Association between estrogen receptor alpha polymorphisms and equol production, and its relation to bone mass.

To investigate the relationship between estrogen receptor polymorphisms and equol production and its effect on bone turnover, 139 workers (mean age 38.3+/-11.1 years) in Japan were recruited. Bone mineral density (BMD), bone turnover markers, and serum equol were measured at a health examination. DNA samples were prepared to detect the estrogen receptor alpha (ERalpha) polymorphism and were digested by PvuII. The number of equol producers was 57. No statistically significant differences were observed in bone mineral density and bone turnover markers between each ERalpha polymorphism and equol production. Since the adjusted odds ratio indicated that interaction itself decreased the risk of osteosono-assessment index (OSI) reduction using logistic regression analysis, further analysis was performed divided by each ERalpha polymorphism. Although the crude odds ratio showed no relationship between equol producers and non-producers, the adjusted odds ratio showed that equol producers with ERalpha pp had a significantly decreased risk of OSI reduction. Although this study was cross-sectional, both equol production and ERalpha polymorphism are closely associated with each other in relation to BMD.

Association between serotonin transporter gene polymorphisms and depressed mood caused by job stress in Japanese workers.

To estimate the genetic factors influencing depressed mood caused by job stress, a total of 243 employees at a manufacturing company and a local hospital in Japan (mean age 40.8+/-10.3 years) were recruited with informed consent. The Brief Job Stress Questionnaire was used to assess the present status of stress. Alcohol consumption and smoking were assessed as lifestyle factors. DNA samples were prepared to detect gene polymorphisms of serotonin transporter (5HTT), aldehyde dehydrogenase 2, D2 dopamine receptor, and cytochrome p450 2A6. The relationship between job stress, lifestyle factors and these polymorphisms was assessed for each gender. The level of depressed mood for female subjects was significantly higher among the carriers of two short (s/s) alleles of the 5HTT regulatory region compared with the carriers of one (s/l) or two (l/l) long alleles (Mann-Whitney U test, p<0.05). The odds ratio of depressed mood also confirmed this relationship for the female subjects, whereas there was no relationship for the male subjects. When social support was taken into consideration, the depressed mood score for those who had high support was significantly lower than for those who had low support, irrespective of 5HTT polymorphisms and gender. Job stress may elicit biological responses that contribute to depressed mood in relation to 5HTT polymorphisms, and social support may reduce depressed mood irrespective of 5HTT polymorphisms.

Mouse model of paraquat-poisoned lungs and its gene expression profile.

Paraquat (PQ)-induced pulmonary toxicity is characterized by initial development of pulmonary edema, infiltration of inflammatory cells, and damage to the alveolar epithelium, which may progress to severe fibrosis. However, the exact role of PQ in the progression of the pathogenesis has not been clearly established. To understand the mechanism of PQ in pulmonary toxicity, we developed an animal model of PQ-induced lung injury by intranasal instillation of PQ solution using C57Black/6J mice. Twenty microliters of PQ solution (0.01, 0.01, and 0.04 mg/mouse) was applied through the nares, and the same amount of vehicle was applied in control mice. The pathological progression of lung pathology in our mouse model was very similar to that of patients suffering from PQ poisoning. The lungs of some animals exposed to PQ showed acute fulmination, resulting in death from 5 days post-exposure, but others showed a more protracted injury, resulting in typical pulmonary fibrosis at 3 weeks. Using this PQ-poisoned mouse model, we examined the gene expression at the initial destructive phase (within 5 days) that fibrosis has not completely developed. We prepared RNAs after 6h, 24h, and 5 days and examined the changes of the expression levels for 45 selected genes. The genes showing >2-fold increase at 6h or a time-dependent decrease during this experimental period may be the early markers for the destructive phase. These genes are Mt1, Mt2, Hmox1, Gcl, GR, IL-6, IL-13, Txn1, Fas, FasL, Lpin2, Mmp1a, Mmp12, Sfp-B, Sfp-D, CAT, EC-SOD, GST, and Pltp. On the other hand, the genes involved in the development of fibrosis, such as procollagen, Fn1, Eln, SMA, and Mmp9, Timp1 were significantly increased on day 5, not at 6h nor at 24h, after PQ treatment (the late marker). The genes showing a significant increase (Mmp3 and Mmp8) or decrease (VEGFA) at 24h and 5 days and not at 6h may be also the late markers. These changes in gene expression, which are equalled to functional activities of proteins, will be the targets for future studies focused on the development on PQ-induced pulmonary damage.

