Teresa Partearroyo

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

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.—Martínez‐Vega, R., Garrido, F., Partearroyo, T., Cediel, R., Zeisel, S. H., Martínez‐Álvarez, C., Varela‐Moreiras, G., Varela‐Nieto, I., and Pajares, M. A. Folic acid deficiency induces premature hearing loss through mechanisms involving cochlear oxidative stress and impairment of homocysteine metabolism. FASEB J. 29, 418‐432 (2015). www.fasebj.org

Vitamin B 12 and Folic Acid Imbalance Modifies NK Cytotoxicity, Lymphocytes B and Lymphoprolipheration in Aged Rats

Different vitamin B12 and folic acid concentrations could exacerbate the immune response. The aim was to evaluate different dietary folic acid and vitamin B12 levels on the immune response in aged rats. Male Sprague Dawley aged rats were assigned to three folic acid groups (deficient, control, supplemented) each in absence of vitamin B12 for 30 days. Several parameters of innate and acquired immune responses were measured. Serum and hepatic folate levels increased according to folic acid dietary level, while vitamin B12 levels decreased. There was a significant decrease in natural killer cell-mediated cytotoxicity in the spleen for the vitamin B12 deficient diet and folic acid control diet groups. Significant changes in CD45 lymphocyte subsets were also observed according to dietary imbalance. Lymphoproliferative response to concanavalin A and phytohemagglutinin did not differ significantly between groups. The spleen response to lipopolysaccharide increased significantly, but was unmodified for the other organs. An imbalance between dietary vitamin B12 and folic acid concentrations alters some immunological parameters in aged rats. Therefore, the ratio between folate and vitamin B12 could be as important as their absolute dietary concentrations.

Maternal folic acid–deficient diet causes congenital malformations in the mouse eye

BACKGROUND The eye is a very complex structure derived from the neural tube, surface ectoderm, and migratory mesenchyme from a neural crest origin. Because structures that evolve from the neural tube may be affected by a folate/folic acid (FA) deficiency, the aim of this work was to investigate whether a maternal folic acid-deficient diet may cause developmental alterations in the mouse eye. METHODS Female C57BL/6J mice (8 weeks old) were assigned into two different folic acid groups for periods ranging between 2 and 16 weeks. Animals were killed at gestation day 17. Hepatic folate was analyzed, and the eyes from 287 fetuses were macroscopically studied, sectioned and immunolabeled with anti-transforming growth factor (TGF)-β2 and anti-TGF-βRII. RESULTS Mice exposed to a FA-deficient diet exhibited numerous eye macroscopic anomalies, such as anophthalmia and microphthalmia. Microscopically, the eye was the most affected organ (43.7% of the fetuses). The highest incidence of malformations occurred from the 8th week onward. A statistically significant linear association between the number of maternal weeks on the FA-deficient diet and embryonic microscopic eye malformations was observed. The optic cup derivatives and structures forming the eye anterior segment showed severe abnormalities. In addition, TGF-β2 and TGF-βRII expression in the eye was also altered. CONCLUSION This study suggests that an adequate folic acid/folate status plays a key role in the formation of ocular tissues and structures, whereas a vitamin deficiency is negatively associated with a normal eye development even after a short-term exposure.

Occurrence of Cleft-Palate and Alteration of Tgf-β3 Expression and the Mechanisms Leading to Palatal Fusion in Mice following Dietary Folic-Acid Deficiency

Folic acid (FA) is essential for numerous bodily functions. Its decrease during pregnancy has been associated with an increased risk of congenital malformations in the progeny. The relationship between FA deficiency and the appearance of cleft palate (CP) is controversial, and little information exists on a possible effect of FA on palate development. We investigated the effect of a 2–8 weeks’ induced FA deficiency in female mice on the development of CP in their progeny as well as the mechanisms leading to palatal fusion, i.e. cell proliferation, cell death, and palatal-shelf adhesion and fusion. We showed that an 8 weeks’ maternal FA deficiency caused complete CP in the fetuses although a 2 weeks’ maternal FA deficiency was enough to alter all the mechanisms analyzed. Since transforming growth factor-β3 (TGF-β3) is crucial for palatal fusion and since most of the mechanisms impaired by FA deficiency were also observed in the palates of Tgf-β3null mutant mice, we investigated the presence of TGF-β3 mRNA, its protein and phospho-SMAD2 in FA-deficient (FAD) mouse palates. Our results evidenced a large reduction in Tgf-β3 expression in palates of embryos of dams fed an FAD diet for 8 weeks; Tgf-β3 expression was less reduced in palates of embryos of dams fed an FAD diet for 2 weeks. Addition of TGF-β3 to palatal-shelf cultures of embryos of dams fed an FAD diet for 2 weeks normalized all the altered mechanisms. Thus, an insufficient folate status may be a risk factor for the development of CP in mice, and exogenous TGF-β3 compensates this deficit in vitro.

