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Life Sciences 95 (2014) 108–117
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Evaluation of differential cytotoxic effects of the oil spill dispersantCorexit 9500
Mengyuan Zheng a, Manuj Ahuja b, Dwipayan Bhattacharya b, T. Prabhakar Clement a,Joel S. Hayworth a, Muralikrishnan Dhanasekaran b,⁎a Department of Civil Engineering, 212 Harbert Engineering Center, Auburn University, Auburn, AL, USAb Department of Pharmacal Sciences, Harrison School of Pharmacy, Auburn University, Auburn, AL, USA
⁎ Corresponding author at: Department of PharmacaPharmacy, Auburn University, Auburn, AL 36849, USA. T334 844 8331.
E-mail address: [email protected] (M. Dhanaseka
0024-3205/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.lfs.2013.12.010
a b s t r a c t
a r t i c l e i n f oArticle history:
Received 24 September 2013Accepted 10 December 2013Keywords:CorexitGulf oil spillEnvironmental toxicityOxidative stressApoptosisMitochondrial dysfunction
Aims: The British Petroleum (BP) oil spill has raised several ecological and health concerns. As the first response,BP used a chemical dispersant, Corexit-9500, to disperse the crude oil in theGulf ofMexico to limit shoreline con-tamination problems. Nevertheless, portions of this oil/Corexit mixture reached the shoreline and still remain invarious Gulf shore environments. The use of Corexit itself has become a significant concern since its impacts onhuman health and environment is unclear.Main methods: In this study, in vitro cytotoxic effects of Corexit were evaluated using different mammalian cells.Key findings:Under serum free conditions, the LC50 value for Corexit in BL16/BL6 cellwas 16 ppm, in 1321N1 cellwas 33 ppm, inH19-7 cellwas 70 ppm, inHEK293was 93 ppm, and inHK-2 cellwas 95 ppm.With regard to themechanisms of cytotoxicity, we hypothesize that Corexit can possibly induce cytotoxicity inmammalian cells byaltering the intracellular oxidative balance and inhibiting mitochondrial functions. Corexit induced increased re-
active oxygen species and lipid peroxide levels; also, it depleted glutathione content and altered catalase activityinH19-7 cells. In addition, therewasmitochondrial complex-I inhibition and increase in the pro-apoptotic factorsincluding caspase-3 and BAX expression.Significance: The experimental results show changes in intracellular oxidative radicals leading to mitochondrialdysfunctions and apoptosis in Corexit treatments, possibly contributing to cell death. Our findings raise concernsabout using large volumes of Corexit, a potential environmental toxin, in sensitive ocean environments.© 2013 Elsevier Inc. All rights reserved.
Introduction
Enormous economic resources and efforts are invested by both pub-lic and private institutions throughout theworld to prevent off-shore oilspill disasters. Despite these efforts, accidents do happen and some-times they release large volume of crude oil into marine environments;unfortunately, the long-term environmental and human health impactsof such large releases aremostly unknown. The recent British Petroleum(BP) Deepwater Horizon (DWH) oil spill disaster is one such event thatreleased over 4.9 million barrels (780,000 m3) of crude oil into the Gulfof Mexico, raising several ecological and health concerns (Hemmeret al., 2011). The release was caused by the explosion of BP's Macondowell #252 (also known as the MC-252 well) on April 20, 2010. Withina month, a considerable amount of partially weathered MC-252 crudeoil began arriving along the white sandy beaches of the Northern Gulfof Mexico. The rate of deposition along the Alabama's shoreline was
l Sciences, Harrison School ofel.: +1 334 844 8327; fax: +1
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ghts reserved.
substantial, at least until about mid-July, 2010 (Hayworth and Clement,2011; Hayworth et al., 2011). The overall shoreline impacts could havebeen even more severe if BP has not used any dispersants to diffusethe oil. It has been estimated that BP used between 1.5 and 1.9 -
million gallons (5678–7192 m3) of dispersants during the oil spillresponse phase (Hayworth and Clement, 2011; Judson et al., 2010).Natural crude oil is a complex mixture containing thousands ofchemicals and has a relatively low specific gravity, ranging from 0.8to 0.9, and hence it floats on water (ATSDR, 1999). Crude oil containsa considerable amount of polycyclic aromatic hydrocarbons (PAHs),and several of these PAHs are known as carcinogen. Several PAHshave also been identified as known environmental toxins, and pub-lished studies have shown a significant decline in human health as a re-sult of PAH exposure (Goldstein et al., 2011; Hayworth and Clement,2011). Rodent studies have shown that PAHs and other hazardouschemical constituents present in crude oil and the dispersants havethe potential to penetrate the cell membrane and cross the bloodbrain barrier to induce biochemical, neurochemical and metabolicchanges (Council NR, 2005; NALCO, 2010).
During the BP oil spill, Corexit 9500 (herein referred to as Corexit)was the major dispersant used by emergency responders. Corexit wassprayed on the ocean surface, andwas also directly injected underwater
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close to the well head (EPA, 2010). Corexit was expected to break thecrude oil into smaller droplets which would facilitate biodegradationby increasing the oil–water surface area; also, dispersion can increasemass removal rates through evaporation and photo-oxidation(Bobra, 1992). Enhanced degradation effects were in-turn expected toreduce the net toxicity of the crude oil. However, from the public accep-tance point of view, the toxicity of Corexit itself was a significantconcern since the impacts of Corexit on human health and the environ-ment were unclear. Preliminary in vitro and in vivo studies conductedby the Environmental Protection Agency (EPA) revealed the toxicity ofCorexit to aquatic species and in cultured hepatic mammalian cells(Judson et al., 2010). These studies evaluated the toxicity of Corexitagainst seven other dispersants and it was determined that Corexitwas a suitable candidate for dispersing the DWH oil. In the EPA study,the cytotoxic potentials of all eight dispersants were assessed andtheir LC50 values were within the concentration range of 100 to1000 ppm. Although this study was conducted in accordance usingstandard guidelines, it only evaluated the cytotoxic potential in limitednumber of cell types; also, the studies primarily focused on the endo-crine effects of these dispersant. Hence, there is a need for a more com-prehensive study that can systemically assess the impacts of Corexit onhuman and environmental health. The primary goal of this study is todevelop a better understanding of potential health and environmentalhazards posed by Corexit.
