Assessing the Toxicity of DNI (3,4-Dichloro-N-isobutyramide) in a Human T-cell Cell Line

Saaketh Kyathari*, Jamie L. McCall, Kensey N. Bergdorf, Casey Hall, Jonathan Soboloff, Bjorn Soderberg and John B. Barnett, Department of Microbiology, Immunology & Cell Biology; Department of Chemistry, West Virginia University, Morgantown, WV 26506; Department of Medical Genetics and Molecular Biochemistry, Temple University, Philadelphia, PA 19140

Field (Broad Category): Medical Sciences (Health Sciences) 

Student’s Major: Immunology and Medical Microbiology 

Propanil (3,4-dichloropropionaniline, DCPA) is a post-emergence herbicide used in crops. Propanil reduces the T cell-dependent antibody response and bone erosion in a mouse model of arthritis. However, its major metabolite, 3,4-dichloroaniline (DCA), is immunotoxic in a C57BL/6 mouse model. Therefore, we sought to identify an analog of DCPA that cannot be metabolized to DCA in attempt to reduce the inflammatory response without inducing the side effects observed with the parent compound metabolite. We first assessed four DCPA analogs for reduced toxicity in a human T-cell cell line, Jurkats. While several compounds reduced cell death, as measured by 7-AAD staining, we identified 3,4-Dichloro-Nisobutyramide (DNI) as the compound with the least toxicity at 48 hours. We hypothesized that DNI has similar anti-inflammatory efficacy but reduced toxicity as compared to the parent compound, DCPA, in vitro. To further define its toxicity profile, we treated Jurkats with increasing concentrations of DNI (25-400 μm) and assessed cell number using trypan-blue exclusion over 96 hours. There was a dose-dependent decrease in cell number with increasing concentrations of DNI. This reduction in cell number may be due to decreased proliferation or increased apoptosis. In future studies, we will assess proliferation using alamar blue, which measures cellular oxidation-reduction (REDOX), and apoptosis by measuring caspase 3/7 activity. Using these data, we will identify a dose that is non-toxic to the cells, which we can then assess for efficacy in a mouse model of arthritis. 

Funding: 

Program/mechanism supporting research/creative efforts: WVU's Research Apprenticeship Program (RAP) & accompanying HONR 297-level course