Age-Related Hearing Loss (ARHL) or presbycusis is one of the most prevalent age-related neurodegenerative disorders. It is estimated that over 40% of the population will experience moderate hearing loss by the age of 65 (40-70 dB), with that number exceeding 70% by age 75 (Bazard et al., 2021a; Dalton et al., 2003; Frisina and Frisina, 1997; Gopinath et al., 2012; Huang and Tang, 2010; Pacala and Yueh, 2012). Aside from affecting communication abilities, ARHL is also highly associated with other disorders of age such as loneliness, depression, and onset of senile dementia (Bazard et al., 2020; Bazard et al., 2021b; Kramer et al., 2002; Lin et al., 2011; Liu and Yan, 2007; Parham et al., 2011; Shukla et al., 2020). In fact, the 2020 Lancet Commission on Dementia named ARHL as its top modifiable risk factor for dementia (Livingston et al., 2020). ARHL contributes to economic burdens through loss of productivity, reductions in quality of life, and additional healthcare costs. Cost estimates have put the negative economic impact between 750 billion and 980 billion dollars in the US alone (Landry et al., 2022; McDaid et al., 2021). Unfortunately, there are no FDA approved drugs/treatments that can slowdown or cure ARHL. In the present report, we investigate the potential role of the novel amino acid L-ergothioneine (EGT) in preventing some key aspects of ARHL in aging mammals.

L-ergothioneine (EGT), appears well suited to tackle some of the challenges of ARHL due to its combination of antioxidant and anti-inflammatory properties which allow it to provide dual anti-aging functions (Cheah and Halliwell, 2012, 2021; Dare et al., 2022; Marone et al., 2016; Tang et al., 2018) related to ARHL. ARHL is characterized by a degradation of important structures throughout the auditory pathways, which can be divided into the peripheral (cochlea) and central auditory system (parts of the brain used for hearing) (Bazard et al., 2022; Frisina et al., 2020; Gates and Cooper, 1991; Gates and Mills, 2005; Howarth and Shone, 2006; Schuknecht and Gacek, 1993; Wang and Puel, 2020; Willott, 1996). Like other aging systems, the degenerations are multifactorial and not limited to just one type of molecular mechanism. Rather, they share similar markers of age-related degradation such as chronic low-grade inflammation (inflammaging), elevated oxidative stress and ROS production, increased apoptosis, and finally DNA and mitochondrial DNA (mtDNA) damage (Bazard et al., 2022; Bazard et al., 2021b; Cheng et al., 2005; Keithley, 2020; Watson et al., 2017; Yamasoba et al., 2013).

In our study, EGT was used as treatment for ARHL in old-aged mice with the hypothesis that EGT's properties will positively impact markers of the aging hearing system. For example, in other models where EGT has been used as a treatment for inflammation, EGT has shown usefulness in preventing induction of pro-inflammatory cytokines as well as a declines in the expression of VCAM-1, ICAM-1, and E-selectin in a human aortic endothelial cell model. EGT effectively prevented monocytes from binding to the endothelium (Martin, 2010). In another study using mouse myoblasts, EGT prevented the upregulation of IL-6 in a study of insulin resistance and type 2 diabetes (Bastard et al., 2002; Laurenza et al., 2008; Pradhan et al., 2001). EGT also works in vivo, as healthy human subjects were administered EGT and it was found to be helpful in reducing some damaging events such as inflammaging, increased ROS, and accumulated mitochondrial damage, analyzed via biomarkers in urine samples (Reduction in the DNA damage product, 8OHdG; protein carbonyls, which indicate protein damage; F2 isoprostanes, indicating non-enzymatic peroxidation; and the inflammatory marker, C-reactive protein) (Cheah et al., 2017). It is also hypothesized that these events are interconnected, acting in a cyclic nature, where one mechanism influences another which ultimately leads to more cellular damage. For example, mitochondrial damage can activate inflammation through damage-associated molecular patterns that recruit neutrophils and cytokines during cellular trauma (Bazard et al., 2021b; Jang et al., 2018; Jara et al., 2019; Strickland et al., 2019). Again, EGT's properties align with possible beneficial effects as they have been shown to protect cells from DNA damage both nuclear and mitochondrial (Lamhonwah and Tein, 2006). This dual protection likely comes from the translocation of EGT into both the nucleoplasm as well as into the mitochondria. Mitochondria are then protected from ROS damage to the electron transport chain, as well as to the unique mtDNA code (Kawano et al., 1982; Paul and Snyder, 2010).Further evidence from Menardo et al. (2011) clearly shows a correlation between oxidative stress, inflammation and mitochondrial damage as critical mechanisms for premature or accelerated ARHL in the SAMP8 mouse. EGT's therapeutic potential lies in its chemical structure as it exists as a thione-thiol tautomer. At physiological pH, EGT is positively charged with a preference for its thione conformation. These attributes allow for EGT to be highly stable and resist auto-oxidation unlike the structurally similar antioxidant glutathione, which is also found in the cochlea (Misra, 1974). Like glutathione, its sulfhydryl group supports a role as an antioxidant, but with a higher redox potential (-0.06 V vs -0.2 to -0.32 V for other antioxidants thiols like glutathione) (Avendaño and Menéndez, 2015). The sulfhydryl group acts by oxidizing and conjugating reactions of peroxides and other ROS until EGT is rapidly reduced back to its original tautomeric state. Glutathione, in contrast, is quickly and completely depleted under the same circumstances and can even generate more free radicals in the process (Cheah et al., 2017; Forman et al., 2009; Misra, 1974; Paul and Snyder, 2010).

