Deep sea minerals prolong life span of streptozotocin‐induced diabetic rats by compensatory augmentation of the IGF‐I‐survival signaling and inhibition of apoptosis

Consumption of deep sea minerals (DSM), such as magnesium, calcium, and potassium, is known to reduce hypercholesterolemia‐induced myocardial hypertrophy and cardiac‐apoptosis and provide protection against cardiovascular diseases. Heart diseases develop as a lethal complication among diabetic patients usually due to hyperglycemia‐induced cardiac‐apoptosis that causes severe cardiac‐damages, heart failure, and reduced life expectancy. In this study, we investigated the potential of DSM and its related cardio‐protection to increase the life expectancy in diabetic rats. In this study, a heart failure rat model was developed by using streptozotocin (65 mg kg^?1) IP injection. Different doses of DSM‐1× (37 mg kg^?1 day^?1), 2× (74 mg kg^?1 day^?1) and 3× (111 mg kg^?1 day^?1), were administered to the rats through gavages for 4 weeks. The positive effects of DSM on the survival rate of diabetes rats were determined with respect to the corresponding effects of MgSO₄. Further, to understand the mechanism by which DSM enhances the survival of diabetic rats, their potential to regulate cardiac‐apoptosis and control cardiac‐dysfunction were examined. Echocardiogram, tissue staining, TUNEL assay, and Western blotting assay were used to investigate modulations in the myocardial contractile function and related signaling protein expression. The results showed that DSM regulate apoptosis and complement the cardiomyocyte proliferation by enhancing survival mechanisms. Moreover DSM significantly reduced the mortality rate and enhanced the survival rate of diabetic rats. Experimental results show that DSM administration can be an effective strategy to improve the life expectancy of diabetic subjects by improving cardiac‐cell proliferation and by controlling cardiac‐apoptosis and associated cardiac‐dysfunction. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 769–781, 2016.     

Role of the cytoskeleton in communication between L‐type Ca2+ channels and mitochondria

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Alpha‐phellandrene‐induced apoptosis in mice leukemia WEHI‐3 cells in vitro

Although reports have shown that α‐phellandrene (α‐PA) is one of the monoterpenes and is often used in the food and perfume industry, our previous studies have indicated that α‐PA promoted immune responses in normal mice in vivo. However, there is no available information to show that α‐PA induced cell apoptosis in cancer cells, thus, we investigated the effects of α‐PA on the cell morphology, viability, cell cycle distribution, and apoptosis in mice leukemia WEHI‐3 cells in vitro. Results indicated that α‐PA induced cell morphological changes and decreased viability, induced G0/G1 arrest and sub‐G1 phase (apoptosis) in WEHI‐3 cells. α‐PA increased the productions of reactive oxygen species (ROS) and Ca²⁺ and decreased the levels of mitochondrial membrane potential (ΔΨ_m) in dose‐ and time‐dependent manners in WEHI‐3 cells that were analyzed by flow cytometer. Results from confocal laser microscopic system examinations show that α‐PA promoted the release of cytochrome c, AIF, and Endo G from mitochondria in WEHI‐3 cells. These results are the first findings to provide new information for understanding the mechanisms by which α‐PA induces cell cycle arrest and apoptosis in WEHI‐3 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1640–1651, 2016.     

Developmental toxic potential of di‐n‐propyl phthalate administered orally to rats

