Oleic acid protects against cadmium induced cardiac and hepatic tissue injury in male Wistar rats: A mechanistic study

Journal Pre-proof Oleic acid protects against cadmium induced cardiac and hepatic tissue injury in male Wistar rats: A mechanistic study

Bharati Bhattacharjee, Palash Kumar Chattopadhyay, Debasish Bandyopadhyay

Pal,

Aindrila

PII:

S0024-3205(20)30071-0

DOI:

https://doi.org/10.1016/j.lfs.2020.117324

Reference:

LFS 117324

To appear in:

Life Sciences

Received date:

24 October 2019

Revised date:

12 January 2020

Accepted date:

13 January 2020

Please cite this article as: B. Bhattacharjee, P.K. Pal, A. Chattopadhyay, et al., Oleic acid protects against cadmium induced cardiac and hepatic tissue injury in male Wistar rats: A mechanistic study, Life Sciences(2020), https://doi.org/10.1016/j.lfs.2020.117324

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

Journal Pre-proof Revised Manuscript Submitted for Publication in: Life Sciences

Title:

of

Oleic acid protects against cadmium induced cardiac and hepatic tissue injury in male Wistar rats: a mechanistic study

Authors:

Bharati Bhattacharjee , Palash Kumar Pala#, Aindrila Chattopadhyayb#, Debasish Bandyopadhyaya*

re

-p

ro

a#

Oxidative Stress and Free Radical Biology Laboratory, Department of Physiology, University of Calcutta, 92, APC Road, Kolkata-700009 Department of Physiology, Vidyasagar College,39, Sankar Ghosh Lane, Kolkata-700006

Jo ur

b

na

a

lP

Affiliations:

# The authors have equal contribution. *Address for Correspondence:

Debasish Bandyopadhyay Professor Department of Physiology University of Calcutta University College of Science and Technology 92, APC Road, Kolkata 700009, India Email:[email protected] Phone- +91-9433072066

Running title: Oleic acid protection of cadmium induced tissue injuries

1

Journal Pre-proof Abstract: Aims: The aim of the present study was to evaluate the possible antioxidant role of oleic acid (OA) against Cd-induced injuries in the heart and liver tissues of male Wistar rats. Main Methods: Rats were treated with either vehicle (control), or OA (10 mg/kg bw., fed orally), or Cd (0.44 mg/kg bw., s.c.), or both (OA+ Cd) for 15 days. Following completion of the treatment period, biomarkers of organ damage and oxidative stress including ROS, activities of antioxidant enzymes and their level, activities of Krebs cycle enzymes and respiratory chain enzymes were measured. Levels of interleukins (IL-1β, IL-6, IL-10), tumor

of

necrosis factor (TNF-α) and nuclear factor kappa B (NFκB) were estimated to evaluate the state of inflammation. In addition, changes in mitochondrial membrane potential and status of

ro

cytochrome c (Cyt c) were also studied.

-p

Key findings: Pre-treatment of rats with OA significantly protected against Cd-induced detrimental changes possibly by decreasing endogenous ROS through regulation of

re

antioxidant defense system, inflammatory responses and activities of metabolic enzymes. Moreover, OA was also found to restore mitochondrial membrane potential possibly by

lP

regulating Cyt c leakage thereby increasing mitochondrial viability. Significance: Our results for the first time demonstrated systematically that OA provided

na

protection against Cd-induced oxidative stress mediated injuries in rat heart and liver tissues through its antioxidant mechanism. The results raise the possibility of using OA singly or in

Jo ur

combination with other antioxidants or diet in the treatment of situations arising due to oxidative stress and may have future therapeutic relevance. Keywords: Cadmium, heart, liver, oxidative stress, oleic acid, antioxidant

2

Journal Pre-proof 1. INTRODUCTION: (499 words; limit is 500 words) Oleic acid (OA), a monounsaturated fatty acid (MUFA), is present in high concentration in olive oil (approx. 72%), the integral source of dietary fat mainly in the traditional Mediterranean diet, and an important component of diverse medicinal plant extracts [1-2]. Our earlier GCMS profiling study of aqueous bark extract of Terminalia arjuna (TA) revealed oleic acid as one of the major phyto-constituents [3]. Steady consumption of olive oil reflects its diverse health benefits in humans including prevention of some cancers, risk of coronary heart diseases and also immune and inflammatory modifications [1-2] which may be due to the presence of OA [4]. Several in vivo and in vitro studies have indicated the

of

antioxidant and anticancer properties of OA [4-5]. It has also been suggested that OA may

ro

participate in blood pressure reduction by improving endothelial function, probably by reducing reactive oxygen species (ROS) [6]. OA is also reported to exert its chemo-protective

-p

role against breast cancer [7] and also reduces the risk of the development of rheumatoid arthritis [8]. Moreover, OA is well documented for its hypoglycemic, hypotension, cardio-

re

protective [9] and hepato-protective roles as well as anti-inflammatory and anti-microbial

lP

properties [10-11] probably due to the presence of one double bond in it, which makes it less susceptible to oxidation and thereby contributing to its antioxidant property against excessive

na

oxidative load [12].

