Simultaneous determination of cyclosporine A, tacrolimus, sirolimus, and everolimus in whole-blood samples by LC-MS/MS.

OBJECTIVES: Cyclosporine A (CyA), tacrolimus (TRL), sirolimus (SIR), and everolimus (RAD) are immunosuppressive drugs frequently used in organ transplantation. Our aim was to confirm a robust sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of CyA, TRL, SIR, and RAD in whole-blood samples. MATERIALS AND METHODS: We used an integrated online solid-phase extraction-LC-MS/MS system and atmospheric pressure ionization tandem mass spectrometry (API-MS/MS) in the multiple reaction monitoring (MRM) detection mode. CyA, TRL, SIR, and RAD were simultaneously analyzed in whole blood treated with precipitation reagent taken from transplant patients. RESULTS: System performance parameters were suitable for using this method as a high-throughput technique in clinical practice. The high concentration of one analyte in the sample did not affect the concentration of other analytes. Total analytical time was 2.5 min, and retention times of all analytes were shorter than 2 minutes. CONCLUSION: This LC-MS/MS method can be preferable for therapeutic drug monitoring of these immunosuppressive drugs (CyA, TRL, SRL, and RAD) in whole blood. Sample preparation was too short and simple in this method, and it permits robust, rapid, sensitive, selective, and simultaneous determination of these drugs.

Investigation of vasoactive ion content of herbs used in hemorrhoid treatment in Turkey.

OBJECTIVES: The aim of the present study is to determine anion and cation contents of the herbals used in Turkish folk remedy to explore the rationale of their use in hemorrhoid treatment in the context of the vasoactivity of these elements. DESIGN: Herbs used in the treatment of hemorrhoid were determined by the way of literature search. These herbs were obtained from certified herb sellers. Ground herb samples were placed in individual tubes containing methanol and incubated for 48 hours at 30 degrees C. At the end of the incubation, supernatants were analyzed for their ion concentrations by using ion chromatography. RESULTS: The difference between ion levels between systemic and locally used herbs, was not statistically significant (p>0.05). Anion concentrations (except nitrate) of locally used herbs were slightly higher than systemically used herbs (p>0.05). Cation levels (except magnesium) of systemically used herbs were slightly higher than locally used herbs (p>0.05). It was shown that the concentration of vasoconstrictor effective ions was higher than the concentration of vasodilator effective ions (p<0.001). While vasoconstrictor ion concentration of systemically used herbs was 88.06 +/- 147.42 mg, vasodilator ion concentration of locally used herbs was 90.15 +/- 136.94 mg. The difference between vasodilator concentrations of groups was more evident; 5.39 +/- 9.80 mg and 14.32 +/- 66.48 mg for locally and systemically used herbs respectively. CONCLUSIONS: This study showed that herbal remedies used for the treatment of hemorrhoid in Turkey contain vasoactive and especially vasoconstrictor ions. Vasoconstrictor agents could amplify each others' effects as it has been previously shown, therefore, it is probable that the vasoconstrictor ion contents could contribute to the curative effects of herbals in the treatment of hemorrhoids.

The concentration-dependent contractile effect of methylene blue in the human internal mammary artery: a quantitative approach to its use in the vasoplegic syndrome.

