Veterinarians exposed to inhaled anesthetic present chromosome damage, apoptosis and cell cycle changes.

This cross-sectional study evaluated, for the first time, DNA damage, viability, and cell death of lymphocytes and cell cycle phases of mononuclear and polymorphonuclear cells in veterinarians exposed to the volatile anesthetic isoflurane. Veterinarians who were occupationally exposed to isoflurane (exposed group; n = 20) and matched-unexposed individuals (volunteers without occupational exposure; n = 20) were enrolled in the study. DNA damage was assessed in lymphocytes by micronucleus (MN) and phosphorylated histone gamma-H2AX (γ-H2AX). Cell viability, cytotoxicity, and the cell cycle were evaluated by flow cytometry. Isoflurane was detected in urine samples by headspace gas chromatography-mass spectrometry. Compared with unexposed subjects, veterinarians occupationally exposed to isoflurane (25.7 ± 23.7 µg/L urine) presented statistically higher MN frequencies, lymphocytic apoptosis rates, and numbers of polymorphonuclear cells in the G0/G1 stage. Additionally, the exposed group presented statistically lower proportions of viable lymphocytes and G2/M polymorphonuclear cells. Our findings indicate that veterinarians who are frequently exposed to inhaled anesthetic exhibit chromosomal and cell damage in addition to changes in peripheral blood cell proliferation.

Ethanol consumption favors pro-contractile phenotype of perivascular adipose tissue: A role for interleukin-6.

Perivascular adipose tissue (PVAT) exerts anticontractile effect, but under non-physiological conditions it may contribute to vascular dysfunction by releasing pro-inflammatory cytokines. Since PVAT is an important source of interleukin (IL)-6, we evaluated whether this cytokine would contribute to ethanol-induced vascular dysfunction. With this purpose, male C57BL/6 wild-type (WT) or IL-6-deficient mice (IL-6-/-) were treated with ethanol for 12 weeks. Increased blood pressure was evidenced after 4 and 6 weeks of treatment with ethanol in WT and IL-6-/- mice, respectively. In WT mice, ethanol increased plasma and PVAT levels of IL-6. Ethanol favoured pro-contractile phenotype of PVAT in mesenteric arteries from WT, but not IL-6-deficient mice. Functional studies showed that tiron [(a scavenger of superoxide (O2-)] reversed the pro-contractile effect of PVAT in mesenteric arteries from ethanol-treated mice. Ethanol increased the levels of O2- in PVAT from WT mice. Ethanol-induced increase in O2- generation was higher in arteries with PVAT from WT mice when compared to IL-6-deficient mice. Treatment with ethanol augmented myeloperoxidase activity in the mesenteric arterial bed (MAB; with or without PVAT) from WT, but not IL-6-deficient mice. In conclusion, IL-6 contributes to the pro-contractile effect of PVAT by a mechanism that involves increase in ROS generation. Additionally, IL-6 mediates intravascular recruitment of neutrophils in response to ethanol and plays a role in the early stages of ethanol-induced hypertension. Collectively, our findings provide novel evidence for a role of IL-6 in the vascular dysfunction induced by ethanol.

Cocaine esterase occurrence in global wastewater microbiomes and potential for biotransformation of novel psychoactive substances.

The analysis of drugs in wastewater for forensic purposes has been constantly increasing and the investigation of the potential interaction between drugs or metabolites and sewage microbiota is important. The results demonstrated that cocaine esterase genes were widely distributed in 1142 global wastewater samples collected from 64 countries and linked to several bacterial species. In addition, in silico predictions indicated that carfentanil, 4F-MDMB-BINACA, 5F-MDMB-PICA, MDMB-4en-PINACA and mitragynine might also undergo microbial hydrolysis, in a similar fashion of cocaine degradation by cocaine esterase. In conclusion, it was demonstrated the microbial potential to hydrolyze drugs of abuse in wastewater environments, contributing to the critical evaluation of potential metabolites as biomarkers for microbial and human transformation of drugs in wastewater.

Inducible nitric oxide synthase (iNOS) mediates ethanol-induced redox imbalance and upregulation of inflammatory cytokines in the kidney.

