Novel cytotoxicity test based on menadione-catalyzed H2O2 productivity for food safety evaluation

Menadione-catalyzed H₂O₂ production by viable cells was proportional to viable cell number, and the assay of this H₂O₂ production was applied to the cytotoxicity test of 17 substances which were used for international validation of fixed-dose procedure as an alternative to the classical LD₅₀ test. The cytotoxicity of substances tested was observed 4 h after the incubation with animal cells, and the viability was determined in 10 min according to menadione-catalyzed H₂O₂ production assay. IC₅₀ of each substance required for 50% inhibition of menadione-catalyzed H₂O₂ production was similar among HepG2, HuH-6KK, HUVE, Vero, Intestine407, NIH/3T3 and Neuro-2a cells. Twelve substances, 3 substances and 2 substances showed the difference of one, two and three orders in the magnitude between LD₅₀ and IC₅₀, respectively. These results show that menadione-catalyzed H₂O₂ production assay is useful for the rapid detection of toxic compounds having the basal cytotoxicity common to various cells, but is unfit for the detection of organ-specific toxic compounds. This revised version was published online in August 2006 with corrections to the Cover Date.     

Application of the chemiluminescent assay to cytotoxicity test: detection of menadione-catalyzed H2O2 production by viable cells.

Menadione-catalyzed H2O2 production by viable cells is proportional to viable cell number. The correlations between the viable cell number and the concentration of H2O2 produced are determined with the rapid chemiluminescent assay (S. Yamashoji, T. Ikeda, and K. Yamashoji, 1989, Anal. Biochem. 181, 149-152). This chemiluminescent assay of viable cells requires only 10 min and is much faster than NR (neutral red) inclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assays, which require 3-5 h. When viable cells are incubated with antitumor drugs, detergents, mycotoxins, and glycoalkaloids for 24-48 h, a decrease in menadione-catalyzed H2O2 production in a dose- or incubation time-dependent manner is observed. In general, the 50% inhibition concentration determined by the chemiluminescent assay is lower than that determined by NR inclusion and MTT reduction assays, and the order of relative cytotoxic effects of agents is the same among these assays. Furthermore, clear cytotoxic effects are observed by the chemiluminescent assay after 1 h exposure of trypsinized cells to toxic compounds. Therefore, the chemiluminescent assay is expected to be more useful for the rapid detection of cytotoxic compounds than NR inclusion and MTT reduction assays.     

Rapid assay of cell activity of yeast cells

Yeast cells produce H₂O₂ in the presence of exogenous menadione, and the extracellular concentration of H₂O₂ produced is proportional to the viable cell number or cell activity. Chemiluminescent assay of menadione-catalyzed H₂O₂ production requires only a few minutes, and is useful for rapid determination of the viability of yeast cells.     

Determination of viable mammalian cells by luminol chemiluminescence using microperoxidase

Chemiluminescent assay for menadione-catalyzed H₂O₂ production by mammalian cells was modified by luminol chemiluminescence with microperoxidase instead of peroxyoxalate chemiluminescence with carcinogenic fluorescent materials. Luminol can be used as a common chemiluminescent reagent for the determination of viable mammalian cells and bacteria.     

Protective effects of cyclosativene on H2O2-induced injury in cultured rat primary cerebral cortex cells

