A. E. Munson

Subchronic 10 Day Immunotoxicity of Polydimethylsiloxane (Silicone) Fluid, Gel and Elastomer and Polyurethane Disks in Female B6C3F1 Mice

Millions of people have been exposed to silicones because of the widespread use in consumer products such as cosmetics and toiletries, food products, household products and paints. Silicones have wide use in medical practice, including lubricants in tubing and syringes, and as implantable devices. The most prevalent silicone in medical use is polydimethylsiloxane. This study was undertaken to determine the subchronic immunotoxicologic potential of the principal constituents of breast implants: silicone fluid, silicone gel and silicone elastomer. An alternative covering for devices containing silicone gels, polyurethane, was also included in the study. Silicone fluid and gel were injected subcutaneously into female B6C3F1 mice (1 ml/mouse) and 6 mm disks of silicone elastomer or polyurethane were implanted subcutaneously. There were no treatment-related deaths or overt signs of toxicity. None of the tested materials had notable effects on body or organ weights, erythrocytes or leukocytes in the blood, blood chemistries such as alanine aminotransferase, urea nitrogen, glucose, albumin or total protein. The cellularity of the bone marrow and responses to CSF-GM and CSF-M were normal. The tested silicones did not alter the distribution of B cells and T cells in the spleen, but polyurethane perturbed the distribution of CD4+CD8+ and CD4-CD8- T cells. The antibody response to sheep erythrocytes was not markedly altered, nor were proliferative responses to concanavalin A, phytohemagglutinin, lipopolysaccharide or allogeneic cells. Reticuloendothelial function was normal, but polyurethane evoked an enhanced phagocytosis of Covaspheres by adherent peritoneal cells. Natural killer cell activity and serum complement were not altered. All silicone materials afforded modest protection to a challenge with Listeria monocytogenes that killed 40 to 58% of control mice. Host resistance to Streptococcus pneumoniae or the B16F10 tumor was not affected by any of the treatments. There is a pattern indicative of some perturbation of T cell differentiation in mice implanted with a polyurethane disk.

A comparative study of antitumour and toxicologic properties of related polyanions

Poly(maleic anhydride) (PMA) homopolymer, DIVEMA copolymer (PCP) and poly(acrylic acid—maleic anhydride) (PAAMA) copolymer were synthesised and separated into discrete molecular weight (MW) fractions. These fractions were tested for antitumour (Lewis lung carcinoma LLC), anti-Friend leukemia (FLV), immunoadjuvant activity, phagocytic activity, sensitization to bacterial lipopolysaccharide (LPS) and inhibition of microsomal mixed functional oxidase enzymes (MMFO). Molecular weight preparations of the three polymers, 10 000 and above, possess pronounced activity against LLC (increased life-span > 35%), FLV (ED50 between 1.6–4.0 mg/kg) and enhanced B lymphocyte activity as measured by increased number of hemolytic plaque forming cells. Molecular weight preparations below 1000 were devoid of these activities. PCP was the most potent inhibitor of MMFO as measured by prolongation of hexabarbital sleeping times. All polymers induced a sensitization to LPS. PCP was the most potent sensitizer followed by PAAMA and PMA. PCP at MW 30 000 and above markedly suppressed phagocytosis as measured by the vascular clearance rate of colloid carbon in mice while MW preparations 2000 stimulated phagocytosis. PMA inhibited phagocytosis to a much lesser degree than PCP. In contrast to PCP, PMA did not stimulate phagocytosis until 7 days after drug administration. Peritoneal exudate cells (PEC) and serum from PCP and PMA-treated mice conferred complete protection against FLV but not LLC in recipient mice. Supernatants from the PEC also transferred protection but not to the same degree as whole cells. These studies begin to delineate the structures and polymer size which possess therapeutic and toxicologic potential.

Immunotoxicity of 180 Day Exposure to Polydimethylsiloxane (Silicone) Fluid, Gel and Elastomer and Polyurethane Disks in Female B6C3F1 Mice

Millions of people have been exposed to silicones which are present in consumer goods such as cosmetics and toiletries, processed foods and household products. In addition, silicones have been used extensively in medical practice as a lubricant in tubing and syringes, and as implantable devices. A silicone widely used in medical practice is polydimethylsiloxane. This study was undertaken to determine the immunotoxicologic potential of long term exposure to the principal constituents of breast implants: silicone fluid, silicone gel and silicone elastomer. An alternative covering for devices containing silicone gels, polyurethane, was also included in the study. Silicone fluid and gel were injected subcutaneously into female B6C3F1 mice (1 ml/mouse) and 6 mm disks of silicone elastomer or polyurethane were implanted subcutaneously. There were no treatment-related deaths or overt signs of toxicity during the 180 day exposure. None of the tested materials had notable effects on body or organ weights, erythrocytes or leukocytes in the blood, blood chemistries such as alanine aminotransferase, urea nitrogen, glucose, albumin or total protein, or serum CH 50 or C3 levels. The cellularity of the bone marrow and responses to CSF-GM and CSF-M were normal. The tested silicones and polyurethane marginally reduced the level of Ig+ cells in the spleen but did not consistently alter the distribution of T cell surface markers. The antibody response to sheep erythrocytes was not markedly altered, nor were proliferative responses to concanavalin A, phytohemagglutinin, lipopolysaccharide or allogeneic cells. Reticuloendothelial function was normal, as was phagocytosis of chicken erythrocytes and Covaspheres by adherent peritoneal cells. Natural killer cell activity was depressed in all silicone treatment groups and in mice implanted with polyurethane. No silicone or polyurethane treatment group displayed altered susceptibility to a challenge with Listeria monocytogenes, Streptococcus pneumoniae or the B16F10 tumor. The only consistent effect of 180 day exposure to silicone materials or polyurethane was a modest depression of natural killer cell activity.

