Prolonged exposure to atrazine through drinking water facilitates splenic apoptosis and induces exhaustion-like checkpoint upregulation in mice

Abstract

Atrazine (ATR) is a widely used herbicide that persists in aquatic environments, raising concerns regarding its long-term immunotoxicity. As a central immune organ, the spleen represents a potential toxicological target, yet the impact of prolonged ATR ingestion via drinking water on splenic apoptosis and immune exhaustion remains unclear. In this study, male C57BL/6 N mice received ATR in drinking water at 0, 1.5 mg/L or 150 mg/L for 24 or 48 weeks. Spleen index, histopathology, peripheral blood leukocytes, serum cytokines, splenocyte apoptosis, T-cell exhaustion-like phenotype-related markers and RIG-I/MAVS signaling were evaluated. Prolonged ATR exposure increased spleen index in a dose- and time-dependent manner and caused progressive white pulp hyperplasia, disruption of splenic architecture, red pulp congestion and macrophage accumulation. Serum IL-6 and TNF-α levels were elevated, accompanied by reduced lymphocyte counts and increased neutrophils and monocytes in peripheral blood. Splenocyte apoptosis was enhanced, with upregulation of Bax, Casp3 and cleaved caspase-3. In parallel, Pdcd1 (PD-1), Ctla4, Lag3 and Tigit expression and PD-1 protein levels were significantly increased, with PD-1-positive regions overlapping with TUNEL-positive cells. RIG-I and Mavs mRNA and RIG-I protein were also upregulated. These results demonstrate that prolonged ATR exposure via drinking water promotes splenic apoptosis and induces an immune exhaustion-like state in mice, associated with the activation of RIG-I/MAVS-linked apoptotic signaling.