Association of interleukin-1 gene polymorphisms with sudden sensorineural hearing loss and Ménière's disease.

Sudden sensorineural hearing loss (SSNHL) and Ménière's disease are the most common inner ear diseases in which the causes are unknown. As recent magnetic resonance imaging has demonstrated disruption of the blood-labyrinth barrier in these inner ear diseases, inflammatory reaction associated with increased permeability of the blood vessels may be involved. The genotypes of interleukin 1A (IL1A) (-889C/T; rs1800587) and interleukin 1B (IL1B) (-511C/T; rs16944) were determined using an allele-specific primer-polymerase chain reaction method in 72 patients with SSNHL, 68 patients with Ménière's disease, and 2202 control subjects living almost in the same area as the patients. A significantly higher prevalence of the IL1A-889T allele was observed in SSNHL and Ménière's disease compared with controls, although no significant difference in distribution of IL1B-511C/T genotypes was observed between the patients and controls. Adjusted odd ratios for SSNHL and Ménière's disease risks in the -889TT genotypes were 25.89 (95% confidence interval (CI) 12.19-54.98) and 18.20 (95% CI 7.80-42.46), respectively, after age and gender were taken as moderator variables. Our results suggested that IL1A is closely associated with susceptibility of SSNHL and Ménière's disease.

Morphological features of elderly patients with obstructive sleep apnoea syndrome: a prospective controlled, comparative cohort study.

OBJECTIVES: To investigate the pharyngeal morphologic features and its pathogenic role on obstructive sleep apnoea syndrome in the elderly population. DESIGN: Prospective controlled, comparative cohort study. SETTING: Territory referral centre. PARTICIPANTS: We enroled 320 consecutive patients with complaints of snoring who visited Nagoya University Hospital from January 2004 to December 2007. We also collected 26 control subjects aged over 60 years from community-dwelling people. MAIN OUTCOME MEASURES: We underwent a morphological evaluation, measurement of nasal resistance, assessment of daytime sleepiness and nocturnal polysomnography. RESULTS AND CONCLUSIONS: Two hundred and ninety-two patients were analysed. The constitution ratio of men, the body mass index and Epworth sleepiness scale were decreased with ageing. Tonsil size was reduced progressively with ageing. Retroglossal space was wider, and soft palate was lower in ≥ 60 year group than in < 40 year group. Retroglossal space was wide in elderly patients with sleep apnoea compared with control subjects. Tonsil size was not correlated to apnoea/hypopnoea index in ≥ 60 year group unlike the other generations. Modified Mallampati Score and tongue size were significantly but mildly correlated only in ≥ 60 year group. Width of fauces was correlated in all the groups. Multiple regression analysis showed that body mass index, age, gender, tonsil size and width of fauces were independent factors for apnoea/hypopnoea index. CONCLUSIONS: Morphologically, the tonsil could play a minor role but the width of fauces could play relatively a major role. Additionally, wide retroglossal space, low positional soft palate and large tongue size may be characteristics for elderly patients of obstructive sleep apnoea syndrome.

NADH-generating substrates reduce peroxyl radical toxicity in RL-34 cells.

There is general agreement that oxidative stress may induce apoptotic and necrotic cell death. Recently it has been shown that NADH can be considered an important antioxidant as it reacts with peroxyl and alkoxyl radicals under in vitro conditions. Therefore, in the present study we hypothesized that an increase in intracellular NADH using specific substrates will protect RL-34 cells against cytotoxicity of 2'-azobis (2-amidinopropane) dihydrochloride (AAPH), which is a peroxyl radical generating compound. Cells treated for 24 hours with 6.0 mM AAPH were severely damaged: mitochondria were vacuolated, and the level of free radicals significantly increased. Both apoptotic and necrotic cells were detected (11.1% and 11.4%, respectively) even after 5 hours of treatment. Pretreatment of the cells with substrates which increase the intracellular level of NADH, such as lactate, beta-hydroxybutyrate, and ethanol, distinctly inhibited AAPH-induced reactive oxygen species (ROS) formation and cell death. On the other hand, acetoacetate (AcA), which decrease the intracellular level of NADH, had opposite effects. Interestingly, NADH-generating substrates augment, while AcA reduced superoxide radical formation induced by AAPH. These results may suggest that although NADH generating substrates may exert some deleterious effects within a cell by inducing reductive stress, they diminish alkoxyl or peroxyl radical cytotoxicity. The protection is associated with a decrease in ROS formation measured by dichlorofluorescein, but with an increase in superoxide radical formation.

