Toxicity Of Low Concentrations Research Articles

Traits and stress: keys to identify community effects of low levels of toxicants in test systems

Community effects of low toxicant concentrations are obscured by a multitude of confounding factors. To resolve this issue for community test systems, we propose a trait-based approach to detect toxic effects. An experiment with outdoor stream mesocosms was established 2-years before contamination to allow the development of biotic interactions within the community. Following pulse contamination with the insecticide thiacloprid, communities were monitored for additional 2 years to observe long-term effects. Applying a priori ecotoxicological knowledge species were aggregated into trait-based groups that reflected stressor-specific vulnerability of populations to toxicant exposure. This reduces inter-replicate variation that is not related to toxicant effects and enables to better link exposure and effect. Species with low intrinsic sensitivity showed only transient effects at the highest thiacloprid concentration of 100 μg/l. Sensitive multivoltine species showed transient effects at 3.3 μg/l. Sensitive univoltine species were affected at 0.1 μg/l and did not recover during the year after contamination. Based on these results the new indicator SPEAR mesocosm was calculated as the relative abundance of sensitive univoltine taxa. Long-term community effects of thiacloprid were detected at concentrations 1,000 times below those detected by the PRC (Principal Response Curve) approach. We also found that those species, characterised by the most vulnerable trait combination, that were stressed were affected more strongly by thiacloprid than non-stressed species. We conclude that the grouping of species according to toxicant-related traits enables identification and prediction of community response to low levels of toxicants and that additionally the environmental context determines species sensitivity to toxicants.

Susceptibility of Four Tick Species, Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Rhipicephalus sanguineus (Acari: Ixodidae), to Nootkatone From Essential Oil of Grapefruit

Toxicity of nootkatone was determined in laboratory assays against unfed nymphs of Amblyomma americanum L., Dermacentor variabilis (Say), Ixodes scapularis Say, and Rhipicephalus sanguineus Latreille. We determined the 50% lethal concentration (LC50) and 90% lethal concentration (LC90) of nootkatone by recording tick mortality 24 h after exposure in treated glass vials. Nymphs were susceptible to nootkatone with LC50 values of 0.352, 0.233, 0.169, and 0.197 microg/cm2, and LC90 values of 1.001, 0.644, 0.549, and 0.485 microg/cm2 for A. americanum, D. variabilis, I. scapularis, and R. sanguineus, respectively. The LC50 value for R. sanquineus was not significantly different from D. variabilis or I. scapularis. Other LC50 comparisons were significantly different. The LC90 for A. americanum was higher when compared with the three other tick species, which were not significantly different. Because nootkatone is volatile, we measured the amount of nootkatone recovered from duplicate-treated vials before tick exposure and from vials after tick exposure. Nootkatone recovered from vials before exposure ranged from 82 to 112% of the expected amounts. The nootkatone recovered after the 24-h exposure period ranged from 89% from vials coated with higher concentrations of nootkatone, down to 29% from vials coated with low nootkatone concentrations. Determination of the nootkatone residue after vial coating demonstrated loss of the active compound while verifying the levels of tick exposure. Toxicity of low concentrations of nootkatone to the active questing stage of ticks reported in this study provides a reference point for future formulation research to exploit nootkatone as a safe and environment-friendly tick control.

Radiosensitizing effect of the histone acetyltransferase inhibitor anacardic acid on various mammalian cell lines

Agents that enhance the effectiveness of ionizing radiation have been investigated over many decades. A relatively new group of potential radiosensitizers consists of agents that inhibit histone acetyltransferases (HATs). This study evaluated the radiosensitizing properties of the HAT inhibitor anacardic acid (AA), used at a low-toxic concentration of 100 μM in V79, SW1573 and U2OS cells. Radiation survival curves were analyzed according to the linear quadratic model. Significant radiosensitization by AA was only obtained in U2OS cells. AA significantly increased the value of the linear parameter α, but not of the quadratic parameter β, indicating fixation of potentially lethal damage and an intact repair function of sublethal damage. The increase of the α value was also observed in SW1573 cells, but was not accompanied by a significant radiosensitization. A likely explanation for the enhancement of the α value may be an increase in the amount of lethal lesions due to the compacted chromatin structure. Despite the conflicting results of the radiosensitizing effect of AA in the three cell lines tested, the ability of AA to increase the α value suggests potential advantages for clinical application.