The objective of this study was to evaluate the developmental toxic potential of di‐n‐propyl phthalate (DnPP) in rats. Pregnant Sprague–Dawley rats were given DnPP at doses of 0 (olive oil), 0.5, 1 and 1.5 g kg^?1 per day, by gavage, on gestation days 6–20. Benchmark doses were calculated for the effects of DnPP on fetal weight and anogenital distance of the male fetuses. Maternal body weight gain was significantly reduced at 1.5 g kg^?1 per day, over gestation days 6–9. DnPP‐treated dams also showed a statistically significant increase in liver weight and a mild but statistically significant peroxisomal enzyme induction at 1 or 1.5 g kg^?1 per day. Male and female fetal body weights were significantly reduced at 1.5 g kg^?1 per day. There was a statistically significant decrease in the anogenital distance of the male fetuses at 1 and 1.5 g kg^?1 per day, and three males (of 75) showed malpositioned testis at the high dose. The mean percentage of fetuses per litter with cervical and thoracic rudimentary ribs was significantly increased at 1 and 1.5 g kg^?1 per day. Delayed ossification was seen at 1 g kg^?1 per day (phalanges) and 1.5 g kg^?1 per day (hyoid, sternebrae, and phalanges). No treatment‐related effects on prenatal viability or on fetal external or visceral malformations or variations were observed at any dose. Thus, there was no evidence of teratogenicity up to the high dose of 1.5 g kg^?1 per day. The no‐observed‐adverse‐effect level (NOAEL) for developmental toxicity was 0.5 g kg^?1 per day. Copyright © 2010 JohnWiley & Sons, Ltd.     

Toxicity of new emerging pollutant tris‐(2,3‐dibromopropyl) isocyanurate on BALB/c mice

The emerging heterocyclic brominated flame retardant tris‐(2,3‐dibromopropyl) isocyanurate (TBC), widely used in reinforced plastics, has demonstrated toxicity to fish. However, little is known about its toxicity in rodents. This study aims to determine the effect of TBC on growth, biochemical parameters in serum, organs and related gene expression of both male and female BALB/c mice after gastro‐gavage administration of 0, 2, 10 and 50 mg kg^?1 TBC for 28 days. Results indicated that exposure to TBC had no effects on basic growth and food intake of mice, but significantly increased serum alanine aminotransferase levels in male mice. Histopathological analyses showed that focal necrosis (2, 10 and 50 mg kg^?1 TBC‐exposed groups) and ballooning degeneration (10 and 50 mg kg^?1 TBC‐exposed groups) were found in mouse liver, whereas transmission electron microscopy revealed dose‐dependent hepatocyte apoptosis, mitochondrial degeneration and endoplasmic reticulum dilation. Histopathological and ultrastructural assessments in the lung showed dose‐dependent hyperplasia of pulmonary alveolar epithelium, bronchial congestion, infiltration of inflammatory cells and mitochondrial swelling following TBC exposure. Our results also indicated that mitochondria are one of the major target cytoplasmic organelles for TBC, suggesting that damage in mitochondria is one of the pathways that led to toxic effects in the liver and lung of TBC‐treated groups. Moreover, TBC effectively activated the gene expression of p53 in mice liver. Our findings provide strong evidence that TBC induces significant toxicity in mice organs, especially in liver and lung, which play vital roles in detoxification and gas exchange, respectively. This research will contribute to characterize the toxic effects of TBC, which was introduced as one of the candidates for brominated flame retardant replacement. Copyright © 2014 John Wiley & Sons, Ltd.     

Influence of α‐Adrenoceptor Agonists and Antagonists on Imipramine‐Induced Hypothermia in Mice

Abstract: Effects of adrenoceptor agonists and antagonists on imipramine‐induced hypothermia in mice were studied. Intraperitoneal injection of imipramine (10–40 mg kg^?1), α₂‐adrenoceptor agonist clonidine (0.05–0.1 mg kg^?1), α₁‐adrenoceptor agonist phenylephrine (6 mg kg^?1) and α₁‐adrenoceptor antagonist prazosin (1–4 mg kg^?1) but not α₂‐adrenoceptor antagonist yohimbine (1–4 mg kg^?1) induced significant hypothermia. The hypothermic response induced by imipramine (10–30 mg kg^?1) was not altered by clonidine (0.05–0.1 mg kg^?1) or phenylephrine (2–6 mg kg^?1). The response of imipramine (10–30 mg kg^?1) was reduced significantly by yohimbine (2 mg kg^?1) and was potentiated by prazosin (1 mg kg^?1). The hypothermic effect of clonidine (0.1 mg kg^?1) and imipramine (20 mg kg^?1) were also decreased significantly by different doses of yohimbine (1–4 mg kg^?1). The hypothermia induced by different doses of prazosin (1–4 mg kg^?1) was not altered by yohimbine (2 mg kg^?1) or by low dose of imipramine (10 mg kg^?1). It is concluded that α₂‐adrenoceptor mechanism may be involved in the hypothermic effect of imipramine.     

