Article

Ameliorative effects of docosahexaenoic acid on the toxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in cultured rat hepatocytes

Toxicology and Industrial Health 1–12 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714547382 tih.sagepub.com

Hasan Turkez1, Fatime Geyikoglu2 and Mokhtar I Yousef3 Abstract The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant toxicant that mediates carcinogenic effects associated with oxidative DNA damage. Docosahexaenoic acid (DHA) with antioxidant functions has many biochemical, cellular, and physiological functions for cells. The present study assessed, for the first time, the ameliorative effect of DHA in alleviating the toxicity of TCDD on primary cultured rat hepatocytes (HEPs). In vitro, isolated HEPs were incubated with TCDD (5 and 10 M) in the presence and absence of DHA (5, 10, and 20 M) for 48 h. The cell viability was detected by 3-(4,5-dimethylthiazol-2-yl) 2,5diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release. DNA damage was analyzed by liver micronucleus assay and 8-oxo-2-deoxyguanosine (8-OH-dG) level. In addition, total antioxidant capacity (TAC) and total oxidative stress (TOS) were assessed to determine the oxidative injury in HEPs. The results of MTT and LDH assays showed that TCDD decreased cell viability but not DHA. On the basis of increasing treatment concentrations, the dioxin caused significant increases of micronucleated HEPs and 8-OH-dG as compared to control culture. TCDD also led to significant increases in TOS content. On the contrary, in cultures treated with DHA, the level of TAC was significantly increased during treatment in a concentration-dependent fashion. DHA showed therapeutic potential against TCDD-mediated cell viability and DNA damages. As conclusion, this study provides the first evidence that DHA has protective effects against TCDD toxicity on primary cultured rat hepatocytes. Keywords Docosahexaenoic acid, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin, liver, cell viability, micronucleus assay, 8-oxo-2deoxyguanosine, antioxidant capacity, oxidative stress, rat

Introduction 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that elicits a broad spectrum of toxic effects in a species-specific manner (Dere et al., 2011). It displays a wide spectrum of toxic effects, including dermal toxicity, immunotoxicity, genotoxicity, carcinogenicity, teratogenicity, neurobehavioral, endocrine, and metabolic alterations (Hung et al., 2006; Turkez et al., 2012d, 2012e). The relative potencies for TCDD have been reported in previous studies (Van den Berg et al., 1998). These studies have suggested that the potential toxic effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR), and oxidative stress is an important

constituent in the mechanism of TCDD toxicity. The exposures of mice and rats to different concentrations of TCDD have resulted in increase in the production

1

Department of Molecular Biology and Genetics, Faculty of Science, Erzurum Technical University, Erzurum, Turkey 2 Department of Biology, Faculty of Science, Atat¨urk University, Erzurum, Turkey 3 Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt Corresponding author: Fatime Geyikoglu, Department of Biology, Faculty of Science, Atat¨urk University, Erzurum 25240, Turkey. Email: [email protected]

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of reactive oxygen species (ROS), lipid peroxidation (LP), and DNA damage (Hassoun et al., 2000; Reyes-Herna´nde et al., 2010; Turkez and Geyikoglu, 2011; Turkez et al., 2012a, 2012b). Hence, TCDD has been shown to be responsible for multisite cancers in experimental animals (Bock, 1994; Grassman et al., 1998). However, in vivo and in vitro studies of human and animal cells have provided inconsistent findings of genetic toxicity of TCDD. On the other hand, there are equivocal findings of chromosomal aberrations in humans exposed in vivo to TCDD (IARC, 1997) and the increases in production 8-OH-dG in the liver of mice and rats (Hung et al., 2006). Recently, it is reported that dioxin-like chemicals alter expression of numerous genes in liver, but it remains unknown which lie in pathways leading to major toxicities such as hepatotoxicity, wasting, and lethality (Forgacs et al., 2010). Docosahexaenoic acid (DHA) is an omega-3 fatty acid. Oils from cold water fish are rich in (n3) polyunsaturated fatty acids, in particular, DHA (Eicher and McVey, 1995). Evidence presented over the past 20 years has shown that long-chain polyunsaturated fatty acids especially the DHA are beneficial for health (Kruger et al., 2010). Diets rich in n3 polyunsaturated fatty acids have been associated with a reduced risk of several types of cancer (Slagsvold et al., 2010). Dietary DHA also reduced the risk of heart disease by reducing the high level of blood triglycerides in humans (Weitz et al., 2010). DHA plays an important role in fetal and infant brain development, including neurocognitive and neuromotor functions (Harper et al., 2011). This fatty acid can act as hepatoprotective agent (Roy et al., 2007). DHA has therapeutic properties such as antimicrobial (Martinez et al., 2009), anti-inflammatory, immunomodulatory (Gapeyev et al., 2011), antioxidant (Arnal et al., 2009), and antitumor (Dupertuis et al., 2007). Despite the valuable biological activities of DHA, however, it always demonstrates considerable biological activity, especially antioxidant activity (Takayama et al., 2010). DHA concentration dependently scavenges the intracellular radical productions induced by oxidative or hypoxic stress (Shimazawa et al., 2009; Turkez et al., 2012b). For this reason, DHA attracts the attention of scientists to search for new therapeutic usage. Antioxidants play an important role in inhibiting and scavenging free radicals, thus providing protection to humans against infectious and degenerative diseases (Nader et al., 2010). According to the above data, TCDD is involved in the production of hepatic oxidative stress, and

