Protective effects of melatonin and N-acetyl cysteine against oxidative stress induced by microcystin-LR on cardiac muscle tissue

Protective effects of melatonin and N-acetyl cysteine against oxidative stress induced by microcystin-LR on cardiac muscle tissue

Toxicon 169 (2019) 38–44 Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Protective effects of ...

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Toxicon 169 (2019) 38–44

Contents lists available at ScienceDirect

Toxicon journal homepage: www.elsevier.com/locate/toxicon

Protective effects of melatonin and N-acetyl cysteine against oxidative stress induced by microcystin-LR on cardiac muscle tissue

T

Leila Ait Abderrahima,∗, Khaled Taïbia, Nawel Ait Abderrahimb, Anas M. Alomeryc, Fatiha Abdellahd, Ayman Saeed Alhazmic, Saad Aljassabie a

Faculty of Life and Natural Sciences, Ibn Khaldoun University, Karman Campus, 14000, Tiaret, Algeria Faculty of Sciences, University of Montpellier, France c Clinical Laboratory Department, Faculty of Applied Medical Sciences, Taif University, Taif, Saudi Arabia d Laboratory of Research on Local Animal Products, Ibn Khaldoun University, Tiaret, Algeria e Institute of Biological Sciences, University of Malaya, Kuala Lampur, Malaysia b

ARTICLE INFO

ABSTRACT

Keywords: Microcystin-LR Oxidative stress Antioxidants Melatonin N-acetyl cysteine Cardiac muscle tissue

Microcystin Leucine-Arginine (MC-LR) is a toxin produced by the cyanobacteria Microcystis aeruginosa. It is the most encountered and toxic type of cyanotoxins. Oxidative stress was shown to play a role in the pathogenesis of microcystin LR by the induction of intracellular reactive oxygen species (ROS) formation that oxidize and damage cellular macromolecules. In the present study we examined the effect of acute MC-LR dose on the cardiac muscle of BALB/c mice. Afterwards, melatonin and N-acetyl cysteine (NAC) were assayed and evaluated as potential protective and antioxidant agents against damages generated by MC-LR. For this purpose, thirty mice were assigned into six groups of five mice each. The effect of MC-LR was first compared to the control group supplied with distilled water, then compared to the other groups supplied with melatonin and NAC. The experiment lasted 10 days after which animals were euthanized. Biomarkers of toxicity such as alkaline phosphatase activity, lipid peroxidation, protein carbonyl content, reduced glutathione content, serum lactate dehydrogenase and serum sorbitol dehydrogenase were assayed. Results showed that toxin treated mice have experienced significant oxidative damage in their myocardial tissue as revealed by noticeable levels of oxidative stress biomarkers and by the reduction in alkaline phosphatase activity. Whereas, melatonin and NAC treated mice manifested lesser oxidative damages. Our findings suggest a potential therapeutic use of melatonin and Nacetyl cysteine as antioxidant protective agents against oxidative damage induced by MC-LR.

1. Introduction Microcystins (MCs) are common intracellular toxic cyclic heptapeptides produced by some species of bloom forming aquatic cyanobacteria often belonging to the genera Microcystis, Planktothrix, Anabaena and Nostoc (Nhoato et al., 2001; WHO, 2003; Manage et al., 2009). These cyanotoxins were first isolated from Microcystis aeruginosa and were primarily associated with hepatotoxicity but they have also been seen to cause damage to other tissues such as kidneys (Chen et al., 2005). High concentrations of these cyanotoxins can be released in waters after cell lysis (Schmidt et al., 2014). In fact, they were reported to be responsible for periodic poisonings of humans and livestock drinking fresh water where the cyanobacteria producing them are endemic (Lone et al., 2015). They can bioaccumulate in aquatic animals and can even persist after boiling indicating that cooking is not



sufficient to destroy the toxins. They also resist to chemical breakdown under conditions found in most natural water bodies (near-neutral pH) demonstrating their high stability (Butler et al., 2009). The chemical structure of MCs includes Cyclo-D-alanine in position (1), two variable L-amino acids in positions (2) and (4), D-β-erythro-βmethylaspartic acid (MeAsp) in position (3), 3-amino-9-methoxy-2,6,8trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) in position (5), DGlutamate in position (6) and N-methyldehydroalanine (Mdha) in position (7) (Izaguirre et al., 2007). So far, more than 90 different types of microcystin have been identified with variation occurring in the nature of the two variable L-amino acids and in the presence or absence of methyl groups on the (MeAsp) and/or MDha residues (Lone et al., 2015). The most common types are microcystin-LR, –RR, and -YR, where the variable amino acids are leucine (L), arginine (R) and tyrosine (Y) (Jungblut et al., 2006; Lone et al., 2015).

