Use of bioassays and biomarkers in Daphnia magna to assess the effect of pharmaceutical residuals in freshwater ecosystems

Widespread occurrence of pharmaceuticals residues has been reported in aquatic ecosystems. However, their toxic effects on aquatic biota remain unclear. Generally, the acute toxicity has been assessed in laboratory experiments, while chronic toxicity studies have rarely been performed. Of importance appears also the assessment of mixture effects, since pharmaceuticals never occur in waters alone. The aim of the present work is to evaluate acute and chronic toxic response in the crustacean Daphnia magna exposed to single pharmaceuticals and mixtures. We tested fluoxetine, a SSRI widely prescribed as antidepressant, and propranolol, a non selective β-adrenergic receptor-blocking agent used to treat hypertension. Acute immobilization and chronic reproduction tests were performed according to OECD guidelines 202 and 211, respectively. Single chemicals were first tested separately. Toxicity of binary mixtures was then assessed using a fixed ratio experimental design with concentrations based on Toxic Units. The conceptual model of Concentration Addition was adopted in this study, as we assumed that the mixture effect mirrors the sum of the single substances for compounds having similar mode of action. The MixTox statistical method was applied to analyze the experimental results. Results showed a significant deviation from CA model that indicated antagonism between chemicals in both the acute and the chronic mixture tests. The study was integrated assessing the effects of fluoxetine on a battery of biomarkers. We wanted to evaluate the organism biological vulnerability caused by low concentrations of pharmaceutical occurring in the aquatic environment. We assessed the acetylcholinesterase and glutathione s-transferase enzymatic activities and the malondialdehyde production. No treatment induced significant alteration of biomarkers with respect to the control. Biological assays and the MixTox model application proved to be useful tools for pharmaceutical risk assessment. Although promising, the application of biomarkers in Daphnia magna needs further elucidation.

Decreased blood pressure response in mice deficient of the α1b-adrenergic receptor

To investigate the functional role of different α1-adrenergic receptor (α1-AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the α1b-AR (α1b−/−). Reverse transcription–PCR and ligand binding studies were combined to elucidate the expression of the α1-AR subtypes in various tissues of α1b +/+ and −/− mice. Total α1-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the α1b −/− as compared with +/+ mice. Because of the large decrease of α1-AR in the heart and the loss of the α1b-AR mRNA in the aorta of the α1b−/− mice, the in vivo blood pressure and in vitro aorta contractile responses to α1-agonists were investigated in α1b +/+ and −/− mice. Our findings provide strong evidence that the α1b-AR is a mediator of the blood pressure and the aorta contractile responses induced by α1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in α1b −/− as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in α1b−/− mice. The α1b-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different α1-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.

Hemodynamic responses to aortic depressor nerve stimulation in conscious L-NAME-induced hypertensive rats

Durand MT, Castania JA, Fazan R Jr, Salgado MC, Salgado HC. Hemodynamic responses to aortic depressor nerve stimulation in conscious L-NAME-induced hypertensive rats. Am J Physiol Regul Integr Comp Physiol 300: R418-R427, 2011. First published November 24, 2010; doi: 10.1152/ajpregu.00463.2010.-The present study investigated whether baroreflex control of autonomic function is impaired when there is a deficiency in NO production and the role of adrenergic and cholinergic mechanisms in mediating reflex responses. Electrical stimulation of the aortic depressor nerve in conscious normotensive and nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats was applied before and after administration of methylatropine, atenolol, and prazosin alone or in combination. The hypotensive response to progressive electrical stimulation (5 to 90 Hz) was greater in hypertensive (-27 +/- 2 to -64 +/- 3 mmHg) than in normotensive rats (-17 +/- 1 to -46 +/- 2 mmHg), whereas the bradycardic response was similar in both groups (-34 +/- 5 to -92 +/- 9 and -21 +/- 2 to -79 +/- 7 beats/min, respectively). Methylatropine and atenolol showed no effect in the hypotensive response in either group. Methylatropine blunted the bradycardic response in both groups, whereas atenolol attenuated only in hypertensive rats. Prazosin blunted the hypotensive response in both normotensive (43%) and hypertensive rats (53%) but did not affect the bradycardic response in either group. Prazosin plus angiotensin II, used to restore basal arterial pressure, provided hemodynamic responses similar to those of prazosin alone. The triple pharmacological blockade abolished the bradycardic response in both groups but displayed similar residual hypotensive response in hypertensive (-13 +/- 2 to -27 +/- 2 mmHg) and normotensive rats (-10 +/- 1 to -25 +/- 3 mmHg). In conclusion, electrical stimulation produced a well-preserved baroreflex-mediated decrease in arterial pressure and heart rate in conscious L-NAME-induced hypertensive rats. Moreover, withdrawal of the sympathetic drive played a role in the reflex bradycardia only in hypertensive rats. The residual fall in pressure after the triple pharmacological blockade suggests the involvement of a vasodilatory mechanism unrelated to NO or deactivation of alpha(1)-adrenergic receptor.

