Search

The objective of the present study was to determine which splenic cell type(s) of B6C3F1 mice was capable of metabolizing B(a)P. Separation of splenocytes based on density by centrifugation through discontinuous Percoll gradients along with immunomagnetic negative selection or antibody-mediated complement lysis was utilized to obtain highly enriched populations of splenocytes for B(a)P metabolism studies. Immunofluorescent cell staining in conjunction with flow cytometry and examination of Giemsa-stained cytospin cell preparations indicated that B- or T-cell populations of greater than 95% purity and an 80–90% pure population of splenic macrophages were routinely attained. Splenic cell populations were incubated with [ 3 H]B(a)P for 24 hr. High-pressure liquid chromatography was used to separate and quantitate the B(a)P metabolites generated by the enriched splenic cell populations. The results of these studies demonstrate that the macrophage is the cell type responsible for the metabolism of B(a)P within the spleen. The major metabolites of B(a)P produced were as follows: an unidentified peak of polar metabolites containing polyhydroxylated metabolites, B(a)P-9,10-dihydroxy-9,10-dihydrodiol, and B(a)P-7,8-dihydroxy-7,8-dihydrodiol. Other splenic cell types examined, including B and T cells, polymorphonuclear cells, or the spleen capsule did not produce amounts of B(a)P metabolites significantly above background levels. Based on these findings, macrophages are the splenic cell types which metabolize B(a)P. As a result, macrophages may be the cell type targeted by B(a)P resulting in suppression of splenic humoral immune responses.

Recent studies have demonstrated that macrophages are the cell types capable of metabolizing benzo[ a ]pyrene (B(a)P) within the spleens of untreated mice. Since repeated exposure to B(a)P results in immunosuppression and B(a)P is known to induce cytochrome P450 levels, the first objective of this study was to investigate whether exposure of mice to B(a)P could increase the amounts of immunosuppressive B(a)P metabolites generated and/or alter the pattern of B(a)P metabolites formed by several different splenic cell types. Mice were dosed with a daily sc dose of 200 mg/kg B(a)P or vehicle for 4 days. Separation of splenocytes based on density by centrifugation through discontinuous Percoll gradients along with immunomagnetic negative selection or antibody-mediated complement lysis was used to obtain different splenic cell populations. Cells were incubated with [ 3 H]B(a)P for 24 hr. High-pressure liquid chromatography was used to separate and quantitate B(a)P metabolites. Results indicate that splenic macrophages of B(a)P-treated mice produced significantly greater amounts of some metabolites compared to those of vehicle-treated mice. The three major metabolites produced were an unidentified peak of polar metabolites containing polyhydroxylated metabolites, B(a)P-9,10- and B(a)P-7,8-dihydrodiols. Other splenic cell types examined did not produce metabolite amounts significantly above (T-cells, PMNs, or the capsule) or just above (B-cells) background. The second objective was to investigate the splenic cell type(s) targeted by B(a)P resulting in suppression of humoral immunity. Separation-reconstitution studies along with in vitro sensitization techniques with several different antigens (sheep red blood cells (SRBC), dinitrophenyl-Ficoll (DNP-Ficoll), lipopolysaccharide (LPS)) were used to identify splenic target cells following exposure of mice to B(a)P (200 mg/kg/day, sc for 4 days). Findings indicate that in vitro plaque-forming cell (PFC) suppression was due to alterations in the adherent (macrophage) cell population. Exposure also suppressed the PFC response to the T-dependent antigen SRBC and the T-independent antigen DNP-Focoll, but did not suppress the PFC response to the polyclonal antigen, LPS. These data suggest that B(a)P is targeting macrophages.

Studies have demonstrated that macrophages are the cell types which metabolize benzo[ a ]pyrene (B[ a ]P) within the murine spleen. B and T cells, polymorphonuclear cells, or the splenic capsule did not produce amounts of B[ a ]P metabolites above those of background. Exposure of mice to B[ a ]P, a known inducer of isozymes of cytochrome P450, resulted in an increase in the amounts of some B[ a ]P metabolites generated by macrophages. Evaluation of the in vitro plaque-forming-cell response to several T-cell and macrophage-dependent antigens following in vivo exposure to B[ a ]P indicated that the macrophage is the cell type responsible for B[ a ]P-induced immunosuppression. While suggestive, the reported data have not definitively established that an enriched splenic macrophage population can generate 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[ a ]P (BPDE) from B[ a ]P-7,8-dihydroxy-7,8-dihydrodiol (B[ a ]P-7,8-diol). These data are critical to our hypothesis that the splenic cell type(s) which from BPDE will be the primary target cell responsible for B[ a ]P-induced immunosuppression. The first objective was to determine if splenic macrophages could generate BPDE. Enriched (80–90% purity) populations of macrophages were incubated with [ 3 H]B[ a ]P for 24 hr. BPDE generated was quantitated by analysis of the cis - and trans -tetrol hydrolysis products of BPDE via HPLC procedures. Splenic macrophages generated BPDE from B[ a ]P. The cis syn was the predominate tetrol detected. Exposure of mice to B[ a ]P increased the amounts of the trans-anti -tetrols 2.2-fold, the trans-syn -tetrols 2.0-fold, the cis-anti -tetrols 1.8-fold, and the cis-syn -tetrols 2.6-fold above those formed by macrophages of vehicle-exposed mice. Both cytochrome P450- and peroxyl radical-dependent pathways are known to oxidize B[ a ]P-7,8-diol to BPDE. Since macrophages were found to generate BPDE, the second objective was to investigate which enzymatic pathway was responsible for its formation. The B[ a ]P-(+)-7,8-diol isomer has been shown to produce different specific BPDE isomers via the cytochrome P450 and peroxyl radical pathways. Macrophage populations were incubated with [ 3 H]B[ a ]P-(+)-7,8-diol for 24 hr and the contribution of the cytochrome P450 and peroxyl radical pathways to BPDE formation determined by detection of syn -BPDE-hydrolysis and anti -BPDE-hydrolysis products, respectively. Based on the ratio of anti/syn BPDE-derived tetrol products, the results demonstrate that splenic macrophages can generate the BPDE by both a cytochrome P450-dependent and -independent (peroxyl radical) pathway. Macrophages are the cells which metabolize B[ a ]P within the murine spleen and may be the cell type responsible for B[ a ]P-induced suppression of humoral immune responses.

