis by spleen cells of bone marrow-reconstituted mice (27). Briefly, 5 x lo5 viable spleen cells were cultured in 0.2 ml in U-shape microculture plates with 5.0 t&ml of concanavalin A (Con A) (Pharmacia Fine Chemicals, Uppsala, Sweden), 50.0 l&ml of phytohemagglutinin (PHA, M form) (Grand Island Biological Company, NY), 50.0 &ml lipopolysaccharide (LPS) (Sigma), 20.0 pg/ml dextran sulfate (DXS) (Sigma), and 25 &ml LPS + 10.0 pg/ml DXS. It was estimated that about 80% of the B cell population was responding to the combination of LPS + DXS (28, 29). The microplates were incubated for 18or 48 hr and pulsed for an additional 18hr with either 0.5 @i [5,6-3H]uridine (UdR) (spec act 45.0 Ci/ m&f, ICN Radiochemicals, Irvine, CA) or 0.5 pCi of [methyl-3H]thymidine (spec act 6.7 CilmM, ICN Radiochemicals), respectively. Cells were harvested and the radioactivity was determined by liquid scintillation, Data are presented as the mean -+ standard deviation (SD). Statistical analysis was performed by independent samples randomization test or Mann-Whitney U test. Experimental groups were compared to vehicle controls only.

RELRTIVE

VOLUME

FIG. 1. Cytoj7uorometric analysis of the prototype of FACS-generated volume vorward scatter) profile corresponding to particle size in horizontal scale and particle frequency on the vertical scale for bone marrow. Peak I, mature RBC; peak II, lymphocytes and late erythroblasts; peak III, myeloid cells, early erythroblasts.

information is provided by this method. A triphasic curve can be observed for murine bone marrow cells, similarly to the results reported by others (16, 19), where each peak represents different cell populations (Fig. 1). The peak on the left-hand side of the profde corresponds to dead cells, mature red blood cells (RBC), and cell debris. Peaks II and III can be associated with lymphoid cells and nonlymphoid cell lineage, respectively (19). The data are expressed for each group as frequency of cells in each of these peaks, as shown in Table 1. No

RESULTS

Effect of Sublethal Aminocarb on Bone Marrow Cells

Exposure

TABLE 1 Frequency of Cells in Peaks II and III in Cytojluorometric Analysis of C57BN6 Mouse Bone Marrow Cells

The effect of the pesticide on mouse bone marrow cells was evaluated in C57BL/6 Peakb Aminocarb” mice gavaged with 5.0, 0.32, 0.08 mg/kg 11 III body wt aminocarb at 10 days prior the cy- b-wh body wt) -(vehicle) 30.61 + 4.87 69.39 f 4.87 tofluorometric analysis. Bone marrow cells 5.00 34.45 ” 1.44 65.55 f 1.44 were collected from each mouse, from fe0.31 32.03 2 3.% 67.97 5 3.% mural and tibial bones, and analyzed inde0.08 33.43 f 6.97 66.57 + 6.97 pendently in vehicle controls and amia C57BL/6 mice (10 animals/group) were gavaged nocarb-exposed animals. with O-5.0 mgikg body wt aminocarb and bone marrow The prototype distribution profile of low cells were collected and analyzed individually at 10 angle (zero degree) forward light scatter is days after treatment. presented in Fig. 1. The amount of light b The results were obtained for gated peaks II and scattered by a particle is related to the size III of the cytofluorometric analysis of bone marrow and shape of that particle. Thus, cell size cells.

IMMUNOTOXICITY

OF AMINOCARE

(II)

39

marked changes in frequency of cells in peaks II and III were observed after the gavage with tL5.0 mglkg body wt aminocarb (Table 1).

diate and low aminocarb doses, introduced by gavage, affected the DNA cell cycle in lymphoid and/or nonlymphoid cells in bone marrow.

