Hardwick, Simon J.; (1990) The role of glutathione in the biochemical mechanism of paraquat toxicity and of other toxicants in the lung. Doctoral thesis (Ph.D.), University College London.

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Abstract

ABSTRACT ................. Paraquat and nitrofurantoin are proposed to exert their effects by redox cycling and oxidative stress. The aim of the studies presented in this thesis was to clarify the role of glutathione status in the toxicity of these agents. Mice and rats were treated with the glutathione reductase inhibitor 1,3 -bis (2-chloroethyl)-l-nitrosourea (BCNU). The lethality of paraquat was potentiated in both experimental species. BCNU was then employed in studies to develop a novel rat lung slice model with a compromised glutathione reductase activity but normal levels of ATP, NADP(H) and glucose oxidation along with a modest reduction in thiol levels in BCNU pretreated slices. The effects of paraquat, nitrofurantoin and 2, 3-dimethoxy-l, 4- naphthoquinone (a control compound, a 'pure' redox cycler) were assessed in the BCNU compromised model. From these studies it was concluded that, on the basis of biochemical (GSH and ATP levels) and functional assessments (oligoamine and 5-hydroxytryptamine uptake) of toxicity, the effects of paraquat, nitrofurantoin and the quinone were potentiated as a result of inhibition of glutathione reductase, thereby implicating GSH in the toxicity. In addition, it was concluded that paraquat selectively affected the alveolar type I and II cells, whereas nitrofurantoin and the quinone exerted effects throughout the lung slice. Furthermore, it was considered on the basis of these studies that much of the pulmonary GSH is in cell types distinct from the alveolar epithelium. The studies highlighted the insights which can be gained from use of theappropriate functional marker and underline the problems faced when investigating biochemical mechanisms of toxicity in a heterogeneous organ such as the lung. Adenosine uptake into rat lung slices was studied in an attempt to develop a method for assessing endothelial function, as an alternative to 5-hydroxytryptamine accumulation. Studies revealed that o<.-naphthylthiourea (ANTU), an endothelial damaging agent, reduced adenosine uptake, whereas paraquat failed to produce a loss of adenosine uptake, suggesting that adenosine uptake was specific for the endothelium. Studies of adenosine uptake into BCNU compromised lung slices confirmed previous studies with regard to the selectivity of paraquat and the quinone. Furthermore, incubation of lung slices with adenosine resulted in an elevation of pulmonary ATP levels, which may provide another exciting tool for toxicological studies, in vitro. In an attempt to raise pulmonary thiols, animals were treated with phorone which resulted in a significant elevation of GSH in lung and liver of rat and mouse after 48 h. Phorone pretreatment did not confer in vivo or inprotection against paraquat-induced lethality in mouse rat jin vivo or in vitro. Elevation of thiols did, however, confer protection against the endothelial damaging agent ANTU as evidenced by the abolition of mortality, hydrothorax and loss of functional markers. It wasj concluded that the lack of protection against paraquat may arise from a failure to elevate thiols in the cell types which constitute a target for paraquat. These studies implicate GSH in the toxicity of these agents whichexert their effects by oxidative stress and suggest that type I and II cells contain relatively small amounts of GSH. Furthermore, the work has established novel methods for investigating mechanisms of pulmonary toxicity.

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