Scott W. Burchiel

Mechanisms for how inhaled multiwalled carbon nanotubes suppress systemic immune function in mice

The potential health effects of inhaling carbon nanotubes are important because of possible exposures in occupational settings. Previously, we have shown mice that have inhaled multiwalled carbon nanotubes have suppressed systemic immune function. Here, we show the mechanisms for this immune suppression. Mice were exposed to 0, 0.3 or 1 mg m(-3) multiwalled carbon nanotubes for 6 h per day for 14 consecutive days in whole-body inhalation chambers. Only those exposed to a dose of 1 mg m(-3) presented suppressed immune function; this involved activation of cyclooxygenase enzymes in the spleen in response to a signal from the lungs. Spleen cells from exposed animals partially recovered their immune function when treated with ibuprofen, a drug that blocks the formation of cyclooxygenase enzymes. Knockout mice without cyclooxygenase enzymes were not affected when exposed to multiwalled carbon nanotubes, further confirming the importance of this enzyme in suppression. Proteins from the lungs of exposed mice suppressed the immune function of spleen cells from normal mice, but not those from knockout mice. Our findings suggest that signals from the lung can activate signals in the spleen to suppress the immune function of exposed mice.

Role of alterations in Ca2+-associated signaling pathways in the immunotoxicity of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. The effects of PAHs on the immune system of various animals and models have been studied for at least 30 yr. Despite these efforts, the mechanism or mechanisms by which PAHs exert their effects on the immune system are still largely unknown. During recent years, the molecular events associated with lymphocyte activation and receptor-mediated signaling have become increasingly clear. Substantial progress has been made in understanding the molecular and cellular bases for toxicant-induced immune cell injury. Understanding mechanisms of drug or chemical effects on the immune system is an important area of research in the field of immunotoxicology, and indeed in all fields of toxicology. Mechanistic toxicology plays an important role in risk assessment and extrapolation of potential human health effects. In this review, we have summarized recent evidence that has examined the effects of PAHs on the immune system of animals and humans. In particular, we have focused on the effects of PAHs on cell signaling in lymphoid cells and have examined the hypothesis that PAHs alter lymphocyte activation via calcium-dependent mechanisms. Previously published reports are discussed, and new data obtained with murine B cells and cell lines are presented demonstrating the relationship between alterations in intracellular calcium and immune dysregulation. These data demonstrate a strong association between PAH-induced alterations in B- and T-lymphocyte activation and changes in calcium homeostasis.

Rapid and efficient purification of mouse monoclonal antibodies from ascites fluid using high performance liquid chromatography.

Techniques for the rapid and efficient purification of mouse monoclonal antibodies from murine ascites are described that utilize anion exchange and gel permeation chromatography using high performance liquid chromatography (HPLC). Anion exchange chromatography was performed at neutral pH using a hydrophilic resin conjugated with a substituted amine (Mono Q column, Pharmacia Fine Chemicals). Various subclasses of mouse IgG monoclonals were assessed for their binding to this matrix, with all of the antibodies tested eluting at relatively low concentrations of sodium chloride. In some instances, a protein tentatively identified as transferrin was co-purified using this anion exchange procedure. However, this protein was easily removed from the IgG using gel permeation chromatography (Bio-Sil TSK-250, Bio-Rad), also performed at neutral pH.

Potential Risks Resulting from Fruit/Vegetable–Drug Interactions: Effects on Drug-Metabolizing Enzymes and Drug Transporters

It has been well established that complex mixtures of phytochemicals in fruits and vegetables can be beneficial for human health. Moreover, it is becoming increasingly apparent that phytochemicals can influence the pharmacological activity of drugs by modifying their absorption characteristics through interactions with drug transporters as well as drug-metabolizing enzyme systems. Such effects are more likely to occur in the intestine and liver, where high concentrations of phytochemicals may occur. Alterations in cytochrome P450 and other enzyme activities may influence the fate of drugs subject to extensive first-pass metabolism. Although numerous studies of nutrient-drug interactions have been published and systematic reviews and meta-analyses of these studies are available, no generalizations on the effect of nutrient-drug interactions on drug bioavailability are currently available. Several publications have highlighted the unintended consequences of the combined use of nutrients and drugs. Many phytochemicals have been shown to have pharmacokinetic interactions with drugs. The present review is limited to commonly consumed fruits and vegetables with significant beneficial effects as nutrients and components in folk medicine. Here, we discuss the phytochemistry and pharmacokinetic interactions of the following fruit and vegetables: grapefruit, orange, tangerine, grapes, cranberry, pomegranate, mango, guava, black raspberry, black mulberry, apple, broccoli, cauliflower, watercress, spinach, tomato, carrot, and avocado. We conclude that our knowledge of the potential risk of nutrient-drug interactions is still limited. Therefore, efforts to elucidate potential risks resulting from food-drug interactions should be intensified in order to prevent undesired and harmful clinical consequences.

