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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009.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 dibenz[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 as thoroughly investigated, even though it has been reported to be more potent than B[a]P (White et al., 1985a; Pufulete, 2004). Four days following a single pharyngeal aspiration of DBZA, systemic immunosuppression of both humoral and cell-mediated immune functions has been reported, with the more significant effects being observed on humoral immunity (Smith et al., 2010).
Previous studies conducted on behalf of the National Toxicology Program evaluated the effects of DBZA in adult female B6C3F1/N mice and demonstrated that DBZA exposure for 28 days produced suppression of humoral, cell-mediated, and innate immunity. Therefore, the NTP requested that a study be conducted to evaluate the effects of perinatal DBZA exposure. These developmental immunotoxicology studies were conducted in female B6C3F1 mice. C57BL/6 dams were exposed to corn oil vehicle or DBZA daily by subcutaneous injection at three dose levels, beginning either at the conclusion of a 5-day mating period with C3H males (Study 1 only) or on gestational day 6 following a single overnight breeding with C3H males (all other studies). Several studies were conducted, utilizing different doses of DBZA. The doses evaluated in these studies were: 2.5, 7.93, 25, 79.3, 250, 793, and 2500 µg/kg. The first day of the mating period was designated as gestational day 0 for the 5-day study, while GD0 was designated as the day following the overnight mating period for the single overnight studies. The first day of DBZA treatment was GD6 for both types of studies. The day of birth for each individual litter was designated as litter post-natal day 0. Dams continued to receive DBZA treatment until the B6C3F1 offspring were weaned on litter PND21. The B6C3F1 offspring were exposed to DBZA through lactation only from birth through weaning on litter PND21. On litter PND21, male offspring were removed from the study and euthanized by carbon dioxide inhalation. Female offspring were weaned on litter PND21 and were exposed to either vehicle or DBZA by s.c. injection once daily from litter PND21 to study termination (between PND45 and PND55). DBZA was prepared weekly as a solution in corn oil. The in-life phase of these studies was carried out between 26 July 2006 and 09 May 2008. No usable sheep red blood cell high-salt release membrane antigen was available to evaluate serum IgM anti-sRBC antibody levels, and pup body weights prior to PND21 were not obtained. No other significant protocol or standard operating procedure deviations occurred during the study that affected the quality of the data and the ability to interpret the data with respect to the developmental immunotoxicity of DBZA.
Overall, body weights in weaned females were unaffected from PND21 to PND42 and at termination of the studies. Furthermore, the weights of the liver, spleen, thymus, lung, and kidney were unaffected at DBZA doses ranging from 25 µg/kg to 250 µg/kg, with the exception of decreases in relative kidney weights in one study at doses ≥ 79.3 µg/kg. In studies where spleen weights were evaluated at higher DBZA exposure levels, significant dose-related decreases were observed following DBZA exposure at the 793 and 2500 µg/kg dose levels. Hematological parameters, spleen cell numbers, and spleen cell differentials were unaffected following DBZA exposure at doses ranging from 25 to 250 µg/kg. At higher doses (i.e., 793 µg/kg and 2500 µg/kg), spleen cell numbers were decreased in animals immunized with sRBC, when compared to the vehicle control group.
The antibody-forming cell response to sRBC was decreased in mice exposed to DBZA at 793 µg/kg in two of three studies, while the 2500 µg/kg dose produced suppression in all three studies utilizing this dose. Inconsistent suppression was observed at the 250 µg/kg dose. Specifically, significant decreases in Specific Activity were observed in only 1 of the 5 AFC studies conducted using this dose, while decreases in Total Spleen Activity were observed in 2 of the 5 studies. Spurious effects were observed in the AFC response at doses < 250 µg/kg. The IgM antibody response to keyhole limpet hemocyanin was not affected following exposure to DBZA at doses up to 25 µg/kg, the highest dose evaluated for this endpoint. In contrast to the effects observed on the AFC response, cell-mediated immunity (as evaluated by assessing the mixed leukocyte response, anti-CD3 mediated spleen cell proliferation, and cytotoxic T-lymphocyte activity) was unaffected. Furthermore, innate immunity, as evaluated by examining the cytolytic activity of natural killer cells, was also unaffected.
In conclusion, perinatal exposure to DBZA resulted in suppression of the humoral immune response, as indicated by the suppression of the antigen-specific AFC response. No effects were observed on cell-mediated or innate immune responses. This is in contrast to the effects observed in studies conducted in adult female B6C3F1/N mice, where DBZA exposure for 28 days produced suppression of humoral, cell-mediated, and innate immunity.
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.