Among diverse stressors, cadmium (Cd)-a Type-1 human carcinogenic transition metal and one of the most toxic environmental pollutants [13] is a naturally non-degradable

Jo ur

toxicant. Therefore incidences regarding food chain mediated serious health hazards to humans are constantly increasing due to enhanced Cd contamination [14-16]. The biological half-life of cadmium is more than 20 years. Therefore, once absorbed it becomes accumulated in the target organ and causes adverse effects in various organs such as heart, liver, kidneys, brain, bones, lungs, reproductive and endocrine systems [3, 15]. Cadmium induces oxidative stress by replacing iron and copper ions from cytoplasmic and mitochondrial membrane proteins, which in turn increases the unbound Fe and copper ion concentrations which participates in the Fenton reaction, resulting in the generation of ROS [17]. Cadmium generated ROS primarily interact with several intrinsic macromolecules, displaying oxidative deterioration of lipids, proteins, and DNA [18]. Numerous animal studies have demonstrated that acute Cd exposure primarily is able to induce diverse hepatotoxicity [4, 19-20] and cardiovascular diseases [21-27] probably by disturbing antioxidative defense system [22-28]. 3

Journal Pre-proof Hence, the aim of the present study was to elucidate the possible protective role of OA against Cd-induced oxidative stress mediated damages in the heart and liver tissues of rat as experimental evidences. The results of the current study reveal that OA has the potential to provide protection against Cd induced oxidative mutilation of the heart and liver tissues through its anti-oxidative mechanisms and indicate toward a possible future use of OA as a therapeutic intervention besides the possibility of using OA as a nutritional supplement or a protective antioxidant either singly or in combination particularly in areas where mankind is exposed to contamination with Cd.

of

2. MATERIALS AND METHODS:

ro

2.1. Chemicals and reagents:

Cadmium acetate [(CH3COO)2Cd.2H2O] was purchased from Qualigens Limited, Mumbai,

-p

India. Oleic acid [CH3(CH2)7CH=CH(CH2)7COOH], JC-1(5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’tetraethylbenzimidazolcarbocyanine iodide) and Janus Green B were purchased from

re

Sigma-Aldrich Co. LLC, St. Louis, MO, USA. Hematoxylins, trichloro acetic acid (TCA),

lP

were obtained from Merck Limited, Delhi, India. Bovine serum albumin (BSA), N-(1naphthyl) ethylenediaminedihydrochloride (NED), sulfanilic acid, sodium nitrite, 2, 2dithiobis-nitro benzoic acid (DTNB), agarose, bromophenol blue, glycerol were obtained

na

from Sisco Research Laboratories (SRL), Mumbai, India. DCFDA (ab113851), different primary [Cu-Zn SOD (ab16831); Mn-SOD (ab13534), catalase (ab16731), GR (ab124995),

Jo ur

GPx (ab108427), NFkβ (ab16502)] and secondary [Goat secondary Ab to Rabbit IgG] antibodies were purchased from Abcam Biotechnology Company, Abcam; USA. β-actin (sc130657) (primary antibody) was obtained from Santa Cruz Biotechnology, Inc., USA. All other chemicals used were of analytical grade. 2.2. Animals: Male Wistar rats (n=56), body weight of 180-220 g, were used in two separate studies. The animals were handled in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. The protocols for the experiments using animals had the approval [Approval Number: IAEC- IV/Proposal/ DB-06/2014/ Dt. 13/03/2014] of the Institutional Animal Ethics Committee (IAEC) of the Department of Physiology, University of Calcutta. Animals were maintained under standard laboratory conditions with access to food and water ad libitum in well-ventilated cages throughout the investigation period. All 4

Journal Pre-proof the experiments were carried out at the animal house facility available with the Department of Physiology, University of Calcutta at its campus located at Rajabazar Science College, 92, APC Road, Kolkata 700 009, India. 2.3. Experimental design: Following proper acclimatization, thirty-two (32) rats were used in the first experiment for the determination of minimum effective dose of OA where significant protection was observed at a dose of 10 mg/kg BW (for details see supplementary file 1; Fig. 1). Beyond this dose, there occurred no further protection. This is the reason for choosing the dose of OA at

of

10mg/kg bw fed orally. Therefore, our subsequent experiments were carried out with OA at a dose of 10mg/kg body weight.

Group 1: Control (Con) rats were injected subcutaneously (s.c.) with normal saline

-p



ro

In the second experiment, rats (n=24) were equally divided into four groups as follow:

(0.9%) for 15 days.

Group 2: Rats were orally treated with oleic acid (OA) at a dose of 10 mg/kg BW (see

re

 

lP

supplementary file 1 for dose-dependent study of OA) once daily for 15 days. Group 3: Rats were injected with cadmium acetate (Cd; 0.44 mg/kg BW) subcutaneously

na

every alternate day for a period of 15 days. The present dose of Cd caused no mortality of animals during the treatment period and our earlier studies demonstrated that this dose of Cd showed the optimum effects [29-31]. Group 4: Rats were administered with OA (10 mg/ kg BW, fed orally), 60 min prior to

Jo ur



subcutaneous injection of Cd (0.44 mg/kg body weight) (OA+Cd). Our dose-response studies demonstrated that this dose of OA provided maximum protection in our experimental conditions (for details supplementary Fig. 1). 2.4. Collection and preparation of blood and tissue samples: After completion of the treatment period, animals were sacrificed through cervical dislocation following mild ether anesthesia. Blood from the individual animal was collected by cardiac puncture for the preparation of serum. Thereafter, the abdomen and thoracic cavity were opened, and the liver and heart were dissected out, weighed and immediately washed using ice-cold saline (0.9% NaCl). For histological studies, an appropriate portion of each of the hepatic and cardiac tissue from the individual animal was immediately placed in appropriate fixative. The rest of the tissues were stored in separate sterile plastic vials at -20ºC for further biochemical analysis. The hepatic and cardiac tissue homogenates (10% w/v) were prepared 5

Journal Pre-proof freshly in 50mM potassium phosphate buffer (pH 7.4), using a Potter–Elvehjem glass homogenizer [31-32]. 2.5. Estimation of different organ damage markers: 2.5.1. Estimation of the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST): Serum ALT and AST were measured according to the method of Reitman and Frankel [33], respectively and expressed as IU/L. 2.5.2. Estimation of the activities of total lactate dehydrogenase (TLDH), lactate

of

dehydrogenase 5 (LDH5) and lactate dehydrogenase1 (LDH1) in serum:

ro

The serum activity levels of TLDH, LDH5 and LDH1 were measured following the method as described by Strittmatter [34], with some modifications [35]. The enzyme activities were

-p

expressed in terms of IU/L.

