Comparative quantitative analysis of agricultural chemicals using a microplate mammalian cell cytotoxicity assay.
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Animals↗
Biomedical subjects
J W Stucki
Publications and source records attributed to J W Stucki.
Transformation of chlorinated aliphatic compounds by ferruginous smectite.
A series of chlorinated aliphatic compounds (RCI, including carbon tetrachloride (PCM), 1,1,1-trichloroethane (TCA), 1,1,2,2-tetrachloroethane (TeCA), pentachloroethane (PCA), hexachloroethane (HCA), trichloroethene (TCE), tetrachloroethene (PCE), trichloronitromethane (chloropicrin, CP), and trichloroacetonitrile (TCAN)) was reacted with ferruginuous smectite (sample SWa-1 from The Source Clays Repository), SWa, in aqueous suspension under anoxic conditions. Compounds highly polarizable or sharing substituents that facilitate charge delocalization adsorbed faster by reduced (SWa-R) than by unaltered (SWa-U) clay, indicating stronger dipole--dipole interactions between the substituents and the clay surface and/or hydrating water molecules. The reduction of the clay accelerated RCI adsorption up to 100-fold. Incubations with SWa-R promoted RCI reduction (CP, TCAN) or dehydrochlorination (TeCA and PCA). The reduction of structural Fe catalyzes the transformation of RCI via Brønsted and Lewis-basic promoted pathways. This study indicates that oxidation state of the structural Fe in SWa greatly alters surface chemistry and has a large impact on clay-organic interactions.
Aluminum Silicates↗
Fate of atrazine and alachlor in redox-treated ferruginous smectite.
The oxidation state of structural iron (Fe) in clay minerals exerts a large influence on clay surface chemistry and may affect the adsorption and degradation of pesticides in the environment. This effect, however, has been little investigated. In the present study, herbicides atrazine and alachlor were reacted with ferruginous smectite (sample SWa-1) in its oxidized, reduced (either chemically or bacterially), and reduced-reoxidized states. In some experiments the herbicide was labeled with 14C. Gas chromatography/mass spectrometry (GC-MS) was also used to detect alachlor degradation products. Compared to oxidized clays, reduction by both chemical and microbial treatments decreased the concentration of both herbicides in the surrounding solution. Reoxidized clay exhibited behavior similar to the oxidized clay. Hydrolysis-dechlorination of atrazine occurred in the presence of chemically reduced SWa-1, and GC-MS analysis of alachlor revealed at least 14 degradation products after treatment with reduced clay and only two with the oxidized clay. Interaction of atrazine and alachlor with the clay may be through a H bond with the waters of hydration surrounding interlayer cations, the extent of which should increase with increasing acidity; but under reduced conditions, the validity of this model is unclear. Reduction of structural Fe may affect pH-dependent phenomena in two ways: The increased surface charge density increases the number of hydrated interlayer cations, thereby enhancing surface acidity, and increased electron density at basal surface oxygens increases their Brønsted basicity. Atrazine could, therefore, adsorb and/or degrade through either acid or alkaline hydrolysis pathways. Increased reduction potential of the reduced clay surfaces may also promote degradation.
Acetamides↗
Inhibition and stimulation of long-chain fatty acid oxidation by chloroacetaldehyde and methylene blue in rats.
The effects of chloroacetaldehyde (CAA) and methylene blue, both alone and together, on mitochondrial metabolism, hepatic glutathione content, and bile flow were investigated in rats. Oxidation of [1-14C]palmitic acid, [1-14C]octanoic acid, and [1,4-14C]succinic acid allowed for the differentiation between carnitine-dependent long-chain fatty acid metabolism, medium chain fatty acid oxidation, and citric acid cycle activity, respectively. CAA, a metabolite of the anticancer drug ifosfamide, which may be responsible for ifosfamide-induced encephalopathy, inhibited palmitic acid metabolism but not octanoic or succinic acid oxidation, depleted hepatic glutathione, and stimulated bile flow. Methylene blue, which is clinically used to either prevent or reverse ifosfamide-associated encephalopathy, markedly stimulated palmitic acid oxidation either in the presence or absence of CAA, but did not affect the oxidation of octanoic and succinic acid or hepatic glutathione. Taken together, this study demonstrates that CAA inhibits palmitic acid metabolism. Methylene blue stimulates long-chain fatty acid oxidation, most likely by facilitating the translocation of fatty acids into mitochondria, and compensates for the CAA effect in vivo.