supplementation with DHA in the animal’s diet may protect from the harmful effects of TCDD. However, no attention was paid to the effects of DHA in hepatoprotection against TCDD; and also the information regarding DHA upon liver micronucleus assays (LMNs) in hepatocyte (HEP) cells remains unknown. Therefore, in our present study, we examined the protective effect of DHA on the viability of HEPs (with lactate dehydrogenase (LDH) and 3-(4,5dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assays) in TCDD-induced liver injury. We also evaluated the role of DHA on antioxidant capacity (with total antioxidant capacity (TAC) and total oxidative stress (TOS) analysis) and DNA damage (with LMN rates and 8-oxo-2-deoxyguanosine (8-OH-dG) levels) after TCDD treatment to the HEP cultures.

Materials and Methods Test compounds and chemicals TCDD (CAS no. 1746-01-6) and DHA (CAS no. 6217-54-5) were purchased from Sigma-Aldrich1 (St Louis, Missouri, USA). All other chemicals used in the experiments were purchased from SigmaAldrich and Fluka1 (Germany).

Animals Male rats of Sprague Dawley strain (from Medical Experimental Research Center, Ataturk University, Turkey), weighing 200–300 g body weight, were used throughout the study. They were allowed water and standard laboratory chow ad libitum and were maintained under standard light, temperature, and relative humidity conditions. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NRC, 1996). The study protocol was approved by the local ethical committee.

HEP isolation and culture Rats were killed by carbon dioxide (CO2) overdose, and the livers were removed immediately. Isolated HEPs from rats were prepared by the collagenase perfusion technique (Wang et al., 2002). The liver was perfused through the hepatic portal vein with calcium-free Hanks balanced salt solution to remove blood for about 10 min at a flow rate of 2.5 mL/min. As soon as the liver became grayish-brown in color, a second buffer solution containing collagenase (Hank’s balanced salt supplemented with 4 mM calcium chloride

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and 0.5 mg collagenase/mL) was perfused at the same rate until the liver appeared to have broken up. After treatment, the liver was minced into 3- to 4-mm pieces with a sterile scalpel. Following mechanical dissociation, the cells were filtered through gauze and centrifuged at 1350 r/min for 5 min. Then, the HEPs were collected in medium containing bovine serum albumin and bovine insulin. The cell suspension was filtered through gauze again and allowed to sediment for 20 min to eliminate cell debris, blood, and sinusoidal cells. The cells were then washed three times, followed by centrifugation at 50g, and tested by trypan blue dye exclusion for viability (always in the range of 82–93%). The HEPs were then suspended in a mixture of 75% Eagle’s minimum essential medium and 25% medium 199, supplemented with 10% fetal calf serum containing streptomycin, penicillin, bovine insulin, bovine serum albumin and sodium bicarbonate (2.2 mg). For the experimental procedure, HEPs were plated in multiwell tissue culture plates (3  105 cells in a well area of 3.8 cm2; 8  105 cells in a well area of 9.6 cm2). The medium was changed 3–4 h later. The effect of TCDD and DHA was studied after 48 h of exposure in cultures maintained with a medium deprived of fetal calf serum but supplemented with hydrocortisone hemisuccinate (7  107 M) (Rakba et al., 1999). HEPs were cultured for an additional 8 h before treatment.