re

2.6. Determination of the level of nitric oxide (NO) in serum: The level of NO in the serum samples was indirectly assessed by measuring the levels of

lP

nitrite following the method of Green et al. [36] by using Griess reagent (1% sulfanilamide and 0.1% NED in 2.5% ortho phosphoric acid) and the optical density was recorded at 540

na

nm [37] (for details refer supplementary file 2). 2.7. Measurement of the levels of inflammatory markers in serum:

Jo ur

Serum levels of IL-1β (ELR-IL1b), IL-6 (ELR-IL6), IL-10 (ELR-IL10) and TNFα (ELRTNFα) were determined through Enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s (Ray Biotech, Norcross, GA) instructions using an UV/visible ELISA reader (Smart Spec Plus spectrophotometer, Bio-Rad). 2.8. Measurement of biomarkers of oxidative stress in tissue - lipid peroxidation (LPO), protein carbonyl content (PCO), reduced glutathione (GSH) and oxidized glutathione (GSSG) contents as well as GSSG: GSH ratio: Lipid peroxides in both the tissue homogenates were measured as thiobarbituric acid reactive substances (TBARS) following the method of Buege and Aust [38] with some modifications [39]. The contents of PCO and GSSG in both the tissues were determined by DNPH assay following the method of Levine et al. [40] and Wendell et al. [41], respectively. GSH content was estimated following the method of Sedlak and Lindsey [42], with some modifications [39]. 6

Journal Pre-proof 2.9. Measurement of the activities of antioxidant enzymes: superoxide dismutases (SOD 1 and SOD 2), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx): Activities of SOD 1 and SOD 2 were measured by the pyrogallol autoxidation method as previously reported [43]. The CAT activity was measured according to the method described by Beers and Sizer [44]. GR activity was determined by the method of Krohne-Ehrich et al. [45], while the activity of GPx was measured according to the method of Paglia and Valentine [46], with some modifications [47] (for details refer supplementary file 2).

of

2.10. Western Blot analysis: Heart and liver tissue homogenates were prepared for western blot analysis according to the

ro

method reported in earlier studies [6, 39]. Following the method as described by Laemmli

-p

[48], an equivalent of 80 µg protein samples was resolved by 10% SDS–PAGE for immunedetection of SOD1, SOD2, CAT, GPx, GR, NFkβ, Cyt c, HSP 70and β-actin (1:2000

re

dilution); while, secondary antibody (Goat anti-rabbit IgG alkaline phosphatase conjugate) was use at a dilution of 1:6000. The pixel density of the each band was analyzed using

lP

ImageJ software (NIH, Bethesda, MD, USA) (for details refer to supplementary file 2).

na

2.11. Measurement of activities of metabolic enzymes: 2.11.1. Measurement of hexokinase (HK) activity:

Jo ur

Hexokinase activity was assayed following the method of Freehold et al. [49] (for details refer to supplementary file 2).

2.11.2. Measurement of the activities of pyruvate dehydrogenase (PDH) and other Krebs cycle enzymes:

The activities of PDH and citrate synthase (CS) were measured according to the method of Chretien et al. [50] and Srere [51], respectively. Activities of isocitrate dehydrogenase (ICDH) and alpha-ketoglutarate dehydrogenase (α-KGDH) were estimated following the method of Duncan et al. [52]. Succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) activities were estimated following the method of Veeger et al. [53] and Mehler et al. [54], respectively (for details refer supplementary file 2). 2.11.3. Measurement of the activities of respiratory chain enzymes: The activities of cytochrome c oxidoreductase and cytochrome c oxidase were estimated following the method of Goyal and Srivastava [55] (for details refer to supplementary file 2). 7

Journal Pre-proof 2.12. Indirect assessment of superoxide anion free radical (O2•-) generation by measuring the activities of xanthine oxidase (XO)/ xanthine dehydrogenase (XDH) system: The activity of XO, by measuring the amount of conversion of xanthine to uric acid was determined spectrophotometrically following the method described by Greenlee and Handler [56] with some modifications as adopted by Mukherjee et al. [32]. Similarly, XDH activity was also measured spectrophotometrically following the method of Mukherjee et al. [32]. The enzyme activities were expressed as milli Units/mg tissue protein. 2.13. Tissue morphological studies of heart and liver:

of

2.13.1. Haematoxylin-Eosin (HE) and PAS staining:

ro

Heart (ventricular region) and liver tissues were fixed in 10% formalin, embedded in paraffin and stained following the routine procedure described by Roy et al. [57]. A required portion

-p

of hepatic tissue section was fixed in 10% natural buffered formalin and processed further for Periodic-Acid Schiff (PAS) staining following routine procedure [32] (for details refer

re

supplementary file 2).

lP

2.13.2. Confocal microscopy of Sirius Red-stained heart and liver tissues: Five μm thick heart and liver tissue sections were stained with Sirius Red (Direct Red 80;

na

Sigma Chemical Co., St. Louis, MO, USA) following the method of Roy et al. [57] (for details refer supplementary file 2).

Jo ur

2.13.3. Scanning electron microscopy (SEM) of cardiac and hepatic tissue sections and isolated mitochondria:

Heart and liver tissue sections and also isolated mitochondria from both the tissues were fixed in 2.5% (w/v) glutaraldehyde at 4ºC for 24-48 h following the method of Watanabe et al. [58] and as followed earlier [6, 59] (for details refer supplementary file 2). 2.14. Determination of mitochondrial intactness by Janus green B staining: Mitochondrial intactness was determined by using a mitochondria-specific supravital basic stain- Janus Green B following a well-calibrated method of Cooperstein et al. [60] (for details refer to supplementary file 2). 2.15. Determination of the mitochondrial membrane potential (Δψm):