Acetaldehyde↗
Permissive role of cAMP in the oscillatory Ca2+ response to inositol 1,4,5-trisphosphate in rat hepatocytes.
Rat hepatocytes respond to alpha-adrenergic stimulation by intracellular production of myo-inositol 1,4,5-trisphosphate (IP3) which stimulates the periodic release and reuptake of intracellular store (IS) Ca2+. The generation of these Ca2+ oscillations was investigated by simultaneously monitoring Ca2+ changes in the cytosol and IS by combined fluorescence microscopy and whole-cell patch clamp. Intracellular IP3 perfusion (1-50 microM in the pipette) produced three types of Ca2+ response: understimulation, oscillations and overstimulation, i.e. with Ca2+ levels not returning to baseline. In a total of 57 experiments, only three displayed oscillations during continuous IP3 infusion, in a narrow range of IP3 concentration centred around 5-8 microM in the pipette. In oscillating cells, cytosolic Ca2+ spikes were synchronized with transient Ca2+ depletions of the IS, consistent with a direct exchange of Ca2+ between the two compartments. Application of 8-Br-cAMP to cells infused with IP3 increased the probability of eliciting Ca2+ oscillations by a factor of 4-5 for IP3 concentrations in the range 1-10 microM, whereas IP3 concentrations above 10 microM always resulted in overstimulation. IP3 photorelease experiments and measurements of IS Ca2+ content indicated that 8-Br-cAMP enhanced the affinity of the IP3 receptor and increased the pool of releasable Ca2+. We propose that cAMP has a permissive role in the generation of IP3-induced Ca2+ oscillations by extending the window of IP3 concentrations able to elicit oscillations.
8-Bromo Cyclic Adenosine Monophosphate↗
Perturbation of myo-inositol-1,4,5-trisphosphate levels during agonist-induced Ca2+ oscillations.
Agonist-induced Ca2+ oscillations in rat hepatocytes involve the production of myo-inositol-1,4,5-trisphosphate (IP3), which stimulates the release of Ca2+ from intracellular stores. The oscillatory frequency is conditioned by the agonist concentration. This study investigated the role of IP3 concentration in the modulation of oscillatory frequency by using microinjected photolabile IP3 analogs. Photorelease of IP3 during hormone-induced oscillations evoked a Ca2+ spike, after which oscillations resumed with a delay corresponding to the period set by the agonists. IP3 photorelease had no influence on the frequency of oscillations. After photorelease of 1-(alpha-glycerophosphoryl)-D-myo-inositol-4,5-diphosphate (GPIP2), a slowly metabolized IP3 analog, the frequency of oscillations initially increased by 34% and declined to its original level within approximately 6 min. Both IP3 and GPIP2 effects can be explained by their rate of degradation: the half-life of IP3, which is a few seconds, can account for the lack of influence of IP3 photorelease on the frequency, whereas the slower metabolism of GPIP2 allowed a transient acceleration of the oscillations. The phase shift introduced by IP3 is likely the result of the brief elevation of Ca2+ during spiking that resets the IP3 receptor to a state of maximum inactivation. A mathematical model of Ca2+ oscillations is in satisfactory agreement with the observed results.
Animals↗
Agonist-specific behaviour of the intracellular Ca2+ response in rat hepatocytes.