Purified c‐phycoerythrin: safety studies in rats and protective role against permanganate‐mediated fibroblast‐DNA damage

We have evaluated in vitro cytotoxicity of cyanobacterial phycoerythrin (C‐PE) on three human cell lines by cell proliferation and neutral red uptake assays. No toxic effects of C‐PE were observed to any of the cell lines tested. The protective role of purified C‐PE to potassium permanganate‐mediated human fibroblast‐DNA damage was assessed by comet assay at 0 (control), 10 and 20 µg C‐PE ml^?1 doses in pre‐, simultaneous and post‐mutagen exposure conditions. Significant DNA damage was detected only in post‐mutagen exposure conditions. Our findings confirmed that the C‐PE is non‐toxic and provides protection against permanganate‐mediated DNA damage. The preliminary acute (2000 mg C‐PE kg^?1 body weight, b.w.) and 90 day sub‐chronic (0, 5, 15 and 25 mg C‐PE kg^?1 b.w./day) oral toxicity studies of purified C‐PE in male albino rats showed no mortality or treatment‐related major clinical signs, and all the doses of C‐PE were well tolerated. The no observed adverse effect level and no observed effect level were found to be 15 and 5 mg C‐PE kg^?1 b.w./day respectively. Copyright © 2010 John Wiley & Sons, Ltd.     

Hyaline droplet accumulation in kidney of rats treated with hexachloro‐1:3‐butadiene: influence of age,dose and time‐course

The present research investigates the occurrence of hyaline droplet (HD) accumulation related to age, dose and time after treatment in male Wistar rats given a single i.p. injection of hexachloro‐1:3‐butadiene (HCBD). In the study on age, rats from 1 to 12 months of age were treated with 100 mg kg^?1 body weight (b.w.) HCBD dose. Rats treated at 2 months of age showed a greater accumulation of HD than the other age groups; HD accumulation was not observed in 1‐month‐old rats. In the dose–response study, the treatment with 25, 50 and 100 mg kg^?1 b.w. at 2 months of age caused HD accumulation in the proximal convoluted tubule at all doses, with the 100 mg kg^?1 b.w. group slightly more affected. Finally, in the time‐course study, rats treated with a 100 mg kg^?1 b.w. dose at 2 months of age and sacrificed at 6, 12, 24, 48, 72 and 96 h post‐dosing showed a time‐related HD accumulation in terms of incidence and severity, after 6 h, with a peak at 24 and 48 h and decreasing at 72 and 96 h. The present results show that HD accumulation is an early finding, and is unrelated to dose level and particularly evident in rats of 2 month of age. These findings in male rats treated with HCBD emphasize the importance of considering the age of rats at the start of a study. The more sensitive model was used in the detection of nephrotoxic effects of chemicals. Copyright © 2011 John Wiley & Sons, Ltd.     

Cross‐talk between L‐type Ca2+ channels and mitochondria

1. Calcium is necessary for myocardial function, including contraction and maintenance of cardiac output. Calcium is also necessary for myocardial energetics and production of ATP by mitochondria, but the mechanisms for calcium regulation by mitochondria are still not fully resolved. 2. The cytoskeleton plays an important role in maintaining a cell’s integrity. It is now recognized that cytoskeletal proteins can also assist in the transmission of signals from the plasma membrane to intracellular organelles. Cytoskeletal proteins can regulate the function of the L‐type Ca²⁺ channel and alter intracellular calcium homeostasis. 3. Recent evidence suggests that calcium influx through the L‐type Ca²⁺ channel is sufficient to alter a number of mitochondrial functional parameters, including superoxide production, NADH production and metabolic activity, assessed as the formation of formazan from tetrazolium salt. This occurs in a calcium‐dependent manner. 4. Activation of the L‐type Ca²⁺ channel also alters mitochondrial membrane potential in a calcium‐independent manner and this is assisted by movement of the auxiliary β₂‐subunit through F‐actin filaments. 5. Because the L‐type Ca²⁺ channel is the initiator of contraction, a functional coupling between the channels and mitochondria may assist in meeting myocardial energy demand on a beat‐to‐beat basis.     

