Effect of potassium (K) supply on the uptake of 137Cs by spring wheat (Triticum aestivum cv. Tonic): a lysimeter study

A lysimeter experiment was carried out on a relatively infertile soil to examine the effect of potassium fertiliser application on the uptake of radiocaesium by spring wheat. Porous ceramic cups were used to obtain samples of soil solution. Results showed that the uptake of radiocaesium by spring wheat was reduced by the addition of potassium. However this inhibitory effect was less marked at later stages of plant growth due to factors such as the spatial variability of potassium within the soil, differences in root distribution down the soil profile and age-related demand for potassium by the plant. There was some evidence that a negative power function could be used to describe the relationship between the concentration of ¹³⁷Cs in the plant and concentrations of potassium or ¹³⁷Cs:K quotients in soil solution over the whole experimental period. Practical implications of potassium fertilisation in terms of reducing uptake of radiocaesium by crops are discussed. Received: 20 April 2000 / Accepted: 16 August 2000     

Effect of magnesium ion (Mg2+) and magnesium adenosinetriphosphate ion (MgATP2-) on pigeon kidney pyruvate carboxylase

Kinetic methods have been used to determine whether Mg²⁺ and MgATP^2? play an important role in regulating pigeon kidney pyruvate carboxylase (pyruvate: CO₂ ligase (ADP), EC 6.4.1.1.). Mg²⁺ not only forms a complex with ATP^4? (MgATP^2?) but is also required for the enzyme activation (and probably for the binding of MgATP^2? to this enzyme). Contrary to the results of other investigators, the MgATP^2? complex was not found to activate pigeon kidney pyruvate carboxylase. We could not demonstrate homotropic cooperativity with MgATP^2? complex. Excess Mg²⁺ induces allosteric stimulation of the enzymatic activity at different concentrations of MgATP^2?. With different Mg²⁺ concentrations, changes also occured in the apparent Km^? and Vmax-values. Without excess of Mg²⁺ only about 2 % of the total enzymic activity available could be demonstrated in the presence of MgATP^2?. It is concluded that Mg²⁺ exhibits a homotropic cooperative effect and is required for the activation of this enzyme. Mg²⁺ may bind either to a specific effector site, at the active site, or at the binding site for MgATP^2? which is capable of functioning as an effector site and in this way facilitates the carboxylation of pyruvate.     

(E)-3-Cyanophosphoenolpyruvate, a new inhibitor of phosphoenolpyruvate-dependent enzymes

(E)-3-Cyanophosphoenolpyruvate has been synthesized by reacting dimethyl chlorophosphate with the potassium enolate of ethyl cyanopyruvate. The resulting trialkyl ester was deesterified with bromotrimethylsilane followed by potassium hydroxide. Subsequent treatment with Dowex-50-H+ resin and cyclohexylamine afforded the tricyclohexylammonium salt; only the E geometric isomer was obtained. This compound can be photoisomerized to a 70:30 E:Z mixture. (E)-3-Cyanophosphoenolpyruvate is an excellent competitive inhibitor of phosphoenolpyruvate carboxylase [KI(Mn2+) = 16 microM, KI(Mg2+) = 1360 microM], pyruvate kinase [KI(Mn2+) = 0.085 microM, KI(Mg2+) = 0.76 microM], and enolase [KI(Mn2+) = 360 microM, KI(Mg2+) = 280 microM]. The compound is a substrate for pyruvate kinase (Vmax approximately 1% of phosphoenolpyruvate rate), but not for the other two enzymes. No irreversible inactivation is observed with phosphoenolpyruvate carboxylase of pyruvate kinase.     

Enzymic assay of 10 to 10 moles of sucrose in plant tissues

Procedures are described for measuring sucrose in plant extracts or freeze-dried tissue in the range between 10⁻⁷ and 10⁻¹⁴ moles. The method is based on the destruction of pre-existing glucose and fructose, followed by the hydrolysis of sucrose and reduction of NADP⁺ by a series of coupled enzymic reactions. Depending on the sensitivity required, the NADPH is determined directly with a spectrophotometer or a fluorometer, or is amplified as much as 30,000 times before fluorometric assay. The procedures suggested for the macro level are simpler than current methods, and those suggested for microanalysis are several orders of magnitude more sensitive.     

