Reina Oshino

GADD34 suppresses lipopolysaccharide-induced sepsis and tissue injury through the regulation of macrophage activation

Growth arrest and DNA damage inducible protein 34 (GADD34) is induced by various cellular stresses, such as DNA damage, endoplasmic reticulum stress, and amino-acid deprivation. Although the major roles of GADD34 are regulating ER stress responses and apoptosis, a recent study suggested that GADD34 is linked to innate immune responses. In this report, we investigated the roles of GADD34 in inflammatory responses against bacterial infection. To explore the effects of GADD34 on systemic inflammation in vivo, we employed a lipopolysaccharide (LPS)-induced murine sepsis model and assessed the lethality, serum cytokine levels, and tissue injury in the presence or absence of GADD34. We found that GADD34 deficiency increased the lethality and serum cytokine levels in LPS-induced sepsis. Moreover, GADD34 deficiency enhanced tissue destruction, cell death, and pro-inflammatory cytokine expression in LPS-induced acute liver injury. Pro-inflammatory cytokine production after LPS stimulation is regulated by the Toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway. In vitro experiments revealed that GADD34 suppressed pro-inflammatory cytokine production by macrophages through dephosphorylation of IKKβ. In conclusion, GADD34 attenuates LPS-induced sepsis and acute tissue injury through suppressing macrophage activation. Targeting this anti-inflammatory role of GADD34 may be a promising area for the development of therapeutic agents to regulate inflammatory disorders.

GADD34 inhibits activation-induced apoptosis of macrophages through enhancement of autophagy

Autophagy is a common physiological function in all eukaryotes. The process is induced by depletion of nutrients including amino acids. GADD34 is expressed following DNA damage, ER stresses and amino acid deprivation. Here, we investigated the effects of GADD34 on autophagy and cell activation in macrophages. The deprivation of tyrosine and cysteine markedly induced the expression of GADD34 in macrophages. LPS stimulation combined with tyrosine/cysteine-deprivation initially activated macrophages, but then shifted to cell death in late phase of stimulation. When LPS stimulation was combined with tyrosine/cysteine-deprivation, a deficiency of GADD34 enhanced cell activation signaling such as Src-family, Erk1/2, p38 MAPK and Akt. In the late phase of stimulation, a deficiency of GADD34 increased apoptosis more than that in wild-type macrophages. Further we found that mTOR-S6K signaling was highly enhanced in GADD34-deficient macrophages compared with wild-type cells when cells were treated by LPS combined with tyrosine/cysteine-deprivation. LC3-II was increased by LPS stimulation combined with tyrosine/cysteine-deprivation. Defective GADD34 reduced LC3-II and autophagosome formation induced by LPS-stimulation and tyrosine/cysteine-deprivation compared with that seen in wild-type macrophages. These results indicates that GADD34 enhances autophagy and suppresses apoptosis stimulated by LPS combined with amino acid deprivation through regulation of mTOR signaling pathway in macrophages.

Manganese-mediated acceleration of age-related hearing loss in mice

Despite the fact that manganese (Mn) is known to be a neurotoxic element relevant to age-related disorders, the risk of oral exposure to Mn for age-related hearing loss remains unclear. In this study, we orally exposed wild-type young adult mice to Mn (Mn-exposed WT-mice) at 1.65 and 16.50 mg/L for 4 weeks. Mn-exposed WT-mice showed acceleration of age-related hearing loss. Mn-exposed WT-mice had neurodegeneration of spiral ganglion neurons (SGNs) with increased number of lipofuscin granules. Mn-exposed WT-mice also had increased hypoxia-inducible factor-1 alpha (Hif-1α) protein with less hydroxylation at proline 564 and decreased c-Ret protein in SGNs. Mn-mediated acceleration of age-related hearing loss involving neurodegeneration of SGNs was rescued in RET-transgenic mice carrying constitutively activated RET. Thus, oral exposure to Mn accelerates age-related hearing loss in mice with Ret-mediated neurodegeneration of SGNs.