The adverse effects of low‐dose exposure to Di(2‐ethylhexyl) phthalate during adolescence on sperm function in adult rats

Di(2‐ethylhexyl) phthalate (DEHP) is the most crucial phthalate derivative added to polyvinyl chloride as a plasticizer. This study examined the effects of low‐dose exposure to DEHP during adolescence on sperm function in adult rats. The male rats were daily gavaged with 30, 100, 300, and 1000 µg kg^?1 of DEHP or corn oil from postnatal day (PND) 42 until PND 105. The selection of DEHP doses ranged from the mean daily intake by the normal‐population exposure levels to no‐observed‐adverse‐effect level of DEHP for the endpoints evaluated until adulthood. Significant increases in the percentage of sperm with tail abnormality, tendency for sperm DNA fragmentation index (DFI) and percentage of sperm with DFI were found in those exposed to 100, 300, and 1000 µg kg^?1 (P < 0.05). We observed a significant increase of hydrogen peroxide (H₂O₂) generation in the sperm of the 1000 µg kg^?1 group compared with the control group (P < 0.05). The excessive production of sperm H₂O₂ coincided with an increase in sperm DFI. In this study, the lowest‐observed‐adverse‐effect level for sperm toxicity was considered to be 100 µg DEHP/kg/day in sperm morphology and chromatin DNA damage. Further research is necessary to clarify the mechanisms of DEHP‐related sperm ROS generation on sperm DNA damage. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 706–712, 2016.     

Preliminary safety evaluation of a taurocholate‐conjugated low‐molecular‐weight heparin derivative (LHT7): a potent angiogenesis inhibitor

In our previous studies, taurocholic acid (TA)‐conjugated low‐molecular‐weight heparin derivative (LHT7) has been proven to be a potent anti‐angiogenic agent by demonstrated successful blockage capability of vascular endothelial growth factors (VEGF). Preliminary safety evaluations were conducted based on its mechanism of action and chemical behavior. For this purpose, acute toxicity study, and hematological and serological evaluations were carried out. Additionally, in order to evaluate mechanism‐related side effects, both blood pressure and the occurrence of proteinuria were measured using a treatment regime of multiple high doses of LHT7 in a biodistribution study. LD₅₀ values for LHT7 in female and male mice were 56.9 and 64.7 mg kg^–1 doses, respectively. There were no vital fluctuations in the serological and hematological parameters, except for the elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 100 and 200 mg kg^–1 doses of LHT7, representing vital changes in the liver function. Moreover, the results of mechanism‐related studies showed that blood pressure at 50 mg kg^–1 did not change but showed elevated levels of protein in urine. In the biodistribution study, a slight accumulation of LHT7 in the kidney and the liver were observed at the 50 mg kg^–1 repeated dose owing to the presence of bile acid. No fatal damage was observed in this study; most observations were related to the chemical composition or the mechanism of action of the material. Copyright © 2014 John Wiley & Sons, Ltd.     

Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3‐E1 cells against antimycin A‐induced cytotoxicity

Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)‐induced toxicity in osteoblastic MC3T3‐E1 cells. Exposure of MC3T3‐E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca²⁺]_i) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A‐induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca²⁺]_i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3‐kinase), Akt (protein kinase B) and CREB (cAMP‐response element‐binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. Copyright © 2011 John Wiley & Sons, Ltd.     