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria.

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.

Free radical-induced megamitochondria formation and apoptosis.

Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H2O2, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H2-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G1 phase, and apoptotic sub-G1 populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H2O2 and O2- from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell.

Z and W chromosomes of chickens: studies on their gene functions in sex determination and sex differentiation.

Since the discovery of SRY/SRY as a testis-determining gene on the mammalian Y chromosome in 1990, extensive studies have been carried out on the immediate target of SRY/SRY and genes functioning in the course of testis development. Comparative studies in non-mammalian vertebrates including birds have failed to find a gene equivalent to SRY/SRY, whereas they have suggested that most of the downstream factors found in mammals including SOX9 are also involved in the process of gonadal differentiation. Although a gene whose function is to trigger the cascade of gene expression toward gonadal differentiation has not been identified yet on either W or Z chromosomes of birds, a few interesting genes have been found recently on the sex chromosomes of chickens and their possible roles in sex determination or sex differentiation are being investigated. It is the purpose of this review to summarize the present knowledge of these sex chromosome-linked genes in chickens and to give perspectives and point out questions concerning the mechanisms of avian sex determination.

Insertion/deletion angiotensin converting enzyme gene polymorphism affects the microvascular structure of the kidney in patients with nondiabetic renal disease.

OBJECTIVE: It has been reported that the deletion allele of the insertion/deletion polymorphism of the angiotensin I converting enzyme gene is associated with increased cardiovascular risk and progressive renal disease, including immunoglobulin A nephropathy. We therefore investigated the relationship between angiotensin converting enzyme polymorphism and intrarenal microvascular structure in 56 patients with nondiabetic renal disease. METHODS AND RESULTS: We determined various cardiovascular hormones of the renin-angiotensin system and angiotensin converting enzyme gene polymorphism in 56 patients with nondiabetic renal diseases who underwent a renal biopsy. The patients were divided into three groups by angiotensin converting enzyme genotype (insertion/insertion, n = 21; insertion/deletion, n = 23; deletion/deletion, n = 12) using polymerase chain reaction methods. The angiotensin converting enzyme insertion/ deletion and deletion/deletion genotypes were associated with a significantly higher interlobular artery wall : lumen ratio than the insertion/insertion genotype (insertion/insertion 0.27 +/- 0.01, insertion/deletion 0.32 +/- 0.01, deletion/deletion 0.33 +/- 0.02; P < 0.05). Afferent arteriolar and tubulo-interstitial injury scores were similar among the three genotypes. Although serum angiotensin converting enzyme activity was higher in the deletion/deletion than in the other two genotypes (insertion/insertion 9.7 +/- 0.7, insertion/deletion 10.7 +/- 0.9, deletion/deletion 14.0 +/- 2.4 IU/I; P < 0.05), other factors of the renin-angiotensin system, including blood pressure and serum creatinine levels, were not different among the three groups. CONCLUSIONS: The angiotensin converting enzyme deletion/deletion genotype may be considered a risk factor for the development of microvascular wall thickening in nondiabetic renal diseases.

Molecular forms of plasma and urinary adrenomedullin in normal, essential hypertension and chronic renal failure.

OBJECTIVES: Human adrenomedullin precursor is converted to glycine-extended adrenomedullin (AM-Gly), an intermediate inactive form of adrenomedullin. Subsequently, AM-Gly is converted to active form of mature adrenomedullin (AM-m). The aim of the present study was to investigate (i) whether sex or age influences plasma and urinary AM-m and AM-Gly levels in normal subjects; (ii) the daytime variability of plasma AM-m and AM-Gly levels in normal subjects; (iii) AM-m and AM-Gly levels and its ratio in plasma and urine in normal subjects, individuals with essential hypertension (HT), and chronic renal failure (CRF); and (iv) the ratio of AM-m and AM-total (T) in plasma of various veins and aorta. METHODS: We measured plasma levels and urinary excretions of AM-m, AM-Gly and AM-T (AM-m + AM-Gly) by recently developed immunoradiometric assay in normal subjects (n = 81), HT (n = 28) and CRF (n = 30). We also determined the molecular forms of plasma adrenomedullin taken from various sites during angiography in patients with suspected renovascular hypertension (n = 9). RESULTS: There were no differences in plasma and urinary excretions of two molecular forms of adrenomedullin among sexes or ages in normal subjects. There was no daytime variation of plasma two molecular forms of adrenomedullin in normal subjects. Plasma AM-m, AM-Gly and AM-T levels were increased in patients with HT and CRF compared with normal subjects, whereas urinary AM-m, AM-Gly and AM-T excretions were decreased in patients with HT and CRF compared with normal subjects. Urinary AM-m: AM-T ratios were significantly higher than plasma AM-m: AM-T ratios. Plasma AM-m and AM-T levels taken from various veins were similar, and they were significantly higher than those of aorta, although there were no differences in plasma AM-Gly levels between aorta and veins. CONCLUSIONS: These results suggest that in normal subjects, and individuals with HT and CRF: (i) plasma and urinary excretions of AM-m and AM-Gly are not affected by age or sex; (ii) AM-m in parallel with AM-Gly is increased; (iii) urine contains a higher percentage of active adrenomedullin than plasma; and (iv) plasma AM-m may be partly metabolized in the lung.