Immunotoxicity of Mono-Nitrotoluenes in Female B6C3F1 Mice: II. Meta-Nitrotoluene

The nitrotoluenes are chemicals used in dyes, agricultural products, pharmaceuticals and explosives. In the present studies, the toxicology and immunotoxicity of meta-nitrotoluene (m-nitrotoluene) were evaluated. Mice, exposed to m-nitrotoluene at dose levels of 200, 400 and 600 mg/kg/body weight for 2 weeks by gastric gavage, gained body weight over the treatment period to a slightly greater extent than the control groups. Of the selected organs weighed, the liver and kidney of mice exposed to m-nitrotoluene were increased in weight while the thymus weight was decreased. The liver of mice exposed to m-nitrotoluene, but not ortho-nitrotoluene, showed slight to moderate swelling of the hepatocytes adjacent to the central veins. The hepatocyte swelling appeared to be reversible and there was no evidence of necrosis. The hematology and serum chemistries examined were unaffected by m-nitrotoluene exposure although there were modest decreases in the percentage of polymorphonuclear leukocytes and eosinophils in differential blood counts. Bone marrow cellularity and the number of CFU/M and CFU/GM were unaffected by m-nitrotoluene exposure. m-Nitrotoluene suppressed the IgM response to sRBC and the DHR response to KLH. There was a slight (8%) decrease in the percentage of B lymphocytes in the spleen. The response to the T cell mitogens was suppressed by as much as 39%. Fc-mediated adherence and phagocytosis of chicken erythrocytes and NK cell activity were increased dose dependently in mice exposed to m-nitrotoluene. Several immune parameters were unaffected by exposure to m-nitrotoluene, including the IgG response to sRBC, responses to the B cell mitogen LPS and to allogeneic cells, and serum interferon levels. Resistance to Streptococcus pneumoniae and Plasmodium yoelii were unaffected also. Resistance to the tumor model PYB6 was increased. Exposure of mice to m-nitrotoluene decreased resistance to Listeria monocytogenes. The decreased resistance to L. monocytogenes may be related to an effect on T cells, evidenced by a decrease in T cell numbers and in the DHR.

Carbon tetrachloride is immunosuppressive and decreases host resistance to Listeria monocytogenes and Streptococcus pneumoniae in female B6C3F1 mice

Carbon tetrachloride (CCl(4)) is an environmental contaminant that has been detected in ambient air, seawater, surface-water and snow. The immunotoxic potential of CCl(4) was evaluated in female B6C3F1 mice. The animals were administered with CCl(4) daily for 14 days at doses of 50, 100, 500 or 1000 mg/kg body weight by gavage with corn oil as a vehicle. Exposure to CCl(4) resulted in an increase of liver weight but not the body weight and the weights of brain, spleen, lungs, thymus and kidneys. Exposure to CCl(4) produced minimal effect on differential hematological parameters; however, it produced a significant increase in serum glutamic-pyruvic transaminase (SGPT) levels in all dose groups while other serum chemistries showed sporadic increases, primarily at the dose level of 1000 mg/kg. Exposure to CCl(4) produced a decreased humoral immune response; the IgM antibody forming cell (AFC) response to sheep red blood cells (sRBC) was suppressed with the maximal decrease (45%) observed at the dose level of 1000 mg/kg. The IgM serum titer to sRBC was also reduced with a maximal decrease (54%) observed at the dose level of 500 mg/kg. Although exposure to CCl(4) had no effects on the mixed leukocyte response (MLR), cytotoxic T lymphocyte activity and natural killer (NK) cell activity, a decrease in both the absolute number and the percentage of CD4(+)CD8(-) at the dose level of 500 mg/kg was observed. The functional activity of the mononuclear phagocyte system was compromised as reflected by a decrease in the vascular clearance of (51)Cr-sRBC and a decrease in the uptake of (51)Cr-sRBC by the liver. Finally, in the two host resistance models evaluated, exposure to CCl(4) decreased host resistance to both Streptococcus pneumoniae and Listeria monocytogenes with greater susceptibility to the latter. Overall, these studies demonstrate that CCl(4) was immunosuppressive in female B6C3F1 mice.

