Hepatic Metabolic Derangements Triggered by Hyperthermia: An In Vitro Metabolomic Study

Abstract

Background and aims: Liver toxicity is a well-documented and potentially fatal adverse complication of hyperthermia. However, the impact of hyperthermia on the hepatic metabolome has hitherto not been investigated.

Methods: In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was applied to assess the in vitro metabolic response of primary mouse hepatocytes (PMH, n = 10) to a heat stress stimulus, i.e., after 24 h exposure to 40.5 °C. Metabolomic profiling of both intracellular metabolites and volatile metabolites in the extracellular medium of PMH was performed.

Results: Multivariate analysis showed alterations in levels of 22 intra- and 59 extracellular metabolites, unveiling the capability of the metabolic pattern to discriminate cells exposed to heat stress from cells incubated at normothermic conditions (37 °C). Hyperthermia caused a considerable loss of cell viability that was accompanied by significant alterations in the tricarboxylic acid cycle, amino acids metabolism, urea cycle, glutamate metabolism, pentose phosphate pathway, and in the volatile signature associated with the lipid peroxidation process.

Conclusion: These results provide novel insights into the mechanisms underlying hyperthermia-induced hepatocellular damage.

Keywords: GC-MS; heat stress; metabolic profile; multivariate statistical analysis; primary mouse hepatocytes.

Figure 1

Cell viability measured by (A) MTT reduction and (B) lactate dehydrogenase (LDH) leakage, 24 h after exposure of primary mouse hepatocytes to normothermic (37 °C) and hyperthermic (40.5 °C) conditions. Results were obtained from 10 independent experiments, performed in triplicate. **** p < 1.00 × 10⁻⁴ (hyperthermic vs. normothermic conditions).

Figure 2

Orthogonal projections to latent structures discriminant analysis (OPLS-DA) score scatter plots obtained for the chromatograms corresponding to cells exposed to normothermic (n = 10, ●) and hyperthermic (n = 10, ▲) conditions, after analysis of the (A) intracellular metabolome, (B) volatile organic compound (VOC) and (C) volatile carbonyl compound (VCC) in extracellular metabolome. (D–F) Statistical validation of the respective OPLS-DA models obtained by permutation tests (500 permutations).

Figure 3

Effect size of the metabolites altered by heat stress, evaluated by comparison of cells exposed to hyperthermic vs. normothermic conditions in the (A) intracellular metabolome and (B and C) extracellular metabolome (VOCs and VCCs, respectively). Unidentified compounds are reported as ‘IM_i’, ‘VOC_i’ and ‘VCC_i’ (i = 1, 2, 3...) according to the ascending order of their retention time (RT) values. Metabolites marked with $\otimes$ are not statistically significant after false discovery rate (FDR) correction (FDR corrected p-value: 3.93 × 10⁻² for intracellular metabolome, 4.84 × 10⁻² for VOCs, and 4.12 × 10⁻² for VCCs). * p < 5.00 × 10⁻², ** p < 1.00 × 10⁻², *** p < 1.00 × 10⁻³, **** p < 1.00 × 10⁻⁴ (hyperthermic vs. normothermic conditions).

Figure 4

Overview of the dysregulated metabolic pathways based on metabolites alteration caused by hyperthermia. The node color is based on the p value, where a dark circle color indicates a more significant pathway. The node radius corresponds to the pathway impact value. Pathways were annotated when p < 0.05 and pathway impact > 0.1.

Figure 5

Heatmap representing the Spearman’s correlations between the metabolites significantly altered (p < 0.01) after a thermal insult. 1. Docosahexaenoic acid; 2. Glycerol monostearate; 3. 1,1-Dimethylpropyl acetate; 4. 4-Methyl-2-pentanone; 5. 3-Ethyl-4-methylpentanol; 6. 2,4,6-Trimethyldecane; 7. 1-(2,4,6-Trimethylphenyl)ethanone; 8. 2,7,10-Trimethyldodecane.