Table 1.

Summary of Information on the Key Events of the AOP

Key Events	Description of Events	Experimental Support and References
MIE: AG accumulation in the inner ear hair cell	The receptor-mediated endocytosis and presence of MET cation channel lead to rapid accumulation and slow elimination of AG in the inner ear	(1) KM was taken up into sensory hair cells via receptor-mediated endocytosis at their apical surfaces because AG molecules are found in vesicles beneath the hair cells from White Leghorn chicks, confirmed by immuno-gold electron microscopy (Hashino and Shero 1995; Richardson et al. 1997)
		(2) MET channels on hair cell functions as open transducer channels that is the main route for aminoglycoside entry. AGs functioned as voltage-dependent MET channel blockers that also rapidly permeate through MET channels into hair cells, which was found in bullfrog model (Steyger et al. 2003), turtle model (Farris et al. 2004), and mouse model (Marcotti et al. 2005). The AG molecules enter the channel and block the ion-conducting pathway, thus such blockage increases voltage. Increased AG entry through the channel pore into the hair cell due to the large electrical driving force also increases the affinity for the blocker. This boosts both the entry of AG into the channel and the channel’s affinity for the drug (Farris et al. 2004; Marcotti et al. 2005; Steyger et al. 2003).
KE 1: Prerequisite events impacting MIE	Nephrotoxicity (KE1-1), hypoalbuminemia (KE1-2), and pre-existing hearing loss (KE1-3) accelerate AG accumulation in the interstitum of hair cell	(1) TDF is mitochondrial toxic, increasing number of abnormal mitochondria including irregular mitochondrial shape, and sparse, fragmented cristae. Abnormal proximal tubule functioning and decreased GFR occurred in patients who had been taking TDF in multiple studies (Calza et al., 2011; Jin et al., 2015; Kenyon et al., 2011; Kohler et al., 2009; Scherzer et al., 2012).
		(2) The albumin-like proteins, including albumin, IgG, IgA, transferrin, antitrypsin, and haptoglobin were the major protein compositions of luminal fluid in inner ear (Kim et al. 2017; Kim et al. 2011). Among patients (n=11) with enlarged vestibular aqueducts, patients with recent hearing loss and increased volume of luminal fluid showed a significantly decreased proportion of the albumin-like proteins in the interstitial space (Kim et al. 2011).
		(3) Pre-existing hearing loss includes mainly noise-induced and age-related hearing loss. Histological evaluation using mice (n=22), received repetitive exposure the acoustic stimuli (~4.5 kHz with 120.5 dB sound pressure level), showed significant structural hair cell damage (Park et al., 2018). It has been found that atrophy of the stria vascularis of cochlear duct was observed in older mice (older than 12 months at least), and that lipofuscin (aging associated pigment granules) accumulation in inner and outer hair cells of the mice were also found from the same age (Saitoh et al. 1994). This structural change is accelerated by the age-related dysfunctions of the systemic immune system accelerated, worsening presbycusis (Cui et al. 2012; Iwai et al. 2008; Kalinec et al. 2017; Lee 2013; Saitoh et al. 1994).