OBJECTIVE: To quantitate the contractile effect of methylene blue on isolated human internal mammary artery (IMA) as used in the vasoplegic syndrome. DESIGN: An in vitro experimental study. SETTING: Cardiovascular Pharmacology Laboratory, Department of Medical Pharmacology. PARTICIPANTS: IMA segments were used from 24 patients undergoing coronary artery bypass surgery. INTERVENTIONS: The responses to methylene blue, norepinephrine, and acetylcholine were recorded isometrically by a force-displacement transducer in an isolated organ bath. MEASUREMENT AND MAIN RESULTS: Methylene blue (10 nmol/L-100 micromol/L) produced concentration-dependent contraction in the arteries. The maximal contraction to methylene blue was 44.2% +/- 3.8% of KCl (68 mmol/L) maximum contraction; the pEC(50) (-log(10) of 50% effective concentration) value was 5.5 +/- 0.1. Methylene blue caused an insignificant leftward shift of the concentration-response curve of norepinephrine. Acetylcholine-induced relaxation in submaximal contracted rings with phenylephrine recovered nearly 6 hours after the methylene blue challenge. CONCLUSION: Methylene blue caused concentration-dependent contraction in human IMAs. Furthermore, the inhibition of ACh-induced relaxation for 6 hours after the methylene blue challenge points out an additional mechanism (ie, receptor occupation). The concentration-dependent contractile effect of methylene blue justifies its use in the vasoplegic syndrome. The findings also suggest that the time course of contraction is longer than the exposure to methylene blue.

The local antinociceptive actions of nonsteroidal antiinflammatory drugs in the mouse radiant heat tail-flick test.

BACKGROUND: While many preclinical models detect the analgesic activity of nonsteroidal antiinflammatory drugs (NSAIDs), the radiant heat tail-flick response has repeatedly been insensitive to this class of drugs. As the tail-flick test involves nociceptive processing at spinal circuits with supraspinal modulation, it seems reasonable to assume that the NSAIDs should not modify strong nociceptive stimuli, since the primary site of action of NSAIDs is likely to be in the periphery. METHODS: We injected 3-300 mug of diclofenac, dipyrone, ketorolac, lysine acetyl salicylate, and sodium salicylate intradermally into mice tails and evaluated the tail-flick response to radiant heat. These results were compared with intraperitoneally injected controls. We also evaluated the ability of naloxone to reverse the observed effects. RESULTS: Intradermal injection of each NSAID produced a dose-dependent increase in tail-flick latency. Intraperitoneal NSAIDs injection produced no antinociceptive effects. Naloxone pretreatment had no effect on the antinociceptive effects of intradermal diclofenac, ketorolac, lysine acetyl salicylate, and sodium salicylate. Naloxone completely blocked the antinociceptive effects of intradermal dipyrone. CONCLUSIONS: Local, but not systemic, administration of NSAIDs produced antinociception in the tail-flick thermal assay. The endogenous opioid system contributes to the peripheral antinociceptive effects of dipyrone, but not to that of diclofenac, ketorolac, lysine acetyl salicylate, or sodium salicylate, suggesting differences in the mechanisms of action among the NSAIDs.

Lipophilic components of different therapeutic mud species.

OBJECTIVE: Mud, which contains organic and mineral ingredients, is used for the treatment of several degenerative diseases. It has been proposed that beneficial effects of mud are not only related to its local thermal effects, but also to its chemical components. Unlike hydrophilic components, the lipophilic components of the mud extract have not been described precisely thus far. Thus, we aimed to determine the lipophilic components of the different mud species. METHODS: Three different mud species (e.g., krenogen, tone, and fango) were analyzed by using gas chromatography-mass spectrometry (GC-MS). RESULTS: There were organic substances with fatty-acid structures found in the structure of mud. Torf mud species contain the most compounds. The compounds of three mud species differ from each other. CONCLUSIONS: The chemical structure of mud does not only contain hydrophilic organic substances, such as humic, fulmic, and ulmic acids, but also low-molecular-weighted organic substances composed of fatty acids in the majority. Moreover, it would not be appropriate to explain mud with a single term, since it has different chemical structures and a new classification of the mud species is required.

Evaluation of potential novel variations and their interactions related to bipolar disorders: analysis of genome-wide association study data.

