Protoplast fusion betweenLentinula edodes andCoriolus versicolor

Protoplast fusion between isoleucine-, arginine- and thymidine-requiring auxotroph (lle, Arg, Thy) ofLentinula edodes and arginine-requiring auxotroph (Arg) ofCoriolus versicolor has been achieved using 30% polyethylene glycol (M.W. 4000) in 10 mMCaCl₂-glycine solution (pH 8.0). Fusion hybrids were selected in the 0.6 M sucrose supplemented minimal media on the basis of nutritional complementation with fusion frequency of 7.4×10⁻⁶. The hybrids included both parental and non-parental types in colony morphology, growth rate and isozyme patterns. We succeeded inter-order protoplast fusion between the auxotrophs ofLentinula edodes and Coriolus versicolor overcoming the natural barriers of incompatibility. We examined the characteristics of the hybrids and clarified the fusion process using electron microscopy.     

Genetic analysis on bioconversion of aniline to acetaminophen inStreptomyces fradiae

S. fradiae showed the highest acetanilide p-hydroxylation activity in the tested strains. AndS. fradiae was well characterized genetically, especially with respect to tylosin production. Two mutants, which lost hydroxylation, were isolated in 140 regenerated colonies from protoplasts. In restriction enzyme digesion of total DNAs, isolation of giant linear plasmid DNA and determination of antibiotic resistances to chloramphenicol, tylosin, hygromycin B and mitomycin C, any differences among mutants and a wild type strain were not detected. These facts suggest that lesion on 6,000 Kb chromosomal DNA was responsible for the lack of p-hydroxylation activity induced by protoplast formation and regeneration.     

Metabolomics of the Bio-Degradation Process of Aflatoxin B1 by Actinomycetes at an Initial pH of 6.0

Contamination of food and feed by Aflatoxin B1 (AFB1) is a cause of serious economic and health problems. Different processes have been used to degrade AFB1. In this study, biological degradation of AFB1 was carried out using three Actinomycete species, Rhodococcus erythropolis ATCC 4277, Streptomyces lividans TK 24, and S. aureofaciens ATCC 10762, in liquid cultures. Biodegradation of AFB1 was optimised under a range of temperatures from 25 to 40 °C and pH values of 4.0 to 8.0. An initial concentration of 20 µg/mL of AFB1 was used in this study. The amount of AFB1 remaining was measured against time by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC), coupled with UV and mass spectrometry (LC-MS). All species were able to degrade the AFB1, and no significant difference was found between them. AFB1 remained in the liquid culture for R. erythropolis, S. lividans and S. aureofaciens were 0.81 µg/mL, 2.41 µg/mL and 2.78 µg/mL respectively, at the end of the first 24 h. Degradation occurred at all incubation temperatures and the pH with the optimal conditions for R. erythropolis was achieved at 30 °C and pH 6, whereas for S. lividans and S. aureofaciens the optimum conditions for degradation were 30 °C and pH 5. Analysis of the degradative route indicated that each microorganism has a different way of degrading AFB1. The metabolites produced by R. erythropolis were significantly different from the other two microorganisms. Products of degradation were identified through metabolomic studies by utilizing high-resolution mass spectral data. Mass spectrometric analysis indicated that the degradation of AFB1 was associated with the appearance of a range of lower molecular weight compounds. The pathway of degradation or chemical alteration of AFB1 was followed by means of high resolution Fourier transform mass spectrometry (HR-FTMS) analysis as well as through the MS² fragmentation to unravel the degradative pathway for AFB1. AFB1 bio-degradation was coupled with the accumulation of intermediates of fatty acid metabolism and glycolysis. A plausible mechanism of degradation of AFB1 by Rhodococcus was hypothesized.     

