Synergistic antibacterial effects of 5-fluorouracil or thioridazine in combination with phytopolyphenols on cultured Porphyromonas gingivalis.

Background/purpose: 5-Fluorouracil (5FU) is a commonly used anticancer drug. However, the severe oral mucositis induced by 5FU in about 60-70% of patients was a major cause of discontinuous therapy. Since oral dysbiosis induced by 5FU was well correlated with severity of oral mucositis and Porphyromonas gingivalis (P.g.) was a keystone pathogen of dysbiosis. Thus, in this study, we aimed to explore the novel regimens of 5FU combined with phytopolyphenols (curcumin, green tea polyphenols) as well as ZnSO4 on antibacterial effects of cultured P.g. growth. In addition, similar regimens containing thioridazine (TRZ) were also tested for their antibacterial efficacy. Materials and methods: The synergistic (Combination Index (CI) < 1) antiproliferation and anti-protease efficacies (IC50) of novel regimens on cultured P.g. were evaluated by OD600 and colorimetric method respectively. Results: The results obtained indicated that both novel regimens of 5FU and TRZ exhibited potent synergistic antibacterial effects against growth and protease of P.g. Conclusion: These novel regimens of 5-FU and TRZ were potent antibacterial agents which merit for further preclinical and clinical trials in management of oral mucositis, cancers and infectious diseases.

Novel regimens of phytopolyphenols with cisplatin or memantine and ZnSO4 for synergistic inhibition of growth and gingipains of the cultured Porphyromonas gingivalis.

Background/purpose: Porphyromonas gingivalis (P.g.) played a keystone pathogen not only in initiation and progression of periodontitis but also as a risk factor involved in systemic diseases (Alzheimer's disease, cancers, diabetes, osteoporosis etc.). Developments of effective and safe drugs to inhibit P.g. growth are urgent. In this study, we aimed at approaching novel regimens so called (PTM) by combination of repurposing drugs including phytopolyphenols (P) (curcumin, tea polyphenols), targeting drugs (T) such as cisplatin or memantine and metal ions(M) (ZnSO4). Materials and methods: The synergistic (combination Index (CI) < 1) antiproliferation and anti-protease efficacies (IC50) of novel regimens on cultured P.g. were evaluated by OD600 and colorimetric method respectively. Results: The results obtained revealed that these novel regimens (PTM) synergistically (combination index, CI < 1) exerted not only antiproliferative but also anti-gingipain protease effects of P.g. The concentrations for 50% inhibition (IC50) of novel regimens on P.g. growth and gingipains were greatly decreased as compared with those of cisplatin and memantine alone. Conclusion: Since these novel regimens exerted potent anti-bacterial effects on both planktonic and biofilm P.g., it is encouraged for further preclinical and clinical trials.

Clinical implications of oxidative stress in schizophrenia: Acute relapse and chronic stable phase.

Several studies have suggested a higher oxidative stress in schizophrenia. However, the implications of oxidative stress on clinical symptoms remain unclear. This study aimed to investigate the platelet oxidative stress in different stages of schizophrenia (i.e., chronic stable and acute relapse) in order to clarify the clinical implications of oxidative stress and the treatment effects. We recruited 43 chronic stable patients with schizophrenia and 48 non-psychiatric controls. Platelets were collected for measuring the levels of nitric oxide (NO), lipid peroxidation (LPO), and glutathione (GSH) and the activity of GSH peroxidase (GPx) and superoxide dismutase (SOD). The levels and activity were compared between patients and controls and were examined for their relationship with clinical severity. Further, we evaluated the changes of levels and activity before and after treatment in an independent sample with acute relapse (N = 19). Patients with chronic stable schizophrenia had lower SOD activity compared to non-psychiatric controls. In chronic stable patients, NO level was positively correlated with positive and disorganized symptoms, while the GPx activity were negatively correlated with excitement. In patients with acute relapse, the levels and activity were not different before and after four weeks of antipsychotic treatment, but LPO level was negatively correlated with pretreatment disorganized symptoms. The change of LPO can also predict the change of disorganized symptoms and negative symptoms. Our findings suggest that platelet SOD was lower in chronic stable schizophrenia. Platelet LPO may be associated with less disorganized symptoms in acute relapse patients and better treatment response.

