lnt. Z lmmunopharmac., Vol. 15, No. 7, pp. 811-819, 1993. Pergamon Press Ltd. Printed in Great Britain. International Society for Immunopharmacology,

CYTOMETRIC PROFILES OF BONE MARROW AND SPLEEN LYMPHOID CELLS AFTER MERCURY EXPOSURE IN MICE

SYLVAIN BRUNET, FRANCE GUERTIN, DENIS FLIPO, MICHEL FOURNIER and KRZYSZTOF KRZYSTYNIAK*

D6partement des Sciences Biologiques and TOXEN, Universit6 du Qu6bec ~ Montr6al, Montr6al, Qu6bec, H3C 3P8 Canada

(Received 18 November 1992 and in final form 22 May 1993)

Abstract -- The potential immunotoxic effects of mercury chloride on murine bone marrow (bm) cell subpopulations, including analysis of maturation patterns for B-cells, were evaluated by flow cytometric analysis. CD-1 outbred mice were exposed for 28 days to relatively low doses of 25- 100 .ppm HgC12 in drinking water and the mercury-related functional cellular changes were validated in a macrophage phagocytosis assay. Lymphocyte subsets from the bone marrow population were stained with PNA lectin and a panel of monoclonal antibodies against cell surface antigens. The incidence of subset-specific staining was also monitored in spleens and thymuses. A dose - effect correlation was noted for the mercury-related activation of macrophage phagocytosis. Subchronic exposure to mercuric chloride resulted in a transient (7 - 14 day) decrease of the lymphoid/total bm cell ratio and affected the incidence of splenic T-cell subsets, however, without a clear dose- response correlation. The B-cell population in spleen and maturation patterns of B-cells in bm appeared to be unaffected by the mercury exposure. Overall, cytometric analysis of lymphoid cell subsets in murine bone marrow revealed transient and subset-non-specific cell fluctuations after subchronic exposure to inorganic mercury.

Nonaccidental exposure to low and very low environ- mental levels of inorganic mercury is pre~licted for large populations, despite an increased awareness of the toxicity of mercuric compounds and decreased use of mercurials (Nielsen, 1992). The exposure results principally from the intake of food items containing mercury, such as fish. For example, the concentration of mercury in fish from unpolluted areas is in the range of 50 - 400/~g Hg/kg, mostly in the form of organomercurials and especially methyl- mercury. It is estimated that approximatively 20 - 40o70 of the mercury in fish is inorganic (Nielsen, 1992).

Mercury chloride accumulates in biological tissues and can interfere with a number of biological processes, including immune responses (Dieter et al., 1983; Burchiel et aL, 1987; Nielsen & Andersen, 1989; Ilback, 1991). Spleen was found to be an important site for the formation of inorganic mercury; spleen macrophages were shown to participate in the biotransformation of organic mercury (Suda & Takahashi, 1986). Humoral,

cellular immune responses and non-specific host defenses were reported to be markedly impaired by mercury exposure in several species (reviewed by Descotes, 1988). Exposure to mercury is considered as a primary factor in the autoimmune glomerulone- phritis (Druet, Pelletier, Rossert, Druet, Hirsh & Sapin, 1989; Hultman & Enestrom, 1992; Hultman & Johansson, 1991). Little is known, however, about the toxic effects of subchronic exposure to HgC12, such as that resulting from industrial exposure or food contamination (Dieter et al., 1983). Non- specific toxicity of orally administered HgCl2 occurred at the 75 ppm dose level, consisting of small differences in body and organ weights, hema- tological changes, and general enzyme inhibition in the bone marrow and spleen. Altered T-cell func- tions were the only immunological defects related to the subtoxic mercury exposure; the bone marrow cellularity and the marrow cell proliferation were not affected (Dieter et ai., 1983).