Corresponding author. E-mail address: [email protected] (L. Ait Abderrahim).

https://doi.org/10.1016/j.toxicon.2019.08.005 Received 18 February 2019; Received in revised form 19 August 2019; Accepted 21 August 2019 Available online 26 August 2019 0041-0101/ © 2019 Elsevier Ltd. All rights reserved.

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Microcystin-LR (MC-LR) is the most frequent and most toxic variant and is the first that was chemically identified (WHO, 2003). It acts by inhibiting serine-threonine phosphatase 1 and 2A; leading to increased protein phosphorylation causing the disruption of many cellular processes and alteration of the cytoskeletal structures (Ding et al., 1998). Studies suggested that oxidative stress also plays a role in the pathogenesis of microcystins; they can induce intracellular reactive oxygen species (ROS) formation that oxidize and damage cellular macromolecules leading to development of degenerative diseases. They also contribute in myocardial cell damage (Valko et al., 2007). Moreover, there is increasing proof that microcystins are genotoxic (Zegura et al., 2006). Even though, living organisms can protect themselves from oxidative stress using enzymatic and non-enzymatic antioxidants as defence mechanisms; the exogenous antioxidants are also important because they enhance antioxidant activity of the organism. They are potential therapeutic agents for conditions ranging from aging to cancer and heart diseases (Mak and Newton, 2001; Valko et al., 2007). Besides, melatonin (5-methoxy-N-acetyltryptamine) is an indolamine synthesized from the amino acid tryptophan primarily in pineal gland. It plays an important role in the regulation of circadian rhythms (Stetinová et al., 2002). It has a wide-ranging of endocrine, neural and immune functions in the body. Melatonin levels are related to the aging process and diseases development (Cornelissen et al., 2000). It has been shown to be an effective antioxidant and free radical scavenger (Al Jassabi and Khalil, 2006). Studies have also demonstrated its anticancer effects (Anisimov et al., 2003). As well, N-acetyl-L-cysteine (NAC) is the acetylated form of the amino acid L-cysteine (Fishbane et al., 2004). It contains a thiol group which confers its antioxidant properties by interacting with ROS. It plays an important role as a glutathione precursor; a key cellular antioxidant and detoxifier (Olofsson et al., 2003; Dekhuijzen, 2004), and has been shown to limit oxidative stress (Kay et al., 2003; Teixeira et al., 2008). While several studies have shown the damages of MC-LR principally on the liver and kidneys, only few ones have been undertaken on the oxidative damage induced by MC-LR on the cardiac tissue (Milutinovic et al., 2006; Wang et al., 2008). As well, finding efficient antidotes against MC-LR wasn't fully addressed. In this context, the present study aims to evaluate the oxidative damages induced by an intraperitoneal (i.p.) injected acute dose of MC-LR on the cardiac muscle of mice then investigate the potential protective effects of melatonin and N-acetyl cystein against such damages.

Fig. 1. Microcystis aeruginosa cells under light microscope (40x).

Fig. 2. Collected sample of blooms.

according to Nhoato et al. (2001), using methanol-butanol-water solution (20:5:75 v-v-v) with constant agitation at room temperature for 15 min followed by sonication during 20 min and then centrifugation at 4000g for 20 min. The supernatant was lyophilized and kept in dark bottle at - 20 °C. Purity of the extract was determined spectrophotometrically based on Oriola and Lawton (2005). 1 mg of the extract was diluted in 2.5 ml methanol and then tested at different wavelength ranging from 200 to 300 nm to determine maximum absorbance. The LD50 of the toxin extract was determined according to up-down method described by Yoshida et al. (1997). In this procedure, mice received intraperitoneal (i.p.) injections of the lyophilized toxin in distilled water at different doses. Briefly, animals are given dose of toxin one at a time and observed for 24 h. If an animal survives, the dose for the next animal is increased; if it dies, the dose is decreased. LD50 value is calculated from the mortality rate observed 24 h after i.p. injection, with minimal animal number.