Studies on a novel regimen for management of orofacial pain and morphine tolerance.

BACKGROUND/PURPOSE: The prevalence of orofacial pain is high but the etiology of orofacial pain is not well understood. Because of clinical treatment is not so effective, it is urgent to explore novel regimens with more effective and less side effects for clinical application. MATERIALS AND METHODS: Male mice (ICR strain) were injected with capsaicin (10µg/5 µl) in vibrissa pad. Spontaneous orofacial pain in 20 min was recorded after receiving capsaicin to quantify the nociceptive level. Green tea polyphenols (GTP 60 mg/kg), memantine (Mem 10 mg/kg), and GTPm (GTP 30 mg/kg plus Mem 3 mg/kg) were dissolved in 2% carboxymethyl cellulose, which was orally administered to mice twice per day and five times per week consecutively for 2 weeks. TruScan photobeam tracking was used to record changes of behavior and locomotor activities. RESULTS: GTPm by itself attenuated orofacial pain induced by capsaicin. Moreover, GTPm enhanced morphine analgesic effects, reduced morphine depressant side effects and delayed morphine tolerance. Along with this experiment, GTPm was tested on the hot plate (52 °C)-induced peripheral thermal pain. It was found that both memantine and GTPm reduced morphine-analgesia in hind paw thermal pain. CONCLUSION: In this study, GTP (60 mg/kg/day) orally administrated produced a significant analgesic effect on capsaicin-induced orofacial pain. Memantine combined with GTP synergistically not only reduced orofacial pain but also enhanced morphine analgesic effects. Thus, a new regimen of GTPm orally administered twice per day attenuated orofacial pain after consecutive 5 days.

Pu-erh tea polysaccharides decrease blood sugar by inhibition of α-glucosidase activity in vitro and in mice.

Type 2 diabetes is mainly induced by environmental factors such as being overweight, decreased physical activity and inbalanced energy metabolism, such as pancreatic beta-cell dysfunction and peripheral insulin resistance. Acarbose, a microbial carbohydrate and an alpha-glucosidase inhibitor, is currently a useful agent for attenuating type 2 diabetes. However, it is usually accompanied by many side effects, such as abdominal distention, flatulence, diarrhea and meteorism. These side effects may be caused by its strong inhibition of alpha-amylase, leading to the accumulation of several undigested carbohydrates. The bacteria residing in the colon can further ferment the undigested carbohydrate to release gas. Finding a new alpha-glucosidase inhibitor with a low inhibitory effect on alpha-amylase is highly anticipated. In this report we describe a group of carbohydrates found in pu-erh tea polysaccharide (PTPS) that can inhibit alpha-glucosidase but have less of an inhibitory effect on alpha-amylase. The preliminary experiments on mice indicate that PTPS might be better than acarbose at suppressing blood glucose after oral administration of a carbohydrate diet; it is recommended that further clinical trials are required in type 2 diabetes in future studies.

Weight reduction effect of Puerh tea in male patients with metabolic syndrome.

Puerh tea has been proposed to promote weight loss and favorably modify glucose, insulin and blood lipids. This study tested the effect of daily Puerh tea consumption for 3 months on weight and body mass index (BMI), and select metabolic parameters. The effect of daily Puerh tea intake on weight, BMI and changes in glucose, HbA1c and lipids was evaluated in patients with metabolic syndrome. The patients (N = 70) were randomized into two groups: those taking Puerh tea extract capsule (333 mg Puerh tea extract) three times a day and those taking a placebo tea for 3 months. There was a decrease in body weight of 1.3 kg in the Puerh tea group (p = 0.077) versus 0.23 kg in the placebo arm (p = 0.186). There was also a slight decrease in BMI 0.47 kg/m(2) in the Puerh tea group (p = 0.076) versus 0.09 kg/m(2) in the placebo arm (p = 0.185), suggesting a trend of weight change, but without statistical significance. Subgroup analysis of the male patients demonstrated statistically significant improvements in body weight reduction (p = 0.004) and BMI (p = 0.004). However, the change in other metabolic parameters (cholesterol or triglyceride) or HbA1c was not statistically significant. Intake of Puerh tea for 3 months was associated with a slight reduction in body weight and BMI, especially in the male patients. Therefore, daily Puerh tea consumption may be an alternative choice to modify body weight.

