An investigation into the contribution of flavin containing monooxygenases to the development and prevention of thiourea-induced pulmonary toxicity in the rat

NR678, a small thiourea-based rodenticide candidate, is lethal to rats when administered orally at relatively small doses (5 and 10 mg/kg) and causes rapid life-threatening respiratory impairment, characterised by severe hydrothorax and pulmonary oedema. However, rats are protected from a normally lethal dose of NR678, after prior exposure to a low dose (0.5 mg/kg). Similar to other molecules containing the thiourea moiety, NR678 is mainly metabolised by flavin containing monooxygenases (FMOs), a class of enzymes involved in the detoxification, or more rarely, bioactivation of N- or S-containing molecules during phase I metabolism. Rat FMO2, the main FMO expressed in the lungs, like the correspondent human isoform, exhibits a genetic polymorphism which may influence the responses of this organ to NR678 and other drugs metabolised by FMOs. The aims of this thesis were to assess the morphological and functional aspects of NR678-induced acute lung injury (ALI) and to investigate the role of the adaptive pulmonary defence response involved in the prevention of the oxidative injury. In addition, the wild rat, which, in contrast to the laboratory rat, possesses a functional pulmonary FMO2, was explored as a possible animal model to evaluate the metabolic and toxicologic consequences of FMO2 polymorphism in humans. It was shown that pulmonary endothelial cells are the main target of acute NR678 oxidative injury and exhibit ultrastructural alterations, which, coupled with a marked depletion of glutathione (GSH) levels, are likely responsible for the perturbation of pulmonary vascular permeability. The lungs of tolerant animals showed mild and transient changes of the vascular permeability and mildly increased cellularity in the lungs, which was characterised by increased numbers of alveolar macrophages and immature pneumocytes within 24 h after dosing, followed by a rise in the number of mature type II pneumocytes one week later. The microscopic changes resolved by the end of the second week, when rats were found to be again susceptible to a lethal dose of NR678. It was speculated that the adaptive response of the lungs to NR678 could be related to irreversible inhibition of FMOs, rather than increased clearance of the oedema fluid by macrophages and type II pneumocytes, down-regulation of FMOs, evaluated by in situ hybridisation and qPCR, or decreased GSH levels. In addition, it was found that the presence of an active FMO2 isoform enhances pulmonary FMO catalytic activity and leads to increased susceptibility to the toxic effects of NR678. In conclusion, the work presented here contributes evidence that FMO2 polymorphism may be relevant to humans and provides an animal model which can be used to study its implications. NR678 represents an interesting and unique approach to investigate the pathogenesis of ALI and, at the same time, understanding the defence mechanisms involved in adaptation may bring new insight into potential therapeutic strategies of lung diseases