Barry R. Blakley

Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam Mya arenaria hemocytes following in vitro exposure to heavy metals

The effectiveness of toxicology biomonitoring programs could be improved by the addition of sensitive biomarkers. In this study the cell viability and sensitivity of phagocytic function of phagocytes from bivalves (Mya arenaria) to selected heavy metals were measured by flow cytometry, a novel approach. Hemocytes (phagocytes) collected from bivalves by puncture of the posterior adductor muscle were incubated in vitro for 18 h in hemolymph containing 10(-9)-10(-3)M of cadmium chloride, zinc chloride, mercuric chloride, methylmercury chloride or silver nitrate, before determining their capacity to phagocytose fluorescent latex beads by flow cytometry. Heterogeneity of the hemocyte cell population was determined by forward scatter (FSC) and side scatter (SSC) cytometric profile which showed two distinct cell populations. At low doses (10(-9), 10(-8) M), all the metal compounds studied stimulated phagocytic activity except silver nitrate. At higher levels of exposure (10(-6), 10(7) M), all metals caused a significant concentration-related decrease in hemocyte phagocytosis activity. From the concentration of each metal inducing 50% suppression (IC50) of the phagocytic activity, the immunotoxic potential of metals with respect to phagocytic function can be ranked in the following increasing order: ZnCl2 < CdCl2 < AgNO3 < HgCl2 < CH3HgCl. Parallel analysis of hemocyte viability showed that suppression of phagocytosis by heavy metals was not solely related to a decreased cell viability. These results reveal the high but different degree of sensitivity of the phagocytosis activity of bivalves with respect to heavy metals, as measured by flow cytometry, and demonstrate that flow cytometry is a potentially useful tool in ecotoxicological monitoring.

Phagocytosis as a Biomarker of Immunotoxicity in Wildlife Species Exposed to Environmental Xenobiotics

*Institut National de la Recherche Scientifique-Institut Armand-Frappier (INRS-IAF), Pointe-Claire, QC H9R 1G6, Canada †Departement of Fisheries and Oceans, Institut Maurice-Lamontagne, Mont-Joli, QC G5H 3Z4, Canada ‡Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada §Biomedical Sciences, Ontario Veterinary College Medicine, Guelph, ON N1G 2W1, Canada ¶Groupe de Recherche en Environnement Côtier, Université du Québec à Rimouski, Rimouski, QC G5L 3A1, Canada

The effect of cadmium on antibody responses to antigens with different cellular requirements.

Six week old BDF1 or CD-1 female mice were exposed to cadmium chloride in the drinking water at concentrations ranging from 0 to 50 ppm cadmium for 3 weeks. The in vivo antibody response against dinitrophenyl-aminoethylcarbamylmethyl-Ficoll (DNP-Ficoll), a T-lymphocyte independent, macrophage dependent response, was enhanced by cadmium. Similarly, the in vivo antibody response against Escherichia coli 0127 (LPS), a T-lymphocyte and macrophage independent response, was also enhanced by cadmium. In contrast, the in vitro antibody response against sheep red blood cells (SRBC), a T-lymphocyte and macrophage dependent response, was suppressed in spleen cell cultures that contained cadmium-exposed non-adherent cells (lymphocytes). Cultures containing cadmium-exposed adherent cells (macrophages) were not suppressed by cadmium. These results suggest that the immunosuppressive effects of cadmium as it relates to humoral immunity involve T-lymphocyte function rather than macrophage or B-lymphocyte activity. The enhanced T-lymphocyte independent antibody responses which accompany suppressed T-lymphocyte-dependent responses following cadmium exposure are an indication of compensatory mechanisms that are associated with the immune system.