Exposure to low dose of cinnabar (a naturally occurring mercuric sulfide (HgS)) caused neurotoxicological effects in offspring mice.

Cinnabar, a naturally occurring mercuric sulfide (HgS), has long been used in Chinese mineral medicine for more than 2000 years. Although mercury is well-known for its toxicity, whether cinnabar induces neurotoxicity, especially in infants and children, is unknown. The purpose of this study was to explore the neurotoxic effects of low-dose of cinnabar (10 mg/kg/day) on developing mice. The results revealed neurobehavioral defects in F1-C-Cin group, which were associated with Hg accumulation, increased NO(x) levels in whole blood, and Na(+)/K(+)-ATPase activities in brain tissues. F1- and F2-Cin-V groups were found to increase brain Hg contents and prominent neurobehavioral defects compared with F1-C-V group, suggesting that the fetal brain was more susceptible to irreversible effects for cinnabar-induced damage. Moreover, F1- and F2-Cin-Cin groups had severely neurobehavioral dysfunctions, closely correlated with the further alteration of NO(x) levels and Na(+)/K(+)-ATPase activities than F1- and F2-C-Cin groups. Effects in F2-Cin-Cin group were more significant than those in F1-Cin-Cin group. In conclusion, this study demonstrates that exposure to low-dose of cinnabar during the perinatal and developmental stages results in irreversible and severe injuries of the neurotoxicity in offspring, and NO(x) and Na(+)/K(+)-ATPase activities may exist potential and useful biomarkers for neurotoxicity-induced by low-doses of mercuric compounds.

Pyrrolidine dithiocarbamate augments Hg(2+)-mediated induction of macrophage cell death via oxidative stress-induced apoptosis and necrosis signaling pathways.

Exposure to mercury can lead to several injuries in mammals, including immune system dysfunction, and pyrrolidine dithiocarbamate (PDTC), as a metal chelator and antioxidant, has been indicated to increase the cytotoxic effects of toxic metals. However, the toxicological effects and possible mechanisms of mercury in combination with PDTC are mostly unclear. In this study, we showed that PDTC dramatically increase the cytotoxic effect of HgCl(2) on cultured murine macrophages (RAW 264.7 cells). PDTC augmented HgCl(2)-induced cytotoxic effects by facilitating the entry of mercury into the cells. The Hg(2+)/PDTC complex significantly and rapidly increased the formation of reactive oxygen species (ROS) and decreased intracellular glutathione (GSH) levels in these cells. Flow cytometry analysis showed that the numbers of sub-G1 hypodiploid cells and annexin V-FITC binding cells increased after Hg(2+)/PDTC complex exposure, and several features of mitochondria-dependent apoptosis were also induced, including mitochondrial membrane depolarization, cytosolic cytochrome c release, poly(ADP-ribose) polymerase (PARP) and caspase 3/7 activation, and DNA fragmentation. Moreover, both apoptotic and necrotic cells were detected using acridine orange/ethidium bromide dual staining. Meanwhile, depleted intracellular ATP levels and increased lactate dehydrogenase (LDH) release were observed, suggesting the induction of necrotic cell death processes. These Hg(2+)/PDTC complex-induced cytotoxicity-related signals could be reversed by pretreatment with the antioxidant N-acetylcysteine. In conclusion, these results suggest that Hg(2+)/PDTC complex-induced oxidative stress causes macrophage cell death via both apoptosis and necrosis. These findings imply for the first time that PDTC dramatically increases the uptake and toxicological effects of Hg(2+) instead of detoxification.

Evidence for improved neuropharmacological efficacy and decreased neurotoxicity in mice with traditional processing of Rhizoma Arisaematis.