The rapidly dividing cell population of bone marrow is considered as one of primary targets for

*Author to whom correspondence should be addressed at: D6partement des Sciences Biologiques, Universit6 du Qu6bec A Montr6ai, C.P.8888, Montr6al, Qu6bec, H3C 3P8, Canada.

811

812 S. BRUNET et al.

chemical immunotoxicity (Burchiel et al., 1987; Martin, Brott, Zandee & McKeel, 1992). For example, significant changes in subpopulations of murine bone marrow were detected within one day following treatment with high doses of cadmium or lead (Burchiel et al., 1987). Furthermore, lead- induced alterations of bone marrow cell responses to the hemapoietic growth factor, CSF-I , were postu- lated to contribute to the decreased host resistance observed in Pb-exposed animals (Kowolenko, Tracy & Lawrence, 1989). Overall, the data suggest that heavy metals can cause immunotoxic effects that are concentration-dependent (Dieter et al., 1983). Thus, we selected one of the cellular functional parameters, i.e. the phagocytic activity of peritoneal macro- phages, as a reference for the dose - response cytometric studies of the cell subpopulations. In these studies we examined the effects of subchronic oral exposure to relatively low doses of HgCI, on murine cellular subsets in bone marrow, thymus and spleen. Immunotyping of the immune cellular subsets is used by many laboratories for immuno- pathological and immunotoxicological purposes (Burchiel et al., 1987; Wong, Fournier, Coderre, Banska & Krzystyniak, 1992). Recent analysis of screening procedures for sensitivity and predict- ability of chemical-related injury of the immune system showed high association of the surface marker test with immunotoxicity (Luster et al., 1992). We report transient, mercury-related, fluc- tuations in splenic T-cell subsets and relatively little sensitivity of lymphoid cell subpopulations in bone marrow to subchronic HgCI2 exposure.

EXPERIMENTAL PROCEDURES

Animals

Female outbred CD-1 mice, 8 - 12 weeks old, were obtained from Charles River Company (St- Constant, Quebec). Upon arrival all mice were quarantined for two weeks prior to use. Animals weighing 1 6 - 1 8 g were selected for experiments. Mice were given distilled drinking water alone or water containing HgC12 (Sigma Chemical Co., St Louis, MO) at 25, 75 and 100 ppm for 28 consecutive days. Control groups received distilled water alone. All animals were fed and maintained in identical conditions. The weekly weight gain was determined for all animals and was analogous for control and experimental groups throughout the experiment (P<0.5). All animals in this study were observed at

least once daily; no mortality and no signs of toxicity were noted throughout the experiment (Dieter et al., 1983).

Cells

Mice were killed by COz and spleens, thymuses and femur and tibia bones were collected in cold Hank's balanced salt solution (HBSS, Flow Laboratories, Toronto, Ont.) (Lombardi, Fournier, Bernier, Mansour, Neveu & Krzystyniak, 1991). There was no overt toxic sign from chemical treatment, including organ cellularity and cell viability, as compared to the untreated controls. Bone marrow cells were collected by flushing from the bone with 2.0 ml HBSS with a 26G needle and syringe (Oliver & Goldstein, 1978). Bone marrow cell suspensions were prepared by several pipetting, strained through fine nylon wool to avoid cell clumps and washed three times, as described previously (Bernier, Fournier, Blais, Lombardi, Chevalier & Krzystyniak, 1989).

Peritoneal exudate macrophages (PEM) were obtained and cultured as described elsewhere (Krzystyniak, Flipo, Mansour & Fournier, 1989). Briefly, PEM were obtained by washing aseptically the peritoneal cavities of mice with a total volume of 8 ml of heparinized HBSS. After collection, cells were centrifuged at 1800revs/min at 4°C for 10 min, resuspended in HBSS, counted and adjusted to 1 × 106 cells/ml.