Environmental adulterants or toxins pose major threat to organismsand have been correlated as potential risk factors in pathophysiology ofvarious diseases. The well-known cases include stimulants or neuro-toxins like MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)'s, her-bicides (paraquat/diquat) and methamphetamine's role in Parkinson'sdisease or nigral degeneration, the role of tobacco smoking in lung cancerandmemory loss (Parameshwaran et al., 2012; Thorgeirsson et al., 2008)and the role of PAHs in inducing carcinogenesis (Mitchell and Holdway,2000). Current studies show that the exposure of organisms to crudeoil anddispersant poses environmental andoccupational hazardwithun-known short and long term consequences (Mitchell andHoldway, 2000).Hence, it is important to study the toxic effects of dispersants on themammalian systems using well-established toxicological experimentalmodels.
During the DWH oil spill, since part of the dispersant (Corexit) wasapplied by the aerial route (spraying from airplanes), onsite workerscould have inhaled the dispersant mist and their skin could have beendirectly exposed to the dispersant. Due to inhalation, the respiratorytract could have been affected and also the inhaled Corexit mist couldhave affected the central nervous system. Due to its lipophilic property,Corexit can potentially cross the blood brain barrier and permeate thebrain through the olfactory system and influence the neurons and theglial cells. In several in vivo studies, scientists have exposed animals(rats) to Corexit (sprayed 27 mg/m3 for 5 h) by the nasal route(Roberts et al., 2011; Sriram et al., 2011) and have shown Corexit toaffect the central nervous system and other organs. Corexit can alsoenter the systemic circulation by dermal permeation. By successfullyentering the systemic circulation, Corexit has the potential to affect sev-eral organs, tissues and cells in the humanbody. In the current study,weused five different types of mammalian cells (cells from different tis-sues) for understanding the toxic effects of Corexit. The cells used are:1) skin cancer cells (B16/BL6); 2) neuronal cells (H19-7) 3); glial cells(1321N1); and 4) two kidney cells (HEK-293 and HK-2). These cellswere exposed to different concentrations of Corexit in the presence orin the absence of serum. Serum-free and serum-fed media representtwo different metabolic conditions that can help directly characterizethe defense mechanisms of a cell and indirectly the respective tissueand organ. Oxidative stress, mitochondrial dysfunction, apoptosis, in-flammation, excitotoxicity and necrosis are themajor known pathwaysof cellular toxicity (Mattson, 2000). In our previous studies with varioustoxins such as MPTP, methamphetamine, paraquat, and nicotine, wehave established the roles of oxidative stress,mitochondrial dysfunction
and apoptosis in causing neurotoxicity (Dhanasekaran et al., 2006;Thrash et al., 2009, 2007). Hence we used a similar procedure to testthe hypothesis that Corexit would exhibit cytotoxic effects by alteringthe oxidative state of a cell and thus inducing oxidative stress, mito-chondrial dysfunction and apoptosis. To elucidate these toxicity mecha-nisms, we studied the effect of Corexit on the generation of reactiveoxygen species, lipid peroxidation, catalase, mitochondrial complex-Iand superoxide dismutase activity, glutathione content, and caspase-3and BAX expression. Thus, the current study is an initial step towardsunderstanding the toxicity effects of Corexit on animal and humanhealth.
Materials and methods
Chemicals and reagents
Thiazolyl Blue Tetrazolium Bromide (MTT), Trypsin-EDTA solution, di-methyl sulfoxide (DMSO) HPLC-grade methanol, Triton-X 100, 4-hydroxyquinoline (4-HQ), bovine serum albumin (BSA), kynuramine, eth-ylenediaminetetraacetic acid (EDTA) sodium salt, O-phthaldialdehyde(OPT), Tris-buffered saline tablet, phosphate buffered saline tablet, re-duced glutathione (GSH), and hydrogen peroxide (H2O2) were purchasedfrom Sigma/Aldrich (St. Louis, MO). Corexit 9500A was obtained fromNALCO (Houston, TX).
Cell culture
Five different types of cell lines were used in our study. These mam-malian cells represented four different tissue types and their details aredescribed below.
Skin cancer cells (B16/BL6)B16/BL6 cell line was obtained from the ATCC (Manassas, VA, USA).
The B16/BL6 carcinoma cell line was obtained originally by injection ofB16 melanoma cells into the derma of both ears of BL6/C57 mice tocreate a primary tumor. For the lab purposes B16/BL6 cells were cul-tured in DMEM supplemented with FBS (10%). Cells were propagatedin 75 cm2 tissue culture flasks, harvested by trypsinization afterreaching 80% confluence (3–4 days), and then plated into 96well platesat a density of 1 × 104 to 5 × 104 cells/ml. Cellswere incubated at 37 °Cand with the supplement of 5% CO2. The cultures were used within 20passages after the cells were received.
Neuronal cells (H19-7)The H19-7 cell line, originally derived from hippocampi dissected
from embryonic day 17 (E17) Holtzman rat embryos and immortalizedby retroviral transduction of temperature-sensitive tsA58 SV40 large Tantigen (Morrione et al., 2000),was obtained from the ATCC (Manassas,VA, USA). H19-7 cells were maintained in Dulbecco's modified Eagle'smedium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodiumbicarbonate and 4.5 g/L glucose supplemented with 10% fetal bovineserum, 200 μg/ml G418, and 1 μg/ml puromycin in flasks coated with15 μg/ml poly-L-lysine. Cells were propagated in 75 cm2 poly-L-lysinecoated tissue culture flasks, harvested by trypsinization after reaching80% confluence (3–4 days), and then plated into poly-L-lysine coated96 well plates at a density of 2 × 105 cells/ml. Cells were incubated at34 °C andwith the supplement of 5% CO2. The cultures were usedwith-in 20 passages after the cells were received.
