Natural Products: Anti-proliferative Effects of Lichen-derived Inhibitors of 5-Lipoxygenase on Malignant Cell-lines and Mitogen-stimulated Lymphocytes

Several lichen species have been used traditionally as medicinal plants. It has previously been shown that two low-molecular-weight lichen metabolites, lobaric acid isolated from Stereocaulon alpinum Laur. and protolichesterinic acid isolated from Cetraria islandica L. (Ach.), have in-vitro inhibitory effects on arachidonate 5-lipoxygenase. We have studied the effects of these compounds on cultured cells from man, including three malignant cell-lines (T-47D and ZR-75-1 from breast carcinomas and K-562 from erythro-leukaemia), as well as normal skin fibroblasts and peripheral blood lymphocytes. Both test substances caused a significant reduction in DNA synthesis, as measured by thymidine uptake, in all three malignant cell-lines; the dose inducing 50% of maximum inhibition (ED50) was between 1.1 and 24.6 μg mL^?1 for protolichesterinic acid and between 14.5 and 44.7 μg mL^?1 for lobaric acid. The breast-cancer cell-lines were more sensitive than K-562. The proliferative response of mitogen-stimulated lymphocytes was inhibited with a mean ED50 of 8.4 μg mL^?1 and 24.5 μg mL^?1 for protolichesterinic acid and lobaric acid, respectively. These concentrations are of the same order of magnitude as the IC50 values in the 5-lipoxygenase assay. Significant cell death (assessed by the MTS (3–4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and trypan blue exclusion) occurred in the three malignant cell-lines at protolichesterinic acid and lobaric acid concentrations above 20 and 30 μg mL^?1, respectively. In K-562 morphological changes consistent with apoptosis were detected. Up to 38% cell death was observed at 20 μg mL^?1 for protolichesterinic acid and 15 μg mL^?1 for lobaric acid in mitogen-stimulated lymphocytes but unstimulated lymphocytes were clearly less sensitive. In contrast, the DNA synthesis, proliferation and survival of normal skin fibroblasts were not affected at doses up to 20 μg mL^?1 for protolichesterinic acid and 30 μg mL^?1 for lobaric acid. We conclude that the anti-proliferative and cytotoxic effects observed might be related to the 5-lipoxygenase inhibitory activity of protolichesterinic acid and lobaric acid. These results open up the opportunity for future studies of these lichen metabolites with regard to their anti-tumour and anti-inflammatory properties.     

Evaluation of cytogenetic and DNA damage induced by the antidepressant drug-active ingredients,trazodone and milnacipran,in vitro

Trazodone and milnacipran are the active antidepressant drugs that are being used in the treatment of psychiatric disorders. In this study, the in vitro genotoxic effects of trazodone and milnacipran have been determined in human peripheral blood lymphocytes by using chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN), and comet assays. 3.13; 6.25; 12.50; 25.00; 50.00; and 75.00?μg/mL concentrations of trazodone and 2.50; 5.00; 10.00; 20.00; 30.00; and 40.00?μg/mL concentrations of milnacipran were used. Trazodone and milnacipran significantly increased the frequency of CAs and SCEs compared with the control. Both of the active ingredients raised the MN frequency in a dose-dependent manner. Mitotic index was significantly decreased, but replication and nuclear division indices were not affected at all treatments. Trazodone was statistically increased the mean comet tail intensity, tail length, and tail moment at three concentrations (6.25; 12.50; and 25.00?μg/mL) compared with control. Two highest concentrations (50 and 75?μg/mL) of trazodone were toxic in the comet assay. Milnacipran increased the comet tail intensity, tail length, and tail moment at all concentrations. It is concluded that trazodone and milnacipran have clastogenic, mutagenic, and cytotoxic effects on human lymphocytes in vitro.     

