Sub-Chronic Methomyl Exposure Induces Oxidative Stress and Inflammatory Responses in Zebrafish with Higher Female Susceptibility

Abstract

The widespread use of carbamate pesticides has raised significant environmental and health concerns, particularly regarding water contamination and the disruption of defense systems in organisms. Despite these concerns, research on the differential impacts of pesticides on male and female organisms remains limited. This study focused on methomyl, investigating sex-specific differences in liver antioxidant defenses and inflammatory response indices in male and female zebrafish after 56 days of exposure to environmentally relevant concentrations (0, 0.05, 0.10, and 0.20 mg/L). Our findings indicate that methomyl exposure significantly increased ROS content in zebrafish livers, inducing oxidative stress and activating enzymatic antioxidant defenses such as SOD, CAT, and GSH-Px activities. Sub-chronic exposure altered the expression of apoptosis-related genes (Bax/Bcl2a and Caspases3a), resulting in liver cell apoptosis in a concentration-dependent manner, with the 0.20 mg/L concentration causing the most severe damage. Additionally, methomyl exposure at environmentally relevant concentrations triggered persistent inflammatory responses in liver tissues, evidenced by increased transcription levels of inflammatory factor genes and the activation of toll-like receptors, heightening susceptibility to exogenous allergens. It is noteworthy that oxidative damage indicators (AST, ROS, MDA) and inflammatory gene expressions (IL-1β, TNF-α) were significantly higher in female livers compared to male livers at 0.10-0.20 mg/L methomyl exposure. Consequently, our study underscores the potential adverse effects of environmental methomyl exposure on aquatic organisms and highlights the need for heightened consideration of the risks posed by environmental endocrine disruptors to female health and safety.

Keywords: inflammation; methomyl; oxidative stress; sex differences; zebrafish.

Figure 1

Apoptosis in the livers of male and female zebrafish after sub-chronic exposure to methomyl. (a) Changes in transcription levels of zebrafish hepatic Bax/Bcl2a and Caspases3a genes compared to the control group (expressed as ratios relative to the levels in the control group). (b) Representative images of TUNEL staining of liver sections from the control group and methomyl-exposed groups under a fluorescence microscope. The green dots represent dead cells, and the blue dots indicate live cells, scale bar: 50 μm. (c) Number of apoptotic cells observed per 1000 cells. Different letters indicate significant differences between concentration groups, p < 0.05.

Figure 2

Changes in hepatic ALT (a), AST (b), and γ-GT (c) activities in zebrafish after sub-chronic exposure to methomyl compared to the control group (expressed as ratios relative to the levels in the control group). Different letters indicate significant differences between concentration groups, p < 0.05.

Figure 3

Changes in zebrafish hepatic CAT (a), GSH-Px (b), SOD (c), MDA (d), and ROS (e) levels after sub-chronic exposure to methomyl compared to the control group (expressed as ratios relative to the levels in the control group). Different letters indicate significant differences between concentration groups, p < 0.05. Asterisks (*) denote significant concentration–sex interaction effects for this concentration group. **: p < 0.01, ***: p < 0.001.

Figure 4

Changes in transcript levels of IL-1β (a), IL6 (b), INF-γ (c), TNF-α (d), Nκap (e), TLR3 (f), and TLR4 (g) genes in zebrafish livers after sub-chronic exposure to methomyl compared to the control group (expressed as ratios relative to the levels in the control group). Different letters indicate significant differences between concentration groups, p < 0.05. Asterisks (*) denote significant concentration–sex interaction effects for this concentration group. **: p < 0.01.

Figure 5

Summary of sub-chronic methomyl exposure in zebrafish livers: sex-susceptible biomarker responses. Key indicators such as AST, ROS, MDA, IL-1β, and TNF-α exhibited heightened sensitivity in females, suggesting that these markers can serve as effective biomarkers for assessing sex differences in response to methomyl exposure. Different letters indicate significant differences between concentration groups, p < 0.05, asterisks (*) denote significant concentration–sex interaction effects for this concentration group. **: p < 0.01, ***: p < 0.001.