Recreational drugs, 3,4-Methylenedioxymethamphetamine(MDMA), 3,4-methylenedioxyamphetamine (MDA) and diphenylprolinol, inhibit neurite outgrowth in PC12 cells

3,4-Methylenedioxymethamphetamine (MDMA) is widely abused as a psychoactive recreational drug. It is well known that MDMA induces neurotoxic damage of serotonergic nerve endings. Although drug abuse is increasing among youths, it is unclear whether recreational drugs affect the development of nerve growth. Thus, the present study examined the effect of recreational drugs, such as MDMA, 3,4-methylenedioxyamphetamine (MDA) and diphenylprolinol, a novel recreational drug with a similar chemical structure as that of psychoactive agent pipradrol, on nerve growth factor (NGF)-induced neurite outgrowth. These recreational drugs induced a dose-dependent cell death in PC12 cells. The IC(50) values of MDMA, MDA, R-diphenylprolinol and S-diphenylprolinol were 4.11 mM, 2.75 mM, 1.00 mM and 0.77 mM, respectively, at 24 hr. To examine the effects of these recreational drugs on NGF-induced neurite outgrowth, PC12 cells were treated with NGF together with MDMA, MDA, S-diphenylprolinol or R-diphenylprolinol at low toxic concentrations. The recreational drugs significantly suppressed neurite outgrowth of PC12 cells induced by NGF. The results suggest that these psychoactive recreational drugs may inhibit neurite growth and thus be implicated in their elicited neurotoxicity.

In Situ Bioremediation of Perchlorate in Groundwater

Perchlorate has been detectable at milligram/liter concentrations in groundwater for decades, and investigations were originally primarily limited to sites where rocket-fuel development or testing had occurred. Other compounds of concern with low-concentration toxicity have been detected at many sites and have dominated the focus of the remedial industry since the 1970s. Once a revised analytical method was developed in 1997 to detect perchlorate concentrations down to 4 µg/L, its widespread presence was recognized at many sites and led perchlorate to claim center stage attention on a national level by the early 2000s. Although perchlorate's early association with military use persists (headlines often equate perchlorate with “rocket fuel,” regardless of the actual source), perchlorate detection at non-military sites has led to the recognition that other potential sources also require consideration. The ability to definitively distinguish potential sources from one another remains a challenge even today. Initial concerns over perchlorate recalcitrance, however, have since been allayed by the repeated demonstrations of successful remediation using a variety of familiar technologies. In this sense, perchlorate remediation strategies have been an outgrowth from chlorinated solvent–focused remediation technologies that had evolved since the 1970s. The fact that in situ technologies were being deployed aggressively—an approach that took many years for the solvent remediation industry to implement with regulatory approval—even as the extent of perchlorate was still be determined at many sites, is evidence that lessons learned from decades of chlorinated solvent remediation have and will continue to benefit perchlorate remediation speed and efficiency. As the debate over risk to human health from perchlorate exposure via food and water continues, it is apparent that in situ bioremediation of soil and groundwater has been demonstrated to be a powerful tool that remedial engineers and scientists would be wise to seriously consider at any site. In Situ Bioremediation of …

Requirement of a dopaminergic neuronal phenotype for toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells

LUHMES cells are conditionally-immortalized non-transformed human fetal cells that can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. After differentiation by GDNF and cyclic adenosine monophosphate, LUHMES were sensitive to 1-methyl-4-phenylpyridinium (MPP +) toxicity at ≤ 5 μM, but resistant to the parental compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The high homogeneity and purity of the cultures allowed the detection of metabolic changes during the degeneration. Cellular ATP dropped in two phases after 24 and 48 h; cellular glutathione (GSH) decreased continuously, paralleled by an increase in lipid peroxidation. These events were accompanied by a time-dependent degeneration of neurites. Block of the dopamine transporter by GBR 12909 or mazindol completely abrogated MPP + toxicity. Inhibition of de novo dopamine synthesis by α-methyl- l-tyrosine or 3-iodo- l-tyrosine attenuated toxicity, but did not reduce the initial drop in ATP. Inhibition of mixed lineage kinases by CEP1347 completely prevented the MPP +-induced loss of viability and intracellular GSH, but failed to attenuate the initial drop of ATP. For the quantitative assessment of neurite degeneration, an automated imaging-based high content screening approach was applied and confirmed the findings made by pharmacological interventions in this study. Our data indicate that inhibition of mitochondrial ATP synthesis is not sufficient to trigger cell death in MPP +-treated LUHMES.