Rhizoma Arisaematis (RA, the rhizome of Pinellia pedatisecta Schott) is a traditional Chinese medicine commonly used in the treatment of convulsions, inflammation, and cancer. Despite the fact that it has been used for more than 2000 years, the pharmacological and toxic effects of traditionally processed products of RA are still unclear. In this study, we attempted to investigate the effects exerted by untreated crude RA and different preparations of RA treated with alumen in combination with ginger juice (Zhinanxing) or bile juice (Dannanxing) in ICR mice. The results showed that both the Zhinanxing and Dannanxing water extracts exerted significantly increased sedative effects, as indicated by the inhibitory effects on ambulatory distances, jumps, vertical-plane entries, and prolonged pentobarbital-induced sleeping time. The extracts also exerted significantly increased analgesic effects (increase of tail flick latency in nociceptive testing) in mice than did the unprocessed crude RA after oral administration for one to three days, and effects persisted 18 days after the cessation of treatment. By contrast, the toxic effects, such as an increase in stereotype-1 episodes of locomotor activities and reduction of the retention time on a rotating rod (motor equilibrium dysfunction), were observed only in mice treated with the unprocessed crude RA for three consecutive days, and effects persisted for 18 days after the cessation of treatment. These neurotoxic effects were accompanied by an increase in plasma lipid peroxidation (LPO), decrease in whole blood nitric oxide (NO(x)) levels, and inhibition of Na(+)/K(+)-ATPase activities in membrane fractions of erythrocytes and in the cerebral cortex. In conclusion, these findings provide scientific evidence that the processed RA indeed possesses not only enhanced neuropharmacological efficacy but also reduced neurotoxic effects as compared to the unprocessed crude RA. The signaling of NO(x)/oxidative stress/Na(+)-K(+)- ATPase activities played a role, at least in part, in the underlying mechanisms of neurotoxic effects induced by the crude RA.

Neurotoxicological effects of low-dose methylmercury and mercuric chloride in developing offspring mice.

Mercury is a well-known toxic metal and potently induces severe neurotoxicological effects, especially in infants and children. The purpose of this study was to explore the underlying mechanisms of neurotoxic effects of mercurial compounds on the different stages of developing mice. Low-doses (the probability of human exposure in mercury-contaminated areas) of methylmercury (MeHg) (M, 0.02mg/kg/day) and mercury chloride (HgCl(2)) (H, 0.5mg/kg/day) were administered to mice of the following groups: (1) treatment with distilled water for 7 consecutive weeks after weaning (control-vehicle (CV)); exposure to mercurial compounds at different stages; (2) for 7 consecutive weeks after weaning (control-MeHg (CM) and control-HgCl(2) (CH)); (3) only during perinatal and weaning stages (MeHg-vehicle (MV) and HgCl-vehicle (HV)); and (4) in all experimental stages (MeHg-MeHg (MM) and HgCl(2)-HgCl(2) (HH)). Results revealed the neurobehavioral defects (increased locomotor activities, motor equilibrium impairment, and auditory dysfunction) that correlated with increasing Hg accumulation in CM and CH groups. However, it revealed a decrease and an increase in locomotor activities in MV and HV groups, respectively; these became more severe in MM and HH groups than in MV and HV groups. Motor equilibrium performance in MV and HV groups remained normal, while that in MM and HH groups was decreased. The most severe auditory defects (altered auditory brainstem response, ABR test) found in MM and HH groups than those in the respective CM and CH, MV and HV, including absolute wave III delays and interwave I-III latencies, which suggested that the irreversible auditory dysfunction caused by mercurial compounds. Furthermore, the alteration of lipid peroxidation (LPO), Na(+)/K(+)-ATPase activities, and nitric oxide (NO(x)) in the brain tissues contributed to the observed neurobehavioral dysfunction and hearing impairment. These findings provide evidence that fetuses were much more susceptible to the effects of mercurial compounds with regard to inducing severely neurotoxicological injuries as that found in human beings. The signaling of ROS/Na(+)-K(+)-ATPase/NO(x) plays a crucial role in the underlying mechanism for mercurial compound-induced toxic effects in offspring.

Enhancing effects of morphine on methamphetamine-induced reinforcing behavior and its association with dopamine release and metabolism in mice.

