Arsenic is among the most ubiquitous of environmental contaminants, originating from natural as well as industrial sources. Human exposure to arsenic can induce multiple acute and chronic illnesses including immune-system disorders. This study is directed towards understanding the molecular mechanisms underlying immunotoxic effects of arsenic using an in vitro model system of human germinal center B-lymphocytes (BLs). Induction of apoptosis in BLs is a potential mediator of immunotoxicity following chemical exposure. However, normal regulation of apoptosis is critical in immune system function. Germinal center BLs undergo apoptosis upon activation of the surface IgM/B-cell receptor (IgM/BCR) to eliminate autoreactive BLs in the process of negative selection, and cells are rescued from IgM/BCR-induced apoptosis after co-stimulation by CD40 in the process of positive selection. The anti-apoptotic effect of CD40 is mediated by the nuclear factor kappa-B (NF-kappaB) pathway, resulting in the activation of the Rel/NF-vJ3 family of transcription factors that induce the expression of multiple genes controlling cell survival and essential BL functions. Preliminary data show that arsenic interferes with the ability of CD40 to rescue BLs from IgM/BCR-induced apoptosis. The current investigation will test the hypothesis that this effect of arsenic is due to inhibition of the NF-kappaB pathway and that key regulatory points within the pathway are differentially sensitive to inhibition by discrete arsenic concentrations. The proposed experiments will utilize a panel of BL cell lines containing defined genetic changes that effect their sensitivity to apoptosis induction or their ability to activate the NF-KappaB pathway. The molecular interactions of arsenic at specific points in the NF-kappaB pathway will be examined, including: the activation of I-kappa B-kinase signaling complexes, the phosphorylation and degradation of the I-kappa B inhibitory protein, and the DNA binding activity of various Rel/NF-kappaB transcription factors. In addition, the relative contribution of reactive oxygen species and protein sulfhydryl damage to inhibition of the NF-kappaB pathway will be assessed. Finally, the effects of arsenic on the profile of Rel/NF-kappaB transcription factors associated with promoters of specific anti-apoptotic genes such as Bcl-2 and A1, immediately after CD40 ligation and at later time points will be determined. Ultimately, the research is expected to identify important mechanisms underlying the inhibitory effects of arsenic on key processes in positive selection in in BLs, including protection from apoptosis and induction of antibody class switching.