Effect of Aminocarb on DNA Content of Bone Marrow Cell Populations

Effect of Aminocarb B Lymphocytes

Bone marrow cells were stained with ethidium bromide that intercalates into the DNA of intact cells and can be used to distinguish between cells of different DNA content by flow cytometric analysis (Table 2). For this purpose the cells in peaks II and III were gated and frequencies of cells in each phase of the DNA cell cycle were evaluated. The data presented in Table 2 were obtained independently from each animal and expressed as the mean + SD (Table 2). It was observed a modification of cell frequency in GdG, phase and S phase of cell cycle for aminocarb-treated groups, as compared to the vehicle controls. At a high concentration of aminocarb, the number of cells in the GdG, phase decreased and the S or G&l phases were normal. The data for intermediate and low aminocarb doses showed a decrease in cell frequency in the G,-,/G,phase, which appeared to be associated with an increased cell frequency in the S phase and normal cell number in the GJM phase. Thus, the data suggest a shift in the cell frequency in aminocarbexpased animals for cells entering the S phase. It appeared, therefore, that interme-

Identification of surface antigens displayed on murine B lymphocytes was employed to assessthe B lymphocyte maturation. Analysis of cells in peak II (Fig. 1) was performed after the labeling of bone marrow cells with antibody or lectin conjugated with fluorescent dye. PNA-FITC was used to discriminate the Ig-bearing cells from immature cells (Table 3). Among the four parameters tested, only the incidence of PNA-positive cells decreased in mice gavaged with 0.31 and 0.08 mg/kg body wt aminocarb (P < 0.05 and P < 0.01, respectively) , whereas other indices remained relatively intact (P < 0.5) (Table 3). Thus the number of immature cells diminished after the exposure to intermediate and low doses of the pesticide. The data for B 220 Ag/18.8 Ab-positive cells, characteristic for pre-B subset and B lineage cells, however, were not different from the vehicle control data (not shown), therefore suggesting no effect of the pesticide exposure on the number of B lymphocytes. In addition, determination of the frequency of mature B cells, expressing the surface IgM, IgM + IgD, did not show any differences between aminocarb-

TABLE Effect

of Sublethal

Oral

Exposure

of C57BLl6

of

2

to Aminocarb

on DNA

Cell

Cycle

in Bone

Marrow

Cells

Percentage of cell-cycle phaseb

Aminocarb” GwAcg body wt) -(vehicle) 5.00 0.31 0.08

Mice

on Maturation

s

GdG

77.8 74.2 72.6 72.8

-r- 1.64 k 2.95* f 1.52** + 1.30**

19.8 23.4 24.4 24.8

2 2 a t

G*M

1.09 4.16 2.40* 3.34**

3.0 2.0 2.8 3.2

+ 5 2 r

0.70 1.22 1.30 2.16

p C57BL/6 mice (10 animals/group) were gavaged with O-5.0 mg/kg body wt aminocarb and bone marrow cells were collected and analyzed individually at 10 days after treatment. b Cytofluorometric analysis was performed with polynomial model of DNA cell-cycle analysis software (Becton-Dickinson). The results are expressed as the mean -C SD. * P < 0.05; **P < 0.01 (U test and randomized test).

40

BERNIER

ET AL.

TABLE 3 Effect of Sublethal Oral Exposure of C57BLl6 Mice to Aminocarb on the Incidence (%) of Cell Surface Markers in Bone Marrow Fraction II Cell surface markef’

Aminocarb” (mg/kg body wt)

PNA

-(vehicle) 5.00 0.31 0.08

52.20 50.78 40.10 37.98

IgM-IgD

W

-t- 5.65 k 4.08 2 7.95* 2 6.13**

38.39 35.81 36.71 39.17

+ + ” ”

5.19 3.00 2.72 2.97

14.81 13.18 15.65 13.92

k f f k

2.18 2.20 4.52 2.37

Ia 39.92 40.52 38.78 40.68

+ 1.99 + 4.49 ‘_ 1.80 zk 2.28

D C57BL/6 mice (10 animals/group) were gavaged with O-5.00 m&g body wt aminocarb and bone marrow cells were collected and analyzed individually at 10 days after treatment. b The results are expressed as the mean -+ SD. * P < 0.05; **P < 0.01 (U test and randomized test).

