The potential health effects of inhaling carbon nanotubes are important because of possible exposures in an occupational setting. Previously, we showed that mice inhaling multiwalled carbon nanotubes (MWCNT) showed suppressed systemic immune function. Here we show the mechanisms for this immune suppression. Mice were exposed to 0, 0.3, or 1 mg/m3 MWCNT for 6h/day for 14 consecutive days in whole-body inhalation chambers. Those exposed to 1 mg/m3 showed compromised systemic immune function. Spleen cells from exposed animals increased gene expression of prostaglandin synthase enzymes and were rescued from immunosuppression when treated with ibuprofen. Cyclooxygenase-2 knockout mice were resistant to MWCNT-induced suppression. Proteins isolated from the lungs of exposed mice contained transforming growth factor-beta, which suppressed immune function of wild-type splenocytes but not those from knockout mice in vitro. This suggests that signals from the lung can activate signals in the spleen to suppress the immune function of exposed mice.

7,12-Dimethylbenz(a)anthracene (DMBA) is a potent carcinogen that induces immunosuppression of both humoral and cell-mediated immunity in mice and other species. Previous studies have shown that CYP1B1 is required for bone marrow toxicity produced by DMBA in mice. Therefore, the purpose of these studies was to determine whether CYP1B1 was required for spleen cell immunotoxicity. Female C57BL/6N wild-type (WT) and CYP1B1 knockout (-/-) mice were treated with 0, 17, 50, or 150 mg/kg (cumulative dose) DMBA in corn oil by oral gavage once a day for five days. Several immunotoxicological assays were used to assess the effects of DMBA on systemic immunity. These included the in vitro T-dependent antibody response to sheep red blood cells (SRBC) measured using a direct plaque forming cell (PFC) assay, T- and B-cell mitogenesis induced by Con A and LPS, and nonspecific cell-mediated immunity was evaluated using an NK cytotoxicity assay. In addition, lymphocyte subpopulations were measured by flow cytometry using specific cell surface markers. Following five days of DMBA treatment, the body weights and spleen cell surface markers of the WT and CYP1B1 (-/-) mice showed no significant changes. A decrease in NK activity was found at the 50 mg/kg DMBA dose in WT mice, but not in the CYP1B1 (-/-) mice. Interestingly, at the 150 mg/kg dose of DMBA, CYP1B1 null mice had decreased NK activity, whereas WT mice did not. The SRBC PFC response demonstrated that the IgM antibody response was suppressed by DMBA in WT mice in a dose-dependent manner (significant at 50 and 150 mg/kg). However, there were no changes in the SRBC PFC responses in any DMBA test group in the CYP1B1 (-/-) mice. Similarly, while DMBA suppressed B- and T-cell mitogenesis at the 50 and 150 mg/kg dose levels in C57BL/6N WT mice, no effect was seen in CYP1B1 (-/-) mice. Thus, CYP1B1 appears to be critical for the immunosuppression of DMBA in mice, suggesting a role for bioreactive metabolites in the spleen cell immunotoxicity produced by DMBA.

Microsomal epoxide hydrolase (mEH, EPHX1) is involved in the metabolism of chemicals to generate dihydrodiol intermediates in the presence of the cytochrome P450. We have previously shown that 7,12-dimethylbenz[a]anthracene (DMBA) can suppress both cell-mediated and humoral immune responses in wild-type (WT) C57BL/6N mice but not in CYP1B1 null mice. In the present studies, we hypothesized the critical metabolite responsible for DMBA-induced immunotoxicity is likely to be the 3,4-dihydrodiol-1,2-epoxide metabolite of DMBA, which requires mEH for formation. Mice were gavaged orally with DMBA (0, 17, 50, and 150 mg/kg) once a day for 5 days. Immune function and other assays were performed on day 7. Our data showed that unlike WT mice, DMBA treatment of mEH null mice produced no alterations in the body weight, spleen weight, or spleen cellularity. Similarly, DMBA treatments did not affect the PFC response in mEH null mice. Natural killer activity was not altered by DMBA treatment in mEH null mice. T-cell mitogenesis was partially suppressed by 50 and 150 mg/kg DMBA treatments of mEH null mice, but B-cell mitogenesis was not affected. Finally, we assessed the biodistribution of DMBA in both C57BL/6N WT and mEH null mice in spleen, thymus, and liver after 24 h and 7 days oral gavage. The concentrations of DMBA in each organ were not significantly different in WT and in mEH null mice. Collectively, these results demonstrate that mEH (EPHX1 gene) is a crucial enzyme for metabolic activation of DMBA in vivo leading to immunosuppression of spleen cells.

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The tumor suppressor protein p53 is a transcription factor that regulates apoptotic responses produced by genotoxic agents. Previous studies have reported that 7,12-dimethylbenz [a]anthracene (DMBA)-induced bone marrow toxicity is p53-dependent in vivo. Our laboratory has shown that DMBA-induced splenic immunosuppression is CYP1B1-and microsomal epoxide hydrolase (mEH)-dependent, demonstrating that the DMBA-3,4-dihydrodiol-1,2-epoxide metabolite (DMBA-DE) is probably responsible for DMBA-induced immunosuppression. DMBA-DE is known to bind to DNA leading to strand breaks. Therefore, we postulated that a p53 pathway is required for DBMA-induced immunosuppression. In the present studies, our data show that activated p53 accumulated in the nuclei of spleen cells in WT and AhR-null mice after DMBA treatment, but not in CYP1B1-null or mEH-null mice. These results suggest that DMBA activates p53 in a CYP1B1-and mEH-dependent manner in vivo but is not AhR-dependent. Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related protein (ATR) are sensors for DNA damage that signal p53 activation. Increased ATM, phospho-ATM (Ser 1987 ), and ATR levels were observed after DMBA treatment in WT, p53-null, and AhR-null mice but not in CYP1B1-null or mEH-null mice. Therefore, ATM and ATR seem to act upstream of p53 as sensors of DNA damage. Ex vivo immune function studies demonstrated that DMBA-induced splenic immunosuppression is p53-dependent at doses of DMBA that produce immunosuppression in the absence of cytotoxicity. High-dose DMBA cytotoxicity may be associated with p53-independent pathways. This study provides new insights into the requirement of genotoxicity for DMBA-induced immunosuppression in vivo and highlights the roles of ATM/ATR in signaling p53.Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are present as products of incomplete combustion of fossil fuels and other organic matter. Most of the members in the PAH family are potent toxicants and can produce cytotoxic, carcinogenic, and immunotoxic effects (Pelkonen and Nebert, 1982;Uno et al., 2004) in various species, tissues, and cell types (Thurmond et al., 1988;Burchiel et al., 1990;Buters et al., 1999Buters et al., , 2003 Sugiyama et al., 2002). 7,12-Dimethylbenz[a]anthracene (DMBA) has been widely used as a model compound for carcinogenic, immunotoxic, and teratogenic studies (Ikegwuonu et al., 1999). Our previous studies have shown that DMBA suppresses both humoral and cell-mediated immune responses (Burchiel et al., 1990). However, the precise molecular mechanism by which DMBA produces immunosuppression is still not well understood.Our laboratory has recently demonstrated that deletion of cytochrome P450 1B1 (CYP1B1) (Gao et al., 2005) and microsomal epoxide hydrolase (mEH) (Gao et al., 2007) protects mice against spleen cell immunosuppression produced by DMBA in vivo. Thus, CYP1B1 and mEH are associated with the formation of DMBA metabolites responsible for immunosuppression. Based on these observations and other report...