PULMONARY AND ASSOCIATED LYMPH NODES ANTHRACOSIS IN HOUSEHOLD DOGS: A SENTINEL FOR ENVIRONMENTAL POLLUTION AND DEGRADATION

Abstract

ABSTRACT Pulmonary anthracosis, the accumulation of carbon particles in the lungs, constitutes a risk factor for cancer. Despite its rising occurrence in stray dogs, there is paucity of information on pulmonary and nodal anthracosis in household dogs and its effects on lung macrophages, bronchiolar epithelial lining, and lymphoid follicles. This study was conducted to investigate the prevalence of pulmonary and lymph node (LN) anthracosis and associated biochemical, pathological and immunohistochemical changes in household dogs in Ogun, Oyo, and Lagos states of Nigeria. The prevalence of pulmonary anthracosis was determined between 2011 and 2020, using post mortem records of selected Veterinary Hospitals. The gross and histopathological changes in lungs and LNs were examined. The levels of some trace metals in freshly collected lung tissues and associated LNs at post-mortem were determined using Atomic Absorption Spectroscopy. Pro-inflammatory (TNF-α, IL-10, TGF-β1, and Gal-9), pro-apoptotic (TP53, Caspase-3), proliferative (Ki-67) and oxidative biomarkers (Nrf-2 and p38) were determined immunohistochemically. RNA Scope in situ hybridization (ISH) was used to determine Ki-69 mRNA, while immunofluorescence was used to determine the DNA (BPDE-DNA) adducts in pulmonary epithelial cells and macrophages, and nodal lymphoid cells. Transmission Electron Microscope (TEM) was used to study the ultrastructural changes. Data were analyzed using Chi-square, one-way ANOVA, and independent t-test considering p < 0.05 as significant. The mean values obtained from immunohistochemical staining intensity were calculated using Image J software and percentage of histopathological changes were calculated. Of the 472 lung tissues examined, 150 (31.8%) were positive for pulmonary anthracosis. Anthracosis was not significantly associated with breed (p = 0.95) or sex (p = 0.98), but age (p = 0.01), with adult dogs being the most prevalent. Significantly higher concentrations of Chromium (p = 0.025), Manganese (p = 0.038), Magnesium (p = 0.038), and Sodium (p = 0.025) were found in anthracotic lungs and LNs compared to non-anthracotic tissues. Histopathologically, bronchiolar smooth muscle hypertrophy (135/150, 90%), severe fibrosis (120/150, 80%), syncytial giant cell formation (75/150, 50%), and accumulation of anthracotic bodies were observed in lungs. Additionally, the LN showed severe effacement of the lymphoid follicles along with the accumulation of anthracotic bodies. The mean values for staining intensity of pro-inflammatory biomarkers (TNF-α (p = 0.001), IL-10 (p = 0.01), TGF-β1 (p = 0.001), Gal-9 (p = 0.01)) were significantly higher in anthracotic lungs and LNs. Anthracotic tissues showed significantly higher mean staining intensity for pro-apoptotic (Caspase-3 (p = 0.038), TP53(p = 0.001)) and proliferative (Ki-67(p = 0.001)) biomarkers. IHC (p = 0.001) showed greater sensitivity than ISH (p = 0.01) for Ki-67 detection in anthracotic and non-anthracotic tissues. Oxidative biomarkers Nrf-2 and p38 were significantly higher in anthracotic tissues (p = 0.001). A significant increase (p = 0.012) in BPDE-DNA adducts was observed in anthracotic lungs and LNs. The TEM revealed nuclear damage, evidenced by marked indentations and infolding nuclear membrane. In conclusion, the prevalence of pulmonary anthracosis in household dogs was high. The pathological changes highlighted the adverse effects associated with the condition and the potential for neoplastic changes in affected organs.