Toxicokinetics and biotransformation of p-nitrophenol in red abalone (Haliotis rufescens)

Red abalone (Haliotis rufescens) were exposed to 3.6 microM (0.5 ppm) 14C-labelled p-nitrophenol (PNP) for 24 h, then were allowed to depurate in clean seawater for another 24 h. Absorption, conditional uptake clearance and elimination rate constants were 0.12+/-0.04 h(-1), 3.2+/-1.1 ml g(-1) h(-1) and 0.05+/-0.02 h(-1), respectively. The sigmoidal shape of the PNP uptake curve suggests a biphasic process. A whole-organism total concentration factor (TCF) of 2.37+/-0.07 was determined from equilibrium tissue and water concentrations, with the highest concentration of PNP plus metabolites found in gill tissue (11.8+/-0.2 nmol g(-1), wet weight). Digestive gland, foot muscle and remaining body tissues accumulated 8.8+/-0.9, 7.7+/-0.6 and 7.5+/-0.6 nmol g(-1) radiolabelled residues, respectively. Abalone depurated 91.6% of absorbed PNP within 24 h, of which 87.5+/-3.1% was unmetabolized parent compound, 13.1+/-3.1% was p-nitrophenylsulfate, 0.32+/-0.09% was p-nitroanisole, and 0.14+/-0.07% was p-acetamidophenol.     

Disposition and biotransformation of pentachlorophenol in the red abalone (Haliotis rufescens)

1. The disposition and biotransformation of pentachlorophenol (PCP) in the red abalone (Haliotis rufescens) have been determined.

2. In a flow-through system, three abalones were exposed to 1˙2?mg/l of [U-¹⁴C]PCP for 5?h to determine bioconcentration and tissue distribution. Retained residues were quantified from radioactivity, while excreted residues were identified and quantified by?h.p.l.c. and determination of radioactivity.

3. The 5-h total concentration factor ranged from 16˙0 to 21˙5; individual tissue concentrations ranged from 133˙4 nmol/g in gill to 17˙5 nmol/g in gonad. Due to its large size, the foot muscle received the largest amount of total retained residue (47˙4%).

4. During a 13-h recovery period the abalones depurated 72˙2% of retained residues; however, residue concentration in gonad increased over 100%. PCP was primarily excreted unchanged (89˙3%), but small amounts of pentachloro-β-D-glucoside (7˙9%), penta-chloroanisole (1˙3%), pentachlorophenylsulphate (0˙9%), and tetrachloro-p-hydroquinone (0˙6%) were also formed.     

Disposition and biotransformation of pentachlorophenol in the red abalone (Haliotis rufescens).

1. The disposition and biotransformation of pentachlorophenol (PCP) in the red abalone (Haliotis rufescens) have been determined. 2. In a flow-through system, three abalones were exposed to 1.2 mg/l of [U-14C]PCP for 5 h to determine bioconcentration and tissue distribution. Retained residues were quantified from radioactivity, while excreted residues were identified and quantified by h.p.l.c. and determination of radioactivity. 3. The 5-h total concentration factor ranged from 16.0 to 21.5; individual tissue concentrations ranged from 133.4 nmol/g in gill to 17.5 nmol/g in gonad. Due to its large size, the foot muscle received the largest amount of total retained residue (47.4%). 4. During a 13-h recovery period the abalones depurated 72.2% of retained residues; however, residue concentration in gonad increased over 100%. PCP was primarily excreted unchanged (89.3%), but small amounts of pentachloro-beta-D-glucoside (7.9%), pentachloroanisole (1.3%), pentachlorophenylsulphate (0.9%), and tetrachloro-p-hydroquinone (0.6%) were also formed.     

Toxic actions of dinoseb in medaka (Oryzias latipes) embryos as determined by in vivo 31P NMR, HPLC-UV and 1H NMR metabolomics