Human astrocytoma cells (1321N1)The 1321N1 cell line was obtained from the ATCC (Manassas, VA,
USA). It is a human astrocytoma cell line isolated in 1972 as a subclone of the cell line 1181N1which in turnwas isolated from the parentline U-118 MG, one of a number of cell lines derived from malignantgliomas. 1321N1 is a neuronal derived inflammatory cell line that wascultured in DMEM supplemented with 10% FBS. Cells were propagated
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in 75 cm2 tissue culture flasks, harvested by trypsinization afterreaching 80% confluence (3–4 days), and then plated into 96well platesat a density of 2 × 104 to 4 × 104 cells per 10 ml. Cells were incubatedat 37 °C and with the supplement of 5% CO2. The cultures were usedwithin 20 passages after the cells were received.
Kidney cell I (HEK-293)HEK-293 (human embryonic kidney) is a hypotriploid human
cell line, which was procured from the ATCC (Manassas, VA, USA).HEK-293 cells were cultured in the DMEM supplemented with 10%FBS. Cellswere propagated in the 75 cm2 tissue cultureflasks, harvestedby trypsin after reaching 80% confluence (3–4 days), and then put into96 well plates at a density of 2 × 105 cells/ml. Cells were incubatedat 37 °C and with the supplement of 5% CO2. The cultures were usedwithin 20 passages after the cells were received.
Kidney cell II (HK2)The HK-2 (human kidney 2) is a proximal tubular cell (PTC) line
derived from normal human kidney. We obtained this cell line fromthe ATCC (Manassas, VA, USA). The main advantage of HK-2 cells isthat they can reproduce experimental results obtained with freshly iso-lated PTCs (Ryan et al., 1994). HK-2 cells were cultured in keratinocyteserum free medium (K-SFM) supplemented with 0.05 mg/ml BPE and5 ng/ml EGF and 5% fetal bovine serum (FBS). Cells were propagatedin 75 cm2 tissue culture flasks, harvested by trypsinization afterreaching 80% confluence (3–4 days), and then plated into 96well platesat a density of 2 × 105 cells/ml. Cells were incubated at 37 °C and withthe supplement of 5% CO2. The cultures were used within 20 passagesafter the cells were received.
Treatment design
Prior to each experiment, Corexit 9500 was diluted to 1000 ppmin double distilled water (ddH2O), and the stock solution was subse-quently diluted to 20 to 200 ppm in the cell culturemedia. Five differentconcentrations of Corexit (20, 40, 80, 160, and 200 ppm) were used toassess cytotoxicity effects in each cell line. Test concentrations were ex-posed to each cell line for 48 h. The 48 hour period represents relativelylong exposure of Corexit in vivo tissue based toxicity testing. After incu-bation, cell viability was measured using the MTT assay under twodifferent metabolic conditions: one with serum representing a well-nourished state, another without serum representing a nutritionallycompromised state. Rationale for using these two states to study thetoxicity effects of Corexit under two different nourishment states.Each concentration of test agent was tested in duplicates per 96-wellplate and every assay was repeated three times.
Cytotoxicity assay
MTT assays were performed using B16/BL6, H19-7, 1321N1, HK2,and HEK293 cells as an indicator of cytotoxicity. The principle of thisassay is the mitochondrial reductive conversion of the yellow coloredMTT, a yellow tetrazole reagent to a blue formazan by livemitochondriaof viable cells (Berridge et al., 1995; Mosmann, 1983). The MTT assaywas performed following the procedure described in Dhanasekaranet al. (2006). Briefly, after incubation of Corexit, with a correspondingcontrol, in serum-fed and serum-free media for 48 h, MTT was addedat a final concentration of 1 mg/ml. The medium was discarded afterincubating the cells for 4 h at 37 °C, and the insoluble dark blueformazan crystals were dissolved in 100 μl of DMSO. Absorbance wassubsequently measured at 570 nm at a reference wavelength of630 nm using a microtiter plate reader (Synergy HT, Bio-Tek Instru-ments Inc., Winooski, VT, USA). We included two different controls forthe two different metabolic conditions, and the results obtained fromthe Corexit treated group (with or without serum) were compared totheir respective control.
Mitochondrial complex-I activity
Mitochondrial complex-I activity (NADH dehydrogenase activity) isbased on the NADH oxidation. Oxidation of NADH by the NADH-dehydrogenase enzyme present in the tissue homogenate was mea-sured spectrophotometrically at 340 nm. The cell homogenate wasadded to the reaction mixture containing NADH, coenzyme Q10 andphosphate buffered saline, to analyze its effect on NADH oxidation bymonitoring the decrease in absorbance at 340 nm. The change in absor-bance indicates the amount of NADH oxidized. Results were expressedas percentage control (Ramsay et al., 1986).
Quantifying reactive oxygen species
Reactive oxygen species (ROS) generation in the cell treatedwith Corexit was estimated by spectro-fluorometerically detectionof conversion of non-fluorescent chloromethyl-DCF-DA (2′, 7-dichlorofluorescindiacetate, DCF-DA) to fluorescent DCF at an exci-tation wavelength of 492 nm and at an emission wavelength of527 nm. About 0.05% w/v solution of DCF-DA in ethanol (10 μl),phosphate buffer (280 μl) and cell homogenate (10 μl) were incu-bated for 1 h at 37 °C. DCFH reacted with ROS to form the fluores-cent product DCF. Intensity was analyzed by BioTek Synergy HTplate reader (BioTek, VT, USA). Results were expressed as percent-age change from the control (Dhanasekaran et al., 2008).
Superoxide dismutase activity
The superoxide dismutase activity was performed using the com-mercially available HT Superoxide Dismutase Assay kit purchasedfrom Trevigen (Gaithersburg, MD, USA).
Catalase activity
Catalase activity was assayed based on H2O2 decomposition moni-tored at 240 nm for 2–3 min (Aebi, 1984). An assay mixture (500 μl)contained suitably diluted enzyme protein (100 μg) in 50 mM phos-phate buffer, pH 7.0. The reaction was started by the addition of H2O2
(30 mM), which provided approximately 0.5 absorbance. The decreasein absorbancewasmonitored and the enzyme activitywas expressed aspercentage control (Muralikrishnan and Mohanakumar, 1998).