L-ergothioneine is a dietary amino acid that can only be obtained in various food products since it is unable to be synthesized in the body with the highest concentrations found in mushrooms (Ey et al., 2007). Additionally, EGT is also unique in that it possesses its own highly specific cell transporter, organic cation transporter novel-1 (OCTN1) (Ben Said et al., 2016; Cheah and Halliwell, 2012; Cheah et al., 2017; Gründemann et al., 2005; Li et al., 2014; Nakamura et al., 2007). Uptake through OCTN1 is largely dependent on temperature, sodium concentrations, and pH, but proceeds as expected under normal physiological conditions and OCTN1 negative cells and knockout animal models, show little uptake of EGT (Cheah and Halliwell, 2012; Nakamura et al., 2008; Taubert et al., 2005). OCTN1 is encoded by the SLC22a4 gene and is found throughout the body with high metabolic activity, as EGT has been shown to reduce oxidative stress in these regions (Ben Said et al., 2016; Cheah et al., 2017; Halliwell et al., 2016), such as the liver and blood, and also found in lesser amounts in the spleen, kidney, lungs, heart, eyes, intestines, and brain (Cheah et al., 2017; Gründemann et al., 2005; Tang et al., 2018). It should be noted that EGT is likely found in high concentrations within whole blood due to its affinity for the divalent ion, Fe2+ (Hanlon, 1971; Motohashi et al., 1976; Motohashi et al., 1974) found in erythrocytes and bone marrow (Gründemann et al., 2005) (Melville, 1959) (Fraser, 1951). These properties then hint towards EGT's high uptake and low excretion which contributes to a long half-life in the body of up to 30 days (Marone et al., 2016; Tang et al., 2018). Even more exciting, EGT has an excellent safety profile as there has been no reported toxicity for any dosage and EGT has been assigned a NOAEL = 800 mg/kg BW/day (Efsa Panel on Dietetic Products et al., 2016).

Recent research has shown EGT's effectiveness in chronic neurodegenerative disorders. For example, EGT declines with age, and lower levels of EGT are associated with incidence of mild cognitive impairment (Cheah et al., 2016). Beyond cognitive function, low serum levels of EGT are associated with other aging pathologies such as coronary artery disease, cardiovascular disease mortality, prostate cancer, breast cancer and total mortality (Beelman et al., 2020; Hatano et al., 2016; Smith et al., 2020; Winkels et al., 2020) (Shin et al., 2010; Zhang et al., 2020). Interestingly, the Smith et al. (2020) longitudinal study found that EGT was an independent biomarker for a lower risk of cardiometabolic disease and mortality among 112 plasma metabolites. As EGT has many benefits and virtually no side effects, the European Food Safety Authority (EFSA) granted its use as a supplement for pregnant and breast-feeding moms, children, and infants (EFSA Panel on Dietetic Products et al., 2017). There are several ongoing clinical studies which are assessing the therapeutic effects of EGT in cognitive decline in Singapore (Phase 3) (Cheah, 2018). A study was registered for EGT clinical trials in USA in Sept. 2020 to find its effect on cognition, mood, and sleep but, unfortunately, it was terminated due to recruitment issues amidst the COVID pandemic (Blue, 2020). We anticipate an increase in the number of EGT clinical trials in the coming years.

EGT has additionally been nicknamed a “longevity” vitamin, because of the human body's specific EGT transporter and its potential physiological and therapeutic roles in many aging diseases. It has the potential to actively decrease ROS levels, impede the transcription pathway of NF-κB, and hinder the generation of proinflammatory cytokines by inhibiting neuroinflammation in a manner dependent on dosage (Jang et al., 2004; Song et al., 2010). Given all these promising actions of EGT, and the many parallels for biological mechanisms of age-related neurological disorders and ARHL, EGT is also a great candidate for therapeutic interventions in the aging auditory system. Surprisingly, a preliminary investigation uncovered gender differences in the accumulation of serum EGT, and the absorption of EGT was correlated with testosterone levels (Mackenzie and Mackenzie, 1957). The EGT transporter ETT (alternate acronym for organic cation/carnitine transporter 1 [OCTN1]; human gene symbol (SLC22A4) is a powerful and highly specific transporter for the uptake of EGT (Gründemann et al., 2022). Later on, it was found that castrated mice exhibited a slight elevation in the mRNA levels of OCTN1 in renal proximal tubules compared to non-surgery controls. However, in ovariectomized (OVX) mice, the mRNA levels for OCTN1 did not show significant changes compared to non-surgery controls (Meetam et al., 2009a, 2009b). These findings suggest a sex difference in the impact of EGT. However, these findings are still pending replication. To the best of our knowledge, there is no study of the effects of EGT on hearing to date, so this is the first report about EGT's possible therapeutic roles for ARHL. Here, aging rodents were treated with EGT, and various hearing tests and initial biomarker measurements were performed in a longitudinal study.