exposed groups and vehicle controls (Table 3). A similar conclusion was obtained for Ia expression in the cell population content in peak II, as no differences were observed between the incidence of Ia-positive cells for control and aminocarb-exposed animals (Table 3). IgM density and a shape of fluorescence distribution were analyzed according to the reported discrimination of El cells as a subpopulation and according to the linear relation of fluorescence to the number of antigens per cell surface (Table 4). For this purpose, IgM-positive cells were discriminated by low and high density expression (Table 4). Interestingly, at low and medium doses, TABLE 4 Effect of Sublethal Oral Exposure of C57BLl6 Mice to Aminocarb on the Incidence (%) of sZgM-Positive Bone Marrow Cells of Low or High Fluorescence Intensity Density of sIgMb

Aminocarb”

@glkgWY Oil 5.00 0.31 0.08

Low

wt)

45.43 48.29 50.38 52.28

-’ 2 ,2

3.60 4.04 1.61*** 6.03*

High 57.50 52.19 49.44 47.72

2 6.83 f 4.01 r+_2.00** + 6.03*

n C57BLl6 mice (10 animals/group) were gavaged with O-5.00 mg/kg body wt aminocarb and bone marrow cells were collected and analyzed individually at 10 days after treatment. b The results are expressed as the mean 2 SD. * P < 0 .05., **P < 0.01; ***P C 0.001 (U test and randomized test).

aminocarb appeared to cause a significant increase of low density IgM, which was correlated with a decreased number of high density IgM-positive cells (Table 4). A marked shift in the distribution profile of IgM density on B cells was observed for 0.31 and 0.08 mg/kg body wt aminocarb doses, but we could not confirm any doserelation phenomenon. Thus, the data on IgM-positive cell density reflect an alteration in B cell subpopulation following exposure to aminocarb. Effect of Aminocarb Bone Marrow

on Regenerating

The effect of aminocarb on regenerating bone marrow was studied in syngeneic marrow transplantation to X-irradiated recipients. Donor C57BL/6 mice were gavaged with aminocarb at 10 days prior transplantation. Two months after reconstitution, mitogenic and allogeneic responsiveness was evaluated. Some mortality was observed in groups reconstituted with marrow cells from donor mice exposed previously to 5.0 and 0.31 mg/kg body wt aminocarb (2/10 and 4/10 dead animals, respectively). This mortality was observed between 7 and 10 days after experiment and the autopsy and visual observation of liver and spleen demonstrated a faded color of these organs (not shown), suggesting that erythropoiesis was affected. Autopsy and observation of spleen and liver organs in the surviving

IMMUNOTOXICITY

recipient mice, however, demonstrated no visual changes in organ color, excluding therefore any pesticide-related damage of erythropoiesis. T cell proliferative responses to PHA and Con A mitogens and to alloantigens were assayed in reconstituted animals by in vitro quantification of cellular RNA and DNA synthesis. Results obtained were compared to normal syngeneic controls reconstituted with bone marrow from untreated mice and oil-treated mice. Exposure to corn oil vehicle did not cause any alteration in regenerating bone marrow (Fig. 2). Furthermore, RNA and DNA synthesis in Con A- and PHA-stimulated cells was not affected in groups reconstituted with bone marrow from aminocarb-exposed mice (Fig. 2). Thus, the T cell subpopulation appeared to

