Synergistic cytotoxic effect of tetrachlorocatechol and sodium azide in Escherichia coli: Toxicity, metabolism, and mechanistic aspects

Abstract

Pentachlorophenol (PCP) is used in industrial and domestic applications, including as a biocide and a wood preservative. Metabolism of PCP undergoes oxidative dechlorination, forming tetrachlorocatechol (TCC) and tetrachlorohydroquinone (TCHQ). Both sodium azide (NaN(3)) and TCC appear naturally in soil. None of them are cytotoxic by themselves or facilitate autooxidation. Here, we show that their combination leads to synergistic cytotoxicity (>6 log bacterial killing) to Escherichia coli. The rate of oxygen consumption in a cell-free system showed that NaN(3) increases TCC oxidation by 520-fold. The synergism coefficient to cells was calculated as 96 or greater, and we have shown the formation of a new compound. It is suggested that the intermediate species, o-tetrachlorosemiquinine, and an unknown, nitrogen-centered free radical, both visualized by electron-spin resonance, are harmful species responsible for the synergistic cytotoxicity of TCC/NaN(3), rather than the endproduct formed during the reaction. Desferrioxamine and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide offered nearly complete protection, but through radical scavenging rather than through chelating properties. The mechanism of damage for TCC compared to its analogue, TCHQ, were investigated, and whereas the cellular damage of TCHQ/NaN(3) is through a site-specific mechanism, in the case of TCC/NaN(3) it is through the accumulation of the component(s) in the bacterial cell membrane, eventually leading to dysfunction, as evidenced by electron microscopy.

Detoxifying Polyhalogenated Catechols through a Copper‐Chelating Agent by Forming Stable and Redox‐Inactive Hydrogen‐Bonded Complexes with an Unusual Perpendicular Structure

“…Our previous work showed that the reaction between TCC and NaN 3 was accompanied with enhanced oxygen consumption (from 100 to 60 %, Figure 3 A: Control) and the formation of O-tetrachlorosemiquinone anion radical (O-TCSQC À ) and other semiquinone radicals (SQC À ; Figure 3 C), which were considered to be the reactive and toxic species responsible for cell death. [15] We found that both the oxygen consumption and semiquinone radicals generation (as monitored by direct ESR) was inhibited by 2,9-Me 2 OP in a dose-dependent manner. When the molecular ratio of [2,9-Me 2 OP]/[TCC] = 1:1, no oxygen consumption was observed and the semiquinone radicals relative value was reduced to the base line, indicating that the reaction between TCC and NaN 3 was completely inhibited by 2,9-Me 2 OP (Figure 3 A and D).…”

“…− ) and other semiquinone radicals (SQ . − ; Figure 3 C), which were considered to be the reactive and toxic species responsible for cell death 15. We found that both the oxygen consumption and semiquinone radicals generation (as monitored by direct ESR) was inhibited by 2,9‐Me 2 OP in a dose‐dependent manner.…”

“…We found recently that the combination of tetrachlorocatechol (TCC) and sodium azide (NaN 3 ) caused a pronounced synergistic cytotoxicity in a bacterial model 14, 15. During our study on the role of transition‐metal ions, especially Cu and Fe, we found, unexpectedly, that only 2,9‐Me 2 OP, but not any other well‐known metal chelating agents, markedly inhibited the synergistic toxicity induced by TCC/NaN 3 .…”

“…These results are in good agreement with our previous finding in the bacterial system. [15] Based on previous studies, [15] we proposed that for TCC to be oxidized to generate the reactive and toxic semiquinone radicals, it is necessary that the hydroxyl group of TCC be first deprotonated to form its anionic form TCC À (Scheme 1).…”

“…[4] We found recently that the combination of tetrachlorocatechol (TCC) and sodium azide (NaN 3 ) caused a pronounced synergistic cytotoxicity in a bacterial model. [14,15] During our study on the role of transition-metal ions, especially Cu and Fe, we found, unexpectedly, that only 2,9-Me 2 OP, but not any other well-known metal chelating agents, markedly inhibited the synergistic toxicity induced by TCC/NaN 3 . Further studies indicate that the protection by 2,9-Me 2 OP may not be due to its chelation of copper, but possibly due to the formation of an unknown supramolecular complex with TCC and/or NaN 3 .…”