KE 2: Prerequisite events impacting initial cellular responses	Immunodeficiency (KE2-1) and antioxidant deficiency (KE2-2) trigger cellular oxidative stress	(1) In comparison between 8 HIV-infected patients (mean CD4+ T-cells count = 280 × 106/L), 7 AIDS patients (mean CD4+ T-cells count = 45 × 106/L), AIDS patients had increased levels of 7,8-dihydro-8-oxoguanine in CD4+ T cells and marked declines in DNA glycosylase activity for the repair of oxidative base lesions in these cells (Aukrust et al. 2005). People living with HIV showed elevated pro-inflammatory cytokines, including interleukin 2 (IL-2), IL-6, and tumor necrosis factor-alpha (TNF-α) and biomarkers associated with inflammation and coagulation, including C-reactive protein (CRP) and D-dimer due to chronic inflammation and immune activation (Abrams et al. 2009; Baker et al. 2010; Deeks et al. 2013; Neuhaus et al. 2010). In cross-sectional human study, the level of lipid peroxidation (LPO) and glutathione were used as a means of determining oxidative stress. The mean LPO levels were significantly higher in HIV-infected patients (n=100; mean= 0.7 ± 0.1 μmol/ml) as compared to healthy controls (n=30; mean= 0.3 ± 0.1 μmol/ml). The mean glutathione level in HIV-infected patients (0.06 ± 0.01 μmol/ml) was significantly lower in compared to healthy controls (0.09 ± 0.01 μmol/ml) (Staal et al. 1992; Wanchu et al. 2009).
		(2) A human study found that compare to younger control subjects, elderly subjects had significantly lower level of glutathione (2.08 ± 0.12 vs. 1.12 ± 0.18 mmol/L RBCs; P < 0.05); glutathione synthesis rates (1.73 ± 0.16 vs. 0.55 ± 0.12 mmol/L RBCs per day; P < 0.01); and higher plasma oxidative stress (304 ± 16 vs. 346 ± 20 Carratelli units; P < 0.05) simultaneously (Sekhar et al. 2011). This indicates that glutathione deficiency in elderly humans resulted from a marked reduction in synthesis, related to oxidative stress.
KE 3: Prerequisite events impacting latter cellular responses	Mitochondrial toxicity (KE3) worsens inhibition of mitochondrial protein synthesis of hair cells	Since reductions in mitochondrial DNA content induced by NRTIs, significantly more HIV-infected patients had or developed persistent hearing loss with/without tinnitus during follow-up (Marra et al. 1997; McNaghten et al. 2001; Simdon et al. 2001a). After short-term exposure to AZT, d4T, ddC, ddI, and FLT (6-72 hours), mtDNA copy numbers were markedly decreased because the NRTIs inhibit mtDNA replication (Smith et al. 2017). Upregulation of glutathione S-transferase 4 expression were significantly increased, which suggests that ROS defense mechanisms likely to be induced by NRTI administration due to mtDNA intoxication (Smith et al. 2017).
AO: SNHL	When programmed cell death-signaling pathways has been activated, hair cells, ancillary sensory cells, and neurons undergo apoptotic cell death, resulting in irreversible SNHL	ROS formation through ototoxicants, including gentamicin and kanamycin, in cochlear tissues of was directly observed in guinea pig by electron paramagnetic resonance spectrometry (Clerici et al. 1996) and in chick by using dichlorofluorescin (Hirose et al. 1997). When chicks and mouse cochlear and vestibular hair cells were exposed to gentamicin, the incorporation of methionine-free medium over 24 hours was reduced by 30–60% compared to control conditions observed by fluorescence microscopy (Francis et al. 2013). This indicates gentamicin inhibited the medium uptake into hair cells by inhibiting protein synthesis in hair cells and activate a c-Jun N-terminal kinase (JNK) pathway as JNKs activate apoptotic signaling (Francis et al. 2013).

Abbreviations: MIE= molecular initiating event; KE= key event; AO= adverse outcome; HIV= human immunodeficiency virus; CD4= cluster of differentiation 4; SNHL= sensorineural hearing loss; MET= mechanoelectrical transducer; NRTI= Nucleoside Reverse Transcriptase Inhibitor; KM= kanamycin; TDF= Tenofovir disoproxil Fumarate; ROS= reactive oxygen species; AZT= zidovudine (3’-Azido-3’-deoxythymidine); d4T= stavudine (2’,3’-didehydro-2’,3’-deoxythymidine); ddC= zalcitabine (2’,3’-dideoxycytidine); ddI= didanosine (2’,3’-dideoxyinosine); FLT= alovudine (3’-deoxy-3’-fluorothymidine)