ToxSci Advance Access originally published online on November 2, 2007
Toxicological Sciences 2008 101(2):263-274; doi:10.1093/toxsci/kfm274
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In Vitro Exposure of Jurkat T-Cells to Industrially Important Organic Solvents in Binary Combination: Interaction Analysis
,1
* Environmental Research Institute
Department of Biochemistry
Department of Pharmacology and Therapeutics
Department of Biochemistry, University College Cork, Lee Maltings, Cork 1, Ireland
1 To whom correspondence should be addressed at Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Cork, Ireland. Fax: +35-32-14-90-42-15. E-mail: j.heffron{at}ucc.ie.
Received August 15, 2007; accepted October 30, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTALRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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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 [Ca2+]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 [Ca2+]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.
Key Words: mixture toxicity; in vitro; organic solvent; isobolographic analysis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Most environmental chemical hazards involve exposure of humans to a number of chemicals in combination or sequentially at low concentrations (Boekelheide, 2007
). Despite this reality, toxicity data on exposure to chemical mixtures are sparse. It has been estimated that most of the resources in toxicology are devoted to studies of single chemicals. However, interest in the area of mixture toxicity has increased in recent years (Feron and Groten, 2002; Yang and Dennison, 2007).
Exposure to chemical mixtures can arise through inhalation of outdoor and indoor air, ingestion of food and beverages, and through contact with numerous consumer products (ATSDR, 2004; USEPA, 1986, 2000). Thus, mixtures may comprise related compounds but often include compounds that are unrelated chemically or commercially. In some instances mixtures are highly complex, consisting of so many compounds that the composition of the mixture is not fully characterized and may be variable (e.g., cigarette smoke, pesticide formulations, and diesel exhaust). Occasionally, the chemical composition of the mixture is well characterized, levels of exposure are known, and toxicological data on the mixture are available (ATSDR, 2004). The main objective of mixture toxicity studies is to assess the risk to human health associated with exposure to chemicals in combination or sequentially. Each component of a mixture has its own toxic potential and may influence the toxicity of the other components of the mixture (Simmons et al., 1994). When assessing mixture toxicity it is important to understand the basic principles of joint action and interaction of chemicals. The three basic types of action are simple similar action, simple dissimilar action, and interaction (Feron and Groten, 2002). Simple similar action, also known as dose addition, is a noninteractive process where each of the chemicals in the mixture acts in the same way, by the same mechanisms, and differ only by their potencies. Simple dissimilar action or response addition is also noninteractive and the chemicals in the mixture do not influence the toxicity of each other, but the modes of action among the chemicals in the mixture differ. Toxic interactions refer to significant deviations of the combined effect of two chemicals from what would be expected under the assumption of additivity (e.g., dose addition, response addition). Synergism occurs when the combined effect is greater than additive, whereas antagonism occurs when the combined effect is less than additive.
The possibility of toxicity due to exposure to two or more chemicals at their no-observed-adverse-effect-level (NOAELs) is of significant public health concern (Van Zorge, 1996). However, it is commonly believed that chemicals are innocuous at low levels and that toxic interaction is highly unlikely at low concentrations such as those found in the environment (Groten et al., 2004). Nonetheless, some studies have suggested that exposure to chemical mixtures at their individual subthreshold level can result in adverse effects (Konemann and Pieters, 1996; Stacey, 1989), for example, acute exposure to trichloroethylene, 1,1,1-trichloroethane and tetrachloroethylene at subthreshold doses produced adverse effects in rat liver. Exposure to heavy metals at their subthreshold levels produced significant decreases in hemoglobin and hematocrit in rats when given in combination (Mahaffey and Fowler, 1977).