A variety of agonists stimulate in hepatocytes a response that takes the shape of repetitive cytosolic free Ca2+ transients called Ca2+ oscillations. The shape of spikes and the pattern of oscillations in a given cell differ depending on the agonist of the phosphoinositide pathway that is applied. In this study, the response of individual rat hepatocytes to maximal stimulation by arginine vasopressin (AVP), phenylephrine and ADP was investigated by fluorescence microscopy and flash photolysis. Hepatocytes loaded with Ca2+-sensitive probes were stimulated with a first agonist to evoke a maximal response, and then a second agonist was added. When phenylephrine or ADP was used as the first agonist, AVP applied subsequently could elicit an additional response, which did not happen when AVP was first applied and phenylephrine or ADP was applied later. Cells microinjected with caged myo-inositol 1,4,5-trisphosphate (IP3) were challenged with the different agonists and, when a maximal response was obtained, photorelease of IP3 was triggered. Cells maximally stimulated with AVP did not respond to IP3 photorelease, whereas those stimulated with phenylephrine or ADP responded with a fast Ca2+ spike above the elevated steady-state level, which was followed by an undershoot. In contrast, with all three agonists, IP3 photorelease triggered at the top of an oscillatory Ca2+ transient was able to mobilize additional Ca2+. These experiments indicate that the differential response of cells to agonists is found not only during Ca2+ oscillations but also during maximal agonist stimulation and that potency and efficacy differences exist among agonists.
Adenosine Diphosphate↗
Stimulation of respiration by methylene blue in rat liver mitochondria.
The effect of methylene blue on isolated rat liver mitochondria in the presence and absence of chloroacetaldehyde was investigated. Fatty acid oxidation was inhibited by chloroacetaldehyde and subsequently stimulated by methylene blue. Assessment of tightly coupled mitochondria revealed decreasing respiratory control ratios induced by increasing concentrations of methylene blue and methylene blue provoked mitochondrial swelling. In uncoupled mitochondria, methylene blue promoted a concentration-dependent stimulation of respiration. These findings provide evidence that methylene blue, the redox dye currently used as an antidote for encephalopathy associated with alkylating chemotherapy, uncouples oxidative phosphorylation and acts as an electron transfer mediator to stimulate mitochondrial respiration.
Acetaldehyde↗
Modulation of hormone-induced calcium oscillations by intracellular pH in rat hepatocytes.
Single isolated rat hepatocytes were used to investigate the influence of intracellular pH (pHi) on hormone-induced cytosolic Ca2+ oscillations, using videofluorescence microscopy. Although pHi did not vary after alpha-adrenergic stimulation, manipulations of pHi induced pronounced alterations in the frequency of oscillations. Increasing the resting pHi with ammonium chloride (5-20 mM), trimethylammonium (2-10 mM), or triethylammonium (1.2-8 mM) reduced the frequency of oscillations. A change in pHi of > 0.25 was sufficient to reversibly inhibit oscillations. This effect could be overcome by increasing the agonist concentration or by adding 8-bromoadenosine 3',5'-cyclic monophosphate, an agent known to potentiate the alpha-adrenergic response. Cellular acidification, obtained by the ammonium prepulse method as well as by application of acetate or the ionophore nigericin, in the continuous presence of agonist was accompanied by a modest frequency increase of the oscillations, leading in some cases to an overstimulated state. This study indicates that pHi, within a range of values expected to occur in vivo (0.1-0.2 pH units), exerts a chronotropic effect on phenylephrine-induced Ca2+ oscillations. In contrast, oscillations induced by ADP or vasopressin were pHi invariant.
Adrenergic alpha-Agonists↗
A critical evaluation of in situ measurement of mitochondrial electrical potentials in single hepatocytes.