Treatments Since we examined the action of co-treatment of DHA on TCDD-induced hepatotoxicity, after 8 h of plating, when primary HEPs got adhered and attained their epithelial morphology, culture media was aspirated and replaced with an equal volume of the media supplemented with different concentrations of TCDD (5 and 10 M) and DHA (5, 10, and 20 M), followed by incubation in CO2 incubator for 48 h (n ¼ 7). This investigation stems from the works of Bechoua et al. (1999) and Katic et al. (2010).

MTT assay Viability of cells was assessed by measuring the formation of a formazan from MTT spectrophotometric assay test, modified after Mosmann’s (1983) study. HEPs were incubated with 0.7 mg/mL MTT for 30 min at 37 C at the end of the experiment. After washing with phosphate-buffered saline, the blue formazan was extracted from cells with isopropanol/

formic acid (95:5) and was photometrically determined at 560 nm (Lewerenz et al., 2003).

LDH assay LDH activity was measured in the culture medium as an index of cytotoxicity, employing an LDH kit (Bayer Diagnostics1, France) adapted to the auto analyzer (ADVIA 1650, MA, USA). Enzyme activity was expressed as the extracellular LDH activity percentage of the total activity on the plates.

TAC and TOS assays The automated Trolox-equivalent TAC and TOS assays were carried out in the culture medium by commercially available kits (Rel Assay Diagnostics1, Turkey). The major advantage of the TAC assay is that it measures the antioxidant capacity of all antioxidants in a biological sample and not just of a single compound. In this test, antioxidants in the sample reduce dark bluish-green colored 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonate radical to its colorless form. The change in absorbance at 660 nm corresponds to the total antioxidant level in a sample. The assay is calibrated with a stable antioxidant standard solution of vitamin E analog, (Trolox equivalent). The TOS assay used here is based on the oxidation of the ferrous ion–chelator complex to ferric ion (Fe3þ), which is mediated by oxidants contained in the tested sample. The reaction is further enhanced by other molecules from the reaction medium. The reaction of Fe3þ with chromogen in an acidic medium produces a colored complex. Its intensity corresponds to the total amount of oxidants in the sample and can be measured spectrophotometrically. The TOS assay is calibrated with hydrogen peroxide (H2O2), and the results are expressed in terms of micromolar H2O2 equivalent per liter (Erel 2004).

LMN assay LMN assay was carried out by using the method described by Suzuki et al. (2009). Immediately prior to evaluation, 10–20 L of HEP suspension was mixed with an equal volume of acridine orange (AO)–40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) stain solution (AO, 0.5 mg/mL; DAPI, 10 g/mL) for fluorescent staining. Approximately 10–20 L of the mixture was dropped onto a glass slide and covered with a cover glass. Samples of well-isolated HEPs were evaluated with the aid of a

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Figure 1. MTT reduction in rat hepatocyte cultures maintained at 48 h in the presence of TCDD, DHA, and their combinations. TCDD 1 (5 M TCDD); TCDD 2 (10 M TCDD); DHA 1 (5 M DHA); DHA 2 (10 M DHA); and DHA 3 (20 M DHA); means (n ¼ 7) in the figure followed by the different letters present significant differences at the p < 0.05 level. MTT: 3-(4,5-dimethylthiazol-2-yl) 2,5diphenyltetrazolium bromide; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; DHA: docosahexaenoic acid.

Figure 2. Extracellular level of LDH in rat hepatocyte cultures maintained at 48 h in the presence of TCDD, DHA, and their combinations. TCDD 1(5 M TCDD); TCDD 2 (10 M TCDD); DHA 1 (5 M DHA); DHA 2 (10 M DHA); and DHA 3 (20 M DHA); means (n ¼ 7) in the figure followed by the different letters present significant differences at the p < 0.05 level. LDH: lactate dehydrogenase; TCDD: 2,3,7,8tetrachlorodibenzo-p-dioxin; DHA: docosahexaenoic acid.

Statistics fluorescence microscope counting the number of micronucleated HEPs (MNHEPs) in 2000 HEPs for each animal. MNHEPs were defined as HEPs with round or distinct micronuclei (MNs)stained like the nucleus, with a diameter one-fourth or less than that of the nucleus and confirmed by focusing up and down, taking into account HEP thickness by one observer.

The experimental data were analyzed using one-way analysis of variance and Fischer’s least significant difference tests to determine whether any treatment significantly differed from the controls or each others. The results presented as mean + SD values, and the level of 0.05 was regarded as statistically significant.

