Generation of active oxygen species in vitro by the interaction of potassium bromate with rat kidney cell

Active oxygen species derived from the interaction of potassium bromate (KBrO3), a rat renal carcinogen, with cells from rat kidney and other organs were examined by electron spin resonance spectrometry using the spin trapping agents 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP). DMPO-OH, an indicator of hydroxyl radical production, was generated from KBrO3 by kidney cells or homogenate, but not by liver preparations and to only a limited extent by heart and brain homogenates, suggesting relative kidney specificity. To assess what chemical components are responsible for production of DMPO-OH, several physiologically related materials were examined. Glucose, saccharose, albumin and methyl linolate were found not to be involved in the KBrO3 reaction, but reduced glutathione and also ferric ions participated to produce DMPO-OH. In addition, DMPO-OH production derived from the reaction of KBrO3 with kidney homogenate was not affected by superoxide dismutase, catalase or hydroxyl radical scavengers such as DMSO or ethanol, but was effectively inhibited by singlet oxygen scavengers such as histidine and NaN3, implying singlet oxygen production. To assess this possibility, TEMP was used as a trapping agent, and TEMPO, derived from singlet oxygen, was found to be produced by the reaction of KBrO3 with homogenates of kidney, but not of liver. Furthermore, singlet oxygen production was confirmed by studies of chemiluminescence using 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2a]pyrazine-3-one. As a control, DMPO-OH was also demonstrated to be produced by a known singlet oxygen source, toluidine blue plus light. The results thus indicate that singlet oxygen is a very probably candidate for the active oxygen species generated in the specific interaction of KBrO3 with rat kidney cells in vitro. This raises the question of its concern with renal carcinogenicity in vivo.