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Abstract

Chronic arsenic exposure has been linked to many health problems including diabetes and cancer. In the present study, we assessed the protective effect of ellagic acid (EA) against toxicity induced by arsenic in isolated rat liver mitochondria. Reactive oxygen species (ROS) and mitochondrial membrane potential decline were assayed using dichlorofluorescein diacetate and rhodamine 123, respectively, and dehydrogenase activity obtained by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion assay. Arsenic increased ROS levels and mitochondrial dysfunction, which led to a reduction in mitochondrial total dehydrogenase activity. Mitochondria pretreated with EA exposed to arsenic at various concentrations led to a reversal of ROS production and mitochondrial damage. Our results showed that mitochondria were significantly affected when exposed to arsenic, which resulted in excessive ROS production and mitochondrial membrane disruption. Pretreatment with EA, reduced ROS amounts, mitochondrial damage, and restored total dehydrogenase activity specifically associated with mitochondrial complex II. EA protective characteristics may be accomplished particularly throughout the mitochondrial maintenance either directly by its antioxidant property or indirectly through its maintaining of complex II. These findings also suggest a potential role for EA in treating or preventing mitochondria associated disorders.

Keywords

Mitochondria arsenic ellagic acid reactive oxygen species

Introduction

Arsenic is one of the most important environmental toxicants in both inorganic and organic forms. Inorganic forms are more toxic than organic compounds.^1,2 Drinking water together with the other environmental pollutions is the major source of inorganic arsenic exposure to many of the people in the world and a public health problem.^3
–5 Reportedly, the prominent exposure route of inorganic arsenic particularly in India, Bangladesh, China, and some Central and South American countries is through contaminated drinking water.⁶ The International Agency for Research on Cancer classified arsenic in group I of human carcinogens.^7,8 The accepted level of arsenic in drinking water issued by the World Health Organization is below 10 µg/L.^2,9,10

Currently, exposure to arsenic has been contributed to complicated diseases including diabetes mellitus, peripheral vascular disease, arteriosclerosis, cardiovascular diseases, hypertension, goiter, hepatomegaly, cancer, and neurological diseases.^11
–13

The mechanism by which arsenic induces its toxicity is not fully recognized. Among the proposed mechanisms are reactive oxygen species (ROS) overproduction and oxidative stress, adenosine triphosphate (ATP) production disruption and carcinogenicity.¹⁴ Overproduction of ROS and antioxidant balance interruption¹⁵ potentially alter the cell signaling pathways including the mitogen-activated protein kinase family (MAPK) particularly p38 and c-Jun NH₂-terminal protein kinase (JNK),^1,16 nuclear factor κB (NF-κB), tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in which NF-κB plays a key role in insulin resistance.¹⁷

Biochemically, arsenate properties are similar to phosphate and can replace phosphate in ATP formation.¹² ATP and other phosphates involved in glucose metabolism are impaired by arsenic interference with the normal metabolism of glucose energy production through the altering of ATP-dependent insulin release from beta cells.^18

–21 Arsenite (trivalent) has a high affinity for binding to sulfhydryl groups in glutathione, glucose transporters, insulin receptors, and pyruvate dehydrogenase and disrupts their normal function associated with oxidative phosphorylation.²² Thus, arsenic exposure would potentially diminish the production of ATP and subsequently insulin secretion eventually leading to insulin resistance.²⁰

There is much evidence suggesting that arsenic-induced mitochondrial membrane permeability is followed by the release of cytochrome c to cytosol and diminution of ATP levels.^23,24 Overproduction of free radicals and oxidative stress and their role in cellular signaling pathway disruption are the most proposed mechanisms of arsenic-induced diabetes and insulin resistance.²⁵

Mitochondrial dysfunction has a key role in ROS generation. The electron transport chain located in the inner mitochondrial membrane and the oxidative phosphorylation are the main sources for the production of intracellular ROS.^5,26 There is much evidence that supports the link between ROS generation from damaged mitochondria and a variety of diseases including cancer, diabetes, and neurodegenerative disorders.^26,27 Mitochondrial dysfunction related to type 2 diabetes has been reported to be linked with impaired insulin action at target tissues and with impaired insulin release. Mitochondrial abnormality in the context of impaired insulin release and action has been reported in different cells and tissues. These include pancreatic β cells, hepatocytes, and skeletal muscles.²

