Effects of Aniline–An Aromatic Amine to Some Freshwater Organisms

We determined the acute and chronic toxicity of aniline to tilapia (Oreochromis mossambicus), cladoceran crusatcea (Moina micrura) and oligochaete worm (Branchiura sowerbyi) using static bioassay tests. The 96h LC₅₀ values of aniline for O. mossambicus, M. micrura and B. sowerbyi were 69.4, 0.6 and 586mg l^–1 respectively. Tilapia responded to even low concentrations of aniline: the fish lost appetite at aniline concentrations as low as 0.02mg l^–1. A 90 d outdoor bioassay with tilapia showed that 0.02mg l^–1 aniline reduced fish yield, specific growth rate and food conversion efficiency. Reproductive functions of fish were affected by aniline at a concentration of 0.5mg l^–1 and above. Dissolved oxygen, primary productivity and plankton population of the test medium also were significantly reduced at 2.65 and 6.94mg l^–1 aniline.     

Pharmacokinetics and safety of ILX23-7553, a non-calcemic-vitamin D3 analogue, in a phase I study of patients with advanced malignancies

Purpose: Differentiation therapy is an alternative to chemotherapy with potentially less toxicity, improved quality of life, and survival. We conducted a phase I trial of ILX23-7553, a formulation of 1,25-dihydroxy-16-ene-23-yne-vitamin D₃, a 1,25-dihydroxyvitamin D₃ analog with preclinically demonstrated antitumor and differentiating effects and diminished hypercalcemic effects. Patients and methods: The protocol consisted of five daily oral treatments during 14-day cycles at 15 dose levels from 1.3 to 45.0g/m²/day. We treated 42 heavily pretreated patients who had a variety of malignancies with 162 treatment cycles, and obtained pharmacokinetics from three patients at the two highest dose levels. Results: There were no grade 3 or 4 toxicities. Grade 1–2 toxicities included diarrhea, nausea, fatigue, constipation, and one grade 1 hypercalcemia. Average day 6 calcium was 9.26 ± 0.55mg/dl in cycle 1 and 9.30 ± 0.67mg/dl in cycle 2. Pharmacokinetics at dose levels 14 (40g/m²/day) (1 patient) and 15 (45g/m²/day) (2 patients) demonstrated an average C _max of 30.4 ± 7.8pg/ml (0.07nM) and 104 ± 38.2pg/ml (0.25nM), and AUCs of 222.5 ± 225.2pg·h/ml and 855 ± 536pgh/ml, respectively. Eight patients (19%) had stable disease. While in vitro effects have been reported at these concentrations, they were at least 10-fold lower than ED₅₀s, and the study was terminated before an MTD was reached. Conclusion: The drug is safe and has potential benefits at serum concentrations where effects begin to be noted in vitro. Further study is needed with a reformulated higher unit dose compound to determine the safety and efficacy of higher serum concentrations.     

Environmental Impacts of Diesel Fuel on Bacteria and Phytoplankton in a Tropical Estuary Assessed Using <Emphasis Type="BoldItalic">In Situ</Emphasis> Mesocosms.

Dissolved or dispersed petroleum hydrocarbon concentrations (DDPH) were monitored in Ponggol estuary, Singapore, fortnightly from July 1999 to June 2000. DDPH concentrations ranged from 4.4 to 248.9gl^–1 and 0.4 to 1099.7gl^–1 for surface and subsurface waters, respectively and with mean concentrations of 41.01gl^–1 in the water column. Absorbed or adsorbed petroleum hydrocarbon (AAPH) concentrations measured in sediments ranged from 20.6 to 541.0 mg kg^–1, with mean concentrations of 148.23 mgkg^–1. In situ mesocosm studies of bacteria and phytoplankton were based on field monitoring ofenvironmentally measured concentrations of petroleum hydrocarbons, using diesel fuel as the source of contaminant. The mesocosm comprised of 25 L clear polycarbonate carboys incubated in situ for 6 days. Water and sediments from a clean site with undetectable levels of petroleum hydrocarbons were used in controls. The treatment mesocosms comprised of mean and highest concentrations of DDPH and AAPH. The study revealed signs of acute toxicity to autotrophs viz., phytoplankton and autotrophic bacteria in treatments simulating concentrations of diesel fuel found in the sediments. A stimulatory effect was seen at lower concentrations. Bacterial heterotrophs responded positively to all concentrations of diesel fuel because of the abundance of a carbon source, reduced grazing pressure and reduced competition for nutrients from phytoplankton.     