2. Material and methods 2.1. Chemicals Chemicals of analytical grades were used in this study and were purchased from Sigma Chemical Co. USA, unless otherwise indicated. In addition, synthetic melatonin (73-31-4) and N-acetyl cysteine (NAC) (616-91-1) were used in this study.

2.4. Experimental design Thirty adult male BALB/c mice of 5–7 weeks old weighting 25–35 g were kept in standard stainless-steel cages and maintained in the animal house under controlled laboratory conditions with free access to water and food ad libitum (Principles of Laboratory Animal Care NIH publication no, 85-23, revised 1985). The experimental protocol used in this study complies with the ARRIVE (Animals in Research: reporting in vivo experiments) guidelines and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). All care was taken to minimize the suffering of the animals. The experimental protocol was approved by the National Ethics Committee. Mice were assigned into six groups of five mice each. The effect of MC-LR was first compared to the control group supplied with distilled water, then compared to the other groups supplied with melatonin and NAC. The experiment lasted 10 days after which animals were euthanized by decapitation. The experimental procedure was designed as

2.2. Cyanobacteria sample and identification Cyanobacteria blooms were collected from King Talal Reservoir, Jordan, during blooming season (early fall). M. aeruginosa cells were identified under light microscope (Fig. 1). According to Al-Jassabi and Khalil (2006), the samples were decanted into glass containers and exposed to sunlight for several days (Fig. 2). After that, the lower water layer was siphoned off; the cells were collected, lyophilized and conserved in dark bottles at −20 °C prior to toxin extraction. 2.3. Microcystin-LR extract preparation MC-LR was extracted from the harvested M. aeruginosa cells, 39

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follow:

serum albumin and 0.1 ml sample solution, were added and immediately mixed by inversion. The decrease in absorbance at 340 nm was recorded for approximately 5 min. The activity was calculated using the following equation:

• Group 1: Control (C): supplemented with distilled water throughout all the experimental period (10 days) and then euthanized. • Group 2: Toxin (T): supplemented with distilled water throughout • • • •

Activity[U/ ml] = [ A340/min x 3 x DF]/[6.22 x 0.1]

all the experimental period. After that, mice were injected intraperitoneally (i.p.) with LD50 of MC-LR then euthanized after 24 h. Group 3: Melatonin (M): supplemented daily, during 10 days, with melatonin (20 mg/kg body weight in distilled water) (Okutan et al., 2004) and then euthanized. Group 4: Melatonin-Toxin (MT): supplemented daily with melatonin (20 mg/kg body weight in distilled water) during all the experimental period. After that, mice were injected (i.p.) with LD50 of MCLR then euthanized after 24 h. Group 5: NAC: supplemented daily, during 10 days, with NAC (10 mM NAC/kg body weight in distilled water) and then euthanized. Group 6: NAC-Toxin (NT): supplemented daily with NAC (10 mM NAC/kg body weight in distilled water) along all the experimental period. After that, mice were injected (i.p.) with LD50 of MC-LR then euthanized after 24 h.

2.6.2. Cardiac tissue 2.6.2.1. - Alkaline phosphatase. Alkaline phosphatase (ALP) is a hydrolase dephosphorylating enzyme present in all tissues throughout the entire body. Decreased activity of ALP indicates its inhibition. Activity of alkaline phosphatase was determined according to the method of Bessey et al. (1946) in which the rate of formation of the yellow colour of p-nitrophenol (p-NP) produced by hydrolysis of pnitrophenylphosphate (p-NPP) in alkaline solution was measured spectrophotometrically at 405 nm and 37 °C. Briefly, 2.8 ml of the buffer (0.3 M 2-Amino −2 Methylpropane −1,3 Diol/0.002M MgCl2, pH 10.25) are mixed with 0.1 ml of the substrate (0.4 M p-nitrophenyl phosphate) in a 3 ml quartz cuvette. The reaction was initiated by the addition of 0.1 ml of the sample and the increase in the yellow colour intensity was followed as the increase in absorbance at 405 nm for 5 min. The activity was calculated using the following equation:

2.5. Serum and tissue sampling

Activity [U/g] = [ A 405/min x 3 x DF ]/[ 18.8 x 0.1] DF: dilution factor.