BACKGROUND: Multifactor dimensionality reduction (MDR) is a nonparametric approach that can be used to detect relevant interactions between single-nucleotide polymorphisms (SNPs). The aim of this study was to build the best genomic model based on SNP associations and to identify candidate polymorphisms that are the underlying molecular basis of the bipolar disorders. METHODS: This study was performed on Whole-Genome Association Study of Bipolar Disorder (dbGaP [database of Genotypes and Phenotypes] study accession number: phs000017.v3.p1) data. After preprocessing of the genotyping data, three classification-based data mining methods (ie, random forest, naïve Bayes, and k-nearest neighbor) were performed. Additionally, as a nonparametric, model-free approach, the MDR method was used to evaluate the SNP profiles. The validity of these methods was evaluated using true classification rate, recall (sensitivity), precision (positive predictive value), and F-measure. RESULTS: Random forests, naïve Bayes, and k-nearest neighbors identified 16, 13, and ten candidate SNPs, respectively. Surprisingly, the top six SNPs were reported by all three methods. Random forests and k-nearest neighbors were more successful than naïve Bayes, with recall values >0.95. On the other hand, MDR generated a model with comparable predictive performance based on five SNPs. Although different SNP profiles were identified in MDR compared to the classification-based models, all models mapped SNPs to the DOCK10 gene. CONCLUSION: Three classification-based data mining approaches, random forests, naïve Bayes, and k-nearest neighbors, have prioritized similar SNP profiles as predictors of bipolar disorders, in contrast to MDR, which has found different SNPs through analysis of two-way and three-way interactions. The reduced number of associated SNPs discovered by MDR, without loss in the classification performance, would facilitate validation studies and decision support models, and would reduce the cost to develop predictive and diagnostic tests. Nevertheless, we need to emphasize that translation of genomic models to the clinical setting requires models with higher classification performance.

A Rare Reason of Hyperinsulinism: Munchausen Syndrome by Proxy.

Hyperinsulinism, one of the most important causes of hypoglycaemia, can be congenital or acquired. Rarely, drug toxicity can be a reason for hyperinsulinism. In the context of Munchausen syndrome by proxy (MSP), toxicity usually occurs in children due to drug administration by a parent or caregiver. A 7-year-old girl was referred to our department due to a hyperglycaemic period and hypoglycaemic episodes. On admission, gliclazide was initiated due to her hyperglycaemia, which we attributed to maturity onset diabetes of the young. However, during follow-up, hypoglycaemic levels were detected. Despite cessation of gliclazide, hypoglycaemic seizures occurred. Even with the medications administered, hypoglycaemia could not be prevented. During follow-up, the mother's affect, characterized by anxiety and interest in her daughter's medical care, appeared discordant with the situation. Due to our suspicion of MSP, we discovered toxic levels of gliclazide in the blood and urine samples which had been sent to the toxicology laboratory to search for hypoglycaemic agents. The patient was isolated, and all medications were stopped. After isolation, her hypoglycaemia disappeared, and she became hyperglycaemic (250 mg/dl). Physicians should consider the possibility of MSP in hyperinsulinaemic patients with discordant laboratory results and clinical symptoms, even if the child's parents display great concern.

The interaction between IL-1beta and morphine: possible mechanism of the deficiency of morphine-induced analgesia in diabetic mice.

It is known that diabetic mice are less sensitive to the analgesic effect of morphine. Some factor(s) derived from mononuclear cells, e.g. interleukin-1beta (IL-1beta), may be responsible for the diminished analgesic effect of morphine in diabetic mice. Therefore, we examined direct effects of IL-1beta, intracerebroventricularly (i.c.v.), on morphine-induced analgesia, subcutaneously (s.c.), in diabetic and control mice by using the tail-flick test. Morphine at doses of 1, 2 and 5 mg/kg (s.c.) produced dose-dependent analgesia in diabetic and control mice but diabetic mice were less sensitive to the analgesic effect of morphine when compared to the controls. IL-1beta at a dose of 0.1 ng/mouse produced analgesia in control mice but not in diabetics, whereas IL-1beta at a dose of 10 ng/mouse produced a hyperalgesic effect both in diabetic and control mice. IL-1beta at a dose of 1 ng/mouse has neither an analgesic nor a hyperalgesic effect in control and diabetic mice. Administration of a neutral (neither analgesic nor hyperalgesic) dose of IL-1beta, 1 ng/mouse (i.c.v.), just prior to administration of morphine (s.c.) abolished the analgesic effect of morphine at doses of 1, 2 and 5 mg/kg in control mice and the analgesic effect of morphine became similar to that in diabetics. The diminished analgesic effect of morphine in diabetes was attenuated further with IL-1beta at a dose of 1 ng/mouse (i.c.v.). These results suggest that the decreased analgesic effect of morphine in diabetes may be related to IL-1beta.

