Osteoclasts are specialized macrophage derivatives that secrete acid and proteinases to mobilize bone for mineral homeostasis, growth, and replacement or repair. Osteoclast differentiation generally requires the monocyte growth factor m-CSF and the TNF-family cytokine RANKL, although differentiation is regulated by many other cytokines and by intracellular signals, including Ca2+. Studies of osteoclast differentiation in vitro were performed using human monocytic precursors stimulated with m-CSF and RANKL, revealing significant loss in both the expression and function of the required components of store-operated Ca2+ entry over the course of osteoclast differentiation. However, inhibition of CRAC using either the pharmacological agent 3,4 dichloropropioanilide (DCPA) or by knockdown of Orai1 severely inhibited formation of multinucleated osteoclasts. In contrast, no effect of CRAC channel inhibition was observed on expression of the osteoclast protein tartrate resistant acid phosphatase (TRAP). Our findings suggest that despite the fact that they are downregulated during osteoclast differentiation, CRAC channels are required for cell fusion, a late event in osteoclast differentiation. Since osteoclasts cannot function properly without multinucleation, selective CRAC inhibitors may have utility in management of hyperresorptive states.

Stimulation of T cells through the T-cell receptor results in the activation of a series of signaling pathways that leads to the secretion of interleukin (IL)-2 and cell proliferation. Influx of calcium (Ca(2+)) from the extracellular environment, following internal Ca(2+) store depletion, provides the elevated and sustained intracellular calcium concentration ([Ca(2+)](i)) critical for optimal T-cell activation. Our laboratory has documented that exposure to the herbicide 3,4-dichloropropionanilide (DCPA) inhibits intracellular signaling events that have one or more Ca(2+) dependent steps. Herein we report that DCPA attenuates the normal elevated and sustained [Ca(2+)](i) that follows internal store depletion in the human leukemic Jurkat T cell line and primary mouse T cells. DCPA did not alter the depletion of internal Ca(2+) stores when stimulated by anti-CD3 or thapsigargin demonstrating that early inositol 1,4,5-triphosphate-mediated signaling and depletion of Ca(2+) stores were unaffected. 2-Aminoethyldiphenol borate (2-APB) is known to alter the store-operated Ca(2+) (SOC) influx that follows Ca(2+) store depletion. Exposure of Jurkat cells to either DCPA or 50 microM 2-APB attenuated the increase in [Ca(2+)](i) following thapsigargin or anti-CD3 induced store depletion in a similar manner. At low concentrations, 2-APB enhances SOC influx but this enhancement is abrogated in the presence of DCPA. This alteration in [Ca(2+)](i), when exposed to DCPA, significantly reduces nuclear levels of nuclear factor of activated T cells (NFAT) and IL-2 secretion. The plasma membrane polarization profile is not altered by DCPA exposure. Taken together, these data indicate that DCPA inhibits T-cell activation by altering Ca(2+) homeostasis following store depletion.

Each year ~1 billion kg of herbicides are used worldwide to control the unwanted growth of plants. In the United States, over a quarter of a billion kg of herbicides are used, representing 28% of worldwide use. (Kiely, T., Donaldson, D., and Grube, A. [2004]. Pesticide Industry Sales and Usage. 2000 and 2001 Market Estimates. Available at: http://www.epa.gov/pesticides/pestsales/01pestsales/market_estimates2001.pdf. Accessed October 25, 2012.) Propanil (3,4-dichloropropionanilide [DCPA]) is a commonly used herbicide in the United States, with 2-4 million kg applied annually to 2 million acres of crop land. The immunomodulatory effects of DCPA have been well documented, but limited data are available on the effects of its metabolites. (Salazar, K. D., Ustyugova, I. V., Brundage, K. M., Barnett, J. B., and Schafer, R. [2008]. A review of the immunotoxicity of the pesticide 3,4-dichloropropionanalide. J. Toxicol. Environ. Health B Crit. Rev. 11, 630-645.) In mammals, hepatic enzymes metabolize DCPA, resulting in the production of 3,4-dichloroaniline (DCA). Further biotransformation of DCA leads to the production of 6-hydroxy-3,4-dichloroaniline (6OH-DCA) and N-hydroxy-3,4-dichloroaniline (NOH-DCA). We report, for the first time, the immunotoxic effects of DCPA metabolites on T-cell function. Human Jurkat T cells were exposed to varying concentrations of DCPA or its metabolites and assayed for effects on T-cell function. In addition, fluorine analogs of DCPA and DCA were investigated to determine the relative role of chlorine substituents on T-cell immunotoxicity. Here we report that exposure of Jurkat T cells to DCPA and DCA alters IL-2 secretion, nuclear factor of activated T cells (NFAT) activity, and calcium influx. However, exposure to 6OH-DCA and NOH-DCA reduces IL-2 secretion and NFAT activity but has no effect on calcium flux. When both chlorines in DCPA and DCA were substituted with fluorines all effects were abrogated. Our data indicate that metabolites of DCPA have differential effects on T-cell function and the presence of chlorines plays an important role in eliciting these effects.