This study focused on the evaluation of the toxicity of MC-LR on the cardiac muscle tissue and the protective effect of melatonin and NAC. Blood was collected immediately after animals’ decapitation then centrifuged at 2000 g for 20 min. The serum was then isolated and stored at −20 °C. Mice cardiac muscles were excised immediately and homogenized separately in phosphate buffer. Homogenates were centrifuged and the supernatants were stored at −20 °C as described by Akcay et al. (2005).

2.6.2.2. Lipid peroxidation. Lipid peroxidation is an oxidative degradation of polyunsaturated fatty acids in cell membranes generating malondialdehyde (MDA) and 4-hydroxynonenal causing cell damage. Measurement of malonaldehyde (MDA) is an indicator of lipid peroxidation. The assay was performed as recommended by Rudnicki et al. (2007). It is based upon the formation of a red adduct between thiobarbituric acid (TBA) and MDA. Briefly, 600 μl 10% trichloroacetic acid were added to 300 μl of the homogenates and centrifuged at 7000 g for 10 min. Then, 400 μl of the supernatant was mixed with 400 μl 0.67% thiobarbituric acid and incubated in a boiling water bath for 30 min then cooled at room temperature. The absorbance was measured at 532 nm using malondialdehyde as an external standard. The data were expressed as malondialdehyde (MDA) equivalents (μmol/g tissue) using the following formula:

2.6. Biochemical assays 2.6.1. Serum 2.6.1.1. - Lactate dehydrogenase. Lactate dehydrogenase (LDH) is a cytosolic enzyme found in many body tissue cells (Valvona et al., 2016). High levels of LDH can be released to bloodstream when cells undergo significant membrane damage or cytolysis. Hence, elevated activity of LDH in the serum indicates tissue damage. The enzyme assay was performed as described by Botha et al. (2004). Briefly, the following reagents were pipetted into a 3 ml cuvette: 2.7 ml (50 mM, pH 7.4) potassium phosphate buffer, 0.1 ml (6 mM) NADH (dissolved in 5 mM buffered water pH 7.4), and 0.1 ml (23 mM) sodium pyruvate solution (dissolved in buffered water), equilibrated at 25 °C for about 5 min then 0.1 ml of sample was added and mixed. The decrease of absorbance at 340 nm was recorded. The blank solution was prepared using potassium phosphate buffer instead of sample. Activity was calculated by using the following formula:

MDA (µmol/g) = [A532/156]x103xDF 2.6.2.3. - Protein carbonyl content. Protein carbonyl content (PCC) are sensitive markers of oxidative injury and indicators of protein oxidation. They were measured as described by Albendea et al. (2007) through colorimetric procedure that measures binding of dinitrophenylhydrazine (DNPH) and protein carbonyls forming a Schiff base to produce the corresponding hydrazone that can be analyzed spectrophotometrically. Briefly, 100 μL of 50 mmol/L tris buffer and 200 μL of 10 mmol/L dinitrophenylhydrazine solution were added to 100 μL of sample and then vortexed followed by incubation at 37 °C for 1 h 325 μL of 20% ice cold trichloroacetic acid was added to the mixture then centrifuged at 3000 g for 10 min. The pellet obtained was washed three times with 1 ml ethanol/ethyl acetate (1:1) and the last pellet was dissolved in 700 μL 6 M guanidine and was carried at 37 °C for 15 min. After centrifugation at 12000g for 10 min, the absorbance of the supernatant was measured spectrophotometrically at 375 nm to quantify protein carbonyl. Guanidine was used as blank. The concentration was expressed as nmol carbonyl groups/g tissue using extinction coefficient 22000 M−1 cm−1.

Activity[U/ml] = [ A340/min x 3 x DF ]/[ 6.2 x 0.1] DF: dilution factor. 2.6.1.2. Sorbitol dehydrogenase. Sorbitol dehydrogenase (SDH) is an enzyme mainly present in the liver. Increased activity of SDH in serum is often confirmation of liver cells damage. The assay was performed according to Gerlach and Hiby (1974). It is based on the rate of decrease in absorbance of the reaction mixture at 340 nm due to the oxidation of NADH, which is directly proportional to the SDH activity. Briefly, 2.35 ml (100 mM) triethanolamine buffer (pH 7.6 at 25 °C), 0.5 ml (1.1 M) D-fructose solution and 0.05 ml (12.8 mM) βnicotinamide adenine dinucleotide (reduced form), were pipetted into 3 ml cuvette, mixed by inversion and equilibrated to 25 °C. The absorbance at 340 nm was monitored until constant, using a suitably thermostatted spectrophotometer. Then 0.1 ml (1% (w/v)) bovine