The effect of exposure to a commercial 2,4-D formulation during gestation on the immune response in CD-1 mice

Pregnant CD-1 mice were administered a commercial 2,4-dichlorophenoxyacetic acid (2,4-D) formulation on days 6-16 days of gestation, in drinking water at concentrations ranging from 0 to 1.0% of the formulated product, equivalent to approximately 0-650 mg/kg per day expressed as the amine derivative. The effect of 2,4-D on immune function was evaluated in offspring 7 weeks after birth. The dams tolerated repeated 2,4-D exposure in drinking water without difficulty. The offspring exhibited decreased body weight with minor reductions in the kidney weights in the 0.1 and 1.0% 2,4-D treatment groups. A generalized suppression of lymphocyte stimulation by concanavalin A (Con A) was observed at high dose of commercial 2,4-D formulation (1.0%). Cytometric studies of the lymphocyte subpopulations demonstrated an increased relative count of B cells and reduced T cytotoxic or suppressor cells in the 1.0% formulation. The humoral immune response, antibody production against sheep red blood cells and peritoneal macrophage phagocytic function, were not altered by 2,4-D. Since the immune alterations in the offspring were observed many weeks after exposure, it appears as though 2,4-D exposure during gestation causes permanent changes in cell types associated with immune function. Since 2,4-D is not considered a persistent chemical, it is unlikely that 2,4-D residues are contributing significantly to the observed immune alterations. The immune alterations were observed only in the higher treatment groups. Therefore, the impact on human and animal health from an immune perspective, which would be encountered following normal application in the environment, would be minimal.

Effect of pentachlorophenol on immune function

The organochlorine compound, pentachlorophenol, was evaluated for effects on immune system function in male Fisher 344 rats. Pentachlorophenol was prepared in an olive oil vehicle and was administered by oral gavage twice weekly for 28 days at a dose of 2.0 mg/kg per treatment. Exposure to pentachlorophenol increased body weight gains (P=0.024) during the treatment period. Liver (P=0.034) and kidney (P=0.012) body weight ratios were also increased. Pentachlorophenol exposure enhanced T-lymphocyte blastogenesis induced by concanavalin A (Con A)(P=0.0001) and phytohemagglutinin (PHA)(P=0.048) evaluated using stimulation indices. Corresponding B-lymphocyte blastogenesis induced by lipopolysaccharide/dextran (LPS/dex)(P=0.0034) was also enhanced by pentachlorophenol exposure. Pentachlorophenol suppressed the antibody response against sheep red blood cells (SRBCs) by 39% when the response was expressed per viable spleen cell (P=0.006). This suppression was not evident when the response was expressed per spleen (P=0.22), suggesting that a compensatory mechanism or extramedullary splenic hemopoiesis was occurring minimizing the overall impact on humoral immunity. The enhanced B- and T-lymphocyte blastogenesis may also reflect compensatory or hemopoietic activity. Pentachlorophenol exposure had no effect on peritoneal macrophage phagocytosis (P=0.31) or lymphocyte cell surface antigen expression. The observed alterations in lymphocyte blastogenesis and humoral immunity subsequent to pentachlorophenol exposure do not appear to be associated with phagocytosis or lymphocyte cell surface antigen expression.

The effect of oral exposure to the n-butylester of 2,4-dichlorophenoxyacetic acid on the immune response in mice

Six week old female BDF1 mice were administered the n-butylester of 2,4-dichlorophenoxyacetic acid (2,4-D) by gastric intubation at dosages ranging from 0 to 200 mg/kg (2,4-D content) in the acute studies and 0 to 100 mg/kg in the subacute studies. Following acute exposure, the antibody production against sheep red blood cells and the induction of DNA synthesis with Escherichia coli lipopolysaccharide, a B-lymphocyte mitogen, were enhanced. Comparable T-lymphocyte mitogen responses induced by concanavalin A, a T-lymphocyte mitogen, were not affected by 2,4-D, though 2,4-D did exhibit a weak, non-specific, dose-related mitogenic effect. Subacute exposure to the 2,4-D ester did not alter antibody production. B-lymphocyte mitogen responses were enhanced, though a linear dose-response relationship was not observed. Histopathological alterations in the brain and spinal cord and clinical symptoms of toxicity were observed in the 200 mg/kg treatment group (acute exposure). Similar effects were not seen in the subacute study. The immunostimulatory effects of 2,4-D were observed at relatively high exposures. It is unlikely that these immune alterations will have any major toxicological significance in agricultural communities utilizing 2,4-D herbicides, though further studies with commercial grade formulations which may contain other compounds in addition to the pure 2,4-D esters must be evaluated at similar levels of exposure.