DMBA induces programmed cell death (apoptosis) in the A20.1 murine B cell lymphoma

The mechanism by which 7,12-dimethylbenz[a]anthracene (DMBA) produces cytotoxicity in lymphocytes was investigated in these studies using the murine A20.1 B cell lymphoma. Results show that in vitro exposure of these cells to 10-30 microM DMBA for 4 hr produced an increase in intracellular Ca2+, DNA fragmentation, and subsequent cell death. Elevation of Ca2+ and DNA fragmentation induced by DMBA were greatly pronounced when the A20.1 cells were exposed at high cell density (10(7) cells/ml). DMBA-induced DNA fragmentation and cell death were inhibited by coexposure of A20.1 cells to a calcium chelator (EDTA), a general nuclease and polymerase inhibitor (aurintricarboxylic acid), and a protein synthesis inhibitor (cycloheximide). These agents have been previously shown to inhibit apoptosis in lymphocytes and other cells exposed to chemical agents. We also found that cyclosporin A, an inhibitor of Ca(2+)-dependent pathways of T and B cell activation, prevented apoptosis in the A20.1 cell line. These results demonstrate that DMBA induces programmed cell death (apoptosis) in the A20.1 murine B cell lymphoma by Ca(2+)-dependent pathways. The increased sensitivity of A20.1 at high cell density to Ca2+ elevation and DNA fragmentation suggests that cell to cell interactions may also be important in this process.

Factors influencing elevation of intracellular Ca2+ in the MCF-10A human mammary epithelial cell line by carcinogenic polycyclic aromatic hydrocarbons

Carcinogenic polycyclic aromatic hydrocarbons and a halogenated aromatic hydrocarbon, 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), were evaluated for their effects on intracellular Ca2+ in the human mammary epithelial cell line MCF‐10A. After two 18‐h incubations with MCF‐10A cells, benzo[a]pyrene (BaP; 1, 3, and 10 μM) produced a dose‐dependent increase in intracellular Ca2+. 7,12‐Dimethylbenz[a]anthracene increased Ca2+ at 10 μM, whereas 3‐methycholanthrene and TCDD did not. The Ca2+‐elevating effect of BaP appeared to be dependent on the influx of extracellular Ca2+, as addition of the Ca2+ chelator EGTA to the extracellular medium prevented the increase in Ca2+. MCF‐10A cells were found by polymerase chain reaction to express cytochrome P4501A and P4501B isozymes as well as the aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator mRNAs associated with cytochrome P450 induction. Certain cytochrome P450–derived metabolites, including benzo[a]pyrene‐7,8‐diol (BP‐diol) and benzo[a]pyrene‐7,8‐diol‐9,10‐epoxide (BPDE), were more effective in increasing Ca2+ than was BaP. The Ca2+‐elevating effect of BP‐diol was prevented by α‐naphthoflavone, a cytochrome P4501A and P4501B inhibitor, but not by the antioxidant N‐acetylcysteine. These results suggest that cytochrome P450–dependent formation of BPDE from BP‐diol is a major mechanism required for elevation of Ca2+ in MCF‐10A cells. Mol. Carcinog. 25:48–54, 1999.

Benzo[a]Pyrene Diones are Produced by Photochemical and Enzymatic Oxidation and Induce Concentration-Dependent Decreases in the Proliferative State of Human Pulmonary Epithelial Cells

Organic components within mixtures of combustion-derived materials may play an important role in the correlation between air pollution and adverse cardio/respiratory health. One class of these organic components, polycyclic aromatic hydrocarbons (PAHs), has been shown to produce a wide variety of adverse health effects. An air toxic and a model PAH, benzo[a]pyrene (BaP), is a component of combustion-derived particulate matter (PM). Although most biological effects associated with BaP have been attributed to the cytochrome P-450 derived BaP 7,8-diol 9,10-epoxide, many other BaP oxidation products are formed in atmospheric and biological reactions and may contribute to PAH-induced adverse health effects. In an ambient environ-ment, BaP and other PAHs undergo oxidation in the presence of ultraviolet light, O 2 , O 3 , NO 2 , or OH ” . Biological peroxidase- and P-450 mediated conversion of BaP produces an extensive metabolic profile of BaP oxidation products that significantly outnumber the 7,8-diol/diol epoxide. The data herein show that in addition to near-ultraviolet light and P-450 isozymes, lactoperoxidase (airway peroxidase) converted BaP into a mixture of three diones, the 1,6-, 3,6-, and 6,12-BaP dione (BPD). In addition, it was found that low concentrations of BPDs induced a concentration-dependent decrease in the proliferation state of human pulmonary epithelial cells in vitro. Nanomolar concentrations of BPDs mediated cell growth inhibition, which was partially reversed by co-incubation with N-acetyl-L-cysteine and ascorbate. BPDs induced the formation of reactive oxygen species as measured by the fluorophore 2,7-dichloro-fluorescein. Together, these results may indicate a role for PAH oxidation products (PAH diones) in the adverse health effects associated with combustion-derived PM and semivolatile organic compounds.