A sensitive radioimmunoassay for the antiviral agent BW248U [9-(2-hydroxyethoxymethyl)guanine

A sensitive radioimmunoassay (RIA) for the new antiviral agent, BW248U [9-(2-hydroxyethoxymethyl)guanine], has been developed. The antisera used in the assay were obtained by immunizing rabbits with a succinyl-BW248U-rabbit serum albumin conjugate. In the assay, a single tube per sample is employed throughout, succinyl-[³H]BW248U is used as the antigen, and only 0.1 ml of sample per tube is required. The procedure does not require sample extraction, and ammonium sulfate is used as the precipitating agent. The RIA has a useful range of 0.05 to 50 nmol of BW248U/ml and the concentrations of the drug determined by this method are in good agreement with those obtained by high-performance liquid chromatography. Naturally occurring purine bases and nucleosides, as well as a wide variety of nonrelated drugs, do not interfere with the assay. When antiserum from one rabbit was employed, the association constant with succinyl-[³H]BW248U was found to be 2.05 × 10⁸ liters/mol while that for [³H]BW248U was 10-fold less.     

Assay for glutamine synthetase activity (Short Communication)

The use of phosphoenolpyruvate plus pyruvate kinase as an ATP-generating system in the assay for glutamine synthetase activity via the formation of gamma-glutamylhydroxamate from glutamate and hydroxylamine with crude tissue preparations is shown to give values far in excess of the true glutamine synthetase activity of the tissue. This is due to the generation of pyruvate, which reacts with hydroxylamine to give a compound that is chromogenic with the ferric chloride reagent used for measuring gamma-glutamylhydroxamate.     

Effect of phenylpyruvate on enzymes involved in fatty acid synthesis in rat brain

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of ¹⁴CO₂ from [1-¹⁴C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K_i of 100μm. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

Transport in C4 mesophyll chloroplasts. Characterization of the pyruvate carrier

1. Evidence is presented for high rates of carrier-mediated uptake of pyruvate into the stroma of intact mesophyll chloroplasts of the C₄ plant Digitaria sanguinalis, but not the chloroplasts of the C₃ plant Spinacea oleracea. Uptake of pyruvate in the dark with the C₄ mesophyll chloroplasts was followed using two techniques: uptake of [¹⁴C]pyruvate as determined by silicon oil centrifugal filtration and uptake as indicated by absorbance changes at 535 nm (shrinkage/swelling) after addition of 0.1 M pyruvate salts.

2. Uptake of the pyruvate anion by an electrogenic carrier is suggested to be the major mode of transport. Chloroplast swelling was observed in potassium pyruvate plus valinomycin and uptake of [¹⁴C]pyruvate was inhibited by membrane-permeant anions. Valinomycin reduced uptake in the absence of external potassium and the inhibition could be reversed by addition of external potassium.

3. Uptake of pyruvic acid (or a pyruvate ⁻/OH⁻ antiport) is ruled unlikely since [¹⁴C]pyruvate uptake was relatively independent of the pH gradient across the envelope and addition of pyruvate to chloroplasts did not result in an alkalization of the medium. The low rate of swelling observed in ammonium pyruvate may be due to non-mediated permeation of pyruvic acid, which is possible only at high pyruvate concentrations.

4. The concentration of pyruvate in the stroma increased with external concentration over the range tested (up to 40 mM) but the concentration ratio (internal/external) was always less than one. The steady-state concentration of [¹⁴C]pyruvate in the stroma was dependent on the ionic strength of the medium, with saturation at roughly I = 0.04 M, while accumulation of the membrane-permeant cation tetraphenylmethylphosphonium decreased with increasing ionic strength. This suggests that ionic strength modifies a membrane potential (inside negative) across the envelope and that pyruvate uptake responds to the magnitude and direction of that potential (−80 mV at low ionic strength).

5. Chloride and inorganic phosphate were potent inhibitors of [¹⁴C]pyruvate uptake. Of the sulfhydryl reagents tested, N-ethylmaleimide was not inhibitory while mersalyl completely blocked [¹⁴C]pyruvate uptake and swelling in potassium pyruvate plus valinomycin. Pyruvate uptake, as measured by valinomycin induced swelling in potassium pyruvate, was highly temperature sensitive, with an energy of activation of 39 kcal/mol above 9 °C.

6. Phenylpyruvate, -ketoisovalerate, -ketoisocaproate, -cyano-4-hydroxycinnamic acid and -cyanocinnamic acid inhibited [¹⁴C]pyruvate but not [¹⁴C]-acetate uptake in the dark and also reduced pyruvate metabolism by the chloroplasts in the light.     