Amitriptyline and Acute Inflammation: A Study Using Intravital Microscopy and the Carrageenan-Induced Paw Edema Model

Background/Aims: Antidepressants are reported to exhibit antiinflammatory effects. However, mechanisms involved in this action have not been elucidated. Thus, the objectives of the present study were (a) to evaluate the effects of amitriptyline on the acute inflammatory process, and (b) to investigate the participation of alpha(1)-adrenergic receptors and glucocorticoids as possible mechanisms implicated in the amitriptyline action on inflammation. Methods and Results: Single and multiple doses of amitriptyline were administered to rats submitted to the carrageenan-induced paw edema model. The results showed a significant antiedematous reaction to amitriptyline, mainly when administered at each elimination half-life. The next step was to evaluate its effects on leukocyte behavior, using intravital microscopy. Amitriptyline produced a significant effect on leukocyte behavior. To investigate possible mechanisms involved, a glucocorticoid receptor antagonist (RU-486) and an alpha(1)-adrenergic receptor antagonist (prazosin) were used. RU-486 administration lacked the ability to decrease the amitriptyline antiinflammatory effects in the carrageenan-induced paw edema model. Prazosin pretreatment potentiated the amitriptyline antiinflammatory effect without presenting an effect per se. Conclusion:The present study shows the ability of amitriptyline to decrease edema and affect leukocyte behavior in an acute inflammatory process; and, for the first time to our knowledge, we suggest the involvement of alpha(1)-adrenoceptors in the antiinflammatory effects of amitriptyline. Copyright (C) 2010 S. Karger AG, Basel

Inhibition of pilocarpine-induced salivation in rats by central noradrenaline

Peripheral treatment with cholinergic or adrenergic agonists results in salivation and the possibility of synergy between cholinergic and adrenergic efferent mechanisms in the control of salivation has been proposed. Central injections of the cholinergic agonist pilocarpine also induce salivation, while the effects of central injections of noradrenaline (norepinephrine) are not known. Here (a) the effects of intracerebroventricular (icv) injection of noradrenaline on the salivation induced by icv or intraperitoneal (i.p.) injection of pilocarpine and (b) the receptors involved in the effects of central noradrenaline on pilocarpine-induced salivation were investigated. Male Holtzman rats with a stainless-steel guide cannula implanted into the lateral ventricle were used. Rats were anaesthetized with tribromoethanol (200 mg/kg body weight) and saliva was collected on small, preweighed cotton balls inserted into the animal's mouth. Noradrenaline (40, 80 and 160 nmol/l mul) injected icv reduced the salivary secretion induced by pilocarpine (0.5 mumol/l mul) injected icv. Noradrenaline (80 and 160 nmol/l mul) injected icv also reduced the salivation induced by pilocarpine (4 mumol/kg) injected i.p. Previous treatment with the alpha(2)-adrenergic receptor antagonists RX 821002 (40, 80 and 160 nmol/l mul) or yohimbine (160 and 320 nmol/l mul) abolished the inhibitory effect produced by icv injection of noradrenaline on pilocarpine-induced salivation in rats. Prazosin (alpha(1)-adrenergic receptor antagonist) injected icv did not change the effect of noradrenaline on pilocarpine-induced salivation. Prior icv injection of only RX 821002 (80 or 160 nmol/l mul) or yohimbine (320 nmol/l mul) increased pilocarpine-induced salivation. The results show that (1) contrary to its peripheral effects, noradrenaline acting centrally inhibits cholinergic-induced salivation in rats; (2) central mechanisms involving alpha(2)-adrenergic receptors inhibit pilocarpine-induced salivation. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Ventromedial hypothalamus lesions increase the dipsogenic responses and reduce the pressor responses to median preoptic area activation