Organic solvents are among the most widely used chemicals in the pharmaceutical, chemical, and food industries, and form an important class of pollutants in the ambient air. Therefore, exposure to solvent mixtures by inhalation is extremely likely given their prevalence. A study assessing the toxic effects of organic solvents in vitro showed that exposure to a mixture of solvents below their individual NOAELs caused cellular toxicity; however, no interaction analysis was carried out (Croute et al., 2002). It would be important therefore in further studies to take into account possible toxic interaction using established methods such as isobolographic analysis.
Our objective in this study was to evaluate the biological effects in vitro of exposure to mixtures of industrially significant organic solvents at low concentrations by investigating the possibility of toxic interaction between organic solvents in binary combination, thereby refining our understanding of the risk associated with environmental exposure to these compounds. Three solvents were studied: toluene and n-hexane as representative hydrophobic solvents (log p > 2) and methyl ethyl ketone (MEK) (log p < 1) as a relatively hydrophilic compound. The range of concentrations of the solvents used was centered on lowest-observed-adverse-effect-levels (LOAELs) previously determined (McDermott et al., 2007a) for the individual compounds tested here. Cells were exposed for 5 days in order to approximate subchronic exposure to the solvents.
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EXPERIMENTAL |
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Materials.
Aluminum crimp caps, ethanol, fetal bovine serum (FBS), metaphosphoric acid, N-ethylmaleimide, glass headspace vials (20 ml), o-pthalaldehyde, RPMI-1640, Teflon-faced butyl rubber septa, TritonX-100, and trypan blue dye were supplied by Sigma Aldrich Ireland, Ltd., Cytotoxicity Detection Kit (LDH [lactate dehydrogenase]) was purchased from Roche Diagnostics Corp., Indianapolis, IN. Analytical grade n-hexane, toluene, and MEK were supplied by BHD Chemicals Ltd., Poole, UK.
Preparation of solvent stock solutions.
Stock solutions were made up on a gravimetric basis in glass chromatography vials (2.0 ml). The vial was filled with ethanol and the desired volume of solvent. Both additions were determined gravimetrically. The vials were immediately sealed with a Teflon-faced butyl rubber septum and aluminum cap. The tightness of the seal was checked manually. Stocks were stored at room temperature (18°C–20°C) for a maximum of 1 month.
Cell culture and solvent exposure system.
Jurkat E6.1 were cultured in RPMI-1640 and, supplemented with 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin, and 25mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (Hepes). Cells were maintained at 37°C and passaged every 2–3 days. Cells were exposed to organic solvents according to the method previously described by McDermott et al. Glass headspace vials (20 ml) were used for Jurkat T-cell exposures (0.1 x 106 cells/ml).
Specific solvent concentrations were chosen based on the original dose–response data for the individual solvents and were centered on the LOAELs (McDermott et al., 2007a). Three doses were chosen for each solvent, giving nine possible binary combinations. Five microliters of each solvent stock solution (in ethanol) was added to the cell suspension using a Hamilton syringe and the vial was rapidly sealed with a Teflon-faced butyl rubber septum and an aluminum crimp cap. An untreated control, an ethanol control (10 µl ethanol added to exposure vessel; final concentration 0.2%), and individual solvent exposures were included in each combination experiment. All vials were maintained for 60 h at 37°C at which time the cells were passaged (1:2) and re-exposed to the solvent mixture. The vials were then maintained for a further 60 h at 37°C (5-day exposure).
To assess the effect of ethanol on the combination response, the 10.2µM toluene/4.08µM n-hexane combination was tested with 10, 15, and 20 µl (final concentration 0.2, 0.3, and 0.4%, respectively) of ethanol present and changes in LDH leakage were examined. Ten, 15, and 20 µl of ethanol alone were also tested.
Quantification of solvent exposure level.