Results Nucleic acid oxidation DNA oxidation was determined by measuring the amount of 8-OH-dG adducts. DNA was digested by incubation with DNAase I, endonuclease, and alkaline phosphatase (Schneider et al., 1993). The amount of 8-OH-dG was measured by high-performance liquid chromatography with multichannel electrochemical detection as described previously (Floyd et al., 1993). The compound was separated on a Waters S-3 4.6  150 mm column with 5% methanol/95% 100 mM sodium acetate buffer (pH 5.2) at a flow rate of 1.0 mL/min. The four electrochemical detector channels were set at 100, 250, 475, and 875 mV.

The results of cell viability measured by MTT assay is shown in Figure 1. When assayed in vitro on the HEP cells using the MTT assay, the values for the 5 and 10 M TCDD-treated cells ranged from 1.3-to 2.4-fold lower than that for the primary rat HEPs, respectively. However, the three concentrations of DHA (5, 10, and 20 M) provided in vitro activities on cell viability against the tested TCDD compound, and no cytotoxicity was reported against the control cells. Then, 5 and 10 M TCDD-induced hepatocellular damages were clearly evidenced by 5- and 12-fold increases in LDH compared with the observations of controls (Figure 2). Although LDH was not affected

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Table 1. Extracellular TAC and TOS levels in cultured rat hepatocytes maintained at 48 h in the presence of TCDD, DHA, and their combinations.a TAC (mmol Trolox equiv./L)

Treatments Control TCDD 1 TCDD 2 DHA 1 DHA 2 DHA 3 TCDD 1 TCDD 1 TCDD 1 TCDD 2 TCDD 2 TCDD 2

þ DHA þ DHA þ DHA þ DHA þ DHA þ DHA

1 2 3 1 2 3

5.04 4.11 3.40 5.94 6.22 6.89 4.76 5.01 5.14 4.32 4.61 4.93

b

+ 0.47 + 0.38d + 0.43c + 0.39e + 0.42e,f + 0.53g + 0.45b,d + 0.48b + 0.39b + 0.47d + 0.50b,d + 0.51b

TOS (mol H2O2 equiv./L) 8.26 + 11.82 + 16.41 + 8.18 + 8.27 + 8.21 + 10.08 + 9.57 + 8.36 + 13.71 + 12.20 + 9.41 +

c

2.14 2.61b,d 2.93e 2.06c 2.19c 2.28c 3.11d 2.43d 2.35c 3.09b 3.14b,d 2.83d

TAC: total antioxidant capacity; TOS: total oxidative stress; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; DHA: docosahexaenoic acid; H2O2: hydrogen peroxide. a TCDD 1(5 M TCDD); TCDD 2 (10 M TCDD); DHA 1 (5 M DHA); DHA 2 (10 M DHA); and DHA 3 (20 M DHA); means (n ¼ 7) in the table followed by the different letters present significant differences at the p < 0.05 level.

by different concentrations of DHA alone, the decrease in the level of enzyme reached statistical significance at 5, 10, and 20 M concentrations of DHA against TCDD toxicity. Table 1 shows the effects of DHA on biochemical parameters in the tissue cells of all experimental groups. The hepatic TAC level decreased (p < 0.05) in the TCDD-administered group. Moreover, TOS levels increased in culture cells due to the effect of TCDD. The HEPs of control groups maintained optimal value of the antioxidant status studied. On the other hand, the cells treated with 5, 10, and 20 M of DHA alone showed increases in the levels of antioxidant capacity. However, TOS levels unchanged in both control and DHA groups. Moreover, application of DHA at all concentrations significantly (p < 0.05) increased the reduced TAC ratio by TCDD. Table 2 shows the results of the LMN assay in rat HEP cultures. The tested concentrations of TCDD induced statistically significant increases in formations of MNHEPs although DHA (at all concentrations) did not change the MNHEPs number as compared to the control group. Moreover, DHA applications minimized the increased MNHEPs rates by TCDD (Figure 3). The status of 8-OH-dG in liver cells of control and experimental groups is presented in Figure 4. First, the

Table 2. Results of liver MN assay in cultured rat hepatocytes maintained at 48 h in the presence of TCDD, DHA, and their combinations.a Treatments Control TCDD 1 TCDD 2 DHA 1 DHA 2 DHA 3 TCDD 1 þ TCDD 1 þ TCDD 1 þ TCDD 2 þ TCDD 2 þ TCDD 2 þ