The range of applicability and the critical parameters involved in the assessment of mitochondrial electrical potential (delta psi mit) using epifluorescence microscopy were evaluated based on both theoretical and experimental analysis. Rat hepatocytes loaded with the potential-dependent fluorescent dye rhodamine 123 exhibited the expected heterogeneity of fluorescence distribution with dark regions corresponding to the nucleus and bright regions corresponding to the mitochondria-rich cytosol. Calibration of the signal was performed by permeabilizing the cell membrane for monovalent cations using nystatin and gramicidin, and equilibrating the cell with a K(+)-free bath solution. A voltage-clamp at defined delta psi mit was then achieved after addition of valinomycin in the presence of different K+ concentrations in the bath. Theoretical analysis indicated that the ratio of fluorescence intensity measured in mitochondria-rich and mitochondria-poor regions of cell was related with delta psi mit and yielded quantitative estimates of electrical potential with an accuracy of 10-20 mV. The ratio tended to plateau at potentials more negative than-140 mV, showing a limitation of the technique. Manoeuvres such as imposing a heavy ATP demand or interfering with the mitochondrial respiration depolarized mitochondria, while delta psi mit was not altered in a measurable manner during Ca2+ oscillations consecutive to alpha 1-agonist stimulation.
Adrenergic alpha-Agonists↗
Simultaneous measurements of Ca2+ in the intracellular stores and the cytosol of hepatocytes during hormone-induced Ca2+ oscillations.
Simultaneous Ca2+ measurements in the cytosol and intracellular stores (IS) of rat hepatocytes were performed using two Ca(2+)-sensitive probes (Fluo-3 and Mag-fura-2), and combined whole-cell patch clamp and fluorescence microscopy. A steady-state Ca2+ concentration of approximately 630 microM was estimated in the IS. alpha 1-Adrenergic stimulation induced periodic elevations of cytosolic Ca2+ and parallel synchronized transient declines in the IS. Subsequent application of the intracellular Ca(2+)-pump inhibitor thapsigargin resulted in a release of Ca2+ from the IS to reach a level of Ca2+ depletion much lower than the lowest transient decline observed during the oscillations.
Aniline Compounds↗
Anti-diabetic biguanides inhibit hormone-induced intracellular Ca2+ concentration oscillations in rat hepatocytes.
Rat hepatocytes respond to glycogenolytic stimuli acting via phosphoinositide breakdown (e.g. alpha 1-adrenergic agonists, vasopressin) by oscillations of the free intracellular Ca2+ concentration ([Ca2+]i). We have investigated the action of metformin and phenformin, two anti-diabetic drugs of the biguanide type, on phenylephrine-induced [Ca2+]i oscillations. Metformin and phenformin lowered the frequency of the [Ca2+]i oscillations in a concentration-dependent manner with an IC50 of 0.1 mM and 1 microM, respectively. Simultaneous addition of the biguanides and insulin resulted in a further reduction of the frequency. By contrast, agents which increase the cellular cyclic AMP (cAMP) concentration (glucagon, forskolin, N,2'-O-dibutyryl-cAMP) reversed this inhibition. Furthermore, we investigated whether biguanides influenced the agonist-induced Ca2+ influx across the plasma membrane. When hepatocytes were loaded with the acetoxymethyl ester of fura-2 (fura-2/AM), addition of Mn2+ led to a quench of cellular fura-2, measured at the isosbestic excitation wavelength of 360 nm, until a new steady state was reached. Surprisingly, however, this addition of Mn2+ caused a marked increase of the fluorescence ratio simultaneously measured at 340 and 380 nm during the approach of the 360 nm signal to a new steady state. This observation can be understood on the basis of a compartmentalization of fura-2/AM into intracellular stores sensing the [Ca2+] therein. Subsequent application of phenylephrine resulted in a further decline of the fura-2 signal at 360 nm and a concomitant decrease of the fluorescence ratio. This second phase of the Mn2+ quench and the decrease of the fluorescence ratio could be diminished by addition of either 3 mM metformin or 30 microM phenformin. By contrast, when hepatocytes were loaded with fura-2/pentapotassium salt via a patch pipette, only the initial Mn(2+)-induced quench, measured at 360 nm, but no change of the fluorescence ratio, could be observed. The subsequent addition of phenylephrine and biguanides during the on-going quench caused no further changes, except for a fading oscillatory response. After loading hepatocytes with fluo-3 acetoxymethyl ester, the cells were permeabilized with 5 microM digitonin. Addition of inositol-1,4,5-trisphosphate (IP3) caused a rapid decrease of the remaining cellular fluorescence which could be effectively inhibited by 20 micrograms/ml heparin, indicating a release of Ca2+ from intracellular compartments mediated by IP3. This IP3-induced release of Ca2+ from intracellular stores could be diminished by prior addition of metformin and phenformin.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
A dialogue on Ca2+ oscillations: an attempt to understand the essentials of mechanisms leading to hormone-induced intracellular Ca2+ oscillations in various kinds of cell on a theoretical level.