Gene expression in rat lungs during early response to paraquat-induced oxidative stress.

Paraquat (PQ) is a well-known pneumotoxicant and provides an established model of oxidative stress. This study focused on the transcriptional response to PQ-driven oxidative stress in rat lungs during an early phase post-injection. Rats were sacrificed at 3 h and 24 h after PQ injection (i.p., 20 mg/kg b.w.), and at 3 h after a second injection (i.p., 20 mg/kg b.w.). The left lungs were rapidly excised and used immediately for RNA preparation. The lung tissues did not show any pathological damage microscopically. Differential expression of RNAs in the lung at 3 h was investigated using a DNA array system. Fifteen genes showed a >1.7-fold change in expression level, which was confirmed by real-time PCR. Five genes related to oxidative stress, TRX, HO-1, GST-Yc, NQO-1, and RL/IF-1, and one gene, CLK3, whose function is unknown, showed a significant increase in their expression due to PQ injection. Two genes, HO-1 and NQO-1, that showed 3- and 2-fold increases at 3-h post-injection, were localized by immunohistochemistry. HO-1 was expressed in the bronchial epithelial cells, some type II cells and macrophages of control lungs, and the cells, especially the bronchial epithelial cells, were strongly stained 3 h following PQ treatment. Immunohistochemical analysis of NQO-1 also showed an increase in positive staining in the bronchial epithelial cells of PQ-treated lung sections. The expression of CYP2C6, 2C7, and 2C12, which are specific to or dominant in female liver, decreased markedly, while the male-specific CYP2C13 and 2C11 showed an increase or no effect. Further investigation is needed to clarify the role of these CYP2C family genes on the early phase of PQ toxicity. These results indicate that the acclimation to oxidative stress is already a highly complex process at the onset of PQ-induced damage and that the genes described herein may prove to be major contributors to the subsequent pulmonary fibrosis.

Changes in gene expression level for defense system enzymes against oxidative stress and glutathione level in rat administered paraquat.

The herbicide paraquat (PQ) forms reactive oxygen species during enzymatic activation. We examined the effect of PQ on the relative levels of gene expression of antioxidant enzymes and glutathione (GSH) status in lungs of rats exposed to 20 mg/kg PQ. At 16 h after PQ intake, the mRNA expression level of glutathione reductase (GR) showed the greatest increase, and those of catalase (CAT) and manganese-superoxide dismutase (MnSOD) showed more modest increases. In contrast, PQ had little or no effect on the levels of mRNAs for copper/zinc-superoxide dismutase (CuZnSOD) and glutathione peroxidase (GPX). These findings indicate that CAT and MnSOD are coordinated and play a major role in removal of oxidants. On the other hand, PQ caused a significant increase in the GSH level in the lungs, but not in the liver. This increase in the lungs was, at least in part, caused by stimulation of the gamma-glutamylcysteine synthetase gene. However, the expression of GPX mRNA was not stimulated as described above. Because GSH is a substrate for GPX and serves as a scavenger of hydroxyl radicals, the increase in GSH as well as GR expression may be insignificant. This imbalance may be a result of oxidative stress due to PQ.

Effect of vitamin K2 on the development of stress-induced osteopenia in a growing senescence-accelerated mouse prone 6 strain.