Immunotoxicity of 2,4-Diaminotoluene in Female B6C3F1 Mice

2,4-Diaminotoluene (DAT) has been demonstrated to be a potent carcinogen. The present studies were carried out to determine the toxic and immunotoxic potential of DAT. Mice exposed to DAT at 25-100 mg/kg per day for 14 days by gavage showed a 42% increase in liver weight and a slight decrease in spleen weight. Histopathologic evaluation of selected organs showed the liver to be the major target with morphological changes which were dose dependent. The high dose (100 mg/kg) was associated with moderate centrilobular necrosis. No abnormal structure was noted in the spleen, lungs, thymus, kidney or mesenteric lymph nodes. The liver toxicity was associated with an elevation in alanine aminotransferase activity. The only change noted in selected hematologic parameters was a 64% increase in peripheral blood leukocytes. Mice exposed to DAT showed a decreased IgM and IgG response to sheep erythrocytes. The decrease was not a function of a decreased number of B cells because the number of B cells increased dose dependently. Proliferative capacity of immunocompetent cells was not impaired by exposure to DAT as measured by the response to several mitogens. The delayed hypersensitivity response to keyhole limpet hemocyanin in mice exposed to DAT was increased. Natural killer cell activity was decreased dose dependently and may represent a spleen cell pool shift because the number of B cells increased in the presence of a decreasing spleen size. Serum C3 was suppressed at the high dose of DAT. Phagocytosis by splenic macrophages, but not peritoneal macrophages, was inhibited by DAT exposure. DAT exposure for 14 days decreased host resistance to the bacteria, Streptococcus pneumoniae and Listeria monocytogenes, while host resistance to the pulmonary tumor model, B16F10, and the PYB6 fibrosarcoma was unaffected by DAT exposure. These data indicate that DAT is hepatotoxic and perturbs the differentiation and maturation of leukocytes.

Effects of Hydroxyethylstarch (HespanR), a Plasma Expander, on the Functional Activity of the Reticuloendothelial System. Comparison with Human Serum Albumin and Pyran Copolymer

The purpose of the present investigation was to determine if RES activity was altered after the infusion (bolus i.v. injection over approximately 3-5 min) of hydroxyethylstarch (HES). RES function was determined by vascular clearance of 51Chromium-labeled sheep erythrocytes and the subsequent uptake into the liver, spleen, lungs, and thymus at 1 hr, 3 hr, 6 hr, 1 day, 3 days, and 7 days post infusion. Infusion with the low doses of HES (20 and 40 ml/kg) produced changes in vascular clearance which were comparable to physiological saline. Infusion with 80 ml/kg HES produced a biphasic response with a modest suppression of vascular clearance (i.e., 151% increase in half life) and hepatic phagocytosis (50%) during the first 6 hours after injection, followed by recovery at 24 hours and a stimulation in hepatic uptake (42%) after 3 days. These effects by HES were compared to those produced by infusion with 80 ml/kg HSA, a comparable colloid and with 80 ml/kg pyran copolymer, a positive control.

OXYMETHOLONE MODULATES CELL-MEDIATED IMMUNITY IN MALE B6C3F1 MICE

Oxymetholone is a synthetic androgen, structurally related to testosterone. It is currently used to treat anemias, but has also been abused as a performance enhancing anabolic steroid by the sport community. Concern about its suspected immunomodulatory properties provided the incentive for a detailed investigation into its effects on the mammalian immune system. In this study, male B6C3F1 mice were treated for 14 d with oxymetholone (0, 50, 150, and 300 mg/kg) by gastric intubation, then evaluated for immunotoxicity using a panel of immunotoxicity assays. Except for an increasing trend in kidney and liver weights, and a dose-dependent increase in serum blood urea nitrogen levels, no other signs of systemic toxicity were observed. Bone marrow DNA synthesis was reduced, though this did not translate into alterations in myeloid or monocyte colony forming units. Spleen B and T cell numbers, antibody response to sheep red blood cells, proliferative response to both mitogen and immunoglobulin receptor immunogens, and NK cell activity were all unaltered in mice treated with oxymetholone. Peritoneal macrophage activity was also unaffected by oxymetholone treatment. A 38% decrease in the spleen cell mixed leukocyte response, and a 15% decrease in cytotoxic T cell activity, measured in the highest oxymetholone treatment group, indicate that cell-mediated immunity was impaired following exposure. This immunomodulation did not however, translate into a change in host resistance to Listeria monocytogenes.

Separation of Murine Splenic B- and T-Lymphocytes for Use in Immunotoxicological Studies

The effects of xenobiotics on the immune system are often diverse and can involve secondary effects on cell types other than those of primary immune origin. Thus, to better understand the effects of chemicals and drugs on immuno-competence, exposure of immune cell types to xenobiotics and assessment of functional activity have been conducted under in vitro culture conditions. Interpretation of data obtained using this approach is reliant on the known composition of the cell populations used in these assay systems as well as on the ability to obtain purified and functionally viable cells from mixed primary cell suspensions. The objective of this study was to develop an effective and reproducible means of separating murine splenic B- and T-cells into purified populations free of monocyte/macrophage and polymorphonuclear cell (PMN) contamination. Cell adherence and discontinuous Percoll gradients in conjunction with immunomagnetic negative selection or antibody-mediated complement lysis were used to obtain B...