Pharmacokinetic interaction study between benazepril and amlodipine in healthy subjects

Pharmacokinetic interaction between benazepril (ACE inhibitor) and amlodipine (calcium channel blocker) was studied in 12 healthy subjects. Single doses of benazepril hydrochloride (10-mg tablet) and amlodipine besylate (tablet equivalent to 5 mg amlodipine) were administered alone or in combination according to a three-way, Latin-Square, randomized crossover design. Serial blood samples were collected following each administration for the determination of benazepril and its active metabolite benazeprilat and amlodipine. The mean values of AUC (0–4 h), C_max andT _max for benazepril given as combination versus given alone were 161 vs 140 ng·h·ml⁻¹, 168 vs 149 ng·ml⁻¹, and 0.5 vs 0.6 h. The mean values of AUC (0–24 h), C_max andT _max for benazeprilat after benazepril given as combination versus given alone were 1470 vs 1410 ng·h·ml⁻¹, 292 vs 257 ng·ml⁻¹, and 1.7 vs 1.5 h. The mean values of AUC (0–144 h), C_max andT _max for amlodipine given as combination versus given alone were 118 vs 114 ng·h·ml⁻¹, 2.5 vs 2.3 ng·ml⁻¹, and 8.3 vs 9.0 h. The differences in these pharmacokinetic parameters between the combination and monotherapy treatments were not statistically significant based on ANOVA. The results of this study indicate that no pharmacokinetic interaction existed between the two drugs.     

Effect ofAlpha-2 adrenergic agonist onBeta adrenoceptor-mediated control of blood glucose in the fasted mouse

Dose-dependent increases in blood glucose were produced by epinephrine and clonidine in fasted male mice. Isoproterenol was ineffective in increasing blood glucose at lower doses (10⁻⁸ M/kg−10⁻⁷ M/kg); with higher dose (10⁻⁶ M/kg) the glucose level was increased. The hyperglycemia induced by epinephrine was inhibited by yohimbine, prazosin and propranolol, indicating that the hyperglycemic effect of epinephrine is mediated byalpha-1,alpha-2 andbeta adrenoceptor. When clonidine (10⁻⁶ M/kg) was administered simultaneously with isoproterenol (10⁻⁶ M/kg), an enhenced hyperglycemic effect was observed. The increment produced by clonidine plus isoproterenol was higher than that by clonidine alone. These results suggest that stimulation ofalpha-2 adrenoceptor may be responsible for the exertion of the hyperglycemic effect bybeta agonists in fasted mice.     

Kanamycin acetyltransferase gene from kanamycin-producingStreptomyces kanamyceticus IFO 13414

A kanamycin producer,Streptomyces kanamyceticus IFO 13414 is highly resistant to kanamycin. Cloning of the kanamycin resistance genes inS. lividans 1326 with pIJ702 gave several kanamycin resistant transformants. Two transformants,S. lividans SNUS 90041 andS. lividans SNUS 91051 showed similar resistance patterns to various aminoglycoside antibiotics. Gene mapping experiments revealed that plasmids pSJ5030 and pSJ2131 isolated from the transformants have common resistant gene fragments. Subcloning of pSJ5030 gave a 1.8 Kb gene fragment which showed resistance to kanamycin. Cell free extracts ofS. lividans SNUS 90041,S. lividans SNUS 91051 and subclone aS. lividans SNUS 91064 showed kanamycin acetyltransferase activity. The detailed gene map is included.     

Lack of enantiomeric influence on the brain cytoprotective effect of ibuprofen and flurbiprofen

R(−) enantiomers of the 2-arylpropionic acid derivatives ibuprofen and flurbiprofen weakly inhibit cyclooxygenase (COX) activity. However, a possible cytoprotective effect has been proposed. The aim of the study is to investigate the possible mechanism of this effect. An in vitro hypoxia–reoxygenation model in rat brain slices was used (n = 6 rats per group). After reoxygenation, we measured LDH efflux (neuronal death), brain prostaglandin E₂ (PGE₂) concentration, interleukins (IL)-1β and 10, oxidative and nitrosative stress (lipid peroxides, glutathione, 3-nitrotyrosine, and nitrites/nitrates). Anti-COX activity was measured in human whole blood. Racemic, R(−), and S(+) enantiomers of ibuprofen and flurbiprofen were tested. All compounds had a cytoprotective effect with IC₅₀ values in the range of 10⁻⁵ M. R(−) enantiomers did not significantly inhibit brain PGE₂. The concentration of IL-1β was reduced by 53.1% by the racemic form, 30.6% by the S(+) and 43.2% by the R(−) enantiomer of ibuprofen. The IL-10 concentration increased significantly only with S(+)-flurbiprofen (33.1%) and R(−)-flurbiprofen (26.1%). Lipid peroxidation was significantly reduced by all three forms of flurbiprofen. Nitrite + nitrate concentrations were reduced by racemic, S(+), and R(−)-flurbiprofen. Peroxynitrite formation (3-nitrotyrosine) was significantly reduced by racemic and S(+)-ibuprofen. COX inhibition is not the main mechanism of cytoprotection for these compounds. Their influence on inflammatory mediators and oxidative and nitrosative stress could account for the potential cytoprotective effect of R(−) enantiomers.     