2.6.2.4. Reduced glutathione. Reduced glutathione (GSH) plays multiple roles in protection against ROS and maintenance of protein thiol groups. Determination of GSH levels in tissue extracts was performed according to Tabassum et al. (2006). The reaction is based on the 40

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interaction of reduced glutathione (GSH) with 5-5′-dithiobis (2nitrobenzoic acid) (DTNB) to form the yellow-colored product 2nitro-5-thiobenzoic acid, which is measured at 412 nm. Briefly, 1 ml sample was precipitated with 1 ml of sulphosalicylic acid (4.0%) and kept at 4 °C for 1 h and then centrifuged at 1200 g for 15 min at 4 °C. The assay mixture contained 0.2 ml of filtered aliquot, 2.6 ml of sodium phosphate buffer (0.1 mol/L sodium phosphate buffer, pH 7.4) and 0.2 ml DTNB (100 mmol/L in sodium phosphate buffer) in a total volume of 3 ml. The absorbance of reaction product was read at 412 nm. The GSH content was expressed as nmol GSH/g tissue using extinction coefficient of 1.36 × 104 M −1cm−1

3.2. Cardiac tissue assays Some biomarkers of MC-LR toxicity were assayed in the cardiac tissue. ALP activity was evaluated in the cardiac cell tissues as the toxin is an inhibitor of phosphatases. As well, lipid peroxidation evaluated by the concentrations of MDA, protein carbonyl content and reduced glutathione concentrations in mice tissues were determined as biomarkers of oxidative stress. Regarding ALP activity in the cardiac tissues, we noted that MC-LR induced a significant decrease of ALP activity (p-value < 0.05*) compared to melatonin (M) and NAC supplemented groups whereas no significant difference was noted in comparison to the other groups (C, MT and NT) (Fig. 5A). Besides, MDA concentration (21.86 ± 2.78 U/ml) was significantly high in the cardiac tissue of mice injected with the toxin compared to the control groups (C, M, NAC), the melatonin-toxin (MT) and NACtoxin (NT) groups (p-value < 0.05*). Besides, when compared to the toxin group (T), melatonin and NAC decreased significantly MDA concentration in the melatonin-toxin (MT) and NAC-toxin (NT) groups. It is important to note that the control groups treated with NAC and melatonin showed significant low MDA concentrations (7.6 ± 0.27 and 9.25 ± 0.17 μmol/g respectively) comparing to control group without any supplementation (11.95 ± 0.22 μmol/g) (Fig. 5B). Likewise, PCC of the cardiac tissues was significantly higher in mice treated with the toxin alone compared to the other groups (p-value < 0.05*). Concentrations of carbonyl derivatives decreased significantly in tissues of mice treated with melatonin (melatonin-toxin group) and with NAC (NAC-toxin group) compared to toxin group. A significant decrease in PCC was observed in MT group compared to NT group (Fig. 5D). Moreover, a significant increase of GSH concentration was noticed in the tissues of mice treated with melatonin and NAC alone compared to control group without any supplementation and to the other groups (p-value < 0.05*). It should also be noted that the MT and the NT groups showed significant increase in GSH levels compared to the toxin group as well as to the control group without any supplementation suggesting that melatonin and NAC increase the production of GSH (Fig. 5C).

2.7. Statistical analysis Results are expressed as means ± standard of deviation. One-way analysis of variance followed by Duncan test was used to compare mean data between the different groups. The results were considered to be statistically significant when P-values were less than 0.05. 3. Results MC-LR was extracted from harvested cells of M. aeruginosa and evaluated for its oxidative damages on the cardiac muscle. Purity of microcystin extract was determined spectrophotometrically by testing the toxin extract absorbance at different wavelengths. The maximum absorbance of the toxin extract was found between 225 and 235 nm (Fig. 3). Regarding the toxicity of the extract, the LD50 of the toxin on mice after 24 h was found to be 34.5 mg toxin/kg mouse body weight. 3.1. Serum assays Lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH) activities were assayed from the sera of mice for the detection of eventual tissue cells damages and particularly liver cells damage which is revealed by high activity of SDH in the serum. MC-LR caused significant increase in serum LDH of mice up to 6.88 ± 0.16 U/ml which is approximately three-folds higher when comparing to control groups (control C, melatonin M and NAC groups) (p-value < 0.05*). By the same, MC-LR induced significant increase in serum SDH of mice (0.65 ± 0.07 U/ml) compared to control groups; by around thirteen-folds compared to control and NAC groups and twenty-sixfolds compared to melatonin group (p-value < 0.01**). Meanwhile, LDH and SDH rates did not differ significantly between control group, melatonin and NAC treated groups. However, when compared to toxin group (T), melatonin and NAC alleviated significantly the adverse effect of MC-LR on LDH and SDH rates in the sera of MT and NT groups (Fig. 4A and 4B).