8

Journal Pre-proof Membrane potential of the mitochondrial samples prepared from both heart and liver tissues was determined by using JC-1 (cationic fluorescent dye) following the method described by Cossarizza et al. [61] (for details refer to supplementary file 2). 2.16. Measurement of the levels of ROS: In order to determine the levels of ROS, cardiac and hepatic tissue samples were stained with 2´,7´–dichlorofluorescein diacetate (DCFDA) followed by washing in PBS. The samples when incubated with the stable non-fluorescent DCFDA, it gets oxidized into a highly fluorescent 2′, 7′-dichlorofluorescein (DCF) in the presence of ROS [62]. The level of ROS

of

were recorded using flow cytometer (BDFACS Versa, USA) and analyzed (104 events/sec) using Flowjo software and expressed as DCF fluorescence intensity (FITC-A Median).

ro

2.17. Determination of modifications in mitochondrial proteins through fluorescence

-p

study:

The methods described by Dousset et al. [63] and Giulivi and Davies [64] were used to study

re

the extent of tryptophan modifications and dityrosine formation through analysis of

lP

fluorescence spectra (for details refer to supplementary file 2). 2.18. Determination of mitochondrial redox status:

na

Mitochondrial redox status was measured following the method of Minezaki et al. [65]. In brief, NADH autofluorescencein the mitochondrial fractions isolated from heart and liver

Jo ur

tissues was evaluated (emission from 400 to 500 nm; slit width 5nm and excitation 340 nm; slit width 5nm) and expressed as fluorescence intensity (arbitrary unit). 2.19. Estimation of cadmium concentration in tissue: Cadmium content in heart and liver tissues was estimated [6, 66] by atomic absorption spectrophotometry and expressed as µg/g (for details refer supplementary file 2). 2.20. Isothermal Titration Calorimetric (ITC) study: The binding study of pure GSH and catalase with Cd and OA was performed by ITC using Microcal ITC-200, Malvern, UK. In this assay, 0.3 mL of GSH (4mM) was titrated separately in the sample cell containing 0.06 mL of Cd (0.1 mM) and/or OA (1 μM) which were used as ligand. In a separate study, 0.35 mL of pure catalase (4 ×10−6mM) was titrated with 0.06 mL of ligands, such as Cd (0.1 mM) and/or OA (1 μM) in the sample cell. Titration was conducted with 20 injections of each ligand with 2 μl volume and 150 sec spacing between

9

Journal Pre-proof two successive injections at 37ºC for approximately 1 h. Data were analyzed using Origin 7.0 software (Microcal LLC, Massachusetts, USA). 2.21. Assessment of generation of free hydroxyl radical (•OH) in rat cardiac and hepatic mitochondria, in vitro: In order to determine the hydroxyl radical scavenging activity of OA, mitochondria was isolated from the heart and liver tissues following the method of Hare et al. [67] with minor modifications [32]. In brief, fifty percent (50%) mitochondrial suspension was incubated in 50mM phosphate buffer (pH 7.4) by using DMSO (500mM) in different combinations with

of

Cd and OA. The final volume of incubation mixture was 1 mL. The four different experimental groups were: (i) Control (with DMSO) (ii) OA (1 µM) + DMSO (iii) Cadmium

ro

(100 µM) + DMSO, and (iv) OA (1 µM) in presence of Cd (100 µM) along with DMSO at 37°C for 60 min. Following completion of 1 hour incubation, the reaction was terminated by

-p

addition of 0.02 mL 35mM EDTA. The level of methane sulfonic acid (MSA) formed during

re

incubation was measured following the method of Babbs and Steiner [68] as modified by Bandyopadhyay et al. [39].

lP

2.22. Protein Estimation:

The protein content of each tissue sample was estimated following the method of Lowry et al.

Jo ur

2.23. Statistical analysis:

na

[69] using the bovine serum albumin as the standard.

All values are expressed as mean ± SEM (Standard error of the mean) of triplicate observations. One-way analysis of variance (ANOVA) was used for determining the statistical significance of the data. The significance of differences between experimental groups was evaluated by post-hoc comparison performed by Tukey’s HSD test. All statistical analyses and data presentation were performed using GraphPad Prism version 5.0, Microcal Origin version 7.0 (Microcal LLC, Massachusetts, USA) and Windows Excel. 3. RESULTS: 3.1. Organ damage markers: 3.1.1. Levels of ALT and AST activities in the serum: Treatment of rats with Cd caused a significant (p<0.001) increase in the serum levels of AST and ALT activities when compared to control value (Fig. 2A). However, Pre-treatment of the animals with OA was found to significantly (p<0.001) decrease their levels of activity when 10

Journal Pre-proof compared to the Cd administered group. Notably, OA alone group did not show any alteration in their AST and ALT levels when compared to control (Fig. 2A). 3.1.2. Activities of TLDH, LDH-5 and LDH-1 in the serum: Cd administration significantly (p<0.001) elevated the activities of TLDH, LDH-5 and LDH1 in the serum in comparison to the control (Fig. 2B), while their activities were significantly protected in Cd+OA group when compared to the Cd-treated group. However, no significant alteration in the activities of TLDH, LDH-5 and LDH-1 was noted in the animals treated with OA alone (Fig. 2B).

of

3.2. Level of NO in the serum:

ro

Administration of Cd caused a significant (p<0.001) elevation in the serum level of NO compared to the control (Fig. 2C). However, NO levels were significantly (p<0.001)

-p

protected from being increased in the Cd+OA group when compared to Cd-treated group. However, OA group did not show any change in the serum level of NO when compared to

re

the control group.

lP

3.3. Levels of inflammatory markers in serum:

A significant (p<0.001) increase in the serum levels of pro-inflammatory cytokines IL-1β, IL-

na

6 and TNF-α in the Cd-treated groups was noted with a concomitant decrease in the level of anti-inflammatory cytokine IL-10 as compared to control (Fig. 2: D-G). Pre-administration of