Testing the homogenizing effect of low copper sulfate concentrations on the size distribution of Portulaca oleracea seedlings in vitro

Traditionally, toxicological bioassays rely upon the differences in mean-based statistical tests between the exposed and unexposed plants, and exceptional plant individuals are treated as statistical outliers. Recently, low toxicant concentrations have been observed to affect gene regulation in exposed plant stands and to change the frequency of the largest individuals even if mean plant size remains unchanged. In this paper, we present the results that the latter phenomenon is not restricted to a single toxicant and plant species. Our data analysis consists of two statistical methods that may be of general interest. We utilized the one-tailed Moses extreme reactions test by comparing exposed groups to control plants with and without the trimming of a certain amount of potential outliers from both treatments compared. We also propose that Mann–Whitney U or other tests at ordinal scale can be utilized to analyze if the largest plant individuals in exposed and control treatments come from a single ‘survivor’ population. We conclude that the results supported the hypothesis that very low toxicant concentrations may have ecological effects on fast-growing plant species. Finally, we discuss the limitations of the statistical methods currently in use.

Long-Term Effects of Ammonia on the Behavioral Activity of the Aquatic Snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca)

An appropriate approach to assess the effect of toxicants on aquatic animals is to monitor behavioral endpoints, as they are a link between physiological and ecological processes. A group that can be exposed long-term to low toxic concentrations is benthic macroinvertebrates, as their mobility in aquatic ecosystems is relatively limited. Therefore, the study of behavioral long-term effects in this group is suitable from an ecological point of view, as behavioral effects can appear before mortality. During the last decades there has been an increase in ammonia concentrations in freshwater ecosystems, threatening aquatic animals. The present study focuses on the long-term effects (40 days) of nonionized ammonia on the behavioral activity of the aquatic snail Potamopyrgus antipodarum. One control and three ammonia concentrations (0.02, 0.07, and 0.13 mg N-NH(3)/L) were used in triplicate, and the activity of snails (as mean time to start normal movement) and immobility were recorded for each treatment after 0, 10, 20, 30, and 40 days of continuous exposure to nonionized ammonia. The results show that P. antipodarum presented a high tolerance to lethal long-term effects of nonionized ammonia, as no animal died during the bioassay. However, the behavioral activity of snails was a very sensitivity endpoint, as a mean nonionized ammonia concentration of 0.07 mg N-NH(3)/L affected P. antipodarum. The results are discussed and compared with the available literature on long-term effects of ammonia on freshwater macroinvertebrates. Additionally, the ammonia water quality criteria, NOECs, LOECs, and long-term LCs are discussed on the basis of the current available data for freshwater macroinvertebrates.

Biological treatment and toxicity of low concentrations of oily wastewater (bilgewater)

The biodegradability and toxicity of low concentrations of oily wastewater (bilgewater) were tested under simulated sanitary wastewater treatment conditions. This was done to establish the feasibility of a combined shipboard oily and nonoily wastewater treatment system. The biodegradability of oily wastewater was determined by proxy; 14C-labeled dodecane, toluene, and phenanthrene (representing alkane, aromatic, and polyaromatic compounds, respectively) were mineralized in petroleum fuels and lubricants. We found that low concentrations of oily wastewater components were mineralized, even in the presence of more abundant substrates (such as synthetic graywater, containing vegetable oil, detergent, gelatin, and starch). The toxic effects of diesel fuel and several other components of oily wastewater (such as surfactants and a synthetic lubricant) on a naïve wastewater assemblage was also tested. In concentrations much higher than would be expected under normal shipboard conditions, we found no evidence of toxic effects of the bilgewater compounds tested. Thus, a combined shipboard bilgewater and sanitary wastewater system might be feasible.

Low toxicant concentrations decrease the frequency of fast-growing seedlings at high densities of annual baby's breath ( Gypsophila elegans)

Very low toxicant concentrations reduce the frequency of most vital seedlings in dense stands in vitro.

