Article

J. Korean Soc. Appl. Biol. Chem. 54(6), 978-985 (2011)

Human HaCaT Cell Toxicity Associated with Oxidative Stress on the Polished Rice Grown Adjacent to Abandoned Mines and Potential Health Risk through Rice Intake Ji-Ho Lee, Eun-Jung Jeong, Geon-Jae Im, Moo-Ki Hong, and Won-Il Kim* Department of Agro-Food Safety, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea Received August 23, 2011; Accepted October 12, 2011 Human HaCaT cell viability, total antioxidants generation, and activities of antioxidant enzymes, i.e. superoxide dismutase (SOD) and catalase (CAT), were estimated on contaminated polished rice grown in abandoned mines. Potential health risk by intake of contaminated polished rice causing cytotoxicity on human HaCaT cell was assessed using the Monte-Carlo simulation. Results showed that the HaCaT cell viability in toxic elements (TEs) -contaminated polished rice was inhibited in a time-dependent manner, ranging from 5−25%. Likewise, time-dependent total antioxidants in contaminated polished rice were significantly decreased to 13−22 mM at 72 h post-incubation than in control demonstrating that the mine-impacted polished rice was associated with oxidative damage and decrement of the total antioxidants. The enzyme activities of scavenging reactive oxygen species, i.e. SOD and CAT were 2−4 folds induced compared to control. Assuming that TEs-contaminated polished rice inducing cytotoxicity on HaCaT cell was ingested by human population, no health risk is expected. Key words: abandoned mines, cytotoxicity, health risk, human HaCaT cell, oxidative stress, polished rice

Waste rocks and tailings generated from mining activity contain large amount of toxic elements (TEs), and hence contaminate the paddy soil through weathering process and runoff during heavy rainfall. Zhu et al. [2008] reported that a widespread use of ground water for irrigation in rice fields leads to detrimental effects on rice growth and yields, and may pose severe toxic effects to animal as well as human being through food chain. Rice has been recognized as a significant dietary source of toxic elements, particularly, arsenic (As) due to the greater bioavailability and accumulation than other agricultural products [Su et al., 2010]. Schoof et al. [1999] reported that rice grain predominantly contains higher inorganic As (Asi) level than other crops. Average content of Asi in rice plant is as high as 83% in mine-impacted paddy fields [Zhu et al., 2008]. Moreover, the cadmium (Cd) and lead (Pb), which are easily absorbed from the soil by *Corresponding author Phone: +82-031-290-0527; Fax: +82-31-290-0506 E-mail: [email protected] http://dx.doi.org/10.3839/jksabc.2011.147

rice roots, are translocated and accumulated in rice grain [Liu et al., 2003; Qian et al., 2010]. The As, Cd, and Pb compounds known as potential toxic elements (PTEs) are reported to cause health risk through the intake of PTEscontaminated rice [Zhu et al., 2008]. The possible toxic mechanism of these toxic elements is associated with oxidative stress [Shi et al., 2004; Sun et al., 2006]. The high concentration of Asi disrupts various metabolic pathways by the interaction with sulfhydryl groups (-SH) and the replacement of phosphate from ATP [Shri et al., 2009]. Moreover, the cell damage is stimulated by interfering with enzyme for trivalent arsenic (As(III)) and oxidative phosphorylation for pentavalent arsenic (As(V)). The Cd and Pb are also involved in accumulation of reactive oxygen species (ROS) caused by oxidative stress, and thereby induce damage in cell structure and function interfering with the redox status [Hatata and Abdel-Aal, 2008]. The increment of ROS due to imbalance between antioxidants and oxidants during metabolism of TEs impairs the mitochondrial function, and thus results in cell toxicity as well as potential carcinogenesis [Shi et al., 2004]. To protect the cells against ROS formation, plants exert an antioxidant