Jo ur

rats with OA was found to significantly (p<0.001) decrease the serum levels of IL-1β, IL-6, and TNF-α, but increased the level of IL-10 when compared to the Cd-treated group. However, OA alone group did not show any significant change when compared to control. 3.4. Biomarkers of oxidative stress in tissue- LPO, PCO, GSH, GSSG, and GSH: GSSG ratio: Administration of Cd caused a significant (p<0.001) increase in the levels of LPO and PCO in the heart and liver tissues when compared to the control values (Fig. 3: A-B). However, the levels of LPO and PCO in both the tissues were found to be significantly (p<0.001) decreased in the Cd+OA group when compared to the Cd-treated group (Fig. 3: A-B). Animals treated with OA alone did not show any alteration when compared to the control group. In contrast, Cd treatment significant (p<0.001) reduced the levels of GSH (Fig. 3C), but increased GSSG level (Fig. 3D) as well as GSSG: GSH ratio (decrease 72.89% in heart and 81.35% in liver vs. control; p<0.001) (Fig. 3E) in the heart and liver tissues when compared 11

Journal Pre-proof to the control. However, levels of GSH, GSSG, and GSSG:GSH ratio in the Cd+OA group were found to be protected from being significantly altered in both the tissues when compared to the Cd-treated group (Fig. 3: C-E). However, irrespective of the studied tissues, OA alone group did not show any change in these parameters when compared to control values. 3.5. Activities and protein levels of antioxidant enzymes and NFκβ and HSP70: Treatment of rats with Cd caused a significant (p<0.001) decrease in the activities of SOD 1 and SOD 2, but a significant increase was noted in the activity of CAT in the heart tissues

of

when compared to the control animals (Fig. 4: A-C). On the other hand, Cd treatment induced significant (p<0.001) elevation in the activities of SOD 1 and SOD 2 in the liver tissue with a

ro

concomitant decrease in the CAT activity. However, in Cd+OA group, the activities of antioxidant enzymes in both heart and liver tissues were found to be significantly (p<0.001)

-p

protected from being altered compared to the Cd-treated groups when the rats were pre-

re

treated with the minimum effective dose of OA (Fig. 4: A-C). OA alone treated group, however, did not show any effect on the activity of these enzymes nor in their protein

lP

expressions when compared to the control group. Notably, western blot analysis of the same tissue samples displayed a similar response in the protein levels of SOD 1, SOD 2 and CAT

na

[(Fig. 5II: A-C) in heart tissue and (Fig. 5III: G-I) in liver tissue]. Moreover, Cd administration to rats caused a significant (p<0.001) elevation in the activities

Jo ur

of GPx with a concomitant decline in GR activities in both the tissues when compared to control values (Fig. 4: D-E). However, when the rats were pre-treated with OA, a significant (p<0.001) protection of both the enzyme activities were observed when compared to the activities observed in the Cd-treated rats. But the rats of the OA alone group did not show any alteration in the activities of these enzymes. Western blot analysis clearly demonstrated a similar response in the protein expression of GPx and GR in the heart (Fig. 5II: D- E) and liver (Fig. 5III: J-K) tissues. In Cd administered group, a significant (p<0.001) elevation in the protein levels of NFκβ (Fig. 5II; F and 5III; L) and HSP70 (Fig. 7II; C and 7III; F) in both heart and liver tissues were observed when compared to the control groups. However, pre-treatment of rats with OA decreased the protein levels of NFκβ and HSP70 in both the tissues when compared to the Cd-treated groups. 3.6. Measurement of activities of metabolic enzymes:

12

Journal Pre-proof 3.6.1. Hexokinase activity: A highly profound significant (p<0.001) decrease in the activity of hexokinase, the first enzyme of the glycolytic pathway, was observed in both the tissues when the rats were treated with Cd. Pre-treatment of rats with OA at a dose of 10 mg/kg BW protected such Cdinduced alterations in HK activity to the control level (Fig. 6A). However, the animals treated with OA alone did not show any significant (p<0.001) effect on the activity of hexokinase in both the tissues. 3.6.2. Activities of PDH and other Krebs cycle enzymes:

of

Treatment of rats with Cd caused significant (p<0.001) decrease in the activities of all the studied Krebs cycle enzymes i.e. PDH, CS, ICDH, α-KGDH, SDH and MDH in both the

ro

tissues when compared to the control. But their activities were significantly (p<0.001)

-p

protected from being altered in the Cd-treated group when the rats were pre-treated with OA (Fig. 6: B-G). The rats treated with OA alone did not show any significant alteration in the

re

activities of Krebs cycle enzymes.

lP

3.6.3. Enzymatic activities of respiratory chain enzymes: A significant (p<0.001) decrease in the activities of cytochrome c oxidase and cytochrome c

na

oxidoreductase in the heart and liver tissues were observed in the animals treated with Cd (Fig. 6: H-I). However, in Cd+OA group, a significant (p<0.001) protection in the activities

Jo ur

of both cytochrome c oxidase and cytochrome c oxidoreductase were found compared to the Cd-treated group when the rats were pre-treated with OA. However, rats treated with OA only did not show any significant change in the activities of respiratory chain enzymes in both the tissues.

In Cd administered group, a significant (p<0.001) elevation in the protein levels of cytoplasmic Cyt c (Fig. 7II; A and 7III; D) in both heart and liver tissues were observed when compared to the control groups. On the other hand, a significant (p<0.001) reduction in the protein levels of mitochondrial Cyt c (Fig. 7II; B and 7III; E) in both heart and liver tissues were observed when compared to the control groups. However, pre-treatment of rats with OA decreased the protein levels of Cyt c in the cytosol of both the tissues when compared to the Cd-treated groups. On the other hand, in comparison with the Cd-treated groups, OA pretreatment was found to increase the levels of Cyt c, although the data was not significant. 3.7. Indirect assessment of superoxide anion free radical (O2•-) generation by the xanthine oxidase (XO)/ xanthine dehydrogenase (XDH) system: 13

Journal Pre-proof A significant enhancement in the generation of O2•- was observed following the treatment of rats with Cd. The activities of XO and XDH, total enzyme activity, i.e., XO+XDH, and XO/XDH ratios in both heart and liver tissues were increased significantly following Cd treatment when compared to the control values. All these parameters, however, in the cardiac and hepatic tissues were found to be significantly protected from being elevated when compared to the Cd treated group (Fig. 8; A-D), indicating its free radical neutralizing property. However, OA only treated group did not show any significant change in their enzyme activities.

