Report Date: November 2012
The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of the NTP or the U.S. Government.
1,2:5,6-Dibenzanthracene, also known as dibenzo[a,h]anthracene, belongs to a class of compounds called polycyclic aromatic hydrocarbons. PAHs are formed as a result of incomplete combustion of organic material. Human exposure to PAHs occurs primarily via the diet, inhalation of polluted air (forest fires, wood-burning stoves, smoking, car exhaust fumes), or through occupational exposure (roofing, smelting, coke ovens). While other PAHs, such as benzo[a]pyrene (B[a]P), have been well-studied for their immunosuppressive effects, DBZA has not been thoroughly investigated, even though it has been reported to be more potent than B[a]P (White et al., 1985a; Pufulete et al., 2004). Four days following a single pharyngeal aspiration of DBZA, systemic immunosuppression of both humoral and cell-mediated immune function has been reported, with the most significant effects being observed on humoral immunity (Smith et al., 2010).
Range-finding studies following 28 days of subcutaneous exposure conducted on behalf of the National Toxicology Program demonstrated that DBZA was a potent immunosuppressive agent. Therefore, the NTP requested that additional studies be conducted to establish the effects of DBZA in disease resistance models. These studies were conducted in female B6C3F1/N mice. Animals were treated subcutaneously with 250, 793, or 2500 µg/kg DBZA daily for 28 days. DBZA was prepared weekly as a solution in corn oil.
Toxicology studies: Following 28 days of exposure to DBZA, no effects were observed on body weights or on body weight gain. Absolute and relative liver weights were increased in mice treated with 2500 µg/kg DBZA. Absolute spleen weights were decreased at the 2500 µg/kg dose, and relative spleen weights were decreased at both 793 µg/kg and 2500 µg/kg. No effects were observed on the weights of the thymus, lungs, or kidneys. Decreases in erythrocyte numbers, hemoglobin concentration, and hematocrit were observed following treatment with 2500 µg/kg DBZA. No other hematological parameters were affected.
Immunology studies: There was a dose-dependent decrease in total spleen cell numbers with significant decreases at doses ≥ 793 µg/kg. The absolute values of multiple spleen cell subpopulations were significantly decreased, including B cells, total T cells and T cell subsets, natural killer cells, and macrophages. The percent values of these phenotypes were unaffected, with the exception of a decrease in the percentage of NK cells in animals treated with 2500 µg/kg DBZA. The T-dependent antibody response was significantly suppressed following DBZA treatment. Specifically, the antibody-forming cell response following immunization with sheep erythrocytes and serum IgM antibody titers to sRBC were both significantly decreased at all doses of DBZA evaluated (i.e., doses ≥ 250 µg/kg). Furthermore, serum IgM anti-keyhole limpet hemocyanin antibody levels were significantly decreased in mice treated with DBZA at the 2500 µg/kg dose. Cell-mediated immune function was also suppressed by DBZA treatment. The proliferation of T cells, following stimulation with either allogeneic lymphocytes (the mixed leukocyte response or anti-CD3 antibody, was decreased at the 2500 µg/kg dose level. Furthermore, the cytotoxic T-lymphocyte response was significantly decreased at three effector:target ratios in mice treated with 2500 µg/kg DBZA. Innate immune function was minimally affected. While the vascular clearance rate of 51Cr-sRBC by the macrophages of the mononuclear phagocytic system was not affected, changes were observed in the percent uptake of the liver and spleen. When evaluated as specific activity, no effects were observed in the liver, while a decrease was observed in the spleen. No effects were observed on the activity of NK cells.
Host resistance assays: Spurious effects only were observed in host resistance to Listeria monocytogenes, B16F10 melanoma, and Streptococcus pneumoniae. However, significant effects were observed in host resistance to Plasmodium yoelii (a non-lethal strain of malaria), which is primarily mediated by humoral immunity. The kinetics of the response was affected, with animals exposed to 2500 µg/kg DBZA demonstrating a delayed onset of the infection. Furthermore, animals exposed to DBZA at doses ≥ 793 µg/kg demonstrated greater maximum parasitemia than that observed in the vehicle control animals. Delayed clearance of the infection was also observed resulting in a dose-responsive increase in the total parasitic burden that reached the level of statistical significance at doses ≥ 793 µg/kg.
White K.L., Lysy H.H., & Holsapple M.P. (1985a). Immunosuppression by polycyclic aromatic hydrocarbons: A structure-activity relationship in B6C3F1/N and DBA/2 mice. Immunopharmacology, 9:155-64.
Pufulete M., Battershill J., Boobis A., & Fielder R. (2004). Approaches to carcinogenic risk assessment for polycyclic aromatic hydrocarbons: A UK perspective. Regul Toxicol Pharmacol, 40:54-66.
Smith D.C., Smith M.J., & White K.L. Jr. (2010). Systemic immunosuppression following a single pharyngeal aspiration of 1,2:5,6-dibenzanthracene in female B6C3F1/N mice. J Immunotoxicol, 7(3):219-31.