Vitamin K2 (VK2) has been used as a therapeutic agent for osteoporosis, since it has been suggested to be able to reduce the frequency of fractures by improving bone quality; however, bone turnover is strictly regulated by various cytokines and hormones. In the present study, the effect of menaquinone-4 (MK-4) on bone turnover was investigated using the senescence-accelerated mouse prone 6 (SAMP6) strain. Since water-immersion restraint stress (WRS) causes a significant decrease in bone mineral density (BMD), WRS was used as the bone resorption model in the SAMP6 strain. Six-week-old SAMP6 male mice were divided into the following three groups: Control, WRS and WRS + MK-4. WRS was performed for 6 h per day, 5 times a week, for 4 weeks. Following WRS, MK-4 (30 mg/kg) was injected subcutaneously 3 times a week for 4 weeks. No growth retardation was observed in the WRS groups as compared with the control group. In the WRS groups, the BMD was significantly lower than that in the control group. The levels of bone formation and resorption markers were increased in the WRS groups, indicating that WRS reduced the BMD by promoting high bone turnover. A bone histomorphometrical examination showed that the trabecular (Tb) bone mass in the secondary spongiosa at the distal femur was significantly reduced in the WRS mice, and this reduction was abrogated by MK-4 treatment. Specifically, the Tb bone reduction was caused by the activation of osteoclasts (Ocs), and Oc activity was suppressed by MK-4. The number of osteoblasts and the mineral apposition rate were significantly increased in the WRS and WRS + MK-4 mice, suggesting that WRS triggered a significantly higher mineral apposition rate. These results indicate that MK-4 can induce recovery from the bone mineral loss caused by WRS treatment. Further studies are required to clarify the association between bone quality and MK-4.

Early differential gene expression of rat lung after exposure to paraquat.

Paraquat (PQ), a quaternary nitrogen herbicide, is highly toxic to humans and animals. Acute poisoning and death due to PQ exposure have been reported over the past few decades. Excessive production of oxygen free radicals has been proposed to play an important role in the pulmonary pathology. The aim of the present work was to evaluate the implications for genes that are regulated by oxidative stress at the early stage of PQ exposure in rat lungs. We performed differential display RT-PCR (DD-PCR) on total RNA extracted from rat lungs after injection of 20mg per kg body weight. The experimental DD-PCR conditions, primer length and annealing temperature, were adjusted to improve reproducibility, and 19 differentiated clones were isolated. Sequence analysis followed by conventional RT-PCR and real-time RT-PCR analyses were used to confirm the results. Four clones were finally determined to be significantly affected. These genes were mRNAs for plasma phospholipid transfer protein (PLTP), CL1BA protein, (latrophilin: LPH), and alphaII-spectrin as well as one unknown gene. We demonstrated the distribution of mRNA expression of one gene, LPH, in lung tissues. The present study suggests that 20mg per kg intraperitoneal PQ affects the expression of numerous genes in the lung at 3 h, the onset of pulmonary injury, and that the four genes specified may be major contributors to serious lung injury due to PQ exposure.

Hypervariable locus of the 3'-flanking region of the neurotensin receptor gene: an effective region for personal identification in forensic practice.

We examined the complex short tandem repeat (STR) locus at the 3'-flanking region of the neurotensin receptor (NTR) gene. The polymorphism of this locus was first reported as a simple tetranucleotide repeat variation by Le et al., but it also offers a surprisingly informative variation, that permits reliable individual identification by two complementary strategies: fluorescent-labelled polymerase chain reaction (PCR)/electrophoresis and direct sequencing of the PCR products. We determined the alleles in 203 Japanese by fluorescent-labelled PCR/electrophoresis. Determination was based on their length with a reliability of +/-1 bp, and the frequency of each allele was very low. Sequencing analysis further grouped these alleles in detail. Sequencing demonstrated that the locus varied by six repetitive units and three insertion/deletion positions of nucleotide fragments. We detected multiple alleles having different structures even in the same allele length. We found structural differences in homozygous alleles having the same base pair size. We also determined that apparently homozygous alleles were heterozygous from sequencing electropherograms showing an overlap of nucleotides or +/-1 bp difference. These results indicate that this locus is structurally hypervariable in addition to having allelic length variations, promising a great advance in individual identification in forensic practice.

Does methamphetamine affect bone metabolism?