Synthesis and antihypertensive activity of stereoisomers of 4-piperidyl-1,3-dihydro-2-oxo-2H-benzimidazoles. Enhanced potencies of (+) isomers.

To elucidate the relationship between the pharmacological activity and stereochemical structure, we resolved 1-[2-(3-,4,5-trimethoxyphenyl)-2-hydroxy-1-methylethyl]-4-(1,3-dihydro-2-oxo-2H -benzimidazol-1-yl)piperidine (1 and 2) and 1-[2-(3,4-dimethoxyphenyl)-2-hydroxy-1-methylethyl]-4-(1,3-dihydro-2-oxo-2H-benzimidazol-yl)piperidine (3), which produced hypotensive effects mainly through their alpha-blocking actions. Threo isomers 1 and 3 were resolved via diastereomeric carbamates. Erythro isomer 2 was obtained by an oxidation and reduction sequence from optically active 1. No significant difference was found between the pharmacological activities of the threo and erythro isomers of the corresponding compounds. However, a clear difference was found between the pharmacological activities of the optical isomers. Difference was most clearly shown in the hypotensive actions of normotensive rats and in alpha-adrenergic blocking activities of isolated rat vas deferens. In these actions, (+) isomers were always more potent than the corresponding (-) isomers.

Positive association of CYP11B2 gene polymorphism with genetic predisposition to essential hypertension.

Predispositions to essential hypertension and cardiovascular diseases are possibly associated with gene polymorphisms of the renin-angiotensin system. Gene polymorphisms of angiotensinogen and angiotensin-converting enzyme genes have been suggested to be risk factors for hypertension and myocardial infarction. Concerning the polymorphism of aldosterone synthase (CYP11B2) gene, earlier studies have shown inconsistent results in terms of its relation to hypertension. In the present case-control study, we investigated the association of -344T/C polymorphism in the promoter region of human CYP11B2 gene with genetic predisposition to hypertension. The genotype of -344T/C polymorphism was determined in essential hypertension subjects (n=250) and normotensive subjects (n=221). The distributions of three genotypes (TT, TC, and CC) were significantly different between the hypertensive and the normotensive groups (chi(2)=9.61, P=0.008). Namely, the frequency of C allele was higher in the hypertensive patients than in the normotensive subjects (34.2 vs 26.5%, P=0.010). Our data suggest that the -344C allele of CYP11B2 gene polymorphism is associated with the genetic predisposition to develop essential hypertension.

Selective synthesis of 4-methoxyestrogen from 4-hydroxyestrogen.

The introduction of an oxygen atom into the C-6 position of 4-hydroxyestrogen allowed for the selective methylation of the two phenolic hydroxyl groups. When the 6-oxo derivative of 4-hydroxyestrone was benzylated in ethanol, only the 3-monobenzyl ether was obtained without formation of the 4-monobenzyl ether. Moreover, the 6-carbonyl group was further reduced to methylene almost quantitatively in the reaction of 4-acetoxy-6-oxoestrone 3-benzyl ether derivative with sodium borohydride. Therefore, 4-methoxyestrogen was synthesized by essentially combining these two reactions.

Characterization of antisera to 2-hydroxyestradiol and 4-hydroxyestradiol using 6-(O-carboxymethyl)oxime- and 17-hemisuccinate-bovine serum albumin conjugates and radioimmunoassay.