Topical cannabinoid antinociception: synergy with spinal sites.

Analgesic effects of cannabimimetic compounds have been known to be related to their central effects. Cannabinoid receptors also exist in the periphery but their role in pain perception has been remained to be clarified. Therefore, we assessed topical antinociceptive effects of WIN 55, 212-2, a mixed CB(1) and CB(2) receptors agonist, in mice using tail-flick test. Immersion of the tail of mouse into the WIN 55, 212-2 solution produced dose-dependent antinociception. This antinociceptive activity was limited to the portion of the tail exposed to WIN 55, 212-2. The antinociceptive response was dependent on duration of exposure to WIN 55, 212-2 solution. The topical antinociceptive effects of WIN 55, 212-2 were dose dependently blocked by topical pretreatment of CB(1) receptor-selective antagonist, AM 251. Thus, topical antinociceptive action of WIN 55, 212-2 involve CB(1) receptors. Intrathecal (i.th.) administration of WIN 55, 212-2 produced a dose-dependent antinociceptive effect. Interestingly, ineffective i.th. doses of WIN 55, 212-2 produced a marked antinociception when combined with topical application of WIN 55, 212-2 and topical antinociceptive effect was potentiated. The dose-response curve of i.th. WIN 55, 212-2 was shifted to the left 15-fold by topical WIN 55, 212-2. This finding suggests that there is an antinociceptive synergy between peripheral and spinal sites of cannabinoid action and it also implicates that local activation of cannabinoid system may regulate pain initiation in cutaneous tissue. Our findings support that cannabinoid system participates in buffering the emerging pain signals at the peripheral sites in addition to their spinal and supraspinal sites of action. In addition, an antinociceptive synergy between topical and spinal cannabinoid actions exists. These results also indicate that topically administered cannabinoid agonists may reduce pain without the dysphoric side effects and abuse potential of centrally acting cannabimimetic drugs.

Topical cannabinoid enhances topical morphine antinociception.

Opioids and cannabinoids produce antinociception through both spinal and supraspinal action. Both opioids and cannabinoids also have important peripheral action. Many previous studies indicate that systemically administered cannabinoids enhance antinociceptive properties of opioids. Experiments were conducted to test the hypothesis that topical cannabinoids would enhance the topical antinociceptive effects of morphine. Antinociception was measured in the radiant tail-flick test after immersion of the tail of mice into a solution of dimethyl sulfoxide (DMSO) containing WIN 55, 212-2, a cannabinoid agonist and morphine, an opioid agonist. Morphine and WIN 55, 212-2 produce time dependent topical analgesic effects limited to the portion of the tail exposed to drugs. WIN 55, 212-2 had a potency lower than that of morphine. The topical antinociceptive effects of WIN 55, 212-2 were blocked by systemic pretreatment of cannabinoid CB1 receptor selective antagonist, AM 251. This suggests that topical antinociceptive effects of WIN 55, 212-2 involve CB1 receptors. Combination of topical WIN 55, 212-2 with topical morphine yielded significantly greater analgesic effects than that of topical morphine alone. The ability of the CB1 receptor antagonist AM 251 to antagonize the enhancement of antinociception of morphine by WIN 55, 212-2 indicates that WIN 55, 212-2 acts through a CB1 receptor to enhance the potency of topical morphine. Additionally, spinally administered ineffective doses of WIN 55, 212-2 potentiated the antinociceptive effects of topical morphine. These results demonstrate an antinociceptive interaction between topical opioids with topical, and spinal cannabinoids. These observations are significant in using of topical combination of cannabinoid and morphine in the management of pain.