Preparation of Heptakis(2,6-di-O-ethyl)-β-cyclodextrin and Its Nuclear Magnetic Resonance Spectroscopic Characterization

Heptakis(2,6-di-O-ethyl)--cyclodextrin (DE--CyD) was prepared and its ¹H and ¹³C nuclear magnetic resonance (NMR) signals in DMSO-d₆ were unequivocally assigned by two-dimensional COSY and ROESY. The results on ¹H coupling constants indicated that all ethylated glucose units are in a ⁴C₁ chair conformation. The average spin-lattice relaxation times (T ₁) of ring carbons of DE--CyD were only slightly shorter, and their standard deviations from the mean T _l value were larger, than those of -cyclodextrin (-CyD) and heptakis(2,6-di-O-methyl)--cyclodextrin (DM--CyD), suggesting the presence of slightly irregular internal motion in the ethylated glucose units. The temperature dependence of chemical shift of DE--CyD in DMSO-d₆ suggested that the C3 hydroxyl protons may participate as proton donor in the intramolecular hydrogen bond to the C2 ethoxyl groups of neighboring glucose, and the intramolecular hydrogen bond of DE- and DM--CyDs is much stronger than that of -CyD, suggesting the stable macrocyclic ring structure of DE--CyD.     

Discriminative stimulus effects of intravenous l-nicotine and nicotine analogs or metabolites in squirrel monkeys

Squirrel monkeys were trained to emit one response after IV administration of l-nicotine (0.4 or 0.2 mol/kg) and a different response after IV administration of saline. After stable discriminative performances were established, subjects were tested with cumulative doses of l-nicotine (0.02–2.2 mol/kg), d-nicotine (0.02–19.7 mol/kg), l-nornicotine (0.2–12.0 mol/kg), l-cotinine (56.8–567.5 mol/kg), and dl-anabasine (0.6–19.7 mol/kg). All of the drugs produced dose-related increases in the percentage of drug-appropriate responses emitted, from predominately saline-appropriate responses after low doses, to predominately drug-appropriate responses at the highest doses studied. Relative potency comparisons indicated that l-nicotine was 28 times more potent than d-nicotine, 29 times more potent than l-nornicotine, and approximately 2000 times more potent than l-cotinine. Each of the drugs also produced decreases in rates of responding, with potency order similar to that obtained for the discriminative effects. The effects of l-cotinine may be attributed to trace amounts of l-nicotine, which existed within the l-cotinine. The effects of dl-anabasine were lethal in one subject and were consequently not studied in the other subjects.     

Behavioural and anticonvulsant effects of Ca2+ channel toxins in DBA/2 mice

This study investigated the behavioural and anticonvulsant effects of voltage-sensitive calcium channel blockers in DBA/2 mice.-Conotoxin MVIIC (0.1, 0.3 g ICV/mouse) and-agatoxin IVA (0.1, 0.3, 1 g ICV), which act predominantly at P- and/or Q-type calcium channels, prevented clonic and tonic sound-induced seizures in this animal model of reflex epilepsy (ED₅₀ values with 95% confidence limits for protection against clonic sound-induced seizures were 0.09 (0.04–0.36) g ICV and 0.09 (0.05–0.15) g ICV, respectively and against tonic seizures 0.07 (0.03–0.16) g ICV and 0.08 (0.04–0.13) g ICV, respectively). The N-type calcium channel antagonists-conotoxin GVIA and-conotoxin MVIIA were also tested in this model.-Conotoxin GVIA was anticonvulsant in DBA/2 mice, but only at high doses (3 g ICV prevented tonic seizures in 60% of the animals; 10 g ICV prevented clonic seizures in 60% and tonic seizures in 90% of the animals), whereas-conotoxin MVIIA did not inhibit sound-induced seizures in doses up to 10 g ICV. Both-conotoxin GVIA and-conotoxin MVIIA induced an intense shaking syndrome in doses as low as 0.1 g ICV, whereas-conotoxin MVIIC and-agatoxin IVA did not produce shaking at any of the doses examined. Finally,-conotoxin GI (0.01–1 g ICV) and-conotoxin SI (0.3–30 g ICV), which both act at acetylcholine nicotinic receptors, were not anticonvulsant and did not induce shaking in DBA/2 mice. These results confirm that blockers of N- and P-/Q-type calcium channels produce different behavioural responses in animals. The anticonvulsant effects of-conotoxin MVIIC and-agatoxin IVA in DBA/2 mice are consistent with reports that P- and/or Q-type calcium channel blockers inhibit the release of excitatory amino acids and are worthy of further exploration.     