Sesquiterpenes have attracted much interest with respect to their protective effect against oxidative damage that may be the cause of many diseases including several neurodegenerative disorders and cancer. Our previous unpublished work suggested that cyclosativene (CSV), a tetracyclic sesquiterpene, has antioxidant and anticarcinogenic features. However, little is known about the effects of CSV on oxidative stress induced neurotoxicity. We used hydrogen peroxide (H₂O₂) exposure for 6 h to model oxidative stress. Therefore, this experimental design allowed us to explore the neuroprotective potential of CSV in H₂O₂-induced toxicity in new-born rat cerebral cortex cell cultures for the first time. For this aim, MTT and lactate dehydrogenase release assays were carried out to evaluate cytotoxicity. Total antioxidant capacity (TAC) and total oxidative stress (TOS) parameters were used to evaluate oxidative changes. In addition to determining of 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels, the single cell gel electrophoresis (or Comet assay) was also performed for measuring the resistance of neuronal DNA to H₂O₂-induced challenge. Our results showed that survival and TAC levels of the cells decreased, while TOS, 8-OH-dG levels and the mean values of the total scores of cells showing DNA damage (Comet assay) increased in the H₂O₂ alone treated cultures. But pre-treatment of CSV suppressed the cytotoxicity, genotoxicity and oxidative stress which were increased by H₂O₂. On the basis of these observations, it is suggested that CSV as a natural product with an antioxidant capacity in mitigating oxidative injuries in the field of neurodegenerative disorders.     

Hydrogen peroxide production is an early event during bicarbonate induced stomatal closure in abaxial epidermis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

The presence of 2 mM bicarbonate in the incubation medium induced stomatal closure in abaxial epidermis of Arabidopsis. Exposure to 2 mM bicarbonate elevated the levels of H₂O₂ in guard cells within 5 min, as indicated by the fluorescent probe, dichlorofluorescein diacetate (H₂DCF-DA). Bicarbonate-induced stomatal closure as well as H₂O₂ production were restricted by exogenous catalase or diphenylene iodonium (DPI, an inhibitor of NAD(P)H oxidase). The reduced sensitivity of stomata to bicarbonate and H₂O₂ production in homozygous atrbohD/F double mutant of Arabidopsis confirmed that NADP(H) oxidase is involved during bicarbonate induced ROS production in guard cells. The production of H₂O₂ was quicker and greater with ABA than that with bicarbonate. Such pattern of H₂O₂ production may be one of the reasons for ABA being more effective than bicarbonate, in promoting stomatal closure. Our results demonstrate that H₂O₂ is an essential secondary messenger during bicarbonate induced stomatal closure in Arabidopsis.     

NO serves as a signaling intermediate downstream of H2O2 to modulate dynamic microtubule cytoskeleton during responses to VD-toxins in Arabidopsis

Although hydrogen peroxide (H₂O₂) and nitric oxide (NO) can act as an upstream signaling molecule to modulate the dynamic microtubule cytoskeleton during the defense responses to Verticillium dahliae (VD) toxins in Arabidopsis, it is not known the relationship between these two signaling molecules. Here, we show that VD-toxin-induced NO accumulation was dependent on prior H₂O₂ production, NO is downstream of H₂O₂ in the signaling process, and that H₂O₂ acted synergistically with NO to modulate the dynamic microtubule cytoskeleton responses to VD-toxins in Arabidopsis.     

Modification of the in vitro Cytotoxicity of Hydrogen Peroxide by Iron Complexes

The effect of a range of iron chelates on the cytotoxicity of H₂O₂ was studied on a mammalian epithelial cell line. Iron complexes which were internalised enhanced the cytotoxicity of H₂O₂ measured by delayed thymidine incorporation. Iron complexed to 8-hydroxyquinoline (Fe/8-HQ) potentiated the cytotoxicity of 50 µM by 38% and Fe/dextran by 23%. Pre-exposure of cells to Fe/dextran at 4°C did not result in any potentiation of H₂O₂-induced cytotoxicity which we ascribe to failure of the Fe/dextran to be endocytosed at low temperature. Iron complexes which are slowly taken up or remain extracellular protected the cells from H₂O₂-induced cytotoxicity. Thus, Fe/EDTA inhibited the cytotoxicity of 50 µM H₂O₂ by 33%; Fe/ADP by 80% and Fe/ATP by 88%, suggesting mutual extracellular detoxification.     

Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on cytotoxicity of PSK-induced peritoneal polymorphonuclear leukocytes (PMNs)

We investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) en hanced the cytotoxicity of PSK-induced polymorphonuclear leukocytes (PMNs) in the peritoneal cavity. Male C3H/He mice, 8- to 10-week-old, received single subcutaneous (s.c.) or intraperitoneal (i.p.) injection of 2.5 µg/animal of rhG-CSF at different time points before or after an i.p. administration of PSK. In other experiments, mice were s.c. or i.p. treated with the same dosage of rhG-CSF every day for 7 or 14 consecutive days and i.p. injected with 2.5 mg/animal of PSK on the last day. Peritoneal PMNs were harvested 6 hrs after the administration of PSK and purified to more than 95% by Ficoll-Paque for in vitro cytotoxic assay.In vitro cytotoxic assays with⁵¹Cr labeled MM46 mammary carcinoma cells were added with 5–20 µg/ml of Nocardia rubra cell wall skeleton (N-CWS) at the beginning of the assay to augment the cytotoxic activity of PMNs.In vitro addition of rhG-CSF to the assay did not enhance the cytotoxicity of PSK-induced PMNs. However, the cytotoxicity was signifi cantly increased when rhG-CSF was s.c. administered 12 hrs before a PSK injection or 2 or 5 hrs after that. On the other hand, the cytotoxicity was rather weak when mice s.c. or i.p. received consecutive injections of rhG-CSF. This cytotoxicity may be mediated by H₂O₂, since H₂O₂ production of PMNs during the cytotoxic assay appears to correlate with the levels of cytotoxicity under suppressed H₂O₂ generation by catalase or enhanced generation by rhG-CSF. These results suggest that rhG-CSF augments the cytotoxicity of PSK-induced PMNs when administeredin vivo timely.     

The involvement of a P38-like MAP kinase in ABA-induced and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-mediated stomatal closure in <Emphasis Type="Italic">Vicia faba</Emphasis> L.

SB203580 is a specific inhibitor of p38 mitogen-activated protein (MAP) kinase and has been widely used to investigate the physiological roles of p38 in animal and yeast cells. Here by using an epidermal strip bioassay, laser-scanning confocal microscopy and whole-cell patch clamp analysis, we assess the effects of pyridinyl imidazoles-like SB203580 on the H₂O₂ signaling in guard cells of Vicia faba L. The results indicated that SB203580 blocks H₂O₂- or ABA-induced stomatal closure, ABA-induced H₂O₂ generation, and decrease in K⁺ fluxing across plasma membrane of Vicia guard cells by application of ABA and H₂O₂, whereas its analog SB202474 had no effect on these events. Thus, these results suggest that activation of p38-like MAP kinase modulates guard cell ROS signaling in response to stress.     

Xylem parenchyma cells deliver the H<Subscript>2</Subscript>O<Subscript>2</Subscript> necessary for lignification in differentiating xylem vessels

Lignification in Zinnia elegans L. stems is characterized by a burst in the production of H₂O₂, the apparent fate of which is to be used by xylem peroxidases for the polymerization of p-hydroxycinnamyl alcohols into lignins. A search for the sites of H₂O₂ production in the differentiating xylem of Z. elegans stems by the simultaneous use of optical (bright field, polarized light and epi-polarization) and electron-microscope tools revealed that H₂O₂ is produced on the outer-face of the plasma membrane of both differentiating (living) thin-walled xylem cells and particular (non-lignifying) xylem parenchyma cells. From the production sites it diffuses to the differentiating (secondary cell wall-forming) and differentiated lignifying xylem vessels. H₂O₂ diffusion occurs mainly through the continuous cell wall space. Both the experimental data and the theoretical calculations suggest that H₂O₂ diffusion from the sites of production might not limit the rate of xylem cell wall lignification. It can be concluded that H₂O₂ is produced at the plasma membrane in differentiating (living) thin-walled xylem cells and xylem parenchyma cells associated to xylem vessels, and that it diffuses to adjacent secondary lignifying xylem vessels. The results strongly indicate that non-lignifying xylem parenchyma cells are the source of the H₂O₂ necessary for the polymerization of cinnamyl alcohols in the secondary cell wall of lignifying xylem vessels.     

