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, 10 (1), 4150

GSTA4 Mediates Reduction of Cisplatin Ototoxicity in Female Mice

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GSTA4 Mediates Reduction of Cisplatin Ototoxicity in Female Mice

Hyo-Jin Park et al. Nat Commun.

Abstract

Cisplatin is one of the most widely used chemotherapeutic drugs for the treatment of cancer. Unfortunately, one of its major side effects is permanent hearing loss. Here, we show that glutathione transferase α4 (GSTA4), a member of the Phase II detoxifying enzyme superfamily, mediates reduction of cisplatin ototoxicity by removing 4-hydroxynonenal (4-HNE) in the inner ears of female mice. Under cisplatin treatment, loss of Gsta4 results in more profound hearing loss in female mice compared to male mice. Cisplatin stimulates GSTA4 activity in the inner ear of female wild-type, but not male wild-type mice. In female Gsta4-/- mice, cisplatin treatment results in increased levels of 4-HNE in cochlear neurons compared to male Gsta4-/- mice. In CBA/CaJ mice, ovariectomy decreases mRNA expression of Gsta4, and the levels of GSTA4 protein in the inner ears. Thus, our findings suggest that GSTA4-dependent detoxification may play a role in estrogen-mediated neuroprotection.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Localization of GSTA4 protein in mouse cochlea. a Western blot analysis of GSTA4 protein levels in the inner ear from young male Gsta4+/+ and Gsta4−/− mice. The full-length blot is presented in the Source Data file. n = 3, *p < 0.05 vs. +/+ (unpaired two-tailed Student’s t test). Error bars represent ± s.e.m. bp GSTA4 staining (green; b, e, h, k, n), DAPI staining (blue; c, f, i, l, o), and merged staining (d, g, j, m, p) were detected in the organ of Corti regions (bd, np), SGNs (eg), organ of Corti (hj), and SVs (km) from 3-month-old WT (bm) and Gsta4−/− (np) males. OC organ of Corti. Scale bar = 100 μm (d, p) or 20 μm (g, j, m)
Fig. 2
Fig. 2
Assessment of ABR hearing thresholds, wave I amplitudes, and latencies. a, b ABR hearing thresholds were measured at 4, 8, 16, 32, 48, and 64 kHz in male (a) and female (b) Gsta4+/+ and Gsta4−/− mice. n = 10, *p < 0.05 vs. +/+ post-cisplatin (two-way ANOVA). Error bars represent ± s.e.m. c The body weight of male and female Gsta4+/+ and Gsta4−/− mice was measured during cisplatin treatment. n = 10. Error bars represent ± s.e.m. d, e ABR wave I amplitudes (d) and latencies (e) were measured in male and female Gsta4+/+ and Gsta4−/− mice. n = 10, *p < 0.05 vs. +/+ (unpaired two-tailed Student’s t test). Error bars represent ± s.e.m. Source data are provided as a Source Data file
Fig. 3
Fig. 3
Assessment of SGN density. an SGN regions in the apical, middle, and basal regions of cochlear tissues from male (af) and female (hm) Gsta4+/+ and Gsta4−/− mice after cisplatin treatment. Scale bar = 20 μm. SGN densities in the apical, middle, and basal regions of cochlear tissues from male (g) and female (n) Gsta4+/+ and Gsta4−/− mice were counted and quantified. The quantification shows a mean of at least three independent experiments (n = 3), *p < 0.05 vs. +/+ (two-way ANOVA). Error bars represent ± s.e.m. Source data are provided as a Source Data file
Fig. 4
Fig. 4
Assessment of SV thickness and capillary number. an SV regions in the apical, middle, and basal regions of cochlear tissues from male (af) and female (hm) Gsta4+/+ and Gsta4−/− mice after cisplatin treatment. Scale bar = 20 μm. The thickness of SV in the apical, middle, and basal region of cochlear tissues from male (g) and female (n) Gsta4+/+ and Gsta4−/− was measured. The quantification shows a mean of at least three independent experiments (n = 3), *p < 0.05 vs. +/+ (two-way ANOVA). Error bars represent ± s.e.m. o, p Numbers of endomucin-positive capillaries in the SVs in each region (apical, middle, and basal region) of cochlear tissues were counted and quantified in male (o) and female (p) Gsta4+/+ and Gsta4−/− mice after cisplatin treatment. The quantification shows mean of at least three independent experiments (n = 3). Error bars represent ± s.e.m. Source data are provided as a Source Data file
