Contribution of reactive oxygen species to para-aminophenol toxicity in LLC-PK1 cells

Abstract

Para-aminophenol (PAP) causes nephrotoxicity by biochemical mechanisms that have not been fully elucidated. PAP can undergo enzymatic or non-enzymatic oxidation to form reactive intermediates. Using modulators of reactive oxygen species (ROS), the role of ROS in PAP toxicity in LLC-PK 1 cells was investigated. ROS formation was determined using a fluorescein derivative and viability using alamarBlue. Following treatment of cells with PAP, ROS formation occurred prior to loss of cell viability. Several modulators of ROS were used to identify the pathways involved in PAP toxicity. Viability was improved with catalase treatment, while viability was decreased when cells were treated with superoxide dismutase (SOD). Both catalase and SOD exert their effects outside of cells in the incubation medium, since there was no evidence of uptake of these enzymes in LLC-PK 1 cells. In cell-free incubations, hydrogen peroxide (H 2 O 2 ) was produced when 0.5 mM PAP was included in the incubation medium. Further, SOD greatly increased and catalase greatly decreased H 2 O 2 production in these cell-free incubations. These data suggest that H 2 O 2 formed in the incubation medium contributes to loss of viability following PAP treatment. When cells were coincubated with 0.5 mM PAP and tiron, pyruvate, bathocuproine, 1, 10-phenanthroline, or dimethylthiourea (DMTU), ROS formation was decreased. However, there was minimal improvement in cell viability. Paradoxically, DMTU exacerbated PAP-induced loss of viability. These data suggest ROS are generated in cells exposed to PAP but these species are not the predominant cause of cellular injury.

4-Amino-2-chlorophenol: Comparative in vitro nephrotoxicity and mechanisms of bioactivation

“…The ability of ascorbate to attenuate 4-A2CP nephrotoxicity in IRCC is not surprising and supports a free radical, and potential oxidative stress component to the mechanism of 4-A2CP cytotoxicity. Similar protection by ascorbate against 4-AP and 4-A2,6DCP nephrotoxicity have also been observed in vivo and in vitro [13,15,16,18,47], and ROS/oxidative stress plays a role in 4-AP and 4-A2,6DCP in vitro nephrotoxicity [18, 25]. The finding that not all antioxidants are protective against 4-A2CP cytotoxicity is also in line with findings using other aminophenol compounds.…”

“…1. Oxidation of the aminophenols to the 1,4-benzoquinoneimine metabolites would be expected to generate reactive oxygen species (ROS) and an arylating metabolite [24,25]. Conjugation of a 1,4-benzoquinoneimine metabolite with glutathione could produce a series of glutathione-derived metabolites that have the potential to redox cycle and generate oxidative stress as well as arylate cellular nucleophiles [45,46].…”

“…Thus, a potential pathway for organ-directed toxicity of aniline compounds could involve formation of aminophenol and aminophenol-derived metabolites. In addition, for 4-AP, oxidative stress appears to play a role in nephrotoxic mechanisms [13,24,25]. However, the ultimate nephrotoxicant metabolite(s), mechanisms of bioactivation, and mechanisms of nephrotoxicity for 4-AP and the phenolic metabolites of the chloroanilines remain to be fully determined.…”

A Cytochrome P450–Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes

“…The possibility of redox cycling of a PAP metabolite was mentioned by Rankin et al (1996), and evidence of lipid peroxidation in rat kidneys and kidney slices has been presented (Kanbak et al, 1996;Harmon et al, 2005Harmon et al, , 2006. In LLCPK1 kidney cells exposed to PAP, generation of reactive oxygen species (ROS) and cytotoxicity occurred; however, ROS scavengers failed to provide protection, suggesting that ROS are not the main cause of PAP-induced cytotoxicity in these cells (Foreman and Tarloff, 2008).…”

Anti-proliferative and pro-apoptotic activities of hydroxytyrosol on different tumour cells: the role of extracellular production of hydrogen peroxide

“…(increasing the pH), which moves the equilibrium to the right. Although the oxidation of catechol can also be due to the transition metals such us Cu and Fe [38] [32,33], myricetin [31] and theaflavin-3 0 3 0 -digallate [39], while it enhances the accumulation of H 2 O 2 induced by para-aminophenol [17] and canned coffee [22]. In this context, it should be underlined that the reactions behind the H 2 O 2 -releasing activity of 3,4-DHPEA may be particularly complex and deeply influenced by the experimental conditions.…”