Glutathione content
GSHwasmeasured fluorimetrically as described earlier by Cohn andLyle (1966). Principally, this method employs o-phthalaldehyde (OPT)condensation reaction with GSH to yield a fluorescent product atpH 8.0. The assay mixture consisted of 0.1% OPT solution in methanol(100 μl), 0.01 M phosphate buffer (1.8 ml) and supernatant of tissue(100 μl) after precipitation of protein by 0.1 M phosphoric acid treat-ment. The assay mixture was incubated for 20 min and readings weretaken at excitation and emission wavelengths of 340 and 420 nm,respectively. A standard curve was prepared from commercially ob-tained GSH. The GSH content was calculated as mmol of GSH/μg pro-tein and reported as percentage control levels (Muralikrishnan andMohanakumar, 1998).
Lipid peroxidation content
The index of lipid peroxidation was estimated by measuring themalondialdehyde (MDA) content in the form of thiobarbituric acid-reactive substances according to the method of Ohkawa et al. (1979).Cell homogenate (100 μl) and 200 μl ice cold TCA (10% w/v in doubledistilled water) were incubated for 15 min on ice. Assay mixture wascentrifuged at 2200 ×g for 15 min at 4 °C temperature. Supernatants(200 μl) of standards and samples were placed into new labeled screw
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top 1.5 ml tubes and equal volumes (200 μl) of 0.67% TBA were added.The reaction mixture was incubated for 20 min in boiling water. Sam-ples after incubation were cooled down to room temperature andplaced in 96-well plate. A standard curve of TBARS reactive substanceswas also prepared by employing different concentrations of MDA.Absorbance was read in plate reader at 532 nm and MDA levels werecalculated as TBARS reactive substances per mg protein. Resultsexpressed as percentage control (Dhanasekaran et al., 2007).
Western blot for caspase-3 and BAX
Total protein was isolated from cells using cell lysis buffer (CellSignaling Technology, Inc., Danvers, MA) containing protease inhibitorcocktail (P8340, Sigma, St. Louis, MO) and phosphatase inhibitors(P 5726, Sigma, St. Louis, MO). Protein concentration was measuredusing the Thermo Scientific Pierce 660 nm Protein Assay reagent kit(Pierce, Rockford, IL). Protein samples were aliquoted into 50 ug frac-tions and were stored in −70 °C. On the day of experiment sampleswere boiled in invitrogen sample loading buffer for 5 min and thenloaded onto a gradient 4–12% NuPAGEBis–Tris Gel (Life Technologies,Grand Island, NY) and electrophoretically transferred onto nitrocellu-lose membrane through semi-dry transfer method. Non-specific bind-ing sites on the membranes were blocked with 5% fat-free milk inTris-buffered saline plus 0.1% Tween-20 (TBST) at pH 7.4. The mem-branes were incubated overnight at 4 °C with specific antibody consti-tuted in 5% BSA in TBST. Primary antibodies used were caspase-3 andBAX (Cell Signaling); β-actin were used as a loading control. Mem-braneswere thenwashedwith TBST (3X, each for 10 min) and incubat-ed with specific species dependent HRP-conjugated secondaryantibodies for 60 min at room temperature. Membranes were againwashed three times for 10 min with TBST after incubation with eachantibody. After washing membranes were incubated with chemilumi-nescent reagent (Enhanced ECL-substrate, Pierce, Rockford, IL) for5 min. Membranes were wrapped in plastic wrap and placed in theX-ray film cassette with protein side oriented up. Chemilumines-cence signal was measured as band density obtained on the X-rayfilm through utilization of Quantity One software (Bio-Rad, Hercules,CA). Band densities for each sample were normalized to its β-actin orGAPDH signal and reported as percentage control.
Statistical analysis
All data are expressed as means ± SEM for each cell line. Statisticalanalyses were performed using one-way analysis of variance (ANOVA)followed by an appropriate post-hoc test including Tukey's and Dunn'smethod (P b 0.05 was considered to indicate statistical significance).All statistical analyses were performed using the Prism-V software (LaJolla, CA, USA).
Results
Corexit exhibits concentration-dependent cell death in differentmammalian cells
In case of cell-based toxicological studies, cancer cell lines are thepreferred option for testing the toxicological potential of any newchemical compound. The primary reason for this is that cancer cellsare highly-specialized cells that have been transformed to a much sim-pler, more primitive stage and thus possess the ability to grow continu-ously by division (Ekwall et al., 1990). Secondly, due to the high activecell division rate, they are more vulnerable than most normal cells toany toxin. Hence, they provide a sensitive cellular model for toxicologi-cal investigations which can be done rapidly and accurately. For estab-lishing the toxic concentrations of Corexit, different concentrations ofCorexit (20, 40, 80, 160, and 200 ppm) were used to incubate B16/BL6cell line for 48 h under two different metabolic conditions. Corexit
exhibited a concentration-dependent toxicity in the mouse-melanomacell line, as shown in Fig. 1 (n = 6, p b 0.05). The toxicity observed inserum-fed condition (nourished) was not as prominent as in theserum-free (unnourished) condition. Almost 100% of cell death wasobserved under unnourished conditions, whereas the same concentra-tion exhibited 40% cell death in nourished condition. The LC50 valuefor B16/BL6 cells, under unnourished conditions, was found to be16 ppm. We did not find 50% or more cell death in serum fed states,which limited our ability to calculate LC50 for Corexit under thiscondition.
