Targeting the Lowest Concentration of a Toxin That Induces a Detectable Metabolic Response in Living Organisms: Time-Resolved In Vivo 2D NMR during a Concentration Ramp

Abstract

In vivo nuclear magnetic resonance (NMR) is a powerful analytical tool for probing complex biological processes inside living organisms. However, due to magnetic susceptibility broadening, which produces broad lines in one-dimensional NMR, 1H–13C two-dimensional (2D) NMR is required for metabolite monitoring in vivo. As each 2D experiment is time-consuming, often hours, this limits the temporal resolution over which in vivo processes can be monitored. Furthermore, to understand concentration-dependent responses, studies are traditionally repeated using different contaminant and toxin concentrations, which can make studies prohibitively long (potentially months). In this study, time-resolved non-uniform sampling NMR is performed in the presence of a contaminant concentration sweep. The result is that the lowest concentration that elicits a metabolic response can be rapidly detected, while the metabolic pathways impacted provide information about the toxic mode of action of the toxin. The lowest concentration of bisphenol A (BPA) that induces a response was ∼0.1 mg/L (detected in just 16 min), while changes in different metabolites suggest a complex multipathway response that leads to protein degradation at higher BPA concentrations. This proof of concept shows it is possible, on the basis of “real-time” organism responses, to identify the sublethal concentration at which a toxin impacts an organism and thus represents an essential analytical tool for the next generation of toxicity-based research and monitoring.

In-cell NMR: Why and how?

“…Simpson and colleagues have established amino-acid specific 2D 1 H- 13 C pulse sequences to monitor the free amino-acid concentrations [1065]. Hence, they measured their increase in D. magna upon exposure to a concentration ramp of bisphenol-A with a 1 minute time-frame allowing a very precise determination of the lowest concentration eliciting a response [1066]. They also introduced 12 C-editing methods to monitor specifically the incorporation of 12 C-molecules in animals grown under a 13 C-labeled diet.…”

“…The field would be impossible to cover entirely and is well beyond our scope. The common INEPT has been the most widely used by the in-cell NMR community, but recent applications of BIRD or TANGO on live material show that these can also yield interesting information [1066,1649].…”

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…This can be executed in a time-resolved fashion through days, , provided that cells are maintained in proper conditions either at low density in hydrogels or using a flow-probe bioreactor. Longitudinal NMR monitoring is also possible with organoids, , plants, or small aquatic animals like C. elegans or water fleas. ,− These objects generate magnetic susceptibility inhomogeneities, which can substantially broaden signals of small molecules and call for the use of advanced pulse sequences. ,− Importantly, the direct observation of the target provides direct proofs of (i) compound binding at the expected protein/nucleic acid site and (ii) of the active/unactive or the phospho/unphospho populations of the studied protein/nucleic acid.…”

“…One-dimensional spectra can yield equivalent information using amino acid-type specific 15 N-labeling at about twice lower concentrations. A number of strategies shall be combined to improve sensitivity: better isotope-labeling schemes, notably when introducing deuteration, better pulse sequences adapted to the cellular viscosity, paramagnetic T1 relaxation enhancement, − continuous readout using time-resolved nonuniform sampling, ,− deep-learning assisted spectral reconstruction, − denoising procedures, ,− deep-learning assisted peak picking, etc. Using all these advanced techniques, it might be possible to record exploitable in-cell 2D NMR spectra of proteins/nucleic acids at intracellular concentrations of a few μmol/L.…”

Unsaturated fatty acids profiling in liveC. elegansusing real-time NMR spectroscopy

“…Moreover, a continuous NMR flow system was used on 13 C-enriched water fleas to assess the effects of growth conditions or stressors on organismal metabolism in real time 27,28 . Other applications of high resolution NMR studies in live animals include the monitoring of enzymatic reductions of exogenously delivered, 15 N isotopically enriched, oxidized methionine derivatives in zebrafish embryos (Danio rerio), a widely used vertebrate model system for biological studies 29 .…”