In this study, we investigated the participation of adrenergic receptors of the median preoptic area (MnPO) and the participation of ventromedial hypothalamus (VMH) in angiotensin II- (ANG II)-induced water intake and presser responses. Male rats with sham or electrolytic VMH lesions and a stainless steel cannula implanted into the MnPO were used. Noradrenaline, clonidine (an alpha(2)-adrenergic receptor agonist), or phenylephrine (an alpha(1)-adrenergic receptor agonist) injected into the MnPO of sham-lesioned rats reduced water ingestion induced by ANG II injected into the same area. In VMH-lesioned rats ANG II-induced water intake increased with a previous injection of noradrenaline, phenylephrine, or isoproterenol. The presser response induced by ANG II injected into the MnPO was reduced in VMH-lesioned rats, whereas the presser response induced by clonidine was abolished. Previous treatment with noradrenaline and phenylephrine into the MnPO of sham-lesioned rats produced a presser response, and a hypotensive response was obtained with the previous administration of noradrenaline, phenylephrine or isoproterenol into the MnPO of VMH-lesioned rats. These results show that VMH is essential for the dipsogenic and presser responses induced by adrenergic and angiotensinergic activation of the MnPO in rats. (C) 1997 Elsevier B.V.

HIPERTENSAO ARTERIAL NA CRIANCA

The objective of this report was to summarily review the concept and the prevalence of arterial hypertension in children, its peculiarities and the difficulties in measuring of blood pressure at this age. Considerations of clinical picture, diagnosis, laboratory and drug-induced test (Captopril) were discussed. The authors presented various therapies utilized in hypertension and hypertensive crisis.

Comparison of medetomidine-ketamine and dexmedetomidine-ketamine anesthesia in golden-headed lion tamarins

The cardiovascular, respiratory, and anesthetic effects of medetomidine-ketamine (20 μg/kg bodyweight [BW] and 10 mg/kg BW) (MK group) or dexmedetomidine-ketamine (10 μg/kg BW and 10 mg/kg BW) (DK group) were studied in golden-headed lion tamarins. Heart rate decreased after administration of both combinations; this reduction was statistically greater in the DK group than in the MK group after 15 and 45 minutes. Systolic arterial pressure decreased in a similar way in both groups, except at 15 minutes, when systolic arterial pressure was significantly lower in the DK group. Diastolic arterial pressure, mean arterial pressure, respiratory rate, and rectal temperature were progressively reduced in all groups. Sedation time was significantly shorter and anesthesia time was significantly longer in the DK group compared with MK group. Anesthetic quality and analgesia scores were significantly greater at 5 and 15 minutes in the DK group compared with the MK group. The administration of dexmedetomidine-ketamine is as safe and effective as the administration of medetomidine-ketamine in tamarins.

Treatment of atherosclerotic renovascular disease

Treatment of atherosclerotic renovascular disease is controversial and revascularization is not a beneficial approach to all patients. Conditions as progressive deterioration of renal function, refractory hypertension or accelerated cardiovascular disease, especially recurrent pulmonary edema, could profit from renal angioplasty with stent placement. Surgical revascularization is a good option for patients who will need concomitant surgical corrections of abdominal aortic lesions. Treatment of all other patients must be individualized. Medical therapy is indicated for all patients with atherosclerotic renovascular disease. Observational studies pointed out to the beneficial effect of controlling blood pressure (<130/80 mm Hg), glucose and lipids profile, lifestyle modifications, specific use of platelet antiaggregant therapy, Angiotensin Conversion Enzyme Inhibitors (ACEI) and statins. All others cardiovascular risk factors must be controlled. The evaluation and management of other systemic atherosclerotic vascular lesions is important, especially coronary, carotid and abdominal aortic. This paper presents a review of evidences to rationale the atherosclerotic renovascular disease treatment. © 2008 Bentham Science Publishers Ltd.