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Animals↗
Unidirectional interaction between two intracellular calcium stores in rat phaeochromocytoma (PC12) cells.
1. A clone of the rat phaeochromocytoma cell line (PC12) was treated with nerve growth factor (NGF) for 4-6 days and used to study caffeine- and bradykinin-induced Ca2+ release from intracellular Ca2+ stores. The caffeine-sensitive store can be depleted by Ca(2+)-induced Ca2+ release (CICR), while the bradykinin-induced release is mediated by inositol 1,4,5-trisphosphate (IP3). The effect of Ca2+ release from these Ca2+ stores on cytosolic free Ca2+ ([Ca2+]i) was measured by means of fura-2 single cell microfluorimetry. 2. Caffeine application caused no or only a small Ca2+ release in untreated cells in normal culture medium. The caffeine-sensitive pool could be filled by Ca2+ entry into cells through either voltage-activated Ca2+ channels or ligand-gated cation channels. 3. Bradykinin application produced substantial Ca2+ release in untreated cells in normal culture medium. The response was enhanced after K(+)-depolarization of the cells. The bradykinin-induced release of Ca2+ also caused depletion of the caffeine-sensitive pool by CICR. However, Ca2+ released from the IP3-sensitive store was not sequestered into the caffeine-sensitive Ca2+ store. 4. The caffeine-induced rise in [Ca2+]i was blocked by ryanodine in a use-dependent manner. In addition, a substantial use-dependent ryanodine block resulted from the bradykinin-induced rise of [Ca2+]i and subsequent CICR. By contrast, the K(+)-induced rise of [Ca2+]i caused only a marginal use-dependent ryanodine inhibition of Ca2+ release. 5. Our results suggest an enhancement of the IP3-induced [Ca2+]i rise in the cytoplasm by CICR from the caffeine-sensitive pool. 6. A mathematical model adequately simulates our experimental data.
Animals↗
Modulation of cytosolic-[Ca2+] oscillations in hepatocytes results from cross-talk among second messengers. The synergism between the alpha 1-adrenergic response, glucagon and cyclic AMP, and their antagonism by insulin and diacylglycerol manifest themselves in the control of the cytosolic-[Ca2+] oscillations.