Cytotoxicity on the Toxic Elements-contaminated Polished Rice and Health Risk Assessment

defense system for minimization of cell damage and death. The ROS-induced cytotoxic effects in rice plants are inhibited by inducing the antioxidant enzymes. Several studies on the cell viability treated with TEs have been documented [Graham et al., 2003; Vega et al., 2003; Zhang et al., 2003; Sun et al., 2006; Chowdhury et al., 2010]. However, little study was done on the effects of TEs-contaminated polished rice on the cytotoxicity, ROS generation, and enzyme activity of antioxidants caused by oxidative stress. The objectives of the present study were to estimate the human HaCaT cell viability on TEscontaminated polished rice grown in nearby abandoned mines, and evaluate the extent of the ROS formation and the activities of antioxidant enzymes i.e. superoxide dismutase (SOD) and catalase (CAT). Moreover, the potential health risk through intake of TEs-contaminated polished rice to induce toxicity on human HaCaT cell was assessed by using the Monte Carlo simulation.

Materials and Methods Chemicals and Reagents. Arsenic trioxide (As2O3) and sodium arsenate dibasic hepta-hydrate (Na2HAsO4 · 7H2O) with a high purity (>99.9%) were purchased from Sigma-Aldrich (St. Louis, MO). Monosodium acid methane arsonate (MMA; CH4AsNaO3) and Dimethylarsinic acid (DMA; C2H7AsO2) with a high purity (>98%) were purchased from Chem Service Inc. (West Chester, PA). One molar stock standard solutions of As(V), MMA, and DMA were prepared by dissolving the appropriate amount with ultrapure Milli-Q® water (Millipore Co. Billerica, MA). As(III) was dissolved with minimum volume of 70% HNO3 solution, stirred overnight, and then neutralized with 1N NaOH. For 3-(4,5-dimethyl thiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with phenol red, fetal bovine serum (FBS), penicillin-streptomycin, EDTA-trypsin, and Dulbecco’s phosphate buffer solution (DPBS) were purchased from Gibco-BRL life technologies (InvitrogenTM, Paisley, UK) and Cellgro® (Mediatech, Manassas, VA). DMEM supplemented with 10% FBS, and 1% penicillinstreptomycin was used for normal cell culture and MTT assay. Sampling. In November 2010, the polished rice was collected from paddy fields adjacent to three abandoned mines (JS, TC, HY) to serve as TEs-contaminated samples (n =5), and from the market to serve as control (n =3). Based on the reports of the Ministry of Environment (2008), the three selected mines have Au-Ag bearing quartz veins, abundant tailings, waste rocks of 10− 200,000 m3, and mine water. Harvested rice samples were

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placed in packing bags, transported to laboratory immediately, and then stored at room temperature. Sample preparation and chemical analysis. The rice samples were air-dried, polished, and then pulverized with a homogenizer (Nihonseiki Kaisha Ltd, Tokyo, Japan) three times for 1 min each time. The standard reference material (SRM) rice flour 1568a (National Standards Institute Technology, NIST, Gaithersburg, MD) and 0.5 g polished rice were transferred into high-pressured polytetrafluoroethylene (PTFE) vessel, and were digested with 8 mL of 70% HNO3 and 1 mL of H2O2 (SigmaAldrich) using microwave digestion system (ETHOS, Milestone Inc., Shelton, CT). After cooling at room temperature, the extracts were filtered with 0.45-μm membrane filter, and adjusted to a final volume of 25 mL. The TEs contents in polished rice were determined by inductively coupled plasma mass spectroscopy (ICP-MS) (Agilent technologies, 7500a, Santa Clara, CA). The SRM accuracy values for As, Cd, and Pb were 0.29±0.05, 0.019±0.001, and 0.01±0.003 mg/kg, with certified values of 0.29±0.03, 0.022±0.002, and <0.01 mg/kg, respectively. Extraction efficiency (%), calculated by dividing the extracted content by total content was 100.58±17.59 for As, 97.40±6.88 for Cd, and 106.15± 1.88 for Pb, respectively. Cell culture and treatment. Human HaCaT cell maintained in DMEM supplemented with 10% FBS, and 1% penicillin-streptomycin was cultured in CO2 incubator under 5% CO2 at 37oC. After growing to 70−80%, confluence, the cell was replaced every 72 h. The DMEM cultured medium was aspirated, and the cell was washed with DPBS, and then treated with 1 mL EDTA-trypsin. The detached cells were counted with hemocytometer (Marienfeld, GMBH & Co. Kg., Lauda-Koenigshofen, Germany), seeded at a density of 1×104/well in DMEM cultured medium, and incubated for 24 h. Polished rice was extracted by modifying the procedure described by Kohlmeyer et al. [2003]. An aliquot of polished rice (0.5 g) was transferred into a 15-mL polypropylene tube, containing 10 mg α-amylase (SigmaAldrich), and dissolved with 3 mL ultrapure Milli-Q® water. The tube was vigorously shaken, and kept at 60oC dry oven for 48 h. The rice extracts were cooled at room temperature, reconstituted into 3 mL with DMEM cultured media, filtered with a 0.2-μm syringe filter and then used for cell toxicity, as well as antioxidants enzyme activity. The rice extracts (100 μL) were exposed to human HaCaT cell, inoculated into 96-well plates with a cell density of 1×104/well and during incubation time kept under the same cultured condition. α-Amylase added into polished rice samples was diluted 1,000 times with