Effect of the Hydroalcoholic Extract of Rauwolfia ligustrina on Smooth and Cardiac Muscles In-vitro

The actions of the hydroalcoholic extract (HE) of Rauwolfia ligustrina (the whole plant) on agonist-induced contractions were analysed in the rat uterus and guinea-pig ileum and trachea, and also in rat atrium contracting spontaneously in-vitro. The HE (100–400 μg mL^?1) caused concentration-dependent rightward shifts of the concentration-response curves and reduced the maximal contraction induced by oxytocin, bradykinin, angiotensin II, prostaglandin F_2α and acetylcholine in the rat uterus. The calculated mean IC50 values were: 300, 147, 158, 197 and 105 μg mL^?1, respectively. In the guinea-pig ileum the HE also caused graded displacement to the right of the concentration—response curves for bradykinin, histamine and carbachol, associated with pronounced inhibition of the agonist-induced maximal contractions. The calculated mean IC50 values were 165, 134 and 241 μg mL^?1, respectively. The HE (100–3000 μg mL^?1) caused graded relaxation (IC50 of 271 μg mL^?1) of strips of guinea-pig trachea precontracted with carbachol (0.2 μM). This effect was not infuenced by propranolol (1 μM), 3-isobutyl-1-methylxanthine (1 μM) or methylene blue (10 μM), but was significantly potentiated (1.5-to 3-fold) by indomethacin (3 μM) and forskolin (1 nM). In addition, N^G-monomethyl-L-arginine (L-NMMA, 100 nM) significantly reduced the HE-induced maximal relaxation, while indomethacin (3 μM) potentiated the HE response. Finally, the HE caused a concentration-dependent and long-lasting inotropic effect in the rat right atrium, contracting spontaneously with a mean EC50 value of 409 μg mL^?1. It is suggested that the effects of the HE of R. ligustrina on smooth and cardiac muscles ‘in-vitro’ may result from its ability to interact, at least partially, with the cAMP pathway.     

Cr and Hg toxicity assessed in situ using the structural and functional characteristics of algal communities

The toxicity of mercury and chromium on algal community structure have been assessed using in situ N₂ase activity, pigment diversity, autotrophic index, and ¹⁴C uptake of algae. The location was in the river Ganga and controlled ecosystem pollution experiment enclosures were used. Maximum inhibition of algal number was observed at 0.8 μg Hg mL^?1 followed by 8.0 μg Cr mL^?1. Unicellular forms, except for Anorthoneis excentrica, were very sensitive to test metals used. The decline in algal number was concentration dependent and metal specific at generic and species levels. Complete elimination of three and six species was observed respectively at 8.0 μg Cr mL^?1 and 0.8 μg Hg mL^?1 after 12 days' exposure. Likewise, a concentration-dependent and metal-specific increase in autotrophic index and pigment diversity of phytoplankton was recorded for Hg and Cr. Inhibition of ¹⁴C uptake of phytoplankton in Ganga water was almost equal (79%) at 0.8 μg Hg mL^?1 and 8.0 μg Cr mL^?1 (78%). Although complete inhibition of in situ N₂ase was observed at 0.8 μg Hg mL^?1, it was only 80% with 8.0 μg Cr mL^?1. Our study suggests that heavy metals inhibit both structural and functional variables of phytoplankton in field microcosms. Hence this technique seems to hold potential for the biomonitoring of heavy metal toxicity in the field.     

A pharmacokinetic evaluation of HIV protease inhibitors,cyclic ureas,in rats and dogs