Cannabinoids blocks tactile allodynia in diabetic mice without attenuation of its antinociceptive effect.

Diabetic neuropathic pain is one of the most commonly encountered neuropathic pain syndromes. However, the treatment of diabetic neuropathic pain is challenging because of partial effectiveness of currently available pain relievers. It is well known that diabetic animals are less sensitive to the analgesic effect of morphine, and opioids are found to be ineffective in the treatment of diabetic neuropathic pain. Cannabinoids are promising drugs and they share a similar pharmacological properties with opioids. It has been reported that cannabinoid analgesia remained intact and to be effective in some models of nerve injury. Thus, we investigated antinociceptive efficacy and the effects of cannabinoids on behavioral sign of diabetic neuropathic pain in diabetic mice by using WIN 55, 212-2, a cannabinoid receptor agonist. Diabetes was induced by streptozotocin (STZ) (200mg/kg) and animals were tested between 45 and 60 days after onset of diabetes. Antinociception was assessed using the radiant tail-flick test. Mechanical and thermal sensitivities were measured by Von Frey filaments and hot-plate test, respectively. Tactile allodynia, but not thermal hyperalgesia developed in diabetic mice. Systemic WIN 55, 212-2 (1, 5 and 10mg/kg) produced a dose-dependent antinociception both in diabetic and control mice. WIN 55, 212-2-induced antinociception were found to be similar in diabetic mice when compared to controls suggesting efficacy of cannabinoid antinociception was not diminished in diabetic mice. WIN 55, 212-2 also produced a dose-dependent antiallodynic effect in diabetic mice. This study suggests that cannabinoids have a potential beneficial effect on experimental diabetic neuropathic pain.

Beneficial effects of N-acetylcysteine on sodium taurocholate-induced pancreatitis in rats.

BACKGROUND: Acute pancreatitis (AP) is a complex disease associated with significant complications and a high rate of mortality. Although several mechanisms are put forward, oxidative stress seems the most important early event in the pathophysiology of AP. Therefore, we evaluated the beneficial effects of N-acetylcysteine (NAC), a strong antioxidant, in experimental AP. METHODS: Forty-nine Sprague-Dawley rats were used. Acute pancreatitis (AP) was induced by the intraductal infusion of sodium taurocholate. Rats were divided into seven groups (each containing seven rats): control, sham-operated (saline-treated, 3.5 and 12 h), non-treated AP (3.5 and 12 h) and NAC-treated AP (3.5 and 12 h). Treated rats received intraperitoneal (i.p.) NAC 1000 mg/kg 24 h before and just before the induction of pancreatitis. RESULTS: Rats with AP had extensive parenchymal and fat necrosis and NAC treatment at 12 h reduced tissue necrosis significantly (P < 0.05). NAC treatment at 12 h reduced leukocytic infiltration significantly (P < 0.05). Edema and hemorrhage were significantly increased in the AP groups when compared to controls (P < 0.001). NAC treatment reduced edema and hemorrhage at both 3.5 and 12 h slightly but not significantly. The total pathological mean score was significantly increased in the AP groups (P < 0.05) and it was reduced by NAC treatment (P < 0.05). NAC treatment decreased plasma amylase and lipase levels significantly (P < 0.05). While glutathione peroxidase (GPx) activity of pancreatic tissue was similar in the NAC-treated and AP groups, hepatic tissue GPx activity was lower in the AP groups, and NAC treatment restored it (P < 0.05). NAC had no effect on pancreatic superoxide dismutase level. In the NAC-treated rats, the serum NO(2)/NO(3) (nitrite/nitrate) level was significantly increased in the 3.5-h group when compared to the respective AP group (P < 0.05). NAC treatment also significantly reduced the serum concentration of the lipid peroxidation product, malondialdehyde, at 12 h (P < 0.05). CONCLUSIONS: NAC treatment had beneficial effects in sodium taurocholate-induced AP in rats. It reduced pancreatic tissue necrosis and lipid peroxidation. In our study, the mechanism underlying the beneficial effects of NAC seemed to be its antioxidant activity, either by increasing hepatic GPx activity, or by a direct scavenging effect on free radicals, thus enhancing the production of and/or inhibiting the degradation of nitric oxide.