Hepatocytes respond to stimulation by glycogenolytic agonists acting via phosphoinositide (PI) breakdown through oscillations of the free cytosolic concentration of Ca2+ ([Ca2+]cyt.). Since the second-messenger repertoire of hepatocytes includes many other factors besides Ca2+, we investigated to what degree the regulation of [Ca2+]cyt. oscillations is integrated into these other signalling systems. [Ca2+]cyt. was recorded in single rat hepatocytes by using the Ca(2+)-indicator fura-2. Parallel stimulation with phenylephrine (an alpha 1-adrenergic agonist of PI breakdown) and glucagon resulted in a synergistic stimulation of [Ca2+]cyt. oscillations. Direct activation of the cyclic-AMP-dependent pathway with several stimuli (forskolin, 8-bromo cyclic AMP, 8-CPT cyclic AMP) mimicked the response to glucagon. In contrast, [Ca2+]cyt. oscillations induced by various combinations of these agonists could be antagonized by the glycogenic hormone insulin. As one of the options in the insulin-signalling network, we tested a diacylglycerol activator of protein kinase C, DiC8. It also acted as an inhibitor of [Ca2+]cyt. oscillations. We investigated how these observations could be reconciled with our previously introduced model of [Ca2+]cyt. oscillations in hepatocytes [Somogyi and Stucki (1991) J. Biol. Chem. 266, 11068-11077]. First of all, the effect of calmodulin inhibitors (calmidazolium and CGS 9343 B), acting at the core of our model on the feedback of Ca2+ on Ins(1,4,5)P3-induced Ca2+ release, was not altered by the new modulators. In addition, all agonists and antagonists could be used interchangeably in combination and introduced no significant change in the oscillatory pattern or spike shape. Since the response was solely limited to frequency modulation, over- or understimulation of the oscillatory system, there is no need to create a new oscillator or to introduce further reaction steps into the core of the model. We conclude that the regulation of [Ca2+]cyt. via the explored second-messenger pathways can be embedded into the oscillatory system as modulation of rate constants already present in this model.
8-Bromo Cyclic Adenosine Monophosphate↗
Non-equilibrium thermodynamic sensitivity of oxidative phosphorylation.
A new method was developed to analyse the dynamic properties of oxidative phosphorylation, in particular the sensitivity of the phosphate potential with respect to fluctuating cellular ATP utilization. This treatment is based on the eigenvalue sensitivity analysis of an experimentally supported non-equilibrium thermodynamic model of oxidative phosphorylation. Such an analysis allows direct access to the kinetic information, while circumventing the awkward conventional numerical integration of a set of nonlinear differential equations. This procedure revealed, for the parameters characteristic for liver of starved rats in vivo, that the sensitivity of oxidative phosphorylation to a fluctuating ATP utilization is minimal at a degree of coupling q = 0.95. This means that the phosphate potential is highly buffered with respect to fluctuating energy demands at the degree of coupling. This value of q agrees well with the degree of coupling qeef, at which net ATP production of oxidative phosphorylation--at optimal efficiency--occurs in the most economic way. This simultaneous maximization of kinetic stability and economic thermodynamic efficiency at the same degree of coupling appears to be a coincidence.
Adenosine Triphosphate↗
Hormone-induced calcium oscillations in liver cells can be explained by a simple one pool model.
Hormone-induced oscillations of the free intracellular calcium concentration are thought to be relevant for frequency encoding of hormone signals. In liver cells, such Ca2+ oscillations occur in response to stimulation by hormones acting via phosphoinositide breakdown. This observation may be explained by cooperative, positive feedback of Ca2+ on its own release from one inositol 1,4,5-trisphosphate-sensitive pool, obviating oscillations of inositol 1,4,5-trisphosphate. The kinetic rate laws of the associated model have a mathematical structure reminiscent of the Brusselator, a hypothetical chemical model involving a rather improbable trimolecular reaction step, thus giving a realistic biological interpretation to this hallmark of dissipative structures. We propose that calmodulin is involved in mediating this cooperativity and positive feedback, as suggested by the presented experiments. For one, hormone-induced calcium oscillations can be inhibited by the (nonphenothiazine) calmodulin antagonists calmidazolium or CGS 9343 B. Alternatively, in cells overstimulated by hormone, as characterized by a non-oscillatory elevated Ca2+ concentration, these antagonists could again restore sustained calcium oscillations. The experimental observations, including modulation of the oscillations by extracellular calcium, were in qualitative agreement with the predictions of our mathematical model.
Animals↗
Hormone-induced intracellular calcium oscillations and mitochondrial energy supply in single hepatocytes.
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Animals↗