Immunotoxicological response of the earthworm Lumbricus terrestris following exposure to cement kiln dusts.

Cement kiln dusts are made of a complex mixture of elements. We have evaluated the potential negative impact of those dusts on the immune system of the earthworm Lumbricus terrestris. We specifically studied cell viability and phagocytic activity of coelomocytes extruded during electrical stimulation. We used two modes of exposures: in vitro, and soil incubation using OECD artificial soil media. Extruded coelomocytes were exposed 18 h in vitro to 10, 100, and 500 mg L(-1) of cement kiln dust particles. The phagocytosis and the cell viability were determined using a double-laser-flow acquisition cytometry system. Using the double laser allows us to use a dichlorofluorescein diacetate (DCFDA) marker to discriminate the biological cells from the cement kiln dusts. Dead cells are marked using propidium iodide (PI). All three exposure levels showed highly significant impacts on cell viability and phagocytic activity. The in vivo soil incubation was performed using 10, 100, and 1000 mg kg(-1) of cement kiln dusts incorporated into the OECD media. Here, to discriminate the biological cells from the mineral dusts we only needed to use PI. The day-to-day variability of the in vivo assay was high and although we can observe an overall reduction in cell viability at the highest concentration tested, no statistically significant effects could be observed on either cell viability or phagocytosis.

Lack of suppressive effects of mixtures containing low levels of methylmercury (MeHg), polychlorinated dibenzo-p-dioxins (PCDDS), polychlorinated dibenzofurans (PCDFS), and aroclor biphenyls (PCBS) on mixed lymphocyte reaction, phagocytic, and natural killer cell activities of rat leukocytes in vitro.

Rat splenocyte mixed leukocyte reaction (MLR), splenic natural killer (NK) cell activity, and phagocytic activities of splenic, peritoneal, and peripheral blood leukocytes (PBLs) were evaluated in vitro to determine the immunotoxicity of mixtures containing low levels of methylmercury (MeHg), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and Aroclor polychlorinated biphenyls (PCBs). The mixtures were based on the concentrations of the chemicals in fish flesh. Leukocytes from male Fischer rats were exposed to MeHg (0.1-2 microg/ml), PCDD/PCDF mixtures (1-15 pg/ml) of three PCDDs (2,3,7,8-tetrachlorodibenzo-p-dioxin, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin) and two PCDFs (2,3,7,8-tetrachlorodibenzofuran and 1,2,3,7,8-pentachlorodibenzofuran), three Aroclor PCB (Aroclor 1242, 1254, and 1260) mixtures (0.01-0.5 microg/ml), or combinations of MeHg/PCB/PCDD/PCDF mixtures for 24 or 72 h before immunological assays. Phagocytosis and NK cell cytotoxicity were evaluated with a flow cytometer, and MLR of Fischer rat responder splenocytes cultured with mitomycin C-treated Long-Evans splenocytes by [3H]thymidine uptake. Exposure to MeHg (2 microg/ml) alone or with PCB/ PCDD/PCDF resulted in significant cytolethality in rat splenocytes, peritoneal leukocytes, and PBLs at 24 h exposure. Treatment with Aroclor PCB mixtures, PCDD/PCDF mixtures, 0.1 microg MeHg/ml (noncytolethal), or PCB/PCDD/PCDF mixtures with 0.1 microg MeHg/ml caused no suppression of splenocyte MLR response, splenic NK cell-mediated lysis of Yac-l cells, or phagocytosis of fluorescent beads by splenic, peritoneal, and peripheral blood phagocytic cells. The results indicate that in vitro exposure of rat leukocytes to low levels of MeHg, Aroclor PCB mixtures, PCDD/PCDF mixtures, or MeHg/PCB/PCDD/PCDF mixtures had no suppressive effects on the immune functions assayed, and thus produced no additive immunotoxicity. However, in order to predict the potential risk of these chemical mixtures to the human immune system, in vivo animal studies with blood (tissue) levels compatible with the levels of MeHg, PCBs, and PCDDs/PCDFs in exposed human populations should be evaluated.

Effects of Great Lakes fish consumption on the immune system of Sprague-Dawley rats investigated during a two-generation reproductive study.