3.8.1. Hematoxylin and Eosin (HE) and PAS staining:

of

3.8. Tissue morphological studies of heart and liver:

ro

Tissue morphological studies of rat heart and liver of Cd administered rats showed marked

-p

cardiac damage including vascular congestion, vacuolization of cytoplasm, capillary dilatation, myocardial fiber necrosis and irregularity of myofibrils when compared to the

re

control rats (Fig. 9: Panel A). Similarly, administration of Cd showed extensive hepatic damage including central as well as portal vein dilatation, vacuolization with necrotic

lP

hepatocytes, sinusoidal congestion, inflammatory hepatic cell infiltration, enlarged nuclei along with degenerative changes when compared to the control rats (Fig. 9: Panel B). On the

na

other hand, PAS-stained liver tissue section showed marked glycogen depletion in the Cdtreated group (Fig. 9: Panel C). However, all the abnormalities in the tissue morphology

Jo ur

induced by Cd in heart and liver tissues were significantly ameliorated when the rats were pre-treated with oleic acid (Fig. 9). The OA alone treated group did not show any change when compared to control.

3.8.2. Confocal microscopy of Sirius Red-stained liver and heart tissues: Picrosirius red staining revealed a significant (p<0.001) decrease in the levels of collagen volume (%) in the cardiac tissue section, but were increased in hepatic ones following the treatment of rats with Cd (Fig. 10: Panel A-C). These changes were found to be protected from being altered when the rats were pre-treated with OA in comparison to Cd administered group. However, OA alone did not show any alteration compared to control. 3.8.3. Scanning electron microscopy (SEM) of cardiac and hepatic tissue sections and isolated mitochondria: Scanning electron microscopic study revealed marked architectural changes in cardiac tissue sections such as irregular branching pattern of cardiac muscle fibers by forming a 14

Journal Pre-proof complicated matrix network following the treatment of Cd. Simultaneously, distorted polyhedral arrangements of hepatocytes with dilated central veins were also observed in Cdtreated liver tissue sections (Fig. 11: Panel A-B). Cd also caused blebbing of mitochondria and distortion indicating disruption of mitochondrial morphology. However, all the Cdinduced alterations were found to be protected when the rats were pre-treated with OA in comparison to Cd-treated group (Fig. 11: Panel C-D). Treatment with OA alone, however, has no significant effect on the tissue and mitochondrial architecture compared to control. 3.9. Assessment of mitochondrial intactness by Janus green B staining:

of

Administration of Cd caused a significant (p<0.001) decrease in the intactness of mitochondria isolated from both heart and liver tissues, when compared to the control (Fig.

ro

12: Panel A-C). The levels of mitochondrial intactness were found to be protected significantly (p<0.001) from being altered when the rats were pre-treated with oleic acid (Fig.

-p

12: Panel A-C). The rats treated with OA only did not show any significant change in the

re

intactness of mitochondria isolated from both heart and liver tissues.

lP

3.10. Determination of the mitochondrial membrane potential (Δψm): Treatment of rats with Cd significantly (p<0.001) increased membrane depolarization in mitochondria isolated from both heart and liver tissues when compared to the control rats

na

(Fig. 12: Panel D-F). Pre-treatment of rats with OA significantly protected the mitochondrial membrane depolarization from being decreased compared to Cd-treated rats. However, OA

Jo ur

alone did not show any significant difference in mitochondrial membrane depolarization compared to control rats.

3.11. Measurement of the levels of ROS: The levels of ROS in heart and liver tissues were measured by flow cytometric analysis using DCFDA. Administration of Cd caused a significant (p<0.001) increase in the DCF fluorescence intensity in both the tissues compared to control group (Fig. 13: A-B). However, this elevation in the levels of DCF intensity was found to be protected significantly (p<0.001) when the rats were pre-treated with OA (Fig. 13: A-B). OA alone has no significant effect on the tissue levels of ROS compared to control. 3.12. Determination of modifications in mitochondrial proteins through fluorescence study:

15

Journal Pre-proof Measurement of tryptophan and dityrosine through fluorescence study provided important information regarding oxidation induced alteration in the respective protein level. In our study, treatment of rats with Cd revealed a significant (p<0.001) decline in the levels of tryptophan with a concomitant elevation in the levels of dityrosine in the mitochondrial samples (isolated from both heart and liver tissues) compared to control (Fig. 14: A-B). However, pre-treatment of rats with OA significantly protected their levels from being altered when compared to Cd administered group (Fig. 14: A-B). However, OA did not show any significant change when compared to control.

of

3.13. Determination of mitochondrial redox status: Redox status of mitochondria isolated from both heart and liver tissues was determined

ro

through NADH autofluorescence study. Treatment of rats with Cd caused a significant (p<0.001) reduction in the levels of NADH compared to the control (Fig. 14C). This

-p

reduction in the level of NADH was found to be significantly protected when the rats were

re

pre-treated with OA (Fig. 14C). However, rats treated with OA alone did not show any

3.14. Tissue Cd concentration:

lP

significant change in the level of NADH compared to control.

na

Cadmium concentration was significantly elevated in both heart and liver tissues following the treatment of rats with this heavy metal, compared with the control group (Fig. 14D).

