Unravelling the metabolic alterations of liver damage induced by Thirdhand Smoke

Objectives: The aim of this study is to investigate the metabolic disorders caused by Thirdhand smoke (THS) exposure in liver of male mice and to evaluate the effects of an antioxidant treatment in the exposed mice. Liver samples were from three mice groups (n=5 for each group): non-exposed mice (CTRL), exposed to THS in conditions that mimic human exposure (THS) and THS-exposed treated with antioxidants (THS-AO). 

Samples: We performed a biphasic extraction of the homogenated liver samples.

LC-MS Analysis: Liver samples were analyzed by liquid chromatography (UHPLC) coupled to high-resolution mass spectrometry (LC-MS) usng 1290 Infinity LC System coupled to a 6230 ESI-QTOF mass spectrometer (both from Agilent Technologies). For both analyses, the mass spectrometer operated in positive electrospray ionization (ESI+) mode in the following conditions: gas temperature, 150 °C; drying gas, 11 L min⁻¹; nebulizer, 35 psi; fragmentor, 120 V; and skimmer, 65 V. The instrument was set to acquire over the m/z range 100-1000 for the aqueous extracts and 50−1200 for the lipidic ones, at an acquisition rate of 3 spectra s^-1

LC-MS files are named following this criteria: 

Sample Code: 

1. First letter correspond to the type of extract, aqueous (A) or lipidic (L).

2. Second letter correspond to the exposure group: C = Control group , T= THS-exposed group, A= THS-exposed group treated with antioxidants and QC=Quality Control formed by a pool of all aqueous extracts.

3. Number of the biological replicate (1 to 5).

MSMS files were acquired using the same LCMS described above in targeted MS2 acquisition mode, m/z range 100-1000, MS/MS scan Rate 5 spectra s^-1 and collision energy fixed at 20 eV.

File code is the commented above, indicating MSMS at the end of the file name, and the acquisition number.

NMR-analysis: ¹H NMR spectra were recorded at 310 K on a spectrometer operating at a proton frequency of 600.20 MHz. For the aqueous extracts, one-dimensional ¹H pulse experiments were carried out using a nuclear Overhauser effect spectroscopy (NOESY)-presaturation sequence to suppress the residual water peak at around 4.7 ppm for aqueous extract. The relaxation delay between scans was set to 5 s. Spectral width was 16 ppm and a total of 256 transients were collected for each spectrum. In the case of lipophilic extracts, a 90° pulse with presaturation sequence (zgpr) was used. Spectral width was 18.6 ppm and a total of 128 transients were collected for each spectrum. The acquired one dimensional (1D) ¹H NMR spectra were phased, baseline-corrected and referenced to glucose signal (5.23 ppm) for the aqueous extracts and to TMS signal for the lipidic extracts with TopSpin from Bruker.

Files are named following the same criteria than LC-MS files, but indicating NMR at the beginning of the file name