Arch Toxicol (2008) 82:165171 DOI 10.

1007/s00204-008-0286-x

M O L EC U L A R T O X I CO L O G Y

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

Catherine McDermott Maria Hutch ODonoghue James J. A. HeVron

Received: 5 December 2007 / Accepted: 15 January 2008 / Published online: 30 January 2008 Springer-Verlag 2008

Abstract 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 timecourse 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 eVects were obtained with TQ. Both EGCG and TQ signiWcantly 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. Keywords n-Hexane Reactive oxygen species Antioxidants Membrane permeability Abbreviations DCFH-DA 2 ,7 -DichlorodihydroXuorescein diacetate EGCG Epigallocatechin-3-gallate FBS Foetal bovine serum LDH Lactate dehydrogenase PBS Phosphate buVered saline ROS Reactive oxygen species TQ Thymoquinone
C. McDermott M. H. ODonoghue J. J. A. HeVron (&) Department of Biochemistry, Biochemical Toxicology Laboratory, University College Cork, Cork, Ireland e-mail: j.heVron@ucc.ie

Introduction Organic solvents, many of which are prominent environmental pollutants, have been shown to cause immunotoxicity in vivo and in vitro but the mechanisms by which they do so are not well understood (Hsieh et al. 1989; Wichmann et al. 2005; McDermott et al. 2007a). Development of oxidative stress has been suggested as a potential cause of cytotoxicity following exposure to environmental pollutants (Dreiem et al. 2002; Olgun and Misra 2006; McDermott et al. 2007a) and it may play a causal role in the development of the toxicity of n-hexane. Oxidative stress develops when there is an imbalance between pro-oxidant [reactive oxygen species (ROS)] and anti-oxidant mechanisms, and can result in lipid peroxidation, DNA damage and degradation of cellular proteins (Halliwell 1993). Antioxidants aim to compensate for increases in ROS formation by delaying or preventing oxidation of substrates (Halliwell et al. 1995). When ROS are generated in living systems a variety of endogenous antioxidants including ascorbic acid, -tocopherol, glutathione peroxidase, and superoxide dismutase come into play (Halliwell et al. 1995). The potential for naturally occurring compounds to protect against the development of disease has received considerable attention in recent years (El-Mahdy et al. 2005). Oxidative damage is frequently involved in the development of disease and toxicity and so these compounds may also protect humans against the toxic eVects of environmental pollutants. Many foods are high in antioxidant compounds such as polyphenols and carotenoid and humans consume appreciable amounts of antioxidants from dietary sources (Ma and Kinneer 2002). Diets high in antioxidantrich foods have been linked to reduced levels of disease (BlomhoV 2005). Polyphenols are believed to be the active constituents of green tea, epigallocatechin-3-gallate

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(EGCG) being the most abundant. Studies have shown that the major polyphenols of green tea possess antimutagenic, antibacterial, anti-inXammatory and anti-tumor properties (Wu and Wei 2002). Thymoquinone (TQ) is the major biologically active component in Nigella sativa seeds, which have been used for centuries all over the world for treatment of conditions including eczema, inXuenza, bronchitis, cough, inXammation, hypertension and fever (Ali and Blunden 2003). Both EGCG and TQ are known for their antioxidant properties (Houghton et al. 1995; Burits and Bucar 2000; Johnson and Loo 2000), which may be one of the major reasons for their beneWcial eVects in vivo. We assessed the role of ROS formation in the development of solvent toxicity in vitro using n-hexane as a model. n-Hexane is a widely used organic solvent in industry, a component of petrol and a common air pollutant. Jurkat T-cells were exposed to n-hexane for 48 h and toxicity assessed in terms of changes in cell proliferation, ROS formation and LDH leakage. Having detected an early increase in ROS formation in the T-cells we examined the potential of natural antioxidants EGCG and TQ to counteract this eVect of n-hexane.