Enantioselective pharmacokinetic and pharmacodynamic properties of carvedilol in spontaneously hypertensive rats: focus on blood pressure variability

The cardiovascular effects and pharmacokinetics of carvedilol were assessed in spontaneously hypertensive (SH) and Wistar Kyoto (WKY) animals with special focus on short-term blood pressure variability (BPV). Male SH and WKY rats were acutely treated with vehicle or carvedilol 1 or 5 mg kg⁻¹ (i.v.), and effects on blood pressure (BP), heart rate (HR) and BPV were recorded. Plasma pharmacokinetics of R- and S-carvedilol was studied by traditional blood sampling. Relationship between carvedilol concentrations and their hypotensive and bradycardic effects was established by pharmacokinetic–pharmacodynamic (PK–PD) modelling. Short-term BPV was assessed by standard deviation of BP recording. Vascular sympatholytic activity of carvedilol was studied by estimation of drug effects on ratio between low frequency (LF) and high frequency (HF) BPV (LF/HF ratio). Although pharmacokinetic properties of carvedilol remained mainly unaffected in SH rats with regard to WKY rats, hypertensive animals showed a reduction in drug clearance of R- and S-carvedilol after administration of 1 mg kg⁻¹ compared with WKY rats. PK–PD analysis of HR changes induced by S-carvedilol showed a greater maximal bradycardic response to carvedilol in SH rats (E _max, −27.6 ± 3.9%; p < 0.05) compared with WKY group (E _max, −13.4 ± 2.5%). SH rats showed a greater hypotensive effect of racemic carvedilol (E _max, −45.5 ± 5.0%; p < 0.05) with regard to WKY group (E _max, −17.9 ± 4.5%). Carvedilol induced a greater reduction of LF/HF ratio in SH rats compared with WKY rats. Short-term BPV was markedly reduced by carvedilol in WKY and SH rats. In conclusion, as a consequence of an enhanced bradycardic response and a greater vascular sympatholytic activity, carvedilol exerts a greater hypotensive response in SH rats compared with WKY animals and dramatically reduces short-term BPV.     

Urotensin II acutely increases myocardial length and distensibility: potential implications for diastolic function and ventricular remodeling

Urotensin II (U-II) is a cyclic peptide that may be involved in cardiovascular dysfunction. In the present study, the acute effects of U-II on diastolic properties of the myocardium were investigated. Increasing concentrations of U-II (10⁻⁸ to 10⁻⁶ M) were added to rabbit papillary muscles in the absence (n = 15) or presence of: (1) damaged endocardial endothelium (EE; n = 9); (2) U-II receptor antagonist, urantide (10⁻⁵ M; n = 7); (3) nitric oxide (NO) synthase inhibitor, N^G-Nitro-l-Arginine (10⁻⁵ M; n = 9); (4) cyclooxygenase inhibitor, indomethacin (10⁻⁵ M; n = 8); (5) NO synthase and cyclooxygenase inhibitors, N^G-Nitro-l-Arginine (10⁻⁵ M) and indomethacin (10⁻⁵ M), respectively, (n = 8); or (6) protein kinase C (PKC) inhibitor, chelerythrine (10⁻⁵ M; n = 9). Passive length–tension relations were constructed before and after a single concentration of U-II (10⁻⁶ M; n = 3). U-II concentration dependently decreased inotropy and increased resting muscle length (RL). At 10⁻⁶ M, active tension decreased 13.8 ± 5.4%, and RL increased to 1.007 ± 0.001 L/L _max. Correcting RL to its initial value resulted in an 18.1 ± 3.0% decrease in resting tension, indicating decreased muscle stiffness, which was also suggested by the down and rightward shift of the passive length–tension relation. This effect remained unaffected by EE damage and PKC inhibition. In contrast, the presence of urantide and NO inhibition abolished the effects of U-II on myocardial stiffness, while cyclooxygenase inhibition significantly attenuated them. U-II decreases myocardial stiffness, an effect that is mediated by the urotensin-II receptor, NO, and prostaglandins. This represents a novel mechanism of acute neurohumoral modulation of diastolic function, suggesting that U-II is an important regulator of cardiac filling. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Presented in part at the American Heart Association Scientific Sessions conference, 2006, in Chicago, Illinois.     