Spleen cells and thymic cells were collected as described before (Lombardi et al., 1991). Briefly, spleens were aseptically removed and splenocyte suspensions were obtained by teasing the spleens into HBSS. Lymphoid spleen cells were cultured at 37°C, 5°'/0 CO2 for 72 h, at a concentration of 5.0 × 106 ceUs/ml in RPMI medium supplemented with 10% fetal calf serum inactivated by heating at 56°C for 30 min, 100 U/ml penicillin, 100/ag/ml strepto- mycin, and 5 × 10-SM 2-mercaptoethanol. Cell viability was determined by the trypan blue exclusion test or by ethidium bromide staining and cytofluo- rometry.

Phagocytosis

The HgC12-related dose -e f f ec t correlation for cellular functional parameters was analyzed for one selected parameter only, i.e. the macrophage phagocytosis, which served further for validation of the dose - response cytometric studies. For this assay we used a flow cytometry quantitation of phago- cytosis of fluorescent microspheres (Steward,

Bone Marrow and Spleen Cytometry after Mercury Exposure

Lehnert & Steinkamp, 1987). Briefly, peritoneal exudates were washed with HBSS medium and incubated for 0 - 6 0 min at 4°C (negative control) and at 37°C (positive control) with fluorescent microspheres (ratio 100 :1 microspheres/cell; cat. No. 17,687, Polyscience, Warrington, PA), as described previously (Krzystyniak et al., 1989). The percentage of cells with engulfed particles was determined by flow cytometry, using FACScan (Becton-Dickinson). Individual particulate burdens within each cell were expressed as total frequency of phagocytic cells and as frequency of phagocytic ceils with three or more internalized particles.

813

IgM) (Fisher Biotech, Orangeburg, NY). The cell labelling was carried out by preparing a mixture of 50/al of the cell suspension (2 × 107 cells/ml) with 50/al of antibody solution (2/ag/ml) or 50/al of P N A - F I T C solution (5/ag/ml) and incubation at 4°C for 3 0 - 4 5 min. Two ml of medium were added, then twice centrifuged at 1200 revs/min for 10 min and the cell pellet was resuspended in 0.5 ml of PBS supplemented with 1 070 BSA and 0.1 °70 sodium azide. To minimize cell adherence, all samples were kept in the dark at 4°C in polystyrene tubes and analyzed on the next day.

Immunotyping of bone marrow lymphoid subsets

Lymphocyte precursors from bone marrow population of CD-1 mice were analyzed by cytofluo- rometric analysis with a FACScan (Becton- Dickinson) flow cytometer (Bernier et al., 1989). For analysis of fluorescein isothiocyanate (FITC) and phycoerythrin (PE)/ethidium bromide, the fluore- scence was monitored through 530 and 585 nm band pass filters, respectively. Frequency of cells in peak II and III of the unstained bone marrow cell population, which have been reported to correspond to lymphoid ceils and non lymphoid/myeloid cell lineage, respectively (Goldschneider, Metcalf, Battye & Mandel, 1980), was determined by direct cyto- metric 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 ceils by direct immunofluorescence. Fiuorescein-labelled peanut agglutinin (PNA-FITC) was used to discriminate Ig-bearing cells from immature cells (Osmond & Owen, 1984; Newman & Boss, 1980). PNA was reported to bind to the cell surface glycoproteins with terminal galactose residues (London & Roelants, 1978) and to a population of surface-IgM-negative and cytoplasmic u-chain- positive cells (Osmond & Owen, 1984; Newman & Boss, 1980). Thus, the PNA binding to the pre-B- cells could be utilized as a surface marker to separate immature B-cells from the mature cells (Osmond & Owen, 1984; Newman & Boss, 1980). Negative isotype controls of the specificity of the labelling were performed for lymphoid cell populations and were shown not to be different from the unstained cell controls used as routine negative controls. Antibody for direct immunofluorescence staining was R-Phycoerythrin-labeled, affinity purified goat anti-mouse IgM, anti-mouse CD45R (GIBCO BRL, Gaithersburg, MD), and anti-mouse Ig (PE-anti-

