Apoptotic and bioenergetic changes in pancreatic cells after exposure to individual toxicants and their mixtures

Abstract

Pancreatic cancer has one of the worst fatality rates in oncology. Despite advances in treatment and recovery rates for other cancers, pancreatic cancer's lack of early symptomology culminates in a dismal five-year survival rate of 3-9% that has remained unchanged for decades. Cadmium has been implicated in the initiation of pancreatic cancer. Its prevalence in the environment make it a candidate for interactions with pesticides that have gone relatively unexplored. In this study, we examined the role of cadmium, pesticides, and their mixtures on the p53 apoptotic pathway and in the adaptive bioenergetics that foretell transition to a cancer state. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to establish a No Observable Adverse Effect Limit (NOAEL) which allowed us to identify sub-toxic chronic exposure concentrations to emulate realistic combination exposures for molecular evaluation of biological endpoints. These concentrations were used to examine p53 recruitment and downstream apoptotic markers phosphatidylserine (PS) and caspase 3/7 activity. Bioenergetic shifts were assessed by mitochondrial toxicity assays in the presence of glucose or galactose and evaluated in conjunction with mitochondrial membrane potential (MMP), oxygen consumption rates, and reactive oxygen species (ROS) production. Non-linear regression analysis led to the employment of 500nM Cd and 1uM Atrazine and Glyphosate test concentrations. Cadmium drove p53 response in HPNE cells while glyphosate and both mixtures increased p53 expression in tumor cells. Interestingly, only cadmium exhibited increased PS by annexin labeling, but mixtures showed significantly less PS, indicating a potential reinforcement of membrane integrity. No treatment groups in either cell line responded to treatment by increases in caspase activity. Taken together, we can surmise that the p53 apoptotic pathway is not initiated by exposure to these toxicants. Although ROS production was elevated in all groups and glutathione response was unaffected, it is unlikely the mild increase is responsible for differences in mitochondrial health. Summation of mitochondrial health using ATP production with cell membrane analysis is the lone parameter where mixtures behaved differently from their parts. Both mixtures distinctly presented as mitochondrial toxins and remodeled metabolism in a manner similar to that found in tumorigenic cells, indicating a potential pathway to carcinogenicity.