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DMEM-cultured medium, and inoculated into 96-well plates at a final concentration of 3.3 mg/L, dose at which no toxic effects were exerted on human HaCaT cell. The fixed cell was dyed with 100 μL MTT solution at 0.5 mg/ mL dissolved in DPBS, and reacted at the same incubation condition for 4 h. After suction of MTT solution, DMSO (Sigma-Aldrich) of 100 μL was added to plates, and shaken for 30 min. Cell viability was quantified by measuring the absorbance value at 595 nm with a Multilabel counter VICTOR 3 (PerkinElmer Inc., Waltham, MA). Total antioxidants formation. Total antioxidant capacity in human HaCaT cell lysate exposed to polished rice extracts was measured using antioxidant assay kit No. 709001 (Caymanchem, Ann Arbor, MI). The amounts of antioxidants in rice extracts required to inhibit the oxidation of 2,2'-Azino-di-(3-ethylbenzthiazoline sulphonate) (ABTS) to ABTS• + by metmyoglobin were estimated. The amount of ABTS• + produced can be determined by measuring the absorbance at 405 nm, at which antioxidants in the polished rice extracts cause the suppression of absorbance under the reaction condition. The capacity of the antioxidants in rice extracts to prevent ABTS oxidation was compared with that of 6-hyroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox, Cayman chemical Co., Ann Arbor, MI), and quantified as Trolox equivalents. Antioxidant enzyme activity. The human HaCaT cells treated with rice extracts were collected by centrifugation at 1,000 g for 10 min and homogenized with a cold buffer. Subsequently, the cell pellets were used for the measurement of antioxidant enzyme activity according to assay kit manual (Caymanchem). Total activity of SOD (Cu, Zn, and Fe SOD) was assayed by detecting the superoxide radicals generated by xanthine oxidase using tetrazolium salt. The cell lysates were homogenized in cold 20 mM [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.2] containing 1 mM ethylene glycolbis(β-aminoethyl ether)-N,N,N'N-tetraacetic acid, 210 mM mannitol, and 70 mM sucrose. One unit of SOD is defined as the amount of enzyme required to exhibit 50% dismutation of the superoxide radicals measured at 450 nm. The CAT activity was assayed by measuring the formaldehyde produced by reaction of methanol with enzyme in the presence of H2O2. The amount of formaldehyde was determined using purpald as a chromogen at 595 nm. One unit of CAT was defined as the amount of enzyme to cause the formation of 1 nmol formaldehyde per min at 25oC. Statistical analysis. Student T-tests were used to evaluate the significant difference in cell viability, ROS generation, and antioxidants (i.e. SOD and CAT) activities between control and TEs-contaminated polished rice.