Combinatorial effect of genistein and female sex‐steroids on uterine fluid volume and secretion rate and aquaporin (AQP)−1, 2, 5, and 7 expression in the uterus in rats

We hypothesized that genistein can interfere with the regulation of uterine fluid volume, secretion rate and expression of aquaporin in the uterus by female sex‐steroids, i.e., estrogen and progesterone. Therefore, the aims of this study were to investigate changes in these parameters in the presence of genistein and female sex‐steroids. Methods: Female Sprague‐Dawley rats were ovariectomized and received 3‐days estradiol‐17β benzoate (E2) plus genistein (25, 50, or 100 mg kg^?1 day^?1) or 3‐days E2 followed by 3‐days E2 plus progesterone with genistein (25, 50, or 100 mg kg^?1 day^?1). A day after last treatment, uterine fluid secretion rate was determined by in vivo uterine perfusion with rats under anesthesia. Animals were sacrificed and uteri were harvested and subjected for histological analyses. Luminal/outer uterine circumference was determined and distribution of AQP‐1, 2, 5, and 7 in endometrium was visualized by immunofluorescence. Expression of AQP‐1, 2, 5, and 7 proteins and mRNAs were determined by Western blotting and Real‐time PCR respectively. Results: Combined treatment of E2 with high dose genistein (50 and 100 mg kg^?1 day^?1) resulted in significant decrease in uterine fluid volume, secretion rate and expression of AQP‐1, 2, 5, and 7 proteins and mRNAs in uterus (p < 0.05). No significant changes in these parameters were observed when 25 mg kg^?1 day^?1 genistein was given with E2 or when genistein was given with E2 followed by E2 plus progesterone Conclusions: Decreased in uterine fluid volume, secretion rate and AQP‐1, 2, 5, and 7 expression in the uterus by high dose genistein in the presence of E2 could potentially affect female fertility. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 832–844, 2017.     

Mitochondrial and liver oxidative stress alterations induced by N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine: relevance for hepatotoxicity

The most significant toxicological effect of nitrosamines like N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) is their carcinogenic activity, which may result from exposure to a single large dose or from chronic exposure to relatively small doses. However, its effects on mitochondrial liver bioenergetics were never investigated. Liver is the principal organ responsible for BBN metabolic activation, and mitochondria have a central function in cellular energy production, participating in multiple metabolic pathways. Therefore any negative effect on mitochondrial function may affect cell viability. In the present work, ICR male mice were given 0.05% of BBN in drinking water for a period of 12 weeks and were sacrificed one week later. Mitochondrial physiology was characterized in BBN‐ and control‐treated mice. Transmembrane electric potential developed by mitochondria was significantly affected when pyruvate–malate was used, with an increase in state 4 respiration observed for pyruvate–malate (46%) and succinate (38%). A decrease in the contents of one subunit of mitochondrial complex I and in one subunit of mitochondrial complex IV was also observed. In addition, the activity of both complexes I and II was also decreased by BBN treatment. The treatment with BBN increases the susceptibility of liver mitochondria to the opening of the mitochondrial permeability transition pore. This susceptibility could be related with the increase in the production of H₂O₂ by mitochondria and increased oxidative stress confirmed by augmented susceptibility to lipid peroxidation. These results lead to the conclusion that hepatic mitochondria are one primary target for BBN toxic action during liver metabolism. Copyright © 2011 John Wiley & Sons, Ltd.     

Regulation of 1‐α, 25‐Dihydroxyvitamin D3 on Interleukin‐6 and Interleukin‐8 Induced by Sulfur Mustard (HD) on Human Skin Cells*

Abstract: The regulatory effects of the active form of vitamin D, 1‐α, 25‐dihydroxyvitamin D₃ (1‐α, 25 (OH)₂D₃) were assessed on the cytokine and chemokine secretion induced by sulfur mustard on human skin fibroblasts and human epidermal keratinocytes. Stimulation of human skin fibroblasts with sulfur mustard (10^?4 M for 24 hr at 37°) resulted in approximately a 5 times increase in the secretion of interleukin‐6 and over a 10 times increase for interleukin‐8, which was inhibited by 1‐α, 25 (OH)₂D₃, at ≤10^?9 M. 1‐α, 25 (OH)₂D₃ also suppressed interleukin‐8 secretion by 5 times and interleukin‐6 by 4 times on sulfur mustard‐stimulated human epidermal keratinocytes at concentrations ≤ 10^?9 M. The effect of 1‐α, 25 (OH)₂D₃ was dose‐dependent for the suppression of interleukin‐6 and interleukin‐8 induced by sulfur mustard on human skin fibroblasts/human epidermal keratinocytes, apparent at nanomolar concentrations. Our results indicate that the suppression of these inflammatory mediators by 1‐α, 25 (OH)₂D₃ is dependent on the source of the primary cultures, cell densities, and kinetics of pretreatments. In contrast to the inhibition of cytokine/chemokine production, cell proliferation was enhanced by almost 1.7 times on treated human epidermal keratinocytes with 1‐α, 25 (OH)₂D₃ (1×10^?9 M) after sulfur mustard‐stimulation (10^?4 M for 24 hr at 37°C). The observed enhancement diversified based on cell density, and kinetics of pretreatment with a maximal synergism (s) observed at 1×10^?9 M. Photomicrographs show typical signs of cellular degeneration caused by sulfur mustard such as chromatin condensation. The observed cellular degeneration was lessened when human epidermal keratinocytes were treated with 1‐α, 25 (OH)₂D₃ (2×10^?9 M). 1‐α, 25(OH)₂D₃ could be an alternative treatment for cutaneous inflammation disorders caused by sulfur mustard because we have demonstrated its ability to suppress inflammatory mediators and enhanced cell proliferation in human skin cells stimulated with sulfur mustard.     

