Cells possess many detoxification enzymes capable of removing thousands of cytotoxic xenobiotics and endogenous toxins such as 4-hydroxynonenal (4-HNE), one of the most abundant cytotoxic end products of lipid peroxidation. The cellular detoxification system involves three phases of enzymatic detoxification. Of these, the glutathione transferase (GST) detoxification system converts a toxic compound into a less toxic form by conjugating the toxic compound to reduced glutathione by GST enzymes. GSTs are a superfamily of enzymes that are divided into several classes on the basis of their primary structure (1-3). Over 20 mammalian GSTs have been identified to date, and the cytosolic GSTs constitute the largest family. The most well characterized GST classes include alpha-class GSTs (GSTAs).
Cisplatin, a platinum-containing compound, is one of the most widely used chemotherapeutic agents (8-10). Evidence indicates that one-third of all cisplatin-treated patients develop hearing loss. Cisplatin-induced hearing loss is generally dose-dependent, irreversible, and associated with loss of sensory hair cells, spiral ganglion neurons and/or stria vascularis cells. Cisplatin is thought to exert its cytotoxic effects through DNA crosslinking and generation of ROS following binding to cytoplasmic proteins, leading to increased oxidative damage and cell death. Our lab has recently shown that GSTA4 immunostaining was prominent in the stria vascularis of Gsta4+/+ mice, suggesting that SV is likely a major site for GSTA4-mediated detoxification of 4-HNE in the cochlea (Park et al., 2019). Cisplatin treatment also stimulated GST activity toward 4-HNE in the inner ear of female Gsta4+/+ mice. In contrast, Gsta4 deficiency resulted in increased levels of 4-HNE and more profound loss of SGNs and SV atrophy in the cochlea of cisplatin-treated female mice. Furthermore, our subsequent study has shown that elevated levels of 4-HNE were observed in the cochlea of aged mice compared to young controls (Park et al., 2020). This was associated with decreased SGN density, reduced SV thickness, and hair cells in the cochlea of aged mice. The central hypothesis of our research proposal is that GSTAs play an essential role in reducing oxidative lipid damage and ototoxicity and slowing cochlear aging. To test this hypothesis, we propose the following research plan:
Aim 1: Determine the role of inner ear GSTA4 detoxification in cochlear aging.
1.1. Investigate whether Gsta4 deficiency accelerates cochlear aging in male and female mice.
1.2. Investigate whether Gsta4 deficiency increases susceptibility to cisplatin ototoxicity in male and female mice across the lifespan.
Aim 2: Determine the role of NRF2 in GSTA4 detoxification in mouse inner ears across the lifespan.
2.1. Investigate whether knockdown of NRF2 results in increased levels of 4-HNE, decreased cell viability, and decreased levels of GSTAs in SH-SY5Y cells under cisplatin treatment.
2.2. Investigate whether Nrf2 deficiency results in increased levels of 4-HNE and decreased levels of GSTA4 in mouse inner ears under cisplatin treatment.
2.3. Investigate whether Nrf2 deficiency accelerates cochlear aging in male and female mice.
2.4. Investigate whether Nrf2 deficiency increases susceptibility to cisplatin ototoxicity in male and female mice across the lifespan.