Changes in metabolism of Japanese medaka (Oryzias latipes) embryos exposed to dinoseb (2-sec-butyl-4,6-dinitrophenol), a substituted dinitrophenol herbicide, were determined by in vivo (31)P NMR, high-pressure liquid chromatography (HPLC)-UV, and (1)H NMR metabolomics. ATP and phosphocreatine (PCr) metabolism were characterized within intact embryos by in vivo (31)P NMR; concentrations of ATP, GTP, ADP, GDP, AMP and PCr were determined by HPLC-UV; and changes in numerous polar metabolites were characterized by (1)H NMR-based metabolomics. Rangefinding exposures determined two sublethal doses of dinoseb, 50 and 75 ppb, in which embryos survived from 1-day post fertilization (DPF) through the duration of embryogenesis. In vivo (31)P NMR data were acquired from 900 embryos in 0, 50, and 75 ppb dinoseb at 14, 62, and 110 h (n = 6 groups) after initiation of exposure. After 110 h, embryos were observed for normal development and hatching success, then either preserved in 10% formalin for growth analysis or flash frozen and extracted for HPLC-UV and (1)H NMR analysis. Dinoseb exposure at both concentrations resulted in significant declines in [ATP] and [PCr] at 110 h as measured by in vivo (31)P NMR (p < 0.01), HPLC-UV (p < 0.001) and NMR-based metabolomics. Reduced eye growth and diminished heart rate occurred in a concentration-dependent fashion. Metabolic effects measured by in vivo (31)P NMR showed a significant increase in orthophosphate levels (P(i); p < 0.05), and significant decreases in [ATP], [PCr] and the PCr/P(i) ratio (p < 0.05). Metabolomics revealed a dose-response relationship between dinoseb and endogenous metabolite changes, with both dinoseb concentrations producing significantly different metabolic profiles from controls (p < 0.05). Metabolic changes included decreased concentrations of ATP, PCr, alanine and tyrosine, and increased concentrations of lactate with medaka embryotoxicity. This study demonstrated that medaka embryos respond to dinoseb with significant changes in metabolism, reduced growth and heart rates, and increased abnormal development and post-exposure mortality. All three analytical methods confirmed similar trends, and utilization of PCr to compensate for ATP loss was found to be a consistent indicator of sublethal stress-one that could be used to quantify stress associated with medaka embryotoxicity.     

In vivo 31P NMR studies of the hepatic response to L-ethionine in anesthetized rats

Phosphorus-31 surface coil spectroscopy has been used to study the effects of L-ethionine administration on the hepatic metabolism of the anesthetized Sprague-Dawley rat. ATP levels were found to decrease by approximately 30% 3 to 4 hr after administration of 1 mg/g body wt of L-ethionine to the anesthetized rat by gastric gavage. ATP levels returned to control values approximately 8 hr postadministration. The relatively small decrease in ATP level was confirmed by extraction and conventional enzyme assay and is a consequence of the mode of administration of the ethionine. Hepatic inorganic phosphate levels rose concomitantly with the ATP fall. There were no significant changes in either cellular pH or Mg2+ levels as monitored by the 31P shifts of sensitive metabolites. In vivo 31P NMR spectroscopy provides a promising approach to study the effects of hepatotoxicants on cellular ATP, pH, and Mg levels.     

Rapid determination of glyphosate in postmortem specimens using 31P NMR

Glyphosate was quantified, using 31P NMR, in postmortem blood, liver, and urine specimens taken from two suicide victims. Apart from addition of D2O to give an NMR lock signal, the only pretreatment required of any of the specimens was an enzymic digestion of the liver. Glyphosate was confirmed by its characteristic 31P chemical shift and proton spin coupling and by a downfield shift on addition of NH4OH. Quantitation can be achieved either by comparison with an external standard or by spiking the specimen with glyphosate. Levels of 1 mg/mL could be detected in less than a minute.     

Inhibition of drug metabolizing enzymes (cytochrome P450) in vitro as well as in vivo by Phyllanthus amarus SCHUM & THONN

An alcoholic extract of Phyllanthus amarus (P. amarus) was found to inhibit cytochrome P450 (P450) enzymes both in vivo as well as in vitro. This was studied using specific resorufin derivatives, as substrate for isoenzymes in the P450 super family. Concentration needed for 50% inhibition of 7-ethoxyresorufin-O-deethylase (EROD), CYP1A1 was 4.6 microg/ml while concentration needed for 7-methoxyresorufin-O-demethylase (MROD) CYP1A2 was 7.725 microg/ml and 7-pentoxyresorufin-O-depentylase (PROD), CYP2B1/2 was found to be 4.18 microg/ml indicating that the extract inhibited the P450 enzymes at very low concentration. Extract also inhibited the activity of aniline hydroxylase (an indicator of CYP 2E1 activity, IC(50) 50 microg/ml) and aminopyrine demethylase (an indicator of CYP 1A, 2A 2B, 2D and 3A activity, IC(50) >1000 microg/ml). Oral administration of the extract was also found to reduce the elevated P450 enzyme activities produced by phenobarbitone by 50% at 250 mg/kg body weight. The implication of these results on the inhibition of carcinogenesis produced by the extract is discussed.     