Apparently, in our daily life, exposure to arsenic is inevitable, with all of us encountering this toxicant at some level.^28
–30 It would, therefore, be of beneficial value to find effective natural substances, potentially originating from our diet, for coping with the abovementioned adverse effects. Ellagic acid (EA) is a remarkable natural substance and phenolic compound found in many fruits, vegetables, and nuts, especially in pomegranate, which comprises potent antioxidant activity^15,31
–33 EA accomplishes its protective effects by inhibiting the activation of NF-κB reduction in the release of nitric oxide (NO) and scavenging of free radicals via strengthening of the cellular antioxidant defense.^{13,32,34
–36} In this study, we tested whether EA could protect hepatic mitochondria against toxicity induced by arsenic.

Materials and Methods

Animals

Male Wistar rats (220–250 g) were housed in polypropylene cages and fed liberally with a standard diet and drinking water. The animals were kept at a controlled temperature (25 ± 2°CC) with a 12-h light/12-h dark cycle. All experiments were carried out in accordance to the standards outlined by the ethical committee of the sponsoring university.

Chemicals

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), mannitol, ethylene glycol tetraacetic acid (EGTA), bovine serum albumin (BSA), 2,7-dichlorofluorescein diacetate (DCFH-DA),^3,4 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), EA, Rhodamine 123, Coomassie Brilliant Blue, rotenone, and arsenic trioxide (ATO) were purchased from Sigma-Aldrich (St Louis, Missouri, USA), and sucrose and dimethyl sulfoxide (DMSO) were obtained from Merck (Darmstadt, Germany).

Isolation of liver mitochondria

Mitochondria were prepared from rat liver by differential centrifugation as previously described with some modifications.³⁷ Briefly, rat liver was removed and washed carefully with buffer and cut into small pieces. Liver pieces were homogenized in an ice-cold isolation buffer containing sucrose 70 mM, 200 mM mannitol, 10 mM HEPES, 1 mM EGTA, and 0.1% BSA (pH = 7.4). The homogenized tissue was centrifuged at 600g for 10 min at 4°C. The supernatant was removed for further centrifugation at 11,000g for 15 min, and the pellet was then resuspended in cold isolation buffer. The previous steps were repeated twice. Finally, the pellet was resuspended in storage buffer. The protein concentration was measured using the Bradford method.¹⁰ All experiments were briefly outlined in Figure 1.

Figure 1.

Schematic view of experiments.

Measurement of mitochondrial ROS

The levels of ROS were measured by adding DCFH-DA to the incubated mitochondria. DCFH-DA penetrates into mitochondria and hydrolyzes to nonfluorescent DCFH, which becomes trapped in the mitochondria. Upon reaction with ROS, it is oxidized to form highly fluorescent 2,7-dichlorofluorescein. The mitochondrial suspension (0.4 mg/mL) was exposed to DCFH-DA and the fluorescence intensity was measured spectrofluorometerically (UV-1650PC SHIMADZU, Kyoto, Japan; Ex = 500 nm, Em = 520 nm).³⁸

MMP assay

The mitochondrial membrane potential (ΔΨm; MMP) collapse was measured using a cationic fluorescent probe, rhodamine 123, which accumulated in the mitochondria by facilitated diffusion and electric gradient force. Healthier mitochondria would accumulate more rhodamine 123 in their matrices. Aqueous rhodamine solution has an emission peak at 535 nm, whereas matrix and under stacking rhodamine undergoes a fluorescence quenching. Upon damage, the ratio of red-to-green fluorescence is decreased compared to healthy mitochondria.¹⁵ Fluorescence intensity was measured spectrofluorometerically (LS50B PerkinElmer, Waltham, Massachusetts, USA; Ex = 490 nm, Em = 535 nm).

MTT conversion (mitochondrial total dehydrogenase activity) assay

The mitochondrial total dehydrogenase activity was assayed by measuring reduction of MTT to formazan. Briefly, the mitochondrial suspensions (0.4 mg protein/mL) were incubated with different concentrations of agents, MTT was added to the medium following the incubation period, formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm by a spectrophotometer (UV-1650PC Shimadzu).^39,40

Statistical analysis

Data were presented as means ± SD for three different experiments. All the results were analyzed using GraphPad Prism (version 5.04). Statistical significance was determined using the one-way analysis of variance with the Tukey’s post hoc test. Statistical significance was set at p ≤ 0.05.