α-Toxin of Staphylococcus aureus overcomes acquired cisplatin-resistance in malignant mesothelioma cells

α-Toxin (α-hemolysin) of Staphylococcus aureus is a pore-forming bacterial toxin which after caveolin-1-dependent assembly induces apoptosis in eukaryotic cells. We investigated if a sub-toxic concentration of staphylococcal α-toxin could enhance cisplatin-induced apoptosis and overcome acquired cisplatin-resistance in cultured malignant pleural mesothelioma (MPM) cells. MPM cells (P31wt) and a cisplatin-resistant sub-line (P31res) was incubated with α-toxin and/or cisplatin followed by determination of cell viability, apoptosis, and signaling pathways. P31res cells were more sensitive to α-toxin than P31 wt cells due to induction of apoptosis. A low-toxic concentration of α-toxin re-sensitized cisplatin P31res cytotoxicity by apoptosis-induced through the mitochondrial pathway without detectable activation of common up-stream apoptosis signaling proteins. The toxin/drug combination should be tested for cisplatin-resistant mesothelioma treatment.

Sub-chronic toxicity of low concentrations of industrial volatile organic pollutants in vitro

Organic solvents form an important class of pollutants in the ambient air and have been associated with neurotoxicity and immunotoxicity in humans. Here we investigated the biological effects of sub-chronic exposure to industrially important volatile organic solvents in vitro. Jurkat T cells were exposed to toluene, n-hexane and methyl ethyl ketone (MEK) individually for 5 days and solvent exposure levels were confirmed by headspace gas chromatography. A neuroblastoma cell line (SH-SY5Y) was exposed to toluene for the same period. Following exposure, cells were harvested and toxicity measured in terms of the following endpoints: membrane damage (LDH leakage), perturbations in intracellular free Ca(2+), changes in glutathione redox status and dual-phosphorylation of MAP kinases ERK1/2, JNK and p38. The results show that sub-chronic exposure to the volatile organic solvents causes membrane damage, increased intracellular free calcium and altered glutathione redox status in both cell lines. However, acute and sub-chronic solvent exposure did not result in MAP kinase phosphorylation. Toxicity of the solvents tested increased with hydrophobicity. The lowest-observed-adverse-effect-levels (LOAELs) measured in vitro were close to blood solvent concentrations reported for individuals exposed to the agents at levels at or below their individual threshold limit values (TLVs).

A Monitoring System of Feeding Rate in Daphnia Magna for Toxicity Test

The monitoring system for feeding rate of Daphnia magna as fluorescent intensity of Chlorella vulgaris was constructed to evaluate toxicities. When 30 daphnids were added to water samples and run the system, the change of the feeding rate by time was measured accuracy. This method revealed that when the toxicants which were potassium dichromate, lead (II) nitrate, Triton X-100, and Benthiocarb were high concentration, the feeding rate decreased compare to the control after 180 minutes exposure. The feeding rate tended to increase linearly after 60 minutes exposure to all toxicants. With low concentrations of toxicants, lead (II) nitrate, Triton X-100, and Benthiocarb, the feeding rate difference 60 minutes exposure and 180 minutes exposure were larger than control. Thus, it was shown that the feeding rate didn't always decrease when toxicants coexisted in low concentration. This method had higher sensitivity than that of acute immobilization test on some toxicants. From these results, it was shown that this system can measure the change of feeding rate. Although it is necessary to improve the sensitivity, conditions, and construction of the system, the toxicity test based on this theory will be used effectively.

Adenylate Cyclase Toxin From Bordetella pertussis Enhances Cisplatin-Induced Apoptosis to Lung Cancer Cells In Vitro

The present study examined the possibility to enhance lung cancer cell cytotoxicity and apoptosis of the anticancer drug cisplatin by exposure with adenylate cyclase (AC) toxin from Bordetella pertussis. A malignant mesothelioma cell line (P31) and a small-cell lung cancer cell line (U1690) were exposed to increasing concentrations of cisplatin and AC toxin, alone or in combination. Cytotoxicity was determined by a fluorescein-based assay and apoptosis by flow cytometry quantification of annexin V binding. Caspase-3, -8, and -9 activities were measured by enzyme activity assays. The cytotoxicity of AC toxin was time and dose dependent with an LD50 value at 72 h of 3 and 7 mg/L for P31 cells and U1690 cells, respectively. Cisplatin showed a similar time- and dose-dependent cytotoxicity, which was increased in the presence of a low toxic concentration (1 mg/L) of AC toxin. Furthermore, cisplatin caused a dose-dependent increase of annexin V binding cells of both cell lines after 24-h incubation, which was also enhanced in combination with AC toxin. AC toxin (1 mg/L) increased cisplatin-induced caspase-3, -8, and -9 activities in U1690 cells. Only minor increases of caspase-8 and -9 were noted for P31 cells. The present results, together with the knowledge that bacterial toxins decrease side effects of traditional cancer treatment, suggest a possibility to use them to enhance the therapeutic effect of cancer chemotherapy with reduced clinical adverse effects.

