Toxicity of industrially relevant chlorinated organic solvents in vitro.

The cytotoxic effects of 4 industrially important chlorinated organic solvents, dichloromethane (DCM), 1,2-dichloroethane (DCE), trichloroethylene (TCE), and tetrachloroethylene (PERC) in vitro, were investigated. Jurkat T cells were exposed to the solvents individually for 72 hours and changes in reactive oxygen species (ROS) formation, cell proliferation, intracellular free calcium concentration ([Ca(2+)]), and caspase-3 activity were measured. There was a concentration-dependent increase in the ROS formation and intracellular free [Ca(2+)] following exposure to each of the solvents. This was accompanied by a decrease in the cell proliferation. Solvent potency decreased in the following order: PERC > TCE > DCM > DCE. Caspase-3 activity was increased in a concentration-dependent manner by TCE and PERC but was not significantly altered by DCM or DCE. n-Acetyl-l-cysteine pretreatment showed that changes in the intracellular free [Ca(2+)] and caspase-3 activity were independent of ROS formation. However, increased ROS formation did play a causal role in the decreased cell proliferation observed.

n-Hexane toxicity in Jurkat T-cells is mediated by reactive oxygen species.

Here we assess the role of reactive oxygen species (ROS) formation in the manifestation of n-hexane toxicity in Jurkat T-cells and the chemo-protective potential of the antioxidants epigallocatechin-3-gallate (EGCG) and thymoquinone (TQ) against n-hexane toxicity in vitro. n-Hexane is an important industrial solvent and ambient air pollutant. Subchronic exposure to n-hexane results in a concentration-dependent increase in ROS formation with a corresponding decrease in Jurkat T-cell proliferation. Results from time-course studies indicate that ROS formation plays a causal role in n-hexane induced alterations in Jurkat T-cell proliferation and membrane integrity. Treatment of cells with EGCG, at a concentration reached in plasma, reduced the ROS formation caused by exposure to n-hexane and inhibited the decrease in cell proliferation. Similar effects were obtained with TQ. Both EGCG and TQ significantly reduced n-hexane-induced LDH leakage to control levels. The combined results show that oxidative stress plays a role in the development of n-hexane toxicity.

In vitro exposure of jurkat T-cells to industrially important organic solvents in binary combination: interaction analysis.

Humans are frequently exposed to mixtures of environmental pollutants at low levels over prolonged periods of time yet most toxicity studies deal with acute exposure to high concentrations of single chemicals. Investigation of the biological effects and possible toxic interactions during long-term exposure to such mixtures is warranted. Here Jurkat T-cells were exposed to toluene, n-hexane and methyl ethyl ketone in binary combination. Concentration ranges were centered on thresholds at which the individual agents caused cell toxicity under otherwise similar conditions, and concentrations were confirmed by headspace gas chromatography. After 5 days cells were harvested and toxicity measured in terms of membrane damage (lactate dehydrogenase [LDH] leakage), perturbations in [Ca(2+)](i) and changes in glutathione redox status. Data for all three endpoints were subjected to isobolographic analysis to test for interaction between components of the solvent mixture. Almost all combinations of toluene and n-hexane elicited greater than additive toxicity in terms of each of the three endpoints, as did methyl ethyl ketone (MEK)/n-hexane and MEK/toluene combinations for the LDH and glutathione endpoints. The main exceptions were the two combinations involving MEK, which caused less than additive effects on perturbations of [Ca(2+)](i). It is concluded that toxicity in immune-derived T cells may exhibit greater than additive effects when there is coexposure to organic solvents. This may have implications for risk assessment of environmental exposure to these agents.

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).

Validation of a method for acute and subchronic exposure of cells in vitro to volatile organic solvents.