MNHEP (%)/2000 HEP

DHA 1 DHA 2 DHA 3 DHA 1 DHA 2 DHA 3

0.25 + 0.86 + 1.74 + 0.17 + 0.26 + 0.23 + 0.65 + 0.52 + 0.44 + 1.28 + 0.91 + 0.78 +

0.08b 0.27c 0.35d 0.16b 0.08b 0.15b 0.23c,e 0.14e 0.26e 0.38c,d 0.41c 0.35c

MN: micronucleus; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; DHA: docosahexaenoic acid; HEP: hepatocyte; MNHEPs: micronucleated hepatocytes. a TCDD 1(5 M TCDD); TCDD 2 (10 M TCDD); DHA 1 (5 M DHA); DHA 2 (10 M DHA); and DHA 3 (20 M DHA); means (n ¼ 7) in the table followed by the different letters present significant differences at the p < 0.05 level.

Figure 3. (a) A sample hepatocyte from 20 M of DHAtreated culture, (b) from control culture, (c) from 10 M of TCDD-treated culture (arrow shows MN formation), and (d) from cultures of hepatocytes co-treated with TCDD and DHA for 48 h. (magnification: 1000). TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; DHA: docosahexaenoic acid; MN: micronucleus.

HEP levels of 8-OH-dG, a sensitive marker of oxidative DNA damage, were quantified with regard to TCDD treatment concentration. It was observed that TCDD significantly increased 8-OH-dG concentrations in the

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Figure 4. 8-OH-dG adducts in rat hepatocyte cultures maintained at 48 h in the presence of TCDD, DHA, and their combinations. TCDD 1(5 M TCDD); TCDD 2 (10 M TCDD); DHA 1 (5 M DHA); DHA 2 (10 M DHA); and DHA 3 (20 M DHA); means (n ¼ 7) in the figure followed by the different letters present significant differences at the p < 0.05 level. 8-OH-dG: 8-oxo-2-deoxyguanosine; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; DHA: docosahexaenoic acid.

liver cell. Whereas DHA did not have any effect on 8OH-dG levels at increasing concentrations. Moreover, DHA significantly decreased 8-OH-dG concentrations in TCDD-treated HEPs in a concentrationrelated manner.

Discussion Experimental studies support the role of oxidative stress in TCDD toxicity (Shertzer, 2010). In this article we demonstrated that TCDD administration in rat HEP cultures caused oxidative stress, cytotoxicity, and also oxidative DNA damages. It is emphasized that the antioxidants (both enzymatic and nonenzymatic) play a central role in cellular oxidant defense systems that protect cells against damage induced by free radicals, such as superoxide anion and H2O2 (Valko et al., 2005). It is observed that TAC is significantly lowered in TCDD-treated rat HEPs. As known, TAC comes from nonenzymes like glutathione (GSH) as well as enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (Murri et al., 2010). GSH is one of the most abundant intracellular antioxidants in animal cells. Since it is considered to be one of the most abundant tripeptides in human organism, and its action against xenobiotics and oxidative radicals is well known (Zachariadis and Rosenberg, 2013). In mice, exposure to TCDD results in significant depletion of GSH by oxidative stress in liver (Slezak et al., 2002). TCDD toxicity can also be decreased by SOD and CAT in neutralizing ROS. SOD

is the primary step of defense mechanism in the antioxidant system against oxidative stress. It
catalyses the dismutation of superoxide radicals O 2 into molecular oxygen (O2) and H2O2 (Kakarla et al., 2005). CAT activity is decreased in HEPs during TCDD treatment, and its activity is inhibited by the superoxide radical (Kono and Fridovich, 1982). The suppression of these enzyme activities has been linked to induction of oxidative stress (El-Tawil and Elsaieed, 2005) and hepatotoxicity. In addition, the observed decreases in GSHrelated enzyme activities indicate that TCDD may induce oxidative stress in rat liver by altering GSH metabolic mechanisms at the cellular level (Twaroski et al., 2001). GSH-Px can protect DNA and lipids of the cell against the peroxidation products (Bukowska, 2004). The observed decline in the activity of GSH-Px in TCDD-treated HEPs may be ascribed to the reduction in the level of GSH and an increase in the level of peroxides (Twaroski et al., 2001). Thus, the balance of this enzyme system may also be essential to remove superoxide anion and peroxides generated in HEPs. The relative potency of TCDD is also estimated by comparing different concentration levels for a particular response. The concentration dependency of TAC suppression in liver is driven by the range of concentration–response data available in our study. The studies provide some evidence that may be concentration dependency of the TCDD and TCDD toxicity in tissues is related to increasing concentration (Slezak et al., 2002).