Carcinogenic risks of dioxin: Mechanistic considerations

Dioxins and dioxin-like chemicals demonstrate high affinity binding to the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor, which mediates most, if not all, of the toxic responses of these agents. Since dioxins are not directly genotoxic their carcinogenic effect is likely the result of their tumor promoting activity produced by activation of the AhR. For the purpose of risk assessment extrapolation from effects in the observable high dose range to background dietary exposure is necessary. In the present review, we discuss various aspects of low-dose–response of receptor-mediated processes in general, including threshold phenomena with regard to tumor promotion during multi-stage carcinogenesis. In this connection the reversibility of tumor promotion plays an important role but this may not be valid for dioxins due to their long half life. The relevance of cytochrome P4501A-induction as biomarker for prediction of carcinogenic effects of dioxins at low doses is considered. Dioxins may act in concert with endogenous ligands of the AhR, an effect which becomes particularly relevant at low toxicant concentrations. At present, however, the nature and role of these postulated ligands are unknown. Furthermore, it is unclear whether dioxins produce synergistic tumor promotional effects with non-dioxin-like chemicals to which humans are also exposed. Dioxins and, e.g., non-dioxin-like PCBs act through different receptors and there is, albeit yet limited, experimental evidence from experimental studies to suggest that they may act on different target cell populations within the same target organ. From the available data the existence of a (physiological) threshold of effects cannot be proven and may not even exist. For regulatory purposes the application of a so called “practical threshold” for the carcinogenic effect of dioxins is proposed. Further mechanistic studies should be conducted to get insight into the dose–response characteristics of relevant events of dioxin-like and non-dioxin-like agents and into the consequences of potential interactions between both group of compounds during carcinogenesis.

Analysis of work on smoke component yields from room-scale fire tests

Smoke Component Yields from Room-Scale Fire Tests (NIST Technical Note TN 1453) has recently been published. This was expected to be an important work in developing concentrations and yields of toxicants that could be used for evaluating the usefulness of small scale smoke toxicity apparatuses (or fire models) for use in the prediction of the toxicity of materials and products in real fires. However, the work has a number of uncertainties that limit its potential for use as a reference. There are three major problems with this work. First, the post-flashover concentrations of CO are too low (as recognized by the authors who recommend that this part of the data not be used). Second, the post-flashover concentrations of the main toxicants measured (HCN and HCl) were much higher than found in most studies. Third, the precision of the data was inadequate. The consequence of the first two issues is that the work seriously overestimates the toxicological importance of gases known to have only minor effects in post-flashover fires, such as HCN and HCl. The very low concentrations of toxicants measured at pre-flashover conditions might have a value not discussed by the authors: an indication that pre-flashover fires of the type conducted here do not generate extremely toxic atmospheres. Accordingly, the report does not provide reliable characteristic room scale combustion gas data that can be used for validating small-scale furnaces.

Acute and chronic toxicity of the pesticide methyl parathion to the rotifer Brachionus angularis (Rotifera) at different algal (Chlorella vulgaris) food densities

Methyl parathion is a commonly used insecticide in Mexico to eradicate insect pests. We evaluated the effects of this insecticide on rotifer B. angularis using both acute and chronic toxicity tests. Median lethal concentration (LC50) of methyl parathion for B. angularis for a 24-h bioassay in the presence and absence of an algal diet was derived. Elevated LC50 due to the survival of a greater number of test individuals in the presence of food was observed. Regardless of the toxicant concentration, population growth curves of the animals maintained at the low food level (0.75×106 cells ml-1) had a longer lag phase than those at the high food level (1.5×106 cells ml-1). Regardless of food level, an increase in the toxicant concentration in the medium resulted in decreased population growth. The lowest peak population density (50 ind. ml-1) was observed at the highest toxicant concentration and the lower food level. The highest population density (200 ind. ml-1) was observed in the controls at high food level. The rates of population increase per day (r) in the controls were higher (from 0.14 to 0.37 depending on the food level). Irrespective of food level, there was a decrease in the r values with increasing pesticide concentration in the medium. In order to detect the effect of population density on the growth rates in relation to the toxicant stress, we plotted the daily growth rate against initial density for the entire duration of the experiment. We observed the existence of a significantly inverse relation at all treatments except at the low food level and high toxicant concentrations (0.625 and 1.25 mg l-1). We discuss the role of algae in the toxicity of methyl parathion to zooplankton.