Evaluation by 31P NMR of the effects of acebutolol on the ischaemic isolated rat heart

We used 31P NMR spectroscopy to study the effect of acebutolol on the metabolic disturbances caused by ischaemia in the isolated rat heart. The intracellular acidosis and high-energy phosphate compound depletion induced by global low-flow ischaemia were significantly attenuated in the presence of acebutolol 2.7 X 10(-5) and 1.35 X 10(-4) M. The drug displayed no significant effect either at the 5.4 X 10(-6) M concentration or when administered as pretreatment to the animals (50 mg X kg-1, 120 and 60 min before isolating the heart). Since acebutolol had no effect on the contractility of the isolated rat heart at the 2.7 X 10(-5) M concentration the present results suggest that the mechanisms through which acebutolol exerts its effects on energy metabolism are not related to a reduction of cardiac work. The possible contribution of the membrane-stabilizing property of the drug is suggested by the fact that high concentrations are needed and that a short pretreatment had no effect.     

Hydrophobic double walled carbon nanotubes interaction with phopholipidic model membranes: (1)H-, (2)H-, (31)P NMR and ESR study

The interactions of carbon nanotubes synthesized by catalytic chemical vapour deposition with phospholipidic bilayers, mimicking biological membranes, have been investigated using solid state (31)P- and (2)H NMR, (1)H- and (31)P NMR in liquids and ESR studies. It was found that carbon nanotubes can integrate the bilayer, depending on the overall cohesion of the membrane used. Whereas no direct interaction can be observed in small unilamellar vesicles or directly in the presence of short-chained phospholipids, carbon nanotubes incorporate into the membrane of multibilayers. The result is a significant 2-3K lowering of the transition temperature in multibilayers of dimyristoyl lecithins, which is more markedly associated with increased fluidity in the most superficial part of the membrane below the transition temperature (292-300K range). However, no ionophoric property was found on large unilamellar vesicles.     

Sublethal responses to endrin in sediment by Stylodrilus heringianus (Lumbriculidae) as measured by a 137cesium marker layer technique

Sediment reworking rates of Stylodrilus heringianus (Oligochaeta: Lumbriculidae) were measured in microcosms containing sediments dosed with the chlorinated pesticide, endrin. Reworking rates were measured at 10°C by monitoring a ¹³⁷cesium marker layer burial in contaminated and uncontaminated microcosms. Endrin concentrations ranged from 3.1 to 42 000 ng/g dry sediment. Alterations in reworking rates were observed at sediment concentrations five and one half orders of magnitude below the LC₅₀ (1 650 μg/g). For the lower concentrations, marker layer burial rate data suggested possible stimulatory effects in the first 300 to 600 h, followed by significant rate decreases relative to controls. For higher concentrations, rates were equal to or slower than control rates in the first 600 h, followed by dramatic decreases in the last 600 h. High final surficial sediment endrin concentrations at the end of experiments implied worm mediated upward transport. Worm mortalities were 9.3 to 28% for the two highest concentrations (42 000 and 11 500 ng/g) and 0 to 6.7% for all other concentrations including controls. Post experimental worm dry weights were inversely related to high concentrations. Bioaccumulation factors ranged from 34 to 67 on a g dry organism to g dry sediment basis.     

Inositolphosphoglycans (IPGs) as mediators of insulin's steroidogenic actions

PCOS is a unique model of insulin resistance in which one tissue (skeletal muscle) is resistant to insulin in terms of glucose metabolism whereas another tissue (ovarian thecal cells) maintains its responsiveness to insulin in terms of testosterone biosynthesis. During the past decade, a series of in vitro studies conducted in human placental cells /25/, swine granulosa cells /28/, and most recently, human thecal cells /17/, have provided compelling evidence that one factor involved in this apparent clinical paradox is the clear demonstration of the utilization of the IPG signal transduction system for insulin's effects on steroidogenesis. Nonetheless, many questions remain to be addressed. For example, it would be instructive to determine simultaneously the effects of insulin on glucose disposal and testosterone biosynthesis in isolated thecal cells. In other words, is the thecal cell itself resistant to insulin in terms of glucose disposal while remaining sensitive to insulin in terms of testosterone biosynthesis? Is the IPG content of human thecal cells from women with PCOS decreased compared with that in matched healthy women? There is substantial evidence to suggest that some IPGs may also be responsible in part for mediating insulin's stimulation of glucose disposal (see paper by Dr. Joseph Larner in this issue)--this raises the question as to whether specific forms of IPGs are responsible for glucose metabolism in vivo, whereas other IPGs are responsible for mediating insulin's effects on steroidogenesis. Is there a disparity in the ratio of different types of IPGs among various tissues (e.g., muscle, liver, ovary), and does that ratio determine the tissue's responsiveness to one or another action of insulin? These questions and others leave IPGs and insulin signal transduction a fruitful area for both clinical and laboratory investigations.     

