Gulf War Illness‐related Chemicals Increase Hepatic Steatosis and Enhance Chronic Ethanol‐induced Liver Damage in Mice

Abstract

Gulf War illness (GWI) affected military veterans of the 1990-1991 Persian Gulf War. GWI symptoms include systemic inflammation, chronic fatigue, fibromyalgia, and memory deficits. Studies on animal models revealed that insecticides (permethrin, DEET) and nerve gas prophylactics (pyridostigmine-bromide, PB) induce symptoms similar to GWI. Previously we showed that rats’ exposure to GWI chemicals enhanced biliary hyperplasia and hepatic fibrosis after BDL-induced cholestasis which was associated with increased Kupffer cells/monocyte-derived macrophages (KC/MoMF). We further investigate the influence of GWI chemicals on liver function in a mouse model of chronic alcohol consumption and assess the role of hepatic inflammation in this process. Male C57BL/6 mice were injected with PB and permethrin (GWI mice) or vehicle (naïve mice) daily for 10 days and allowed to recover for 4 months. Then, GWI and naïve mice were treated with either vehicle or Pexidartinib (PLX3397) to reduce hepatic macrophage infiltration, followed by a chronic-plus-single-binge ethanol (EtOH) feeding challenge. Serum biochemistry and liver histopathology were assessed using ELISA, H&E, and IHC. Oil Red O was used for lipid droplet (LD) detection. Reactive oxygen species (ROS) were assayed using a commercial kit. Gene markers for mitochondrial fatty acid oxidation (FAO) and steatosis were assessed by RT-qPCR array. Genes of fibrosis (TGFb, CTGF, aSMA, Col1A1) and inflammation (IL-1b, IL-6, CCL2, TNFa) were assayed by RT-qPCR. GWI chemicals induced hepatic microvesicular steatosis and increased KC (CD68+/Clec4f+) and MoMF (CD68+/CD11b+) in the liver. GWI exposure resulted in increased ROS and impaired FAO coupled with LD formation. Mitochondrial FAO genes including PPARa, CPT1A, CPT2 were downregulated while genes of LD formation, e.g. PLIN1-3, were upregulated by GWI chemicals. Upon treatment with chronic-EtOH-plus-binge feeding, mice exposed to GWI chemicals had increased levels of serum AST, ALT, and increased fibrosis markers compared to EtOH-mice without GWI treatment. Macrophage markers CD68, Clec4f, and CD11b/c that were slightly augmented by the GWI chemicals, were significantly enhanced in GWI+EtOH compared to EtOH mice. PLX3397 treatment reduced KC/MoMF infiltration induced by GWI chemicals and attenuated the GWI-induced increase of hepatic serum markers and steatosis observed after EtOH exposure. These data suggest that exposure to GWI chemicals causes long- term changes in liver pathology, resulting in low level inflammation, oxidative stress and microvesicular steatosis. These GWI-induced changes contribute to increased susceptibility to alcohol-induced liver damage that is driven, at least in part, by an increased macrophage infiltration.