cin and 25 mM Hepes. Cells were maintained at 37C and passaged every 23 days. Cell viability was assessed by trypan blue exclusion; cell viability was 95% for all experiments. Cells were exposed to organic solvents in a closed system according to the method previously described by McDermott et al. (2007b). BrieXy, 10 l solvent stock solution was added by Hamilton syringe to Jurkat T-cell suspensions (0.1 106 cells/ml) in glass headspace vials (20 ml), then the vial was rapidly sealed with a TeXonfaced butyl rubber septum and an aluminium crimp cap. Cells were maintained at 37C for 48 h. Control and methanol control (10 l methanol added to exposure vessel) cells were included in each experiment. The equilibrium liquid phase n-hexane concentrations to which the cells were exposed were previously determined (McDermott et al. 2007b) and did not change over the exposure period. ROS Xuorimetry Following 48 h n-hexane exposure total formation of ROS by the cells was estimated using DCFH-DA. Jurkat T-cells were suspended in phosphate-buVered saline (PBS, pH 7.4) loaded with DCFH-DA (10 M) for 40 min at 37C, washed twice in PBS, and resuspended in RPMI-1640 culture medium. Fluorescence due to oxidized dye (excitation at 485 nm, emission at 535 nm) was measured using a Tecan InWnite M200 plate reader and Tecan i-Control software. DCF Xuorescence was normalized to controls using corresponding resazurin reduction data. Resazurin proliferation assay Reduction of the redox dye resazurin to resoruWn was used to measure the proliferation of cell cultures (OBrien et al. 2000). After solvent exposure resazurin (Wnal concentration 44 M) was added to cell suspensions, and resoruWn formation was measured Xuorometrically at 560 and 590 nm using a Tecan InWnite M200 plate reader and Tecan i-Control software. LDH leakage assay LDH leakage from cells to the culture medium was determined using a Cytotoxicity Detection Kit. LDH leakage was expressed as a percentage of total LDH release from cells lysed with TritonX-100. At the end of exposure cultures were diluted with culture medium, then cell-free supernatant was collected. A measure of 100 l supernatant was incubated with 100 l reaction mixture for 30 min at room temperature, protected from light, after which absorbance was measured at 490 nm using a Tecan InWnite M200 plate reader and Tecan i-Control software.

Materials and methods Chemicals and reagents 2 ,7 -DichlorodihydroXuorescein diacetate (DCFH-DA) was purchased from Molecular Probes, Leiden, The Netherlands. Aluminium crimp caps, dimethyl sulfoxide (DMSO), epigallocatechin gallate (EGCG), foetal bovine serum (FBS), 20 ml glass headspace vials, l-glutamine, methanol, penicillin/streptomycin resazurin, RPMI-1640, teXon faced butyl rubber septa, thymoquinone (TQ) and trypan blue solution were supplied by Sigma Aldrich Ireland Ltd. Analytical grade n-hexane was supplied by BDH Chemicals Ltd., Poole, England. Preparation of n-hexane stock solutions Stock solutions were made up on a gravimetric basis using glass chromatography vials (2.0 ml). The vial was Wlled with methanol and the desired volume of n-hexane. Both additions were determined gravimetrically. The vials were immediately sealed with a TeXon-faced butyl rubber septum and aluminium cap. The tightness of the seal was checked manually. Stocks were stored at room temperature (1820C) for a maximum of 1 month. Cell culture and exposure protocol Jurkat E6.1 were cultured in RPMI-1640, supplemented with 10% FBS, 1% L-glutamine, 1% penicillin/streptomy-

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Statistical analysis Results are expressed as mean standard deviation (SD). Data were analyzed using one-way ANOVA with the TukeyKramer multiple comparisons test, using Graphpad Instat software (San Diego, CA). SigniWcance levels were deWned as P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).

linked to ROS formation (Dreiem et al. 2003) and so we examined ROS formation in Jurkat T-cells following nhexane exposure. There was a concentration-dependent increase in DCF Xuorescence in Jurkat T-cells following 48 h exposure to n-hexane (Fig. 1b) indicating an increase in ROS production. No signiWcant change in cell proliferation or ROS formation was noted in the methanol control cells (P > 0.05; data not shown). Time-course of the action of n-hexane The time-course of these eVects of n-hexane was determined by exposing Jurkat T-cells to 34 M n-hexane and changes in resazurin reduction, LDH leakage and DCF Xuorescence were measured at 6, 18, 24, 30 and 48 h (Fig. 2). There was a signiWcant increase in DCF Xuorescence after 18 h, which was followed by a signiWcant increase in LDH leakage at 24 h. Exposure to 34 M n-hexane produced a signiWcant decrease in resazurin reduction after 30 h. Thus the increase in ROS formation precedes the increase in LDH leakage and altered cell proliferation. EVect of EGCG and TQ on Jurkat T-cell proliferation Before examining the antioxidant and chemoprotective potential of EGCG and TQ on n-hexane toxicity we tested their eVects on Jurkat T-cell proliferation following 48 h treatment. Both compounds produced a concentration dependent decrease in Jurkat T-cell proliferation above a threshold level of 5 and 25 M for TQ and EGCG, respectively (Fig. 3). One concentration of TQ (1 M) and EGCG (100 nM) was then chosen from below the threshold levels (Fig. 3) and tested for their ability to protect Jurkat T-cells from the toxic eVects of n-hexane. Evaluation of antioxidant eVects of EGCG and TQ Jurkat T-cells were treated with 100 nM EGCG and DCF Xuorescence measured following n-hexane exposure to assess its antioxidant potential (Fig. 4a). When EGCG treated samples were compared to untreated controls, 88.8 M n-hexane was the only exposure level that still produced a statistically signiWcant increase in DCF Xuorescence (P < 0.01). However, this was still signiWcantly lower that the response seen in cells exposed to the same concentration of n-hexane in the absence of EGCG. A total of 100 nM EGCG reduced DCF Xuorescence to control values over the remainder of the n-hexane exposure range investigated. To determine if the ROS formation observed (Figs. 1b, 2b) plays a causal role in the decreased cell proliferation seen following n-hexane exposure (Fig. 1a) cells were treated with 100 nM EGCG and exposed to n-hexane for