Importance of spontaneous alpha-ketoacid decarboxylation in experiments involving peroxide

The potential role of spontaneous alpha-ketoacid decarboxylation as a source of interference in experiments involving peroxide was investigated. The assay of pyruvate dehydrogenase activity in isolated renal mitochondria was employed as an example. Spontaneous peroxide-induced pyruvate decarboxylation competed significantly with enzymatic decarboxylation at peroxide concentrations greater than 50 microM. Corrected values for enzymatic decarboxylation could be obtained by subtracting spontaneous decarboxylation rates from rates obtained in the presence of mitochondria. At higher peroxide concentrations (greater than 200 microM), reaction product accumulates (acetoacetate) to levels which may have regulatory effects on mitochondrial metabolism. The divalent cations, Ca2+ and Mg2+, both accelerate spontaneous peroxide-induced pyruvate decarboxylation while other components of the assay medium had an inhibitory effect on the reaction. The results are discussed in relation to the currently accepted reaction mechanism. Investigators who perform experiments involving reactive oxygen species should be familiar with this often overlooked reaction.     

Dimethylglyoxime (DMG) staining for semi-quantitative mapping of Ni in plant tissue

Determination of the nickel (Ni) distribution in tissues of hyperaccumulator plants aids in understanding the strategies and mechanisms used by these plants to take up Ni from soils. Commonly used methods for measuring Ni distribution in plant tissues require expensive equipment and complex sample preparation. We tested a suite of staining methods consisting of dimethylglyoxime (DMG) dissolved in a range of solvents for the mapping of Ni distribution in the Ni hyperaccumulator Berkheya coddii Roessler. The best solution was DMG (10 g l⁻¹) dissolved in borax (25 mM) and KOH (30 mM). Plant tissue cross-sections were imaged under a microscope immediately after DMG application. A Karhunen-Loeve transformation was applied to the images to minimize interference from colours of other origin, e.g. from chlorophyll. The distribution of Ni could be determined at the cellular level and consistent patterns were obtained for replicates. Staining of Ni dissolved in agar at various concentrations was used to calibrate the method. Concentrations as low as 50 mg kg⁻¹ (fresh weight) could be detected. Averaged over several cross-sections the DMG method systematically gave lower concentrations than ICP-OES analysis of the respective plant part, indicating that not all Ni in the tissue reacted with DMG, but only Ni that is readily available. The DMG method may be used in conjunction with spectroscopic methods to resolve biologically active Ni.     

Determination of myrosinase (thioglucoside glucohydrolase) activity by a spectrophotometric coupled enzyme assay

The hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system was used to assay for plant thioglucoside glucohydrolase (myrosinase, EC 3.2.3.1) by measuring the rate of glucose released during hydrolysis of glucosinolates. This coupled assay was compared with two other assays for myrosinase: a pH-stat assay that measures the rate of acid released during glucosinolate hydrolysis, and a spectrophotometric assay in which the decrease in the absorbance at 227.5 nm is used to measure the disappearance of the substrate, 2-propenylglucosinolate (DSA assay). The coupled and pH-stat assays were found to give comparable activities and were linear with enzyme concentration over the range 0 to 30 micrograms. The DSA assay gave lower myrosinase activity in comparison to the coupled and pH-stat assays. This is due to the lower concentrations of substrate and activator (ascorbate) which must be used in the assay. The DSA assay was found to give a nonlinear relationship with enzyme concentration over the range 2 to 30 micrograms. For these reasons this assay was found to be unsatisfactory. The coupled assay was found to be more sensitive and more widely applicable than the pH-stat assay as a routine continuous assay for myrosinase activity.     

Properties of pyruvate kinase from soybean nodule cytosol

The properties of pyruvate kinase from soybean (Glycine max L.) nodule cytosol were examined to determine what influence the N₂ fixation process might have on this supposed key control enzyme. A crude enzyme preparation was prepared by chromatography of cytosol extract on a diethylaminoethyl-cellulose column. ATP and citrate at 5 mm concentrations inhibited pyruvate kinase 27 and 34%, respectively. Enzyme activation was hyperbolic with respect to both K⁺ and NH₄⁺ concentrations. In the presence of physiological concentrations of K⁺ and high phosphoenolpyruvate (PEP) concentrations, NH₄⁺ inhibited enzyme activity. Comparisons of kinetic parameters (V_max and apparent K_a) for NH₄⁺ and K⁺ with inhibition curves indicated that inhibition was very likely a result of competition of the ions for activation site(s) on the pyruvate kinase. In addition, apparent K_a (monovalent cation) and K_m (PEP) were influenced by PEP and monovalent cation concentrations, respectively. This effect may reflect a fundamental difference between plant and animal pyruvate kinases. It is concluded that control of cytosol pyruvate kinase may be closely related to reactions involved in the assimilation of NH₄⁺.     

