title: Investigating potential mitochondrial mechanisms of, and understanding the effect of mitochondrial genome on, antimicrobial-induced liver injury
Creators: name: Kiy, Robyn
Publisher: name: University of Liverpool Repository
Year: 2024

description: As the primary site of drug metabolism, the liver is exposed to a substantial amount of chemical insult over the average person’s lifetime. Drug-induced liver injury (DILI) is the leading cause of safety-related preclinical drug attrition as well as post-marketing withdrawals, and thus represents a substantial issue for pharmaceutical development and beyond. The mechanisms underlying DILI can be multifactorial and influenced by intrinsic and extrinsic factors, resulting in significant interindividual variation in susceptibility to, and severity of, DILI. This is particularly true of idiosyncratic DILI (iDILI), which despite its rarity remains a contemporary drug-safety issue due to the unpredictable nature of this adverse drug reaction (ADR). iDILI is characterised by complex dose-dependence, with incidences commonly appearing to be independent of treatment dose, duration, or route of administration. 

Mitochondrial dysfunction is recognised as a key mechanism of DILI. Mitochondria play a central role in ATP generation, making them essential in the maintenance of physiological cellular functioning and signalling. Therefore, drugs that induce mitochondrial perturbations can cause the deterioration of physiological cellular function, ultimately resulting in cell death. Mitochondria are ubiquitous, and present at levels related to bioenergetic demand throughout the body. The metabolic role of the liver is energy-intensive, and thus hepatocytes are mitochondria-rich and particularly susceptible to drug-induced mitochondrial dysfunction (DIMD). Mitochondria are highly complex organelles and are able to utilise a plethora of stress-management mechanisms. This combination of multiple potential toxicity routes and protective mechanisms means that the identification of mitotoxic drugs can be challenging. The functional complexity of mitochondria is further compounded by the additional factor of interindividual variation in mitochondrial DNA (mtDNA) sequence. This intricacy contributes to the fact that mechanisms underlying iDILI are not understood for many compounds. Investigating and elucidating these mechanisms, and identifying genetic risk factors, is vital in improving the safety profiles of drugs commonly implicated in iDILI. 

Antiribosomal antibiotics are frequently associated with DIMD as a result of structural similarities between bacterial and mitochondrial ribosomes. For some sub-classes, including the macrolides, this association is largely hypothetical based on the mechanism of action of the compounds. For other drugs, including linezolid, this association is well-characterised and drug safety has been shown to be influenced by mtDNA variation. Due to the structural similarities between bacterial and mitochondrial constituents, thorough investigation of mitochondrial safety is imperative when developing antibiotics. This is particularly pertinent as the antimicrobial resistance (AMR) crisis continues, and the development of novel antibiotics remains a global priority. Therefore, the aims of this research were to investigate the assumed mitochondrially-mediated mechanisms of macrolide-induced hepatotoxicity in physiologically- and clinically-relevant in vitro models (enabling dosing periods of up to 28 days), and to use HepG2 transmitochondrial cybrids to assess the influence of mtDNA variation on antiribosomal antibiotic-induced mitochondrial dysfunction. 

Initial investigations using the positive controls chloramphenicol and linezolid confirmed that HepaRG cells are a suitable in vitro model for assessing antiribosomal antibiotic-induced mitoribosome inhibition, and enable the ranking of mitotoxic compounds. Following this validation, the mitotoxic potential of six macrolides were assessed. It was revealed that mechanisms of macrolide toxicity are not consistent across the class, with only solithromycin eliciting significant reductions in mitoribosome-dependent protein expression. Respirometry revealed potential electron transport chain (ETC) dysfunction following treatment with several of the macrolides, with the most significant dysfunction observed following solithromycin treatment. These bioenergetic changes were frequently non-monotonic, highlighting the resilience of mitochondria which is exemplified by the threshold effect. Next, HepG2 transmitochondrial cybrids were confirmed as a suitable in vitro model for studying the effects of mtDNA variation on susceptibility to iDILI. Linezolid treatment typically resulted in greater hepatic mitochondrial dysfunction in cell lines containing the risk-associated variants, m.2706A&gt;G and m.3010G&gt;A, although outcomes varied between endpoints. However, metabolomic investigation may suggest that the process of ‘cybridisation’ induces substantial changes to intracellular metabolome, in the absence of observable phenotypic changes. Finally, clinically-relevant investigations in HepaRG cells confirmed the mitochondrial safety of the active metabolite of molnupiravir, β-d-N4-hydroxycytidine (NHC), providing additional assurance over the published findings in HepG2 cells. 

In conclusion, DILI is a multi-mechanistic toxicity with mitochondrial dysfunction commonly implicated in its onset. As a result of its clinical rarity, the mechanisms underlying DILI are not fully elucidated for many compounds. Following the confirmation of HepaRG as a suitable in vitro model for the assessment of antiribosomal antibiotic-induced hepatotoxicity, research within this thesis revealed that whilst toxicity mechanisms vary across the macrolide class, several macrolides appear to be capable of inducing hepatic mitochondrial dysfunction. Additionally, HepG2 transmitochondrial cybrids were identified as a useful translational model in recapitulating increased susceptibility to linezolid-induced mitochondrial dysfunction in the presence of risk-associated mtDNA variants. Overall, the incorporation of intrinsic and extrinsic features known to affect drug safety into in vitro models is pivotal in improving the predictive power of preclinical DILI assessment, ultimately benefitting patients, healthcare professionals, and the pharmaceutical industry.