Cell detachment and growth of fibroblasts as parameters for cytotoxicity of inorganic metal saltsin Vitro

Eight inorganic metal compounds (AlCl₃, Al(OH)₃ gel, Al(OH)₃ salt, SnCl₂, ZnSO₄, K₂Cr₂O₇, CdCl₂, HgCl₂) were tested for their cytotoxic effect on an established hamster fibroblast line (BHK-21/C13) in vitro using a cell detachment assay and two different growth assays, the cloning effieciency and the cell number after 2 days subconfluent culture as parameters. The test conditions for these assays were optimized, including incubation period, application of test substance, growth conditions and data analysis. Aluminum, zinc and tin compounds showed low cytotoxic effects when compared to potassium, cadmium and mercuric compounds. Potassium dichromate was highly toxic in both growth assays (0.0001–0.01 mM, with a clear dependency on the incubation time), whereas it proved to be only slightly toxic in the detachment assay (0.1–5 mM). Cadmium and mercuric chlorides were the most toxic compounds in the growth (0.00001–0.001 mM) and the cell detachment assays (0.01–0.1 mM). Variable incubation periods barely affected the cytotoxicity of mercuric chloride. Ranking of these cytotoxicity data was found to be identical to the ranking of LD₅₀ values (oral, rat) as well as to the ranking according to threshold limit values for human workroom environment, and of human eye irritation data.Abbreviations HK-21/C13 Baby, hamster kidney fibroblasts, clone 21/C13 - CH_low lowest concentration at which a significant increase in number of detached cells was measured - CE_low lowest concentration at which a significant decrease in cloning efficiency was measured - GI_low lowest concentration at which growth was significantly inhibited under sunconfluent culture conditions - MEM Eagle's minimum essential medium - TLV/TWA Threshold limit values (time weigthed average, USA) for human workroom environment     

The quantitative measurement of H2O2 generation in isolated mitochondria

Adequate methods to measure the rate of mitochondrial oxygen radical generation are needed since oxygen radicals are involved in many pathologies. A fluorometric method appropriate to measure the rate of generation of H₂O₂ in intact mitochondria is described. Just after isolation of functional mitochondria from fresh tissues, rates of generation of H₂O₂ are kinetically measured by fluorometry in the presence of homovanillic acid and horseradish peroxidase. The method is specific for H₂O₂ and is sensitive enough to assay mitochondrial H₂O₂ generation in the presence of respiratory substrate without inhibitors of the respiratory chain. Simultaneous measurement of mitochondrial oxygen consumption allows calculation of the free radical leak: the percentage of electrons out of sequence which reduce oxygen to oxygen radicals along the mitochondrial respiratory chain instead of reducing oxygen to water at the terminal cytochrome oxidase. The method shows instantaneous response to H₂O₂. This makes it appropriate to study the quick effects of different inhibitors and modulators on the rate of mitochondrial oxygen radical production. Its application to the localization of the sites where caloric restriction decreases mitochondrial oxygen radical generation in heart mitochondria is described.     

Oligochitosan induced Brassica napus L. production of NO and H2O2 and their physiological function

NO (nitric oxide) and H₂O₂ (hydrogen peroxide) are important signaling molecule in plants. Brassica napus L. was used to understand oligochitosan inducing production of NO (nitric oxide) and H₂O₂ (hydrogen peroxide) and their physiological function. The result showed that the production of NO and H₂O₂ in epidermal cells of B. napus L. was induced with oligochitosan by fluorescence microscope. And it was proved that there was an interaction between NO and H₂O₂ with L-NAME (N^G-nitro-l-arg-methyl eater), which is an inhibitor of NOS (NO synthase) in mammalian cells that also inhibits plant NO synthesis, and CAT (catalase), which is an important H₂O₂ scavenger, respectively. It was found that NO and H₂O₂ induced by oligochitosan took part in inducing reduction in stomatal aperture and LEA protein gene expression of leaves of B. napus L. All these results showed that oligochitosan have potential activities of improving resistance to water stress.     