OF

AMINOCARB

be unaffected in X-irradiated recipient animals receiving bone marrow transplants from aminocarb-exposed donors. This was confirmed by the assay of one-way MLR with reconstituted groups of mice receiving marrow transplant from aminocarb-exposed donors (Fig. 3). Splenocytes from animals receiving marrow transplants from untreated- or aminocarb-treated donors responded to alloantigens, demonstrating therefore a good, specific T lymphoproliferative potential (Fig. 3). The allogeneic responses for groups receiving marrow from donor mice treated with 0.31 and 0.08 mg/ kg body wt aminocarb were more variable and lower when compared to other groups (Fig. 3). Nevertheless, these effects were not significant and therefore aminocarb appeared to have no effect on the reconstitution phenomenon. The effect of aminocarb on B cell proliferating responses of the regenerating bone marrow was assessed by in vitro stimulation with the B cell-specific mitogens, LPS, DXS, and DXS-LPS (Fig. 4). Stimulation by DXS, expressed as RNA and DNA synthesis, was similar in all aminocarb and vehicle groups (Fig. 1). LPS stimulation was

2w

60.00

200 ;;160

t

50.W

%

b

41

(II)

x

40.00

1 g

30.00

t 0100

20.00 10.00 so I 0

N/N

o/N

5.0/N

0.31/N

0.06/N

Donor/Reclplont FIG. 2. Proliferative responses expressed in dpm of (crossed bars) [‘HlTdR and (open bars) [‘H]UdR incorporation of reconstituted recipient C57BL16 mouse spleen cells cultured with (A) PHA and(B) Con A. The recipient animals were X irradiated and reconstituted with syngeneic bone marrow cells from donor mice gavaged with O-5.0 mglkg body wt aminocarb at IO days prior to transplantation. N, normal mice; 0, vehicle-treated mice.

0.00

o/n

5.0/N Donor

/

0.31/N

mclptmnt

FIG. 3. One-way MLR response of spleen cells from reconstituted recipient C57BLl6 mice expressed in dpm of [3H]TdR incorporation. Recipient mice were X irradiated and reconstituted with syngeneic bone marrow cells from donor mice gavaged with O-5.00 mglkg body wt aminocarb at 10 days prior to the experiment. Cells were cultured for 96 hr either alone (open bars) or cocultured with X-irradiated A/J spleen cells (crossed bars). N, normal mice; 0, vehicle-treated mice.

42

BERNIER

ET AL.

In addition, stimulation with DXS-LPS resulted in normal responses in all groups (Fig. 4). Generally then, exposure of donor mice to aminocarb did not affect B cell subpopulation in regenerating bone marrow in the recipient animals. DISCUSSION

FIG.

4. Spleen lymphocyte proliferative

responses

of reconstituted recipient C57BLl6 mice, expressed in dpm of (crossed bars) r3HJTdR or (open bars) [3mlJdR incorporation, cultured with (A) DXS; (B) LPS; and (C) LPS-DXS! Recipient mice were X irradiated and reconstituted with syngeneic bone marrow cells from donor mice gavaged with O-5.00 mglkg body wt aminocarb at 10 days prior to transplantation. N, normal mice; 0, vehicle-treated mice.

slightly higher, when expressed as DNA synthesis, in groups reconstituted with marrow from oil-treated donors, when compared to normal donors. Similar effects were observed for donor groups injected with 0.31 and 0.08 mglkg body wt aminocarb; however, no aminocarb-related, significant changes were noted (Fig. 4). Thus, the slight effect on LPS stimulation appeared to be related to the vehicle only.