To evaluate the cytotoxic effect on mammalian hippocampal neuro-nal cells, following Corexit treatment the undifferentiatedH19-7 hippo-campal neurons were incubated with Corexit (20, 40, 80, 160, and200 ppm). Corexit significantly reduced the cell viability concentrationdependently in both nourished and unnourished states of neuronalcells (Fig. 1, n = 6; p b 0.05). However, the toxicity of Corexit wasmore prominent in serum-free state with a reduction in cell viabilityby 80%, compared to the 20% reduction observed under serum-fed con-ditions (Fig. 1). Additionally, in the serum-free media a more conspicu-ous concentration-dependent effect of Corexit was observed in terms ofneuronal cell death. Serum-free media represent an unnourished statethat compromises the defense mechanisms of a cell and the respectivetissue and organ. Thus, in an already compromised mammalian systemthe toxicity of Corexit to central nervous system would increase bymany folds. The LC50 for H19-7 cell was 70 ppm. Similar to the cancercell line, in serum fed states we were unable to deduce LC50 valueseven for the H19-7 cells. The morphological change of H19-7 cell linetreated with different concentrations of Corexit is shown in Fig. 2.With increasing concentration of Corexit the shrinkage of neuronalcells increases which implicates extensive cell death. Fig. 2A showsthat Control group has the least shrinkage and cell death. However,Fig. 2B (80 ppm), C (160 ppm) and D (200 ppm) represents theincreasing concentration of Corexit inducing respective escalation inneuronal cell shrinkage and cell death.
The human astrocytoma cells (1321N1) used in this studywere orig-inally isolated from primary cultures of cerebral glioma multiforme(Ponten et al., 1968). These are neoplastic cells (tumor cells) from a spe-cific type of neoplasm of the brain. Being a cancer cell line of neuronalinflammatory cells, 1321N1 cells proved to be a valuable toxicologicalin-vitro model for studying the toxic effects of Corexit. Similar to theother cells, Corexit was incubated at different concentrations with thiscell. Results show a trend similar to the onewe observed for the rat hip-pocampal H19-7 cell (see Fig. 1, n = 6; p b 0.05). However, the glialcells were found to be more vulnerable to Corexit induced cell deathat much lower concentrations ranging from 20 ppm to 40 ppm. Thehigher level of toxicity could be attributed to the neoplastic nature ofthis cell. For 1321N1 cells, the LC50 was 33 ppm (Fig. 1). Serum-freeconditions exacerbated Corexit toxicity in 1321N1 cells to many-foldresulting in almost 100% cell death even at 40 ppm concentration(Fig. 1). Indirectly, Corexit toxicity to astroglial cells poses a threat tothe CNS, as these are inflammatory cells of the CNS and play a vitalrole in protecting neuronal cells.
Liver and kidneys are the two key organs that remove toxic sub-stances from mammalian bodies; therefore, they are ideal candidatesfor toxicity testing. Toxic-byproducts of cellular metabolism, foreignparticles and transformed metabolites of xenobiotics are efficientlyremoved by kidneys. We selected a well-established and extensivelyused human kidney cell line (HK-2) (Gunness et al., 2010). HK-2 is animmortalized proximal tubule epithelial cell line from normal adulthuman kidney. It retains the phenotypic characteristics of a wellgrown differentiated kidney cell. In addition, HK-2 cells possess func-tional characteristics of proximal tubular epithelium (Na+ dependent/phlorizin sensitive glucose transport; adenylate cyclase responsivenessto parathyroid, but not to antidiuretic hormone). These cells thus helpus to identify the toxicological effects of Corexit on adult kidney cells.Previous studies have tested the Corexit toxicity in liver cell lines
Fig. 1. Effects of Corexit on skin tumor cells (B16/BL6); neuronal cells line (H19-7); glial cells (1321N1); and two kidney cells (HEK 293 and HK-2). Cells were treated with different con-centrations of Corexit for 48 h. TheMTT assaywas used to assess cell viability. Control cellswere incubated in Corexit freemedia run inparallel to treatment groups. The data are expressedas mean ± SEM of three independent experiments (duplicates in each experiment). (*) indicates a statistically significant difference compared to controls (p b 0.05).
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(Judson et al., 2010), but no reports have been published for kidney celltoxicity. To evaluate Corexit toxicity on human kidney cells, we incubat-ed HK-2 cell lines with different concentrations of Corexit (20, 40, 80,160, and 200 ppm) for 48 h in both serum-free and serum-fed states.Results revealed that HK-2 shares H19-7's reduced sensitivity toCorexit-induced cell death under serum-fed condition. Only 20% ofcell deathwas observed in serum-fed states at thehighest concentrationof Corexit. In serum-free states, Corexit-induced cell toxicity effect wasprominent with 160 ppm of Corexit resulting in 80% cell death (see
Fig. 1). These results imply that Corexit can disrupt normal kidney func-tions at higher concentrations and thus could cause kidney failure andother systemic problems. The LC50 of HK-2 cell was 95 ppm. The LC50of all the five cell lines used in this study is listed in Table 1.
Corexit induces oxidative stress
Oxidative stress caused by the generation of ROS can damage organ-elles and biological molecules (protein, DNA, mitochondria and lipids).
Fig. 2.Morphological characterizations of neuron cell. Cellswere treatedwith different concentrations of Corexit for 48 h. At the end of 48 h the cells werewashedwith PBS and visualizedunder microscope (magnification 10×). (A) Control; (B) 80 ppm of Corexit; (C) 160 ppm of Corexit; (D) and 200 ppm of Corexit.
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When ROS is generated in the cell, the self-protective mechanisms inthe cell activate the antioxidants (enzymes, proteins and tripeptides)to protect against the deleterious effects of the highly active and toxicradicals. The toxic reactive radicals mainly affect the lipids and increasethe formation of lipid peroxides. Therefore, we tested the activities andthe content of antioxidants: superoxide dismutase (SOD) activity,catalase activity, and glutathione content (GSH) in the Corexit treatedH19-7 cells (Fig. 3). Corexit concentration dependently (20 and80 ppm) increases the ROS generation in H19-7 cells (Fig. 4). Corexitat 20 ppm increased ROS generation by 16%, and Corexit at 80 ppmincreased ROS generation by 26%, as compared to the control (n = 6,p b 0.05). These results correlated positively with the mitochondriacomplex-I activity deficits observed in the current study. In our previousstudy,methamphetamine also inhibitedmitochondrial complex-I activ-ity and increased the generation of ROS (Thrash et al., 2010). Similarwork done by Wilk et al. (2013) has demonstrated that non-toxic andtoxic concentrations of polycyclic hydrocarbons can trigger a significantamount of ROS generation and cause oxidative DNAdamage. Corexit didnot affect SOD activity at low concentration, but higher concentrationsof Corexit (at 80 ppm) increased the SOD activity by 1.25 fold as com-pared to the control (n = 6, p b 0.05, Fig. 4); this correlated positivelywith ROS generation. Similarly, Corexit at 20 ppm did not alter the
Table 1LC50 values for Corexit evaluated using skin tumor cell line (B16/BL6); neuronal cell line(H19-7); glial cell line (1321N1); and two kidney cell lines (HEK-293 and HK-2).