Results

Effect of ATO on mitochondrial ROS generation

ROS was determined throughout DCFH-DA oxidation. The relative DCF fluorescence intensity reflects different ROS amounts in different groups. Rat’s liver mitochondria (0.4 mg/mL protein) were isolated, purified, and incubated in buffer containing mannitol, sucrose, HEPES, and EGTA (pH = 7.4) for 1 h, followed by 1 h exposure to ATO. Fluorimetric measurements were read at λ_excitation = 500, λ_emission = 520 nm. As shown in Figure 2(a), as expected, different concentrations of ATO provoked ROS generation significantly more than control. ATO (20 μM) increased ROS level approximately 17.6%, mitochondria treated with 40 and 100 μM elevated ROS formation by 29.6% and 15.3%, respectively. It was also observed that the concentration of 40 μM ATO significantly increased ROS amounts in comparison with 20 and 100 μM.

Figure 2.

Mitochondrial ROS generation under different concentrations of ATO and EA. ROS determined using DCFH-DA oxidation. Relative DCFH fluorescence intensity reflects different ROS amount in different groups. Rat liver mitochondria were isolated, purified (0.4 mg/mL protein), and incubated in buffer containing mannitol, sucrose, HEPES, and EGTA (pH = 7.4), followed by 1 h exposure to ATO. Fluorimetric measurements were read at λ_excitation = 500, λ_emission = 520 nm. *p < 0.05: significant difference as compared to the control; ^$p < 0.05: significant difference as compared to ATO (20, 40, and 100 µM)); ⁺ p < 0.05: significant difference as compared to rot (1 µM); ^$$p < 0.05: significant difference as compared to ATO (100 µM); ^&p < 0.05: significant difference as compared to EA (20 and 40 µM). ROS: reactive oxygen species; ATO: arsenic trioxide; rot: rotenone; Cntl: control; EA: ellagic acid; DCFH-DA: 2,7-dichlorofluorescein diacetate; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

Effect of EA on mitochondrial ROS formation

In the second step, we investigated whether EA was solely active against ROS produced by mitochondria. The isolated mitochondria were treated with EA (20, 40, and 80 μM) for 1 h, and the results were compared with intact mitochondria. The comparative results are drawn in Figure 2(b) showing that different concentrations of EA decreased mitochondrial ROS generation. The EA concentration of 20 μM reduced ROS levels by approximately 32.8%, 32.5%, and 46% when mitochondria were treated with 40 μM and 80 μM, respectively.

Effect of EA on arsenic-induced mitochondrial ROS generation

To determine whether EA pretreatment influenced ROS overproduction by ATO in mitochondria, we tested the effect of different concentrations of EA at a constant ATO concentration. The mitochondria were pretreated with EA (20, 40, and 80 μM) for 1 h and subsequently exposed to constant concentrations of ATO (20, 40, and 100 μM) each time separately. As shown in Figure 2(c), the addition of 20 μM ATO to pretreated mitochondria did not elevate ROS amounts to the extent observed in nontreated mitochondria. The decline in ROS generation for 20, 40, and 80 μM EA pretreatment was at 7.5%, 26.4%, and 32.7%, respectively.

The same results with different intensities were observed using 40 and 100 μM of ATO (Figure 2(d) and (e)). When pretreated mitochondria (20, 40, and 80μM EA 1 h) were exposed to 40 μM of ATO, the results were a 16%, 17.7%, and 23.7% reduction in ROS generation, respectively, while for 100 μM of ATO exposure, the results were at 8.6%, 9.7%, and 17%, respectively.

Effect of ATO on MMP

MMP was determined using cationic fluorophore dye Rhodamine 123. Higher fluorescence intensity reflects the higher collapse of MMP. Rat liver mitochondria (0.4 mg/mL protein) were isolated, purified, and incubated in buffer containing mannitol, sucrose, HEPES, EGTA (pH = 7.4) for 1 h, followed by a 1 h exposure to ATO. Fluorimetric measurements were read at λ_excitation = 490 and λ_emission = 535 nm.