The pharmacokinetics of a series of novel cyclic, non-peptide inhibitors of HIV protease were studied in rats or dogs after intravenous and oral administration. Six symmetrically substituted cyclic urea compounds (XK234, XM311, XM320, XM321, XM323, and XM412), which effectively inhibited HIV virus replication, with IC₉₀, values of 0.03–1.0 μM (0.017–0.76 μg mL^?1), were evaluated. Plasma concentrations were measured in rats and dogs using specific and sensitive HPLC methods. In rats, the maximum plasma concentrations of 0.21–1.88 μg mL^?1 were detected within 1 h of oral administration of 10 mg kg^?1 of the compounds. The elimination half-lives ranged from 1.25 to 3.3 h in rats and the absolute oral bioavailability ranged from 18 to 100%. In dogs, the maximum plasma concentration and absolute oral bioavailability were 4.37 μg mL^?1 and 48%, 1.07 μg mL^?1 and 16%, and 1.48 mg mL^?1 and 38% for XK234, XM311, and XM323, respectively. The data demonstrated that the maximum plasma concentrations of these cyclic ureas were several times higher than the IC₉₀ for inhibition of viral replication after single doses of 10 mg kg^?1 in rats and dogs. With this combination of high potency against virus replication and good oral bioavailability, these cyclic ureas represent a new class of compounds that are suitable for development as therapeutic agents for the treatment of HIV-associated diseases.     

In vitro cytotoxicity and genotoxicity of Furia®180 SC (zeta-cypermethrin) and Bulldock 125®SC (β-cyfluthrin) pyrethroid insecticides in human peripheral blood lymphocytes

In the present study, human peripheral blood lymphocytes were exposed in vitro to 0, 6, 12, 18, 24, and 30?μg/mL Furia^®180 SC (zeta-cypermethrin) and 0, 6.3, 12.5, 18.8, 25, and 31.3?μg/mL Bulldock^®125 SC (β-cyfluthrin). Exposure to 32?µg/mL bleomycin for 24?h served as a positive control. The cytotoxic and genotoxic effects of each insecticide were analyzed using alkaline comet and trypan blue dye exclusion assays. DNA damage was evaluated through three genotoxicity parameters: tail length (TL), tail moment (TM) and tail intensity (TI). Furia^®180 SC and Bulldock^®125 SC pyrethroid insecticides and bleomycin significantly increased DNA damage in a concentration-dependent manner. Bulldock^®125 SC induced more DNA damage than Furia. Lymphocyte viability did not change after exposure to different concentrations of the two pyrethroid insecticides and bleomycin. Moreover, genotoxic results demonstrated that Furia^®180 SC and Bulldock^®125 SC insecticides caused in vitro DNA damage in human peripheral lymphocytes.     

Airway Reactivity to Inhaled Spasmogens 18–24 h after Antigen-challenge in Sensitized Anaesthetized Guinea-pigs

The anaesthetized allergic guinea-pig was used to assess changes in airway reactivity to four different inhaled spasmogens: methacholine, 5-hydroxytryptamine (5-HT), histamine and the thromboxane A₂ mimetic, 9,11-dideoxy-9α,11α-methano-epoxy-PGF_2α (U-46619). Reactivity was determined 18 to 24 h after challenge of ovalbumin-sensitized guinea-pigs with inhaled ovalbumin. This time coincides with the appearance of a late-phase bronchoconstriction in these animals. Sensitivity to the spasmogen was assessed from the concentration-response curve for the increase in pulmonary inflation pressure (PIP) in ovalbumin- and saline-challenged sensitized animals. When methacholine, 5-HT or histamine were the spasmogens there was no hyper-reactivity. The geometric mean EC50 values (i.e. the concentrations inducing half the maximum effect) obtained from the dose-response curves for methacholine (73 (42–129) and 94 (66–134) μg mL^?1), 5-HT (1.5 (0.81–3.03) and 1.1 (0.51–2.24 μg mL^?1) and histamine (39 (21–75) and 72 (32–162) μg mL^?1) did not differ significantly (P > 0.05) between saline- and ovalbumin-challenged animals, respectively. However, when U-46619 was the spasmogen, ovalbumin-induced airway hyper-reactivity was observed as a leftwards shift of the concentration-response curve and the EC50 value for ovalbumin-challenged animals (8.1 (5.1–13) ng mL^?1) was significantly (P < 0.05) less than the value for control animals (39 (21–75) ng mL^?1). Our findings suggest that airway hyper-reactivity is not ‘non-specific’, but instead depends on the chosen spasmogen. The absence of hyper-reactivity with certain spasmogens was not a result of poor delivery, because all spamogens caused a bronchoconstriction by the inhaled route. It was also not associated with the model because ozone has been shown to induce hyper-reactivity to inhaled methacholine and 5-HT. Because airway hyper-reactivity to both inhaled histamine and agonists at muscarinic receptors is regularly seen in man, the anaesthetized guinea-pig might not be the ideal model for assessing airway hyper-reactivity after antigen challenge and its modification by anti-asthma drugs.     