Exposure to 7,12-dimethylbenz[a]anthracene (DMBA) has been demonstrated by numerous investigators to result in suppression of both humoral and cell-mediated immune responses of mice and cultured splenocytes. The mechanism(s) of this DMBA-induced immunosuppression, however, is not well characterized. PAHs must be converted to reactive metabolites via cytochrome P450-dependent monooxygenase systems to exert their carcinogenic and mutagenic effects. Thus, we have hypothesized that immunosuppression seen upon exposure to DMBA may also be mediated by its reactive metabolites. The objective of this study was to determine if DMBA metabolites can suppress the in vitro, T-dependent humoral immune response to sheep red blood cells. Compounds were evaluated in the in vitro plaque-forming cell (PFC) response at concentrations of 10−9 to 10−5 m . DMBA and benzo[a]pyrene (B[a]P) were also evaluated for their ability to suppress the in vitro PFC response. Addition of either of these PAHs to splenocyte cultures produced a concentration-dependent suppression of the PFC response, in which B[a]P was found to be 17.5-fold more potent that DMBA. These results are in contrast to those found in vivo, where DMBA has been shown to be more potent than B[a]P at suppressing humoral immunity. The 3,4-diol metabolite of DMBA produced a concentration-dependent suppression (10−8 to 10−5 m ) of the in vitro PFC response and was found to be 65-fold more potent than the parent compound DMBA. In contrast, the 5,6-diol metabolite of DMBA had no effect on the PFC response or cell viability. Both the 3-OH-DMBA and 7-hydroxymethyl-12-methyl-benz[a]anthracene (7-OHMe-12-Me-BA) metabolites were found to be immunosuppressive at concentrations of 10−6 m . Furthermore, suppression by 7-OHMe-12-Me-BA was observed at concentrations as low as 10−8 m . Immunosuppression by the 7-Me-12-OHMe-BA and the di-OHMe-BA metabolites was only observed at high (10−5 m ) concentrations. The cytochrome P450 inhibitor, α-naphthoflavone (ANF), was utilized to determine if cytochrome P450-mediated metabolism is involved in DMBA-induced suppression of the in vitro PFC response. ANF (10−5 m ) reversed the DMBA-induced immunosuppression seen at 10−5 m and attenuated the immunosuppression at 3 × 10−5 and 10−4 m . The results of these studies demonstrate that several metabolites of DMBA which can be generated by the cytochrome P450-dependent monooxygenase systems are immunosuppressive in the in vitro PFC response assay. Furthermore, the cytochrome P450 inhibitor, ANF, was able to reverse DMBA-induced immunosuppression. However, since the degree of DMBA-induced and B[a]P-induced suppression of humoral immunity in vitro does not correlate with that observed in vivo, DMBA metabolites generated within the spleen may not be responsible for or may only partially contribute to the immunosuppression observed in the whole animal.

Times Cited: 0 1 45th Congress of the European-Societies-of-Toxicology Oct 05-08, 2008 Rhodes, GREECE European Soc Toxicol

The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding the relationship between sensitization potential and allergy disease. To address these and other issues, an April 2012 international symposium was held in Prague, Czech Republic, to review and discuss the state-of-the-science of sensitizing properties of protein allergens. The symposium, organized by the Protein Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary scientists from academia, government, and industry participated in the symposium. Experts provided overviews on known mechanisms by which proteins in food may cause sensitization, discussed experimental models to predict protein sensitizing potential, and explored whether such experimental techniques may be applicable in regulatory settings. Three accompanying reviews address critical factors and methods for assessing allergic sensitization: 1) food-and protein-related factors; 2) host-specific factors and 3) screening methods, i.e., the ability of experimental models to predict the sensitizing potential of proteins and whether such models are applicable within regulatory settings.

Experimental in silico, in vitro, and rodent models for screening and predicting protein sensitizing potential are discussed, including whether there is evidence of new sensitizations and allergies since the introduction of genetically modified crops in 1996, the importance of linear versus conformational epitopes, and protein families that become allergens. Some common challenges for predicting protein sensitization are addressed: (a) exposure routes; (b) frequency and dose of exposure; (c) dose-response relationships; (d) role of digestion, food processing, and the food matrix; (e) role of infection; (f) role of the gut microbiota; (g) influence of the structure and physicochemical properties of the protein; and (h) the genetic background and physiology of consumers. The consensus view is that sensitization screening models are not yet validated to definitively predict the de novo sensitizing potential of a novel protein. However, they would be extremely useful in the discovery and research phases of understanding the mechanisms of food allergy development, and may prove fruitful to provide information regarding potential allergenicity risk assessment of future products on a case by case basis. These data and findings were presented at a 2012 international symposium in Prague organized by the Protein Allergenicity Technical Committee of the International Life Sciences Institute’s Health and Environmental Sciences Institute.