Amplification of sumatriptan-induced contractions with phenylephrine, histamine and KCl in the isolated human mesenteric artery: in-vitro evidence for sumatriptan-induced mesenteric ischaemia.

Sumatriptan, a 5-HT(1B/1D/1F) receptor agonist, is used to relieve migraine headache. Sumatriptan contracts some arteries either directly or after modest precontraction with non-serotonergic agonists. Sumatriptan can cause myocardial ischaemia and myocardial infarction. While previous in-vitro studies have shown that sumatriptan has no or only weak contractile activity in human omental arteries, recent clinical studies suggest that sumatriptan may induce mesenteric ischaemia. The aim of this study was to investigate the presence of contractile 5-HT(1B) receptors in the human mesenteric artery and to establish whether the weak sumatriptan-induced contractions are amplified by precontraction with various contractile substances. The study was performed in organ baths using endothelium-denuded isolated human mesenteric arteries. Sumatriptan induced concentration-dependent contractions in some mesenteric arteries [ E(max) 61+/-10% of the maximum contraction induced by 80 mM KCl, pD(2) (-log(10)EC(50)) 6.56+/-0.20, n=9]. In the other mesenteric arteries, sumatriptan induced only very weak ( E(max) <5%) or no contraction ( n=13). GR127935 (3 nM), a selective 5-HT(1B) receptor antagonist, antagonized sumatriptan-induced contractions insurmountably in sumatriptan-sensitive arteries. When the resting tension of the arterial rings was increased moderately by threshold concentrations (EC(10)-EC(20) of maximum contraction induced by 80 mM KCl) with the non-5-HT receptor agonists phenylephrine (10-100 nM), histamine (100 nM-1 microM) or the depolarizing agent KCl (4-10 mM), 5-HT(1B) receptor-mediated responses were amplified in sumatriptan-insensitive arteries (with phenylephrine E(max) 82+/-17%, pD(2) 6.64+/-0.20, n=7; with histamine E(max) 107+/-26%, pD(2) 6.16+/-0.14, n=6; with KCl E(max) 78+/-16%, pD(2) 6.45+/-0.15, n=7). These results show that sumatriptan induced concentration-dependent contractions in sumatriptan-sensitive mesenteric arteries and that 5-HT(1B) receptors were present and active in these vessels. However, in sumatriptan-insensitive arteries, precontraction is required for sumatriptan to induce concentration-dependent contractions. These findings suggest that sumatriptan may induce ischaemia in human mesenteric vasculature directly or in the presence of precontractile risk factors.

Discovering missing heritability and early risk prediction for type 2 diabetes: a new perspective for genome-wide association study analysis with the Nurses' Health Study and the Health Professionals' Follow-Up Study.