Immunophenotyping of splenic and thymic cell subsets

Monoclonal antibodies (mAb) coupled with fluorescein isothocyanate (FITC) and phyco- erythrine (PE) were used for direct staining of splenic and thymic cells (Krzystyniak et al., 1992): anti- L3T4-PE (CD4+), anti-Lyt2-FITC (CD8÷), anti-Thy 1.2-FITC (T-cells) and anti-Ig-FITC (B-cells) (Becton-Dickinson Immunocytometry Systems, Mountain View, CA). Splenic and thymic cells were depleted from red blood cells by osmotic shock, washed three times with PBS, adjusted to 106 cells/ 0.1 ml in PBS + 0.1070 NaN3 and 1070 BSA, stained with appropriate mAb reagent for 30 min at 4°C, washed three times with PBS, and resuspended in 0 .5ml of P B S - B S A - N a N 3 . After a second incubation of 30 min at 4°C, and three washings, cells were fixed with 107o formaldehyde. Fluorescence was analyzed by a FACScan (Becton-Dickinson) flow cytometer. Specific fluorescence for lymphoid cell subsets was determined and expressed as a percent of mAb marker-positive ceils inside the lymphoid cell aquisition gate. For this purpose, a light scatter aquisition gate was optimized for recovery of lymphoid cells (Loken, Brosnan, Bach & Ault, 1990).

Data handling and statistics

Data have been expressed as arithmetical mean +_. S.D. Analysis of variance (ANOVA) was used to establish significant differences between control and experimental groups. Significance was concluded at P<0.05 and was denoted by **. For the significantly different groups, the Sheffe's comparison of means was carried out between the control and experimental groups. For the statistical analysis of percentages, the X 2 (Chi 2) test was used (Lutz, 1987).

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Fig. 1. Cytometric assay of a dose-related activation of the peritoneal macrophage phagocytosis of fluorescent microspheres by in vivo exposure of CD-1 mice to HgCI2 in drinking water: open bars = controls, right-crossed bars = 25 ppm, pointed bars = 75 ppm, left-crossed bars = 100 ppm. The data are mean of six animals per

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peak III: myeloid cells, early erythroblasts.

RESULTS

S. BRUNET et al.

Dose-dependent stimulation o f macrophage phagocytosis by subchronic HgC12 exposure

Potential mercury-related functional alterations of macrophage phagocytic activities were analyzed by cytometric assay of the uptake of fluorescent microspheres by the cells. The uptake of three and

more microspheres was counted; this procedure discriminated the efficient phagocytic activity of the cells. Good phagocytic activity of control macro- phages was noted during a 30-min incubation period (Fig. 1). As shown in Fig. 1, the percentage of phagocytosing macrophages increased significantly after exposure to 25 - 100 ppm HgCl2. Furthermore, a dose-effect correlation was noted for. the mer- cury-related stimulation of macrophage phagocyto- sis; the correlation coefficients were 0.99 at 7 days, 0.91 at 14 days, 0.91 at 21 days and 0.80 at 28 days of the exposure, respectively. Thus, oral uptake of relatively low, subchronic doses of HgCl2 markedly stimulated phagocytic activity of peritoneal exudate macrophages, possibly affecting the non-specific immunity in mice.

