Uses and future applications of flow cytometry in immunotoxicity testing.

Flow cytometry is an emerging technology that has numerous applications to immunotoxicity testing. The use and development of high-speed single-cell laser-based assays capable of quantitation of fluorescence, light scatter, and electrical impedance measurements can provide important information on xenobiotic-induced toxicity in defined target cell populations. The purpose of this article is to briefly review established and emerging immunotoxicology assays that use flow cytometry. In the coming years it is likely that many new flow cytometry assays will be developed and validated that will improve the sensitivity and perhaps specificity of immunotoxicity testing. Since flow cytometry is readily adaptable to high-throughput screening, it is also likely that this technology will increasingly find its place in the preclinical testing of drugs and chemicals in the pharmaceutical and chemical industries.

The extracellular signal-regulated kinase pathway contributes to mitogenic and antiapoptotic effects of peroxisome proliferators in vitro.

Peroxisome proliferators are a class of nongenotoxic rodent hepatocarcinogens thought to induce tumors by altering the balance between mitosis and apoptosis. Previous studies suggest mitogenic growth factors that act through the extracellular signal-regulated kinase (ERK) pathway, including insulin and epidermal growth factor (EGF), modulate peroxisome proliferator-activated receptor alpha activation as well as the mitogenic activity of peroxisome proliferators. We have investigated whether the ERK pathway plays a role in regulating the growth and survival altering properties of peroxisome proliferators in primary mouse hepatocytes. Exposure of hepatocytes to Wy-14,643 and trichloroacetate resulted in a dose-dependent phosphorylation and activation of ERK. Peroxisome proliferator-induced ERK phosphorylation was blocked when cells were pretreated with the MEK (ERK kinase) inhibitor, PD098059, or the phosphatidyl-inositol 3-kinase (PI3K) inhibitors, LY294002 and apigenin, suggesting that both MEK and PI3K are involved in the initial response. The pathway leading to peroxisome proliferator-induced ERK activation is different than that induced by phorbol ester or EGF, since the PI3K inhibitors had no effect on ERK phosphorylation induced by these agents. Under defined culture conditions, Wy-14,643 increased the level of BrdU incorporation in primary hepatocytes and suppressed the incidence of apoptosis induced by transforming growth factor beta 1. In contrast, concentrations of PD098059 that block Wy-14,643-induced ERK phosphorylation also blocked the stimulation of DNA replicative synthesis and suppression of apoptosis by Wy-14,643. These studies indicate that activation of the ERK pathway through a PI3K-dependent mechanism may play a significant role in the tumor-promoting properties of peroxisome proliferators.

Alterations in human B cell calcium homeostasis by polycyclic aromatic hydrocarbons: possible associations with cytochrome P450 metabolism and increased protein tyrosine phosphorylation.

Previous studies performed in this laboratory have shown that certain benzo(a)pyrene (BaP) metabolites, such as benzo(a)pyrene-7,8-dihydrodiol (BaP-7,8-diol) and benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), were more effective in elevating intracellular Ca2+ in normal human peripheral blood mononuclear cell (HPBMC) T and B cells than was BaP. Additionally, it has been shown that the suppression of human T cell mitogenesis produced by polycyclic aromatic hydrocarbons (PAHs) and certain BaP metabolites is reversed by treatment with alpha-naphthoflavone (ANF), a cytochrome P450 1A and 1B inhibitor. ANF also diminishes the elevation in intracellular calcium (Ca2+) produced by BaP in HPBMC. In the present studies, we further defined the relationships between intracellular Ca2+ elevation produced by BaP and two immunotoxic P450-derived metabolites, BaP-7,8-diol and BPDE in the Daudi human B cell line. At 1, 4, and 18 h, both BaP-7,8-diol and BPDE produced a significant rise in intracellular Ca2+. This effect, however, was not observed with BaP or benzo(e)pyrene (BeP), a nonimmunotoxic PAH. To evaluate the potential role of cytochrome P450 metabolism in PAH-induced Ca2+ elevation, Daudi cells were pretreated with ANF for 4 h, followed by treatment with BaP metabolites for 18 h. ANF completely reversed the rise in Ca2+ produced by BaP-7,8-diol, but had no effect on the Ca2+ elevation produced by BPDE. These results suggest that BPDE may be the ultimate P450 metabolite responsible for Ca2+ elevation in human B cells. BaP-7,8-diol and BPDE were found to increase tyrosine phosphorylation in Daudi whole cell lysates and to increase tyrosine phosphorylation of two important Src-related protein tyrosine kinases (PTKs), Lyn and Syk. Inhibition of tyrosine phosphorylation by herbimycin A was found to largely prevent the increase in intracellular Ca2+ produced by BaP-7,8-diol and BPDE, suggesting that Ca2+ elevation is coupled to increased tyrosine phosphorylation in Daudi. BPDE was found to produce a statistically significant increase in tyrosine phosphorylation of Lyn and Syk within 10 min of exposure. Collectively, these data demonstrate that certain P450-derived metabolites of BaP may be responsible for PTK activation and an increase intracellular Ca2+, which may alter antigen receptor signaling in human B cells.