Statistical analysis used was performed using SPSS program ver. 18.0 (Chicago, IL), with a probability value of 0.05 or 0.01 being considered statistically significant. Human health risk assessment. Human health risk due to intake of TEs-contaminated polished rice inducing toxicity at the cellular level was assessed by the hazard index (HI) and target hazard quotient (THQ) as noncarcinogenic health risk. The HI was summed to THQ value for estimation of overall health risk for specific receptor or pathway, assuming the additive and interactive effects among TEs constituents in polished rice using the following equation (1) [Hang et al., 2009]. HI=ΣTHQi; i = 1….n

(1)

If HI is less than 1.0, the tested sample is believed to be safe with no cancer toxic risk. If it exceeds 1.0, the health risk due to human dietary exposure of TEs through rice consumption may be assumed to be significant, thus increasing the probability of health risk as the THQ value increases [Zheng et al., 2007]. The THQ value was determined by comparing the estimated average daily dose (ADD) values with oral reference dose (RfD; mg/kg-day) for As, Cd, and Pb exposures using the following equation (2) [Hang et al., 2009]. ADD THQ = ------------RfD

(2)

The RfD values of As, Cd, and Pb were determined using the following equation (3) [Hang et al., 2009]. PTWI RfD = --------------7

(3)

where PTWI is the provisional tolerable weekly intake of As, Cd, and Pb, i.e. 2.1 µg Asi/kg bw/day (FAO/WHO, 1989), 1 µg Cd/kg bw/day (FAO/WHO, 2003), and 3.6 µg Pb/kg bw/day (FAO/WHO, 1993), respectively. The ADD values were determined by both rice contents of As, Cd, and Pb and the amount of rice consumption using the following modified equation (4) [Qian et al., 2010]. Average daily dose (ADD; mg/kg-days) C × IR × AR × EF × ED = ---------------------------------------------------------W AB × AT

(4)

where, C is total contents of As, Cd, and Pb in polished rice (mg/kg); IR is the intake rate of polished rice (kg/ day); AR is absorption rate for rice intake (unitless;1); EF is exposure frequency (365 days/year); ED is the exposure duration (70 years); WAB is the average body weight of exposed person (kg); and AT is the average time

Cytotoxicity on the Toxic Elements-contaminated Polished Rice and Health Risk Assessment

of exposure to polished rice (days). The ADD value was estimated by Monte Carlo simulation using Crystal ball program ver. 11.1.0 (Denver, CO). Probability distribution was randomly sampled to produce various scenarios performing 10,000 iterations. Published input parameters used for estimation of ADD values for the exposures of As, Cd, and Pb were obtained as follows: Average body weight and daily consumption of polished rice were obtained from the fourth Korean National Health and Nutrition Examination Survey database in Korea Centers for Disease Control and Prevention (2008) and from the third Korean National Health and Nutrition Examination Survey-Nutrient survey (I) in Korea Health Industry Development Institute (2006). The proportion of inorganic As (Asi) of 57.4% to total As content was used in estimating the ADD value for As exposure [Paik et al., 2010].

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Results and Discussion

Fig. 1. Human HaCaT cell toxicity in polished rice adjacent to abandoned mines and control. Values indicate the mean ± SD in three independent experiments. Asterisk indicates that significant difference in HaCaT cell viability between samples and control (*p <0.05, **p <0.01).

Effects of contaminated polished rice on human HaCaT cell. The effects of the polished rice grown in adjacent abandoned mines to human HaCaT cell viability were estimated during various exposure times (Fig. 1). The HaCaT cell viability in TEs-contaminated polished rice decreased in a time-dependent manner. The contamination of polished rice inhibited the HaCaT cell viability by 5− 25%. A slight reduction in HaCaT cell viability was observed in TEs-contaminated polished rice at 24 and 48 h, and no significant difference was shown between control and samples (p >0.05). The viability of HaCaT cell exposed to TEs-contaminated polished rice at 96 h was significantly inhibited to the maximum value of 25% than that exposed to control (p <0.05, p <0.01). Particularly, the TEs-contaminated polished rice cultivated in adjacent to JS mine significantly reduced the HaCaT cell viability to 20−25% at 72 and 96 h (p <0.01). These results implied that human intake of polished rice, which induces such toxicity on the HaCaT cell exert potential health risk. The polished rice samples selected in our study were contaminated with high As level over the range of 238− 369 μg/kg, which were 5−7 times higher than those in control. These levels were as high as those in heavily Ascontaminated district of Bangladesh [Alam et al., 2002], and exceeded the global normal As content in the range of 0.082−0.202 mg/kg [Zavala and Duxbury, 2008]. On the contrary, the average contents of Cd and Pb respectively ranged from 10−140 and 2−160 μg/kg, which were below the safety guideline (0.2 mg/kg) established by Korea Food Drug Administration (2006). Moreover, the Cd and Pb contents in control were estimated to be low at 40 and 70 μg/kg, respectively. Highly As-contaminated polished