BACKGROUND/AIM: Despite the rise in type 2 diabetes prevalence worldwide, we do not have a method for early risk prediction. The predictive ability of genetic models has been found to be little or negligible so far. In this study, we aimed to develop a better early risk prediction method for type 2 diabetes. MATERIALS AND METHODS: We used phenotypic and genotypic data from the Nurses' Health Study and Health Professionals' Follow-up Study cohorts and analyzed them by using binary logistic regression. RESULTS: Phenotypic variables yielded 70.7% overall correctness and an area under the curve (AUC) of 0.77. With regard to genotype, 798 single nucleotide polymorphisms with P-values of lower than 1.0E-3 yielded 90.0% correctness and an AUC of 0.965. This is the highest score in the literature, even including the scores obtained with phenotypic variables. The additive contributions of phenotype and genotype increased the overall correctness to 92.9% and the AUC to 0.980. CONCLUSION: Our results showed that genotype could be used to obtain a higher score, which could enable early risk prediction. These findings present new possibilities for genome-wide association study analysis in terms of discovering missing heritability. These results should be confirmed by follow-up studies.

Pediatric reference intervals for plasma and urine essential amino acids in a Turkish population.

AIM: To establish age- and sex-specific reference intervals for essential amino acids in a healthy Turkish pediatric population. MATERIALS AND METHODS: A total of 945 clinically healthy children (531 boys and 414 girls, ranging in age from birth to 14 years) were enrolled. Plasma and urine amino acids' concentrations were measured by high-performance liquid chromatography. RESULTS: Concentrations of essential amino acids in plasma were higher in girls than in boys in the age groups of 0-1 months and 7-14 years; however, there was no difference in the other age groups. Concentrations of essential amino acids in urine were higher in girls than in boys in the age group of 0-1 months; however, there was no difference in the other age groups. Our results demonstrated the sex-related differences in concentrations ofleucine, isoleucine, valine, phenylalanine, lysine, and histidine in plasma, which increased with age in boys but not in girls. The concentrations of leucine, tryptophan, methionine, and lysine in urine declined with age in girls but not in boys, which were sex-related differences, too. CONCLUSION: We defined essential amino acids' reference intervals in a Turkish pediatric population.

Detection of PIGO-deficient cells using proaerolysin: a valuable tool to investigate mechanisms of mutagenesis in the DT40 cell system.

While isogenic DT40 cell lines deficient in DNA repair pathways are a great tool to understand the DNA damage response to genotoxic agents by a comparison of cell toxicity in mutants and parental DT40 cells, no convenient mutation assay for mutagens currently exists for this reverse-genetic system. Here we establish a proaerolysin (PA) selection-based mutation assay in DT40 cells to identify glycosylphosphatidylinositol (GPI)-anchor deficient cells. Using PA, we detected an increase in the number of PA-resistant DT40 cells exposed to MMS for 24 hours followed by a 5-day period of phenotype expression. GPI anchor synthesis is catalyzed by a series of phosphatidylinositol glycan complementation groups (PIGs). The PIG-O gene is on the sex chromosome (Chromosome Z) in chicken cells and is critical for GPI anchor synthesis at the intermediate step. Among all the mutations detected in the sequence levels observed in DT40 cells exposed to MMS at 100 µM, we identified that ∼55% of the mutations are located at A:T sites with a high frequency of A to T transversion mutations. In contrast, we observed no transition mutations out of 18 mutations. This novel assay for DT40 cells provides a valuable tool to investigate the mode of action of mutations caused by reactive agents using a series of isogenic mutant DT40 cells.

Formation of hydroxymethyl DNA adducts in rats orally exposed to stable isotope labeled methanol.

Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol's well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [¹³CD4]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [¹³CD4]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account.

The efficiency of proanthocyanidin in an experimental pulmonary fibrosis model: comparison with taurine.