Nonenzymatic exchange of tritium from (5-3H) deoxyuridylate in the thymidylate synthetase assay

When 2′-[5-³H] deoxyuridine-5′-phosphate and 2-mercaptoethanol are used to assay thymidylate synthetase activity, the tritium of 2′-[5-³H] deoxyuridine-5′-phosphate exchanged nonenzymatically with water. The rate of the isotope exchange depended on the concentrations both 2-m2-mercaptoethanol and amine buffer and the pH of the assay mixture. Such an exchange is a possible source of serious error in the assay when low activity of enzyme is assayed with highly radioactive 2′-deoxyuridine-5′-phosphate but can be minimized by maintaining a low concentration of 2-mercaptoethanol and unprotonated amine buffer in the assay mixture.     

High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc -) Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells

The cystine-glutamate antiporter (system x_c ^-) is a Na⁺-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system x_c ^- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system x_c ^- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system x_c ^- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system x_c ^- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system x_c ^-. Human glioma cells were chosen based on their high system x_c ^- activity. Using these cells, [¹⁴C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well ¹⁴C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of cystine uptake and glutamate release previously reported in normal human fibroblast cells.     

The atypical velocity response by pyruvate carboxylase to increasing concentrations of acetyl-coenzyme A

An investigation was made of the interaction of pyruvate carboxylase with its allosteric effector, acetyl-CoA, and the velocity profile of the deacylation of acetyl-CoA as a function of acetyl-CoA concentration indicated that this ligand does not bind to this enzyme in a positive homotropic co-operative manner. An examination was therefore made of the factors that contribute to the sigmoidicity of the rate curves obtained for pyruvate carboxylation with various concentrations of acetyl-CoA. Hill coefficients for acetyl-CoA obtained with both sheep and chicken liver pyruvate carboxylases were found to be dependent on the fixed pyruvate concentration used in the assay solution. Thus, by varying the acetyl-CoA concentration, the degree of saturation of the enzyme by pyruvate was also changed. A further consequence of non-saturating concentrations of pyruvate was that the non-productive hydrolysis of the enzyme- carboxybiotin complex increased, resulting in an under-estimate of the reaction velocity measured by oxaloacetate formation. Another factor contributing to the sigmoidicity is that, at non-saturating concentrations of acetyl-CoA, the enzyme undergoes inactivation upon dilution to low protein concentrations, again resulting in an under-estimate of the reaction velocity. Under conditions where none of the above factors was operating and the only effect of varying acetyl-CoA concentrations was to alter the proportion of the enzyme catalysing the carboxylation reaction at acetyl-CoA-dependent and -independent rates, the sigmoidicity of the acetyl-CoA velocity profile was completely eliminated.     

The mitochondrial pyruvate carrier (MPC) complex mediates one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here, we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its putative orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis thaliana mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration. ¹³C-pyruvate feeding of leaves lacking MPC showed metabolic homeostasis was largely maintained except for alanine and glutamate, indicating that transamination contributes to the restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases when MPC1 is absent resulted in extremely retarded phenotypes in Arabidopsis, suggesting all pyruvate-supplying enzymes work synergistically to support the TCA cycle for sustained plant growth.

Pyruvate is supplied by three independent processes that act synergistically to maintain metabolic flux and support plant respiration in a variety of circumstances.     

Cationic responses of organs and haemolymph of Biomphalaria pfeifferi (Krauss), Biomphalaria glabrata (Say) and Helisoma trivolvis (Say) (Gastropoda: Planorbirdae) to cationic alterations of the medium

The cationic responses of haemolymph, mantle collar, headfoot, gut + digestive diverticulum and ovotestis + albumin gland of three planorbids to cationic changes in the media are explored. Body organs exhibited cationic homeostasis, although attained with difficulty under very low Ca : Mg and Ca : Na ratios. Haemolymph imbalances were obtained at very low calcium concentrations and at very low Ca : Mg and Ca : Na ratios in the medium; this may be linked to competition for Ca²⁺ uptake sites in the epithelium by Mg²⁺ and possibly Na⁺ ions.Fecundity and shell growth in relation to calcium concentrations and to Ca : Mg and Ca : Na ratios are examined.Normal internal cationic levels, under optimal conditions, were obtained for each species. Significant interspecific differences for haemolymph sodium were found; magnesium levels were slightly higher in Biomphalaria spp. than in Helisoma trivolvis; potassium levels were all similar. Amounts of tissue calcium decreased as follows: Mantle collar head-foot ovotestis + albumin gland-gut + diverticulum. Tissue magnesium levels in the gut were low. H. trivolvis had highest tissue calcium and sodium; B. glabrata had highest tissue potassium.     