Overexpression of the inducible isoform of the enzyme nitric oxide synthase (iNOS) has been associated to pathological processes in the kidney. Ethanol consumption induces the renal expression of iNOS; however, the contribution of this enzyme to the deleterious effects of ethanol in the kidney remains elusive. We examined whether iNOS plays a role in the renal dysfunction and oxidative stress induced by ethanol consumption. With this purpose, male C57BL/6 wild-type (WT) or iNOS-deficient (iNOS-/-) mice were treated with ethanol (20% v/v) for 10 weeks. Treatment with ethanol increased the expression of Nox4 as well as the concentration of thiobarbituric acid reactive substances and the levels of tumor necrosis factor α in the renal cortex of WT but not iNOS-/- mice. Augmented serum levels of creatinine and increased systolic blood pressure were found in WT and iNOS-/- mice treated with ethanol. WT mice treated with ethanol showed increased production of reactive oxygen species and myeloperoxidase activity, but these responses were attenuated in iNOS-/- mice. We concluded that iNOS played a role in ethanol-induced oxidative stress and pro-inflammatory cytokine production in the kidney. These are mechanisms that may contribute to the renal toxicity induced by ethanol.

Nebivolol prevents vascular oxidative stress and hypertension in rats chronically treated with ethanol.

BACKGROUND AND AIMS: Chronic ethanol consumption is associated with hypertension and atherosclerosis. Vascular oxidative stress is described as an important mechanism whereby ethanol predisposes to atherosclerosis. We hypothesized that nebivolol would prevent ethanol-induced hypertension and vascular oxidative stress. METHODS: Male Wistar rats were treated with ethanol 20% (vol./vol.) or nebivolol (10 mg/kg/day, p. o., gavage), a selective ß1-adrenergic receptor antagonist. RESULTS: Ethanol-induced increase in blood pressure and in the circulating levels of adrenaline and noradrenaline was prevented by nebivolol. Similarly, nebivolol prevented ethanol-induced increase in plasma levels of renin, angiotensin I and II. Chronic ethanol consumption increased the aortic levels of superoxide anion (O2-), thiobarbituric acid reactive species (TBARS) as well as the expression of Nox1 and nitrotyrosine immunostaining in the rat aorta. Treatment with nebivolol prevented these responses. The decrease in aortic levels of nitrate/nitrite (NOx) induced by ethanol was prevented by the treatment with nebivolol. Finally, nebivolol attenuated ethanol-induced increase in phenylephrine- and noradrenaline-induced contraction of endothelium-intact and endothelium-denuded aortic rings. CONCLUSIONS: The novelty of our study is that nebivolol prevented ethanol-induced hypertension and vascular oxidative stress. Additionally, we showed that the sympathetic nervous system (SNS) and the renin-angiotensin system (RAS) are important endogenous mediators of the cardiovascular effects of ethanol.

Ethanol withdrawal increases blood pressure and vascular oxidative stress: a role for angiotensin type 1 receptors.

We evaluated the possible mechanisms underlying the oxidative stress induced by ethanol withdrawal. With this purpose, we verified the role of AT1 receptors in such response. Male Wistar rats were treated with ethanol 3%-9% (vol./vol.) for 21 days. Ethanol withdrawal was induced by abrupt discontinuation of the treatment. Experiments were performed 48 hours after ethanol discontinuation. Increased plasma levels of angiotensin II were detected after ethanol withdrawal. Losartan (10 mg/kg; p.o. gavage), a selective AT1 receptor antagonist, impeded the increase in blood pressure induced by ethanol withdrawal. Increased lipoperoxidation and superoxide anion (O2-) levels were detected in aortas after ethanol withdrawal, and losartan prevented these responses. Decreased hydrogen peroxide and nitrate/nitrite concentration were detected in aortas after ethanol withdrawal, and losartan prevented these effects. Nitrotyrosine immunostaining in the rat aorta was increased after ethanol withdrawal, and AT1 blockade impeded this response. Increased expression of PKCδ and p47phox was detected after ethanol withdrawal, and treatment with losartan prevented these responses. Our study provides novel evidence that ethanol withdrawal increases vascular oxidative stress and blood pressure through AT1-dependent mechanisms. These findings highlight the importance of angiotensin II in ethanol withdrawal-induced increase in blood pressure and vascular oxidative damage.

Chronic ethanol consumption increases vascular oxidative stress and the mortality induced by sub-lethal sepsis: Potential role of iNOS.