Acute coronary syndrome associated with continuous 5-fluorouracil infusion in a patient with metastatic colorectal cancer-a case report with a discussion on this clinical dilemma

Introduction: 5-Fluorouracil (5-FU) is considered to be the backbone of colorectal cancer (CRC) systemic therapy since the great majority of recommended regimens include its administration. A clinical picture consisting of chest pain, sometimes cardiac enzyme elevation, electrocardiogram abnormalities consistent with myocardial ischemia, and normal coronary angiogram associated with 5-FU administration have been infrequently reported. The clinical dilemma is: Which chemotherapy regimen should we use in CRC patients with a previous acute coronary syndrome (ACS) associated with 5-FU? Case Report: We describe the case of a 55-year-old otherwise healthy woman with metastatic colon adenocarcinoma who presented an ACS probably secondary to arterial vasospasm while receiving continuous intravenous 5-FU infusion (mFOLFOX6 regimen). After the ACS, the patient was treated with raltitrexate plus oxaliplatin (TOMOX) and subsequently with irinotecan plus cetuximab with no other cardiac event. Conclusion: The risk of cardiotoxicity associated with 5-FU is low but real. The probable mechanism is arterial vasospasm, as suggested by our case report. Both the use of the TOMOX regimen and irinotecan plus cetuximab seems to be safe regimens to be considered in this clinical scenario. © 2009 Humana Press Inc.

Nitric oxide synthase content of hypothalamic explants: increase by norepinephrine and inactivated by NO and cGMP.

Release of luteinizing hormone (LH)-releasing hormone (LHRH), the hypothalamic peptide that controls release of LH from the adenohypophysis, is controlled by NO. There is a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers in the lateral median eminence, intermingled with terminals of the LHRH neurons. To study relations between NOS and LHRH in this brain region, we measured NOS activity in incubated medial basal hypothalamus (MBH). NOS converts [14C]arginine to equimolar quantities of [14C]citrulline plus NO, which rapidly decomposes. The [14C]citrulline serves as an index of the NO produced. NOS basal activity was suppressed by incubation of the tissue with an inhibitor of NOS, nitroarginine methyl ester (NAME) (10(-5) M). Furthermore, incubation of MBH explants for 30 min with norepinephrine (NE) increased NOS activity and the increase was prevented by prazosine (10(-5) M), an alpha 1-adrenergic receptor blocker; however, direct addition of NE to the tissue homogenate or to a preparation of MBH synaptosomes did not alter enzyme activity, which suggested that NE increased the content of NOS during incubation with the tissue. After purification of NOS, the increase in enzyme content induced by NE was still measurable. This indicates that within 30 min NE increased the synthesis of NOS in vitro. Incubation of MBH or the MBH homogenate with various concentrations of sodium nitroprusside (NP), a releaser of NO, reduced NOS activity at high concentrations (> or = 0.9 mM), which were associated with either a reduction of stimulation or a plateau of LHRH release. Finally, incubation of either MBH or the homogenate with cGMP, a major mediatior of NO action, at concentrations that increased LHRH release also reduced NOS activity. These results indicate that NO at high concentrations can inactivate NOS and that cGMP can also inhibit the enzyme directly. Therefore, the increased NOS activity induced by activation of alpha 1 receptors by NE is inhibited by NO itself and a principal product of its activity, cGMP, providing negative feedback on NOS. In central nervous system (CNS) infections with high concentrations of inducible NOS produced by glial elements, the high concentrations of NO and cGMP produced may suppress LHRH release, resulting in decreased gonadotropin and gonadal steroid release.

Ezrin directly interacts with the alpha1b-adrenergic receptor and plays a role in receptor recycling.

Using the yeast two-hybrid system, we identified ezrin as a protein interacting with the C-tail of the alpha1b-adrenergic receptor (AR). The interaction was shown to occur in vitro between the receptor C-tail and the N-terminal portion of ezrin, or Four-point-one ERM (FERM) domain. The alpha1b-AR/ezrin interaction occurred inside the cells as shown by the finding that the transfected alpha1b-AR and FERM domain or ezrin could be coimmunoprecipitated from human embryonic kidney 293 cell extracts. Mutational analysis of the alpha1b-AR revealed that the binding site for ezrin involves a stretch of at least four arginines on the receptor C-tail. The results from both receptor biotinylation and immunofluorescence experiments indicated that the FERM domain impaired alpha1b-AR recycling to the plasma membrane without affecting receptor internalization. The dominant negative effect of the FERM domain, which relies on its ability to mask the ezrin binding site for actin, was mimicked by treatment of cells with cytochalasin D, an actin depolymerizing agent. A receptor mutant (DeltaR8) lacking its binding site in the C-tail for ezrin displayed delayed receptor recycling. These findings identify ezrin as a new protein directly interacting with a G protein-coupled receptor and demonstrate the direct implication of ezrin in GPCR trafficking via an actin-dependent mechanism.