Fig. 5
Fig. 5
Assessment of GST activity. a, b GST activities toward 4-HNE were measured in the cytosol of inner ear tissues from male (a) and female (b) Gsta4+/+ and Gsta4−/− mice prior to and after cisplatin treatment. n = 3, *p < 0.05 vs. precisplatin (two-way ANOVA). Error bars represent ± s.e.m. c, d 4-HNE-positive SGNs were counted and quantified in the apical, middle, and basal regions of cochlear tissues from male (c) and female (d) Gsta4+/+ and Gsta4−/− mice after cisplatin treatment. The quantification shows a mean of at least three independent experiments (n = 3), *p < 0.05 vs. +/+ (two-way ANOVA). Error bars represent ± s.e.m. Source data are provided as a Source Data file
Fig. 6
Fig. 6
Assessment of mRNA expression levels of detoxification genes in the inner ears of CBA/CaJ mice. a mRNA expression levels of nine genes involved in the enzymatic processes of Phase II detoxification were analyzed by PCR arrays in the inner ears from male and female CBA/CaJ mice. n = 5, *p < 0.05 vs. M (unpaired two-tailed Student t test). Error bars represent ± s.e.m. b mRNA expression levels of Nrf2 were measured by qRT-PCR in the inner ears from male and female CBA/CaJ mice. n = 5, *p < 0.05 vs. M (unpaired two-tailed Student t test). Error bars represent ± s.e.m. The relative mRNA levels were normalized to levels of B2m. M: male, F: female. Source data are provided as a Source Data file
Fig. 7
Fig. 7
Assessment of mRNA-expression levels of detoxification genes in the inner ears of CBA/CaJ mice. a mRNA expression levels of 25 genes involved in the enzymatic processes of Phase II detoxification were analyzed by PCR arrays in the inner ears from female and OVX CBA/CaJ mice. n = 5, *p < 0.05 vs. F (unpaired two-tailed Student t test). Error bars represent ± s.e.m. b mRNA expression levels of Nrf2 were measured by qRT-PCR in the inner ears from female and OVX CBA/CaJ mice. n = 5, *p < 0.05 vs. F (unpaired two-tailed Student t test). Error bars represent ± s.e.m. The relative mRNA levels were normalized to levels of B2m. F: female, O: ovariectomized female. Source data are provided as a Source Data file
Fig. 8
Fig. 8
Assessment of GSTA4 protein levels in the inner ears of CBA/CaJ mice. a, c GSTA4 protein levels were measured in the inner ear tissues from male and female CBA/CaJ mice. n = 5. Error bars represent ± s.e.m. b, c GSTA4 protein levels were measured in the inner ear tissues from female and OVX CBA/CaJ mice. n = 5, *p < 0.05 vs. F (unpaired two-tailed Student t test). Error bars represent ± s.e.m. The relative protein levels were normalized to levels of GAPDH. The full-length blot is presented in the Source Data file. M: male, F: female, O: ovariectomized female. Source data are provided as a Source Data file
Fig. 9
Fig. 9
Assessment of oxidative DNA damage in the inner ears of Gsta4+/+ and Gsta4−/− mice. a Levels of 8-oxoguanine as an oxidative DNA damage marker were measured in the inner ear tissues from cisplatin-treated male and female Gsta4+/+ and Gsta4−/− mice. n = 5, *p < 0.05 vs. +/+ (unpaired two-tailed Student t test). Error bars represent ± s.e.m. b Levels of 8-oxoguanine were measured in the inner ears from cisplatin-treated male and female Gsta4−/− mice. The quantification shows a mean of at least four independent experiments (n = 4). Error bars represent ± s.e.m. c Levels of 8-oxoguanine were measured in the inner ears from cisplatin-treated female and OVX Gsta4−/− mice The quantification shows a mean of at least four independent experiments (n = 4). Error bars represent ± s.e.m. M: male, F: female, O: ovariectomized female. Source data are provided as a Source Data file

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