Cell Description LC 50
B16/BL6 Skin Cancer 16 ppm1321N1 Glial Cancer 33 ppmH19-7 Brain 70 ppmHEK-293 Kidney 93 ppmHK-2 Kidney 95 ppm
catalase activity, but at the higher concentration increased the catalaseactivity by 65%. The data also indicated that oxidative stress occursthrough the generation of ROS and the catalase activity was increasedto counteract the oxidative stress, thereby protecting the cells fromthe ROS induced toxic effects (Shirahata et al., 1997). GSH plays animportant role in the cellular metabolism of reactive oxygen species asan antioxidant and radical scavenger. It is commonly believed that thedepletion of GSH may lead to oxidative stress (Mytilineou et al., 2002)and the decreased cellular GSH level is associated with ROS-mediatedapoptosis (Circu and Aw, 2010). Interestingly, Corexit at 80 ppm signif-icantly depleted the GSH (n = 6, p b 0.05, Fig. 4). In our previous study,MPTP also depleted GSH and increased the generation of ROS(Mohanakumar et al., 2000; Muralikrishnan and Mohanakumar,1998). ROS can cause damage to mitochondria, DNA, and the lipids inthe cell membrane (Cabiscol et al., 2000). The lipid peroxidation assaywas employed to assess the degradation of lipids which serves as anindicator of membrane damage. Corexit at 80 ppm caused a significant(n = 6, p b 0.05) elevated level of lipid peroxide which clearly indicat-ed damage of cell membrane.
Corexit inhibits mitochondrial complex-I activity
Mitochondria are the powerhouse of a cell. The function of mito-chondrial complex-I decreases with aging and in many neurodegenera-tive diseases (Moreno et al., 2012). The decreased activity of complex-Imay cause mitochondrial dysfunction, therefore leading to the forma-tion of the various reactive oxygen species. In the current study, anon-toxic concentration level of Corexit (at 20 ppm) did not altercomplex-I activity, however, a toxic concentration level of Corexit (at80 ppm) resulted in significant decrease in mitochondria complex-Iactivity, as shown in Fig. 5 (n = 6, p b 0.05). This result indicate therelation between increased Corexit cytotoxicity and decreased complex-Iactivity, which may contribute to the generation of ROS (Ide et al.,1999) and induce apoptosis (Hartley et al., 1994).
Fig. 3.Effects of Corexit (control, 20 ppm, 80 ppm)on reactive oxygen species generation, superoxide dismutase activity, catalase activity, glutathione and lipid peroxide content inneuroncells (H19-7 cells). Corexit (80 ppm) significantly increased reactive oxygen speices (n = 6, p b 0.05), superoxide dismutase activity (n = 6, p b 0.05), catalase activity (n = 6, p b 0.05),glutathione content (n = 6, p b 0.05), and lipid peroxidation (n = 6, p b 0.05) as compared to control. Note (*) indicates a statistically significant difference compared to controls(p b 0.05).
Fig. 4. Effects of Corexit (20 ppm, 80 ppm) on complex-I activity in neuronal cells (H19-7cells). Corexit (80 ppm) showed significant decrease in complex-I activity (n = 6,p b 0.05) as compared to control. Note (*) indicates a statistically significant differencecompared to controls (p b 0.05).
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Corexit induces apoptosis
Generally cell death can be characterized as apoptosis or necrosis(Majno and Joris, 1995). Apoptosis (programmed cell death) involvescaspase activation (specifically caspase-3 appears in the execution-phase of cell apoptosis), expression of various pro-apoptotic and/oranti-apoptotic proteins, nuclear condensation, fragmentation and cleav-age of chromosomal DNA without causing inflammation. Necrosis in-volves breakdown of the plasma membrane and the release of cellularcontents. Caspases-3 as a frequently activated death protease is an im-portant mediator of apoptosis. It acts by catalyzing the specific cleavageof many proteins and acts prior or at the stage when commitment toloss of cell viability occurs (Porter and Janicke, 1999). Bax is aproapototic protein that belongs to Bcl-xl family; the over-expressionof BAX is related to apoptosis (Porter and Janicke, 1999). Thus, for theconfirmation of apoptotic stimulation by the Corexit, the expression ofcleaved caspase-3 and Bax proteins was quantified using Western
Fig. 5. Expression of Bax and cleaved Capase3 inH19-7 cells. The cellswere harvested afterexposing them to two concentrations of Corexit (20 ppm, 80 ppm for 48 h). Blots weredeveloped using 1:1000 dilution for cleaved Caspase3, and Bax. β-Actin (1:1000) wasused as a loading control. Chemiluminescence signal was measured as band densityobtained on the X-ray film using Quantity One software (Bio-Rad, Hercules, CA). Note(*) indicates a statistically significant difference compared to controls (p b 0.05).
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blot. Corexit (80 ppm) significantly increased the expression of cleavedcaspase-3 and BAX indicating Corexit-induced apoptosis (Fig. 5, n = 6,p b 0.05).