Pulmonary fibrosis (PF) is a progressive fatal disorder. Bleomycin (BLM) is a widely used chemotherapeutic agent causing PF. Numerous agents have been investigated to prevent the progression of PF so far, but there is still a need to find more efficacious agents. Proanthocyanidin (PA) is a strong antioxidant, the main ingredient of grape seed extract. Since PA is ready for use in practice, we aimed to compare the preventive effect of PA in comparison with taurine (Tau) in BLM-induced PF. Forty Wistar male albino rats were used in the study and were divided into four groups: group 1, control; group 2, BLM-induced PF group; group 3, BLM-induced PF and treated with PA group; and group 4, BLM-induced PF and treated with Tau group. Treatments were begun 10 days before and continued 21 days after BLM injection. PA and Tau effectively inhibited inflammation, edema, severity of fibrosis, fibrosis extension, inflammatory cell accumulation, iNOS staining, and hydroxyproline level as well (p < 0.05). Total histological scores of the PA group were similar to the control group; Tau was significantly higher than the control group but lower than the BLM group (p < 0.05). We believe that PA could be a new treatment choice for PF, but further studies need to be conducted to verify the findings of the current study.

The protective effects of ozone therapy in a rat model of acetaminophen-induced liver injury.

OBJECTIVES: Acetaminophen (APAP) overdose may cause acute liver injury. Ozone therapy (OT) is shown to reduce inflammation and necrosis in several entities. Thus, we have designed this study to evaluate the efficacy of OT in a rat model of APAP-induced liver injury. METHODS: Twenty-seven Sprague-Dawley rats were divided into three groups: sham, APAP and APAP+OT groups. In the APAP and the APAP+OT groups, liver injury was induced by oral administration of 1 g/kg APAP. The APAP+OT group received a single dose ozone/oxygen mixture (0.7 mg/kg) intraperitoneally 1h after APAP administration. All animals were killed at 24 hour after APAP administration. Blood samples and liver tissues were harvested to determine liver injury and oxidative stress parameters. Liver tissues and blood samples were obtained for biochemical and histopathological analyses. RESULTS: APAP administration caused necrosis in the liver after 24h. The degrees of liver necrosis of the APAP group were higher than the other groups (in both p<0.05, respectively). In the APAP+OT group, liver antioxidant enzymes activities were significantly higher than the APAP group (p<0.05), but were lower than the sham group (p<0.05). In the sham group, serum neopterin, a marker of cell-mediated immunity, concentrations (4.8±1.2 nmol/L) were lower than the APAP (14.7±1.4 nmol/L) and APAP+OT groups (7.5±2.4 nmol/L) (in both p<0.05, respectively). CONCLUSION: Our results showed that OT prevented liver necrosis in rats and reduced neopterin levels. These findings suggest that the use of OT as an adjuvant therapy which might improve the outcome in APAP induced liver injury.

Systemic and spinal administration of etanercept, a tumor necrosis factor alpha inhibitor, blocks tactile allodynia in diabetic mice.

Painful diabetic neuropathy is one of the most common forms of neuropathic pain syndromes. Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine that has been implicated as a key pain mediator in the development and maintenance of neuropathic pain conditions. Recent studies showed that endogenous TNF-alpha production was also accelerated in neural tissues and spinal cord under chronic hyperglycemia. Thus, in this study, we investigated whether pharmacological inhibition of TNF-alpha by etanercept, a TNF-alpha antagonist, could block behavioral sign of diabetic neuropathic pain. Diabetes was induced by streptozotocin (STZ) (200 mg/kg, i.p.) in Balb-c mice and behavioral tests were performed between 45 and 60 days after STZ administration. Mechanical and thermal sensitivities were measured by a series of calibrated Von Frey filaments and hot plate test, respectively. Etanercept was given by either intravenous (i.v.), intrathecal (i.th.) or intraplantar (i.pl.) routes to the diabetic mice. Tactile allodynia, but not thermal hyperalgesia, developed in diabetic mice. Both i.v. (1, 10 and 20 mg/kg) or i.th. (1, 5 and 10 µg/mouse) treatments with etanercept produced dose dependent reversal of tactile allodynia in diabetic mice. However, etanercept was found to be inactive against allodynia when given i.pl. (1, 5 and 10 µg/mouse). Our results suggest that etanercept has promising effects on diabetic neuropathic pain with antiallodynic effects when given systemically or intrathecally.