The most interesting observation in these studies is relative alteration of bone marrow lymphocyte populations after sublethal exposure to the carbamate pesticide aminocarb. Several parameters remained unaffected or were only slightly affected: (i) the number of bone marrow cells remained unaffected: (ii) the number of B cells was unchanged; (iii) reconstitution with bone marrow from aminocarb-exposed animals did not affect regenerating bone marrow in the recipient animals. Therefore, these data demonstrated no direct pesticide-related damage of marrow cells. Characterization of bone marrow cell population demonstrated, however, a modification in B cell subpopulation, following sublethal exposure to aminocarb. The following observations lead to these conclusions: (i) adult bone marrow cells from aminocarb-treated mice labeled with PE-anti-IgM displayed a decrease of the fluorescence profile in the population of B lymphocyte with a low density of surface IgM; (ii) this was associated with decreased relative frequency of B lymphocytes with a high density expression of IgM. Aminocarb exposure, however, did not affect the number of B cells in bone marrow since the data show any changes in frequency of IgM+ cells or IgM +IgD + . In addition, aminocarb-related alteration of bone marrow microenvironment cannot be excluded as microenvironment-dependent maturation of B lymphocytes was not affected in normal, X-irradiated syngeneic recipients receiving bone marrow transplants from aminocarb-exposed donors. Many authors reported that B lymphocyte could be subdivided on the basis of IgM expression and that the level of IgM on surface cell could be used as an indicator

IMMUNOTOXICITY

for the maturation level of functional B cells (20, 21, 30). Moreover, immature distribution of total surface Ig and IgM on B lymphocytes in CBA/N mice was attributed to an abnormality in the development of mature B lymphocytes from their immature precursors. In addition, the abnormality in distribution of adult splenic CBA/N cells with a given density of total surface Ig and IgM was associated with a number of defects in B lymphocyte functions (30). According to these findings, our data suggest that aminocarb could induce an augmentation of one population with the weakest IgM density. Osmond and Nossal (19) reported that these cells illustrate the youngest cohort among postmitotic IgMf marrow lymphocytes. This population of cells was found to be preponderant IgD negative (21). Our results demonstrated no changes in the incidence of B lymphocytes bearing only the IgM marker. Nevertheless, this observation was important and the latter findings did not contradict the data since the cells appeared to leave bone marrow at various stages of maturation (19). PNA binds to glycoproteins with terminal galactose residues (31) and was reported to bind to a population of surface IgM-negative and cytoplasmic k-chainpositive cells (l&22). Thus, PNA binding is related to pre-B cells and can be utilized as a surface marker to separate immature B cells from the mature cells (18, 22). Our interpretation of the data for the B cell subpopulation is that, at the intermediate and lower doses, aminocarb appeared to decrease the number of pre-B cells, likely due to a shift in B cells with low density of surface IgM. Interestingly, a decrease in the cell number in GdG,, associated with an increased number of cells entering the S phase, was observed for the exposure doses of aminocarb which modulated the number of the pre-B or B lymphocytes. Therefore, we can assume that the pesticide induced an augmentation of premitotic events in bone marrow, but not at the level of the mitose rate. Luster et al. (8) demon-

OF

AMINOCARE

(II)

43

strated increased proliferative responses of pluripotent stem cells (CFU-S) after exposure to methyl carbamate. An alternative explanation can be an arrest in the GJM phase, observed, for example, for many antineoplastic drugs (32). This supposition was partially supported by pharmacological studies for antileukemic drug with the carbamate moiety (9). Other studies on the carbamate pesticide furadan showed a depression in bone marrow cell population (13) and this observation is different from our data for aminocarb . Nevertheless, it appears that carbamate-related changes in bone marrow are significant and the mechanism remains to be elucidated. Our previous data demonstrated that oral exposure to aminocarb augmented a humoral response in mice, without affecting the cellular response (5, 7). Another carbamate pesticide, carbaryl, was shown to induce an increased IgG antibody production after parenteral immunization (33) or, if administered intraperitoneally, augmented the IgM humoral response (6). On the other hand, carbaryl induced the suppression of the humoral response at the systemic level when administered at nearlethal doses (34). Thus, the exposure route was possibly an important factor in mediation of the effect of carbamate pesticide (57, 34). Overall, the data confirm an activating potential of aminocarb on the immune system. ACKNOWLEDGMENTS

The authors thank Mme Monique Morin for her excellent technical help in fluorocytometric analysis. This work was supported by the Natural Sciences and Engineering Research Council of Canada and Toxen. Universite du Quebec a Montreal. REFERENCES