4. Discussion The occurrence of cyanobacterial toxic water blooms is a worldwide concern (Zegura et al., 2006). Fawell et al. (1999) and Hoger (2003) demonstrated that liver is the main target organ for microcystin MC-LR. Acute exposure to the toxin causes severe intrahepatic hemorrhage (Solter et al., 1998) while chronic exposure can promote liver tumor formation. In terms of oxidative stress, some acute MC-LR exposure tests showed serious oxidative damage in different organs in mammals primarily in liver and kidneys (Prieto et al., 2007). This study aimed to determine the effect of microcystin-LR on cardiac muscle in terms of oxidative damage and whether melatonin and N-acetyl cysteine confer antioxidant and protective effect against such damages. Regarding microcystin extract purity, the spectrophotometric analysis showed an absorption maximum around 225–235 nm. Studies have shown that most microcystins present a characteristic ultraviolet absorbance maximum at 238 nm due to the two conjugated ð-bonds present in the Adda residue (Akin-Oriola and Lawton, 2005). Toxicity of MC-LR extract on the cardiac tissue was evaluated by assaying ALP activity and some important biomarkers of cell oxidative damage. It is known that once absorbed into the blood, MCs can be transported via the bloodstream rapidly and distributed to various organs (Wang et al., 2008). Researchers suggested a potential cardiogenic effect of MCs in addition to the effect on the liver (Dawson, 1998). It was

Fig. 3. Absorbance spectrum of the extracted toxin. 41

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Control

b,c

a,b,c

Toxin

c

A a,b,c

a

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Melatonin

24

Control

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d c

C c,d

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12 b

8 4 0

a

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20 15

B

c b

b a

10

a

5 Control

8

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NAC

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7 6 5 c

4 3 2

b a

a

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1 0

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Toxin

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30

MDA concentration (µmol/g)

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

PCC concentration (nmol/g)

GSH conce ntration (µmol/g)

ALP activity (U/g)

Fig. 4. LDH (A) and SDH (B) concentrations in the sera of mice before and after supplementation with treatments. * Alphabetic letters indicate the homogeneous groups; groups with the same alphabetic letter indicate no significant difference between them.

Control

Melatonin

NAC

Fig. 5. ALP activity (A), MDA concentrations (B), GSH concentrations (C) and PCC (D) in the cardiac muscle tissues before and after supplementation with treatments.* Alphabetic letters indicate the homogeneous groups; groups with the same alphabetic letter indicate no significant difference between them.

also reported that MC-LR induced myocardial cell damage in the heart muscle after 24 h (Milutinovic et al., 2006). MC-LR inhibits protein phosphatases altering the phosphorylation state of proteins (hyperphosphorylation) leading to rearrangement of the cell's intermediate filament network and destruction of the cytoskeleton causing disintegration of cell structure (Moore et al., 2016). Besides the inhibition of phosphatases, several studies have demonstrated the implication of oxidative stress in MCs mechanisms of toxicity leading to apoptosis, necrosis and genotoxicity (Campos and Vasconcelos, 2010; Martins et al., 2011). Jiang et al. (2013) showed a significant induction of hydroxyl radicals (•OH) in carp liver after exposure to MC-LR. Reactive oxygen species ‘ROS’ production in mammalian cells is primarily associated with mitochondrial metabolism. Ding et al. (2001) attributed the increased ROS formation after exposure to MC-LR to a surge of Ca2+ level in the mitochondria resulting in the onset of membrane permeabilization transition (MPT) leading to cell death. The MPT leads to the production of ROS, loss of the mitochondrial membrane potential and the release of apoptotic factors causing apoptosis. Moreover, several studies have demonstrated the implication of reactive oxygen species (ROS) in the onset of cardiac toxicity such as