Review of Lithium in the Aquatic Environment: Distribution in the United States,Toxicity and Case Example of Groundwater Contamination

Lithium is found at low concentrations in the major rivers of the United States (about 0.002mgl^–1) and as a mineral or salt in pegmatites and brines. The United States produces many lithium materials and consumes the greatest amount of Li in the world for use in ceramics, glass, aluminum, pharmaceuticals, batteries, etc. From 1950 to 1970, Li was central to many nuclear-related US Department of Energy (DOE) activities. The historical and current use of Li has not prompted many studies of the toxicity of this element to aquatic organisms. Here, we review the distribution and use of Li in the US with emphasis on usage by DOE. We also summarize information on the toxicity of lithium to aquatic biota. A case-example is provided which demonstrates the potential for contamination of groundwater with Li, evaluates the toxicity of the Li-contaminated groundwater, and identifies a treatment alternative.     

Modification of the noradrenergic cyclic AMP generating system in the rat limbic forebrain by amphetamine: Role of its hydroxylated metabolites

Summary The cyclic AMP responses to norepinephrine (NE) in slices of the rat limbic forebrain after the administration of (S)-amphetamine and the role of its para- and -hydroxylated metabolites were investigated. The chronic but not acute administration of (S)-amphetamine to rats causes a significant reduction in the sensitivity of the cyclic AMP generating system to NE without changing the basal level of the nucleotide. This change in the sensitivity of the system is not associated with a change in the EC₅₀ value for NE but reflects mainly a decrease in the maximal response. After withdrawal of the drug, the cyclic AMP response to NE returned to control values within 4 days. In vitro, (S)-p-hydroxyamphetamine (POH) and all stereoisomers of p-hydroxynorephedrine (PHN) except (S,R)-PHN enhanced the cyclic AMP response to low concentrations of NE. Since (S,R)-PHN [like the other stereoisomers of PHN and (S)-POH] inhibited in a dose-dependent manner the high affinity uptake of ³H-NE into crude synaptosomal fractions of the limbic forebrain, the results might suggest that the presumably physiological enantiomer of PHN also exerts receptor blocking properties. The inhibition by (S,R)-PHN of the cocaine induced potentiation of the cyclic AMP response to NE supports this supposition. The results provide evidence that the hydroxylated metabolites of (S)-amphetamine, (S)-POH and (S,R)-PHN, modify the action of the parent drug on central noradrenergic function at the level of the NE receptor coupled adenylate cyclase system.     

Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β)

Purpose. Two recombinant IFN- products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN--la) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN--lb). The structural basis for activity differences between IFN--la and IFN--lb, is determined. Methods. In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN- products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences. Results. IFN-- la was 10 times more active than IFN-- Ib with specific activities in a standard antiviral assay of 20 × 10⁷ lU/mg for IFN--la and 2 × 10⁷ lU/mg for IFN--lb. Of the known structural differences between IFN--la and IFN--lb, only glycosylation affected in vitro activity. Deglycosylation of IFN--la produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN--lb revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 × 10⁷ lU/mg). Crystallographic data for IFN--la revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure. Conclusions. Together these results suggest that the greater biological activity of IFN--la is due to a stabilizing effect of the carbohydrate on structure.     

