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Mitochondria are affected by chemical substances and play a critical role in drug-induced liver injury (DILI). Chemical substances can have a significant impact on various cellular processes, such as the disruption of oxidative phosphorylation, oxidative stress, and alteration of glucose metabolism. Given the consequences of these effects, it is crucial to understand the molecular pathways of chemical substances in the context of hepatotoxicity to prevent and treat DILI. In this regard, the Seahorse XFe24 Analyzer is a valuable tool for assessing mitochondrial bioenergetics and glucose metabolism. The Mito Stress Test and Glycolytic Rate Assay allow real-time assessment of the metabolic state after chemical exposure. Additionally, HepG2 spheroids have emerged as an important alternative tool for assessing hepatotoxicity, as they provide results that are more comparable to those found in humans than monolayer cultures or animal tests (such as rodent tests). By integrating these two powerful tools, it is possible to bridge the gap between animal and human tests, resulting in more reliable results in the assessment of human hepatotoxicity and DILI. However, because of the high variability in characteristics between 3D cultures (such as spheroids and organoids), XF analyzer assays are not well optimized for use with HepG2 spheroids. Here, we describe a streamlined and optimized protocol for performing the Mito Stress Test and Glycolytic Rate Assay using HepG2 spheroids cultured in alginate microcapsules in the Seahorse XFe24 Analyzer.

Diabetes and associated conditions are now considered a worldwide epidemic, with increasing costs and burdens with no cure yet developed. The chitin-derived glucosamine biopolymer chitosan has shown promising results when supplied to diabetic patients. However, no study has investigated the possible toxic side effects of chitosan treatments, in particular when regarding the most important bioenergetic organelle, mitochondria. As such, we aimed to understand if supplementation of chitosan to the diet of normal and diabetic rats could compromise mitochondrial function on two of the major organs involved in diabetes, obesity, and metabolic regulation, the liver and skeletal muscle. We supplemented the drinking water of normal Wistar and diabetic Goto-Kakizaki rats with 0.5% chitosan for 6 weeks. We show here that, in terms of hepatic bioenergetics, chitosan was relatively inert and had no major side effects. However, regarding skeletal muscle bioenergetics, chitosan significantly affected various bioenergetic parameters. As such, we conclude that chitosan, at the tested doses, is relatively safe for treatment of diabetic situations. Nonetheless, the potential for adverse toxicological side effects appears to be present, which might be relevant if higher doses are utilized.

The reductive power provided by nicotinamide adenine dinucleotides is invaluable for several cellular processes. It drives metabolic reactions, enzymatic activity, regulates genetic expression and allows for the maintenance of a normal cell redox status. Therefore, the balance between the oxidized (NAD(+)) and the reduced (NADH) forms is critical for the cell's proper function and ultimately, for its survival. Being intimately associated with the cells' metabolism, it is expected that alterations to the NAD(+)/NADH ratio are to be found in situations of metabolic diseases, as is the case of diabetes. NAD(+) is a necessary cofactor for several enzymes' activity, many of which are related to metabolism. Therefore, a decrease in the NAD(+)/NADH ratio causes these enzymes to decrease in activity (reductive stress), resulting in an altered metabolic situation that might be the first insult toward several pathologies, such as diabetes. Here, we review the importance of nicotinamide adenine dinucleotides in the liver cell and its fluctuations in a state of type 2 diabetes mellitus.

Environmental pollutants such as TCDD and tetraethyl lead are extremely toxic and related with pulmonary disease development. Lung mitochondria are primary cellular targets for dioxins exposure-induced toxicity. TCDD showed a delay in the repolarization after a phosphorylative cycle and a decrease on state 3 respiration, suggesting alterations at the phosphorylative system level. The ATPase activity showed no differences between control and lung mitochondria incubated with TCDD, implying alterations in other components of the phosphorylative system. Tetraethyl lead also showed a delay in the repolarization after a phosphorylative cycle and a decrease on RCR. These data suggest that lung mitochondria incubated with TCDD and tetraethyl lead showed impaired mitochondrial function, reflecting the loss of oxidative phosphorylation capacity.