Presynaptic α2‐adrenoceptor‐mediated modulation of adenosine 5′ triphosphate and noradrenaline corelease: differences in canine mesenteric artery and vein

1 The modulatory effects of agonists and antagonists of prejunctional α₂‐adrenoceptors on the electrical field stimulation (EFS, 0.3 ms, 12 V)‐induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5′ triphosphate (ATP) were measured in endothelium‐denuded segments of canine inferior mesenteric artery and compared with effects in mesenteric vein. The overflow of NA and ATP was evoked by long‐duration (2 min) EFS at low frequency (4 Hz) and high frequency (16 Hz) of stimulation and was analysed using HPLC techniques with electrochemical detection and fluorescence detection, respectively. 2 The EFS‐evoked overflow of both NA and ATP was significantly reduced by tetrodotoxin (1 μM ) and guanethidine (10 μM ) in the artery and vein. Desipramine (10 μM ), a blocker of neuronal uptake of NA, increased the EFS (4 and 16 Hz)‐evoked overflow of NA in both artery and vein. EFS‐evoked overflow of NA in vein exceeded the NA overflow in artery at both 4 and 16 Hz in control preparations as well as in the presence of desipramine. However, the EFS‐evoked overflow of ATP was equal in the artery and vein. 3 Stimulation of α₂‐adrenoceptors with clonidine (0.1 μM ) and oxymethazoline (0.3 μM ) reduced the EFS evoked overflow of NA in both artery and vein at 4 Hz, whereas the NA overflow at 16 Hz remained unchanged in both blood vessels. The overflow of ATP as well as of ADP (and hence ATP:ADP ratio) was unaffected by the α₂‐adrenoceptor agonists in the artery and vein. 4 In artery, blockade of α₂‐adrenoceptors with yohimbine at a concentration of 0.1 μM caused no effect on the NA overflow neither at 4 Hz nor at 16 Hz of EFS. Yohimbine at a concentration of 1 μM increased the overflow of NA at 4 Hz but not 16 Hz of EFS. In vein, however, yohimbine (0.1 and 1 μM ) increased NA overflow at both 4 and 16 Hz of stimulation. Idazoxan (1 μM ) increased the NA overflow in artery only at 4 Hz, whereas in vein idazoxan increased the NA overflow at both 4 and 16 Hz. No changes of EFS‐evoked ATP overflow were observed in the presence of 0.1 μM yohimbine in both artery and vein. Greater concentration of yohimbine (i.e. 1 μM ) increased the overflow of ATP in both the artery and vein only at 4 Hz EFS. Idazoxan (1 μM ) enhanced the ATP overflow only at 16 Hz in vein. The overflow of ADP was affected by both yohimbine and idazoxan in a similar manner to the ATP overflow so that the ATP:ADP ratios were not changed. 5 In conclusion, sympathetic nerves in both mesenteric arteries and veins appear to release ATP along with NA. Release of NA in veins exceeds release of NA in arteries, whereas both the canine artery and vein release equal amount of ATP. At long‐duration nerve stimulation (as might occur during stress) the α₂‐adrenoceptors appear to rather modulate release of NA than release of the cotransmitter ATP. The prejunctional autoinhibition of NA release is more effective at lower frequencies of nerve stimulation. The α₂‐adrenoceptor‐mediated neuromodulation plays a greater role in veins than arteries. Quantitative differences in α₂‐adrenoceptor‐mediated neuromodulation in the arteries and veins may participate to differing contributions of mesenteric blood vessels to the control of blood flow and volume distribution in splanchnic circulation.     

