SIN-1 cytotoxicity to PC12 cells is mediated by thiol-sensitive short-lived substances generated through SIN-1 decomposition in culture medium

Kanako Konishi, Nobuo Watanabe, Takao Arai

Research output: Contribution to journalArticlepeer-review

22 Citations (Scopus)

Abstract

As a generator of peroxynitrite (ONOO-), 3-morpholinosydnonimine (SIN-1) is widely used in the study of oxidative/nitrosative stress in cultured cells, although controversy exists regarding active species responsible for cytotoxicity. In this study, we report that unstable thiol-sensitive substances, generated from the reaction of SIN-1 with components in culture medium, play a crucial role in SIN-1 cytotoxicity in PC12 cells. Exposure of cells to culture medium obtained after almost complete SIN-1 decomposition at 37 °C for 2 h demonstrated almost the same degree of cytotoxicity as did fresh SIN-1. The cytotoxicity of SIN-1-decomposed medium largely depended on serum, decayed with time, and could be completely abolished by the addition of thiols. Degradation of synthetic ONOO- in the culture medium did not reproduce the unstable cytotoxicity. The presence of superoxide dismutase (SOD) during SIN-1 decomposition prevented the formation of the cytotoxic substances, whereas SOD had no protection against the cytotoxicity itself, suggesting a crucial role of simultaneously generated superoxide and nitric oxide in the formation of the toxicants, but not in their cytotoxic action. The cytotoxicity of fresh SIN-1 is dramatically suppressed in a basal medium (Hanks balanced salt), suggesting that the cytotoxicity of fresh SIN-1 also requires components of culture medium. These results suggest that SIN-1 cytotoxicity in PC12 cells is mediated via the generation of cytotoxic substances in the medium during its decomposition.

Original languageEnglish
Pages (from-to)270-278
Number of pages9
JournalNitric Oxide - Biology and Chemistry
Volume20
Issue number4
DOIs
Publication statusPublished - 2009 Jun 1

Keywords

  • NO donor
  • Nitrosation
  • Oxidative/nitrosative stress
  • Peroxynitrite

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Clinical Biochemistry
  • Cancer Research

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