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.

Toxicokinetics and tolerance of a high energy material 3,4,5-trinitropyrazole (TNP) in mice.

The high-energy compound 3,4,5-trinitropyrazole (TNP) was developed as an alternative to other less energetic and more sensitive explosive materials, in particular 1-methyl-2,4,6-trinitrobenzene (TNT). However, the level of toxicity of TNP remains understudied. Here using an in vivo CD1 mouse model, we mimicked an acute exposure (24 h) to TNP, given either orally or intravenously, and determined the maximum administrable doses (190 mg/kg and 11 mg/kg, respectively), as well as the lethal dose for 50% (LD50) of female or male mice (390 mg/kg for both) treated intravenously with TNP alone. Several metabolites including nitroso-dinitro-pyrazole, hydroxylamino-dinitro-pyrazole, hydroxyl-dinitro-pyrazole and amino-dinitro-pyrazole were identified in urine. TNP is quickly metabolized and eliminated via urine as two main amino-dinitro-pyrazole metabolites. A comparison of the transcriptomic effects of TNP and TNT after 10 days exposure enabled us to demonstrate no major induction of transcripts involved both in cell death mechanisms (apoptosis, necrosis, autophagy) and physiological pathways (glycolysis, ATP production). Finally, subchronic exposure to TNP was replicated in female mice, fed 16.8-52.8 mg/kg/day of TNP for one month, to study the impact on cellular functions. Although blood TNP levels remained high, a lower rate of TNP accumulation in the liver and lungs were observed than during an acute exposure. Conversely, cellular stress functions explored using the RT2 Profiler™ PCR Array Mouse Molecular Toxicology PathwayFinder remained unaltered after this chronic exposure. These findings demonstrate that TNP can be rapidly eliminated in vivo without accumulating in vital organs.

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.

Impact of interleukin-6 on drug transporters and permeability in the hCMEC/D3 blood-brain barrier model.

The blood-brain barrier (BBB) is a highly selective membrane composed predominantly of brain capillary endothelial cells expressing drug efflux transporters that prevent substrates from accessing the brain. Inflammation is associated with central nervous system diseases and can impair BBB permeability via several mechanisms, including altered transporter and cell junction expression. This can modify the brain's exposure to drugs. However, comprehensive genomic analysis of the impact of interleukin (IL)-6, which plays a key role in the inflammatory response, on the BBB is lacking. In the present study, we analyzed the effects of exposure of hCMEC/D3 cells to 20 ng/mL IL-6 for 72 h. We performed RNA sequencing and ABC transporter efflux assays. Physiologically based pharmacokinetics (PBPK) simulations were conducted to evaluate the potential impact of IL-6 on the digoxin pharmacokinetics profile and brain exposure by decreasing BBB ABCB1 efflux activity. Exposure of hCMEC/D3 cells to IL-6 triggered the deregulation of numerous genes involved in barrier permeability, such as cell junctions, focal adherens complex, and cell adhesion molecules. We observed mild modification of the mRNA expression and efflux activities of ABC transporters. PBPK simulation showed that, if we only consider the impact of IL-6 on ABCB1 transporter, the modification of the digoxin pharmacokinetics profile and brain exposure is slight. IL-6 slightly affected the gene expression levels and activities of ABC transporters on BBB cells, exhibiting a weaker effect than on hepatic cells. However, inflammation may cause other modifications, such as altered BBB permeability, that could modify drug pharmacokinetics.

Impact of Interleukin-6 on Drug-Metabolizing Enzymes and Transporters in Intestinal Cells.

Inflammatory response is characterized by an increase of several cytokines. Some are known to modify drugs pharmacokinetic by reducing the expression levels of drug-metabolizing enzymes (DMEs) and transporters. This impact of inflammatory signaling is well established in hepatic cells, but not in intestinal cells. EpiIntestinal is a 3D human small intestinal tissue model with epithelial polarity, allowing good evaluation of metabolism and drug transport. This study aimed to analyze the effect of IL-6 on this tissue model. RNA sequencing was performed in cells incubated with 5, 10, or 20 ng/mL IL-6 for 8 h to 72 h to study the impact of IL-6 on drug metabolism and pharmacokinetics gene expression. The influence of IL-6 on the activity of cytochromes P450 (CYPs) was studied by measuring metabolite formation of specific substrates with LC-HRMS. Its impact on ATP-binding cassette (ABC) transport was evaluated by measuring intra- and extracellular substrates using spectrofluorometry. Exposure of EpiIntestinal cells to IL-6 resulted in reduction of some CYP mRNAs, such as CYP2C19, CYP2C9, and CYP3A4, by 40% to 50%. Activities of these CYPs were also decreased in EpiIntestinal cells by 20% to > 75%. IL-6 exposure did not modify ABCB1 and ABCCs transporter activities in this model. This study shows that gene expression levels and activities of drug-metabolizing enzymes and ABC transporters may be altered by the pro-inflammatory cytokine IL-6 in intestinal cells. If these results are confirmed in vivo, it may result in pharmacokinetic modifications, such as pre-systemic metabolism, with clinical effects, and require dosage adaptation.

Metabolomics and cytotoxicity of monomethylhydrazine (MMH) and (E)-1,1,4,4-tetramethyl-2-tetrazene (TMTZ), two liquid propellants.

Hydrazine-based liquid propellants are routinely used for space rocket propulsion, in particular monomethylhydrazine (MMH), although such compounds are highly hazardous. For several years, great efforts were devoted to developing a less hazardous molecule. To explore the toxicological effects of an alternative compound, namely (E)-1,1,4,4-tetramethyl-2-tetrazene (TMTZ), we exposed various cellular animal and human models to this compound and to the reference compound MMH. We observed no cytotoxic effects following exposure to TMTZ in animal, as well as human models. However, although the three animal models were unaffected by MMH, exposure of the human hepatic HepaRG cell model revealed that apoptotic cytotoxic effects were only detectable in proliferative human hepatic HepaRG cells and not in differentiated cells, although major biochemical modifications were uncovered in the latter. The present findings indicate that the metabolic mechanisms of MMH toxicity is close to those described for hydrazine with numerous biochemical alterations induced by mitochondrial disruption, production of radical species, and aminotransferase inhibition. The alternative TMTZ molecule had little impact on cellular viability and proliferation of rodent and human dermic and hepatic cell models. TMTZ did not produce any metabolomic effects and appears to be a promising putative industrial alternative to MMH.