Results Evaluation of cell proliferation and ROS formation in vitro following n-hexane exposure n-Hexane toxicity was Wrst assessed in terms of its ability to induce changes in Jurkat T-cell proliferation. This was measured by assessing the cells ability to reduce resazurin (non-Xuorescent) to resoruWn (highly Xuorescent). Fortyeight hours exposure to n-hexane resulted in a concentration-dependent decrease in Jurkat T-cell proliferation (Fig. 1a). Organic solvent toxicity has previously been

Fig. 1 EVect of 48 h n-hexane exposure on a cell proliferation as assayed by resazurin reduction and b ROS formation in Jurkat T-cells. Data are expressed as means SD of Wve determinations. Data were analyzed using one-way ANOVA with TukeyKramer multiple comparisons test. SigniWcance levels were deWned as P < 0.05 (*),P < 0.01 (**) and P < 0.001 (***)

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Fig. 3 EVect of 48 h EGCG or TQ treatment on Jurkat T-cell proliferation as assayed by resazurin reduction. Data are expressed as means SD of four determinations. Data were analyzed using one-way ANOVA with TukeyKramer multiple comparisons test. SigniWcance levels were deWned as P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***)

We then tested TQ for its antioxidant and chemoprotective potential and found that 1 M TQ treatment reduced DCF Xuorescence to the control value up to 34 M n-hexane exposure (Fig. 5a). This antioxidant action protected Jurkat T-cells from changes in cell proliferation from 9.16 to 18.2 M n-hexane exposure (Fig. 5b).
Fig. 2 EVect of 34 M n-hexane on a resazurin reduction, b DCF Xuorescence and c LDH leakage at 6, 18, 24, 30 and 48 h. Data are expressed as means SD of four determinations. Data were analyzed using one-way ANOVA with TukeyKramer multiple comparisons test. SigniWcance levels were deWned as P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***)

EVect of EGCG and TQ on membrane damage in vitro following n-hexane exposure A previous study found that sub-chronic n-hexane exposure causes a concentration dependent increase in membrane damage as indicated by LDH leakage (McDermott et al. 2007a). To see if ROS formation was responsible for nhexane-induced LDH leakage, cells were exposed to 34 M n-hexane in the absence and presence of EGCG and TQ. Forty-eight hours exposure to 34 M n-hexane resulted in a signiWcant increase in LDH leakage from Jurkat T-cells (from 3.9 1.6% to 13.3 2.5%; P < 0.001; Fig. 6). Neither EGCG nor TQ signiWcantly altered basal LDH leakage levels (P > 0.05; Fig. 6). However, treatment with the test

48 h. ECGC had a protective eVect on cells exposed to 3.6 34 M n-hexane; however, higher concentrations still produced a signiWcant decrease in resazurin reduction in EGCG treated cells (P < 0.001) (Fig. 4b). This suggests that ROS formation is responsible for the decrease in Jurkat T-cell proliferation observed at lower n-hexane exposure levels but at higher levels of exposure other factors may be involved.

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Fig. 4 EVect of 100 nM EGCG treatment on a ROS formation (DCF Xuorescence) induced in Jurkat T-cells and b alterations in cell proliferation (resazurin reduction) following exposure to n-hexane for 48 h. Data are expressed as means SD of four determinations. Data were analyzed using one-way ANOVA with TukeyKramer multiple comparisons test. SigniWcance levels were deWned as P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***)

Fig. 5 EVect of 1 M TQ on a ROS formation (DCF Xuorescence) induced in Jurkat T-cells and b alterations in cell proliferation (resazurin reduction) following exposure to n-hexane for 48 h. Data are expressed as means SD of four determinations. Data were analyzed using one-way ANOVA with TukeyKramer multiple comparisons test. SigniWcance levels were deWned as P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***)

compounds signiWcantly reduced n-hexane-induced membrane damage (Fig. 6). Presence of the methanol solvent did not signiWcantly alter LDH leakage over the exposure period (P > 0.05), data not shown.