Polydrug abuse has become a significant problem worldwide, and the combined use of methamphetamine (MA) and morphine (M) is now highly prevalent among addicts. In the present study, we investigated the neurobehavioral effects of repeated treatment regimens of these drugs (i.p. administration of 0.75 mg/kg/day MA, 5 mg/kg/day M, and their combination for five consecutive days followed by once weekly for five consecutive weeks) in mice. In addition, we used an in vivo microdialysis technique to study the changes in extracellular concentrations of dopamine (DA) and its metabolites in the mouse striatum after challenge administration of these drugs. The results showed that systemic M increased MA-induced conditioned place preference (CPP), as revealed by higher CPP values which were also maintained for a longer duration compared with those induced by an identical dose of MA or M alone. Subsequent to challenge with combined MA and M, mice exhibited an increase in stereotyped behavior, which appeared to be associated with an elevation of extracellular concentration of DA in the striatum. Our findings suggest that M not only produces synergistic effects on MA-induced CPP, but also interacts with MA to induce stereotyped behavioral sensitization which is mediated by an increase in DA outflow in the striatum. These findings provide insight into the behavioral and neurochemical basis responsible for the combined abuse liability of MA and M.

Ursolic acid inhibits IL-1beta or TNF-alpha-induced C6 glioma invasion through suppressing the association ZIP/p62 with PKC-zeta and downregulating the MMP-9 expression.

Ursolic acid (UA), a constant constituent of Rosmarinus officinalis extracts, is a triterpenoid compound which has been shown to have antioxidant and anticarcinogenic properties. In the present study, we found that UA was able to reduce interleukin-1 beta (IL-1beta) or tumor necrosis-alpha (TNF-alpha)-induced rat C6 glioma cell invasion, which was examined by a reconstituted basement membrane in a set of transwell chambers. However, the inhibitory effect of UA did not influence cell proliferation or cause cell cytotoxity. The results analyzed by zymography assay and Western blotting revealed that the activity and expression of matrix metalloproteinase-9 (MMP-9) was eliminated by UA in a dose-dependent manner. Because MMP-9 is the target gene of the transcription factor nuclear factor-kappaB (NF-kappaB), we further investigated the effect of UA on the activity of NF-kappaB. As expected, UA upregulated the levels of IkappaBalpha (IkappaBalpha) and attenuated the nuclear translocation of p65. Furthermore, UA suppressed the IL-1beta or TNF-alpha-induced activation of protein kinase C-zeta (PKC-zeta). Our data showed UA potently inhibited the association of ZIP/p62 and PKC-zeta. Taken together, we demonstrated that UA could efficiently inhibit the interaction of ZIP/p62 and PKC-zeta. It also further suppressed the activation of NF-kappaB and downregulation of the MMP-9 protein, which in turn contributed to its inhibitory effects on IL-1beta or TNF-alpha-induced C6 glioma cell invasion. These results all showcase the potential UA has in the chemoprevention and treatment of cancer metastasis and invasion.

Involvement of activating transcription factors JNK, NF-kappaB, and AP-1 in apoptosis induced by pyrrolidine dithiocarbamate/Cu complex.

Pyrrolidine dithiocarbamate (PDTC) is a metal chelator. Biologically, slight toxic affects EC50, 100+/-5.9 microM are observed when added to cultured HL-60 cells. CuCl2 at a physiological concentration (1 microM), but not FeCl2, Pb potentiated the cytotoxic effect of PDTC by 700 fold (EC50, 0.14+/-0.02 microM). Furthermore, results indicated that the PDTC/Cu complex induced an apoptotic process, evidenced by apoptotic bodies, DNA ladder and hypodiploidy cells. Additional studies showed that PDTC/Cu complex significantly decreased mitochondrial membrane potential, increased cytochrome c release, and reactive oxygen species production, and depleted reduced non-protein thiols in a time-dependent manner. Following oxidative stress, the PDTC/Cu complex sequentially activated JNK, NF-kappaB and AP-1 signaling pathways while IkappaB kinase activity was enhanced. The apoptotic process was eventually induced by caspase 3 activation and PARP degradation. The non-permeable copper-specific chelator-bathocuproine disulfonate (BCPS) and vitamin C were able to inhibit apoptosis and the elevation of intracellular Cu. Based on these findings; we conclude that PDTC/Cu complex-induced apoptosis is mediated by activation of JNK, NF-kappaB, AP-1 and caspase 3. Due to its high potency, PDTC may be useful as a therapeutic anti-cancer drug.

Novel regimen through combination of memantine and tea polyphenol for neuroprotection against brain excitotoxicity.