Jo ur

However, pre-treatment of the animals with OA significantly reduced the concentration of Cd in both the tissues when compared to the Cd-treated animals (Fig. 14D). Treatment of rats with OA only did not have any effect on tissue Cd level compared to control. 3.15. Isothermal Titration Calorimetric (ITC) study: The ITC data chart illustrates that the binding pattern of pure GSH individually with Cd and OA. The titration profile showed that a three-site binding sequential reaction when GSH directly bind with Cd, indicating a spontaneous reaction with high intensity of binding energy. However, titration of OA with GSH displayed a high initial heat change with significant linear one site binding pattern (Fig. 15: A-C). Interestingly, an endothermic reaction was observed upon titration of Cd and OA with GSH. In the sample cell both the ligands competes for the same binding site of GSH and a significant one site binding pattern with a decline in the reaction was observed where the net energy change was positive. Thermodynamic parameter of 1 mM Cd interaction with catalase at a constant temperature was also analyzed by ITC. Upon titration of pure catalase with Cd only, an exothermic 16

Journal Pre-proof interaction with a significant one site binding pattern was observed (Fig. 15: D-F). The titration profile showed that a high initial heat change occurred which ultimately decline with an increase in time. However, pure catalase with OA when titrated in a sample cell, a one site binding with high initial heat change was observed along with sequential titration with time. Interestingly, titration profile showed that both the ligands such as OA and Cd compete for the same binding site of catalase when 1 µM OA and 1 mM Cd was injected as a ligand in the catalase containing sample cell. An endothermic titration reaction was observed which ultimately slowed down with the increase in time. 3.16. Assessment of generation of free hydroxyl radical (•OH) in mitochondria from

of

heart and liver: an in vitro study:

ro

Incubation of isolated mitochondrial fractions of the heart and liver tissues with 100µM of Cd was found to significantly (p<0.001) increase (Fig. 8E) the generation of free •OH radical.

-p

Such elevations were protected from being altered when mitochondria of both the tissues

re

were co-incubated with OA at a concentration of 1 µM, indicating a probable direct free radical scavenging activity of OA (Fig. 8E).

lP

4. DISCUSSION: (1582 words; limit is 1500 words) The importance of oleic acid as an antioxidant and hence its potentiality to prevent Cd-

thoroughly studied.

na

induced oxidative stress-mediated [25-27] injuries to rat heart and liver tissues has been

Jo ur

Cardiac and hepatic dysfunction is associated with increased levels of tissue specific marker enzymes in the serum indicating loss of membrane integrity [6]. In our study levels of tissue-specific serum biomarker enzymes viz. AST, ALT, T-LDH, LDH-5, and LDH-1 in the Cd-treated groups were elevated which is clearly indicative of cardiac and hepatic toxicity. Cd-induced such were protected when rats were pre-treated with oleic acid (10 mg/kg BW). Similarly, cadmium induced marked elevation in the serum levels of different proinflammatory cytokines (IL-1β, IL-6, and TNF-α) along with decrease in the level of antiinflammatory cytokine (IL-10) suggested the potentiality of Cd to induce inflammation through immunomodulation of these cytokines [70]. However, pre-treatment of OA protected the levels of the inflammatory cytokines in Cd-treated rats which possibly resulted from the anti-inflammatory property of OA. Our histological findings showed profound disruption and degeneration in the histoarchitecture of both the cardiac and hepatic tissues following Cd treatment. Similarly, PAS-

17

Journal Pre-proof stained hepatic tissues sections revealed depletion of glycogen content in the cadmium treated rats probably due to rapid glycogenolysis [71, 72]. Glycogen depletion may also result from inhibition of oxidative phosphorylation due to reduced oxygen consumption induced by cadmium [73]. Pre-treatment with OA protected the tissues from undergoing such histopathological alterations, indicating that OA can help overcome such intracellular oxidative stress. Following picrosirus staining marked depletion of collagen content in cardiac tissue sections and deposition in the hepatic tissue sections were noted indicating mature fibrosis with ongoing inflammation [74]. However, pre-treatment with OA inhibited such deleterious effects of Cd on both the tissues.

of

Cd, a non-Fenton metal induces nitrosative stress indirectly by the generation of

ro

various oxygen radicals including nitric oxide [75]. In our study, Cd induced elevation in serum NO level were restricted and furthermore decreased following OA pre-treatment,

-p

arguing in favor of its antioxidant capacity. Moreover, Cd treatment increased the accumulation of this metal in both the tissues which might be due to its long half-life [2].

re

However, in our experiments, pre-treatment with OA decreased such accumulation possibly through its heavy metal chelating property.

lP

Lipid peroxidation, a potent indicator of oxidative damage [76] and PCO content which signifies oxygen radical mediated damage of protein molecules [77] were increased in

na

both cardiac and hepatic tissues indicating profound oxidative damage. However, pretreatment with OA at a dose of 10 mg/kg BW successfully decreased the levels of LPO and

Jo ur

PCO possibly by scavenging the harmful free radicals generated during Cd toxicity, thus protecting the tissues from undergoing oxidative damage. Reduced glutathione plays a crucial role in detoxification of ROS for cellular protection. In our study, Cd administration reduced the levels of GSH in both heart and liver tissues possibly due to formation of cadmium-glutathione complex or, increased utilization of GSH (free radical scavenger) by the cell [31, 78]. Under stressful condition, GSSG accumulates in the cells with a concomitant decrease in the ratio of GSH/GSSG which serves as an indicator of oxidative stress [78]. Present investigation revealed that Cd-induced stress increased the levels of GSSG, thereby decreasing the GSH/GSSG ratios in both tissues. OA pre-treatment, however, protected the tissue levels of GSH, GSSG as well as redox status (GSH/GSSG ratio) indicating that OA treatment is able to mitigate Cd-induced oxidative stress. The intrinsic fluorescence of tryptophan and di-tyrosine is a sensitive marker of oxidative damage which determines the modification of specific amino acids [79]. In the 18

Journal Pre-proof present study, Cd-caused reduction in the tryptophan levels but elevated di-tyrosine levels in both heart and liver mitochondrial samples. However, their levels were protected in both the tissues when the rats were pre-treated with OA which may be attributed to its antioxidant property. Cadmium is well known to alter the activities of numerous antioxidant enzymes either by direct binding to their active site or, by displacing a beneficial metal (iron or, zinc) from their catalytic site [80, 81]. In our study, Cd-treatment reduced the activities of SOD enzymes (SOD 1 and SOD 2) in cardiac tissues which indicated inactivation of these antioxidant enzymes either by Cd-induced excess accumulation of superoxide anion radicals or, directly

of

though antagonistic effect of Cd with copper and zinc, resulting in loss of its catalytic

ro

function through conformational changes [82]. On the other hand, increased activities of both isoforms of SODs in liver tissues following Cd treatment might be an adaptive response of

