(337) Understanding Mechanisms of Atrazine Induced Damage in the Ovary

Ngoc T. T. Nguyen¹, Zaahida Abdul-Jalil¹, Bethany Finger², Qiaochu Wang¹, Xuebai Cai¹, Nadeen Zerafa¹, Lauren R. Alesi¹, Yujie Cao¹, Simon Chu³, Jodi Flaws⁴, Amy L. Winship¹, Mark Green², Jessica M. Stringer¹, Karla J. Hutt¹

1. Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Melbourne, Australia


2. School of BioSciences, University of Melbourne, Melbourne, Australia


3. Centre for Endocrinology and Metabolism, Hudson Institute of Medical Research, Melbourne, Australia


4. College of Veterinary Medicine, University of Illinois (Urbana-Champaign), Illinois, USA

In the United States and Australia, heavy use of the herbicide atrazine has led to its widespread accumulation in the environment, leading to the contamination of waterways and reservoirs used for drinking and irrigation. Consequently, humans and animals may be exposed to small amounts of atrazine, for their entire lives. Concerningly however, the reproductive health impacts of long-term low-level exposures to atrazine on females, are unknown. To gain some insight into this issue, female C57BL/6 mice were continuously exposed to environmentally relevant concentrations of atrazine (0.02 ppb), via drinking water, for 3 generations. From the second generation onwards, there was a significant decrease in the number of primordial follicles and an increase in atretic antral follicles in the ovaries of atrazine-exposed mice compared to vehicle controls at 3 or 6 months of age. Interestingly, although atrazine is a proposed endocrine disruptor, no differences in serum oestradiol or testosterone levels were observed between exposed and control mice. However, the percentage of follicles positively stained for markers of oxidative stress-induced damage in DNA (8-Oxo-2'-deoxyguanosine), protein (nitrotyrosine), and lipids (4-hydroxynonenal), were significantly increased in atrazine-exposed mice in generation 2 and 3, compared to controls. The mRNA levels for antioxidant genes Cat, Sod2 and Gpx3 were also altered in the ovaries of atrazine-exposed mice compared to controls. These data suggest that continuous multi-generational exposure of mice to low environmental levels may damage the ovary through the induction of intra-ovarian oxidative stress. To further understand the mechanisms of atrazine-mediated ovarian damage, granulosa-like KGN cells were exposed to varying concentrations of atrazine in vitro and reactive oxygen species within cells were measured using a DCFDA ROS assay. Unexpectedly, acute (4-hours) atrazine exposure (0.1-100 μM) reduced reactive oxygen species levels compared to untreated cells. Cells were subsequently treated with a combination of tert-Butyl hydroperoxide (oxidative stress inducer) and atrazine (0.1-100 μM), under the same conditions. At all concentrations, atrazine mitigated the impact of the oxidative stress inducer. Using a Seahorse XF cell mitochondrial stress test, it was found that the mitochondrial health of acutely exposed cells was unchanged when compared to controls. These data suggest that atrazine behaves as an antioxidant acutely. To evaluate the effect of chronic atrazine exposure, cells were exposed to atrazine (0.001nM, 0.1nM) for 3-28 days and analysed as described above. Oxidative stress levels were significantly elevated after long-term atrazine exposure relative to the short-term acute exposure, and reactive oxygen species accumulated with the duration of exposure time. When treated with tert-Butyl hydroperoxide, cells exposed to atrazine for longer had significantly more ROS accumulation, relative to cells exposed for less. The Seahorse mitochondrial stress test revealed that the mitochondrial function of cells exposed for 28 days was significantly altered, with increases in basal respiration, proton leakage, oxygen consumption and ATP production, compared to unexposed controls. Taken together, these data suggest that atrazine's effects on granulosa cells depend on both concentration and exposure duration, with chronic exposure—but not acute exposure—leading to oxidative stress. Overall, this study suggests that chronic multigenerational exposure to environmentally relevant concentrations of atrazine depletes primordial and increases follicle atresia, potentially through oxidative stress. Understanding the mechanisms of damage caused by pervasive environmental toxicants like atrazine is crucial for identifying targets to mitigate reproductive disorders in current and future generations.