Table 3.

Studies addressing histone modifications induced by noise exposure

HISTONE MODIFICATIONS
Study location	Experimental settings	Study design	Results	References
IN VIVO STUDIES
USA	Male CBA/J mice assigned to 3 experimental groups (n. 20 each group): the control group; the DMSO group, that was exposed to noise and received an intraperitoneal injection of DMSO (10%) 3 days before the exposure; the SAHA group, that was exposed to noise and received an intraperitoneal injection of SAHA (25 mg/kg) 3 days before noise exposure.	Noise exposure	✓ Noise exposure resulted in PTS at 4, 8, 12, 24, 32 and 48 kHz, 2 weeks following exposure to noise. Compared with the DMSO-injected group, pre-treatment with the HDAC inhibitor, SAHA, significantly reduced PTS.	Wen et al.[24]
		Mice were exposed to broadband noise with a frequency spectrum from 2 to 20 kHz for 2 h/day for 2 weeks at 110 dB SPL to induce a PTS with loss of cochlear OHCs. Control animals were not exposed to noise.	✓ The expression of HDAC1 (2-fold) and HDAC4 (3 fold) increased 1 h following noise exposure compared to controls. The H3-AcK9 levels decreased 1 h following noise exposure.
		Biological analysis The expression levels of H3K9ac and HDAC1 and HDAC4 were assessed by western blot analysis on cochlear tissue homogenates 1 h after exposure.	✓ The number of OHCs loss decreased following SAHA pre-treatment compared to the DMSO pre-treatment. SAHA pre-treatment doubled the number of survival OHCs and attenuated cilia damage.
USA	Male CBA/J mice (max n.7 per group) were exposed to noise and cochlear tissues were analysed 1 h after the exposure completion	Noise exposure	✓ Immunolabeling for H3K9ac decreased in the nuclei of OHCs (60%) and strial marginal cells of the cochlear basal turn 1 h after noise exposure compared to control mice.	Chen et al.[25]
	Control group: mice not exposed to noise	Mice were exposed to broadband noise with a frequency spectrum from 2 to 20 kHz for 2 h at 98 dB SPL to induce a PTS with loss of OHCs.	✓ Noise exposure increased HDAC1, 2, and 3 in cochlear tissues (organ of Corti, spiral ganglion cells and stria vascularis) 1 h after noise exposure compared to control mice.
		Biological analysis	✓ Blockade of HDAC1, 2, or 3 alone in cochlear tissues resulted not sufficient to reduce PTS.
		The expression levels of H3K9ac was assessed by immunohistochemistry on cryosection 1 h after the exposure. The levels of HDAC1, 2, and 3 using paraffin-embedded cochlear sections from the mid-modiolar region were assessed 1 h after the exposure. Two week following noise exposure, treatment with SAHA (50 mg/kg intraperitoneal injection) was applied to the animals and noise-induced OHCs loss, PTS and H3K0ac distribution in the nuclei was assessed.	✓ Treatment with SAHA significantly decreased noise-induced OHCs loss (40% at 4 mm, 50% at 4.5 mm, and 20% at 5 mm), and significantly attenuated PTS by about 20 dB at 16 kHz compared to vehicle-treated mice. After 1 h SAHA treatment there was an enrichment of H3K9ac at the nuclear periphery.
China	Male albino guinea pigs (n. 120) divided into 3 groups: control group (no noise exposure, n. 40); noise only group (n. 40); noise + SB (intraperitoneal injection at a dose of 600 mg/kg once per day on the 3 days before and after noise exposure, as well as 30 min before and 2 h after the noise exposure) group (n. 40).	Noise exposure	✓ Noise exposure induced PTS in both the noise only group and the noise with SB group. However, the ABR threshold shifts in this latter group were significantly lower than those in the noise only group.	Yang et al.[26]
	In the noise-exposed group: 10 animals underwent hearing test at 1 d before and 14 d after exposure and then were euthanized; 30 animals were euthanized at 2 h after noise exposure	Animals were exposed to 1/3-octave-wide narrowband noise, centered at 4kHz at 122 dB SPL for 3 h.	✓ Noise exposure significantly decreased H3‑AcK9 expression, and increased HDAC1 expression, in the nuclei of OHCs, IHCs, and Hensen’s cells. SB treatment partially reversed these changes.
		Biological analysis Cochlear tissues were collected for immunofluorescence staining to assess H3AcK9, HDAC1 and 3NT levels. Homogenized cochlear tissues were used for protein total extraction and western blot analysis	✓ The 3-NT was significantly increased in OHCs in the noise only group compared to the control group. SB treatment significantly reduced the noise-induced increase of 3-NT
USA	Male CBA/J mice (max n.6 per group) were exposed to noise and cochlear tissues were analysed 1 h after the exposure completion	Noise exposure	✓ Immunoreactivity of G9a protein increased in the nuclei of OHCs, IHCs, supporting cells of organ of Corti, the nuclei of SGNs and the nuclei of marginal cells in lateral wall tissues in the basal turn of the cochlea	Xiong et al. [27]
	Control group: mice not exposed to noise	Mice were exposed to broadband noise with a frequency spectrum from 2 to 20 kHz for 2 h at 101 dB SPL to induce a PTS with loss of cochlear OHCs.	✓ Noise exposure increased immunolabelling for H3K9me2 levels in nuclei of OHCs, strial marginal cells, and SGNs from the basal turn of the cochlea.
		Biological analysis	✓ Noise exposure reduced the expression of KCNQ4 in OHCs of the basal turn of the cochlea (75%).
		The levels of the lysine dimethyltransferase G9a in cochlear cells were assessed by immunohistochemistry. Since a primary function of G9a is to demethylate lysine 9 of histone 3, levels of H3K9me2 in the cochlea 1 h after completion of noise exposure was assessed through immunolabelling. Expression levels of potassium channel genes KCNQ4 was assessed 1 h after noise exposure completion.	✓ Treatment with BIX 01294 (a specific inhibitor of G9a prevented the noise-induced decrease of KCNQ4 immunolabelling in OHCs, the loss of OHCs and reduces auditory threshold shifts.

3NT, 3-nitrotyrosine; DMSO, Dimethyl Sulfoxide; H3K9me2, histone H3 lysine 9 dimethylation; HDAC, histone deacetylases; IHCs, inner hair cells; KCNQ4, potassium voltage-gated channel subfamily Q member 4; OHCs, outer hair cells; PTS, permanent threshold shift; SAHA, Suberoylanilide hydroxamic acid; SB, sodium butyrate; SGNs, spiral ganglion neurons; SPL, sound pressure level.