Effect o f HgC12 exposure on bone marrow cells

The effect of mercury on mouse bone marrow cells was evaluated in Cd-I mice exposed to 25, 75 and 100 ppm HgC12 for 28 days and cytofluorometric analysis. Bone marrow cells were collected from each mouse, from femoral and tibial bones, and analyzed independently in controls and mercury-exposed animals. The prototype distribution profile of low angle (zero degree) forward light scatter of the heterogeneous bone marrow cell population is presented in Fig. 2. The amount of light scattered by a particle is related to the size and shape of that particle. Thus, cell size information is provided by this method. A triphasic curve was observed for murine bone marrow cells, where each peak represented different cell populations (Fig. 2). The peak I on the left-hand side of the profile corresponded to dead cells, mature red blood cells (RBC) and cell debris and peak III, representing non- lymphoid (myeloid) cell lineage, were not analyzed. Peak II, corresponding to the bone marrow lymphoid cells, was analyzed throughout the experiment and compared to control data from untreated animals (Table 1). The relative cell number in gated bone marrow lymphoid cells (peak II) was relatively stable for control animals (mean: 4446 _+ 769), including the lymphoid/total bone marrow cell ratio (Table 1). Significant changes in relative frequency of cells in peak II were observed after the exposure to 75 ppm (3614 _+ 703, P~<0.05) and 100 ppm HgC12 (2910 + 572, P~<0.05), at 7 and 14 days of treatment. No dose-response relation- ship, however, could be concluded. Furthermore, the lymphoid/total bone marrow data for 21 and 28 days of the mercury exposure were not different from the control data (P>0.05) (Table 1). Furthermore, none

Bone Marrow and Spleen Cytometry after Mercury Exposure 815

Table 1. Effect of subchronic HgCI2 exposure of CD-I mice on the lymphoid/total cell ratio and specific staining of lymphoid subset in bone marrow. Lymphoid cell fraction of bone marrow*

Lymphoid % Specific staining Days of cell ratio * exposure HgCI2 t (fraction II/total) CD45R ÷ PNA ÷ Ig + IgM ÷

7 days

14 days

21 days

28 days

-(control) 0.34 ± 0.04 59.48 +- 10.52 52.59 ___ 10.79 32.31 ± 5.91 32.95 ± 6.06 25 ppm 0.31 ± 0.05 68.41 ± 14.95 55.06 ± 13.50 35.08 +_ 4.65 35.17 ± 5.55 75 ppm 0.24 ± 0.05 ~ 50.83 -4- 9.55 42.95 + 18.54 34.86 ± 9.94 34.04 ± 9.33 100 ppm 0.32 _ .05 65.83 ± 11.98 59.14 ± 8.34 40.69 ± 7.48 35.87 ± 3.76

-(control) 0.28 +_ 0.07 60.34 _+ 10.82 42.23 ± 4.51 33.09 ± 8.13 33.22 ± 6.60 25 ppm 0.28 ± 0.08 66.85 _+ 7.88 50.26 + 13.33 34.21 _+ 3.89 35.63 ± 2.69 75 ppm 0.22 ± 0.04 58.61 _ 8.99 46.74 _+ 8.73 41.49 +_ 9.16 26.80 ± 3.89 100 ppm 0.19 +__ 0.04 ~ 55.95 +_ 4.78 43.87 _ 5.28 40.60 ± 10.52 26.70 ± 8.52

-(control) 0.24 _ 0.06 62.28 _+ 12.98 55.68 ± 10.48 35.29 ± 9.45 33.27 ± 1.82 25 ppm 0.24 ± 0.08 68.45 ± 9.20 66.20 _ 8.01 35.96 ± 4.79 32.93 ± 7.90 75 ppm 0.26 ± 0.07 72.07 _ 9.45 69.25 ± 13.43 40.23 _ 9.46 33.40 +__ 11.12 100 ppm 0.26 ± 0.07 52.04 ± 7.74 49.02 ± 9.05 30.32 ± 14.41 31.27 ± 4.59

-(control) 0.33 ± 0.04 63.21 + 5.52 50.20 ± 6.34 28.47 ± 5.05 25.80 +_ 4.98 25 ppm 0.33 _+ 0.04 62.51 ___ 4.99 43.52 ± 10.01 34.89 +_ 9.98 28.63 ± 4.29 75 ppm 0.28 _ 0.05 62.82 _ 6.92 53.22 ± 7.71 35.66 _ 16.92 27.44 _ 2.60 100 ppm 0.33 _ 0.04 63.37 _ 5.11 49.91 ± 4.53 31.49 + 7.71 24.20 _+ 3.56