Headspace GC was employed to determine the equilibrium gas (CG) phase solvent concentration in the exposure vessels (McDermott et al., 2007b; McIntosh and Heffron, 2000). A series of headspace standards was prepared by the total vaporization technique (Kolb and Ettre, 1997). Sample vials were set up as described above. To determine the gas phase solvent concentration in the exposure, vessels were incubated at 37°C and an aliquot of the gas phase was taken manually using a 1-ml gastight Luer-lock glass syringe. This was then manually injected into the gas chromatograph equipped with a flame ionization detector. A HP-1 capillary column (30 m x 0.53 mm internal diameter [I.D.], 0.88-µm film thickness) was used with nitrogen carrier gas to measure toluene and n-hexane gas phase concentrations. The injector and detector temperatures were 170°C and 250°C, respectively. The oven temperature was set to 40°C for 1 min followed by a ramp to 80°C at 10°C/min. Total run time was 5 min. A ZB-WAX (30 m x 0.32 mm I.D., 0.50-µm film thickness) capillary column was used to measure MEK gas phase concentrations. The gas chromatographic conditions were the same as for toluene and n-hexane measurements. Medium/air partition coefficients (2.05, 0.59, and 198 for toluene, n-hexane, and MEK respectively determined using RPMI-1640 culture medium) (McDermott et al., 2007b) were used to calculate equilibrium liquid phase solvent concentrations.
LDH leakage assay.
LDH leakage from cells to the culture medium was determined using the Roche cytotoxicity detection kit. Control experiments showed that solvent exposure did not affect total LDH activity, so LDH leakage was expressed as a percentage of LDH release from cells lysed with TritonX-100. At the end of exposures, cultures were diluted to a uniform cell density, then cell-free supernatant was collected. One hundred microliters of supernatant was incubated with 100 µl of reaction mixture for 30 min at room temperature, protected from light, after which absorbance was measured at 490 nm using a Tecan microtiter plate reader.
Measurement of intracellular free calcium ion concentration ([Ca2+]i).
Following exposure, cells were washed twice in phosphate-buffered saline (PBS) and resuspended in physiological buffer (pH 7.4) containing (mM), NaCl 115, KCl 5, NaHCO3 10, Hepes 25, MgCl2 0.5, CaCl2 1, and glucose 5.6. Thirty micromolars of Fura-2/AM (from a 1mM stock in dimethyl sulfoxide) was added to the cell suspension and then incubated for 30 min at 37°C protected from light to prevent photobleaching. Cells were then centrifuged, washed, and resuspended in PBS. After a further 10-min incubation at 37°C in the absence of Fura-2/AM to allow complete de-esterification of the dye, cells were again centrifuged, washed, and resuspended to 1 x 106 cells/ml in physiological buffer for measurement of dye fluorescence.
Fluorescence intensity of fura-2 loaded cells was measured using an Aminco Bowman Series 2 Luminescence Spectrometer with a xenon arc lamp, excitation at 340 and 380 nm, and emission at 510 nm. To correct for dye leakage, 50µM MnCl2 was added to the cuvette immediately before measurement of fluorescence ratio (Prentki et al., 1987). [Ca2+]i and their changes were expressed as fluorescence emission ratio (340/380 nm).
Measurement of reduced and oxidized glutathione.
Intracellular reduced and oxidized glutathione (GSH and GSSG) were measured in exposed cells using the fluorescent dye o-phthalaldehyde (OPA) and a modification of the method of Hissin and Hilf (1976). After fluorescence excitation at 350 nm, emission was measured at 420 nm. GSH and GSSG standard solutions in either 0.1M sodium phosphate—5mM ethylenediaminetetraacetic acid buffer (pH 8.0) or 0.1M NaOH, respectively, was mixed with 100 µl of OPA (1 mg/ml) and diluted to a final volume of 2 ml. Total GSH and GSSG in samples was expressed as nmol/mg cell protein. Protein concentration was measured using Biorad protein assay with bovine serum albumin standards.
Isobolographic analysis.