There is a close relationship between the central nervous system activity and bone metabolism. Therefore, methamphetamine (METH), which stimulates the central nervous system, is expected to affect bone turnover. The aim of this study was to investigate the role of METH in bone metabolism. Mice were divided into 3 groups, the control group receiving saline injections, and the 5 and 10mg/kg METH groups (n=6 in each group). All groups received an injection of saline or METH every other day for 8 weeks. Bone mineral density (BMD) was assessed by X-ray computed tomography. We examined biochemical markers and histomorphometric changes in the second cancellous bone of the left femoral distal end. The animals that were administered 5mg/kg METH showed an increased locomotor activity, whereas those receiving 10mg/kg displayed an abnormal and stereotyped behavior. Serum calcium and phosphorus concentrations were normal compared to the controls, whereas the serum protein concentration was lower in the METH groups. BMD was unchanged in all groups. Bone formation markers such as alkaline phosphatase and osteocalcin significantly increased in the 5mg/kg METH group, but not in the 10mg/kg METH group. In contrast, bone resorption markers such as C-terminal telopeptides of type I collagen and tartrate-resistant acid phosphatase 5b did not change in any of the METH groups. Histomorphometric analyses were consistent with the biochemical markers data. A significant increase in osteoblasts, especially in type III osteoblasts, was observed in the 5mg/kg METH group, whereas other parameters of bone resorption and mineralization remained unchanged. These results indicate that bone remodeling in this group was unbalanced. In contrast, in the 10mg/kg METH group, some parameters of bone formation were significantly or slightly decreased, suggesting a low turnover metabolism. Taken together, our results suggest that METH had distinct dose-dependent effects on bone turnover and that METH might induce adverse effects, leading to osteoporosis.

Cardiotoxicity of methamphetamine under stress conditions: comparison of single dose and long-term use.

Methamphetamine (METH) abuse continues to be a worldwide problem, damaging the myocardial tissues, as well as the brains of individual users. In addition, stressors that increase drug cravings also contribute to cardiovascular diseases. The aim of the present study was to examine the myocardial effects of METH, including METH­stress interactions and particularly, the effect of METH RNA expression in the heart. The study also aimed to compare single dose (acute) and long-term (chronic) treatments. Mice were divided into the control (C), METH injection (M), stress exposure (S) and METH plus stress (MS) groups and subjected to an acute water­immersion restraint stress or a mixed chronic stress composed of restraint, electric foot­shock and temperature change. METH was injected at 30 mg/kg (the acute study) or 10 mg/kg intraperitoneally (i.p.) three times per week (the chronic study). The results demonstrated that METH induced more deleterious effects in the myocardial tissues during acute and chronic administrations when under stress conditions. Heat shock proteins (Hsps) played a critical role in the acute phase, while numerous genes, including anti­oxidant, anti­apoptotic and physiological function genes, played significant roles in the chronic phase. These results indicate that METH abuse, ranging from episodes of binge abuse to chronic abuse over several years, may cause severe myocardial damage in human users under stress.

The effect of CAG repeat polymorphism in the glucocorticoid receptor on stress responses of mice exposed to water-immersion restraint stress.

Exposure to stress activates the hypothalamus-pituitary-adrenal (HPA) axis, which is followed by an increase in production of its end product, corticosterone, considered to be the most important glucocorticoid (GC) in rodents. Glucocorticoid receptor (GR) signaling has been suggested as a potential mechanism responsible for the pathogenesis of many clinical disorders. Here, we investigated the involvement of the GR polymorphism in stress response. ICR mice were screened by genomic PCR, bred, and divided into 3 groups according to the GR polymorphism, GRwt/wt, GRwt/Qn, and GRQn/Qn. Mice were exposed to water-immersion restraint stress (WRS), and then examined for gastric mucosal lesions, serum corticosterone, serum cytokines and serum Hsp70 levels. Male mice with GRQn/Qn exhibited a significantly greater gastric lesion index than those with GRwt/wt at 6 h of WRS. Stress-induced corticosterone output achieved peak levels at 3 h, after which it was downregulated. The serum level in the control group was GRwt/wt >GRwt/Qn >GRQn/Qn, whereas the order at 6 h of WRS was reversed, GRQn/Qn >GRwt/Qn >GRwt/wt, suggesting that the GRwt allele responded rapidly to stress. The IL-6 levels of each polymorphic line increased at 3 h and particularly at 6 h. On the contrary, the IL-10 levels in GRwt/wt and GRwt/Qn increased following exposure to WRS, whereas that in GRQn/Qn showed no change. The Hsp70 levels in mice with GRQn allele particularly increased at 6 h of WRS, and the concentration in GRQn/Qn significantly increased as compared to that in GRwt/wt. These results suggest that the GR gene polymorphism has a significant impact on the stress-induced output, including the gastric lesion index, corticosterone, cytokines, and Hsp70 levels in serum. The present study provides insights into the role of GR in individual responses to stress.