The isolated mitochondria were treated with ATO for 1 h. Figure 3(a) shows different concentrations of ATO exposed to mitochondria. Concentration of 20 μM ATO increased mitochondrial damage by approximately 4.6% as compared to control. When mitochondria were exposed to 40 μM of ATO, their damage elevated to 7.1%, whereas 100 μM of ATO elevated mitochondrial damage to 2.7%. Moreover, 40 μM of ATO increased mitochondrial damage more prominently than 20 and 100 μM. Collectively, this suggests that mitochondrial damage had not elevated through a dose-dependent manner upon exposure to different ATO concentrations.

Figure 3.

Mitochondrial membrane damage under different concentrations of ATO and EA. MMP (ΔΨm) determined using Rhodamine 123. Relative fluorescence intensity reflects different quantity of damage in different groups. Rat liver mitochondria were isolated, purified (0.4 mg/mL protein), and incubated in buffer containing mannitol, sucrose, HEPES, and EGTA (pH = 7.4) for 2 h. Fluorimetric measurements were read at λ_excitation = 490, λ_emission = 535 nm. *p < 0.05: significant difference as compared to the control; ^$p ≤ 0.05: significant difference as compared to ATO (20, 40, and 100 µM); ⁺ p < 0.05: significant difference as compared to rot (1 µM); ^$$p < 0.05: significant difference as compared to ATO (100 µM); ^&p < 0.05: significant difference as compared to EA (20 µM); and ^#p < 0.05: significant difference as compared to ATO 40 + EA 20 (µM). ATO: arsenic trioxide, rot: rotenone; Cntl: control; EA: ellagic acid; MMP: mitochondrial membrane potential; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EGTA: ethylene glycol tetraacetic acid.

Effect of EA on MMP

The isolated mitochondria were treated with EA (20, 40, and 80 μM) for 1 h. As shown in Figure 3(b), different concentrations of EA decreased fluorescence intensity, resembling mitochondrial protection. The 20 μM EA decreased fluorescence intensity by approximately 5.8% as compared to control. This parameter was reduced to 7.3% and 9.4% when mitochondria were treated with 40 μM and 80 μM of EA, respectively.

Effect of EA on arsenic-induced mitochondrial damage

For combination evaluations, we tested the effect of different concentrations of EA at a constant ATO concentration. The mitochondria were pretreated with EA (20, 40, and 80 μM) within 1 h and then exposed to different concentrations of ATO. As shown in Figure 3(c), EA decreased mitochondrial damage in the presence of the ATO at all concentrations. MMP collapse induced by 20 μM ATO was decreased 5.5%, 6.4%, and 10% when pretreated with EA 20, 40, and 80 μM, respectively.

As shown in Figure 3(d) and (e), when pretreated mitochondria (20, 40, and 80 μM EA 1 h) were exposed to 40 μM of ATO, the MMP were diminished by 4.7%, 5.8%, and 7%, respectively.

Effect of ATO on MTT conversion

MTT conversion was measured using MTT reduction by mitochondrial dehydrogenases. Rat liver mitochondria (0.4 mg/mL protein) were isolated, purified, and incubated in buffer containing mannitol, sucrose, HEPES, and EGTA, (pH = 7.4) for 1 h, followed by 1 h exposure to ATO. Absorbance were read at λ_ma = 570 nm.

The isolated mitochondria were treated with ATO for 1 h. As shown in Figure 4(a) in the maximum absorbance proportional with MTT conversion, different concentrations of ATO were significantly less than control. The 20 μM ATO decreased MTT conversion approximately 16.1%; moreover, when mitochondria were exposed with 40 μM and 100 μM ATO, absorbance decreased to 21.7% and 25.4%, respectively. Indeed, the concentration of 40 μM arsenic significantly decreased viability in comparison with 20 and 100 μM.

Figure 4.