Isobolographic analysis provides a two-dimensional representation of the interaction between two compounds in a binary mixture (Gessner, 1995). Specific solvent concentrations were chosen based on the original dose–response data for the individual solvents and were centered on LOAELs. Three doses were chosen for each solvent, giving nine possible binary combinations. Jurkat T-cells were exposed to solvents in binary combination and effects measured in terms of LDH leakage, perturbations in intracellular free [Ca2+], and changes in glutathione redox status. Isobolographic analysis was used to test binary mixture response data for interaction.
An isobol was constructed for each mean response magnitude obtained and where the same response magnitudes were found for different combinations (as in Fig. 1) one isobol was constructed. Using the dose–response curves for the individual solvents (McDermott et al., 2007a) the concentration of each solvent individually, which elicited a response equal in magnitude to that of the binary combination, was established. These individual solvent concentrations were plotted and the line, which connects them, is called the isobol; this is illustrated by the dark line in Figure 1. The region bounded by the broken lines is known as the region of additivity. The broken lines on either side of the isobol represent the 95% confidence intervals of the isobol and were obtained from the dose–response relationships of the individual solvents. The 95% confidence limits of the isobol were calculated from the 95% confidence limits of the individual solvent concentrations that elicit a given response magnitude individually. The data point for the binary combination with its 95% confidence intervals was then plotted on the isobologram to ascertain if a given response magnitude is indicative of additive action or an interaction; this is illustrated in Figure 1. This method of constructing isobols is similar to that described by Gessner (1995) in Fig. 11 in which the interaction of midazolam and dexmetomidine is illustrated.
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Analysis of interaction was based on the position of the data point and its 95% confidence intervals relative to the region of additivity. If the combination data point and its 95% confidence intervals lie within the region of additivity the response is deemed to be additive. It the data point and its confidence intervals lie below the region of additivity, closer to the origin and do not overlap with the 95% confidence band for the line of addition this provides evidence of a greater than additive response. On the other hand, if the combination data point lies above the region of additivity and again its 95% confidence intervals do not overlap the region of additivity the response is less than additive.
Berenbaum's combination index.
Berenbaum's combination index was used as a measure of interaction (Berenbaum, 1989). For a binary solvent mixture the combination index is defined as
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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To investigate the effects of binary solvent exposure in vitro, Jurkat T-cells were exposed to solvents in binary combination for 5 days and the effects on LDH leakage, [Ca2+]i, the levels of GSH and GSSG, and GSH/GSSG ratio were measured. Specific solvent concentrations were chosen based on the original concentration–response data for the individual solvents. Three concentrations were chosen for each solvent, giving nine possible binary mixtures. These solvent concentrations were centered on concentration thresholds at which the individual agents caused cell toxicity under otherwise similar conditions. For each binary exposure experiment eight control vials were included—untreated cells, ethanol exposed cells, and cells exposed to the three concentrations of both solvents individually. The resulting data were subjected to both isobolographic and Berenbaum's combination index analysis to test for interaction.
Alterations in Solvent-Induced LDH Leakage due to Coexposure to a Second Solvent
Subchronic exposure to binary combinations of toluene, n-hexane, and MEK produced an increase in LDH leakage from Jurkat T-cells (Table 1). Isobolographic analysis and Ic values were used to evaluate interaction in binary solvent mixtures (Table 1). The LDH leakage response to almost all combinations of toluene/n-hexane, toluene/MEK, and n-hexane/MEK was greater than additive (Table 1 and Fig. 1). The only exceptions were two combinations of toluene/n-hexane, where an additive response was noted in each case. One of the toluene/MEK combinations that exhibited greater than additive interaction used solvent concentrations that were below their individual LOAELs.
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To examine the role of ethanol in the combination response, Jurkat T-cells were exposed to the 10.2µM toluene/4.08µM n-hexane combination in the presence of 0.2, 0.3 and 0.4% ethanol. There was no significant difference in LDH leakage from Jurkat T-cells exposed to toluene/n-hexane combinations in the presence of 0.2, 0.3 or 0.4% ethanol, LDH leakage was 35.6 ± 3.97%, 31.1 ± 4.68%, and 35.39 ± 7.15%, respectively (p > 0.05, n = 4). 0.2, 0.3, or 0.4% ethanol alone did not significantly alter LDH leakage (data not shown).