The effects of Great Lakes fish contaminants on several quantitative and functional aspects of the immune system were investigated in the first (F1) and second (F2) generations of Sprague-Dawley rats. The F0 rats were fed either a control diet or diets containing 5 or 20% lyophilized chinook salmon from the Credit River of Lake Ontario (LO) and Owen Sound point of Lake Huron (LH). The F1 and F2 pups were exposed to fish in utero, through the dam's milk to 21 days old, and through the dam's respective diets to 13 weeks of age. The study included an F1-reversibility (F1-R) phase in which rats at 13 weeks of exposure to fish or control diets were switched to the control diet for 3 months. The most outstanding finding was a statistically significant increase in absolute spleen leukocytes and absolute and percentage lymphocytes in the F2 male rats fed the LH fish diets compared to the control and to those fed the LO fish diets with the 20% fish diets having higher cell numbers compared to the LO-5% fish diets. A parallel increase in the T-helper/inducer T-lymphocyte subset numbers was observed. Increased but statistically insignificant plaque-forming cell (PFC) numbers were obtained in the F2 male rats fed the LH fish diets compared to those fed the LO fish diets and in the F1-R female group of rats fed the LH fish diet compared to those fed the LO fish diets. Phagocytosis by resident peritoneal macrophages was significantly increased in the F1 male and F2 female rats fed the fish diets compared to the control. The phagocytic activity was significantly higher in the F2-generation male and female rats fed the LO diets compared to those fed the LH diets. Other parameters including lymphocyte transformation in response to mitogens, the number of Listeria monocytogenes bacteria surviving in the rat spleens, and the natural killer cell activity were not affected significantly by any of the treatments. Overall, the effects of diets containing chinook salmon from the LO and LH sources on the immune system of rats were minimal and were on quantitative rather than on functional aspects of the system. Further focused research would be required in order to establish conclusively that the immune system of cohorts who ingest Great Lakes fish frequently is at a greater risk for adverse effects.

Increased apoptosis, changes in intracellular Ca2+, and functional alterations in lymphocytes and macrophages after in vitro exposure to static magnetic field.

Electromagnetic-related alteration of cellular functions is well documented for extremely low-frequency low-energy pulsing electromagnetic fields (ELF-EMF). In this study we examined the in vitro effects of static magnetic fields (SMF) on the cellular immune parameters of the C57BI/6 murine macrophages, spleen lymphocytes, and thymic cells. The cells were exposed in vitro for 24 h at 37 degrees C, 5% CO2, to 250-1500 G SMF. Exposure to the SMF resulted in the decreased phagocytic uptake of fluorescent latex microspheres, which was accompanied by an increased intracellular Ca2+ level in macrophages. Exposure to SMF decreased mitogenic responses in lymphocytes, as determined by incorporation of [3H]thymidine into the cells. This was associated with the increased Ca2+ influx in concanavalin A-stimulated lymphocytes. Furthermore, exposure to SMF produced markedly increased apoptosis of thymic cells, as determined by flow cytometry. Overall, in vitro exposure of immunocompetent cells to 250-1500 G SMF altered several functional parameters of C57BI/6 murine macrophages, thymocytes, and spleen lymphocytes.

In vitro lymphotoxicity and selective T cell immunotoxicity of high doses of acyclovir and its derivatives in mice.

The antiviral drug acyclovir [9-(2-hydroxyethoxymethyl)guanine (ACV)], its 7-isomer (7-ACV) and its two derivatives: N2-acetyl ACV (ac-ACV) and N2,O-diacetyl ACV (diac-ACV) were examined for their potential in vitro lymphotoxicity and in vivo immunotoxicity in mice. In vitro lymphotoxicity of ACV and its acetylated derivatives was low, whereas the 7-ACV isomer enhanced the in vitro cell proliferation in PHA-stimulated cultures. Addition of 2'-deoxyguanosine (dGuo) did not exhibit any inhibitory potential of ACV. However, reduction in the absolute number of CD3+, CD8+, and CD25+ cells, but not Ig+ cells, was noted at high concentrations of ACV and its derivatives, suggesting a selective T cell cytotoxicity. Similarly, the in vivo exposure revealed selective T cell immunotoxicity of ACV and its derivatives since the reduced number of Thy 1.2+ and CD8+ cells was not accompanied with any marked changes in the Ig+ population. The CD4+/CD8+ ratio was affected both in vitro and in vivo by high concentrations of ACV.

Immune functions in beluga whales (Delphinapterus leucas): evaluation of phagocytosis and respiratory burst with peripheral blood leukocytes using flow cytometry.

Flow cytometric assays using peripheral blood were developed to study phagocytosis and respiratory burst, the two major functions of neutrophils and among the most important non-specific defense mechanisms, in beluga whales. The use of flow cytometry avoids the problems associated with the isolation and purification of different cell types, and allows the measurement of a large number of cells (10,000) in a very short period of time. The methods described will be used to compare these functions in blood samples from highly contaminated beluga whales from the St. Lawrence and from relatively clean arctic beluga whales.

Cytometric profile of molybdenum-induced contact sensitization versus a strong allergen reaction to oxazolone in murine auricular lymph node (ALN) test.