Discussion Concentration dependent changes in Jurkat T-cell proliferation and ROS formation were found following 48 h exposure to n-hexane. The solvent also caused increased membrane permeability as indicated by LDH leakage. Timecourse studies showed that a signiWcant increase in ROS formation (18 h) preceded changes in LDH leakage (24 h) and resazurin reduction (30 h). EGCG and TQ proved to have signiWcant chemo-protective properties against the toxicity of n-hexane, an archetypal organic solvent, in Jurkat T-cells. The generation of ROS beyond the antioxidant capacity of the biological system gives rise to oxidative stress, which can damage the major molecular targets including lipids, proteins and DNA. When ROS are generated in vivo

Fig. 6 EVect of 1 M TQ and 100 nM EGCG on n-hexane induced LDH leakage from Jurkat T-cell following 48 h exposure. Data are expressed as means SD of at least four determinations. Data were analyzed using one-way ANOVA with TukeyKramer multiple comparisons test. SigniWcance levels were deWned as P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***)

a wide variety of endogenous antioxidants comes in to play. The type of antioxidant employed depends on which ROS is generated and the target substrate. While a particular

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antioxidant can protect one substrate it may fail to protect another (Halliwell et al. 1995). Some of the foods and beverages, which we consume contain antioxidants which, if absorbed into the body, may aid endogenous antioxidants avoid or reduce the development of oxidative stress. Tests have shown that EGCG and TQ exhibit not only anti-oxidant (Houghton et al. 1995; Guo et al. 1996; Burits and Bucar 2000; Johnson and Loo 2000) but also pro-oxidant (Johnson and Loo 2000; Kanadzu et al. 2006) properties depending on the concentration. One study found that in individuals given 200 ml green tea, peak EGCG plasma concentrations of 174 49.5 nM were reached after 1.31.6 h (Lee et al. 2002). The elimination half-life was 3.4 0.3 h. The concentration of EGCG (100 nM) tested in our study for its chemoprotective eVects is lower than the peak plasma concentrations reached in individuals who drink green tea. A recent study examining the levels of TQ and the other major constituents of Nigella sativa seeds in Wve diVerent brands on sale in Saudi Arabia (Al-Saleh et al. 2006) found that the daily intake of TQ ranged from 2.55 to 6.19 mg/day depending on the brand consumed. No data on TQ plasma concentrations could be found in the literature and so we tested a concentration that did not alter Jurkat T-cell proliferation (Fig. 3). Treatment with TQ or EGCG prevented increases in ROS formation caused by exposure to n-hexane. In turn this prevented nhexane induced changes in Jurkat T-cell proliferation. Our results show that development of oxidative stress plays a role in the development of n-hexane toxicity. Organic solvents are known for their ability to interact with biological membranes causing damage and alteration of function of integral membrane proteins (Thti 1992; Urban 2004). Previously we found that sub-chronic exposure to organic solvents including n-hexane results in a signiWcant decrease in GSH/GSSG ratio in Jurkat T-cells coupled with an increase in membrane damage (McDermott et al. 2007a). These endpoints had similar sensitivity in n-hexane exposed cells. An increase in ROS formation is the most likely cause of these changes. Our results indicate that ROS formation plays a causal role in n-hexane-induced membrane damage in Jurkat T-cells as treatment with EGCG or TQ at levels that reduced ROS to control values prevented changes in membrane permeability (Figs. 4, 5, 6). Other studies have also indicated that ROS formation may play an important part in the development of solvent toxicity in a number of target organs (Dreiem et al. 2002; Baydas et al. 2003; Dreiem et al. 2003). The fact that EGCG and TQ inhibited ROS formation is further evidence for the involvement of ROS formation in n-hexane toxicity. Research suggests that diets rich in antioxidants can reduce risk of diseases associated with free radicals (Johnson and Loo 2000). Our data show that EGCG, at plasma concentrations, and TQ are chemoprotective against

n-hexane toxicity in immune derived T-cells, which manifests as increased ROS formation, membrane damage and decreased cell proliferation. The immune system is considered to be sensitive to chemicals at concentrations lower than those causing toxicity in other systems (Olgun and Misra 2006). Changes in immune system function due to exposure to environmental pollutants is thought to be the cause of increased allergy, diminished resistance against infection and tumour formation, and hypersensitivity (Johnson and Loo 2000; Palermo-Neto et al. 2001). The potential for long-term exposure to pollutants such as n-hexane to result in injury to the immune system is of considerable importance to occupational and public health.
Acknowledgments Supported by the Higher Education Authority (HEA), of Ireland, under the Programme for Research in Third Level Institutions, Grant number 3443.

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