Pharmacokinetic Drug–Drug Interaction of the Novel Anticancer Agent E7070 and Acenocoumarol

E7070 is a novel sulfonamide anticancer agent that arrests cancer cells at the G1/S boundary of the cell cycle. Three patients receiving chronic therapy with the oral anticoagulant acenocoumarol experienced bleeding and/or a prolonged prothrombin time after treatment with E7070 at a dose of 700mg/m² given as a 1-h infusion. In vitro studies have shown that E7070 has the potential to inhibit several cytochrome P450 (CYP)-enzymes, including CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. The major enzyme involved in the metabolism of acenocoumarol in man is CYP2C9. This study was performed to investigate the interaction between E7070 and acenocoumarol.Blood samples were obtained from two patients receiving daily oral maintenance treatment with acenocoumarol both prior to and following treatment with E7070. In addition, we incubated acenocoumarol enantiomers with pooled human microsomes with and without E7070 and measured the in vitro plasma protein binding of acenocoumarol after incubation with E7070. Pharmacokinetic parameters of acenocoumarol were calculated by noncompartmental analysis and revealed that in both patients the area under the concentration–time curve up to 24h after the acenocoumarol administration was higher following E7070 (2.56 and 1.58h*mol/L) compared to the systemic exposure in the absence of E7070 (1.87 and 1.23h*mol/l). The formation of acenocoumarol metabolites was retarded by E7070 at already low concentrations (2.1M). The plasma protein binding of acenocoumarol was reduced at higher concentrations of E7070 (259M).These results indicate that E7070 may primarily interact with acenocoumarol by reducing its systemic clearance. Displacement of acenocoumarol's plasma protein binding by E7070 may also occur but to a minor extent. In the absence of careful monitoring this drug–drug interaction may result in hypoprothrombinemia and a hemorrhagic tendency.     

Studies on the chemical constituents from the roots of <Emphasis Type="Italic">Platycodon grandiflorum</Emphasis>

Three known monodesmosidic saponins: 3-O--d-glucopyranosyl-2,3,16,23,24-pentahydroxyolean-12-ene-28-oic acid, 3-O--d-glucopyranosyl polygalacic acid, and 3-O--d-glucopyranosyl-(13)--d-glucopyranosyl polygalacic acid; and two known nonsaponin compounds: a mixed compound of n-tetracosanoic acid (lignoceric acid), n-hexacosanoic acid (cerotic acid), and n-octacosanoic acid, and -monopalmitin; were isolated for the first time from the root of Platycodon grandiflorum A. DC. together with another seven known compounds: platycoside G₁ (3-O--d-glucopyranosyl-(16)--d-glucopyranosyl-(16)--d-glucopyranosyl-2,3,16,23,24-pentahydroxyolean-12-ene-28-oic acid 28-O--d-xylopyranosyl-(14)--l-rhamnopyranosyl-(12)--l-arabinopyranoside), deapio-platycodin D, Polygalacin D, deapio-platycodin D₃, platycoside A, -spinasterol, and -spinasteryl-3-O--d-glucopyranoside. Alkaline hydrolysis of platycoside G₁ afforded a new monodesmosidic prosaponin: 3-O--d-glucopyranosyl-(16)--d-glucopyranosyl-(16)--d-glucopyranosyl-2,3,16,23,24-pentahydroxyolean-12-ene-28-oic acid. Their chemical structures were elucidated on the basis of their spectral data and chemical evidence.     

8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29

8-(N, N-diethyl amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) is a widely used pharmacological tool to investigate the involvement of intracellular Ca²⁺ stores in cellular responses. In this study we investigate the effect of TMB-8 as a putative inhibitor of Ca²⁺ signalling in single fura-2 loaded HT₂₉ coIonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 mol/l intracellular Ca²⁺ ([Ca²⁺]_i) transient with an IC₅₀ of 20 mol/l. However, [Ca²⁺]_i transients induced by other phospholipase C coupled agonists ATP (10 mol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mol/l). The agonist-induced [Ca²⁺]_i transients remained equally unaffected by 100 mol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mol/I for ATP (n = 4) or 1 nmol/l for NT (n = 4). The competitive nature of the TMB-8-induced inhibition of the CCH-induced [Ca²⁺]_i transient was demonstrated by examining the agonist at various concentrations in absence and presence of the antagonist. High TMB-8 concentrations (100 mol/l) alone induced a small [Ca²⁺]_i increase ([Ca²⁺]_i: 40 ± 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization ( pH: 0.1 ± 0.02, n = 7) occurring simultaneously with the increase in [Ca ⁺]_i. From these results we draw the following conclusions: (1) In sharp contrast to a large number of other studies, but in agreement with studies in other types of cells, these results substantially challenge the value of the tool TMB-8 as an intracellular Ca²⁺ antagonist; (2) TMB-8 acts a muscarinic receptor antagonist at the M₃ receptor; (3) TMB-8 does not influence the release of Ca²⁺ from intracellular stores when IP₃ signal transduction is activated by ATP or NT; (4) TMB-8 as a weak organic base alkalinizes the cytosol at high concentrations; and (5) TMB-8 induces small [Ca²⁺]_i transients at higher concentrations.     