*The results presented are the mean value of six mice _+ S.D. ~Concentration of HgCI2 in drinking water. *The cell ratio was calculated as the relative cell number in gated (fraction II) lymphoid cells versus total bone marrow cell population. ~P~0.05.

o f the specifically-stained CD45R +, P N A +, Ig + and IgM + bone marrow cellular subsets appeared to be markedly affected by the exposure (Table 1). In addit ion, no marked changes for the light scattering at right angles, associated with chromat in deconden- sation, were noted th roughout the experiment for the bone marrow cell popula t ion (not shown). It appeared therefore that the mercury-related decrease in the l y m p h o i d / b o n e marrow cell ratio appeared to be general for all examined cell subsets o f the peak II. Generally then, a transient decrease in the bone marrow lymphoid cell popula t ion was noted for 75 and 100 ppm HgC12, however, no marked fluctu- ations were noted for the CD45R ÷, P N A ÷, Ig ÷ and IgG + bone mar row cellular subsets after subchronic mercury exposure.

P N A - F I T C staining was used to discriminate the Ig-bearing cells f rom immature cells and anti-CD45R for the determinat ion of the total lymphoid cell number (Table 1). Despite a marked decrease in a total cell number for 75 and 100 ppm HgC12, no significant changes were observed for any of the four parameters tested. Thus, the relative cell distr ibution for different matura t ion steps in bone marrow was intact after subtoxic exposure to mercury chloride. In addit ion, determinat ion of the frequency of mature B-cells, expressing the surface IgM, did not show any differences between the mercury-exposed groups and the controls. Overall, it could be concluded that HgCI2 had a transient and non- specific effect on total bone marrow lymphoid cell populat ion.

Effect o f mercury on maturation o f B-lymphocytes

Identif icat ion of surface antigens displayed on murine B-lymphocytes were employed to assess the B-cell matura t ion . The gated bone mar row lymphoid cell popula t ion in peak II (Fig. 2) was fur ther analyzed by specific staining of bone marrow cell subsets with f luorescent ant ibodies and lectin. The

Effect o f HgC12 exposure on thymic and splenic cell subsets

We studied the effect o f mercury on thymic and splenic cells in addit ion to cytometric analysis o f bone marrow lymphoid cells in Cd-I mice exposed to 2 5 - 1 0 0 ppm HgC12 for 28 days. Thymuses and spleens were collected and analyzed independent ly in

816 S. BRUNET et al.

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Fig. 3. Incidence of specific staining of splenic cell subsets after subchronic exposure to (11) 25 ppm, (A) 75 ppm and (O) 100 ppm HgCI2: (A) Ig + cells, (B) Thy 1.2 ÷ cells, (C) L3T4 ÷ cells, (D) Lyt 2 + cells; dotted line represents mean for

controls throughout the experiment _+ S.D. (crossed space). The data are mean of six animals per group; **P~0.05.

controls and mercury-exposed animals. The specific staining of thymic cells with Thy 1.2 fluorescent antibody in controls was relatively high (86.6 _ 4.4°70 -99 .4 __. 1.6070) and was not different from the percentage of Thy 1.2 ÷ cells in mercury- exposed animals (88.9 _+ 6.5°70- 99.0 _+ 0.5070), throughout the experiment (P>0.5). Overall, no mercury-related pathological changes were noted in thymuses from the animals exposed for 28 days to 25 -100 ppm HgCl2. Similarly, no pathological changes, including spleen cellularity, were noted for spleen organs from control- and mercury-exposed animals. As shown in Fig. 2, the specific staining of spleen cell subsets in control animals was stable throughout the experiment and was 19.7 _+ 3.907o for PNA + cells, 50.8 -+ 6.507o for Ig + cells, 30.9 _+ 7.20/0 for Thy 1.2 + cells, 23.7 _+ 5.3°70 for L3T4 ÷ cells and 5.2_+ 1.0070 for Lyt 2 + cells (mean values), respectively. Specific staining of the Ig + cell subpopulation in mercury-exposed animals was not different from the controls (P>0.5) throughout the experiment (Fig. 3). Exposure to 2 5 - 1 0 0 p p m HgC12 at 7 - 14 days appeared to affect the splenic