1. NRCC Publication, “Aminocarb: The Effects of Its Use on the Forest and Human Environment,” National Research Council Canada Publications, NRCCICNRR, No. 18979, Ottawa, 1982. 2. R. Vincent, J. Boudreau, S. Lapare, D. Nadeau, B. Trottier, M. Fournier, K. Rrzystyniak, and G. Chevalier, Biochemical responses in rat

44

BERNIER

lungs following acute exposure to aerosolized oil-formulated aminocarb insecticide. Submitted. J. Aero. Med. Vo12 No 4 in press. 3. I. Vassilieff and D. J. Ecobishon, Acute toxicity of aminocarb in male rats and inhibition of tissue esterases, Bull. Environ. Contamin. Toxi-

ET AL.

16.

col. 31, 326 (1983).

4. M. Blaszczyk, S. Boileau, M. Foumier, G. Chevalier, and K. Krzystyniak, Growth of facultative anaerobic population of human intestinal bacteria with the carbamate insecticide aminocarb, Pestic. Biochem. Physiol. 29, 233 (1987). 5. J. Bemier, M. Foumier, Y. Blais, P. Lombardi, G. Chevalier, and K. Krzystyniak, Immunotoxicity of aminocarb. I. Comparative studies of sublethal exposure to aminocarb and diekhin in mice, Pestic. Biochem. Physiol. 30, 238 (1988). 6. M. Foumier, J. Bemier, D. Flipo, and K. Krzystyniak, Evaluation of pesticide effects on humoral response to sheep erythrocytes and mouse hepatitis virus 3 by immunosorbent analysis. Pestic. Biochem. Physiol. 26, 353 (1986). 7. J. Bemier, D. Gerard, M. Rola-Pleszczynski, G. Chevalier, K. Krzystyniak, and M. Foumier, Immunotoxicity of aminocarb. III. Exposure route-dependent immunomodulation by aminocarb in mice. Submitted. 8. M. I. Luster, J. H. Dean, G. A. Boorman, M. P. Dieter, and H. T. Hayes, Immune functions in methyl and ethyl carbamate treated mice, Clin. Exp. Immunol. 50, 223 (1982). 9. W. K. Anderson and M. J. Halat, Antileukemic activity of derivatives of 1,2-dimethyl-3,Cbis(hydroxymethyl)-5phenylpyrrole bis(N-methylcarbamate), J. Med. Chem. 22, 977 (1979). 10. G. Renoux, Immunopharmacologie et pharmacologie du diethyldithiocarbamate (DTC). J. Pharmacol. (Paris) 13, Suppl. 1, 95 (1982). 11. L. J. Olson, The immune system and pesticides. J. Pestic. Ref. Summer, 20 (1986). 12. L. J. Olson, B. J. Erickson, R. D. Hinsdill, J. A. Wyman, W. P. Porter, L. Binning, R. Bidgood, and E. Nordheim, Immunosuppression of mice by a pesticide groundwater contaminant, in “Proc. Algime Pest Management Conference, Madison, WI, January 21-23,” Vol. 25, p. 155, 1986. 13. M. Gupta, G. Bagchi, S. Bandyopadhyay, D. Sasmal, T. Chatterjee, and S. N. Dey, Hematological changes produced in mice nuvacron or furadan, Toxicology 25, 255 (1982). 14. P. J. Oliver and A. L. Goldstein, Rapid method for preparing bone marrow cells from small laboratory animals, J. Immunol. Methods 19, 289 (1978). 15. I. W. Taylor, A rapid single step staining tech-