NMDA receptors are abundant, ubiquitously distributed throughout the brain, fundamental to excitatory neurotransmission, and critical for normal CNS function. However, excessive glutamate overstimulates NMDA receptors, leading to increased intracellular calcium and excitotoxicity. Mitochondrial dysfunction associated with loss of Ca(2+)homeostasis and enhanced cellular oxidative stress has long been recognized to play a major role in cell damage associated with excitotoxicity. In this experiment, we attempted to explore whether treatment with memantine (an NMDA receptor antagonist) and tea polyphenol (an antioxidant and anti-inflammatory agent), either alone or in combination, is effective in neuroprotection in a mouse excitotoxic injury model. Memantine (10 mg/kg/day), tea polyphenol (60 mg/kg/day), or a combination (memantine 5 mg/kg/day plus tea polyphenol 30 mg/kg/day) was administered by oral gavage for 2 consecutive days before causing excitotoxic injury. Mice received a 0.3-microL NMDA [335 mM (pH 7.2)] injection into the left striatum. Locomotor activity was assessed 24 hr before and after excitotoxic injury. Brain synaptosomes were harvested 24 hr after excitotoxic injury for assessment of Na(+), K(+)-ATPase and Mg(2+)-ATPase activity, reactive oxygen species production, mitochondrial membrane potential (Delta Psi m), mitochondrial reductase activity (MTT test), and Ca(2+)concentration. The results showed that treatment with memantine could significantly rescue mitochondrial function by attenuating the decreased mitochondrial membrane potential (Delta Psi m) and mitochondrial reductase activity in mouse excitotoxic injury. Treatment with tea polyphenol could significantly decrease the increased production of synaptosomal reactive oxygen species (ROS) and thus reduced the deteriorative ROS-sensitive Na(+), K(+)-ATPase and Mg(2+)-ATPase activity. However, neither memantine nor tea polyphenol alone could significantly improve the impaired locomotor activity unless treatment was combined. Combined treatment with memantine and tea polyphenol could significantly protect mice against excitotoxic injury by reducing the increased synaptosomal ROS production, attenuating the decreased Na(+), K(+)-ATPase and Mg(2+)-ATPase activity, the mitochondrial membrane potential (Delta Psi m), the mitochondrial reductase activity, and the increased synaptosomal Ca(2+)concentration. In addition, the impairment in locomotor activity was also significantly improved. Therefore, the combined treatment of memantine and tea polyphenol is more effective in neuroprotection than either memantine or tea polyphenol alone in mouse excitotoxic injury. These findings provide useful information about the potential application of memantine and tea polyphenols in preventing clinical excitotoxic injury such as brain trauma, brain ischemia, epilepsy, and Alzheimer's disease.

Ototoxicity induced by cinnabar (a naturally occurring HgS) in mice through oxidative stress and down-regulated Na(+)/K(+)-ATPase activities.

Cinnabar, a naturally occurring mercuric sulfide (HgS), has long been used in Chinese mineral medicine for more than 2000 years; currently it is still used as a sedative for infants in Asian countries. Since methylmercury is potently ototoxic, whether cinnabar also induces hearing impairment is awaited for delineation. In this study, we attempted to explore the toxic effects of cinnabar on the auditory brainstem response (ABR) system during 2-10 weeks administration at a clinical oral dosage of 10mg/kg/day in mice. The results showed that Hg contents of the brainstem were significantly increased accompanied with gradually progressive abnormality of ABR during 4-10 weeks of cinnabar administration. The progressive increase in hearing thresholds, prolonged absolute and interwave latencies of ABR apparently exhibited a gender difference. Male mice were more sensitive to cinnabar in producing hearing impairment correlated with the biochemical alterations in plasma and brainstem, e.g. an increase of lipid peroxidation (LPO), altered Na(+)/K(+)-ATPase activities and decrease of nitric oxide (NO(x)) levels. Moreover, accumulation of Hg contents in brainstem with a greater extent was found in male mice. These findings provide important information that the clinical dosage of cinnabar (10mg/kg/day) still exhibited ototoxicity after continuously long-term exposure. The signaling pathway of oxidative stress/Na(+)-K(+)-ATPase activities/NO of brainstem (a central auditory regulatory system) probably plays an important role in the toxic mechanisms of cinnabar-induced ototoxicity. The gender difference in cinnabar-induced neurotoxic effects merits further investigation.