Mitochondrial total dehydrogenase activity (MTT conversion). Dehydrogenase activity was assayed using the relative amount of formazan formation. Rat liver mitochondria were isolated, purified (0.4 mg/mL protein), and incubated in buffer containing mannitol, sucrose, HEPES, and EGTA (pH = 7.4) for 3 h. Absorbance was read at 570 nm by spectrophotometer. *p < 0.05: significant difference as compared to the control. ^$p ≤ 0.05: significant difference as compared to ATO (20, 40, and 100 µM); ^&p ≤ 0.05: significant difference as compared to EA(20 µM); and ^#p < 0.05: significant difference as compared to ATO 40 + EA 20 (µM). ATO: arsenic trioxide; Cntl: control; EA: ellagic acid; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ; EGTA: ethylene glycol tetraacetic acid; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

Effect of EA on MTT conversion

In the next step, we investigated whether EA alone could cause an increase in MTT conversion. The isolated mitochondria were exposed with EA (20, 40, and 80 μM) for 1 h, and the results were compared with the control. The comparative results (Figure 4(b)) show that different concentrations of EA increased MTT conversion. The concentration of 20 μM of EA increased MTT conversion by approximately 4.16%. The addition of 40 and 80 μM of EA to isolated mitochondria increased mitochondrial viability by approximately 6% and 7.3%, respectively.

Effect of EA on arsenic-decreased MTT conversion

To investigate an additive effect of EA alone on MTT conversion by ATO, we checked the effect of various concentrations of EA with a constant ATO concentration. The mitochondria were pretreated with EA (20, 40, and 80 μM) for 1 h and then exposed to separate constant concentrations of ATO (20, 40, and 100 μM). As shown in Figure 4(c), the 20 μM ATO concentration exposed to mitochondria was decreased to 16.1% compared with the control. However, with the addition of the pretreated mitochondria with EA (20, 40, and 80 μM), decreases were observed at 5.8%, 10.4%, and 13.5%, respectively. Figure 4(d) and (e) shows the results of the addition of 40 and 100 μM of ATO. When pretreated mitochondria (20, 40, and 80 μM EA 1 h) were exposed to 40 μM of ATO, the results indicated a reduction in MTT conversion at approximately 6.8%, 13.7%, and 15.8%, respectively, whereas results obtained for 100 μM of ATO exposure were decreased to 11.6%, 20.3%, and 22%, respectively.

Discussion

Arsenic is one of the most significant toxic pollutants and is a prooxidant for biological systems. Arsenic-contaminated drinking water is increasingly becoming a source of some chronic diseases worldwide. It is widely reported that arsenic increases oxidative stress and lipid peroxidation in liver and brain mitochondria and is a hepatotoxic heavy metal.^3,6,41
–43 Growing evidence suggests that oxidative stress and overproduction of oxygen-free radicals are among the most important mechanisms for arsenic toxicity.^11,44

Mitochondria play a key role in energy production, metabolism, apoptosis, and cellular signaling.⁴⁵ Dysfunction of mitochondria leads to excessive production of reactive oxygen and nitrogen radicals and subsequently results in oxidative stress.⁴⁶ Apparently, mitochondria are the first targeted organelles for arsenic toxicity with the accumulation of ROS and oxidative stress induction, condensation of the mitochondrial matrix, mitochondrial permeability transition (MPT) pore opening, and eventually apoptosis (Figure 5).^11,47 Studies report that arsenic exposure at concentrations higher than 100 µM were associated with a direct inhibitory effect on the mitochondrial complex I through oxidation of pyruvate and malate, decreased phosphorylation of adenosine diphosphate and MPT pore opening, and disruption of MMP.³⁷ On the other hand, it was suggested that complete inhibition of complex I abrogated the ROS amounts.⁴⁸ However, mitochondrial complex II might also be involved in this dilemma. In our study, all concentrations of arsenic (Figure 2(a)) dramatically increased the level of ROS in which a higher rate of ROS production was detected at the concentration of 40 µM. Surprisingly, the highest concentration of arsenic (100 µM) generated a lower level of ROS (below 40 µM). The reason why the higher concentration of arsenic produced a diminished level of ROS may be related to the complete inhibition of complex II by arsenic, as it was reported previously for complex I.⁴⁸ In other words, partially inhibited complex II by arsenic produces higher amounts of ROS than full inhibition (Figure 3(a)). In all arsenic-treated groups, when complex I was totally inhibited by rotenone, the same levels of ROS were detected except for arsenic at 100 µM. Current thinking emphasizes that rotenone as well as arsenic are complex I inhibitors.^27,47,49 When a combination of these two inhibitors is applied on mitochondria, the result is a full inhibition of complex I and a diminished amount of ROS. Interestingly, our results (Figure 2(a)) did not confirm this sort of expectation. It seems that either rotenone or arsenic might be contributed to the inhibition of another complex, likely complex II. To confirm the belief above, we applied a higher concentration of arsenic to rotenone-treated mitochondria. Our results were unexpected, in comparison with arsenic alone at 100 µM, higher significant ROS levels were detected. The reason here can be explained due to the full inhibition of complex II by arsenic at 100 µM. The excessive amount of ROS when rotenone was used prior to the addition of arsenic (100 µM) may be related to complex I inhibition, although the ROS level was lower than what was detected for rotenone alone. This may be moderately due to an unidentified inhibitory effect of rotenone on complex II.⁵⁰ Arsenic disrupts the function of complex II by binding to succinate substrate and mitochondrial complex II, which is a direct link between the tricarboxylic acid (TCA) cycle and the respiratory electron transport chain for ATP production. Hence, any disruption in complex II activity leads to the accumulation of succinate and increase in ROS. The mitochondrial complex II can be a major regulator and modulator for ROS production in pathological and physiological conditions. It was shown that the complex II could be associated to ROS levels insofar as or exceeding the maximum level of complex I and III contributions. Complex II probably plays a key role in the mitochondrial ROS production under pathological circumstances.^50
–52