Discussion
Unlike natural disasters such as hurricanes and earthquakes, oilspills can negatively affect ecosystems several years after the event.Apart from affecting the neighboring ecological niches, oil spills canimpact several aspects of human activities. The Exxon Valdez oil spill(EVOS), which occurred in Prince William Sound, Alaska, on March 24,1989, revealed that in addition to affecting sea birds and marineanimals, the spill also impacted socio-economic conditions of peopleresiding in nearby areas (Goldstein et al., 2011). Alaska oil spill causedeconomic losses for fishermen and supporting businesses, induced so-cial conflicts, declined community cohesiveness, and increased alcoholand drug abuse in some of the local populations (Picou et al., 1992).Additionally, oil spill events such as the DWH oil spill lead to a large-scale environmental contamination that impacts the recreational andecological value of the entire ecosystem (Hayworth and Clement,2011). The USEPA has developed guidelines that support the use of dis-persants to manage certain types of oil spills. However, studies havesuggested that dispersed crude oil poses a greater threat to aquaticand terrestrial systems than undispersed oil (Rico-Martínez et al.,2013). In an earlier study, rats were orally treated for 5 weeks withcrude oil, Corexit, or Corexit with crude oil (George et al., 2001). Thestudy mainly investigated the mutagenic properties and quantified theeffects on the intestinal microflora and enzymes. The study showedthat prolonged exposure of rats to oil, dispersant or combination altersthe intestinalmetabolism,which ultimately could impact detoxificationof oil constituents and lead to toxicity. Recent comparative toxicitystudies of eight dispersants on mysid shrimp (Americamysis bahia)and inland silversides (Menidia beryllina) have shown that Corexit9500A demonstrated a similar toxicity to the other seven dispersants,and all eight dispersants were less toxic (LC50: 3 to N5600 μl/L) thandispersant + Louisiana sweet crude oil mixtures (0.4 to 13 mg TPH/L)in these two marine species (Hemmer et al., 2011). George-Ares andClark (2000) have shown that Corexit has low (LC50 N 100 ppm) tomoderate (LC50 from 1 to 100 ppm) acute toxicity to most aquaticorganisms in laboratory. Moreover, evidence for human toxicologicaleffects from Corexit exposure can be found from the workers involvedin the DWH oil spill cleanup efforts, who were reported to haveacute pulmonary and dermatological health effects (Andersonet al., 2011). Rodents exposed to dermal applications of Corexit
exhibited concentration-responsive increases in dermal irritation,ear swelling and lymphocyte proliferation, suggesting Corexit as apotent sensitizer (Anderson et al., 2011). Thus, individuals withhypersensitive immune response exposed to Corexit could poten-tially exhibit severe allergic reactions. Furthermore, there are reportsof Corexit inducing neurotoxicity in rodents (Sriram et al., 2011).Corexit administration altered the levels of various synaptic andneuronal intermediate filament proteins in specific brain areas.Reactive astrogliosis, as evidenced by increased expression of glialfibrillary acidic protein, was observed in the hippocampus andfrontal cortex (Sriram et al., 2011). Collectively, these findings aresuggestive of disruptions in olfactory signal transduction, axonalfunction, and synaptic vesicle fusion, which are events that couldresult in an imbalance in the neurotransmitter signaling.
To the best of our knowledge, our study is the first to consider thetoxicity of Corexit to different mammalian cells. The DWH oil spill is arelatively recent event, only a few studies considered the short andlong-term impacts of this human-caused disaster. Our study is a prelim-inary step in understanding the toxicity of Corexit on the health ofhumans. We have investigated the effect of Corexit as a function oftwo different cell metabolic conditions: one with normal amount ofgrowth factors (i.e. serum-fed media), and another at a relativelycompromised state where the cells have limited or no access to growthfactors present in the serum (i.e. serum-free conditions). It has beenaccepted that the serum concentration can affect the result of the MTTassay, and serum itself may cause false positive results (Funk et al.,2007). Interestingly, the type and the quantity of serum can also influ-ence the result of cytotoxicity studies (Gülden et al., 2006). This is be-cause, serum contains protein, nutrients and lipids that can bind to thepotential toxins used in the MTT assay, therefore decrease the bioavail-ability of the target toxins (Gulden and Seibert, 2003; Seibert et al.,1989). The lipophilicity of a potent toxin is another factor that can influ-ence the result of cytotoxicity assays. Researchers have shown that thecytotoxicity potency of chemicals in general is positively correlatedwith the lipophillicity (Ahlers et al., 1991; Gülden and Seibert, 2005).Furthermore studies have shown that there is an increased correspon-dence between acute in vivo toxicity and in vitro toxicity data whenusing serum-free media. Therefore, the use of serum-free media in thein vitro study can have a better prediction of in vivo effects (Ekwallet al., 1990), and to quantify the deleterious effects of toxins in differentcells. Therefore, to better understand a potent toxin, use of a selectivemedium which is serum-free and supplemented with basic nutrientsand growth factors is recommended (Barnes and Sato, 1980). Basedon these recommendations, we used serum free media supplementedwith basic nutrients to quantify the cytotoxicity effects and to under-stand the toxicity mechanisms.
Cell-based toxicity models can provide a sensitive and reliabletoxicity assessment, while avoiding complications arising through theuse of conventional animal-based toxicological screening studies(Bandele et al., 2012). Biologically relevant endpoints and biomarkerscan be used to deduce the toxicity of complex mixtures and to identifythe constituent(s) that may adversely affect the physiological activities.The advantages of these toxicological systems include accumulatingaccurate and valuable information over a short time interval, whichcan assist in the development of further experimental strategies. Inour study, we found that differentmammalian cells have variable abilityto resist Corexit toxicity effects. Cells were found to bemore defenselessunder serum-free states when compared to serum-fed states. In theserum-free state, the order of sensitivity towards Corexit induced celldeath was found to be: B16/BL6 N 1321 N1 N H19-7 N HEK293 N HK2.The average value of LC50 for each cell line was found to be: 16 ppmfor BL16/B6; 33 ppm for 1321N1; 70 ppm for H19-7 cell; 93 ppm forHEK-293 and 95 ppm for HK-2 cell. These results imply that highly pro-liferative cells are more susceptible to Corexit induced cell death, ascompared to normally transformed adult cells of neuronal and kidneytissues. Previous studies have reported the LC50 of Corexit in two
116 M. Zheng et al. / Life Sciences 95 (2014) 108–117
human liver cell lines (HepG2 and HepG2/C3A) to be 120 ppm and250 ppm, respectively (Judson et al., 2010). The difference in thesereported LC50 values may be due to the difference in the assay methodused to study the cell proliferation effects, as well as the inherent differ-ences in the cell lines (HepG2 being a parental cell line, and HepG2/C3Aa subclonal cell line) (Bandele et al., 2012). In our study, we haveemployed a variety of cells to study the concentration dependent toxic-ity effects of Corexit. In order to compare the cellular-level toxicity ofCorexit with other environmental toxins, we have summarized theliterature-derived LC50 values of various environmental toxins eval-uated using different types of cells (Table 2). These results indicatethat Corexit LC50 values are similar to environmental toxins. Overall,Corexit was found to be less potent than some highly toxic compounds(e.g. 6-hydroxydopamine, diquat, Tributyltin oxide and Trimethyltinchloride). From neurotoxicity perspective, Corexit appears less harmfulthan other well-known neurotoxins (such as Beta-amyloid) but it mayhave other long term toxic effects. Similarly, acute toxicity of nicotineis low (LC50 N 20,000 ppm) but it has been shown to have morelong-term deleterious developmental effects than many other toxins(Parameshwaran et al., 2012). Therefore, although Corexit appears tobe less acutely toxic, its long-term toxicity to mammals is currentlyunknown.