Multiple reactions in vanadyl-V(IV) oxidation by H2O2

Oxidation of vanadyl sulfate by H₂O₂ involves multiple reactions at neutral pH conditions. The primary reaction was found to be oxidation of V(IV) to V(V) using 0.5 equivalent of H₂O₂, based on the loss of blue color and the visible spectrum. The loss of V(IV) and formation V(V) compounds were confirmed by ESR and⁵¹V-NMR spectra, respectively. In the presence of excess H₂O₂ (more than two equivalents), the V(V) was converted into diperoxovanadate, the major end-product of these reactions, identified by changes in absorbance in ultraviolet region and by the specific chemical shift in NMR spectrum. The stoichiometric studies on the H₂O₂ consumed in this reaction support the occurrence of reactions of two-electron oxidation followed by complexing two molecules of H₂O₂. Addition of a variety of compounds—Tris, ethanol, mannitol, benzoate, formate (hydroxyl radical quenching), histidine, imidazole (singlet oxygen quenching), and citrate—stimulated a secondary reaction of oxygen-consumption that also used V(IV) as the reducing source. This reaction requires concomitant oxidation of vanadyl by H₂O₂, favoured at low H₂O₂:V(IV) ratio. Another secondary reaction of oxygen release was found to occur during vanadyl oxidation by H₂O₂ in acidic medium in which the end-product was not diperoxovanadate but appears to be a mixture of VO ₃ ⁺ (–546 ppm), VO³⁺ (–531 ppm) and VO ₂ ⁺ (–512 ppm), as shown by the⁵¹V-NMR spectrum. This reaction also occurred in phosphate-buffered medium but only on second addition of vanadyl. The compounds that stimulated the oxygen-consumption reaction were found to inhibit the oxygen-release reaction. A combination of these reactions occur depending on the proportion of the reactants (vanadyl and H₂O₂), the pH of the medium and the presence of some compounds that affect the secondary reactions.     

Ozone-mediated cytotoxicity after short-term exposure and its relation to the production of cellular metabolites (NO,H2O2)

Alveolar macrophages secrete numerous mediators, playing an important role in host defence. Among these mediators, nitric oxide (NO) and hydrogen peroxide (H₂O₂) are both involved in bactericidal killing and trigger the release of other cellular metabolites. We have analyzed the effect of an atmosphere polluted with ozone (0.03–0.5 ppm v/v) on the monocytic cell line THP-1, as a model for alveolar macrophages,in vitro. NO and H₂O₂ were chosen to evaluate cell response to ozone. Cell injury was evaluated using lactate dehydrogenase (LDH) liberation into the medium. An exposure to 0.5 ppm ozone proved to be more toxic to the cells, than 0.1 or 0.03 ppm, evidenced by more LDH being liberated and cytotoxicity reaching values up to 64%. For all ozone concentrations, H₂O₂ production reached a peak value after 10–15 min of exposure, after which the concentration of extracellular H₂O₂ production diminished rapidly. The highest NO concentrations were measured with 0.5 ppm ozone, reaching a maximum value of 1460 nmol/L per 5×10⁶ cells, which is 1.55 times higher than for nonexposed cells. Lower concentrations barely induced higher NO concentrations compared to nonexposed cells. The results indicate that ozone effects not only the viability of human monocytes but also the release of antibacterial and defense signaling molecules and suggest that ozone-mediated cytotoxicity may be related to the secretion of NO and H₂O₂. This revised version was published online in July 2006 with corrections to the Cover Date.     

d-β-Hydroxybutyrate inhibited the apoptosis of PC12 cells induced by H2O2 via inhibiting oxidative stress