Figure 5.

By partially inhibiting mitochondrial complex I and/or II, arsenic-triggered ROS overproduction leading to MPTP opening and mitochondrial damage. EA interrupts arsenic toxicity apparently via activating TCA cycle and antioxidant defense. ROS: reactive oxygen species; EA: ellagic acid; MPTP: mitochondrial permeability transition pore; TCA: tricarboxylic acid.

Arsenic by oxidizing thiol groups of adenine nucleotide translocase (ANT) and by MPT pore opening decreases MMP and liberates cytochrome c into the cytosole, which is followed by the activation of caspases 9 and 3 and finally inducing apoptosis. There is no evidence for direct action of arsenic upon MPT. Conversely, it seems that complex I inhibition by arsenic yields high ROS levels by which MPT opening ensues in rat liver mitochondria.^41,53 Arsenic exposure in brain increased ROS generation and led to membrane damage, MPT opening, and mitochondrial potential disruption.⁵ Our results showed that arsenic in different concentrations disrupted MMP (Figure 3(a)). These results were parallel to what was obtained for ROS generation with arsenic at 40 µM showing the greatest damage.

In isolated liver and heart mitochondria, arsenic, decreased succinate dehydrogenase (SDH), isocitrate dehydrogenase (IDH), pyruvate dehydrogenase, and α-ketoglutarate TCA cycle component activities mediated through a ROS-dependent mechanism, led to liver and cardiac dysfunction.^8,54 Our results presented in Figure 4(a) demonstrated that arsenic deteriorated the mitochondrial total dehydrogenase activity. By those inhibitions of TCA cycle enzymes, arsenic disrupts oxidative phosphorylation, enhances ROS production, and induces MMP collapse. Mitochondrial dysfunction induced by arsenic is believed to have a key role in the pathogenesis of various diseases including cardiovascular and neurodegenerative disorders as well as cancer and diabetes.¹ Oxidative stress has been extensively linked to diabetes mellitus.¹⁷ Mice exposed to arsenic in drinking water expressed glucose intolerance and insulin resistance, which suggested to be mediated by ROS activity.² In vitro studies have shown that the interrelation between diabetes and arsenic might be the transmitting signals and translation factors related to the insulin pathway, glucose uptake, and metabolism.¹⁹ Arsenic, via increasing ROS levels, inducing oxidative stress, inflammation, and apoptosis, diminished antioxidant defense including glutathione contents^18,23,55 that as mentioned connected to diabetes development.^17,22,55

In point of fact, exposure to arsenic in our daily life is unavoidable, and in many ways we are at the risk of injury from this toxicant.^28
–30 EA is a natural substance and a phenolic compound found in many fruits, vegetables, and nuts, which comprises potent antioxidant activity.^31
–33 The substance is hepatoprotective and a potent inhibitor for superoxide anion and hydroxyl radicals, as well as having a protective effect on lipid peroxidation.^54,56,57 By affecting signaling pathways, EA diminishes MAPK, NF-κB, P38, JNK, and inflammatory markers presumably via attenuating the ROS levels. Thus, it seems that EA, through its antioxidative and inflammatory properties, hinders the adverse effects of arsenic exposure that in parallel modulates predisposing factors in diabetes.^{36,58

–62} The antioxidant effect of EA on atherosclerosis leads to reduced oxidative stress linked with cardiovascular diseases and decreases in the lipid peroxidation in macrophages.^60,63 On endothelial cells, the administration of EA showed a reduction in ROS generation.