T-cell subpopulations since the incidence of specific staining of the Ly2 ÷, L3T4 ÷ and Thy 1.2 + was altered at 7 - 1 4 days of the exposure, however, without any clear dose - effect relationship (Fig. 3). These mercury-related changes in lymphocyte cell subsets were transient as no fluctuations were noted at 2 1 - 2 8 days of the exposure (/'>0.5) (Fig. 3). Thus, the transient shift in the cell subsets were noted at the same time interval for both bone marrow and spleen lymphoid cells, i.e. at the early stage of the mercury exposure (Table 1, Fig. 3). Since none of these cell subsets appeared to be selectively discriminated in a dose-response pattern, it could be concluded that the transient ( 7 - 1 4 days) and non-specific toxicity of mercury exposure appeared to affect the total bone marrow and splenic T-cell populations.

DISCUSSION

Cytometric profile of bone marrow lymphoid cell subsets in mice, including the B-cell maturation profile, was unaffected by mercury employed at

Bone Marrow and Spleen Cytometry after Mercury Exposure

relatively low doses. Our studies revealed a normal cytometric profile in the bone marrow of mercury- exposed animals: (i) the relative distribution of cell subsets in bone marrow cells remained unaffected; (ii) the proportion of B-cells was unchanged; (iii) the process of differentiation/maturation of B lymphoid bone marrow population was intact. At low mercury doses, the animals exhibited intact bone marrow characteristics (Dieter et al., 1983). Furthermore, manifestations of the mercury-related general toxicity did not include any signs of specific immunotoxicity. Subchronic exposure to relatively low concentrations of HgCl2 in drinking water resulted in a time-related and dose-related accumulation of the toxicant in kidneys, however, the retention of mercury was only 0.14070 of the estimated daily intake (0.3/~g at 75 ppm HgC12) (Dieter et al., 1983). No detectable mercury was noted for the immune system organs, such as in thymus and bone marrow, with the exception for spleen only, which contained a 3- to 5-fold less of the toxicant than in the kidney (Dieter et al., 1983). Regarding maturation of B-lymphocytes the B-cell population can be recognized according to the IgM÷IgD + and IgM÷Ia + surface expression (Lala, Johnson, Battye & Nossal, 1979). In other words, the IgM+IgD ÷ and IgM+Ia + cells represent the final steps in the B-cell maturation in bone marrow. Since the premature IgM + bone marrow cells were unaltered, it could be concluded that the early stages of B-cell maturation were unaffected by the mercury exposure. Furthermore, the absence of any marked changes in light scattering at right angles for cytometric profile of the bone marrow cell population could be interpreted as a lack of any mercury-related activation of bone marrow cells. The increased light scattering at right angles was shown to be associated with chromatin deconden- sation, a marker of the highest activated state within GO phase prior to entry into cell cycle (Walker, Guy, Brown, Rowe, Milner & Gordon, 1986; Krzystyniak et al., 1992). Overall, we concluded no direct mercury-related damage of bone marrow lymphoid cells after the exposure to low levels of inorganic mercury. It should be stressed that our data were obtained from an environmentaUy-relevant model, i.e. exposure of heterogeneous population to low, nonaccidental doses of inorganic mercury. However, genetic factor(s) can play an important role in the sensitivity of the immune system to the effects of mercury exposure. Other studies using genetically homogeneous animals demonstrated strain dif- ferences in the effects of mercury on the immune response, such as mercury-related autoimmunity and

817

immune-complex disease in mice and rats (Hultman & Enestrom, 1992; Hultman & Johansson, 1991; Aten et al., 1991; Dubey et al., 1991).