Stability,tissue metabolism,tissue distribution and blood partition of azosemide

Stability of azosemide after incubation in various pH solutions, human plasma, human gastric juice, and rat liver homogenates, metabolism of azosemide after incubation in 9000g supernatant fraction of various rat tissue homogenates in the presence of NADPH, tissue distribution of azosemide and M1 after intravenous (IV) administration of azosemide, 20 mg kg^?1, to rats, and blood partition of azosemide between plasma and blood cells from rabbit blood were studied. Azosemide seemed to be stable for up to 48 h incubation in various pH solutions ranging from two to 13 at an azosemide concentration of 10 μg mL^?1; more than 93.4% of azosemide was recovered and a metabolite of azosemide, M1, was not detected. However, the drug was unstable in pH 1 solution: 75.8% of azosemide was recovered and 2.16 μg mL^?1 of M1 (expressed in terms of azosemide) was formed after 48 h incubation in pH 1 solution at an azosemide concentration of 10 μg mL^?1. Azosemide was stable in both human plasma and rat liver homogenates for up to 24 h incubation at an azosemide concentration of 1 μg mL^?1, and in human gastric juice for up to 4 h incubation at an azosemide concentration of 10 μg mL^?1. However,-all rat tissues stdied had metabolic activity for azosemide in the presence of NADPH, with heart having a considerable metabolic acitivity: approximately 22% of azosemide disappeared and 9.32 μg of M1 was formed per gram of heart (expressed in terms of azosemide) after 30 min incubation of 50 μg of azosemide in 9000g supernatant fraction of heart homogenates. The tissue to plasma ratios of azosemide (T/P) were greater than unity only in the liver (1.26) and kidney (1.74); however, M1 showed high affinity for all tissues studied except the brain and spleen when each tissue was collected at 30 min after IV administration of azosemide to rats. The equilibrium plasma to blood cell concentration ratios of azosemide were independent of azosemide blood concentrations: the values were 2.78–4.25 at azosemide blood concentrations of 1, 10, and 20 μg mL^?1 three rabbits. There was negligible ‘blood storage effect’ of azosemide, especially at low blood concentrations of azosemide, such as 1 and 10 μg mL^?1.     

Chromosomal aberrations in cultured human lymphocytes treated with the fungicide,Thiram

In vitro effects of different concentrations of Thiram were tested on human lymphocytes to determine, by means of the chromosome aberrations (CAs) assay, whether this fungicide could induce clastogenic damage. Evidences of the effect of Thiram on human lymphocytes were limited to sister chromatid exchange, micronuclei formation, and comet assays. We evaluated 0.01, 0.1, 1.2, and 12.0 μg/mL of Thiram, where 0.01 μg/mL represent the acceptable daily intake dose set by the World Health Organization and the Food and Agriculture Organization for fruit and vegetables, whereas 0.1, 1.2, and 12.0 μg/mL are its multiple values. Results indicated that human lymphocytes treated in vitro with Thiram at concentrations of 1.20 and 12.0 μg/mL significantly increased CAs frequency, compared with the negative control, whereas at lower concentrations (0.01 and 0.1 μg/mL), this effect was not observed. However, Thiram showed a clastogenic effect also at the concentration value of 1.2 μg/mL that represents a lower value with respect to the residue limits found in Italy for grapes, strawberries, potatoes, tobacco, and other fruits and vegetables. Finally, according to some evidence obtained from the study of other fungicides, Thiram produced a significant reduction in the mitotic index with increasing concentration.     