Environmental polycyclic aromatic hydrocarbons, benzo(a) pyrene (BaP) and BaP-quinones, enhance IgE-mediated histamine release and IL-4 production in human basophils

Polycyclic aromatic hydrocarbons (PAHs) are major components of diesel exhaust particles found in pollutant respirable particles. There is growing evidence that these fossil fuel combustion products exacerbate allergic inflammation. Basophils contribute to allergic inflammation through the release of preformed and granule-derived mediators. To determine whether allergens and PAHs interact, we incubated human basophils with PAHs and measured the release of histamine and IL-4 with and without added antigen. None of the PAHs induced mediator release by itself and none affected total cellular histamine levels. However, several PAHs enhanced histamine release and IL-4 production in response to crosslinking the high-affinity IgE receptor, Fc epsilon RI. The enhancement seen with 1,6-BaP-quinone involved an increase in tyrosine phosphorylation in several different substrates, including the Fc epsilon RI-associated tyrosine kinase, Lyn, and elevated reactive oxygen species (ROS) levels detected by dichlorofluorescein fluorescence and flow cytometry. The PAH-induced enhancement of mediator release and ROS production could be inhibited with the antioxidant N-acetylcysteine. These data provide further evidence that environmental pollutants can influence allergic inflammation through enhanced Fc epsilon RI-coupled mediator release from human basophils.

Inhibition of lymphocyte activation in splenic and gut-associated lymphoid tissues following oral exposure of mice to 7,12-dimethylbenz[a]anthracene

The hypothesis that 7,12-dimethyl-benz[a]anthracene (DMBA) suppresses immune function in mice via an inhibition of lymphocyte activation was examined in these studies. Daily exposure of B6C3F1 mice to DMBA (cumulative doses of 1.4 to 140 mg/kg) via the oral route for 14 days was found to inhibit phytohemagglutinin (PHA) and lipopolysaccharide mitogen responses in lymphoid cells obtained from the spleen. Peyers Patches, and mesenteric lymph nodes. The 14 mg/kg cumulative dose of DMBA produced no significant decrease in the number of recovered viable cells, yet mitogen responses were suppressed by approximately 50% in the spleen and mesenteric lymph nodes, and by greater than 70% in the Peyers Patches. DMBA inhibited PHA-induced Ca+2 mobilization measured by flow cytometry in each of these three lymphoid tissues. There was no change in the percentage of T cells recovered from the spleen, mesenteric lymph nodes, or Peyers Patches. Peyers Patch lymphocytes obtained from the GI tract appeared to be slightly more sensitive to inhibition of mitogen responsiveness and perhaps Ca+2 mobilization, potentially due to the oral route of exposure to DMBA. These studies provide evidence that DMBA inhibits early events associated with lymphocyte activation in mice.

Differential binding of monomethylarsonous acid compared to arsenite and arsenic trioxide with zinc finger peptides and proteins

Arsenic is an environmental toxin that enhances the carcinogenic effect of DNA-damaging agents, such as ultraviolet radiation and benzo[a]pyrene. Interaction with zinc finger proteins has been shown to be an important molecular mechanism for arsenic toxicity and cocarcinogenesis. Arsenicals such as arsenite, arsenic trioxide (ATO), and monomethylarsonous acid (MMA(III)) have been reported to interact with cysteine residues of zinc finger domains, but little is known about potential differences in their selectivity of interaction. Herein we analyzed the interaction of arsenite, MMA(III), and ATO with C2H2, C3H1, and C4 configurations of zinc fingers using UV–vis, cobalt, fluorescence, and mass spectrometry. We observed that arsenite and ATO both selectively bound to C3H1 and C4 zinc fingers, while MMA(III) interacted with all three configurations of zinc finger peptides. Structurally and functionally, arsenite and ATO caused conformational changes and zinc loss on C3H1 and C4 zinc finger peptide and protein, respectively, whereas MMA(III) changed conformation and displaced zinc on all three types of zinc fingers. The differential selectivity was also demonstrated in zinc finger proteins isolated from cells treated with these arsenicals. Our results show that trivalent inorganic arsenic compounds, arsenite and ATO, have the same selectivity and behavior when interacting with zinc finger proteins, while methylation removes the selectivity. These findings provide insights on the molecular mechanisms underlying the differential effects of inorganic versus methylated arsenicals, as well as the role of in vivo arsenic methylation in arsenic toxicity and carcinogenesis.