Inhibition of CYP2C9 by selective serotonin reuptake inhibitors: in vitro studies with tolbutamide and (S)-warfarin using human liver microsomes

Objective: To investigate the in vitro potential of selective serotonin reuptake inhibitors (SSRIs) to inhibit two CYP2C9-catalysed reactions, tolbutamide 4-methylhydroxylation and (S)-warfarin 7-hydroxylation. Methods: The formation of 4-hydroxytolbutamide from tolbutamide and that of 7-hydroxywarfarin from (S)-warfarin as a function of different concentrations of SSRIs and some of their metabolites was studied in microsomes from three human livers. Results: Both tolbutamide 4-methylhydroxylation and (S)-warfarin 7-hydroxylation followed one enzyme Michaelis-Menten kinetics. Kinetic analysis of 4-hydroxytolbutamide formation yielded a mean apparent Michaelis-Menten constant (K_m) of 133 μM and a mean apparent maximal velocity (V_max) of 248 pmol · min⁻¹ · mg⁻¹; formation of 7-hydroxywarfarin yielded a mean K_m of 3.7 μM and a mean V_max of 10.5 pmol · min⁻¹ ·  mg⁻¹. Amongst the SSRIs and some of their metabolites tested, only fluvoxamine markedly inhibited both reactions. The average computed inhibition constant (K_i) values and ranges of fluvoxamine when tolbutamide and (S)-warfarin were used as substrate, were 13.3 (6.4–17.3) μM and 13.0 (8.4–18.7) μM, respectively. The average K_i value of fluoxetine for (S)-warfarin 7-hydroxylation was 87.0 (57.0–125) μM. Conclusion: Amongst the SSRIs tested, fluvoxamine was shown to be the most potent inhibitor of both tolbutamide 4-methylhydroxylation and (S)-warfarin 7-hydroxylation. Fluoxetine, norfluoxetine, paroxetine, sertraline, desmethylsertraline, citalopram, desmethylcitalopram had little or no effect on CYP2C9 activity in vitro. This is consistent with in vivo data indicating that amongst the SSRIs, fluvoxamine has the greatest potential for inhibiting CYP2C9-mediated drug metabolism. Received: 20 July 1998 / Accepted in revised form: 6 October 1998     

Evaluation of protective efficacy of Spirulina platensis against collagen-induced arthritis in rats

Aim  To assess the protective efficacy of Spirulina platensis against collagen-induced arthritis (CIA) in female Wistar rats based on the changes in paws thickness, serum albumin, cholesterol, lipid peroxidation, alkaline phosphatase and acid phosphatase activities and histology of paw joints. Methods  Arthritis was induced by intradermal injection of Collagen and Freund’s adjuvant incomplete suspension at several sites on the back with a dose of 2 mg kg⁻¹ of body weight and boosted with 0.1 ml intradermally at the base of the tail. CIA rats were orally treated with 200 and 400 mg kg⁻¹ per oral of S. platensis from 0 to 45th day. Results   S. platensis at 400 mg kg⁻¹ per oral significantly elevates serum albumin and decreases the serum cholesterol, alkaline phosphatase and acid phosphatase activities, lipid peroxidation, paw thickness as well as normalize the joint histopathology of CIA rats. Conclusions   S. platensis (400 mg kg⁻¹) significantly normalizes changes observed in arthritic rats to near normal conditions, indicates that S. platensis has promising protective efficacy against CIA rats.     