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The relative potency of TCDD in cells may be modulated by binding to proteins (De vito et al., 1995). This study supports that the TCDD toxicity is highly correlated with the decreasing TAC in liver. In the present study, the decreased antioxidant status against increased TOS may be due to TCDDinduced protein dysfunction in rat HEPs as a result of oxidative stress or direct inhibition of the enzyme activities by ROS. The protein damage may cause the decline in enzyme activities of the antioxidant system and probably the electron-transporting system, thus leading to excessive ROS generation in HEPs (Meister and Anderson, 1983). TCDD is a human carcinogen affecting target organs (Pelclova et al., 2011). TCDD-induced oxidative stress causes 8-OH-dG and single-strand breaks in DNA from liver and brain tissues of rats (Hassoun et al., 2000; Hung et al., 2006). In our investigation, 8OH-dG significantly increased by the effect of TCDD. In addition, MN test is performed for the first time and is revealed that exposure to TCDD increases the rate of MNHEPs formation in HEPs. The MN assay is a mutagenic test for the detection of chemical agents which induce the formation of small membrane-bound DNA fragments, that is, MNs in the cytoplasm of cells (Sisman and Turkez, 2010; Turkez, 2008, 2011; Turkez et al., 2007), and MNHEPs production as 8-OH-dG is one of the key pieces of evidence for the possible involvement of antioxidant activity in oxidative DNA damage (Tung-Kwang et al., 2010; Turkez et al., 2010). The MN count also gives a measure of cytogenetic damage induced by genotoxic agents (Fenech, 2000). ROS can alter vital cell components like polyunsaturated fatty acids, proteins, and nucleic acids (Halliwell and Gutteridge, 1990). The increased production of ROS, LP, and DNA and membrane damage are always associated with TCDD exposure (Hung et al., 2006; Turkez et al., 2012c, 2012f, 2012g, 2012h). If 8-OH-dG remains unrepaired, it can cause high levels of guanine: cytosine to thymine: adenine transversion mutations (Valavanidis et al., 2009). The detrimental effects in hepatic tissue of TCDD may also lead to disruption in the functional integrity of HEPs (Czepiel et al., 2010). In the present study, the TCDD elicits severe instances of liver damage (increasing LDH). The LDH in serum as a biological marker for liver damage reflected as increase in the serum level of LDH (Park et al., 2010). The LDH released into the medium provides an index of cell death and membrane permeability to LDH, and an

increase in LDH activity in the medium occurs as a result of cell membrane disintegration and enzyme leakage (Yokogawa et al., 2004). In addition, cytotoxicity, the degree to which a chemical can cause cell damage, is assessed in this study by means of MTT assay. As shown in Figure 1, the MTT assay results revealed that TCDD is cytotoxic to human liver. Overall, TCDD significantly decreases the viability of HEPs. Consistent with our finding, MTT assay demonstrated that the viability of human adrenocortical, pancreatic, and mammary cells is significantly decreased after TCDD treatment (Andersson et al., 2005). The effective antioxidants are free radical scavengers that interfere with radical chain reactions; it is possible to protect cellular DNA from oxidative stress by supplementation with antioxidants (Lee et al., 2010). Studies have been carried out on the role of antioxidant components in the detoxification of TCDD, and it has been established that limited efficacy of AhR agonists was unable to provide sufficient protection without using antioxidants (Hung et al., 2006). In our study, DHA, which has not previously been used as detoxifier, is demonstrated to display beneficial effects on MNHEPs and 8-OH-dG, by returning their values close to those of the control group after TCDD insult. Because DHA alone led to increase in TAC and decrease in TOS levels significantly. This suggests the antioxidant activity of DHA. DHA may be protective against oxidative stressinduced cell damages. The 8-OH-dG appears to play a role in tissue cell injury via the induction of apoptotic cell death (Tsuruya et al., 2003). One way to prevent oxidative biomarker 8-OH-dG is to augment antioxidant enzyme activity in the liver and respiratory system (Yen et al., 2011). It was evident from a previous study that the supplementation of DHA protected subjects against oxidative DNA damage indicated by a reduction of the 8-OH-dG (Dawczynski et al., 2010). Analysis of the main endogenous antioxidant defenses suggested the prevalence of metabolic oxidative pathways leading to the more reactive *OH on exposure to TCDD. The highest degree of genotoxic damage was consistently observed in tissues in which the capacity to scavenge radical was the lowest. These data suggested a general relationship between oxidative stress and loss of DNA integrity (Regoli et al., 2003). Therefore, it is necessary to evaluate the genotoxic potencies of free radicals (Edwards, 1977). The MNHEPs increase with the genotoxic and the prooxidant potential of chemicals (Kim et al.,