Several aspects of the oral exposure to low inorganic mercury should be discussed: first, the toxicokinetics studies of orally administered inor- ganic mercury, possibly a major route for human exposure to inorganic mercury compounds, revealed an increased organ disposition of the toxicant, as compared to the parenteral administration (Nielsen & Andersen, 1989; Nielsen, 1992). Next, the cytotoxic effects of mercurials administered at relatively low doses included alteration of protein and nucleic acid synthesis and inhibition of several membrane-bound and cytosolic enzymes in different cells (Lachapelle, Guertin, Marion, Fournier & Denizeau, 1993). For example, our recent studies showed that in vitro treatment of hepatocytes with a low, 5/aM HgCl2 dose resulted in reduced albumin secretion and drastic morphological changes, such as reduced number of ribosomes associated with the rough endoplasmic reticulum and dilatation of the Golgi apparatus (Lachapelle et al., 1993). Regarding functional cellular parameters at minimal mercury doses, our data demonstrated a clear dose-related functional change in peritoneal macrophages, such as augmented phagocytic activity. This observation is interesting in spite of the absence of any modifications of functional activities of spleen lymphoid cells at minimal mercury doses and requires further investigation. For example, different cellular sensitivity to mercury exposure can be possible for macrophages and spleen lymphoid cells. Alternatively, differences in the level of mercury bioaccumulation in tissues can possibly result in marked differences in the HgCI2 exposure of peritoneal macrophages and spleen lymphocytes. Other authors reported mostly data on the in vitro effects of mercury on macrophage phagocytic activity (Contrino, Kosuda, Marucha, Kreutzer & Bigazzi, 1992; Christensen, Morgensen & Rungby, 1988; Tam & Hinsdill, 1985). These data, however, were not consistent and demonstrated either no effects of noncytotoxic doses of HgCl2 on the macrophage phagocytosis (Tam & Hinsdill, 1985) or impaired phagocytic activity after in vitro exposure to mercuric chloride (Contrino et al., 1992; Christensen et al.,, 1988).

Mercury-related transient shift in total bone marrow lymphoid population could be possibly attributed to the injury of early steps of stem cell differentiation. It may be noted that for other heavy metals, such as cadmium and lead, an increase in the number of progenitor cells (CFU-C) obtained from

818 S. BRUNET et al.

bone mar row, was observed as early as 5 days af ter the exposure of mice to the toxicant (Burchiel et al., 1987).

Dieter et al. (1983) suggested tha t preferent ia l accumula t ion o f mercury in spleen and the resul tant enzymat ic inh ib i t ion o f glucose me tabo l i sm might only reflect mercury- induced shifts in splenic cellular compos i t ion . Our data , however , demons t r a t ed only a t rans ien t shif t in splenic T-ceU subpopula t ions , wi thou t a clear d o s e - e f f e c t re la t ionship. Overall , f low cy tomet ry subset analysis o f lymphoid cells, which have been p roven as a useful and sensitive measu remen t o f immunotox ic i ty , revealed t rans ien t f luc tua t ions in splenic T-cell subsets and intact cytometr ic profi les o f bone m a r r o w and thymus upon subchron ic exposure to relatively low doses of

inorganic mercury in dr inking water. However , the ques t ion remains open for more toxic fo rms of organic mercury and for a chronic exposure to low, env i ronmenta l ly re levant mercury doses. Most toxicokinet ic studies o f mercur ic c o m p o u n d s have used ei ther single-dose admin i s t ra t ion or shor t - t e rm exposures, despite the fact tha t mos t h u m a n s are chronical ly or subchronical ly exposed to low or very low doses of mercurials .

Acknowledgements - - The authors thank Mr Saad Mansour for his excellent technical help in cytometric analysis. This work was supported by the Natural Sciences and Engineering Research Council of Canada and Toxen, Universit6 du Qu6bec h Montr6al and Canadian Network of Toxicology Centres (CNTC).

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