Chronologic changes of nitric oxide concentration in the cochlear lateral wall and its role in noise-induced permanent threshold shift.

OBJECTIVE: The objective of this study was to investigate the chronologic changes of nitric oxide (NO) concentration in the cochlear lateral wall and to explore its possible role in permanent threshold shift (PTS) after intense noise exposure. MATERIALS AND METHODS: Seventeen guinea pigs were subjected to a single continuous exposure to broadband white noise at 105 +/- 2 dB sound pressure level (SPL) for 40 hours and were divided into four groups according to various postnoise recovery periods. Another 12 guinea pigs were not exposed to noise and served as controls. The hearing status of all animals was evaluated with auditory brainstem responses (ABR) evoked by condensation "click" sounds. ABR were recorded both prior to noise exposure and immediately before killing the animal. After death, NO concentration in the cochlear lateral wall was directly measured with an NO/ozone chemiluminescence technique. RESULTS: An approximately 1.7-fold increase in NO concentration was observed immediately postnoise exposure, which persisted for up to 28 days. The threshold of ABR elevation (mean, 30 dB SPL) peaked immediately after cessation of noise exposure and gradually resolved to a PTS (mean, 14.5 dB SPL) 56 days after noise exposure when NO concentration had returned to its prenoise exposure level. CONCLUSION: Noise-induced threshold shift, which resolved to a mild PTS, can be partially attributed to NO elevation in the cochlear lateral wall. Our results revealed a nonlinear correlation between ABR recovery and depletion of NO, indicating that the mechanisms of NO changes in the cochlear lateral wall may be more complicated than previously conceived and that other pathophysiologic mechanisms may also play important roles in noise-induced PTS.

Neurotoxicological mechanism of methylmercury induced by low-dose and long-term exposure in mice: oxidative stress and down-regulated Na+/K(+)-ATPase involved.

Methylmercury (MeHg), a potent neurotoxicant, easily passes through the blood-brain barrier (BBB), accumulates in the brain regions and causes severe irreversible damage. However, the neurotoxic effects and action mechanisms of MeHg are still unclear, especially in low-dose and long-term exposure. In this study, we attempted to explore the toxic effects of low-dose MeHg (0.05 mg/kg/day), which was the possible exposed dose by ingestion in MeHg-contaminated areas, on the time course of changes in locomotor activities and auditory brainstem response (ABR) system after administration for 7 consecutive weeks in mice. The results showed that the retention time on the rotating rod (60 rpm) was preferentially decreased after 1-week oral administration with MeHg. The locomotor activities parameters of ambulatory distances and stereotype-1 episodes were significantly increased and vertical-plane entries were progressively decreased after MeHg exposure in 3 consecutive weeks. Gradually progressive abnormality of ABR (increase in hearing thresholds, prolonged absolute and interwave latencies) was found during 4-6 weeks administration of MeHg. These impairments correlated with significant Hg accumulation and biochemical alterations in brain regions and/or other tissues, including the increase of lipid peroxidation (LPO) production, influence of Na+/K(+)-ATPase activities and nitric oxide (NO) levels were found. These findings provide evidence that the signaling of oxidative stress/Na+/K(+)-ATPase/NO plays a role in the underlying mechanisms of the neurotoxic effects induced by low-dose and long-term exposure of MeHg.

Effects of methylprednisolone on nitric oxide formation and survival of facial motor neurons after axotomy.

The aims of this study were to evaluate the effect of a high dose (160 mg/kg) of methylprednisolone sodium succinate (MPSS) on the formation of endogenous nitric oxide (NO) in the brainstem after facial nerve transection and to explore whether this effect has relevance to the survival of facial motor neurons. Guinea pig facial nerves were transected at the tympanic segment, and half were administered with MPSS, while the other half were given a vehicle of saline solution. Post operation NO formation in the brainstem at different time points was directly measured with a NO/ozone chemiluminescence technique. The surviving motor neurons were counted in histological coronal frozen sections of facial motor nuclei. The present results revealed that facial nerve transection induced a significant increase in NO formation in the brainstem by 1 week in both MPSS and saline treated groups and lasted to the end of the study (4 weeks). Compared to the saline treated group, it appeared that MPSS administration could delay the increase of nitric oxide synthase (NOS) expression and NO formation during the first 1 approximately 2 weeks after facial nerve transection. The survival rate of facial motor neurons was significantly higher in the MPSS treated group than in the saline treated group when examined at 3 or 4 weeks after facial nerve transection. These results indicate that a high dose of MPSS elicited a delayed increase of NO formation and thus may concomitantly enhance the survival time of motor neurons after facial nerve transection.