Induction of contact hypersensitivity (CH) by molybdenum chloride (MoCl5) was determined by auricular lymph node (ALN) test in C57B1/6 mice. The ALN test was further improved by immunophenotyping and cytometric analysis of subset-specific cell in the draining node. Skin sensitization was induced by topical ear exposure to 1.0-50% oxazolone and resulted in a strong dose-related ALN reaction. Analogous exposure to MoCl5 resulted in a weaker but marked dose-related reaction, also manifested as an increase in cell number/ALN. Other differences between the oxazolone-induced strong sensitization and the MoCl5-related ALN reaction were: (1) an increase in the total number of Ig+ cells, which was, however, unchanged in the MoCl5-exposed mice; (2) a significant increase in the total number of large/activated T-cell subsets; and (3) a marked shift in the relative percentage of gated large/activated subsets of ALN cells, which was not observed in the MoCl5-exposed animals. Thus, it appeared that the molybdenum exposure induced a nonspecific increase in the cell number/ALN and was not accompanied by any marked activation of the T-cell subsets. Immunotoxicity of a 14 day subchronic exposure to MoCl5 at 1-100 ppm in food was studied by quantification of splenic humoral IgM response to sheep erythrocytes (SRBC). Plaque-forming cells (PFC) and enzyme-linked immunosorbent assay (ELISA) revealed unchanged humoral exposure in MoCl5-exposed mice. Cytometric assay of fluorescent beads uptake showed unchanged phagocytic activity of peritoneal macrophages from the MoCl5-exposed mice. Immunophenotyping of CD4+, CD8+, Thy 1.2+ and Ig+ cells revealed no effect of MoCl5 exposure on the total count of cell subsets in the ungated populations of spleen, lymph nodes and peripheral blood cells. Molybdenum chloride should thus be considered as a non-immunotoxic and a weak, nonspecific contact irritant.

Cytometric profiles of bone marrow and spleen lymphoid cells after mercury exposure in mice.

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 HgCl2 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.

Fast separation of macrophages by retention on cross-linked amylose and release by enzymatic amylolysis of the chromatographic material.

Macrophages from mice peritoneal exudate were isolated on basis of specific adherence on epichlorohydrin cross-linked amylose (CLA), a chromatographic gel presenting a high susceptibility to advanced amylolysis with alpha-amylase. The cell suspension, containing predominantly macrophages and lymphocytes, was applied onto the column and incubated for 30 min at 37 degrees C for the adherence of macrophages. After this interval the non-adherent cells were eluted with buffered medium and the CLA support was incubated in the column with an alpha-amylase-buffered solution liquefying the matrix and releasing, in situ, the adherent cell population containing 90% macrophages with a viability higher than 90%.

Immunotoxicity of subchronic versus chronic exposure to aldicarb in mice.

In this study we compared the immunotoxicity of subchronic vs chronic exposure to the aldicarb insecticide at a relatively low, 0.1-10 ppb, level in drinking water. The immunotoxicity of aldicarb was evaluated in 28- and 90-day studies by determination of the humoral, cellular and nonspecific immunity in inbred C57BL/6 mice. Quantification of splenic plaque-forming cells (PFC) to sheep erythrocytes (SRBC), mitogen activation of spleen lymphocytes, mixed lymphocyte reaction (MLR) and the cytofluorometric assay of the phagocytic uptake of fluorescent beads were among the parameters studied. Neither the cell viability nor the splenic cell count was affected by the insecticide exposure. Immunophenotyping and cytometric determination of L3T4+, Lyt2+ and Ig+ cells revealed no effect of the insecticide exposure on the total count of cell subsets in the ungated splenocyte population. However, a marked shift in the percentages of L3T4+ and Lyt2+ cells was noted after subchronic exposure to 1 and 10 ppb aldicarb, possibly indicating activation of these splenic T-cell subsets. Subchronic aldicarb exposure significantly suppressed the splenic PFC response to SRBC at 1 ppb dose, however, no dose-effect correlation could be concluded. Similarly, no dose-effect correlation was observed for subchronic aldicarb-related changes in mitogen responses. Subchronic exposure to aldicarb had no statistically significant effect on the mixed lymphocyte reaction (MLR) or on the macrophage phagocytosis. Chronic exposure to 0.1-10 ppb aldicarb did not affect any of the parameters measured, including the cell subsets. Thus, aldicarb-related changes in immune parameters, noted after a 28-day exposure, were compensated over chronic exposure to the insecticide.

Combined effects of selected insecticides on humoral immune response in mice.