Characterization of intracellular calcium responses produced by polycyclic aromatic hydrocarbons in surface marker-defined human peripheral blood mononuclear cells.

Previous studies have demonstrated that polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP) and 7,12-dimethybenz[a]anthracene (DMBA), and possibly 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD), may exert their immunosuppressive effects by altering intracellular Ca2+ homeostasis in lymphocytes. In these studies, we examined the effects of two immunosuppressive PAHs (BaP and DMBA), two nonimmunosuppressive PAHs (benzo[e]pyrene (BeP) and anthracene (ANTH)), and TCDD on intracellular Ca2+ levels in surface marker-defined human peripheral blood mononuclear cells (HPBMC). BaP and DMBA, but not BeP and ANTH, were found to produce a time-dependent increase in intracellular Ca2+ with maximal effects achieved following 42- to 66-hr exposures. In a series of studies with HPBMC obtained from 10 donors exposed in vitro for 42 hr, BaP and DMBA were found to produce a significant increase in Ca2+ in CD3+ T cells, CD19+ B cells, and CD14+ monocytes. BeP and ANTH did not produce a statistically significant increase in Ca2+ in the group of donors, but occasionally produced an apparent nonspecific elevation of Ca2+ in HPBMC from individual donors. Interestingly, TCDD produced a small and statistically significant increase in Ca2+ only in B cells analyzed for the pooled 10 donors. Certain BaP metabolites, such as the 7,8-dihydrodiol and the 7,8-diol-9,10-epoxide, were more effective in elevating Ca2+ in HPBMC lymphocytes at 20 hr than was BaP. These results demonstrate in normal HPBMC that immunosuppressive PAHs alter intracellular Ca2+ homeostasis in B cells, T cells, and monocytes, and suggest that P450 metabolism may play an important role in the immunotoxicity of certain PAHs.

Depletion of glutathione by benzo(a)pyrene metabolites, ionomycin, thapsigargin, and phorbol myristate in human peripheral blood mononuclear cells.

Previous studies in this laboratory have shown that polycyclic aromatic hydrocarbons (PAHs) alter Ca2+ homeostasis and inhibit activation of both B and T lymphocytes obtained from rodents and humans. In the present studies, we demonstrate that alpha-naphthoflavone (ANF), an inhibitor of cytochrome P4501A activity, reduced the Ca2+ elevation produced by BaP in human peripheral blood mononuclear cell (HPBMC) lymphocytes. These results suggested that BaP metabolites may play a role in intracellular Ca2+ homeostasis in human lymphocytes. Reactive oxidative intermediates of BaP produced in HPMBC are known to be highly carcinogenic and have also been shown to be immunosuppressive. We examined the effects of benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA), benzo(e)pyrene (BeP), and anthracene, as well as certain BaP metabolites, on the levels of intracellular Ca2+ and glutathione in HPBMC. While BaP, DMBA, BeP, and anthracene did not cause a statistically significant decrease in GSH in HPBMC at concentrations of 1 or 10 microM following a 6-, 48-, or 72-hr exposure, reactive BaP metabolites including 4,5-epoxide BaP and 7,8-diol-9,10-epoxide BaP consistently produced a 20-30% depletion of glutathione in HPBMC following a 6-hr treatment period. These BaP metabolites also elevated intracellular Ca2+ in HPBMC during a 6-hr incubation. Results of these experiments suggest that metabolism of BaP to certain epoxide metabolites may be responsible for sulfhydryl damage leading to transient GSH depletion and Ca2+ elevation. These results are consistent with the hypothesis that sulfhydryl damage by certain PAH metabolites may lead to altered Ca2+ homeostasis, leading to inhibition of cell activation and proliferation in HPBMC.