Effect of nitrate on human cell lines in culture

Abstract

Nitrate is a ubiquitous drinking water contaminant with potential adverse effects on human health. However, little is known about nitrate toxicity at the cellular and molecular level. The purpose of this study was to examine the effects of environmentally relevant concentrations of nitrate on cytotoxicity and protein expression in human cell lines. To determine if tissue-specific responses occurred, a human hepatoma cell line (HepG2) and a human embryonic kidney cell line (HEK293) were used. Both potassium and ammonium salts of nitrate were used to determine salt-specific toxicity. Test concentrations of nitrate varied from 1 μg/L to 5000 mg/L. Cells were exposed to a nitrate salt for 24, 48, or 72 hours and then examined for effects on viability (using the Neutral Red assay) or proliferation (using the BrdU ELISA assay). To determine the effects of nitrate on protein expression, levels of PCNA, Hsp70, Hsc70, and VEGF protein were monitored using Western blotting in HepG2 and HEK293 cells exposed to KNO₃ or NH₄NO₃ for 24 hours. Nitrate was cytotoxic to both cell types at high concentrations, with EC₅₀ values between 1557 mg/L – (approximately) 5852mg/L for viability, and ~2.5 mg/L – 3631 mg/L for proliferation. Several EC₅₀ values were not calculable based on the available data, but appeared to be far greater than 5000 mg/L. Ammonium nitrate was generally more toxic than potassium nitrate, and increasing exposure time generally resulted in greater toxicity. The HepG2 and HEK293 cells displayed similar responses for most assays, except the 24 hour KNO₃ Neutral Red assay. Here, HEK293 viability increased with increasing KNO₃ concentrations, while HepG2 viability decreased. The reason for this finding is unknown, but may involve cell-specific homeostatic mechanisms. A hormetic-like effect was observed in both cell types in several of the proliferation assays; the biological significance of this effect remains unknown. No significant changes in protein expression were observed under these experimental conditions. Some subtle trends were present, such as a slight increase in Hsp70 expression with increasing nitrate concentration in both cell types. In HepG2 cells, PCNA expression increased slightly with increasing nitrate concentrations; however, the opposite effect was observed in HEK293 cells. This may be due to transcriptional or translational regulation. In summary, environmentally relevant concentrations of nitrate did not appear to evoke significant cytotoxicity or changes in protein expression. Cell viability and proliferation effects were observed at higher concentrations of nitrate. Private water supplies may contain nitrate concentrations above the EC₅₀ values in these experiments. More research is required to determine if this poses a direct threat to human health.