Biological effects data with single insecticides are far more abundant than with mixtures. These data cannot be used directly to predict the effects of insecticide mixtures. Three insecticides of different chemical classes: organochlorine; dieldrin, organophosphate; malathion, and carbamate; carbofuran, previously evaluated for their immunotoxic potential, were selected for studies of combined acute exposure in C57B1/6 inbred mice. The humoral response to sheep red blood cells (SRBC) and the functional activities of peritoneal macrophages, such as phagocytosis of fluorescent beads and presentation of a single protein antigen, avidin, were examined after in vivo exposure of mice to different combinations of the selected pesticides and compared with the vehicle controls. Regarding exposure to single substances, the data confirmed the immunosuppressive potential of dieldrin and carbofuran and the immunopotentiating effect of malathion. Following the acute concomitant exposure to dieldrin/carbofuran mixture, however, values for the parameters of antigen presentation, primary IgM antibody response to SRBC antigen, and macrophage phagocytosis, returned to control or above-control values, indicating a lack of any synergistic or additive effects of the chemicals on the immune response. Thus, it was concluded the dieldrin/carbofuran mixture had an antagonistic effect on the humoral response to SRBC and the macrophage phagocytic activity, in comparison with the action of administration of each of the insecticides alone.

Suppression of avidin processing and presentation by mouse macrophages after sublethal exposure to dieldrin.

The molecular events in macrophage antigen processing and presentation were examined to determine the possible site(s) of cell-xenobiotic interaction. Antigenic processing by mouse peritoneal macrophages of a single protein antigen, avidin, was significantly suppressed following sublethal exposure of animals to an organochlorine pesticide, dieldrin. Exposure of C57B1/6 female mice to dieldrin affected the in vitro uptake of [methyl-14C]avidin by peritoneal macrophages and markedly decreased phagocytosis of fluorescein-labelled microspheres and Salmonella typhimurium. Release of the processed avidin, determined by immunochemical quantification of immunogenic avidin and by bioassay of immunogenicity of the released antigen, was also markedly affected. Dieldrin markedly affected presentation of avidin on the macrophage surface, observed by cytoimmunochemical staining of the antigen with fluorescent antibody and flow cytometry. Inhibition of the release of processed avidin was dieldrin dose- and time-dependent, following single sublethal intraperitoneal (ip) exposure to the pesticide. The antigenic properties of processed avidin, determined by biological assay using lymphocyte cultures of normal C57B1/6 mice primed with avidin, were proportional to the antigen concentration in supernatants of macrophage cultures, for both vehicle controls and dieldrin-exposed animals. This observation and analysis of the kinetics of release of processed avidin by macrophages from control and dieldrin-exposed animals suggested that the release of processed avidin, but not the immunogenicity of the antigen itself, was affected by the pesticide exposure. Generally, impairment of avidin processing and presentation appeared to be more dramatic than other pesticide-related injuries to macrophages, such as the uptake of the antigen. In conclusion, antigen processing could be a sensitive target for dieldrin-related injury of macrophage functional activities, which, in consequence, could produce suppression of the humoral immune response.

Increased susceptibility to mouse hepatitis virus 3 of peritoneal macrophages exposed to dieldrin.

Interaction of a single dose (36 mg/kg body wt) of the organochlorine pesticide dieldrin with mouse peritoneal macrophages was examined in C57Bl/6, (C57Bl/6 X A/J)F1, and A/J strains of different genetic resistance to mouse hepatitis virus 3 (MHV3) infection. In vivo studies showed increased susceptibility to MHV3 acute disease of C57Bl/6 and (C57Bl/6 X A/J)F1 animals challenged with the pesticide. Significant decrease of mean time of death in dieldrin-exposed, MHV3-infected susceptible C57Bl/6 mice was observed similarly upon po or ip administration of a single, sublethal dose of dieldrin. In addition, decrease of humoral response to the virus was quantified by determination of anti-MHV3 IgG antibodies in spleen cell supernatant fractions and in blood sera of dieldrin-exposed C57Bl/6 mice. A single dose of dieldrin did not alter the in vivo resistance of A/J animals to acute MHV3 disease. The resistant A/J mice, however, showed increased mortality upon two subsequent exposures to dieldrin followed by infection with high lethal doses of MHV3. Phagocytic activity, cell adherence capacity, and attachment and uptake of 3H-radiolabeled MHV3 by C57Bl/6 peritoneal macrophages were determined by in vitro studies. These affector activities of peritoneal macrophages were slightly decreased or unchanged in cells originating from animals exposed to the pesticide. However, the intrinsic activity of MHV3 restriction appeared to be affected in macrophages derived from dieldrin-treated animals: (i) peritoneal C57Bl/6 macrophages collected from the early phase of acute MHV3 disease contained increased MHV3 antigen and (ii) increased cytolysis was observed after in vitro MHV3 infection of macrophages originating from dieldrin-exposed C57Bl/6 mice.