Alterations in Solvent-Induced Perturbations in [Ca2+]i due to Coexposure to a Second Solvent
Jurkat T-cells exposed to combinations of toluene/n-hexane, toluene/MEK, and n-hexane/MEK for 5 days showed concentration-dependent increase in [Ca2+]i with respect to the control values. These response magnitudes were analyzed to assess toxic interaction (Table 2). Apart from one additive response, all combinations of toluene/n-hexane investigated elicited greater than additive toxicity. Combinations involving MEK caused less than additive effects on changes in [Ca2+]i. The effect of n-hexane/MEK was uniformly less than additive for all except one combination, whereas toluene/MEK combinations exhibited less than additive effects at higher MEK concentrations. It is possible that this disparity reflects a specific rather than generalized toxicity of MEK on calcium homeostasis.
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Alterations in Solvent-Induced GSH Depletion due to Coexposure to a Second Solvent
Jurkat T-cells exposed to toluene/n-hexane, toluene/MEK, and n-hexane/MEK combinations for 5 days showed a decrease in the level of GSH with respect to untreated control values (Table 3). The effect of exposure to these solvents in binary combination was uniformly greater than additive for all combinations investigated. This was true for combinations where both solvent concentrations were at or below their individual NOAELs.
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Alterations in Solvent-Induced GSSG Increase due to Coexposure to a Second Solvent
All combinations of toluene, n-hexane, and MEK investigated produced a significant increase in GSSG in Jurkat T-cells following 5 days exposure. Greater than additive interaction was noted for all but two combinations, where possible additive responses were found (Table 4). Several combinations investigated used solvent concentrations which were at or below their individual NOAELs; greater than additive toxicity was noted in each case.
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Alterations in Solvent-Induced GSH/GSSG Ratio Decrease due to Coexposure to a Second Solvent
Subchronic exposure to toluene, n-hexane, and MEK in binary combination resulted in a decrease in GSH/GSSG ratio in Jurkat T-cells. Isobolographic analysis of the resulting GSH/GSSG ratio data indicated that exposure to these solvents in combination resulted in greater than additive interaction (Table 5).
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Isobolographic analysis of response magnitudes from binary solvent exposure studies showed that almost all combinations of toluene and n-hexane produced greater than additive toxicity in terms of LDH leakage, perturbation in [Ca2+]I, and changes in the level of GSH and GSSG. Exposure to combinations of n-hexane/MEK and toluene/MEK also resulted in greater than additive effects for LDH and glutathione endpoints. The only exceptions were the combinations involving MEK, which caused less than additive effects on perturbations in [Ca2+]i. These interactions were confirmed using Berenbaum's combination index (Ic), there was excellent agreement between the two methods. The possibility of toxicity due to exposure to chemical mixtures at subthreshold levels is of significant health concern. A number of the combinations studied here used solvent concentrations that were below their individual LOAELs. Greater than additive interaction was noted in each case.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Here we found that almost all combinations of toluene and n-hexane investigated elicited greater than additive toxicity in terms of the three independent endpoints used. Greater than additive toxicity was also seen for toluene/MEK and n-hexane/MEK combinations for the LDH and glutathione endpoints. The principal exceptions were for combinations involving MEK, which caused less than additive effects on perturbations in [Ca2+]i. Our results indicate that toxicity in immune-derived T-cells exhibit greater than additive toxicity when there is exposure to more than one solvent at a time, even at levels below their individual LOAELs. The overall patterns of interaction are summarized in Figure 2. It was also shown that the vehicle ethanol, at the concentration used, did not significantly alter the effect of the solvent combination in terms of LDH leakage suggesting that ethanol has no detectable effect on the interaction between the solvents.
