Folic acid deficiency induces premature hearing loss through mechanisms involving cochlear oxidative stress and impairment of homocysteine metabolism

Abstract

Nutritional imbalance is emerging as a causative factor of hearing loss. Epidemiologic studies have linked hearing loss to elevated plasma total homocysteine (tHcy) and folate deficiency, and have shown that folate supplementation lowers tHcy levels potentially ameliorating age-related hearing loss. The purpose of this study was to address the impact of folate deficiency on hearing loss and to examine the underlying mechanisms. For this purpose, 2-mo-old C57BL/6J mice (Animalia Chordata Mus musculus) were randomly divided into 2 groups (n = 65 each) that were fed folate-deficient (FD) or standard diets for 8 wk. HPLC analysis demonstrated a 7-fold decline in serum folate and a 3-fold increase in tHcy levels. FD mice exhibited severe hearing loss measured by auditory brainstem recordings and TUNEL-positive-apoptotic cochlear cells. RT-quantitative PCR and Western blotting showed reduced levels of enzymes catalyzing homocysteine (Hcy) production and recycling, together with a 30% increase in protein homocysteinylation. Redox stress was demonstrated by decreased expression of catalase, glutathione peroxidase 4, and glutathione synthetase genes, increased levels of manganese superoxide dismutase, and NADPH oxidase-complex adaptor cytochrome b-245, α-polypeptide (p22phox) proteins, and elevated concentrations of glutathione species. Altogether, our findings demonstrate, for the first time, that the relationship between hyperhomocysteinemia induced by folate deficiency and premature hearing loss involves impairment of cochlear Hcy metabolism and associated oxidative stress.

Keywords: apoptosis; dietary restriction; hair cell loss; hyperhomocysteinemia; methionine cycle.

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Figures

Figure 1.

Pathways involved in Hcy metabolism and systemic metabolite levels. A) Scheme of Hcy metabolism and related pathways. Metabolites appear in white boxes, and the enzymes listed include the following: ADA, ADK, AdoHcy hydrolase (AHCY), BHMTs, AdoMet-dependent methyltransferases (MTs), MATs, vitamin B6-dependent CBS, MTHFR, and vitamin B12-dependent MTR. B) Serum and plasma metabolite levels from all animals in both normal (NF) and FD groups are shown. Serum folic acid [50.85 ± 22.03 (n = 10) vs. 7.46 ± 3.88 μg/L (n = 11)], tHcy [5.05 ± 2.45 (n = 33) vs. 14.70 ± 3.19 μM (n = 69)], and plasma vitamin B6 levels [40.00 ± 15.71 (n = 11) vs. 43.71 ± 8.74 μg/L (n = 18)] were measured as specified under Materials and Methods. The figure shows the mean ± SEM for each group, and differences were considered significant when P ≤ 0.05; ***P < 0.0001.

Figure 2.

FD mice show early signs of hearing loss. The auditory response was analyzed in mice of normal (NF) and FD groups. A) Scheme of the auditory pathway adapted from Murillo-Cuesta et al. (32). B) Representative ABR recordings obtained in response to click stimuli of NF (showing normal hearing) and FD mice (showing profound hearing loss). C) ABR thresholds in response to click and tone burst stimuli in NF (○) and FD (•) mice, after 8 wk of diet (n = 21 for each group). D) Latency-intensity functions for wave I of NF and FD mice showing a delay in the appearance of the wave. Data are shown as the mean ± SEM. ***P < 0.001.

Figure 3.

FD mice showed altered cochlear morphology and apoptotic cells. Cochlear morphology from normal (NF) and FD mice was studied by histochemistry and immunohistofluorescence. A) Representative micrographs show basal and middle turn details of the organ of Corti in sections of NF (a–e; n = 6) and FD mice (f–j; n = 9). Phalloidin (Phal) staining of the organ of Corti appears in panels d, e, i, and j. Asterisks denote the flat epithelium of the organ of Corti in panel g. Asterisks indicate the absence of hair cells in panel i. B) Representative images and quantification of cell death by TUNEL assay of the organ of Corti per region of interest (ROI) from the middle turn of both groups. C) Percentage of TUNEL-positive cells/ROI in the stria vascularis from FD mice compared to NF (n = 6 mice studied per experimental group). IHC, inner hair cell; OHC, outer hair cell; SV, scala vestibulae; ST, scala tympani; SM, scala media. Scale bars, 500 µm (a and f), 50 µm (d, e, i, and j), and 25 µm (b, c, g, and h).