Alterations in bone turnover by isoflavone aglycone supplementation in relation to estrogen receptor α polymorphism.

Soybean isoflavones have structural similarity to estrogen and have attracted much attention due to their prevention of postmenopausal symptoms. It is critical for women to maintain a high bone mineral density (BMD) prior to menopause to prevent osteoporosis. In the present study, the effect of isoflavone aglycone (IA) supplementation on bone turnover was examined in relation to the estrogen receptor α (ERα) polymorphism. Natural isoflavones are glycosides that must be hydrolyzed to aglycones by intestinal microflora to have an effect. To avoid interference by flora, IA (30 mg/day) (but not isoflavones) or a placebo were administered as a supplement for 3 months to a Japanese population consisting of 81 premenopausal women. Due to variations in the intestinal flora, some but not all subjects were able to further metabolize IA into equol. Differences between equol producers and non-producers were also considered. To estimate BMD, the osteo-sono-assessment index (OSI) was determined by measuring bone density at the calcaneus and levels of bone biochemical markers (bone-specific alkaline phosphatase, α-carboxylated osteocalcin, undercarboxylated osteocalcin and deoxypridinoline) before and after supplementation. DNA samples from the subjects were examined for the presence of the XbaI restriction fragment length polymorphism (RFLP) in intron 1. According to univariate analysis, IA had a favorable effect on the OSI of subjects with the X allele, with X designated RFLP undigested by XbaI, although the difference was not statistically significant. Alterations in the levels of bone biochemical markers were also not significant. Thus, a further logistic regression analysis was performed. This indicated that subjects with the XX homozygote administered the IA supplement were less likely to have reduced OSI values. Although equol has been proposed to have the highest phytoestrogen activity, its effect was not apparent. Thus, low-dose IA supplementation is useful for maintaining BMD in premenopausal XX subjects, independent of equol.

Methamphetamine protects against stress-induced gastric mucosal lesions in mice.

Stress-induced gastric mucosal injury is a common clinical entity. On the other hand, abuse of methamphetamine (MA) represents a growing social problem. MA users are frequently in stressful situations. In this study, we examined the effects of MA on gastric injury, corticosterone level and immunomodulation using a water-immersion restraint stress (WRS) mouse model that is well known to induce gastric lesions. Mice were randomly divided into five groups: (1) the normal group, (2) the 3 hour (3 h)-WRS group, (3) the 6 hour (6 h)-WRS group, (4) the MA (3 mg/kg) plus 6 h-WRS group and (5) the MA (30 mg/kg) plus 6h-WRS group. As expected, most animals examined (above 90%) showed gastric injury after the WRS exposure. However, administration of MA at both 3 and 30 mg/kg resulted in significant suppression of the injury. The corticosterone levels were increased by exposure to the stress and/or MA, but there was no difference between these groups. The levels of the serum cytokines IL-6, IL-10 and TNF were increased by WRS, and were markedly increased by MA plus WRS; in particular, the level of IL-6 was synergistically increased. On the contrary, the level of IL-1beta was significantly decreased by WRS and MA plus WRS. This is the first report showing the protective effect of MA on stress-induced gastric injury, although further study is necessary to resolve the mechanism of MA-driven suppression of the injury.

5HTT polymorphisms are associated with job stress in Japanese workers.