High doses of 2,2′‐dithienyl diselenide cause systemic toxicity in rats: an in vitro and in vivo study

Organoselenium compounds have important pharmacological properties. However, these compounds can cause toxicity, typically related to oxidation of endogenous thiols. The aim of this study was to investigate whether 2,2′‐dithienyl diselenide (DTDS) has potential toxicity in vitro and in vivo. Therefore, sulfhydryl‐containing enzyme activities, δ‐aminolevulinic acid dehydratase (δ‐ALA‐D) and Na⁺–K⁺‐ATPase were used to predict DTDS toxicity in rat brain homogenate in vitro. In in vivo experiments, a DTDS administration (50 or 100 mg kg^?1, p.o.) to rats was performed and toxicological parameters were determined. DTDS inhibited δ‐ALA‐D (IC₅₀ 2 µm ) and Na⁺–K⁺‐ATPase (IC₅₀ 17 µm ) activities in vitro. The inhibitory effect of DTDS on δ‐ALA‐D and Na⁺–K⁺‐ATPase activities was restored by dithiothreitol. DTDS (5–25 µm ) elicited a thiol oxidase‐like activity. In vivo, DTDS (50 and 100 mg kg^?1) caused systemic toxicity, evidenced by a decrease in water and food intakes and body weight gain, as well as the death of rats. DTDS at the dose of 100 mg kg^?1 increased plasma alanine and aspartate aminotransferase activities and decreased urea levels. At 50 and 100 mg kg^?1, it increased lipid peroxidation levels. At the highest dose, DTDS inhibited δ‐ALA‐D activity. By contrast, Na⁺–K⁺‐ATPase activity and antioxidant defense were not altered in the brains of rats exposed to DTDS. In conclusion, interaction with the cisteinyl residues seems to mediate the inhibitory effect of DTDS on sulfhydryl‐containing enzymes in vitro. In addition, high oral doses of DTDS induce toxicity in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Lack of specificity of [35S]‐ATPγS and [35S]‐ADPβS as radioligands for ionotropic and metabotropic P2 receptor binding

Adenosine‐5′‐O‐3‐thio[³⁵S]triphosphate ([³⁵S]‐ATPγS) has been reported to specifically bind several P2X receptor subtypes, including P2X₁, P2X₂, P2X₃, and P2X₄. Similarly, adenosine‐5′‐O‐2‐thio[³⁵S]diphosphate ([³⁵S]‐ADPβS) has been reported to label putative P2Y receptors. To address whether these radioligands selectively label P2 receptors, the functional activity of various P2 ligands was compared with their ability to compete for [³⁵S]‐ATPγS and [³⁵S]‐ADPβS binding to cell membrane preparations from rat brain, HEK293 cells, and to native and P2X₄ transfected 1321N1 astrocytoma cells. [³⁵S]‐ATPγS (0.2 nM) and [³⁵S]‐ADPβS (0.1 nM) displayed a high percentage of specific binding to membranes prepared from 1321N1 human astrocytoma cells, which were found to be devoid of detectable P2X and P2Y functional activity. [³⁵S]‐ATPγS and [³⁵S]‐ADPβS also exhibited equivalent high percentages of specific binding to HEK293 cell membranes, which endogenously express the P2Y₁ and P2Y₂ receptor subtypes, to 1321N1 cells stably transfected with the human P2X₄ receptor, and to rat brain membranes, which have previously been shown to contain both P2X and P2Y receptor subtypes. The potency order of P2 agonists to compete for radioligand binding to these cell membrane preparations was significantly different from the functional rank order potencies determined in HEK293 cells and 1321N1 cells expressing the P2X₄ receptor, as measured by cytosolic calcium flux. These data indicate that [³⁵S]‐ATPγS and [³⁵S]‐ADPβS appear to bind sites that do not correspond to known functional P2 receptor subtypes. The apparent lack of specificity of these radioligands for labeling P2 receptors is similar to that reported for other radiolabeled nucleotides and illustrates the need for caution in interpreting the apparent pharmacology of native P2 receptors on the basis of binding data alone. Drug Dev. Res. 48:84–93, 1999. © 1999 Wiley‐Liss, Inc.     