In vitro assessment of organic solvents can be problematic as the volatile nature of these compounds makes maintaining a constant exposure level difficult. However, a stable exposure level must be maintained if reliable dose response data are to be obtained. Here we describe a gas-tight glass exposure system which allows prolonged exposure of cultured cells to constant concentrations of volatile organic solvents. The system permits convenient sampling of gas and liquid phases for reliable quantification of solvent concentration. We determined medium/air partition coefficients (K) for toluene, n-hexane and methyl ethyl ketone which can be used to calculate liquid phase solvent exposure levels in an in vitro system specifically designed for organic solvent exposure. Cultured cells were exposed to these compounds for five days and toxicity assessed by trypan blue exclusion. Headspace gas chromatography was used to determine K in RPMI-1640 and EMEM tissue culture medium at 37 degrees C. The presence of cells in the system at levels normally used in in vitro exposure systems did not significantly alter solvent partitioning. Equilibrium liquid phase solvent concentrations were measured by gas chromatography for two of the compounds to confirm that exposure levels calculated using K were correct. Results show that sub-chronic exposure to volatile organic solvents causes a dose dependent decrease in Jurkat T-cells and SH-SY5Y viability. Solvent potency increased with lipophilicity (n-hexane>toluene>MEK).

3,4-Methylenedioxymethamphetamine (ecstasy) activates skeletal muscle nicotinic acetylcholine receptors.

Adverse 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) effects are usually ascribed to neurotransmitter release in the central nervous system. Since clinical features such as fasciculations, muscle cramps, rapidly progressing hyperthermia, hyperkalemia, and rhabdomyolysis point to the skeletal muscle as additional target, we studied the effects of MDMA on native and cultured skeletal muscle. We addressed the question whether malignant hyperthermia (MH)-susceptible (MHS) muscle is predisposed to adverse MDMA reactions. Force measurements on muscle strips showed that 100 microM MDMA, a concentration close to that determined in some MDMA users, regularly enhanced the sensitivity of skeletal muscle to caffeine-induced contractures but did not cause contractures on its own. The left-shift of the dose-response curve induced by MDMA was greater in normal than in MHS muscle. Furthermore, MDMA did not release Ca(2+) from isolated sarcoplasmic reticulum vesicles. These findings do not support the view of an MH-triggering effect on muscle. However, MDMA induced Ca(2+) transients in myotubes and increased their acidification rate. Surprisingly, alpha-bungarotoxin, a specific antagonist of the nicotinic acetylcholine receptor (nAChR), abolished these MDMA effects. The nAChR agonistic action of MDMA was confirmed by patch-clamp measurements of ion currents on human embryonic kidney cells expressing nAChR. We conclude that the neuromuscular junction is a target of MDMA and that an activation of nAChR contributes to the muscle-related symptoms of MDMA users. The drug may be of particular risk in individuals with abundant extrajunctional nAChR such as in generalized denervation or muscle regeneration processes and may act on central nAChR.

Increased sensitivity of the ryanodine receptor to halothane-induced oligomerization in malignant hyperthermia-susceptible human skeletal muscle.

Mutations in the skeletal muscle RyR1 isoform of the ryanodine receptor (RyR) Ca2+-release channel confer susceptibility to malignant hyperthermia, which may be triggered by inhalational anesthetics such as halothane. Using immunoblotting, we show here that the ryanodine receptor, calmodulin, junctin, calsequestrin, sarcalumenin, calreticulin, annexin-VI, sarco(endo)plasmic reticulum Ca2+-ATPase, and the dihydropyridine receptor exhibit no major changes in their expression level between normal human skeletal muscle and biopsies from individuals susceptible to malignant hyperthermia. In contrast, protein gel-shift studies with halothane-treated sarcoplasmic reticulum vesicles from normal and susceptible specimens showed a clear difference. Although the alpha2-dihydropyridine receptor and calsequestrin were not affected, clustering of the Ca2+-ATPase was induced at comparable halothane concentrations. In the concentration range of 0.014-0.35 mM halothane, anesthetic-induced oligomerization of the RyR1 complex was observed at a lower threshold concentration in the sarcoplasmic reticulum from patients with malignant hyperthermia. Thus the previously described decreased Ca2+-loading ability of the sarcoplasmic reticulum from susceptible muscle fibers is probably not due to a modified expression of Ca2+-handling elements, but more likely a feature of altered quaternary receptor structure or modified functional dynamics within the Ca2+-regulatory apparatus. Possibly increased RyR1 complex formation, in conjunction with decreased Ca2+ uptake, is of central importance to the development of a metabolic crisis in malignant hyperthermia.