Subcellular/tissue distribution and responses to oil exposure of the cytochrome P450-dependent monooxygenase system and glutathione S-transferase in freshwater prawns (Macrobrachium malcolmsonii, M. lamarrei lamarrei)

Subcellular fractions (mitochondrial, cytosolic and microsomal) prepared from the tissues (hepatopancreas, muscle and gill) of freshwater prawns Macrobrachium malcolmsonii and Macrobrachium lamarrei lamarrei were scrutinized to investigate the presence of mixed function oxygenase (MFO) and conjugating enzymes (glutathione-S-transferase, GST). Cytochrome P450 (CYP) and other components (cytochrome b₅; NADPH-cytochrome c (CYP) reductase and NADH-cytochrome c-reductase activities) of the MFO system were predominantly present in the hepatic microsomal fraction of M. malcolmsonii and M. lamarrei lamarrei. The results are in agreement with the notion that monooxygenase system is mainly membrane bound in the endoplasmic reticulum, and that the hepatopancreas is the major metabolic tissue for production of biotransformation enzymes in crustaceans. Further, the prawns were exposed to two sublethal (0.9 ppt (parts per thousand) and 2.3 ppt) concentrations of oil effluent. At the end of 30th day, hydrocarbons and detoxifying enzymes were analysed in the hepatopancreas. The accumulations of hydrocarbon in the tissues gradually increased when exposed to sublethal concentrations of oil effluent and were associated with significantly enhanced levels of cytochrome P450 (180.6±6.34 pmol mg⁻¹ protein (P<0.05 versus control, 136.5±7.1 pmol mg⁻¹ protein) for 2.3 ppt and 305.6±8.5 pmol mg⁻¹ protein (P<0.001 versus control, 132.3±6.8 pmol mg⁻¹ protein] for 0.9 ppt of oil exposed M. malcolmsonii; 150±6.5 pmol mg⁻¹ protein (P<0.01 versus control, 84.6±5.2 pmol mg⁻¹ protein) for 2.3 ppt and 175±5.5 pmol mg⁻¹ protein (P<0.01 versus control, 87.6±5.4 pmol mg⁻¹ protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei), NADPH cytochrome c-reductase activity (14.7±0.6 nmol min⁻¹ mg⁻¹ protein (P<0.05 versus control, 6.8±0.55 nmol min⁻¹ mg⁻¹ protein) for 2.3 ppt and 12.1±0.45 nmol min⁻¹ mg⁻¹ protein (P<0.01 versus control, 6.9±0.42 nmol min⁻¹ mg⁻¹ protein) for 0.9 ppt of oil exposed M. malcolmsonii; 12.5±0.31 nmol min⁻¹ mg⁻¹ protein (P<0.001 versus control, 4.6±0.45 nmol min⁻¹ mg⁻¹ protein) for 2.3 ppt and 9.6±0.32 nmol min⁻¹ mg⁻¹ protein (P<0.01 versus control, 4.9±0.41 nmol min⁻¹ mg⁻¹ protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei) and cytochrome b₅ (124.8±3.73 pmol mg⁻¹ protein (P<0.01 versus control, 76.8±4.2 pmol mg⁻¹ protein) for 2.3 ppt and 115.3±3.86 pmol mg⁻¹ protein (P<0.01 versus control, 76.4±4.25 pmol mg⁻¹ protein) for 0.9 ppt of oil exposed M. malcolmsonii and 110±3.11 pmol mg⁻¹ protein (P<0.01 versus control, 63.7±3.24 pmol mg⁻¹ protein) for 2.3 ppt and 95.3±2.63 pmol mg⁻¹ protein (P<0.01 versus control, 61.4±2.82 pmol mg⁻¹ protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei). The enhanced levels of biotransformation enzymes in oil-exposed prawns demonstrate a well-established detoxifying mechanism in crustaceans, and the response offers the possibility of use as a biomarker for the early detection of oil pollution.Special Issue on Biomarkers of Marine Pollution and Bioremediation     

Studies on protoplast formation and regeneration ofGanoderma lucidum

To obtain a new strain ofGanoderma lucidum by protoplast fusion technique, its protoplast formation and regeneration were studied. Several factors affecting the protoplast formation and regeneration were investigated to find their optimum conditions. The mycelium was grown for four days on the cellophane membrane placed onG. lucidum complete medium (GCM). When various commercial lytic enzymes were examined for protoplast isolation, the combination of Novozym 234 and β-glucuronidase was found to be effective. An osmotic stabilizer, 0.6 M sucrose in 20 mM phosphate buffer pH 5.8, gave the highest yield of protoplasts. Three-hour incubation in shaking incubator was most suitable for releasing protoplasts. To increase the protoplast yield, pretreatment with 2-mercaptoethanol was carried out. The regeneration frequency in GCM containing 0.6 M MgSO₄·7H₂O was shown to be 0.66%.     