Direct human DNA protection by Coriolus versicolor (Yunzhi) extract

Abstract

Context and objective: Scientific evidence has shown Coriolus versicolor (L. ex Fr.) Quel (also known as Yunzhi) has the role of immunomodulator in therapeutic effect. The aim of this in vitro study was to investigate the antioxidative effect of Yunzhi and to explore the mechanisms behind its DNA protection.

Materials and methods: Commercial Yunzhi extract was dissolved in water and diluted in five concentrations (10¹–10⁵?μg/L) with appropriate buffers. Lymphocytes harvested from three healthy subjects were incubated with Yunzhi extract for 30?min. Cells were then subjected to 5?min oxidant challenge by 45?μM hydrogen peroxide. The standard alkaline comet (SAC) assay and lysed cell comet (LCC) assay were performed in parallel. DNA damage of each treatment was scored under a fluorescence microscope and compared with the cells without Yunzhi pretreatment.

Results: U-shaped dose–response was seen in both versions of the comet assay. Yunzhi at 10⁴?μg/L demonstrated a genoprotective effect against oxidative damage in the SAC assay (25% decrease in comet score). In the LCC assay, a trend of protection in lymphocytes was observed but it did not reach statistical significance.

Conclusion: A direct antioxidant effect of Yunzhi against oxidant challenge on the DNA of lymphocytes was evidenced. The active component in Yunzhi was likely to be membrane permeable.     

The effects of chromium on the growth and activity of a pentachlorophenol-degrading bacterium

The effects of chromium (or chromate, as supplied by CrO₃) on a pentachlorophenol-degrading Flavobacterium sp. (ATCC 53874) were examined in a liquid bacterial growth medium. Cr⁶⁺ concentrations ? 5.0 μg mL^?1 caused the complete inhibition of bacterial growth. The EC₂₅, EC₅₀, and EC₇₅ calculated after 96 h of incubation were 0.44, 1.44, and 3.82 μg mL^?1, respectively. Cr⁶⁺ caused an irreversible reduction in total cell yield during the 21-day incubation. Cr⁶⁺ also elicited an increase in the lag time recorded before there was measurable pentachlorophenol (PCP) degradation by this bacterium. There was also an increase in the overall time required for complete degradation of PCP to nondetectable levels. A similar response was noted with all PCP concentrations examined from 10 to 100 μg mL^?1. However, a more pronounced response occurred at the lower PCP concentrations. The significance of these data relative to the in situ use of PCP-degrading bacteria for site bioremediation is outlined. © 1994 by John Wiley & Sons, Inc..     

Amiodarone and desethylamiodarone tissue uptake in rats chronically treated with amiodarone is non-linear with the dose

Four groups of rats were given amiodarone chronically at 25, 37m?5, 50, 75 mg kg^?1/12 h for 3 weeks; on day 21 the animals were killed and blood, plasma, heart, lung, liver and fat were collected and assayed for amiodarone and desethylamiodarone. Amiodarone plasma concentrations ranged from 0m?74 to 4m?68 μg mL^?1 and desethylamiodarone from 0m?08 to 2m?05 μg mL^?1. Plasma, blood and tissue concentrations of amiodarone and desethylamiodarone increased significantly with the dose. Blood/plasma and tissue/plasma partition ratios of amiodarone and desethylamiodarone increased significantly at the higher doses. Blood/plasma ratio was a good predictor of tissue/plasma ratios of amiodarone and its metabolite, except in fat. Total phospholipid concentrations in lung were correlated with amiodarone and desethylamiodarone concentrations in plasma, blood and lung.     