Figure 4.

FD mice showed altered cochlear cytoarchitecture. Sections of cochleae from mice on normal (A–H; n = 6) or FD (J–Q; n = 9) diets were stained with Masson’s Trichrome, H&E, or used for immunofluorescence. Basal and middle turn details of the ligament (Spl), cochlear ganglion (CG), and stria vascularis (StV) are shown. Immunohistochemistry results of the cochlear ganglion neurons labeled with myelin protein zero are shown at higher magnification in the insets to panels (D), (E), (M), and (N). Details of the stria vascularis immunolabeled with Kir4.1 are included as insets to panels (G), (H), (P), and (Q). Arrows indicate the absence of cells (J and M). Panel (C) depicts a scheme of the cochlea where basal and middle turns are indicated by squares. The intensity of the signals (0–256 gray scale) was quantified for normal (NF; n = 4) and FD n = 6) samples, and the results are shown as the mean ± SEM in (F), (I), (L), (O), and (R) histograms. **P < 0.01; ***P < 0.001. Scale bars, 25 µm (A–H, J–Q, and insets to G, H, P, and Q) and 10 µm (insets to D, E, M, and N).

Figure 5.

Cochlear Hcy metabolism in control mice. Cochleae from mice receiving an NF diet were used to characterize Hcy metabolism and related reactions. A) Expression levels of the genes of interest were determined by RT-qPCR using total cochlear RNA; the data were normalized using the Rn18s gene as reference (n = 6). B–D) Total cochlear proteins from wild-type and Bhmt−/− null mice (200 µg), were used to verify the specificity of bands detected on Western blots. Membranes were incubated with (B) rabbit anti-BHMT (37) and mouse anti-tubulin, (C) preimmune serum, and (D) goat anti-AHCY. Cytosols from hepatoma H35 cells (3 μg) were used as reference for the expected mobility. Size of the prestained markers is indicated on the left side of the blots.

Figure 6.

Folate deficiency alters expression of genes involved in Hcy metabolism. Total cochlear RNA of mice on normal (NF, n = 11) or FD (n = 12) diets was used to analyze expression levels of the genes of interest by real-time RT-PCR using the Rn18s gene as reference. The results are shown as the mean ± SEM of determinations made in triplicate for each animal sample. Statistical analysis by Student’s t test was carried out using GraphPad Prism, and data are considered significant when *P ≤ 0.05.

Figure 7.

Effects of folate deficiency on cochlear protein levels of enzymes involved in Hcy metabolism. Total cochlear protein (200 μg/lane) of animals on normal (NF; n = 11) and FD (n = 20) diets was analyzed by Western blot using the following antibodies: A) anti-AHCY, B) anti-CBS, C) anti-BHMT, D) anti-MTR, E) anti-ADK, and F) anti-ADA. Representative immunoblots for each antibody are shown, together with quantifications (mean ± SEM) carried out with ImageJ software, normalized using tubulin as the loading control. For graphic purposes, the mean of the NF group ratio was considered 100% in each case. G) Representative image of anti- Hcy Western blot of NF and FD mice and its densitometric analysis. Mean data of the NF group are presented as 100% for graphic purposes; ratios are 3.92 ± 0.80 (NF; n = 4) and 6.28 ± 1.06 (FD; n = 5). Statistical analysis was carried out using Student’s t test and differences were considered significant when P ≤ 0.05. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 8.

FD mice showed oxidative imbalance in the cochlea. Cochleae from mice on normal (NF) and FD diets were used to evaluate several oxidative stress markers. A) Schematic representation of the role of the oxidative stress markers analyzed in this work. B) Representative immunoblots for NOX4 (n = 3 NF and n = 4 FD), MnSOD (n = 6 NF and n = 9 FD), and p22phox (n = 5 NF and n = 9 FD). The histograms show the mean ± SEM of densitometric scanning results after normalization using β-actin levels. C) Expression levels of Cat, Gpx1, GPx4, Gsr, Gclc, and Gss evaluated by real-time RT-PCR using Rplp0 as reference. D) Evaluation of cochlear GSSG and GSH levels (n = 6 mice per group). E) Representative images of 3-NT levels detected by immunohistochemistry in the stria vascularis (StV; a and b) and cochlear ganglion (SG; c and d) of NF (a and c; n = 3) and FD (b and d; n = 6) mice. e) Quantification of the 3-NT signal is shown in the histograms as the mean ± SEM. Statistical evaluation of the data was performed by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.