Formaldehyde Damages PC12Cells Via Inhibition Of Endogenous Hydrogen Sulfide Generation By Activating Nitric Oxide Synthase

【Backgroud and Objective】Formaldehyde (FA), the simplest chemical structure in aldehydes molecules, can causenerotoxcity by elevating intracellular reactive oxygen species (ROS) levels and oxidativestress. Increasing evendence has shown that hydrogen sulfide (H2S), the third endogenous gassignaling molecule, has the neuroprotective and antioxidant effects. Thus, we suggested thatformaldehyde induces neurotoxicity and oxidative stress through inhibiting the generation ofendogenous H2S.Inaddition, it has been confirmed that excessive nitric oxide (NO) generation in thecentral nervous system inhibits the activity of cystathionine-Bata-synthase (CBS), which isthe key enzyme to build H2S in central nervous system. Therefore, we suggested thatformaldehyde inhibits endogenous production of H2S by promoting NO excessive generation.So that, in this work, we will use formaldehyde-induced damage in PC12cells as themolde of formaldehyde neurotixcity to study whether formaldehyde inhibits endogenousproduction of H2S by promoting excessive NO generation.【Methods】We used PC12cells exposed to formaldehyde as the cell model of formaldehydeneurotoxicity, the viability of PC12cells was observed by the trypan blue dye exclusion assay,MTT assay, and Cell Counting Kit-8. The apoptosis of PC12cells were detected by Hoechst33258staining and flow cytometry after PI staining. The level of intracellular ROS in PC12cells was dertmined using fluorescence microscopy after DCFH-DA staining. The production of H2S and the activity of CBS in PC12cells were detected by methylene bluespectrophotometric method and the expression of CBS in PC12cells was determined byWestern Blot. RNA interference was used to inhibit the expression of CBS. NOS activity andNO contents were evaluated using NOS and NO kit, respectively.【Results】1. Formaldehyde inhibits the viability and endogenous H2S generation in PC12cellsTreatment of PC12with FA (60,120,240μmol/L) for24h inhibited the viability ofPC12cells and decreased the content of H2S in cell culture medium in PC12cells in aconcentration-dependent manner. FA, at the concentrations of120and240μmol/L,down-regulated the expression of CBS protein and inhibted the activity of CBS. These dataimply that the neurotoxicity of FA is associated with inhibition of endogenous H2S generation,resulting from down-regulation of the expression and activity of CBS.2. Inhibiton of endogenous H2S by CBS-siRNA deteriorates formaldehyde-inducedtoxicity to PC12cellCBS in PC12cells was silenced by CBS-siRNA using RNA interference (RNAi)technology and endogenous H2S generation was inhibited by transfection with CBS-siRNA.Pretreatment with CBS-siRNA aggravates the inhibition of viability and H2S generation,apoptosis, and accumulation of ROS in PC12cells induced by formaldehyde (120μmol/L) for24h. These results suggest that inhibition of H2S generation by CBS-siRNA deterioratesformaldehyde neurotoxicity.3. Formaldehyde inhibits endogenous H2S generation in PC12cells by promoting theoverproduction of NO.Treatment of PC12cells with FA (120and240μmol/L, for24h) significantly increasedthe activation of NOS as well as the content of NO in cell culture medium in PC12cells,respectively. Pretreatment with160μmol/L of ADMA, an endogenous NOS inhibitor, for30min markedly reversed FA (120μmol/L, for24h)-induced increase in the activity of NOS andthe generation of NO in PC12cells. These findings suggested that formaldehyde inhibits endogenous H2S generation by excessive activation of NOS and overproduction of NO.4. Inhibition of NO generation prevents the neurotoxicity of formaldehydePretreatment with160μmol/L of ADMA, an endogenous NOS inhibitor, for30minmarkedly prevented the inhibition of cell viability, the induction of apoptosis, and theaccumulation of ROS in PC12cells caused by FA (120μmol/L, for24h). These datasuggested that inhibition of NO generation reversed formaldehyde-induced neurotoxicity toPC12cells.【Conclusion】1. The neurotoxicity of formaldehyde is asscociated with the inhibited endogenous H2Sgeneration.2. Formaldehyde inhibits the generation of endogenous H2S through excessive NOS activityand overproduction of NO.