We hypothesized that long-term ethanol consumption would increase the mortality and aggravate the deleterious effects of sub-lethal cecal ligation and puncture (SL-CLP) in the vasculature by inducing the expression of inducible nitric oxide (NO) synthase (iNOS). Male C57BL/6J wild-type (WT) or iNOS-deficient mice (iNOS-/-) were treated with ethanol (20% v/v) for 12 weeks and then subjected to SL-CLP. Mice were killed 24 h post-operatively or followed six days for survival. Septic ethanol-treated mice showed a higher mortality than septic WT mice. However, septic iNOS-deficient mice treated with ethanol showed a decreased mortality rate when compared to ethanol-treated WT mice. Ethanol and SL-CLP augmented superoxide anion (O2-) generation in the mesenteric arterial bed (MAB) of both WT and iNOS-deficient mice. Treatment with ethanol and SL-CLP enhanced lipoperoxidation in the MAB of WT, but not iNOS-deficient mice. SL-CLP enhanced nitrate/nitrite (NOx) concentrations in the MAB of WT, but not iNOS-deficient mice. Both, ethanol and SL-CLP increased TNF-α and IL-6 levels in the MAB. Treatment with ethanol as well as SL-CLP up-regulated the expression of iNOS in the MAB of WT mice. The major finding of our study is that chronic ethanol consumption increases the mortality induced by SL-CLP and that iNOS plays a role in such response. Although ethanol led to vascular alterations, it did not aggravate the vascular injury induced by SL-CLP. Finally, iNOS mediated the increase in oxidative stress and pro-inflammatory cytokines induced by SL-CLP in the vasculature.

Tumor necrosis factor-α receptor 1 contributes to ethanol-induced vascular reactive oxygen species generation and hypertension.

We evaluated the contribution of tumor necrosis factor-α receptor 1 (TNFR1) to ethanol-induced hypertension and vascular oxidative stress and the possible role of perivascular adipose tissue (PVAT) in such responses. Male C57BL/6 wild-type (WT) or TNFR1-deficient mice (TNFR1-/-) were treated with ethanol (20% vol/vol) for 12 weeks. Ethanol induced an increase in blood pressure in WT mice and TNFR1-/- at 4 and 5 weeks of treatment, respectively. Treatment with ethanol increased tumor necrosis factor-α and interleukin-6 levels in aortas with or without PVAT (PVAT+ and PVAT-, respectively) from WT mice, but not TNFR1-/-. Ethanol increased superoxide anion (O2-) generation, thiobarbituric acid reactive substance concentration, and the activity of superoxide dismutase and catalase in aortas (PVAT- and PVAT+) from WT mice, but not TNFR1-/-. Conversely, ethanol consumption decreased the concentration of nitrate/nitrite in aortas (PVAT- and PVAT+) from WT mice, but not TNFR1-/-. Treatment with ethanol increased myeloperoxidase activity in aortas (PVAT- and PVAT+) from WT mice, but not TNFR1-/-. The major finding of our study is that TNFR1 contributes to ethanol-induced hypertension and oxidative stress in the vasculature. Additionally, TNFR1 plays a role in ethanol-induced increase in proinflammatory cytokines and neutrophils migration. However, PVAT does not counteract or aggravate the effects induced by ethanol.

Protective Effects of Cannabidiol against Seizures and Neuronal Death in a Rat Model of Mesial Temporal Lobe Epilepsy.

The present study reports the behavioral, electrophysiological, and neuropathological effects of cannabidiol (CBD), a major non-psychotropic constituent of Cannabis sativa, in the intrahippocampal pilocarpine-induced status epilepticus (SE) rat model. CBD was administered before pilocarpine-induced SE (group SE+CBDp) or before and after SE (group SE+CBDt), and compared to rats submitted only to SE (SE group), CBD, or vehicle (VH group). Groups were evaluated during SE (behavioral and electrophysiological analysis), as well as at days one and three post-SE (exploratory activity, electrophysiological analysis, neuron density, and neuron degeneration). Compared to SE group, SE+CBD groups (SE+CBDp and SE+CBDt) had increased SE latency, diminished SE severity, increased contralateral afterdischarge latency and decreased relative powers in delta (0.5-4 Hz) and theta (4-10 Hz) bands. Only SE+CBDp had increased vertical exploratory activity 1-day post SE and decreased contralateral relative power in delta 3 days after SE, when compared to SE group. SE+CBD groups also showed decreased neurodegeneration in the hilus and CA3, and higher neuron density in granule cell layer, hilus, CA3, and CA1, when compared to SE group. Our findings demonstrate anticonvulsant and neuroprotective effects of CBD preventive treatment in the intrahippocampal pilocarpine epilepsy model, either as single or multiple administrations, reinforcing the potential role of CBD in the treatment of epileptic disorders.

Ethanol-induced erectile dysfunction and increased expression of pro-inflammatory proteins in the rat cavernosal smooth muscle are mediated by NADPH oxidase-derived reactive oxygen species.