For radioimmunoassay of the catechol estrogens, four hapten-bovine serum albumin (BSA) conjugates were prepared from 6-oxo-2-hydroxyestradiol 6-(O-carboxymethyl)oxime, 2-hydroxyestradiol 17-hemisuccinate, 6-oxo-4-hydroxyestradiol 6-(O-carboxymethyl)oxime and 4-hydroxyestradiol 17-hemisuccinate by coupling with BSA, employing the mixed anhydride method. The antisera elicited in rabbits by immunization with these antigens showed high affinity and specificity for 2-hydroxyestradiol or 4-hydroxyestradiol with cross-reactivities to a few structurally related estrogens. The specificity of antisera obtained is discussed in relation to the site of attachment of the hapten to BSA.

Age-related changes in the susceptibility to clofibric acid, a hypolipidemic agent, of male rat liver.

Age-related changes in the susceptibility to clofibric acid were investigated in male F344 rats of 8, 52, and 117 weeks old. Hepatomegaly, decrease of serum total cholesterol and triglyceride, increase of the total cytochrome P-450 contents, induction of the activities of microsomal omega-hydroxylation and peroxisomal beta-oxidation, proliferation of smooth endoplasmic reticulum and peroxisomes were detected in 8- and 52-week-old rats. In 117-week-old rats clofibric acid treatment resulted in decrease of serum total cholesterol, elevation of the activities of microsomal and peroxisomal enzymes, and slight proliferation of peroxisomes. These results suggest that the susceptibility of the male F344 rat liver to clofibric acid decreases in 117-week-old rats, though the effect is still recognizable.

Change of the sex-dependent response to clofibrate in F344 rat liver during postnatal development.

Although it has been reported that male rats are more responsive than females to peroxisome proliferation induced by clofibrate, these sex differences have been confirmed in young adult rats. Using 4-, 8-, and 12-week-old F344 rats, postnatal change of the sex-dependent response to clofibrate was investigated. These animals were administered 200 mg/kg body wt./day clofibrate by gavage for 7 days. In 4-week-old rats clofibrate-dependent changes (hepatomegaly, induction of hepatic microsomal and peroxisomal enzymes, proliferation of smooth endoplasmic reticulum and peroxisomes of hepatocytes) were slight in both sexes. In 8- and 12-week-old rats clofibrate-induced changes of males were moderate, whereas those of females were slight. These results suggest that the responsiveness of immature rat to clofibrate is weak and in males the susceptibility is gradually strong during postnatal development.

Effects of alpha-tocopherol on cisplatin-induced ototoxicity in guinea pigs.

Cisplatin (CDDP), an antitumor agent widely used in the treatment of head and neck cancers, has dose-limiting side effects such as ototoxicity and nephrotoxicity. Recently, evidence has been accumulated to demonstrate that these side effects are closely related to oxidative stress. In the present study, we attempted to suppress CDDP-induced ototoxicity and nephrotoxicity in guinea pigs by administering alpha-tocopherol, a naturally occurring antioxidant. Hartley albino guinea pigs (250 approximately 300 g) were treated with CDDP (4 mg/kg intraperitoneally (I.P.)) for 3 days in the presence and absence of alpha-tocopherol (50 mg/kg I.P.) injection for 6 days. The combined treatment of animals with alpha-tocopherol distinctly improved the CDDP-induced side effects. These were: loss of Preyer's reflex at high frequencies; distinct elevation of auditory brain stem response threshold at 16 kHz; increased lipid peroxidation in the cochlea determined by the malondialdehyde-thiobarbituric acid method; substantial losses of outer hair cells in the basal and second turns of the cochlea; fragmentation of nuclear DNA detected by the TUNEL method in cochlear hair cells and cells in the stria vascularis; and increases in serum BUN and Cr. These results strongly suggest that alpha-tocopherol suppresses CDDP-induced ototoxicity and nephrotoxicity via the suppression of the increased production of reactive oxygen species.

Spontaneous globoid mineralization in the cerebellum of rats.

Cerebellar globoid mineralization in two rats was examined by light and electron microscopy, and by X-ray microanalysis. The mineralization was round to oval in shape; it varied in size and was positive for the periodic acid-Schiff and von Kossa reactions. Ultrastructurally, a concentric lamellar structure was prominent in moderately electron-dense depositions. Elemental analysis revealed the presence of large amounts of calcium and phosphorus, and small amounts of zinc, potassium and aluminum.

