With ever-increasing production and use of nanoparticles (NPs), there is a necessity to evaluate the probability of consequential adverse effects in individuals exposed to these particles. It is now understood that a proportion of NPs can translocate from primary sites of exposure to a range of secondary organs, with the liver, kidneys and spleen being some of the most important. In this study, we carried out a comprehensive toxicological profiling (inflammation, changes in serum biochemistry, oxidative stress, acute phase response and histopathology) of Ag NP induced adverse effects in the three organs of interest following acute exposure of the materials at identical doses via intravenous (IV), intratracheal (IT) instillation and oral administration. The data clearly demonstrated that bioaccumulation and toxicity of the particles were most significant following the IV route of exposure, followed by IT. However, oral exposure to the NPs did not result in any changes that could be interpreted as toxicity in any of the organs of interest within the confines of this investigation. The finding of this study clearly indicates the importance of the route of exposure in secondary organ hazard assessment for NPs. Finally, we identify Connexin 32 (Cx32) as a novel biomarker of NP-mediated hepatic damage which is quantifiable both (in vitro) and in vivo following exposure of physiologically relevant doses.