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

nique for DNA analysis by flow cytofluorometry, J. Histochem. Cytochem. 28, 1021 (1980). I. Goldschneider, D. Metcalf, F. Battye, and T. Mandel, Analysis of rat hemopoietic cells on the fluorescence activated cell sorter. I. Isolation of pluripotent hemopoietic stem eels and granulocyte-macrophage progenitor cells, J. Exp. Med. 152, 419 (1980). M. R. Loken and L. A. Herzenberg, Analysis of cell population with a fluorescence-activated cell sorter, Ann. N.Y. Acad. Sci. 254, 163 (1975). D. G. Osmond and J. J. T. Owen, Pre-B cells in bone marrow: Size distribution profile, proliferative capacity and peanut agglutinin binding of cytoplasmic p chain-bearing cell populations in normal and regenerating bone marrow, Zmmunology 51, 333 (1984). D. G. Osmond and G. J. V. Nossal, DitTerentiation of lymphocytes in mouse bone marrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studies by double labeling, Cell. Zmmunol. 13, 132 (1974). P. K. Lala, G. R. Johnson, F. L. Battye, and G. J. V. Nossal, Maturation of B lymphocytes. I. Concurrent appearance of increasing Ig, Ia, and mitogen responsiveness, J. Immunol. 122, 334 (1979). P. K. Lala, J. E. Layton, and G. J. V. Nossal, Maturation of B lymphocytes. II. Sequential appearance of increasing IgM and IgD in adult bone marrow. Eur. J. Zmmunol. 9, 39 (1979). R. A. Newman and M. A. Boss, Expression of binding sites for peanut agglutinin during murine B lymphocyte differentiation. Immunology 40, 193 (1980). J. W. Goding, “Monoclonal Antibodies: Principles and Practice,” 2nd ed., p. 256, Academic Press, New York, 1986. J. J. Mond, S. Kessler, F. D. Finkelman, W. E. Paul, and I Scher, Heterogeneity of Ia expression on normal B cells, neonatal B cells, and on cells from B cell-defective CBA/N mice. J. Immunol. 124, 1675 (1980). D. M. Wier, “Handbook of Experimental Immunology: Cellular Immunology,” 3rd ed., Vol. 2, Blackwell, Oxford, 1979. P. Hugo, J. Bernier, K. Krzystyniak, and M. Foumier, Transient inhibition of mixed lymphocyte reactivity by dieldrin in mice, Toxicol. Lett. 41, 1 (1988). L. Viheneuve, P. Brousseau, J. Chaput, and R. Elie, Role of adherent cells in graft-versus-host induced suppression of the humoral immune response, &and. 1. Zmmunol. 12, 321 (1980). G. D. Wetzel and J. R. Kettman, Activation of murine B cells. II. Dextran sulfate removes the

IMMUNOTOXICITY requirement for cellular interaction during lipopolysaccharide-induced mitogenesis, Cell. Immunol. 61, 176 (1981). 29. G. D. Wetzel and J. R. Kettman, Activation of murine B lymphocytes. III. Stimulation of B lymphocyte clonal growth with lipopolysaccharide and dextran sulfate, J. Immunol. 126, 723 (1981). 30. I. Scher, S. 0. Sharrow, and W. E. Paul, X-linked B-lymphocyte defect in CBA/N mice. III. Abnormal development of B-lymphocyte populations defined by their density of surface immunoglobulin, J. Exp. Med. 144, 507 (1976). 31. J. London and G. E. Roelants. Terminal galactosyl residues as marker of lymphoid maturation

OF AMINOCARB

(II)

45

studied by peanut agglutinin, in “Protide of the Biological Fluids” (H. Peeters, Ed.), p. 611, Pergamon, Oxford, 1978. 32. X. Ronot and M. Adolphe, Update on the concept of cell cycle: The contribution of flow cytometry, Biol. Cell. 58, 113 (1986). 33. F. Andre, J. Gillon, C. And&, S. Lafont, and G. Jourdan, Pesticide-containing diets augment anti-sheep red blood cell nonreaginic antibody responses in mice may prolong murine infection with Giadia muris, Environ. Res. 32,145 (1983). 34. R. W. Wiltrout, C. D. Ercegovich, and W. S. Ceglowski, Humoral immunity in mice following oral administration of selected pesticides, Bull. Environ. Toxicol. 20, 423 (1978).