In Australia, heavy use of the herbicide atrazine has led to widespread environmental contamination(1). The reproductive health impacts of long-term low-level exposures to this environmental toxicant are unknown. We have demonstrated that chronic multi-generational exposure of female mice to environmentally relevant levels of atrazine increases intra-ovarian oxidative damage and induces follicle death. To further understand the mechanisms of atrazine-mediated ovarian damage, granulosa-like KGN cells were exposed to varying concentrations of atrazine or metabolites in vitro and cell viability and ROS assessed.
Interestingly, acute exposure to ATZ (0.1-100uM) or metabolites for 4 hours reduced ROS levels compared to untreated cells. Cells were subsequently treated with a combination of tert-Butyl hydroperoxide (TBHP; ROS inducer) and ATZ (0.1-500uM) for 4 hours. At low concentrations (0.1-0.5uM), ATZ mitigated the impact of TBHP, whereas at moderate ATZ concentrations (1-100uM). ROS levels were similar to the ROS inducer alone, whilst at high ATZ concentrations (500uM) ROS levels surpassed the ROS treated control (ATZ+TBHP mean=3.17±1.74, TBHP mean= 1.67±0.72; t-test p=0.007).
To evaluate the effect of chronic exposure, cells were exposed to ATZ (0.001nM, 0.1nM or 10uM) for 3-14 days. ROS levels were significantly elevated after chronic exposure relative to the short-term acute exposure. Moreover, ROS levels accumulated with the duration of time the cells were exposed to ATZ (ATZ 3 days=-0.365±0.027, ATZ 14D=-0.167±0.0263, t-test P<0.05).
Collectively, these data suggest that at very low concentrations, ATZ initially functions as an antioxidant, while prolonged exposure lead to accumulation of ROS and oxidative damage. This study highlights that environmental toxicants can act in a non-canonical manner with different impacts depending on the concentration and duration of exposure. Understanding the mechanism of damage caused by pervasive environmental toxicants, like atrazine, is crucial to understand and identify potential targets to mitigate and prevent disorders and diseases in current and future generations.