impairment of the blood pumping-function, as the heart may be more susceptible to oxidative damage due to the low activity of the antioxidant system there compared to other tissues (Kannan et al., 2004). In this study, high activity of sorbitol dehydrogenase (SDH) in the serum of mice treated with MC-LR was noted. This is primarily an indication of liver cells damage, as this enzyme is a specific liver cytoplasmic and mitochondrial enzyme (Dawson, 1998). In addition, high activity of lactate dehydrogenase (LDH) was also observed in the serum of toxin injected mice. LDH is a cytoplasmic enzyme found mainly in the kidneys, myocardium, skeletal muscles and liver. High concentration in the serum indicates cell damage and generally is accepted as a sign of necrosis (Botha et al., 2004; Jovanović et al., 2010). However, the activity of SDH and LDH significantly decreased in the sera of mice treated with melatonin and NAC demonstrating the protective effect of these substances against damages to the cells. Regarding ALP activity in the cardiac tissue, a numerical decrease was observed in tissue of mice treated with MC-LR though not significant compared to the control group and to MT and NT groups. However, it is interesting to note that mice supplemented with melatonin and NAC showed high activity of ALP compared to mice without any supplementation suggesting that these substances increase ALP 42

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activity. Moreover, increased protein carbonyl groups and MDA content in the cardiac tissue of mice injected with MC-LR, indicate important oxidative damage. It is well known that ROS can react with different molecules such as lipids, proteins and sugars generating byproducts (Guzman and Solter, 2002). Lipid peroxidation is a consequence of chain reaction within all membranes initiated by interaction of hydroxyl radicals and polyunsaturated acids that produce peroxyl radical (ROO) which attacks structures found in cell membrane (Izaguirre et al., 2007). Reactive carbonyl derivatives are products of protein oxidation by ROS. However, carbonyl groups may be introduced into proteins by reactions with carbonyl-containing oxidized lipids such as 4-hydroxy-2-nonenal and malondialdehyde resulting from lipid peroxidation (Berlett and Stadtman, 1997). Carbonyl groups can also be a product of the reaction of carbonyl derivatives produced by glycoxidation reactions with the primary amino group of lysine residues (Dalle-Donne, et al., 2006). Mice treated with NAC and melatonin before injection with MC-LR showed a significant decrease in MDA contents and PCC demonstrating efficacy against oxidative damage induced by the toxin. Besides, mice treated with NAC and melatonin showed increased levels of GSH compared to those injected with the toxin. In fact, reduced levels of GSH are the result of the interaction with the toxin and the free radicals produced from oxidative alterations of molecules. GSH is one of the key components of the antioxidant defense system in living cells. A central nucleophilic cysteine residue is responsible for high reductive potential of GSH. It is the most abundant non protein thiol in most cells. It protects the cell by removing potential harmful molecules such as ROS (Blokhina et al., 2003). Studies suggest that MCs conjugate with GSH in a reaction catalyzed by glutathione-S-transferase, as the first step in their detoxification process (Billam, 2006). The results obtained from the present study clearly demonstrate that the heart suffers the same oxidative damage as the liver from a single acute MC-LR dose administration. It is important to note that some substances can stimulate the synthesis of cellular antioxidants and/or act as external antioxidants. Studies had shown that melatonin, characterized by the presence of an amino group, has an important role in reducing free radicals (TomasZapico and Coto-Montes, 2007). In the present study, melatonin was found to have the highest antioxidant activity in comparison to NAC. It is an electron-rich molecule, able to interact with free radicals generating many stable compounds that can be excreted by urine. Melatonin has been shown to be effective in reducing LPO. The reason is that melatonin counteracts lipid peroxidation by preventing the initiation of the process as well as by breaking the lipid peroxide chain reaction (Zhao et al., 2007). Melatonin can also reduce oxidative stress by activating anti-oxidative pathways such as glutathione synthesis, stimulating antioxidant enzymes such superoxide dismutase, glutathione peroxidase and glutathione reductase. It was reported to protect cerebral nerve cells against oxidative damage as well protects or repairs DNA damage (Liu et al., 2018). The facts that it’s widely distributed in all tissues and is very lipophilic substance, allow it to act on all cells (Okutan et al., 2004; Lee et al., 2009). The study of Xu et al. (2018) showed that MC-LR has a profound influence on modulation of the circadian clock system of cardiomyocytes. Melatonin play a key role as an endogenous synchronizer regulating seasonal and circadian rhythms along with its sleep-inducing effects. Studies have shown that diminished melatonin production at night has consistently been reported in severely hypertensive patients and in patients with coronary diseases (Zisapel, 2018). More interesting is that current findings from experimental studies support the potential use of melatonin as preventive and adjunctive curative therapy in cardiovascular diseases (Sun et al., 2016). These findings combined with our results support the idea of melatonin as protective agent against MC-LR damages induced in the cardiac muscle. In addition, NAC was shown to have an important role as