Renal Excretion and Accumulation Kinetics of 2-Methylbenzoylglycine in the Isolated Perfused Rat Kidney

The effect of protein binding on kidney function has been studied by investigating the renal accumulation and secretion of the hippurate analogue 2-methylbenzoylglycine in the isolated perfused rat kidney in the absence and presence of bovine serum albumin (BSA). Experiments were performed with either 2.5% pluronic or a combination of 2.2% pluronic and 2% BSA as oncotic agents; a wide concentration range (1–190 μg mL^?1) of 2-methylbenzoylglycine was studied. Tubular secretion appeared to be a function of the amount of unbound drug in the perfusate and was best described by a model consisting of a high and low affinity Michaelis-Menten term. Parameters obtained after the analysis of renal excretion data were maximum transport velocity for the high affinity site (T_M,H) = 3.0 ± 2.8 μg min^?1, Michaelis-Menten constant for tubular transport for the high affinity site (K_T,H) = 0.5 ± 0.8 μg mL^?1, maximum transport velocity for the low affinity site (T_M,L) = 250 ± 36 μg min^?1, and Michaelis-Menten constant for tubular transport for the low affinity site (K_T,L) = 62 ± 17 μg mL^?1. The compound accumulated extensively in kidney tissue, ratios up to 175 times the perfusate concentration were reached. Accumulation data were best analysed by a two-site model similar to the model used to describe renal excretion. Calculated parameters were theoretical maximum capacity of the high affinity site (R_M,H) = 26 ± 23 μg g^?1, affinity constant for renal accumulation at the high affinity site (K_A,H) = 0.2 ± 0.4 μg mL^?1, theoretical maximum capacity of the low affinity site (R_M,L)= 1640 ± 1100 μg g^?1 and affinity constant for renal accumulation at the low affinity site (K_A,L) = 60 ± 58 μg mL^?1. The very high accumulation in kidney tissue could be explained by active tubular uptake, mediated by the secretory mechanisms involved, and dependent on the amount of free drug in the perfusate. This study shows that anionic drugs, subject to active secretion, may reach high concentrations in tubular cells even at low plasma concentrations.     

The effect of carbenicillin on the haemostatic mechanism

Carbenicillin has no effect on the thrombin time, partial thromboplastin time, prothrombin time, platelet factor 3 availability, fibrinogen and plasminogen levels, Factor XIII, fibrin plate lysis or euglobulin lysis time in vitro in concentrations from 20–1280 μg ml^?1. After incubation with plasma, carbenicillin inhibited platelet aggregation to ADP at all the concentrations examined (20–1280 μg ml^?1). The same coagulation tests were unaffected 30 min and 4 h after intravenous administration of 5 g of carbenicillin to six normal subjects, though some impairment of platelet response to ADP occurred in three subjects. This impairment of platelet function is considered unlikely to contribute towards a bleeding tendency in normal subjects but might perhaps be a contributory factor in haemostatic failures in patients with uraemia.     

Delivery of atovaquone and proguanil across sublingual membranes,in vitro

Malarone^?, a combination of atovaquone (AT) and proguanil (PR), is indicated for the prophylaxis and treatment of uncomplicated Plasmodium falciparum malaria. This study aimed to determine in vitro the feasibility of delivering the combination of AT and PR as a spray formulation via the sublingual route, using Franz diffusion cells incorporating porcine sublingual mucosa. Firstly, 1?mg mL^?1 of each drug in 20% 1,8-Cineole in ethanol was used; and secondly, 5?mg mL^?1 AT and 1?mg mL^?1 PR in 20% 1-methyl-2-pyrrolidone in ethanol was examined, dosed every 2?h over a 12-h period and receptor phase samples were analyzed by HPLC. From the first study, mean fluxes for AT and PR were 12.89?±?1.2 and 5.88?±?0.9 µg cm^?2 h^?1 respectively; pharmacokinetic calculations indicated that these fluxes were insufficient to achieve the target plasma concentrations for AT and PR of 1.4 µg mL^?1 and 200?ng mL^?1 respectively, in the treatment of falciparum malaria. However, in the second study, the fluxes of AT and PR increased to 50.92?±?20.8 and 12.01?±?1.5 µg cm^?2 h^?1 respectively, and pharmacokinetic calculations indicated that therapeutic plasma concentrations are attainable for pediatric application.     