rice may cause chronic toxic effects via intake, i.e. skin lesion, abnormal pigmentation, skin manifestation, keratosis, and skin cancer development [Sun et al., 2006]. In this respect, cell proliferation on arsenic species exposed to human HaCaT cell was evaluated at 48 h (Supplementary material Fig. 1). The proliferation in HaCaT cell was inhibited in a concentration-dependent manner for As species, and a significant relationship was observed between their concentrations and HaCaT cell proliferation (p <0.0001). As the concentration of various arsenic species increased from 10−6 to 10−2 M, the HaCaT cell proliferation dramatically decreased in As(III) and As(V) forms, whereas slowly decreased in MMA and DMA species. Effective concentration to cause 50% inhibition (EC50) on the proliferation in human HaCaT cell was as follows: As(III) (0.09 mM) > As(V) (0.18 mM) > MMA (2.28 mM) > DMA (4.57 mM). The lowest EC50 value for As (III) was observed at 0.09 mM, indicating the greatest inhibition of human HaCaT cell. The As(III) concentration to cause 50% reduction in the viability of HaCaT cell exposed at 72 h postincubation is reported to be 0.05 mM [Graham-Evans et al., 2003]. Based on EC50, As(V) showed 2-fold lower toxicity than As(III). Vega et al. [2003] reported that the human keratinocytes cell proliferation after exposure to As(V) was inhibited at 2fold higher concentration than that to As(III). As(III) and As(V) did not inccur the cell proliferation at a low concentration of 1 mM; however, at same exposure concentration, MMA and DMA presented the HaCaT cell proliferation of above 75%. On the other hand, a significant decrease of 50% in cell proliferation was

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Fig. 2. Total antioxidants formation in human HaCaT cell lysate exposed to toxic elements (TEs)-contaminated polished rice and control. Values indicate the mean ± SD in three independent experiments. Asterisk indicates the significant difference in total antioxidants production (mM) between samples and control at 0.01 probability level.

observed at 25−50-fold higher concentrations in MMA and DMA than those of As(III) and As(V). Total antioxidant generation. Total antioxidants were measured to examine whether the inhibition of HaCaT cell viability in TEs-contaminated polished rice was associated with oxidative stress (Fig. 2). Total antioxidants in HaCaT cell treated with TEs-contaminated polished rice more decreased in a time-dependent manner than those of control. The concentrations of total antioxidants in polished rice extracts showed a slight decrement at 24 h, postincubation and were significantly reduced to 13− 22 mM at 72 h (p <0.01) in the following order: Control (37.8 mM) >> HY-2 (22.4 mM) > HY-1 (20.7 mM) > TC2 (19.4 mM) > TC-1 (15.9 mM) > JS (13.4 mM). This result demonstrated that the viability of HaCaT cell exposed to TEs-contaminated polished rice was involved in oxidative damage, with decreasing antioxidant capacity. Several studies have reported that heavy metal-induced plants cause cellular damage in the structure and function by the mechanism of oxidative stress [Mascher et al., 2002; Gratão et al., 2005; Shri et al., 2009; Singh et al., 2009]. Singh et al. [2009] reported that the root of Oryza sativa (rice) due to As exposure at 25−50 μM significantly enhances the formation of superoxide as well as hydrogen peroxide compared to untreated control. Antioxidant enzyme activity. Antioxidant enzymes, i.e. SOD and CAT activities, to defend against ROS generation in TEs-contaminated polished rice were evaluated (Fig. 3). The SOD activities in TEscontaminated polished rice were observed in a timedependent manner (Fig. 3A). SOD activities were similar to those of control at 24 h; however, showed significant

