Subchronic γ-hexachlorocyclohexane exposure is associated with altered TLR4-linked pulmonary immune responses following endotoxin challenge: Insights from a two-hit mouse model

Abstract

γ-Hexachlorocyclohexane (γ-HCH) is a persistent organochlorine pesticide widely used in agriculture and public health. While its neurotoxicity in humans and animals is well documented, its toxic effects on non-target organs, particularly the lungs, remain poorly understood. In the general population, diet is the primary route of γ-HCH exposure; however, the long-term effects of dietary exposure on pulmonary immunity remain poorly understood. This study employed a two-hit murine model to determine whether prolonged oral exposure of γ-HCH alters pulmonary immune responses to a secondary inflammatory challenge. Swiss albino male mice were administered γ-HCH orally (0.12 mg/kg body weight) for 60 or 90 days, followed by an acute intranasal lipopolysaccharide (LPS) challenge at the respective terminal time points. Systemic and local inflammatory responses were assessed by quantifying cytokine levels (IL-1β and TNFα) in serum and bronchoalveolar lavage fluid using ELISA. Lung tissues were assessed for TLR4 and TNFα expression by immunohistochemistry and examined ultrastructurally by transmission electron microscopy. Subchronic γ-HCH exposure led to a significant increase in IL-1β and TNFα levels, accompanied by robust expression of these markers in pneumocytes, alveolar epithelial cells, and vascular endothelium. Upon LPS challenge, γ-HCH-treated mice showed exacerbated pulmonary inflammation with histopathological changes. Interestingly, the inflammatory response in γ-HCH + LPS groups was not consistently greater than that in the LPS-only group; in some cases, cytokine levels were moderately reduced. These findings suggest that subchronic dietary γ-HCH exposure primes the immune system and alters pulmonary inflammatory responsiveness to a subsequent endotoxin challenge. This pattern is consistent with immune modulation rather than amplified protective immunity. While the observed responses are associated with activation of innate immune signaling pathways, including TLR4-linked mechanisms, further functional studies are required to establish causal relationships. Overall, this study underscores the importance of cumulative and sequential environmental exposures when assessing immunotoxic potential of persistent organic pollutants.