Hepatotoxicity Associated with Dietary Iron Overload in Mice

1 Weanling male CD-1 mice were fed 120 (control), 5000 and 8000 mg of iron kg-1 for seven weeks. The haematocrit (P=0.265), water consumption ( P=0.170) and percentage body weight ratios of kidney, spleen and heart were not affected by iron supplementation. 2 Iron supplementation reduced weight gain (P=0.023), increased weight of liver (P=0.0001), the iron deposition index and concentration of iron in the liver (P<0.01). A strong correlation between liver iron concentration and level of iron in the diet (r=0.989) was observed. Histologically, the deposition of iron was restricted to the hepatocytes, Kupffer cells and splenic macrophages. 3 Consumption of 5000 and 8000 mg of iron kg-1 resulted in hepatic damage, as judged by elevated serum alkaline phosphatase and alanine aminotransferase activities (P<0.05). 4 This study indicates that prolonged feeding of excess dietary iron has the potential to cause hepatic accumulation of iron with resultant liver toxicity, and that mice may be a suitable model to study the mechanisms of dietary iron overload.

The effects of thiamin on lead metabolism: whole body retention of lead-203

The effects of thiamin on the whole body retention of led were evaluated in CD-1 mice treated intragastrically or intraperitoneally while exposed to a single dose of lead acetate (100 micrograms) containing 100 mu Ci lead-203. They were administered thiamin (25 or 50 mg/kg body wt.), calcium ethylenediamine tetraacetic acid (CaEDTA) (50 mg/kg body wt.) or their combination in pretreatment or posttreatment regimens for 13 days. Both pre- and posttreatment with thiamin reduced the lead retention compared to the untreated lead-exposed mice, although the different patterns of lead retention were observed. The combined pretreatment (thiamin 50 mg/kg and CaEDTA) and the CaEDTA treatment alone reduced the whole body retention of lead most effectively. Thiamin, CaEDTA and the combined treatments decreased the absorption of lead-203 and the biological half-life of retained lead-203 following oral or intraperitoneal lead exposure. The reduced absorption and enhanced excretion of lead associated with thiamin administration may have therapeutic application for the treatment of lead poisoning.

Equine toxicoses: Investigative strategies and approaches for performance horses

Abstract The investigation of suspect toxicosis is one of the most complex diagnostic problems faced by equine practitioners, requiring a wide range of clinical skills. When poisoning occurs, there is often no clear evidence of exposure to a toxic agent. Establishing an accurate diagnosis of poisoning requires a systematic approach, beginning with a comprehensive history, followed by clinical examination of affected horses, clinical pathologic testing, postmortem examinations, and analytic toxicology testing. The diagnosis of potential toxicosis in performance horses especially in the racetrack environment presents unique challenges to the veterinarian. Economic concerns including wagering, competition, inflated animal values, and insurance issues combine to create a situation in which intentional poisonings using obscure toxicants and unusual routes of exposure may occur. Collection of an accurate history is frequently confounded under these circumstances. This problem, coupled with the wide range of potential malicious toxic agents available, reinforces the requirement to approach these cases in a thorough and systematic fashion. Throughout the investigative process, potential medicolegal issues should be kept in the forefront. Care must be taken to maintain complete and accurate medical records and to maintain chain of custody on all antemortem and postmortem samples submitted to the diagnostic laboratory, because suspicious or unexplained deaths frequently end up in court.

Effect of dietary iron supplementation on the body burden of iron in mice

Abstract Weanling male CD-1 mice were fed 120 (control), 250, 500 and 800 ppm iron (Fe) in diets or 120 ppm Fe and injected subcutaneously (s.c) weekly with 0.5 mg Fe as iron sorbital for 7 weeks. Body weight gain, hematocrit and water consumption were not altered by iron exposure ( p =0.598, p =0.266, p =0.664 respectively). Dietary iron concentrations above 120 ppm or injection of 0.5 mg Fe s.c weekly increased liver iron stores in the mice ( p =0.0001) in a dose and time-dependent manner. Mice fed control diet (120 ppm Fe) maintained a constant concentration of iron in the liver and serum indicating that body iron level is normally maintained within narrow physiological limit. Significant positive correlations were observed between concentrations of iron in the liver and dietary levels of iron r=0.998, p =0.0001). In contrast, dietary iron concentrations correlated poorly with serum iron concentrations (r=0.024). Serum iron concentrations increased during the first 3 weeks of growth ( p p