Alterations in T cell function and activation during exposure to the herbicide 3,4 dichloropropionanilide (DCPA) and its metabolites Tricia L. Lewis Approximately 5. 3 bi llion po unds of pes ticides ar e a pplied ann ually ac ross t he U nited States and 15 of the top 25 most used pesticides are herbicides. Total herbicide use the in the U nited S tates r epresents 28% of all w orldwide her bicide us e. DCPA (3,4 dichloropropionanilide, c ommon name pr opanil) i s a pos t em ergent her bicide t hat i s used extensively for control against several broadleaf plants and grasses. I t is the 17 th most c ommon her bicide i n t he U nited States and 6-9 m illion pounds are appl ied annually to 2 million acres of rice fields. In mammals, DCPA is metabolized in the liver and pr oduces 3, 4-dichloroaniline (DCA) and pr opionic ac id as i ts m ajor m etabolites. DCA is further biotransformed leading to the production of 2 hydroxylated metabolites; 6-hydroxy-3,4-dichloroaniline (6OH-DCA) a nd N-hydroxy-3,4-dichloroaniline (NOH-DCA). The i mmunomodulatory e ffects of D CPA ar e w ell doc umented but onl y l imited data i s av ailable on the ef fects of i ts metabolites. Previous s tudies hav e s hown t hat DCPA al ters t ranscription f actors i nvolved i n t he ex pression o f I L-2 and dec reases mRNA a nd I L-2 pr otein i n hum an and m ouse T c ells. I L-2 i s an early c ytokine t hat i s secreted by activated T c ells and plays an important role in the activation, proliferation and di fferentiation of s everal i mmune c ells. E xpression of I L-2 r elies on activation an d influx of calcium through channels in the plasma membrane. This study was conducted to examine the effects of DCPA and its metabolites on T cell activation and function and to propose a m echanism for the observed effects. H uman Jurkat T c ells, a m odel cell line f or T cell s ignaling, w ere exposed t o i ncreasing c oncentrations of DCPA or its metabolites and T cell function was assessed by measuring IL-2 secretion. DCPA and its metabolites all inhibit IL-2 secretion in a concentration-dependent manner, however, NOH-DCA is t he m ost pot ent i nhibitor, f ollowed by D CPA. To better under stand t he mechanism by w hich t hey s uppress I L-2, N FAT ac tivity and calcium i nflux w ere investigated. Interestingly, D CPA and D CA i nhibited I L-2 i n a c alcium-dependent manner w hereas t he hy droxylated m etabolites i nhibited I L-2 i n a c alcium-independent manner. The c alcium-dependent al terations i n I L-2, N FAT an d c alcium influx ar e influenced by t he pr esence of c hlorines, a s s ubstitution w ith f luorines abr ogated al l effects. F urther s tudies i nvestigating t he r ole of D CPA i n c alcium r elease-activated calcium (CRAC) channels revealed that activation of a key protein, Stromal interaction molecule-1 (Stim1), is i nhibited by D CPA. C ollectively, t his dat a supports t he conclusion that DCPA suppresses IL-2 production by inhibiting Stim1. This mechanism describes a novel pathway for immunosuppression. iii DEDICATION I de...