Oxidative stress has an important role in neurodegenerative diseases and cerebral ischemic injury. It is reported that d-β-hydroxybutyrate (DβHB), the major component of ketone bodies, is neuroprotective in recent studies. Therefore, in the present work the neuroprotective effects of DβHB on H₂O₂-induced apoptosis mediated by oxidative stress was investigated. PC12 cells were exposed to H₂O₂ with different concentrations of H₂O₂ for different times after DβHB pretreatment. MTT assay, apoptotic rates, intracellular reactive oxygen species (ROS) level, GSH content, mitochondrial membrane potential (MMP) and caspase-3 activity were determined. The results showed that DβHB inhibited the decrease of cell viability induced by H₂O₂ in PC12 cells. DβHB decreased the apoptotic rates induced by H₂O₂. The changes of intracellular ROS, GSH, MMP and caspase-3 activity due to H₂O₂ exposure were partially reversed in PC12 cells. So DβHB inhibited the apoptosis of PC12 cells induced by H₂O₂ via inhibiting oxidative stress.     

The molecular mechanisms of diallyl disulfide and diallyl sulfide induced hepatocyte cytotoxicity

Diallyl disulfide (DADS) and diallyl sulfide (DAS) are the major metabolites found in garlic oil and have been reported to lower cholesterol and prevent cancer. The molecular cytotoxic mechanisms of DADS and DAS have not been determined.The cytotoxic effectiveness of hydrogen versus allyl sulfides towards hepatocytes was found to be as follows: NaHS > DADS > DAS. Hepatocyte mitochondrial membrane potential was decreased and reactive oxygen species (ROS) and TBARS formation was increased by all three allyl sulfides. (1) DADS induced cytotoxicity was prevented by the H₂S scavenger hydroxocobalamin, which also prevented cytochrome oxidase dependent mitochondrial respiration suggesting that H₂S inhibition of cytochrome oxidase contributed to DADS hepatocyte cytotoxicity. (2) DAS cytotoxicity on the other hand was prevented by hydralazine, an acrolein trap. Hydralazine also prevented DAS induced GSH depletion, decreased mitochondrial membrane potential and increased ROS and TBARS formation. Chloral hydrate, the aldehyde dehydrogenase 2 inhibitor, however had the opposite effects, which could suggest that acrolein contributed to DAS hepatocyte cytotoxicity.     

Potent Upregulation of Glutathione and NAD(P)H:Quinone Oxidoreductase 1 by Alpha-lipoic Acid in Human Neuroblastoma SH-SY5Y Cells: Protection Against Neurotoxicant-elicited Cytotoxicity

Alpha-lipoic acid (LA) has recently been reported to afford protective effects in neurodegenerative disorders. However, the mechanisms underlying LA-mediated neuroprotection remain to be investigated. This study was undertaken to determine whether LA treatment could increase endogenous antioxidants and phase 2 enzymes in cultured human neuroblastoma SH-SY5Y cells, and whether such increased cellular defenses could afford protection against cytotoxicity induced by neurotoxicants. Incubation of SH-SY5Y cells with micromolar concentrations of LA for 24 h resulted in a significant increase in the levels of reduced glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQQ1) in a concentration-dependent fashion. Treatment of the cells with LA also led to an increased mRNA expression of γ-glutamylcysteine ligase catalytic subunit (GCLC) and NQO1. To determine the protective effects of the LA-induced cellular defenses on neurotoxicant-elicitedl cell injury, SH-SY5Y cells were pretreated with LA for 24 h and then exposed to acrolein, 4-hydroxy-2-nonenal (HNE), H₂O₂ and the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1). We observed that LA pretreatment of SH-SY5Y cells led to a marked protection against acrolein, HNE, H₂O₂ and SIN-1-mediated cytotoxicity, as detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction assay. Taken together, this study demonstrates for the first time that LA can induce GSH and NQO1 in cultured human neuroblastoma cells and LA-upregulated cellular defenses are accompanied by a markedly increased resistance to cytotoxicity induced by various neurotoxicants. The results of this study may have important implications for the neuroprotective effects of LA.