Study of intracellular anabolism of 5-fluorouracil and incorporation in nucleic acids based on an LC-HRMS method.

5-Fluorouracil (5-FU) is an anticancer drug extensively used for different cancers. Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on multiple cellular targets such as enzymes, DNA and RNA. In this paper, we describe the development of a method based on liquid chromatography coupled with high resolution mass spectrometry suitable for the simultaneous determination of the ten anabolic metabolites (nucleoside, nucleotide and sugar nucleotide) of 5-FU. The chromatographic separation was optimized on a porous graphitic carbon column allowing the analysis of the metabolites of 5-FU as well as endogenous nucleotides. The detection was performed on an Orbitrap® tandem mass spectrometer. Linearity of the method was verified in intracellular content and in RNA extracts. The limit of detection was equal to 12 pg injected on column for nucleoside metabolites of 5-FU and 150 pg injected on column for mono- and tri-phosphate nucleotide metabolites. Matrix effect was evaluated in cellular contents, DNA and RNA extracts for nucleoside and nucleotides metabolites. The method was successfully applied to i) measure the proportion of each anabolic metabolite of 5-FU in cellular contents, ii) follow the consequence of inhibition of enzymes on the endogenous nucleotide pools, iii) study the incorporation of metabolites of 5-FU into RNA and DNA, and iv) to determine the incorporation rate of 5-FUrd into 18 S and 28 S sub-units of rRNA.

Study of intracellular anabolism of 5-fluorouracil and incorporation in nucleic acids based on an LC-HRMS method / 药物分析学报

5-Fluorouracil(5-FU)is an anticancer drug extensively used for different cancers.Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on multiple cellular targets such as enzymes,DNA and RNA.In this paper,we describe the development of a method based on liquid chromatography coupled with high resolution mass spectrometry suitable for the simultaneous determination of the ten anabolic metabolites(nucleoside,nucleotide and sugar nucleotide)of 5-FU.The chromatographic separation was optimized on a porous graphitic carbon column allowing the analysis of the metabolites of 5-FU as well as endogenous nucleotides.The detection was performed on an Orbitrap? tandem mass spectrometer.Linearity of the method was verified in intra-cellular content and in RNA extracts.The limit of detection was equal to 12 pg injected on column for nucleoside metabolites of 5-FU and 150 pg injected on column for mono-and tri-phosphate nucleotide metabolites.Matrix effect was evaluated in cellular contents,DNA and RNA extracts for nucleoside and nucleotides metabolites.The method was successfully applied to i)measure the proportion of each anabolic metabolite of 5-FU in cellular contents,ii)follow the consequence of inhibition of enzymes on the endogenous nucleotide pools,iii)study the incorporation of metabolites of 5-FU into RNA and DNA,and iv)to determine the incorporation rate of 5-FUrd into 18 S and 28 S sub-units of rRNA.

Structure-activity relationship study: Mechanism of cyto-genotoxicity of Nitropyrazole-derived high energy density materials family.

Stringent toxicological tests have to be performed prior to the industrial development of alternative chemicals particularly high energy dense materials (HEDMs) such as explosives. The properties (e.g., power, stability) of these compounds are constantly being improved, the current axis of research being the nitration of nitrogen heterocycles leading to HEDMs such as nitropyrazole-derived molecules. However, except for 3,4,5-trinitropyrazole (3,4,5-TNP), which was shown to be highly toxic in mice, the toxicological impact of these HEDMs has so far not been investigated. Furthermore, as industrials are strongly advised to develop alternative safety testing assays to in vivo experiments, we herein focused on determining the cytotoxic and genotoxic effects of seven Nitropyrazole-derived HEDMs on three rodent cell lines (mouse embryonic BALB/3T3 clone A31 cells, Chinese hamster ovary cells CHO-K1 and mouse lymphoma L5178Y TK +/- clone (3.7.2C) cells), two human fibroblast lines (CRC05, PFS04062) and on the human hepatic HepaRG model (both in proliferative and differentiated cells). A stronger cytotoxic effect was observed for 1,3-dinitropyrazole (1, 3-DNP) and 3,4,5-TNP in all cell lines, though differentiated HepaRG cells clearly displayed fewer likely due to the metabolism and elimination of these molecules by their functional biotransformation pathways. At the mechanistic level, the sub-chronic cytotoxic and genotoxic effects were linked to ROS/RNS production (experimental assays), HA2.X and to transcriptomic data highlighting the increase in DNA repair mechanisms.