Oral exposure to arsenic causes hearing loss in young people aged 12–29 years and in young mice

There is no information on the association between oral exposure to arsenic (As) and hearing loss in humans or mice. In this combined epidemiological study and experimental study, the association of oral exposure to As with hearing loss in people aged 12–29 years and young mice was examined. Subjects in the exposure group (n = 48), who were drinking tube well water contaminated with As, showed significantly higher risks of hearing loss at 4 kHz [odds ratio (OR) = 7.60; 95% confidence interval (CI): 1.56, 57.88], 8 kHz (OR = 5.00; 95% CI: 1.48, 18.90) and 12 kHz (OR = 8.72; 95% CI: 2.09, 47.77) than did subjects in the control group (n = 29). We next performed an experiment in which young mice were exposed to As via drinking water at 22.5 mg/L, which is a much greater concentration than that in human studies. The exposure group showed hearing loss and accumulation of As in inner ears. Ex vivo exposure of the organ of Corti from mice exposed to As significantly decreased the number of auditory neurons and fibers. Thus, our combined study showed that oral exposure to As caused hearing loss in young people and young mice.

Arsenic levels in cutaneous appendicular organs are correlated with digitally evaluated hyperpigmented skin of the forehead but not the sole in Bangladesh residents

There has been no report showing the effect of arsenic level on digitized skin pigmentation level, a typical diagnostic marker for arsenicosis. Correlations among history of drinking well water, arsenic levels in hair and toenails, and digitalized skin pigmentation levels (L*-value) in sunlight-exposed (forehead) and unexposed (sole) skin areas digitally evaluated by using a reflectance spectrophotometer were examined in 150 residents of Bangladesh. Univariate analysis showed that arsenic levels in hair and toenails of subjects with a history of drinking well water were 10.6-fold and 7.1-fold higher, respectively, than those in subjects without a history of drinking well water. The mean L*-value of foreheads, but not that of soles, in subjects with a history of drinking well water was 1.15-fold lower (more pigmented) than that in subjects without a history of drinking well water. Significant correlations were found between duration of drinking well water and arsenic concentrations in hair (r=0.63; P<0.01) and toenails (r=0.60; P<0.01). Multivariate analysis showed that the arsenic levels in hair and toenails and the duration of drinking well water were strongly correlated with the digitized pigmented level of the forehead but not that of the sole. An increase in the duration of drinking well water may increase hyperpigmentation in the forehead, but not that in the sole, through an increased arsenic level in the human body as shown in cutaneous appendicular organs (hair and toenails).

Risk Assessment of Neonatal Exposure to Low Frequency Noise Based on Balance in Mice

General electric devices and ventilation systems are known to generate low frequency noise (LFN) with frequencies of <100 Hz. Previous studies showed that exposure to LFN caused impairments of balance in humans and mice during adulthood. On the other hand, a previous study showed that noise levels in the neonatal intensive care unit (NICU) were greater than those in general home or office environments. Therefore, it is possible that neonates have a potential risk to be exposed to LFN in the NICU. However, the risk of neonatal exposure to LFN remains unclear in humans and mice. In this study, male ICR mice were exposed to LFN at 100 Hz for 4 weeks after birth and then subjected to rotarod and beam crossing tests in order to assess LFN-mediated risk of imbalance during the neonatal period. Exposure to LFN at 70 dB, but not exposure to LFN up to 60 dB, during the neonatal period significantly decreased performance scores for rotarod and beam crossing tests compared to the scores of the control group. The number of calbindin-positive hair cells in the saccule and utricle was decreased in mice exposed to LFN at 70 dB for 4 weeks in the neonatal phase. Cessation of exposure for 10 weeks did not result in recovery of the decreased performance in rotarod and beam crossing tests. Thus, our results suggest that 70 dB is a possible threshold for exposure to LFN for 4 weeks during the neonatal period causing unrecoverable imbalance in mice.