The protective effects of EA may potentially be through inhibiting the superoxide production of NADPH oxidase, suppressing the release of NO by downregulation of inducible nitric oxide synthase and increasing the antioxidant defense capacity.^64,65 Our results confirm previous studies that have shown the protective effect of EA (Figure 1(b) to (e)). EA has a dose-dependent effect in the induction of arsenic-induced ROS formation in mitochondria isolated from rat liver. EA protected cells against oxidative stress and mitochondrial membrane permeability.^32,38

Comparison of the results in Figures 3(b) and 4(b) with Figure 2(b) shows that there is no significant oxidative stress insult in the mitochondria. Figures 3(b) and 4(b) display a good quality for dehydrogenase activity and mitochondrial membrane intactness and indicate that the mitochondria were not under oxidative stress. For oxidative stress counterpart, that is, reductive stress, if it exists, we should notice its deleterious effect on the mitochondria as well. We have no evidence for pathogenic mitochondrial oxidation at decreased levels of ROS in our in vitro model. On the other hand, reductive stress may increase ROS production by mitochondria^66,67 and paradoxically lead to oxidative stress. In Figure 2(b), a decline in ROS is observed resembling a mitohormesis pattern.⁶⁸ However, for hormesis, to be established in the mitochondria, longer duration of exposure than what was applied in our study (30–60 min) is needed. Further studies will be required to address whether EA exposure is linked to reductive stress in the mitochondria.

Our results confirmed the majority of reports from previous studies showing the connection in increased ROS generation and mitochondrial membrane collapse in arsenic-treated isolated mitochondria. However, we have also demonstrated that when mitochondria were pretreated with EA, ROS generation and mitochondrial membrane damage were inhibited, which significantly increased mitochondrial protection (Figure 3(b) to (e)). Mari Kannanand Darlin Quine showed that heart mitochondria treated with EA exhibited a lower lipid peroxidation and mitochondrial damage as well as higher antioxidant enzyme and TCA cycle enzyme activities (IDH, SDH, malate dehydrogenase, and α-ketoglutarate dehydrogenase).⁶⁹ Parallel to what were observed in ROS and MMP results, pretreatment with EA restored total mitochondrial dehydrogenase activity that were disrupted by the application of arsenic (Figure 4(a) to (d). Previous studies have indicated that EA increased the glutathione content in mitochondria via the pentose phosphate pathway.³¹

In conclusion, our results clearly confirm that the mitochondria are targets of the toxic effect of arsenic. Although there is much evidence suggesting the toxic effect of arsenic upon mitochondrial complex I, arsenic apparently influences the activity of mitochondrial complex II as well that plays a crucial role in arsenic-induced mitochondrial damage. Both arsenic and EA seem to affect the same target in the mitochondrion that would apparently be the complex II. EA protective characteristics may be accomplished particularly throughout the mitochondrial maintenance. EA protects mitochondria against arsenic toxicity either directly by its antioxidant property or indirectly through maintaining the complex II. Our results suggest that EA or its chemically modified derivatives might be useful for preventing or treating the mitochondria-associated disorders.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by a grant (D-9210) from the Diabetes Research Center, Ahvaz Jundishapur University of Medical Sciences.

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Ellagic acid protects against arsenic toxicity in isolated rat mitochondria possibly through the maintaining of complex II

Abstract

Keywords

Introduction

Materials and Methods

Animals

Chemicals

Isolation of liver mitochondria

Measurement of mitochondrial ROS

MMP assay

MTT conversion (mitochondrial total dehydrogenase activity) assay

Statistical analysis

Results

Effect of ATO on mitochondrial ROS generation

Effect of EA on mitochondrial ROS formation

Effect of EA on arsenic-induced mitochondrial ROS generation

Effect of ATO on MMP

Effect of EA on MMP

Effect of EA on arsenic-induced mitochondrial damage

Effect of ATO on MTT conversion

Effect of EA on MTT conversion

Effect of EA on arsenic-decreased MTT conversion

Discussion

Footnotes

Declaration of Conflicting Interests

Funding

References