Hormetic effects of noncoplanar PCB exposed to human lung fibroblast cells (HELF) and possible role of oxidative stress

Hormesis, a biphasic dose–response phenomenon, which is characterized by stimulation of an end point at a low‐dose and inhibition at a high‐dose. In the present study we used human lungs fibroblast (HELF) cells as a test model to evaluate the role of oxidative stress (OS) in hormetic effects of non coplanar PCB 101. Results from 3‐(4,5‐dime‐thylthiazol‐2‐yl)‐2,5‐diphenyltetrazo‐lium bromide (MTT) assay indicated that PCB101 at lower concentrations (10^?5 to 10^?1 μg mL^?1) stimulated HELF cell proliferation and inhibited at high concentrations (1, 5, 10, and 20 μg mL^?1) in a dose‐ and time‐dependent manner. Reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) (except 48 h) showed a significant increase at higher concentrations of PCB 101 than those at the lower concentrations with the passage of time. Antioxidant enzymes such as glutathione peroxidase (GSH‐Px) exhibited decreasing trends in dose and time dependent manner. Lipid peroxidation assay resulted in a significant increase (P < 0.05) of MDA level in PCB 101‐treated HELF cells compared with controls, suggesting that OS plays a key role in PCB 101‐induced toxicity. Comet assay indicated a significant increase in genotoxicity at higher concentrations of PCB 101 exposure compared to lower concentrations. Overall, we found that HELF cell proliferation was higher at low ROS level and vice versa, which revealed activation of cell signaling‐mediated hormetic mechanisms. The results suggested that PCB 101 has hormetic effects to HELF cells and these were associated with oxidative stress. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1385–1392, 2015.     

The Effects of N-Benzoyl-β-alanine,a New Nephroprotective Drug,on the Distribution and Renal Excretion of Enprofylline in Rats

Abstract— The effects of the new nephroprotective drug N-benzoyl-β-alanine (BA) on the disposition and renal excretion of the bronchodilator enprofylline, which is actively secreted in urine, were investigated in rats. Enprofylline was administered intravenously at a dosage of 2·5 mg kg^?1 under three different steady-state plasma BA concentrations (100,200 and 400 μg mL^?1) which were achieved by constant infusion rates. Pharmacokinetic parameters for both total and unbound enprofylline were estimated by model-independent methods. The presence of BA (400 μg mL^?1) increased the systemic clearance by 25% and the volume of distribution at steady-state by 90%. A significant increase in the dissociation constant, which is the protein binding parameter of enprofylline was observed in the presence of BA (400 μg mL^?1), indicating that BA competitively inhibits the protein binding of enprofylline. However, BA significantly decreased the systemic clearance and volume of distribution for unbound enprofylline. These results suggest that BA, the organic anion transport inhibitor, inhibits renal excretion of enprofylline with a high affinity for renal tubular secretion, although the unbound concentration of enprofylline increases with administration of BA. We conclude that BA decreases the renal tubular secretion of enprofylline probably by reducing the affinity of the tubular transport system, and that these changes have marked effects on the pharmacokinetic behaviour of enprofylline.     

Toxicity thresholds for juvenile freshwater mussels Echyridella menziesii and crayfish Paranephrops planifrons,after acute or chronic exposure to Microcystis sp