Arsenic level in toenails is associated with hearing loss in humans

Arsenic (As) pollution in drinking water is a worldwide health risk for humans. We previously showed hearing loss in young people who live in areas of As-polluted drinking water and in young mice orally treated with As. In this study, we epidemiologically examined associations between As levels in toenails and hearing in 145 Bangladeshi aged 12–55 years in 2014. Levels of As in toenails, but not those in urine, were shown to be significantly correlated with hearing loss at 4 kHz [odds ratio (OR) = 4.27; 95% confidence interval (CI): 1.51, 12.05], 8 kHz (OR = 3.91; 95% CI: 1.47, 10.38) and 12 kHz (OR = 4.15; 95% CI: 1.55, 11.09) by multivariate analysis with adjustments for age, sex, smoking and BMI. Our experimental study further showed a significant association between As levels in inner ears and nails (r = 0.8113, p = 0.0014) in mice orally exposed to As, suggesting that As level in nails is a suitable index to assess As level in inner ears. Taken together, the results of our study suggest that As level in nails could be a convenient and non-invasive biomarker for As-mediated hearing loss in humans.

Increased expression level of Hsp70 in the inner ears of mice by exposure to low frequency noise

&NA; Previous studies showed that people in urban areas are possibly exposed to 60–110 dB of low frequency noise (LFN) defined as noise of ≤100 Hz in their daily life. Previous studies also showed increased health risks by exposure to high levels (130–140 dB) of LFN in animals. However, little is known about the health effects of exposure to an ordinary level of LFN. We biochemically and immunohistochemically assessed the effects of exposure to inaudible LFN for mice (12 h/day of 100 Hz LFN at 95 dB for 5 days), at a level to which people are possibly exposed in daily life, on a murine inner ear by targeting 9 stress‐reactive molecules. There was more than a 5‐fold increased transcript level of heat shock protein 70 (Hsp70) in the whole inner ear exposed to LFN. However, the transcript levels of the other 8 stress‐reactive molecules including Hsp27 and Hsp90 were comparable in LFN‐exposed and unexposed murine inner ears. Only the transcript level of Cebp&bgr; among the previously reported 4 transcriptional activators for Hsp70 expression was more than 3‐fold increased by LFN exposure. Hsp70 transcript expression levels in the inner ears 3 days after LFN exposure were comparable to those in unexposed inner ears. The protein level of Hsp70, but not the levels of Hsp27 and Hsp90, was also increased in the vestibule by LFN exposure. However, hearing levels as well as expression levels of Hsp70 protein in the cochleae were comparable in LFN‐exposed mice and unexposed mice. Our results demonstrated that the inner ear might be one of the organs that is negatively affected by stress from inaudible LFN exposure. Moreover, LFN exposure might increase Hsp70 expression level via Cebp&bgr; in the inner ear. Thus, Hsp70 and Cebp&bgr; levels could be candidates of biomarkers for response to LFN exposure. HighlightsEffects of exposure to an ordinary level of low frequency noise (LFN) were examined.Hsp70 level in the whole inner ear exposed to LFN was increased >5‐fold.The increased Hsp70 had returned to the basal level 3 days after LFN exposure.Effects of LFN on hearing and Hsp70 expression in cochleae are limited.Hsp70 and Cebp&bgr; levels may be biomarkers for response to LFN exposure.

Impairments of Inner Ears Caused by Physical Environmental Stresses.

The inner ears contain the organ of Corti, vestibule and semicircular canal. The organ of Corti is crucial for hearing, while the vestibule and semicircular canal play important roles in maintaining balance. Exposure to noise at excessive levels is known to cause damages of the inner ears, resulting in noise-induced hearing loss. On the other hand, noise levels (dB) are used for the evaluation of health risks by exposure to noise, although noise consists of sound with broad frequencies (Hz). Thus, information about the frequency-dependent effect of noise on health is largely unknown. In this review, we first introduce noise-induced hearing loss caused by exposure to audible noise. We then describe the imbalance in mice exposed to low-frequency noise (100 Hz).