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There are obvious limitations when approaching mixture toxicity studies such as the number of combinations that could be investigated. Three concentrations were chosen for each solvent, centered on their individual LOAELs (McDermott et al., 2007a
), which gives nine binary combinations. The individual LOAELs were comparable with blood solvent concentrations reported for individuals exposed to the agents at levels at or below their individual threshold limit values (McDermott et al., 2007a). When studying chemical mixtures it is of primary importance to be able to identify deviations from additivity. Isobolographic analysis, determination of combination index, and response-surface analysis are the methods most frequently used to detect interaction (Carter, 1995; Groten et al., 2004; Tallarida, 2002). We showed that exposure to combinations of toluene/n-hexane, toluene/MEK, and n-hexane/MEK resulted in an increase in LDH leakage from Jurkat T-cells. Only two additive responses were observed for toluene/n-hexane combinations, at the low toluene concentrations. Greater than additive toxicity was noted for almost all solvent combinations investigated including a toluene/MEK combination where both solvent concentrations were below their individual LOAELs.
Based on [Ca2+]i data, the effect of exposure to toluene/n-hexane combinations was mostly greater than additive; only one combination showed an additive response. Toluene/MEK combinations showed less than additive effects on [Ca2+]i at higher MEK concentrations (132 and 319µM). Lower MEK concentrations in combination with toluene produced additive and greater than additive responses. This pattern of toxic response resembles the results obtained in in vitro exposure to binary mixtures of polychlorinated biphenyls and methyl mercury (Bemis and Seegal, 2000). In that study, exposure to low doses of the mixture produced greater than additive increases in [Ca2+]i in rat cerebellar granule cells. However, when [Ca2+]i was measured following exposure to higher doses of this mixture a less than additive response was noted. Hence, both may be examples of a general phenomenon where beyond a certain threshold concentration toxic interaction changes from greater than additive to less than additive. Such responses were described by Berenbaum; he classified such contours of constant response as not consistent (Berenbaum, 1989).
The effect of n-hexane/MEK combinations was uniformly less than additive for all concentrations investigated except one suggesting that the less than additive response noted for MEK/toluene and MEK/n-hexane combinations reflects a specific rather than generalized toxicity of MEK on calcium homeostasis in immune derived cells. There is growing evidence to suggest that anesthetics chemically resembling the organic solvents used here involve direct interaction between the anesthetic and membrane proteins (Franks, 2006). Toluene and n-hexane are very hydrophobic solvents with log p values of 2.7 and 3.9, respectively, compared with 0.29 for MEK. Further, MEK has a high polar surface charge compared with that for toluene or n-hexane and the carbonyl group of MEK is strongly polarized (calculated using Chemdraw 9.0 software, Cambridge Software Corp., Cambridge, MA). It is therefore probable that MEK may be interacting at a different site on the membrane proteins involved in Ca2+ transport resulting in the observed less than additive effects.
In terms of changes in GSH and GSSG, the effect of exposure to combinations of the solvents was consistently greater than additive. The only exception was that of n-hexane/MEK where an additive response was noted. Greater than additive toxicity was noted in terms of both GSH and GSSG for combinations where both solvent doses were below their individual LOAEL. GSH/GSSG ratio response magnitudes also proved to be greater than additive.
McDermott et al. (2007a) found significant correlation between the increase in membrane permeability, elevation of intracellular free [Ca2+], and decrease in the GSH/GSSG ratio. Thus, the toxic outcomes are dependent on one another, although it is likely that the primary toxic outcome is increased membrane permeability in view of the hydrophobicity of the respective solvents.
