L. Baecher-Steppan

Immunologic and endocrine effects of the flame-retardant pentabromodiphenyl ether (DE-71) in C57BL/6J mice

Polybrominated diphenyl ethers are manufactured for use as flame retardants in commercial plastics and textiles in Europe and North America. These studies investigated the acute and subchronic immunotoxicity and endocrine effects of a commercial pentabromodiphenyl either mixture, DE-71, in female C57BL/6 mice. Mice were orally exposed to acute single doses of DE-71 of 0, 0.8, 4.0, 20, 100, or 500 mg/kg, or to subchronic daily doses totaling 0, 250, 500, or 1000 mg/kg over a 14 day period. Immunotoxicity was assessed by measuring the plaque-forming cell response to sheep erythrocytes (SRBC) and natural killer cell (NKC) activity (basal and poly I:C stimulated) to YAC-1 target cells. Liver cytochrome P450 content and activities (ethoxyresorufin-o-deethylase (EROD) and pentoxyresorufin-o-deethylase (PROD)) as well as corticosterone (CS) and thyroxine (T4) concentrations were also measured. PROD activity was induced 3-5-fold in mice exposed acutely or subchronically to DE-71 at doses > 250 mg/kg. EROD activity and total microsomal cytochrome P450 content were significantly induced only in mice treated subchronically with DE-71; maximum induction of EROD was 3.3-fold. Total serum T4 concentrations were significantly lower in mice treated acutely with DE-71 at all doses except the 100 mg/kg dose. Total and free T4 concentrations were dose-dependently decreased in DE-71-treated mice following subchronic exposure. Plasma CS levels were elevated following subchronic exposure to DE-71. The elevation of CS was correlated with order of capture at necropsy, suggesting an interactive effect of DE-71 and stress. In regard to immunotoxicity, significant suppression of the anti-SRBC response was seen only in mice exposed subchronically to 1000 mg DE-71/kg, an exposure that also resulted in decreased thymus weight. NKC activity was not altered by exposure to DE-71.

Immunotoxicity of pentachlorophenol (PCP): Increased susceptibility to tumor growth in adult mice fed technical PCP-contaminated diets☆

Adult male C57B16 mice were fed diets containing 50 or 500 ppm pure (99+%) or technical grade (86%) pentachlorophenol (PCP) for 10–12 weeks prior to assessment of immunocompetence. Assays for immunocompetence included in vivo host susceptibility to virus infection and tumor growth and in vitro quantitation of T-cell cytotoxicity and macrophage phagocytosis. Exposure of mice to technical PCP resulted in profound enhancement of tumor susceptibility. The incidence of progressive methylcholanthrene (MCA)-induced transplanted tumors increased from 35% in controls to 67 and 82% in animals exposed to 50 and 500 ppm technical PCP, respectively. Mortality following primary Moloney sarcoma virus (MSV) inoculation and secondary MSB challenge increased from 19% in controls to 45 and 73% in animals exposed to 50 and 500 ppm technical PCP, respectively. An additional 50% of the MSVMSB challenged mice exposed to 50 ppm technical PCP exhibited tumor dissemination in the spleen. Animals exposed to pure PCP did not exhibit enhancement of primary MCA- or MSV-induced tumor growth. However, splenic tumor development was observed in 22 and 44% of the MSVMSB challenged mice exposed to 50 and 500 ppm pure PCP, respectively. Control mice did not develop splenic tumors. In contrast to the enhancement of tumor susceptibility, mortality associated with encephalomyocarditis virus infection tended to be reduced by technical PCP exposure. In vitro immune functional assessment indicated significant depression in T-cell cytolytic activity and enhancement of macrophage phagocytosis in animals exposed to technical PCP. No significant changes in in vitro immune response were noted with cells from pure PCP-exposed animals.

Immunotoxicology studies on lead: Effects of exposure on tumor growth and cell-mediated tumor immunity after syngeneic or allogeneic stimulation

Chronic exposure of C57BL/6 mice to lead acetate in the drinking water enhanced the growth of primary Moloney sarcoma virus (MSV)-induced tumors. Regression of MSV-induced tumors was not prevented by lead exposure and lead-treated animals were more resistant to late sarcoma development following primary tumor regression. The primary cell-mediated cytotoxic response in the spleen or lymph nodes of MSV-tumor bearing mice was significantly augmented by lead exposure. This augmentation appeared to reflect the increased antigenic stimulation resulting from the enhanced primary tumor growth in lead-exposed animals. Using an allogeneic tumor model, under conditions of similar antigenic stimulation, little effect of lead on T cell-mediated cytotoxicity was observed. On the other hand, macrophage phagocytic activity was significantly depressed in lead-exposed mice. Coupled with a decrease in the total number of macrophages recovered from lead-exposed mice, the results suggested significant impairment of macrophages function by lead. The influence of lead-induced macrophage dysfunction on tumor growth is discussed.