Protection from noise-induced temporary threshold shift by D-methionine is associated with preservation of ATPase activities.

OBJECTIVE: The present study was designed to test whether noise-induced temporary threshold shift (TTS) could be attenuated by D-methionine and its possible relation to the biochemical changes of cochlear lateral walls such as ATPase activities and oxidative stress in guinea pigs. DESIGN: Thirty-two normal-hearing male guinea pigs were randomly divided into saline-treated and D-methionine-treated (300 mg/kg) experimental groups. One hour after treatment, they were exposed to a continuous broadband white noise at 105 +/- 2 dB sound pressure level for 10 min, causing TTS. Each group was then divided into four subgroups based on the number of survival days after noise exposure (0, 1, 2, and 7 days). Each subgroup had four animals and eight ears included. By means of click-evoked auditory brain stem responses (ABR), auditory thresholds of guinea pigs were measured before noise exposure, immediately after noise exposure, and before killing. After animals were killed, cochlear lateral walls were immediately harvested and assayed for enzyme-specific activities of Na+, K+-ATPase and Ca2+-ATPase, lipid peroxidation, and nitric oxide. RESULTS: A 15.31 +/- 3.80 dB threshold shift was found immediately after noise exposure in saline-pretreated guinea pigs. In contrast, ABR threshold shift was significantly attenuated to 4.06 +/- 2.35 dB in D-methionine-treated animals. Furthermore, D-methionine enhanced the restoration of ABR threshold to baseline level by 1 day. In addition, noise significantly decreased Na+, K+-ATPase, and Ca2+-ATPase activities and increased lipid peroxidation and nitric oxide levels of the cochlear lateral walls. D-methionine significantly protected against all of these changes. CONCLUSIONS: Noise not only induced TTS but also inhibited ATPase activities as well as increased oxidative stress in guinea-pig cochlear lateral walls; all of these changes could be attenuated by d-methionine through its antioxidative property. These results suggest the potential usefulness of d-methionine in protecting from noise-induced ototoxicity.

Attenuation by methyl mercury and mercuric sulfide of pentobarbital induced hypnotic tolerance in mice through inhibition of ATPase activities and nitric oxide production in cerebral cortex.

This study is aimed at exploring the possible mechanism of hypnosis-enhancing effect of HgS or cinnabar (a traditional Chinese medicine containing more than 95% HgS) in mice treated with pentobarbital. We also examined whether the effect of HgS is different from that of the well-known methyl mercury (MeHg). After a short period (7 days) of oral administration to mice, a nontoxic dose (0.1 g/kg) of HgS not only significantly enhanced pentobarbital-induced hypnosis but also attenuated tolerance induction; while a higher dose (1 g/kg) of HgS or cinnabar exerted an almost irreversible enhancing effect on pentobarbital-hypnosis similar to that of MeHg (2 mg/kg) tested, which was still effective even after 10 or 35 days cessation of administration. To study comparatively the effects of different mercury forms from oral administration of MeHg and HgS on membrane ATPase activities of experimental mice, analysis of the Hg content in the cerebral cortex revealed that correlated with the decrease of Na(+)/K(+)-ATPase and Ca(2+)-ATPase activities. Furthermore, NO levels of blood but not that of cerebral cortex were also decreased by mercuric compounds. Although pentobarbital alone enhanced cytochrome p450-2C9 in time dependent manner, all of mercurial compounds tested had no such effect. All of these findings indicated that the mercurial compounds including cinnabar, HgS and MeHg exert a long-lasting enhancing hypnotic activity without affecting pentobarbital metabolism, which provides evidence-based sedative effect of cinnabar used in Chinese traditional medicine for more than 2,000 years. The nontoxic HgS dosing (0.1 g/kg/day) for consecutive 7 days is perhaps useful for delaying or preventing pentobarbital-tolerance.