Elucidating the antidepressant actions of substance P (NK1 receptor) antagonists

Substance P (NK1 receptor) antagonists (SPAs) are currently the best validated and clinically most advanced novel approach to treating major depressive disorder (MDD), and several compounds are in advanced clinical development. Three years since the discovery of the antidepressant efficacy of MK-869 (Merck & Co Inc), the first in a new class of drugs that act by selectively blocking the actions of substance P, the principle that blocking the NK1 receptor can alleviate major depression has recently been replicated in a placebo-controlled, blinded study. SPAs are active in a range of preclinical assays that detect clinically used antidepressant drugs, but they have a pharmacological profile that is distinct from established drugs. There is preliminary evidence that substance P and NK1 receptor density may be altered in MDD, suggesting a possible link between substance P and depressive pathophysiology. Studies in animals indicate that the psychotherapeutic effects of SPAs may be mediated at least partly through stimulation of hippocampal neurogenesis, by direct blockade of NK1 receptors in the amygdala and its associated output projections, and also via interactions with monoamines. Additional studies are needed to explore these hypothesesfurther.     

Dimerization region of soluble guanylate cyclase characterized by bimolecular fluorescence complementation in vivo

The ubiquitously expressed nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in signal transduction. Binding of NO to the N-terminal prosthetic heme moiety of sGC results in approximately 200-fold activation of the enzyme and an increased conversion of GTP into the second messenger cGMP. sGC exists as a heterodimer the dimerization of which is mediated mainly by the central region of the enzyme. In the present work, we constructed deletion mutants within the predicted dimerization region of the sGC alpha(1)- and beta(1)-subunit to precisely map the sequence segments crucial for subunit dimerization. To track mutation-induced alterations of sGC dimerization, we used a bimolecular fluorescence complementation approach that allows visualizing sGC heterodimerization in a noninvasive manner in living cells. Our study suggests that segments spanning amino acids alpha(1)363-372, alpha(1)403-422, alpha(1)440-459, beta(1)212-222, beta(1)304-333, beta(1)344-363, and beta(1)381-400 within the predicted dimerization region are involved in the process of heterodimerization and therefore in the expression of functional sGC.     

Effects of propentofylline on energy metabolism of the ischemic brain studied by in vivo 31P nuclear magnetic resonance spectroscopy

The effects of 3-methyl-1-(5'-oxohexyl)-7-propylxanthine (propentofylline, HWA 285) on transient cerebral ischemia were studied in Mongolian gerbils by measuring the in vivo 31P nuclear magnetic resonance (NMR) spectra and cerebral water content. Transient ischemia was produced by bilateral common carotid artery occlusion for 30 min, which was followed by 60 min of reperfusion. Propentofylline (1, 2.5 or 30 mg/kg) or normal saline was administered intravenously at 2 min after the reperfusion. The 31P spectra during the occlusion showed a marked reduction in adenosine triphosphate (ATP) and phosphocreatine (PCr) with elevation of inorganic phosphate (Pi) in all groups. The intracellular pH (pHi) calculated from the chemical shift of Pi was markedly reduced in all groups. After the reperfusion, ATP, PCr, Pi and pHi gradually recovered towards the normal levels in the control group. In the 2.5 mg/kg propentofylline group, the energy recovery was significantly faster than in the controls. The cerebral water content measured at the end of reperfusion was significantly lower in the 2.5 mg/kg propentofylline group than in the controls. However, such cerebral protective effects were not observed in the 1 mg/kg and 30 mg/kg groups. The present results suggest that propentofylline may accelerate the energy recovery of the transiently ischemic brain and suppress the development of post-ischemic cerebral edema. The effects, however, were not dose-dependent in manner. The detailed mechanism of the effects requires further investigation.     