Involvement of Multidrug Resistance-Associated Protein 1 in Intestinal Toxicity of Methotrexate

Purpose  Methotrexate (MTX) causes dose-limiting gastrointestinal toxicity due to exposure of intestinal tissues, and is a substrate of the multidrug resistance-associated protein (MRP) 1. Here we examine the involvement of MRP1, which is reported to be highly expressed in the proliferative crypt compartment of the small intestine, in the gastrointestinal toxicity of MTX. Methods  MTX was intraperitonealy administered to mrp1 gene knockout (mrp1 ^(−/−)) and wild-type (mrp1 ^(+/+)) mice. Body weight, food and water intake were monitored, intestinal histological studies and pharmacokinetics of MTX were examined. Results   mrp1 ^(−/−) mice more severely decreased body weight, food and water intake than mrp1 ^(+/+) mice. Almost complete loss of villi throughout the small intestine in mrp1 ^(−/−) mice was observed, whereas the damage was only partial in mrp1 ^(+/+) mice. Plasma concentration and biliary excretion profiles of MTX were similar in mrp1 ^(−/−) and mrp1 ^(+/+) mice, though accumulation of MTX in immature proliferative cells isolated from mrp1 ^(−/−) mice was much higher compared to mrp1 ^(+/+) mice. Immunostaining revealed localization of Mrp1 in plasma membrane of the intestinal crypt compartment in mrp1 ^(+/+) mice, but not in mrp1 ^(−/−) mice. Conclusion  Mrp1 determines the exposure of proliferative cells in the small intestine to MTX, followed by gastrointestinal toxicity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dose-dependent influence of buspirone on the activities of selective serotonin reuptake inhibitors in the mouse forced swimming test

Recent clinical data suggest that buspirone may enhance the efficacy and/or reduce the latency to therapeutic effect of selective serotonin reuptake inhibitors (SSRIs) in unipolar major depressive disorder. The present study, using the mouse forced swimming test, was performed to investigate further the mechanisms involved in the potential antidepressant-enhancing effects of buspirone. Prior administration of buspirone (0.06 mg kg⁻¹, IP) significantly enhanced the anti-immobility effects of subactive doses of fluvoxamine (4 mg kg⁻¹, IP; P < 0.01), paroxetine (4 mg kg⁻¹, IP; P < 0.01), citalopram (4 mg kg⁻¹, IP; P < 0.01) and sertraline (2 mg kg⁻¹, IP; P < 0.01) in the forced swimming test. However, pretreatment with buspirone did not induce antidepressant-like effects when tested in combination with fluoxetine (4 mg kg⁻¹, IP). Each antidepressant tested reduced immobility time in the forced swimming test [citalopram (16 mg kg⁻¹, IP; P < 0.01), fluoxetine (32 mg kg⁻¹, IP; P < 0.01), fluvoxamine (32 mg kg⁻¹, IP; P < 0.01), paroxetine (16 mg kg⁻¹, IP; P < 0.01) and sertraline (16 mg kg⁻¹, IP; P < 0.01)]. Pretreatment with buspirone (0.5 mg kg⁻¹, IP), or its major metabolite 1-PP (0.5 mg kg⁻¹, IP), attenuated all SSRI-induced anti-immobility effects (P < 0.01). Concomitant studies of locomotor activity ruled out any stimulant or sedative effects of the interactions. The results of the present study suggested that low dose buspirone enhanced the activity of subactive doses of SSRIs in the mouse forced swimming test, probably via an action at 5-HT_1A receptors. On the other hand, a high dose of buspirone attenuated the antidepressant-like effects of active doses of these drugs, possibly via the generation of an active metabolite (1-PP) acting at alpha₂-adrenoreceptors. Received: 19 October 1997 / Final version: 16 December 1997     