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1997). DHA does not induce MN formation in in vitro and in vivo assays (Hammond et al., 2002). Moreover, DHA concentration dependently scavenges the intracellular radical productions induced by H(2)O(2) radical, O(2)*(), and *OH. In the present study, the DHA has also a pronounced effect on the relative potency of TCDD. Noteworthy, it prevents liver damages (with LDH and MTT assays). Liver damage associated with enhanced LP and LDH efflux shows a linear correlation with concentrations of lipid peroxides (Li et al., 2012). The degradative effect of DHA is determined by the abundance of fatty acids that can be attacked to form lipid peroxides in liver. Thus, the dietary supplementation with DHA induces the expected significant changes in plasma and mitochondrial membrane fatty acid composition (Andreo et al., 2011). It also significantly influences their resistance to calcium ions and prooxidative agents (Stavrovskaya et al., 2012). Cell viability was measured by the MTT (metabolic activity) assay estimation for the mitochondria structural integrity (Knazicka et al., 2013). TCDD-mediated oxidative stress is responsible for mitochondrial damage and thus apoptosis (Forgacs et al., 2010; Shen et al., 2005). TCDD significantly increases LP in hepatic mitochondria and enhanced LP occurs with the increased oxidative stress (Alsharif et al., 1994). In conclusion, TCDD induces increases in mitochondrial respiration and decrease mitochondrial membrane fluidity (Shertzer, 2010). On the contrary, brain, cardiac, and liver mitochondria fatty acids are highly regulated by diet. The DHA induces dramatic beneficial changes with the inhibition of cell death cascades that involve mitochondria (Pepe, 2005). Some authors have underlined the occurrence of alterations in enzyme activities (Kim and Chung, 2007). DHA increased mitochondrial SOD activity in the liver, uterus, and cerebrum of rats. On the other hand, Richard et al. (2008) showed that DHA is more active as superoxide radical scavengers than other polyunsaturated fatty acids in human endothelial cell cultures. Moreover, supplementation with fish oils resulted in low formation of ROS and in high inhibition of superoxide anion. Ambrozova et al. (2010) investigated the effect of DHA on the free radicals, and they reported that DHA exhibits antioxidant effects. Di Nunzio et al. (2011) found that DHA was the most effective in improving antioxidant defenses without showing any adverse effect on cell viability. Other authors have suggested that fish oils with higher DHA ratios enhance the activity of some antioxidant enzymes in

mice. Another study investigated the impacts of DHA on antioxidant activities in vitro and the beneficial effects of feeding with DHA showed on antioxidant activities in brain and liver tissues and the cognitive functions of the developing brain. Results indicated that DHA significantly enhanced antioxidant activities and increased cell viabilities in vitro (Chaung et al., 2013). DHA has been tested for the galactosamine-induced liver damage in mice, and the results indicated that DHA is able to present hepatoprotective effects (Roy et al., 2007). The oxidation patterns of cytosolic proteins from kidney and liver also indicated protective effects on proteins for the fish oil treatments (Me´ndez et al., 2012). Again, the dietary supplementation of DHA in obese mice reduced hepatic lipid content, with concomitant antioxidant and anti-inflammatory responses (Valenzuela et al., 2012). In summary, our data clearly show that TCDD induces generation of DNA damages and also leads to cell deaths together with HEP damages by a mechanism involving oxidative stress. Our results show that DHA in increasing concentrations is a promising source for the development of potential protectors against TCDD-induced oxidative stress and, especially, against MNHEPs and 8-OH-dG; thus, DHA may reduce the risk of carcinogenesis in liver. Because of the effects on TAC and TOS of DHA, this finding may provide new insight into the development of therapeutic and preventive approaches against TCDD toxicity. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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