Fig. 3. Superoxide dismutase (A) and catalase (B) activity induced by TEs-contaminated polished rice and control. Values indicate the mean ± SD in three independent experiments. Asterisk indicates that significant difference in HaCaT cell viability between samples and control (*p <0.05, **p <0.01).

enhancement at 72 h postincubation compared to control (p <0.05; p <0.01). The SOD activities were induced in the following order: JS (175.8 U/mL) > HY-1 (171.5 U/ mL) > HY-2 (164.2 U/mL) > TC-1 (163.7 U/mL) > TC-2 (153.8 U/mL), showing a significant 4-fold induction compared to that of control (46.5 U/mL). The activities of SOD enzyme significantly increase in rice roots treated with As concentrations of 25 and 50 μM [Singh et al., 2009]. Time-dependent CAT activities were likewise induced in TEs-contaminated polished rice (Fig. 3B). The activities of CAT at 72 h postincubation were as follows: JS (24.7 mmol/min/mL) > HY-1 (24.1 mmol/min/mL) > TC-1 (22.9 mmol/min/mL) > HY-2 (22.8 mmol/min/mL) > TC-2 (19.7 mmol/min/mL) >> control (13.0 mmol/min/ mL), indicating that TEs-contaminated polished rice significantly stimulated the CAT activity to a maximum of 2-fold higher than that of control (p <0.01). The greater the CAT activity was generated in TEs-contaminated polished rice samples, the more the total antioxidants were decreased. Increased activity of various scavenging

Cytotoxicity on the Toxic Elements-contaminated Polished Rice and Health Risk Assessment

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Table 1. The average daily dose (ADD) value through human intake of TEs-contaminated polished rice and health risk Mines

As-ADD (mg/kg-day) Mean

P95b

Cd-ADD (mg/kg-day) Mean

P95b

Pb-ADD (mg/kg-day) Mean

P95b

As-THQa

Cd-THQa

Pb-THQa

HI c

Mean

P95b

Mean

P95b

Mean

P95b

Mean

P95b

0.27 0.34 0.34 0.37 0.42 0.06

0.43 0.54 0.55 0.59 0.67 0.09

0.28 0.09 0.04 0.56 0.14 0.17

0.44 0.14 0.06 0.90 0.22 0.27

0.00 0.02 0.15 0.18 0.08 0.07

0.00 0.04 0.24 0.29 0.12 0.12

0.55 0.45 0.54 1.11 0.63 0.30

0.88 0.72 0.86 1.77 1.01 0.47

JS 5.6×10-4 9.0×10-4 2.8×10-4 4.4×10-4 7.8×10-6 1.2×10-5 TC-1 7.1×10-4 1.1×10-3 8.7×10-5 1.4×10-4 7.9×10-5 1.3×10-4 TC-2 7.2×10-4 1.2×10-3 3.9×10-5 6.3×10-5 5.6×10-4 8.8×10-4 HY-1 7.7×10-4 1.2×10-3 5.6×10-4 9.0×10-4 6.6×10-4 1.0×10-3 HY-2 8.7×10-4 1.4×10-3 1.4×10-4 2.2×10-4 2.8×10-4 4.4×10-4 Control 1.2×10-4 1.9×10-4 1.7×10-4 2.7×10-4 2.7×10-4 4.3×10-4 a

95th percentile The THQ was determined by dividing the ADD value of As, Cd, and Pb into respective provisional tolerable daily intake (PTDI), which are 2.1 µg Asi/kg bw/day (FAO/WHO, 1989), 1 µg Cd/kg bw/day (FAO/WHO, 2003), and 3.6 µg Pb/kg bw/ day (FAO/WHO, 1993) c The HI was summed to THQ value for estimation of overall health risk for specific receptor or pathway, assuming the additive and interactive effects among TEs constituents in polished rice b