Comparative toxicity of hydrophobic contaminants to microalgae and higher plants

To enable rapid and sensitive screening of phytotoxic compounds in terrestrial system, a 4 day solid-phase microalgal bioassay was developed. Three species of microalgae (Selenastrum capricornutum, Chlorococcum hypnosporum and Chlorococcum meneghini) were chosen to investigate their responses to DDTs (DDT, DDD and DDE) and PAHs (naphthalene, phenanthrene and pyrene) spiked sands. The bioassay results showed that PAHs and DDTs were toxic to microalgae in a 4-day exposure tests but not to seed germination of ryegrass (Lolium perenne). Phenanthrene was the most phytotoxic. Among three investigated endpoints, fluorescence emissions by microalgae were less sensitive than cell density (optical density OD₆₅₀) and chlorophyll a concentration as endpoints. In general, S. capricornutum was the most sensitive species for PAHs (EC₅₀ for phenanthrene = 9.4 mg kg⁻¹), while C. meneghini for DDTs (EC₅₀ for DDE = 20.0 mg kg⁻¹). Comparison of the microalgal tests with US EPA standard seed germination/root elongation test (using Lolium perenne) demonstrated the superior screening potential of phytotoxic hydrophobic compounds using the proposed bioassay. Using OD₆₅₀ as the endpoint, EC₁₀ of selected microalgae for PAHs and DDTs were 0.43–64.3 mg kg⁻¹ and 0.67–117 mg kg⁻¹ respectively, which were much lower than the EC₁₀ of L. perenne for both PAHs (94–187 mg kg⁻¹) and DDTs (113–483 mg kg⁻¹). The results encourage further studies involving wider types of vascular plants and more comparison with standard phytotoxicity tests from different authorities using contaminated soils to verify the effectiveness of the microalgal bioassay.     

Autoradiographic mapping of μ opioid receptor changes in rat brain after long-term haloperidol treatment: relationship to the development of vacuous chewing movements

Brain opioid systems modulating basal ganglia function may be involved in the development of neuroleptic-induced orofacial dyskinesias. This study examined changes in μ opioid receptors labeled with [³H]D-Ala², N-MePhe⁴, Gly-ol⁵-enkephalin ([³H]DAMGO) in 79 different brain regions of rats showing vacuous chewing movements after 21 weeks of treatment with haloperidol decanoate (HAL). Dopamine D₂ receptors labeled with [³H]raclopride were also examined in the adjacent sections of the same brains. For brain analyses HAL-treated rats were divided into a group showing high incidence of vacuous chewing movements (VCMs) and a group showing low incidence of VCMs. As expected, long-term HAL resulted in a pronounced elevation of D₂ receptors in caudate-putamen, n. accumbens, globus pallidus and olfactory bulbs (range: 27–70% increases) compared to controls. These changes were equal in magnitude in both HAL-treated groups, irrespective of the frequency of VCMs. In HAL-treated rats [³H]DAMGO was significantly decreased in several parts of the basal ganglia, including n. accumbens (−21%, P < 0.01), patchy area of the anterior caudate-putamen (−12%, P < 0.05), ventral pallidum (−27%, P < 0.01) and globus pallidus (−21%, P < 0.02). Statistically significant decreases were also seen in the subthalamic nucleus (−12%, P < 0.05) and ventrolateral thalamus (−21%, P < 0.05), both of which are targets of basal ganglia output. However, as in the case of [³H]raclopride binding, [³H]DAMGO changes were generally seen both in the High VCM and the Low VCM groups. These results confirm that long-term haloperidol leads to a decrease in μ-opioid binding in basal ganglia and related structures, similar to what is seen after 6-OHDA denervation. The observed μ-receptor binding changes may be a contribution factor, but do not appear sufficient to account for the differential development of neuroleptic-induced vacuous chewing movements. Received: 2 February 1996 / Final version: 10 June 1996     

Binding of a new multidrug resistance modulator,S9788, to human plasma proteins and erythrocytes

Summary The interactions of S9788 with human plasma proteins have been investigatedin vitro by an erythrocyte partitioning technique that allows an estimation of the plasma proteins and erythrocytes binding parameters. S9788 was 98% bound to plasma and blood. Lipoproteins bound S9788 with high affinities (binding constants of 0.645, 12.8 and 87.0×10⁶M⁻¹ for, HDL, LDL and VLDL, respectively) and accounted for more than 55% of the total circulating S9788. Albumin and alpha₁-acid glycoprotein also bound S9788 with lower binding constants of 0.022 and 0.245×10⁶ M⁻¹. S9788 was mainly distributed in the plasma blood compartment (75–80%) with blood-to-plasma concentrations ratio of 0.6 to 0.7. These results indicate that,in vivo, the fraction of blood S9788 available for tissue diffusion,i.e., the free drug fraction in blood, should depend on lipoprotein concentration in plasma.     