Enhancement of Solubility and Bioavailability of β-Lapachone Using Cyclodextrin Inclusion Complexes

Purpose. To explore the use of cyclodextrins (CD) to form inclusion complexes with -lapachone (-lap) to overcome solubility and bioavailability problems previously noted with this drug. Methods. Inclusion complexes between -lap and four cyclodextrins (-, -, -, and HP-CD) in aqueous solution were investigated by phase solubility studies, fluorescence, and ¹H-NMR spectroscopy. Biologic activity and bioavailability of -lap inclusion complexes were investigated by in vitro cytotoxicity studies with MCF-7 cells and by in vivo lethality studies with C57Blk/6 mice (18-20 g). Results. Phase solubility studies showed that -lap solubility increased in a linear fashion as a function of -, -, or HP-CD concentrations but not -CD. Maximum solubility of -lap was achieved at 16.0 mg/ml or 66.0 mM with HP-CD. Fluorescence and ¹H-NMR spectroscopy proved the formation of 1:1 inclusion complexes between -CD and HP-CD with -lap. Cytotoxicity assays with MCF-7 cells showed similar biologic activities of -lap in -CD or HP-CD inclusion complexes (TD₅₀ = 2.1 M). Animal studies in mice showed that the LD₅₀ value of -lap in an HP-CD inclusion complex is between 50 and 60 mg/kg. Conclusions. Complexation of -lap with HP-CD offers a major improvement in drug solubility and bioavailability.     

Influence of neuronal uptake on the presynaptic α-adrenergic modulation of noradrenaline release

Summary Conditions required for the inhibitory feedback modulation of noradrenergic neurotransmission were studied in isolated atria of the rat.The alpha adrenergic antagonist, yohimbine, 0.8 M, or phentolamine, 1 M, did not affect the chronotropic response to 4 or 8 shocks at 0.8 Hz but increased it when a higher number of shocks was applied. When neuronal uptake was inhibited by cocaine, 2.9 M, or desipramine, 0.1 M, the enhancement of neurotransmission by yohimbine or phentolamine was higher than that observed in the presence of -adrenergic antagonists alone.In atria preincubated with ³H-noradrenaline, the effect of the drugs on the ³H-overflow evoked by 240 shocks at 2.0 Hz was studied. Cocaine 2.9 M, did not increase the evoked overflow but yohimbine, 0.8 M, did. The ³H-overflow obtained in the group of yohimbine plus cocaine was significantly higher than was expected from the effects of both drugs alone.It is concluded that yohimbine or phentolamine enhance the chronotropic response in rat atria only when the concentration of noradrenaline in the biophase is sufficiently high to activate presynaptic receptors. In this tissue, the efficiency of the neuronal uptake influences the degree of -adrenergic autoinhibition.     

ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors

Summary Effects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow were investigated in the rat isolated iris.ATP and adenosine inhibited the evoked overflow of [³H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (–log K_B = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 mol/l) and DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 50 mol/l) but was antagonized by the P_2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 mol/l, –log K_B = 4.7)and ,-methylene-ATP (10 mol/l). Whereas the evoked [³H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and ,-methylene-ATP caused a small and transient increase. Cibacron blue at 30 mol/l failed to antagonize the inhibition of evoked [³H]-noradrenaline overflow that adenosine produced in the absence of DPCPX. Basal [³H]-noradrenaline overflow was enhanced by cibacron blue, not changed by ,-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A₁ and P_2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by ,-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A₁- but also via these P_2Y-receptors. Correspondence to: H. Fuder at the above address     

Anti-cholinergic effect of verapamil on the muscarinic acetylcholine receptor-gated K+ channel in isolated guinea-pig atrial myocytes