Recently, the number of workers who suffer from job stress was increasing in Japan because of a prolonged recession, increasing number of elderly workers, and structural reorganization of companies. On the other hand, polymorphism associated with depression or alcoholism was detected. Relationship between job stress and these polymorphisms were investigated. Brief job stress questionnaire was assessed for 243 employees who worked at a manufacturing company and a local hospital in Japan (mean age 40.8 years). Alcohol consumption and smoking habit were assessed as lifestyle factors. DNA samples were prepared to detect polymorphisms of 5HTT, aldehyde dehydrogenase 2 (ALDH2), D2 dopamine receptor (DRD2), and cytochrome p450 2A6 (CYP2A6) genes. The level of depressed mood by job stress was significantly higher among carriers of two short alleles of the 5HTT regulatory region compared with carriers of one or two long alleles (Mann-Whitney U, p<0.05). In a logistic regression analysis, the s/s allele of the 5HTT had a tendency to be a risk of depressed mood. When subjects had high supervisor's support, depressed mood was significantly lower irrespective of 5HTT polymorphism. Job stress may elicit biological responses that contribute to depressed mood in relation to 5HTT polymorphism.

Paraquat-induced gene expression in rat kidney.

Paraquat, one of the most widely used herbicides, is highly toxic to humans and animals. There is much information regarding its toxic effects on the lungs, but less is known about its toxicity in other organs. Paraquat is thought to play pivotal roles in the pathophysiology of acute renal failure and the progression of chronic kidney disease. We investigated the effects of paraquat on gene expression in the kidneys of rats treated with paraquat using a DNA array system, and the gene up-regulation observed was confirmed by quantitative real-time RT-PCR. Rats were sacrificed at 3, 24 h after the first injection (20 mg/kg), and at 3 h after the second injection. Expression of six genes had increased significantly by 3 h after the first injection: metallothionein-1 (MT-1), phosphoenolpyruvate carboxykinase, Na/K-transporting ATPase beta1 subunit, glutamate oxaloacetic transaminase, glutathione-S-transferase, and heme oxygenase-1 (HO-1). The transcription levels of MT-1 and HO-1 showed the biggest increases, but the increases did not continue until 24 h after injection, and the second injection had less effect than the first. Up-regulation of MT-1 and HO-1 mRNA levels was confirmed at the protein level. We observed a paraquat-induced increase of these proteins at 3 h post-injection, whereas this level did not continue until 24 h, as observed in RNA levels. The MT-1 protein in kidneys had been consumed. In addition, the protein level due to the second injection did not increase to the same level as that due to the first injection. These results suggest that protection against paraquat injury is mediated by induction of expression of some genes, and suppression on the induction of MT-1 and HO-1 may explain the injury observed due to paraquat intake. This is the first report of inducible pathways of defense against paraquat-induced oxidative stress in the kidney.

Paraquat-induced gene expression in rat lung tissues using a differential display reverse transcription-polymerase chain reaction.

Increased formation of reactive oxygen species is a cause of paraquat (PQ)-induced injury and also provides a link between the signaling pathways and transcriptional events that regulate the expression of a large number of genes. However, the molecular mechanisms involved in PQ-induced injury remain unclear. To investigate the changes in gene expression at the onset of PQ injury, we used the differential display-polymerase chain reaction (PCR) method. Rats were treated intraperitoneally with 20 mg/kg PQ, and after 3 h the lungs were immediately excised. Samples of mRNA from normal and treated rats were used to prepare radiolabeled cDNAs, which were electrophoresed. Then the transcription levels were compared. We isolated 26 fragments of cDNA that were potentially affected by PQ, and determined their nucleotide sequences. Six clones of interest were selected and analyzed further. The reverse transcript-PCR based on their sequence information confirmed the differential expression for five clones: four clones were up-regulated and one was down-regulated. We were particularly interested in two genes that had homology with the known gene: TATA box-binding protein-associated factor, RNA polymerase II, B, 150 kDa (TAFIIB), and a candidate gene for lipodystrophy, Lpin2. Both genes were significantly up-regulated within 3 h of PQ intake and the stimulation continued during our 24-h observation period. In addition, up-regulation of Lpin2 was observed in the lungs, but not in the liver and kidneys. In situ hybridization using lung sections showed that the expression of both genes was strongly visualized in Clara cells and in alveolar macrophages. These findings suggest a stimulation of transcription levels and changes in lipid metabolism in Clara cells and in macrophages in the lungs, which result in their playing a crucial role at the onset of PQ-driven pulmonary injury.