In the current study, our aim was to develop a better understandingof acute Corexit toxicity at the cellular level. The data obtained from thisstudy will be useful for quantifying both the environmental and humanhealth risks posed by Corexit. Furthermore, the results obtained fromthis study help better understand underlying toxicity mechanismswhich can be used to develop novel drug therapies to protect againstCorexit-induced toxicity effects. Mechanistically, the electron transpor-tation chain serves as the means of oxidative phosphorylation ineukaryotes. Through electron transportation chain, ATP is produced toprovide the energy for living cells and this procedure occurs on themembrane of the mitochondria. The decreased activity of complex-Ican lead to the dysfunction of mitochondria. Previous studies havemapped the potential relation between complex-I and ROS generation(Beers and Sizer, 1952). ROS includes free radicals such as hydroxyl(•OH), alkoxyl (RO), and peroxyl (ROO) ions, it also includes non-radicals such as hydrogen peroxide (H2O2). ROS are the by-products ofelectron transportation chain reactions in the mitochondria (Wallace,1999). It is also believed that the generation of superoxide anion is relat-ed to the alteration of complex-I and complex-III activities (Beers andSizer, 1952).When superoxide anion is formed, the superoxide dismut-ase will be activated and converts superoxide anion to hydrogen perox-ide. Glutathione and catalase are the two antioxidants which caneliminate the deleterious effects of hydrogen peroxide by converting it
Table 2Comparison of LC50 values of various environmental and endogenous neurotoxins.
Toxin Cell line LC50(ppm)
Citation
Beta-amyloid PC-12 0.05 Conte et al. (2003)TBT Kidney cells 1.05 Thompson et al. (1996)6-Hydroxydopamine Dopaminergic neurons 1.7–17 Michel and Hefti (1990)TET Kidney cells 3.3 Thompson et al. (1996)Diquat Hepatocyte 3.4 Rikans and Cai (1993)TMT Kidney cells 12.9 Thompson et al. (1996)Corexit 9500A B16/BL6 16 Our current studySalsolinol Neuronal cells 17.9 Wang et al. (2007)Paraquat Neuronal cells 26.75 Ortiz-Ortiz et al. (2011)Cupric sulfate HepG2 cell 28.686 Ponsoda et al. (1995)Corexit 9500A 1321 N1 33 Our current studyIron sulfate Brain endothelial cells 59.982 Lockman et al. (2012)Corexit 9500A H19-7 70 Our current studyCorexit 9500A HEK293 93 Our current studyCorexit 9500A HK-2 95 Our current studyIron sulfate Neuronal cells 159.952 Lockman et al. (2012)Formaldehyde EpiAirway cell 545.4 Balharry et al. (2008)Nicotine EpiAirway cell 20,265 Balharry et al. (2008)
to water and oxygen. If formed in excess, hydrogen peroxide being astable chemical will be rapidly degraded to hydroxyl radicals (OH•).The hydroxyl radical thus formed attacks cell membrane and initiatelipid peroxidation, causing DNA cleavage, and protein degradation.
Our results show decreased complex-I activity in cells treated withCorexit (80 ppm) which indicated mitochondria dysfunction. ROS con-centrations increased significantly after cells were treated with Corexit(80 ppm) for 48 h, indicating that the cells were under stress. Mean-while, the antioxidants also responded to the stress triggered byCorexit.The SODactivitywas elevated indicating a self-protectionmechanismofthe cell, while catalase activity was also increased. However, the antiox-idant GSH contentwas depleted in cells treatedwith high concentrationof Corexit (80 ppm). It has been reported in the published literaturethat the depletion of GSH may be the reason for accumulation of ROSwithin cells (Sakon et al., 2003). Our data is consistent with this litera-ture since we also observed increased level of ROS and decreased levelof GSH, indicating Corexit induced depletion of GSH. The increasedLPO activity in cells treated with Corexit (80 ppm) suggests the lipidson the surface of the cell membrane were degraded; and this couldexplain cell death. The apoptotic maker caspase-3 and Bax were alsoshown to increase in response to higher levels of Corexit exposure.Overall, our experimental data support our hypothesis that Corexitinduced cell death was caused by apoptosis and oxidative stress.
Conclusion
Using the cell viability assay (MTT), we have shown that highly pro-liferating cells are more susceptible to Corexit-induced toxicity. Basedon our study, Corexit exhibits its toxicity by inducing oxidative stress,mitochondrial dysfunction, and apoptosis. The use of antioxidants andmitochondrial energy enhancers could potentially decrease both acuteand chronic toxicity effects of Corexit. The current observations alsoraise the concerns that this toxic effect will be more pronouncedwhen crude oil is mixed with Corexit and our ongoing research willaddress these concerns.
Conflict of interest statement
None.
Acknowledgment
This study was supported by the grant from the Office for VicePresident for Research (OVPR), Auburn University. We would also liketo sincerely thank Dr. Amin, Dr. Hussain and Dr. Shen for their valuablecontributions with the cells.
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