Male reproductive toxicity study of nitrazepam in rats.

The main focus of this study is the optimal administration period concerning toxic effects on male fertility in rats. To assess functional and morphological changes induced in the testis by nitrazepam, male rats were administered the drug at doses of 0, 20, 40 or 80 mg/kg during pre-mating periods of 2, 4 or 9 weeks and then the 2 weeks of mating. At the end of the administration period the animals were sacrificed and sperm number, motility, abnormalities and histopathological changes in the testis were examined. Decreases in testis weight, epididymis weight, number of sperm in the testis and sperm motility were observed in the 40 and 80 mg/kg sections of the 2, 4 and 9 week pre-mating treated groups. Mating with untreated females revealed no adverse effects on copulation rate in any group; however, a remarkable decrease in pregnancy rate was noted in the 80 mg/kg section of the 2, 4 and 9 week treated groups. On histological examination, various degrees of localized necrosis in the seminiferous epithelium and Leydig cell hyperplasia were observed in the testis. No clear changes were observed in the 20 mg/kg section of the 2 week pre-mating administration group, but at the 4 week time point, necrosis of spermatogenic cells began to appear. The primary morphological event was evident in spermatocytes with necrosis of the cytoplasm observed from 4 weeks after administration of nitrazepam, although sperm motility and sperm head counts were unaffected. From these findings, examination of sperm characteristics and histopathological changes in the testis are important parameters for evaluation of drugs inducing testicular damage. We conclude that a 4 week administration period is sufficient to detect effects of nitrazepam on male fertility.

Age-related changes in thyroid lesions and function in F344/DuCrj rats.

Age-related thyroid changes, such as those in weight, histology, morphometry, and hormonal concentrations were evaluated in 460 male and 460 female F344 rats from 9 to 109 weeks of age. The absolute weight of the thyroid increased with age in both sexes, but the relative weight was unaffected by age. Ectopic thymus and ultimobranchial cyst were observed in rats of both sexes from 9 to 109 weeks of age. The incidence of both congenital anomalies decreased with age. The incidence of follicular cyst, which was first observed in rats at 20 weeks of age, increased at 109 weeks. Hyperdistention of the follicle with infiltration of macrophages in the lumen and deposition of brown pigments in the cytoplasm of epithelial cells were observed in a few rats at 59 weeks of age. These follicular lesions were found in 29% of the males and 7% of the females at 109 weeks. The first thyroid tumor was seen at 59 weeks of age. In 109-week-old rats, 5% of males had follicular tumors, and 18% of males and 10% of females had C-cell tumors. At 82 weeks of age, the follicular area and the area of the follicular lumen increased, and the height of follicular epithelial cells decreased. Serum T3, T4 and TSH concentrations decreased with age and were significantly reduced at 82 weeks. These results suggest that in F344 male rats the thyroid structural and functional changes occurred with age, and thyroid function decreased after 82 weeks of age.

Vestibular and cochlear toxicity of aminoglycosides--a review.

Recently, there have been many reports describing the efficacy of intratympanic aminoglycoside injection for the treatment of intractable vertigo in patients with Ménière's disease. However, the number of injections and the amount of drug injected varies, with concomitant variation in the side-effect of hearing deterioration. To identify drugs that are more selectively vestibulotoxic, we have reviewed the ototoxicity of aminoglycosides, focusing on differences between vestibulo- and cochleotoxicity. At present, the basis for the different effects of each drug is unknown. The mechanisms of vestibulo- and cochleotoxicity are deemed worthy of further study.

A case of intracytoplasmic edema of follicular epithelial cells in rat thyroid.

Hydropic change of follicular epithelial cells in the thyroid was observed in a female Fischer 344 rat. Microscopically follicular epithelial cells were characterized by edematous swelling with weakly eosinophilic and homogeneous cytoplasm. The cytoplasm was negative for periodic acid-Schiff reaction, and thyroxine- and thyroglobulin-immunohistochemical reactions. Electron-microscopically, a small amount of amorphous substance was noted in remarkably dilated rough-surfaced endoplasmic reticulum (r-ER), and slight regressive changes of cytoplasmic organella were also observed. These morphological changes may indicate that focal intracytoplasmic edema was occurred in r-ER, and that the change belonged to hydropic degeneration of the thyroid follicular cells in the thyroid.