enzyme is required to prevent cellular damages by ROS formation [Gratão et al., 2005]. This was consistant with the previous results that As-induced stress plants significantly stimualted the activities of scavenging enzymes to overcome the oxidative damage [Mascher et al., 2002]. Stimulated activities of SOD and CAT enzymes were positively related to the changes in ROS generation, confirming that accumulation of TEs in polished rice induced these enzyme activities and thereby prevented the oxidative damage. The catalysis of SOD invokes scavenging of the superoxide radicals (O2− •) into hydrogen peroxide (H2O2), which can be partially converted into H2O and O2 via CAT activity in ascorbateglutathione cycle of the cytosol and chloroplasts. Antioxidant enzyme plays an important role in regulating the level of ROS, which induces damages of cell structure and function [Noctor and Foyer, 1998]. Health risk assessment of contaminated polished rice intake. Health risk probability was assessed under the assumption that TE-contaminated polished rice causes cytotoxicity and enhances the antioxidants enzyme activity when consumed by human population. Human exposure via intake of TE-contaminated polished rice to induce oxidative stress on HaCaT cell was estimated by using the ADD value, and corresponding health risk was also determined by THQ as well as HI (Table 1). The health risk was evaluated for respective As, Cd, and Pb, which are classified into human carcinogen and probable human carcinogen based on United States Environmental Protection Agency Integrated Risk Information System (US EPA IRIS) database. Such compounds are recognized as potentially toxic elements (PTEs) to be persistent in the environment [Zhu et al., 2008]. The contents of As, Cd, and Pb in polished rice and the amount of rice

consumption were mainly used in estimating the ADD values. The ADD values at mean and 95th percentile via consumption of contaminated polished rice ranged from 0.56 to 1.40 for As, from 0.04 to 0.90 for Cd, and from 0.008 to 1.00 for Pb μg/kg bw-day, all of which are well below the provisional daily tolerable daily intake (PTDI) value for each toxic elements (FAO/WHO, 1989; 1993; 2003). For human exposure to As, the contaminated polished rice showed the seven times greater ADD value than that of control estimated to be at 0.12 mg/kg-day. The THQ values for polished rice polluted with As, Cd, and Pb ranged from 0.27 to 0.42, 0.04 to 0.56, and 0.00 to 0.18, respectively. On the contrary, THQ values of the polished rice cultivated in control were estimated to be 0.06, 0.17, and 0.07 in As, Cd, and Pb, respectively. THQ values of As, Cd, and Pb showed decreasing trend, indicating that there is a greater health risk from human intake of polished rice with high As content and minimum risk for Pb intake. Human exposure to polished rice contaminated with As, Cd, and Pb may not cause non-carcinogenic toxic effects if THQ is below 1.0. Such inorganic compound exists in mixtures, allowing additive or antagonistic response by their interactions according to US EPA reports (2004). Should this be similar mechanism for human beings exposed to PTEs, the mean HI values summed to respective THQ value of As, Cd, and Pb from nearby HY-1 sites exceeded 1.0. Moreover, the excessive consumption of polished rice produced from nearby HY2 sites may also pose potential toxic effects. This demonstrated that polished rice cultivated around HY mine will lead to possible non-carcinogenic health risk. In other abandoned mines, intake of TEs-contaminated polished rice was not expected to cause non-carcinogenic health risk by triggering toxicity on human HaCaT cell.

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Because the HI result in control fell below 1.0, there was definitely little possibility of its toxic effects on humans. These results could be overestimated or underestimated in assessing actual health risk through intake of the TEcontaminated polished rice. The bioavailable TEs can be released during digestion process in gastric and gastrointestinal phases, and cause adverse toxic risk. On the contrary, they may be transformed into a less toxic form by enzyme activity during metabolic process. Considering these aspects, bioaccessibility research group of Europe (BARGE) has developed the unified bioaccessibility method (UBM) and applied bioaccessibility (%) using gastric and gastrointestinal extraction method to assess reliable health risk [Cave et al., 2006]. Our results are thought to be reliable in considering the proportion of the bioavailable form and its absolute bioavailability reported by Juhasz et al. [2006]. Acknowledgment. This study was supported by the 2011 Post Doctoral fellowship Program (PJ006446042011) of the National Academy of Agricultural Science, Rural Development Administration, Republic of Korea.

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