The effects of adenine nucleotides on pyruvate metabolism in rat liver

1. The effects of adenine nucleotides on pyruvate metabolism by isolated liver cells and isolated mitochondria have been investigated. The amount of pyruvate carboxylated has been estimated by determining the tricarboxylic acid-cycle intermediates, glutamate and aspartate accumulating in the incubation medium. The extent of pyruvate oxidation has been assessed by measuring oxygen uptake and the yield of ¹⁴CO₂ from [1-¹⁴C]pyruvate and [2-¹⁴C]pyruvate. 2. When catalytic amounts of adenine nucleotides (1–2mm) were added to suspensions of isolated liver cells incubated with pyruvate an ATP:ADP ratio greater than 6:1 was maintained. Both pyruvate oxidation to acetyl-CoA and the oxidation of acetyl-CoA through the tricarboxylic acid cycle were stimulated but pyruvate carboxylation was not affected. The production of acetyl-CoA exceeded the capacity of the cells for the oxidation of acetyl-CoA and the excess was converted into ketone bodies. 3. If a low ATP:ADP ratio was maintained in isolated cells or mitochondria by incubating them with dinitrophenol or hexokinase, pyruvate carboxylation was grossly inhibited, oxygen uptake depressed and ketone-body formation stimulated. Measurement of oxaloacetate concentrations confirmed that under these conditions oxaloacetate was rate-limiting for the oxidation of acetyl-CoA via the tricarboxylic acid cycle. The inclusion in the incubation medium of fumarate (1·25mm) completely prevented the ketogenic action of dinitrophenol or hexokinase. 4. When ADP (5mm) was added to a suspension of isolated liver cells incubated with pyruvate an actual ADP concentration of about 1mm was attained. This brought about effects on pyruvate metabolism similar to those obtained with dinitrophenol or hexokinase. 5. These results support the concept that the relative concentrations of adenine nucleotides within the liver cell may play a role in governing the rates of pyruvate oxidation and carboxylation. In addition, they provide further evidence that the availability of oxaloacetate in the liver cell can play a key role in determining whether acetyl-CoA arising from pyruvate is oxidized through the tricarboxylic acid cycle or converted into ketone bodies.     

A direct fluorometric assay of benzo[a]pyrene hydroxylase.

A technique to measure the activity of pyruvate carboxylase spectrophotometrically in crude liver homogenates is described. The assay is based on the transformation of oxaloacetate, which is formed during the carboxylation reaction, into citrate in the presence of excess acetyl CoA and citrate synthase. After removal of pyruvate with KBH₄ and of protein with HClO₄, citrate is cleaved with citrate lyase into oxaloacetate and acetate, and oxaloacetate then is measured spectrophotometrically. Optimal concentrations of pyruvate, Mg²⁺, ATP, and KHCO₃ for the carboxylation reaction and the V_max were in good correlation with the data found by others using [¹⁴C]pyruvate.     

Hsp72 mRNA production in cultured human cells submitted to nonlethal aggression by heat, ethanol, or propanol. Application to the detection of low concentrations of chromium(VI) (potassium dichromate)

The HT29 and HepG2 human cell lines have been shown to express stress proteins (heat shock proteins, HSP) when submitted to a variety of sublethal environmental aggressions. In the present study, these cells were submitted to standardized mild aggression by heat, ethanol, or propan-1-ol in vitro. Subsequent formation of the hsp72 mRNA was measured by a very specific RNase protection method using a radiolabeled antisense RNA probe. The accumulation of the mRNA coding for the HSP72 stress proteins was found to be maximum within 3 h after the aggression. Results were obtained faster and were much more interpretable than those from the classical method involving the autoradiography of electrophoretically separated ³⁵ S-labeled proteins, especially in the case of very weak, threshold-level, aggressions. When this model was used as a biological system for the detection of low concentrations of chromium(VI) (Cr₂O₇ ^2-), it was possible to detect concentrations as low as 0.5 µmol/L. This indicates that measuring indices of stress induction in human cultured cells can be several orders of magnitude more sensitive than the commercial Microtox assay used for detecting low levels of pollution.