Acute liver injury induces nucleocytoplasmic redistribution of hepatic methionine metabolism enzymes

AIMS The discovery of methionine metabolism enzymes in the cell nucleus, together with their association with key nuclear processes, suggested a putative relationship between alterations in their subcellular distribution and disease. RESULTS Using the rat model of d-galactosamine intoxication, severe changes in hepatic steady-state mRNA levels were found; the largest decreases corresponded to enzymes exhibiting the highest expression in normal tissue. Cytoplasmic protein levels, activities, and metabolite concentrations suffered more moderate changes following a similar trend. Interestingly, galactosamine treatment induced hepatic nuclear accumulation of methionine adenosyltransferase (MAT) α1 and S-adenosylhomocysteine hydrolase tetramers, their active assemblies. In fact, galactosamine-treated livers showed enhanced nuclear MAT activity. Acetaminophen (APAP) intoxication mimicked most galactosamine effects on hepatic MATα1, including accumulation of nuclear tetramers. H35 cells that overexpress tagged-MATα1 reproduced the subcellular distribution observed in liver, and the changes induced by galactosamine and APAP that were also observed upon glutathione depletion by buthionine sulfoximine. The H35 nuclear accumulation of tagged-MATα1 induced by these agents correlated with decreased glutathione reduced form/glutathione oxidized form ratios and was prevented by N-acetylcysteine (NAC) and glutathione ethyl ester. However, the changes in epigenetic modifications associated with tagged-MATα1 nuclear accumulation were only prevented by NAC in galactosamine-treated cells. INNOVATION Cytoplasmic and nuclear changes in proteins that regulate the methylation index follow opposite trends in acute liver injury, their nuclear accumulation showing potential as disease marker. CONCLUSION Altogether these results demonstrate galactosamine- and APAP-induced nuclear accumulation of methionine metabolism enzymes as active oligomers and unveil the implication of redox-dependent mechanisms in the control of MATα1 subcellular distribution.

Iron intake and dietary sources in the spanish population: findings from the ANIBES study

Background: Iron deficiency is one of the most common nutritional problems in the world. It is frequent in both developed and developing countries and mainly affects women of childbearing age and children. Methods: Results were derived from the ANIBES cross-sectional study using a nationally-representative sample of the Spanish population (9–75 years, n = 2009). A three-day dietary record, collected by means of a tablet device, was used to obtain information about food and beverage consumption and leftovers. Results: Total median dietary iron intake was 9.8 mg/day for women and 11.3 mg/day for men. Highest intakes were observed among plausible adolescent reporters (13.3 mg/day), followed by adults (13.0 mg/day), elderly (12.7 mg/day), and children (12.2 mg/day). Prevalence of adequacy for iron intakes as assessed by EFSA criteria was higher than for the Spanish Recommended Iron Intake values in all age groups. Females had lower adequacy than males for both criteria, 27.3% and 17.0% vs. 77.2% and 57.0% respectively. Cereals or grains (26.7%–27.4%), meats and derivatives (19.8%–22.7%), and vegetables (10.3%–12.4%) were the major iron contributors. Conclusion: Higher iron intakes were observed in adolescents and were highest for non-heme iron. The prevalence of adequate iron intake according to EFSA criteria was higher than compared to national recommendations, and women had the lowest intakes. Therefore, there is a need to define standard dietary reference intake to determine inadequate iron intakes in the Spanish population.

Cochlear homocysteine metabolism at the crossroad of nutrition and sensorineural hearing loss

Hearing loss (HL) is one of the most common causes of disability, affecting 360 million people according to the World Health Organization (WHO). HL is most frequently of sensorineural origin, being caused by the irreversible loss of hair cells and/or spiral ganglion neurons. The etiology of sensorineural HL (SNHL) is multifactorial, with genetic and environmental factors such as noise, ototoxic substances and aging playing a role. The nutritional status is central in aging disability, but the interplay between nutrition and SNHL has only recently gained attention. Dietary supplementation could therefore constitute the first step for the prevention and potential repair of hearing damage before it reaches irreversibility. In this context, different epidemiological studies have shown correlations among the nutritional condition, increased total plasma homocysteine (tHcy) and SNHL. Several human genetic rare diseases are also associated with homocysteine (Hcy) metabolism and SNHL confirming this potential link. Accordingly, rodent experimental models have provided the molecular basis to understand the observed effects. Thus, increased tHcy levels and vitamin deficiencies, such as folic acid (FA), have been linked with SNHL, whereas long-term dietary supplementation with omega-3 fatty acids improved Hcy metabolism, cell survival and hearing acuity. Furthermore, pharmacological supplementations with the anti-oxidant fumaric acid that targets Hcy metabolism also improved SNHL. Overall these results strongly suggest that cochlear Hcy metabolism is a key player in the onset and progression of SNHL, opening the way for the design of prospective nutritional therapies.