Soil Pollution Alters ATP and Chlorophyll Contents in Pisum sativum Seedlings

Pisum sativum L. cv. Phenomen plants were grown in pots in greenhouse and their growth, and ATP and chlorophyll (Chl) a and b contents were assessed after 9-d exposure to sodium arsenate [0.04 and 0.07 mmol kg−1(soil)], or to lead acetate [2.0 and 4.0 mmol kg−1(soil)], or zinc acetate [5.3 and 9.3 mmol kg−1(soil)]. The luciferin-luciferase method was used for ATP analyses. Soil pollution reduced significantly the growth, but the low toxicant concentrations elevated the cotyledon and shoot ATP concentrations per fresh matter content. The ATP/Chl ratio was increased in the zinc-treated seedlings as compared with the respective controls. The ATP concentration and a number of growth parameters were negatively correlated, and thus the high ATP content might contribute to the significantly reduced growth of seedlings.

Oxidant-induced hepatocyte injury from menadione is regulated by ERK and AP-1 signaling

Oxidative stress has been implicated as a mechanism for a variety of forms of liver injury. Although reactive oxygen species (ROS) may damage cellular macromolecules directly, oxidant-induced cell death may result from redox effects on signal transduction pathways. To understand the mechanisms of hepatocyte death from oxidative stress, the functions of the mitogen-activated protein kinases (MAPKs) were determined during oxidant-induced hepatocyte injury from menadione. Low, nontoxic, and high toxic concentrations of the superoxide generator menadione were established in the RALA255-10G rat hepatocyte cell line. Death from menadione was blocked by catalase and ebselen, indicating that death was secondary to oxidant generation and not arylation. Treatment with a nontoxic menadione concentration resulted in a brief activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In contrast, treatment with a toxic menadione concentration induced a prolonged activation of both ERK and JNK. Chemical inhibition of ERK function sensitized RALA hepatocytes to death from previously nontoxic menadione concentrations in association with sustained JNK activation. Adenoviral expression of a dominant-negative protein for c-Jun, a downstream substrate for JNK, blocked death from menadione. The pro-apoptotic effect of c-Jun was not mediated through the mitochondrial death pathway. In conclusion, RALA hepatocyte resistance to oxidant-induced death from menadione is dependent on ERK, whereas cell death is mediated by AP-1 activation. These findings identify signaling pathways that may be therapeutic targets in the prevention or treatment of oxidant-induced liver injury. (H epatology 2003;37:1405-1413.)

Gene and protein expression profiles of anti- and pro-apoptotic actions of dopamine, R-apomorphine, green tea polyphenol (-)-epigallocatechine-3-gallate, and melatonin.

Significant evidence has been provided to support the hypothesis that oxidant stress may be responsible for degeneration of dopaminergic neurons in the substantia nigra pars compacta in Parkinson's disease. Dopamine (DA), R-apomorphine (R-APO), green tea polyphenol (-)-epigallocatechine-3-gallate (EGCG), and melatonin are neuroprotective and radical scavenger compounds. The aim of this study was to establish the mechanism of the concentration-dependent neuroprotective and pro-apoptotic action of these drugs via gene expression and protein determination. cDNA microarrays provide new prospects to study and identify various mechanisms of drug action. We employed this technique for the study reported in this paper. Total RNA was extracted from SH-SY5Y cells exposed to low neuroprotective and high toxic concentrations of the drugs, followed by synthesis of cDNA, and hybridization to a microarray membrane related to apoptosis, survival, and cell cycle pathways. We demonstrated a concentration and time-dependent correlation between R-APO, DA, EGCG, and melatonin in modulation of cell survival/cell death-related gene pathways. The results were confirmed by quantitative real-time PCR and protein profiles. Unlike the effects of low concentrations (1-10 micro M), where an antiapoptotic response was manifest, a proapoptotic pattern of gene expression was observed at high toxic concentrations (50-500 micro M) of the antioxidants (e.g., increase in caspases, fas, and gadd45). Our results have provided novel insights into the gene mechanisms involved in both the neuroprotective and proapoptotic activities of neuroprotective drugs. We have shown that DA, R-APO, EGCG, and melatonin exhibit similar gene expression and protein profiles.