Greater than additive responses are known to be involved in virtually all levels of cell function. However, the molecular mechanisms that underlie many greater than additive responses remain unresolved (Barrera et al., 2005). Greater than additive response can be distinguished based on the nature of the interaction: pharmacodynamic or pharmacokinetic. Pharmacodynamic responses result from two compounds directed at a similar target or system. Specific interaction with receptors, channels, or transcription factors may account for greater than additive responses and cross-talk between signal transduction pathways can also produce responses that are significantly greater than additive responses. Greater than additive effects might reflect interactions with specific proteins. For example, a conformational change caused by binding of one solvent may make the protein more responsive to interaction with the second solvent. The greater than additive effects noted may also reflect pharmacokinetic interaction whereby the processes of compound distribution or biotransformation may be altered. Coexposure may result in increased bioavailability due to competition for albumin binding or cause alterations in metabolic pathways resulting in increased concentration, thus producing greater than additive effects.
Occupational exposure to organic solvents has been shown to result in immune alterations. A significant decrease in serum immunoglobulin levels was observed in 35 male workers occupationally exposed to n-hexane (Karakaya et al., 1996). The proliferative response of peripheral lymphocytes in humans was stimulated after chronic exposure to toluene, n-hexane, and MEK (Karakaya et al., 1999). Also animals exposed to toluene and n-hexane in combination for 37 days showed impaired resistance to Mycobacterium bovis induced infection (Palermo-Neto et al., 2001). Oxidative stress is thought to underlie the altered pathogenic behavior of lymphocytes in some human diseases. Transient increase in reactive oxygen species plays a vital role in optimizing transcriptional and proliferative responses in T-lymphocytes, whereas chronic oxidative stress leads to decreased lymphocyte proliferation and increased transcription of inflammatory gene products (Remans et al., 2004). Increases in [Ca2+]i are critical for T-cell activation and interleukin-2 production (Choudhry et al., 1999) and so altered calcium homeostasis may result in altered immune response. Here we found that 5-day exposure to organic solvents in binary combination resulted in significant oxidative stress and increases in [Ca2+]i even at levels below their individual LOAELs. Such biochemical effects may contribute to immune alterations in vivo.
Although it is highly unlikely that occupational exposure limits will be based on in vitro data, in vitro toxicity testing may play a significant role in assessing the risks of chemical mixture exposure for humans by increasing the possibilities of extrapolating data derived from animal studies through providing invaluable mechanistic information (Eisenbrand et al., 2002). Making comparison of the dose applied in vitro with that at the target organ in vivo is difficult because in vitro test systems lack normal absorptive, distributive, and excretory pathways. To overcome these problems, physiologically based pharmacokinetic modeling has been used to convert in vitro toxic concentrations into equivalent in vivo exposures, which can then be compared with actual exposure levels (DeJongh et al., 1998, 1999). For comparison of in vitro and in vivo toxicity data differences in the availability of the chemicals in the biological systems must also be taken into account (Gülden and Seibert, 2003). These authors presented an extrapolation model for estimating serum concentrations of chemicals equivalent to in vitro effective concentrations. Combes (2005) stated that the use of human cell lines as a biological model strengthens any extrapolations made to the human situation and eliminates some uncertainties associated with interspecies extrapolation.
Our results may be useful in providing further understanding of the relationship between "interaction thresholds" for chemical mixtures and "thresholds" of toxicity for individual chemicals as recently discussed by Yang and Dennison (2007). Insight is needed into the combined action of chemicals to help protect humans from the harmful effects of exposure to chemical mixtures (Feron et al., 2004; Konemann and Pieters, 1996). Our results show that toxic interaction is likely in low dose exposures to chemical mixtures and suggest that interaction should be considered when outlining occupational exposure limit values for these compounds. Relying solely on exposure limits for the individual components is clearly not suitable. Investigation of the combined effects of chemicals at environmentally relevant concentrations is crucial.
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SUPPLEMENTARY DATA |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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FUNDING |
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Higher Education Authority (HEA) of Ireland, Programme for Research in Third Level Institutions, Cycle 2 (HEA PRTLI), National Development Plan.
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