antioxidant and glutathione precursor as it can penetrate cells easily (Zegura et al., 2006). Our results demonstrated an increase of GSH concentrations in mice treated with NAC. In addition, we noted that NAC attenuated the oxidative damage in cardiac tissue. These results are in accordance with those obtained by Jiang et al. (2013) who showed that pre-injection of NAC to carp exposed to MC-LR has a significant protective effect on liver cytoskeleton. Studies showed that NAC is efficiently transported into the cell where it is converted to cysteine for GSH synthesis thus improving the intracellular detoxification and reducing the liberation of ROS (Laurent et al., 1996). The protective effect of NAC can be attributed to its sulfhydryl group (Sprong et al., 1998). The free thiol group of NAC is capable of interacting with ROS, forming intermediate NAC thiol, with NAC disulphide as a major end product (Dekhuijzen, 2004). 5. Conclusion Reactive oxygen species play an important role in the onset of cardiac toxicity and the heart may be more susceptible to oxidative damage due to the low activity of the antioxidant system there compared to other tissues. The results obtained from the present study demonstrate clearly that the heart suffers the same way from oxidative damages as the liver from an acute dose administration of MC-LR. Besides, to date, there is still no effective antidote against oxidative damages induced by toxic molecules in general and by microcystin-LR particularly. Our work demonstrated that the supplementation of mice with melatonin and NAC reduced significantly the damages elicited by MC-LR on the cardiac tissue. Thus, the therapeutic use of melatonin and N-acetyl cysteine as antioxidant protective agents should be further explored. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.toxicon.2019.08.005 References Akcay, Y.D., Yalcin, A., Sozmen, E.Y., 2005. The effect of melatonin on lipid peroxidation and nitrite/nitrate levels, and on superoxide dismutase and catalase activities in kainic acid-induced injury. Cell. Mol. Biol. Lett. 10, 321–339. Akin-Oriola, G.A., Lawton, L.A., 2005. Detection and quantification of toxins in cultures of Microcystis aeruginosa (PCC 7820) by HPLC and protein phosphatase inhibition assayffect of blending various collectors at bulk. Afr. J. Sci. Technol. (AJST). Sci. Eng. Series 6 (1), 1–10. https://doi.org/10.4314/ajst.v6i1.55157. Al-Jassabi, S., Khalil, A.M., 2006. Microcystin-induced 8-hydroxydeoxyguanosine in DNA and its reduction by melatonin, vitamin C and vitamin E in mice. Biochemistry 71 (10), 1115–1119. Albendea, C.D., Gomez-Trullen, E.M., Fuentes-Broto, L., Miana-Mena, F.J., Millan-Plano, S., Reyes-Gonzales, M.C., Martinez-Ballarin, E., Garcia, J.J., 2007. Melatonin reduces lipid and protein oxidative damage in synaptosomes due to aluminium. J. Trace Elem. Med. Biol. 21, 261–268. Anisimov, V.N., Alimova, I.N., Baturin, D.A., Popovich, I.G., Zabezhinski, M.A., Manton, K.G., Semenchenko, A.V., Yashin, A.I., 2003. The effect of melatonin treatment regimen on mammary adenocarcinoma development in HER-2/neu transgenic mice. Int. J. Cancer 103, 300–305. https://doi.org/10.1002/ijc.10827. Berlett, B.S., Stadtman, E.R., 1997. Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272 (33), 20313–20316. Bessey, O.A., Lowry, O.H., Brock, M.J., 1946. A method for the rapid determination of alkaline phosphatise with five cubic millimetres of serum. J. Biol. Chem. 164, 321–329. Billam, M., 2006. Development and Validation of Microcystin Biomarkers for Exposure Studies. (Dissertation in Environmental Toxicology. Submitted to the Graduate Faculty of Texas Tech University in partial fulfillment of the requirements for the degree of Doctor of Philosophy).

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