The oxidative metabolism of metoprolol in human liver microsomes: inhibition by the selective serotonin reuptake inhibitors

Objective: Biotransformation of metoprolol to α-hydroxymetoprolol (HM) and O-demethylmetoprolol (ODM) is mediated by CYP2D6. The selective serotonin reuptake inhibitors (SSRIs) are known to inhibit CYP2D6. The aim was to study in vitro the potential inhibitory effect of SSRIs on metoprolol biotransformation. Methods: Using microsomes from two human livers, biotransformation of metoprolol to α-hydroxymetoprolol (HM) and O-demethylmetoprolol (ODM) as a function of the concentrations of the SSRIs and of some of their metabolites was studied. Results: The kinetics of the formation of both metabolites are best described by a biphasic enzyme model. The estimated values of V_max and k_M for the high affinity site are for the α-hydroxylation in human liver HL-1 32 pmol mg⁻¹ min⁻¹ and 75 μmol · l⁻¹ respectively, and in human liver HL-9 39 pmol mg⁻¹ · min⁻¹ and 70 μmol · l⁻¹ respectively; for the O-demethylation in HL-1 131 pmol mg⁻¹ min⁻¹ and 95 μmol · l⁻¹ respectively, and in HL-9 145 pmol mg⁻¹ min⁻¹ and 94 μmol · l⁻¹ respectively. Quinidine is for both pathways a potent inhibitor of the high-affinity site, with K_i values ranging from 0.03 to 0.18 μmol · l⁻¹. Fluoxetine, norfluoxetine and paroxetine are likewise potent inhibitors, with K_i values ranging from 0.30 to 2.1 μmol · l⁻¹ fluvoxamine, sertraline, desmethylsertraline, citalopram and desmethylcitalopram are less potent inhibitors, with K_i values above 10 μmol · l⁻¹. Conclusion: The rank order of the SSRIs for inhibition of metoprolol metabolism is comparable to that reported in the literature for other CYP2D6 substrates, with fluoxetine, norfluoxetine and paroxetine being the most potent. These findings need further investigation to determine their clinical relevance. Received: 24 October 1997 / Accepted: 17 January 1998     

Effects of an anionic surfactant (FFD-6) on the energy and information flow between a primary producer (<Emphasis Type="Italic">Scenedesmus obliquus</Emphasis>) and a consumer (<Emphasis Type="Italic">Daphnia magna</Emphasis>)

The effects of a commercially available anionic surfactant solution (FFD-6) on growth and morphology of a common green alga (Scenedesmus obliquus) and on survival and clearance rates of the water flea Daphnia magna were studied. The surfactant-solution elicited a morphological response (formation of colonies) in Scenedesmus at concentrations of 10–100 μl l⁻¹ that were far below the No Observed Effect Concentration (NOEC) value of 1,000 μl l⁻¹ for growth inhibition. The NOEC-value of FFD-6 for colony-induction was 3 μl l⁻¹. Daphnia survival was strongly affected by FFD-6, yielding LC_50–24h and LC_50–48h of 148 and 26 μl l⁻¹, respectively. In addition, clearance rates of Daphnia feeding on unicellular Scenedesmus were inhibited by FFD-6, yielding a 50% inhibition (EC_50–1.5h) at 5.2 μl l⁻¹ with a NOEC of 0.5 μl l⁻¹. When Daphnia were offered FFD-6-induced food in which eight-celled colonies (43 × 29 μm) were most abundant, clearance rates (~0.14 ml ind.⁻¹ h⁻¹) were only 25% the rates of animals that were offered non-induced unicellular (15 × 5 μm) Scenedesmus (~0.56 ml ind.⁻¹ h⁻¹). As FFD-6 concentrations in the treated food used in the experiments were far below the NOEC for clearance rate inhibition, it is concluded that the feeding rate depression was caused by the altered morphology of the Scenedesmus moving them out of the feeding window of the daphnids. The surfactant evoked a response in Scenedesmus that is similar to the natural chemically induced defensive reaction against grazers and could disrupt the natural information conveyance between these plankton organisms.