Summary Effects of verapamil on the acetylcholine (ACh)-induced K⁺ current were examined in single atrial cells, using the tight-seal whole-cell clamp technique. The pipette solution contained guanosine-5-triphosphate (GTP) or guanosine-5-O-(3-thiotriphosphate) (GTP-S, a non-hydrolysable GTP analogue). In GTP-loaded cells, ACh induced a specific K⁺ current, which is known to be mediated by pertussis toxin-sensitive GTP-binding (G) proteins. Verapamil (0.1–100 M) depressed the ACh-induced K⁺ current in a concentration-dependent fashion. In GTP-S-loaded cells, the K⁺ current remained persistently after wash-out of ACh, probably due to irreversible activation of G proteins by GTP-S. Verapamil (0.1–100 M) also depressed the intracellular GTP-S-induced K⁺ current. However, the magnitude of verapamil-depression of the K⁺ current in GTP-S-loaded cells was significantly smaller than that in GTP-loaded cells at concentrations between 1 and 10 M of the drug. From these results, it is suggested that verapamil may block not only the function of muscarinic ACh receptors but also of G proteins and/or the K⁺ channel itself and thereby depress the ACh-induced K⁺ current in isolated atrial myocytes.Supported by grants from the Ministry of Education, Science and Culture of Japan and the Research Program on Ca Signal Control Send offprint requests to Y. Kurachi at the above address     

Toxicity of Organic Compounds to Marine Invertebrate Embryos and Larvae: A Comparison Between the Sea Urchin Embryogenesis Bioassay and Alternative Test Species

This study investigated the toxic effects of the insecticides lindane and chlorpyrifos, the herbicide diuron, the organometallic antifoulant tributyltin (TBT), and the surfactant sodium dodecyl sulfate (SDS) on the early life stages of Paracentrotus lividus (Echinodermata, Euechinoidea), Ciona intestinalis (Chordata, Ascidiacea), Maja squinado and Palaemon serratus (Arthropoda, Crustacea) in laboratory acute toxicity tests. The assays studied embryogenesis success from fertilized egg to normal larvae in P. lividus (48 h incubation at 20 °C) and C. intestinalis (24 h incubation at 20 °C), and larval mortality at 24 and 48 h in M. squinado and P. serratus. For P. lividus, the median effective concentrations (EC₅₀) reducing percentages of normal larvae by 50% were: 350 g l^–1 for chlorpyrifos, 5500 g l^–1 for diuron, 4277 g l^–1 for SDS, and 0.309 g l^–1 for TBT. For C. intestinalis, the EC₅₀ values affecting embryogenesis success were 5666 g l^–1 for chlorpyrifos, 24,397 g l^–1 for diuron, 4412 g l^–1 for lindane, 5145 g l^–1 for SDS, and 7.1 g l^–1 for TBT. The median lethal concentrations (LC₅₀) for M. squinado larval survival were 0.84 g l^–1 (24 h) and 0.79 g l^–1 (48 h) for chlorpyrifos, 2.23 g l^–1 (24 h) and 2.18 g l^–1 (48 h) for lindane, and 687 g l^–1 (48 h) for SDS. For P. serratus the LC₅₀ values obtained were 0.35 g l^–1 (24 h) and 0.22 g l^–1 (48 h) for chlorpyrifos, 3011 g l^–1 (24 h) and 3044 g l^–1 (48 h) for diuron, 5.20 g l^–1 (24 h) and 5.59 g l^–1 (48 h) for lindane, and 22.30 g l^–1 (24 h) and 17.52 g l^–1 (48 h) for TBT. Decapod larvae, as expected, were markedly more sensitive to the insecticides than sea urchins and ascidians, and SDS was the least toxic compound tested for these organisms. Lowest observed effect concentrations (LOEC) of TBT for sea urchin and ascidian embryos, chlorpyrifos and lindane for crustacean larvae, and SDS, were similar to those found in many coastal areas indicating that there would be a risk to invertebrate embryos and larvae from exposure in the field to these pollutants.     

The binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties

Summary Four groups of narcotic analgesic drugs have been assessed for their opiate activities by using three binding assays and three pharmacological bioassays. In the binding assays, their inhibition constants (K _I, nM) were determined against the binding of the -ligand, [³H]-[d-Ala ² ,MePhe ⁴ , Gly-ol⁵]enkephalin, of the -ligand, [³H]-[d-Ala ² ,d-Leu ⁵]enkephalin and of the -ligand, [³H]-(±)-ethylketazocine after suppression of - and -binding by 100 nM of the unlabelled -ligand and 100 nM of the unlabelled -ligand. The pharmacological agonist or antagonist activities were assayed on the guinea-pig ileum, mouse vas deferens and rat vas deferens.The first group of compounds were pure agonists in all three pharmacological bioassays. The majority of the compounds showed preference to -binding but phenazocine and particularly etorphine had also high affinities to the - and -binding sites.The second group consisted of N-allyl and N-cyclopropylmethyl homologues of the morphine, 3-hydroxymorphinan and normetazocine series which had agonist and antagonist activities in the guinea-pig ileum and mouse vas deferens but were pure antagonists in the rat vas deferens. In the binding assays, -binding and -binding were prominent.The third group was made up by the ketazocine-like compounds which in the guinea-pig ileum and mouse vas deferens were pure agonists and in the rat vas deferens pure antagonists. The binding spectrum showed particularly high binding to the -binding site.The fourth group was the antagonists which were devoid of agonist activity with the exception of diprenorphine and Mr 2266 which had retained some agonism. The binding spectrum showed considerable variation, naloxone in low concentration being a selective -antagonist, Mr 2266 having high affinities to the - and -binding sites and diprenorphine having considerable affinities to the -, - and -binding sites.Since each of the four groups of compounds, whether pure agonists, agonist-antagonists, ketazocine-like drugs or pure antagonists, shows independent varittions in the affinities to the - and -binding sites, their different pharmacological behaviour cannot be solely due to difference in the binding spectra.     

Studies on the constituents of Scutellaria species (XXI): constituents of the leaves of Scutellaria strigillosa Hemsley

From the leaves of Scutellaria strigillosa, 14 compounds, chrysin, apigenin, 5,7,2-trihydroxyflavone, norwogonin, ursolic acid, 6-hydroxy-4-stigmasten-3-one, 6-hydroxy-4,22-stigmastadien-3-one, 2 R,4 R,8 R--tocopherol, (S)-5,5 -bi--tocopheryl, (R)-5,5 -bi--tocopheryl, solanachromene, tocopherylquinone, jodrellin T, and 14,15-dihydrojodrellin T were isolated. The structures were determined on the basis of chemical and spectral data.     

Cellular effects of reactive intermediates: Nephrotoxicity of S-conjugates of amino acids

Several cysteine S-conjugates are potent nephrotoxins and require enzymatic activation to produce cytotoxicity. Strategies based on the knowledge that renal cysteine conjugate -lyase is apparently a pyridoxal phosphate (PLP)-dependent enzyme have been exploited to test the hypothesis that a -lyase-dependent activation is required for the expression of cysteine S-conjugate-induced toxicity. First, the toxicity of the model conjugate S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is blocked both in vivo and in isolated, renal proximal tubular cells by aminooxyacetic acid, an inhibitor of PLP-dependent enzymes. Second, the nonmetabolizable -methyl analogue S-(1,2-dichlorovinyl)-DL--methylcysteine is not toxic. Third, to test the hypothesis that the toxicity of DCVC is associated with the metabolic formation of a reactive thiol, S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC), which may undergo a PLP-dependent -elimination reaction to produce an identical thiol, was studied. DCVHC is a potent nephrotoxin, and, similar to DCVC, its toxicity was blocked by aminooxyacetic acid and the -methyl analogue S-(1,2-dichlorovinyl)-DL--methylhomocysteine was not toxic. Moreover, exposure of renal proximal tubular cells to propargylglycine, a suicide substrate for PLP-dependent enzymes that catalyze -elimination reactions, blocked the toxicity of DCVHC. Fourth, the renal mitochondrial -lyase is localized in the outer membrane; therefore, although DCVC was toxic to mitochondria, no toxicity was produced in mitoplasts, which shows that a suborganelle site of activation is involved in the mitochondrial toxicity of DCVC. Finally, the toxicity of both DCVC and DCVHC was blocked by probenecid, indicating a role for the anion transport system. DCVC and DCVHC inhibit cellular and mitochondrial respiration, indicating that mitochondria are primary intracellular targets for nephrotoxic S-conjugates. Thus, the nephrotoxicity of cysteine and homocysteine S-conjugates is dependent on enzymatic activation to produce a reactive thiol, which is involved in the production of cytotoxicity.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthdayThis research was supported by National Institute of Environmental Health Sciences grant ES03127 to M. W. A.L. H. L. was supported by N. I. E. H. S. Institutional Research Service Award ES07026