Ethanol consumption is associated with an increased risk of erectile dysfunction (ED), but the molecular mechanisms through which ethanol causes ED remain elusive. Reactive oxygen species are described as mediators of ethanol-induced cell toxicity/damage in distinctive tissues. The enzyme NADPH oxidase is the main source of reactive oxygen species in the endothelium and vascular smooth muscle cells and ethanol is described to increase NADPH oxidase activation and reactive oxygen species generation. This study evaluated the contribution of NADPH oxidase-derived reactive oxygen species to ethanol-induced ED, endothelial dysfunction and production of pro-inflammatory and redox-sensitive proteins in the rat cavernosal smooth muscle (CSM). Male Wistar rats were treated with ethanol (20% v/v) or ethanol plus apocynin (30mg/kg/day; p.o. gavage) for six weeks. Apocynin prevented both the decreased in acetylcholine-induced relaxation and intracavernosal pressure induced by ethanol. Ethanol increased superoxide anion (O2-) generation and catalase activity in CSM, and treatment with apocynin prevented these responses. Similarly, apocynin prevented the ethanol-induced decreased of nitrate/nitrite (NOx), hydrogen peroxide (H2O2) and SOD activity. Treatment with ethanol increased p47phox translocation to the membrane as well as the expression of Nox2, COX-1, catalase, iNOS, ICAM-1 and p65. Apocynin prevented the effects of ethanol on protein expression and p47phox translocation. Finally, treatment with ethanol increased both TNF-α production and neutrophil migration in CSM. The major new finding of this study is that NADPH oxidase-derived reactive oxygen species play a role on chronic ethanol consumption-induced ED and endothelial dysfunction in the rat CSM.

NADPH Oxidase Plays a Role on Ethanol-Induced Hypertension and Reactive Oxygen Species Generation in the Vasculature.

AIMS: Investigate the role of NADPH oxidase on ethanol-induced hypertension and vascular oxidative stress. METHODS: Male Wistar rats were treated with ethanol (20% v/v). RESULTS: Apocynin (10 mg/kg/day, i.p.) prevented ethanol-induced hypertension. The increased contractility of endothelium-intact and endothelium-denuded aortic rings from ethanol-treated rats to phenylephrine was prevented by apocynin. Ethanol consumption increased superoxide anion (O2 (-)) generation and lipid peroxidation and apocynin prevented these responses. The decrease on plasma and vascular nitrate/nitrite (NOx) levels induced by ethanol was not prevented by apocynin. Treatment with ethanol did not affect aortic levels of hydrogen peroxide (H2O2) or reduced glutathione (GSH). Ethanol did not alter the activities of xanthine oxidase (XO), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Ethanol increased the expression of Nox1, PKCδ, nNOS, SAPK/JNK and SOD2 in the rat aorta and apocynin prevented these responses. No difference on aortic expression of Nox2, Nox4, p47phox, Nox organizer 1 (Noxo1), eNOS and iNOS was detected after treatment with ethanol. Ethanol treatment did not alter the phosphorylation of SAPK/JNK, p38MAPK, c-Src, Rac1 or PKCδ. CONCLUSIONS: The major new finding of our study is that the increased vascular generation of reactive oxygen species (ROS) induced by ethanol is related to increased vascular Nox1/NADPH oxidase expression. This mechanism is involved in vascular dysfunction and hypertension induced by ethanol. Additionally, we conclude that ethanol consumption induces the expression of different proteins that regulate vascular contraction and growth and that NADPH oxidase-derived ROS play a role in such response. SHORT SUMMARY: The key findings of our study are that ethanol-induced hypertension is mediated by NADPH oxidase. Moreover, increased vascular Nox1 expression is related to the generation of reactive oxygen species (ROS) by ethanol. Finally, ROS induced by ethanol increase the expression of the regulatory vascular proteins.

Direct Analysis of Amphetamine Stimulants in a Whole Urine Sample by Atmospheric Solids Analysis Probe Tandem Mass Spectrometry.

Amphetamine-type stimulants (ATS) are among illicit stimulant drugs that are most often used worldwide. A major challenge is to develop a fast and efficient methodology involving minimal sample preparation to analyze ATS in biological fluids. In this study, a urine pool solution containing amphetamine, methamphetamine, ephedrine, sibutramine, and fenfluramine at concentrations ranging from 0.5 pg/mL to 100 ng/mL was prepared and analyzed by atmospheric solids analysis probe tandem mass spectrometry (ASAP-MS/MS) and multiple reaction monitoring (MRM). A urine sample and saliva collected from a volunteer contributor (V1) were also analyzed. The limit of detection of the tested compounds ranged between 0.002 and 0.4 ng/mL in urine samples; the signal-to-noise ratio was 5. These results demonstrated that the ASAP-MS/MS methodology is applicable for the fast detection of ATS in urine samples with great sensitivity and specificity, without the need for cleanup, preconcentration, or chromatographic separation. Thus ASAP-MS/MS could potentially be used in clinical and forensic toxicology applications.