Immunotoxicity of Technical Pentachlorophenol (PCP-T): Depressed Humoral Immune Responses to T-dependent and T-independent Antigen Stimulation in PCP-T Exposed Mice

The effects of chronic dietary exposure to technical pentachlorophenol (PCP-T) on humoral immune responses in mice were examined. Primary and secondary splenic antibody responses to the T-dependent antigen, sheep red blood cells (SRBC), were examined in Swiss-Webster mice using our recently developed screening technique, the Hemolytic Antibody Isotope Release (HAIR) assay. To assess direct effects of PCP-T on B cells, the splenic plaque-forming cell response and serum antibody titers to the T-independent antigen, dinitrophenyl (DNP)-Ficoll, were examined. PCP-T exposure altered both the kinetics and the magnitude of the humoral antibody responses to SRBC and DNP-Ficoll. Peak splenic antibody production and serum antibody titers were delayed and the magnitude of the antibody responses were dose-dependently suppressed by PCP-T exposure. IgM responses appeared to be more sensitive to PCP-T-induced suppression than the IgG response. Significant depression of the IgM anti-SRBC splenic HAIR response was apparent as early as 2 weeks after initiation of PCP-T exposure and persisted for at least 8 weeks after termination of PCP-T feeding. Liver weight and serum lactate dehydrogenase (LD-L) and alanine aminotransferase (ALT) levels were significantly elevated during PCP-T exposure and returned to control levels after a 4-6 week recovery period. The immunotoxic effect of PCP on humoral immunity was observed only in animals exposed to technical grade PCP known to be contaminated with significant levels of other chlorinated phenols as well as non-phenolic impurities including chlorinated dioxins, furans, and diphenyl ethers. Animals exposed to analytical grade PCP did not exhibit depressed humoral immunity.

Effects of dietary technical pentachlorophenol exposure on T cell, macrophage and natural killer cell activity in C57B1/6 mice☆

The effects of technical grade pentachlorophenol (T-PCP) exposure on several immunological parameters were examined in adult C57Bl/6 mice following eight weeks of dietary exposure. Immune function tests included mitogen-induced lymphocyte blastogenesis, mixed lymphocyte reactivity (proliferation and cytotoxicity), spontaneous and boosted levels of natural killer (NK) cytotoxicity, and phagocytic activity of resident, thioglycollate-induced, and P815-tumor activated peritoneal macrophages. Thymic and splenic weights, spleen cellularity, percentages of splenic T and B cells, and bone marrow cellularity were also determined. The only statistically significant functional alteration observed in T-PCP exposed mice in these studies was a reduction in the lymphoproliferative response in mixed lymphocyte culture which occurred in the absence of any apparent effect on the generation of cytotoxic cells. Mitogen responses, NK cytotoxicity and macrophage phagocytosis were unaltered by exposure to T-PCP. No changes were observed in spleen or thymus weights or in spleen or bone marrow cellularity. A dose-responsive trend toward reduced T cell and increased B cell percentages in the spleen of T-PCP exposed mice was noted. The apparent functional resistance of T cells, macrophages, and NK cells to T-PCP is in contrast to the marked sensitivity of the humoral immune response to T-PCP induced suppression. The results are discussed in relation to the dioxin contaminants present in T-PCP.

The broiler chicken as a model for immunotoxicity assessment: 1. Standardization of in vitro immunological assays

The broiler chicken was developed as an alternative animal model to laboratory rodents for immunotoxicologic assessment. In vivo treatment with 100–200 mg/kg cyclophosphamide (CY) was used as a known immunosuppressive treatment to standardize the assay systems. Protocols for assessing specific immunological functions were developed in specific pathogen-free (SPF) broilers to measure lymphocyte blastogenesis to T-cell (concanavalin A and phytohemagglutinin) and B-cell (Staphylococcus aureus cells) mitogens, delayed-type hypersensitivity (DTH) to tuberculin, natural killer (NK) cell cytotoxicity, plaque-forming cell (PFC) response to sheep red blood cells (SRBC), and serum antibody titers to SRBC. CY was an effective immunosuppressant in the broiler system for assessment of lymphocyte responsiveness to mitogenic stimulation, DTH reactivity, and the antibody response to SRBC as assessed by PFC and serum antibody titers. NK cytotoxicity was not altered on a cellular level following treatment with CY at a dose that produced greater than 75% depletion of spleen cellularity. However, under these conditions, it must be assumed that the capacity of CY-treated birds to mediate NK effector functions would be reduced. These results demonstrate the applicability of the broiler chicken as an animal model for immunotoxicity testing.