Inhibition by substance P of some peripheral actions of acetylcholine in the cat

1 The effect of substance P on contractions of the nictitating membrane and pressor responses to acetylcholine (ACh) and dimethylphenyl-piperazinium (DMPP) which were mediated via nocotinic receptors was studied in cats anaesthetized with chloralose.

2 Substance P (2-20 nmol) injected into the lingual artery giving estimated concentrations in arterial blood of 10^-6 to 10^-5 M, or intravenously giving estimated concentrations in blood of 10^-8 to 10^-7 M, reduced hexamethonium-sensitive but not atropine-sensitive responses.

3 The pressor effects of ACh and DMPP injected intra-arterially in atropinized and non-atropinized cats respectively were consistently attenuated by substance P given intra-arterially or intravenously.

4 The contractile effect of ACh in atropinized and of DMPP in non-atropinized cats was attenuated by substance P injected intra-arterially but only rarely when the polypeptide was injected intravenously.

5 The depressor effects of substance P per se were variable in magnitude and duration as were the inhibitory effects upon nicotinic receptors. The depressor and inhibitory effects of substance P were unrelated.

6 There was desensitization to all of these effects of substance P which probably contributed to the variation in the magnitude of the effects observed.

7 Substance P had no effect on muscarinic actions of acetyl-β-methylcholine on the nictitating membrane or blood pressure.

8 The results are discussed in relation to the ubiquity of the modulatory actions of substance P on nicotinic receptors and in relation to the possible physiological significance of the action.

     

Sublethal responses to endrin in sediment by Limnodrilus hoffmeisteri (Tubificidae), and in mixed-culture with Stylodrilus heringianus (Lumbriculidae)

Sediment reworking by Limnodrilus hoffmeisteri (Tubificidae) alone, and with Stylodrilus heringianus (Lumbriculidae) were measured in sediments dosed with endrin by monitoring the burial of a ¹³⁷cesium marker layer. Endrin concentrations ranged from 16.1 to 81 400 ng/g dry sediment weight. Alterations in reworking rates were observed at sediment concentrations two to five orders of magnitude below LC₅₀ values. In single species experiments with L. hoffmeisteri at low endrin concentrations, marker layer burial rate data did not suggest stimulation of reworking, as had previously been found for S. heringianus. At higher concentrations, reworking rates were equal to or slower than control rates early in experiments, followed by dramatic decreases thereafter. Reworking rates with mixed species (1:1 species ratio) suggested that the presence of S. heringianus enhanced the reworking response of L. hoffmeisteri.Post-experimental worm dry weights were inversely related to high sediment concentrations for both species. Reductions in post-experimental L. hoffmeisteri mortalities and increases in L. hoffmeisteri dry weights in mixed species tests at high endrin concentrations implied that L. hoffmeisteri benefits from the presence of S. heringianus, although the reverse was not observed.High final sediment endrin concentrations in the upper three cm implied worm mediated upward contaminant transport. Bioaccumulation factors for S. heringianus ranged from 9.7 to 43.8 and were consistently three to four times greater than bioaccumulation factors for L. hoffmeisteri (1.7 to 13.6).     

Proposed model for in vitro interaction between fenitrothion and DNA,by using competitive fluorescence, 31P NMR, 1H NMR,FT-IR,CD and molecular modeling

In this work we proposed a model for in vitro interaction of fenitrothion (FEN) with calf thymus-DNA by combination of multispectroscopic and two dimensional molecular modeling (ONIOM) methods. The circular dichroism results showed that FEN changes the conformation of B-DNA and caused some changes to C-DNA form. The FT-IR results confirmed a partial intercalation between FEN and edges of all base pairs. The competitive fluorescence, using methylene blue as fluorescence probe, in the presence of increasing amounts of FEN, revealed that FEN is able to release the non-intercalated methylene blue from the DNA. The weak chemical shift and peak broadening of ¹H NMR spectrum of FEN in the presence of DNA confirmed a non-intercalation mode. The ³¹P NMR showed that FEN interacts more with DNA via its –NO₂ moiety. The ONIOM, based on the hybridization of QM/MM (DFT, 6.31++G (d,p)/UFF) methodology, was also performed by Gaussian 2003 package. The results revealed that the interaction is base sequence dependent, and FEN interacts more with AT base sequences.