A novel 7‐azaisoindigo derivative‐induced cancer cell apoptosis and mitochondrial dysfunction mediated by oxidative stress

This research focused on a novel 7‐azaisoindigo derivative [namely N¹‐(n‐butyl)‐7‐azaisoindigo, 7‐AI‐b], and investigated its molecular antitumor mechanism by exploring the means of cell death and the effects on mitochondrial function. 7‐AI‐b inhibited cancer cell proliferation in a dose‐ and time‐dependent way. The morphological and nuclei changes in H₂B‐GFP‐labeled HeLa cells were observed using a live cell system. The results suggested that cell death induced by 7‐AI‐b is closely related to apoptosis. 7‐AI‐b induced release of cytochrome C from mitochondria to cytosol and activation of caspase‐3, showing that the apoptosis is mediated by the mitochondrial pathway. Furthermore, our data indicated that 7‐AI‐b triggers apoptosis through reactive oxygen species (ROS): cellular ROS levels were increased after 3 h exposure of 7‐AI‐b, which was reversed by the ROS scavenger N‐acetyl‐l ‐cysteine. As a consequence, 7‐AI‐b‐mediated cell death, mitochondrial transmembrane potential collapse and ATP level were partly blocked by N‐acetyl‐l ‐cysteine. Further study showed that 7‐AI‐b could induce mitochondrial dysfunction: collapse of the mitochondrial transmembrane potential and reduction of intracellular ATP level. In summary, the novel synthesized 7‐AI‐b was demonstrated to be effective in killing cancer cells via an ROS‐promoted and mitochondria‐ and caspase‐dependent apoptotic pathway. Copyright © 2010 John Wiley & Sons, Ltd.     

Attenuation of gentamicin‐induced nephrotoxicity: trimetazidine versus N‐acetyl cysteine

Gentamicin (G) is a highly nephrotoxic aminoglucoside. It was used to experimentally induce nephrotoxicity in male Wistar rats. To find a drug capable of protecting the nephron we assayed a cardioprotector (trimetazidine, TMZ) and a hepatoprotector (N‐acetyl cysteine, NAC). The rats were divided into six groups (n = 8): (A) control without drugs; (B) treated with 50 mg kg^?1 per day (i.p.) of G for 7 days; (C) diet supplemented with 20 mg kg^?1 per day of TMZ for 7 days; (D) treated with 10 mg kg^?1 per day (i.p.) of NAC for 7 days; (E) pretreated for 7 days with 20 mg kg^?1 per day of TMZ and during the following 7 days with G + TMZ; (F) pretreated for 7 days with 10 mg kg^?1 per day (i.p.) of NAC and during the following 7 days with G + NAC. Urea and creatinine as well as the excretion of urinary γ‐glutamyl transpeptidase (GGT_u) and urinary N‐acetyl‐glucosaminidase (NAG_u) were determined and structural and ultrastructural studies were carried out. Group B was used as a G‐induced nephrotoxicity control. Pretreatment with TMZ (E) showed a protector effect against induced nephrotoxicity, with no biochemical or functional changes nor alterations in histoarchitecture or ultrastructure. Pretreatment with NAC (F) showed no protector effect against G‐induced nephrotoxicity since no statistically significant differences were found with respect to the control group with G. We conclude that G‐induced nephrotoxicity is attenuated by the cytoprotective effect of TMZ. We may infer that TMZ inhibits the reabsorption and consequently the accumulation of G in the proximal tubule cell. Copyright © 2010 John Wiley & Sons, Ltd.