Survival of juvenile freshwater mussels (Echyridella menziesii (Gray, 1843) formerly known as Hyridella menziesi) and crayfish (Paranephrops planifrons, White, 1842) decreased after four days exposure to microcystin‐containing cell‐free extracts (MCFE) of Microcystis sp. at concentrations typical of severe cyanobacterial blooms. Crayfish survival was 100, 80, and 50% in microcystin concentrations of 1339, 2426, and 11146 μg L^?1 respectively, and shade‐ and shelter‐seeking behavior was negatively affected when concentrations were ≥2426 μg L^?1. Mussel survival decreased to 92% and reburial rates decreased to 16% after exposure for 96 h to MCFE containing microcystins at concentrations of 5300 μg L^?1. Crayfish survival was 100% when fed freeze‐dried Microcystis sp. incorporated into an artificial diet (6–100 μg microcystin kg^?1 ww) at dietary doses from 0.03 to 0.55 μg g^?1 body weight d^?1 for 27 days. Specific growth rate was significantly lower in crayfish fed ≥0.15 μg g^?1 body weight day^?1 compared with controls, but not compared with a diet incorporating nontoxic cyanobacteria. Microcystins accumulated preferentially in crayfish hepatopancreas and mussel digesta as MCFE or dietary concentrations increased. These laboratory data indicate that, assuming dissolved oxygen concentrations remain adequate, and no simultaneous exposure to live Microcystis sp. cells, cell‐free microcystins will only be a significant stressor to juvenile crayfish and mussels in severe Microcystis sp. blooms. In contrast, crayfish were negatively affected by relatively low concentrations of microcystins in artificial diets compared with those measured locally in benthic cyanobacterial mats. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 487–502, 2014.     

Pharmacokinetics of a novel retinoid AGN 190168 and its metabolite AGN 190299 after intravenous administration of AGN 190168 to rats

The pharmacokinetics of AGN 190168, a novel synthetic retinoid, and its major metabolite, AGN 190299, in rat blood after intravenous administration was investigated. Approximately 4.4 mg kg^?1 (high dose) or 0.49 mg kg^?1 (low dose) of AGN 190168 was administered to rats via the femoral vein. Blood was collected from the femoral artery at various time points during an 8 h period. Blood concentrations of AGN 190168 and AGN 190299 were determined by a specific and sensitive high-pressure liquid chromatographic (HPLC) method. AGN 190168 was rapidly metabolized in rats. The only detectable drug-related species in the blood was AGN 190299. Therefore, only pharmacokinetics of AGN 190299 were calculated. Elimination of AGN 190299 appeared to be non-linear after administration of the high dose, and linear after administration of the low dose. The maximum elimination rate (V_max) and the concentration at half of the V_max (k_m), as estimated by a Michaelis—Menten one-compartment model, were 7.58 ± 2.42 μg min^?1 (mean ± SD) and 6.10 ± 1.58 μg mL^?1, respectively. The value of the area under the blood concentration time curve (AUC) was 9.54 ± 1.68 μg h mL^?1 after administration of the high dose and 0.594 ± 0.095 μg h mL^?1 after administration of the low dose. The clearance value was 7.79 ± 1.20 mL min^?1 kg^?1 after the high dose, statistically significantly different from that after the low dose (p < 0.05), 14.0 ± 2.2 mL min^?1 kg^?1. The terminal half-life (t_1/2) was 1.25 ± 0.74 h for the high-dose group and 0.95 ± 0.16 h for the low-dose group. Study results demonstrate rapid systemic metabolism of AGN 190168 to AGN 190299, non-linear pharmacokinetics of AGN 190299 after the 4.4 mg kg^?1 dose, and the lack of difference in disposition profiles between sexes after intravenous administration of AGN 190168 to rats.     

Cytotoxicity and Genotoxicity Induced by the Pesticide Profenofos on Cultured Human Peripheral Blood Lymphocytes

Profenofos is a broad-spectrum organophosphate insecticide and acaricide used widely for agricultural and household purposes. The aim of this investigation was to determine its toxicity profile in vitro, using lymphocytes from peripheral blood samples of healthy human donors. We found the IC₅₀ of profenofos to be 3.5 μM as measured by Trypan blue dye exclusion method. Chromosomal analyses of the metaphase plates of the samples treated with sublethal concentrations of profenofos revealed satellite associations and chromatid breaks and gaps, indicating its effect on chromosomes. The results were further supported by comet assay, where single-strand breaks in DNA were observed as comet tail lengths. The results were statistically significant (p < 0.01, ANOVA). Hence, it may be proposed that in vitro assays like the comet assay and chromosomal aberrations test, which indicate genetic damage, could be used to study the effect of organophosphorus pesticide poisoning in humans.