Ethanol withdrawal increases oxidative stress and reduces nitric oxide bioavailability in the vasculature of rats.

We analyzed the effects of ethanol withdrawal on the vascular and systemic renin-angiotensin system (RAS) and vascular oxidative stress. Male Wistar rats were treated with ethanol 3-9% (v/v) for a period of 21 days. Ethanol withdrawal was induced by abrupt discontinuation of the treatment. Experiments were performed 48 h after ethanol discontinuation. Rats from the ethanol withdrawal group showed decreased exploration of the open arms of the elevated-plus maze (EPM) and increased plasma corticosterone levels. Ethanol withdrawal significantly increased systolic blood pressure and plasma angiotensin II (ANG II) levels without an effect on plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, or plasma angiotensin I (ANG I) levels. No differences in vascular ANG I, ANG II levels, and ACE activity/expression and AT1 and AT2 receptor expression were detected among the experimental groups. Plasma osmolality, as well as plasma sodium, potassium, and glucose levels were not affected by ethanol withdrawal. Ethanol withdrawal induced systemic and vascular oxidative stress, as evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels and the vascular generation of superoxide anion. Ethanol withdrawal significantly decreased plasma and vascular nitrate/nitrite levels. Major new findings of the present study are that ethanol withdrawal induces vascular oxidative stress and reduces nitric oxide (NO) levels in the vasculature. Additionally, our study provides novel evidence that ethanol withdrawal does not affect the vascular ANG II generating system while stimulating systemic RAS. These responses could predispose individuals to the development of cardiovascular diseases.

Treatment with cannabidiol reverses oxidative stress parameters, cognitive impairment and mortality in rats submitted to sepsis by cecal ligation and puncture.

Oxidative stress plays an important role in the development of cognitive impairment in sepsis. Here we assess the effects of acute and extended administration of cannabidiol (CBD) on oxidative stress parameters in peripheral organs and in the brain, cognitive impairment, and mortality in rats submitted to sepsis by cecal ligation and perforation (CLP). To this aim, male Wistar rats underwent either sham operation or CLP. Rats subjected to CLP were treated by intraperitoneal injection with "basic support" and CBD (at 2.5, 5, or 10mg/kg once or daily for 9days after CLP) or vehicle. Six hours after CLP (early times), the rats were killed and samples from lung, liver, kidney, heart, spleen, and brain (hippocampus, striatum, and cortex) were obtained and assayed for thiobarbituric acid reactive species (TBARS) formation and protein carbonyls. On the 10th day (late times), the rats were submitted to the inhibitory avoidance task. After the test, the animals were killed and samples from lung, liver, kidney, heart, spleen, and brain (hippocampus) were obtained and assayed for TBARS formation and protein carbonyls. The acute and extended administration of CBD at different doses reduced TBARS and carbonyl levels in some organs and had no effects in others, ameliorated cognitive impairment, and significantly reduced mortality in rats submitted to CLP. Our data provide the first experimental demonstration that CBD reduces the consequences of sepsis induced by CLP in rats, by decreasing oxidative stress in peripheral organs and in the brain, improving impaired cognitive function, and decreasing mortality.

Development and validation of a disk solid phase extraction and gas chromatography-mass spectrometry method for MDMA, MDA, HMMA, HMA, MDEA, methamphetamine and amphetamine in sweat.

We describe the development and validation of a method for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3-hydroxy-4-methoxymethamphetamine (HMMA), 3-hydroxy-4-methoxyamphetamine (HMA), 3,4-methylenedioxyethylamphetamine (MDEA), methamphetamine (MAMP) and amphetamine (AMP) in sweat. Drugs were eluted from PharmChek sweat patches with sodium acetate buffer, extracted with disk solid phase extraction and analyzed using GC/MS-EI with selected ion monitoring. Limits of quantification (LOQ) for MDMA, MDEA, MAMP and AMP were 2.5 ng/patch, and 5 ng/patch for MDA, HMA and HMMA. This fully validated procedure was more sensitive than previously published analytical methods and permitted the simultaneous analysis of multiple amphetamine analogs in human sweat.