Long-Term Dietary Folate Deficiency Accelerates Progressive Hearing Loss on CBA/Ca Mice

Dietary folic acid deficiency induced early hearing loss in C57BL/6J mice after 2-months, corroborates the epidemiological association previously described between vitamin deficiency and this sensory impairment. However, this strain is prone to early hearing loss, and hence we decided to analyze whether the effects exerted by folate deprivation follow the same pattern in a mouse strain such as CBA/Ca, which is resistant to hearing impairment. Here, we show results of a long-term study on hearing carried out on CBA/Ca mice subjected to dietary folate deprivation. Systemic changes included decreased serum folate levels, hyperhomocysteinemia and signs of anemia in the group fed with folate-deficient (FD) diet. Initial signs of hearing loss were detected in this strain after 8-months of vitamin deficiency, and correlated with histological damage in the cochleae. In conclusion, the data presented reinforce the importance of adequate folic acid levels for the auditory system and suggest that the impact of dietary deficiencies may depend on the genetic background.

Long-term omega-3 fatty acid supplementation prevents expression changes in cochlear homocysteine metabolism and ameliorates progressive hearing loss in C57BL/6J mice

Omega-3 polyunsaturated fatty acids (PUFAs) are essential nutrients well known for their beneficial effects, among others on cognitive development and maintenance, inflammation and oxidative stress. Previous studies have shown an inverse association between high plasma levels of PUFAs and age-related hearing loss, and the relationship between low serum folate and elevated plasma homocysteine levels and hearing loss. Therefore, we used C57BL/6J mice and long-term omega-3 supplementation to evaluate the impact on hearing by analyzing their auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) thresholds. The omega-3 group showed significantly lower ABR hearing thresholds (~25 dB sound pressure level) and higher DPOAE amplitudes in mid-high frequencies when compared to the control group. These changes did not correlate with alterations between groups in plasma homocysteine or serum folate levels as measured by high-performance liquid chromatography and a microbiological method, respectively. Aging in the control group was associated with imbalanced cytokine expression toward increased proinflammatory cytokines as determined by quantitative reverse transcriptase polymerase chain reaction; these changes were prevented by omega-3 supplementation. Genes involved in homocysteine metabolism showed decreased expression during aging of control animals, and only alterations in Bhmt and Cbs were significantly prevented by omega-3 feeding. Western blotting showed that omega-3 supplementation precluded the CBS protein increase detected in 10-month-old controls but also produced an increase in BHMT protein levels. Altogether, the results obtained suggest a long-term protective role of omega-3 supplementation on cochlear metabolism and progression of hearing loss.

Dietary sources and intakes of folates and vitamin B12 in the Spanish population: Findings from the ANIBES study

Background Folates and vitamin B12 are key nutrients in one-carbon metabolism and related diseases. Updated and plausible information on population intakes and their major dietary sources is scarce and urgently needed in Spain in order to increase the knowledge that can lead as previous step to prevention by fortification and supplementation policies. Aims The present study aims to evaluate main dietary folate and vitamin B12 sources and intakes in the Spanish population. Materials and methods Results were derived from the ANIBES cross-sectional study using a nationally representative sample of the Spanish population (9–75 years, n = 2,009). Results Food groups with the highest mean proportional contribution to total folate intakes in both males and females were vegetables (21.7–24.9%) and cereals (10.7–11.2%), while meat and meat products (26.4%) and milk and dairy products (27.3%) were for B12. Total median folate and B12 intakes amongst women were 156.3 μg/d and 4.0 μg/d while for men were 163.6 μg/d and 4.5 μg/d, respectively. In all age groups, vitamin intakes were significantly higher in plausible than in non-plausible energy reporters. Conclusion A limited number of participants had adequate folate intakes, whereas vitamin B12 intakes were adequate for practically the entire population. There is a clear need for improving folates intake in the Spanish population.