-p

the hepatic cells to excessive production of H2O2 [83]. Similarly, Cd induced alterations in the catalase activity in cardiac and hepatic tissues may also be an adaptive response to

re

oxidative stress [84]. Such contention may be supported by similar responses in the protein levels of these enzymes subjected to Cd treatment. However, Cd-induced alterations in the

lP

levels or, activities of all these enzymes were protected when the rats were pre-treated with OA indicating the indirect antioxidant property of OA. Interestingly, results of ITC study

na

indicated that Cd thermodynamically binds predominantly via electrostatic forces to GSH at one site and catalase at the other site which exhibits sequential binding. The observed

Jo ur

thermodynamically favored interaction of Cd with GSH might be due to thiol affectionate property of Cd- a crucial event in Cd-toxicity [81]. Cd-catalase interaction altered the enzyme activity possibly by misfolding its structure [85]. Interestingly, OA protected the GSH level and catalase activity from being altered by Cd either by quenching the harmful free radicals or, by its direct binding to Cd or, by protecting the vulnerable sites from oxidative damage. These present observations again supported our in vivo results. Cadmium induced increase in the activity of GPx in both heart and liver tissues might be due to elevation in the levels of intracellular H2O2, hydroperoxides and LPO [86]. In contrast, decrease in GR activity in both the tissues might result from increased usage of this enzyme in scavenging Cd-induced free radicals through formation of Cd-SH complex [87]. Such diverse response in the activities of these enzymes to Cd toxicity might be due to the abundance of their respective proteins. However, both the activities and protein levels of GPx and GR were found to be protected when the rats were pre-treated with OA suggesting its potent indirect antioxidant property. 19

Journal Pre-proof Activity of hexokinase, a crucial enzyme in glycolysis, was reduced following Cdtreatment suggesting an impairment of glucose metabolism and energy production, thereby ensuring oxidative stress within the concerned tissue. Pre-treatment of Cd-treated rats with OA protected the activity of hexokinase from being altered. Superoxide radicals are mostly formed and dismutated to H2O2 in mitochondrial ETC complex, primarily in complex II and complex III [81] where various transition metals such as Cu, Zn, and Fe are vital for the catalytic function of several enzymes to rescue the antioxidant defense mechanism [88]. Cd is already demonstrated to replace such redox active metals from the active site of these catalytic enzymes and generate O2•- anion radical [80],

of

thus inhibiting their activities in ETC. In our study, Cd exposure interrupted mitochondrial functionality by reducing the activity of PDH and some of the Krebs cycle enzymes such as

ro

CS, ICDH, α-KGDH, SDH and MDH in both heart and liver tissues. However, pre-treatment

-p

with OA rescued the activities of these enzymes possibly through its free radical scavenging capacity. Similarly, Cd administration also reduced the activities of NADH cytochrome c

re

reductase and cytochrome c oxidase in both heart and liver tissues [89]. OA pre-treatment, however, protected the activities of these respiratory chain enzymes from being altered,

lP

probably by inhibiting Cd-induced over production of oxidative reactive radicals. In contrast, increase in the protein levels of HSP70, a stress activated protein, in both the cytosolic and

na

mitochondrial fractions following Cd treatment might be responsible for altering the cascade of ETC and ATP production, thus triggering over production of ROS mediated damages to

Jo ur

intracellular essential matrix enzymes [90]. Similarly, Cd treatment elevated mitochondrial membrane potential and pre-treatment with OA protected any such alteration in membrane potential. Increased activities of XO and XDH with a concomitant increase in their cumulative activities (XO+XDH) and ratio of XO and XDH (XO/XDH) following Cd treatment indicate, although indirectly, generation of large amount of O2•- anion radical in both the tissues [6]. Pre-treatment of rats with OA, however, protected such alterations in the activities of these enzymes. Intracellular co-enzyme NADH plays a crucial role in cellular oxidation-reduction reactions [91]. NADH auto-fluorescence study revealed marked decline in the NADH levels in both heart and liver mitochondrial following Cd –induced oxidative stress resulting in the altered mitochondrial surface topology. However, pre-treatment with OA protected the levels of NADH. Similar observations were reported following JG-B and SEM studies [59]. Pretreatment with OA in Cd administered rats, however, protected the mitochondria and prevented the structural alterations of the mitochondrial surfaces. On the other hand, Cd 20

Journal Pre-proof treatment at a dose of 0.44 mg/kg BW increased the protein level of NFkB transcription factor in both the tissues which might be responsible for alterations in the expression of oxidative stress related genes [92]. Cadmium induced enhancement in the production of hydroxyl radical and ROS, as is evident from the results of the in vitro study, and it might be a probable cause for this upregulation of NFkB protein. In either case, oleic acid pre-treatment, however, decreased the levels of cellular ROS as well as protected the transcription factor NFkB from being activated in both heart and liver tissues, indicating free radical scavenging property of OA in mitigating Cd-induced oxidative stress. Thus, the present studies, and other similar studies carried out by different authors only can

of

significantly indicate the deleterious effects of cadmium exposure and possibilities of

ro

providing protection against such situation. However, long term observational and clinical studies are warranted in the days to come to explain how a human being would be affected

re

Conclusion: (149 words; Limit 150 words)

-p

following life time exposure either occupationally or non-occupationally.

The present study revealed the novel potentiality of oleic acid in providing protection against

lP

cadmium-induced tissue injury. OA may directly regulate the synthesis of different antioxidant enzymes as well as their activities to restrict Cd-induced oxidative damage or

na

may interact with Cd, restricting its binding to endogenous functional proteins, such as enzymatic or non-enzymatic antioxidants; thus demonstrating its chelating property. It may

Jo ur

also possibly inhibit the generation of Cd-induced ROS either by inhibiting structural modulation of mitochondrial proteins or, by protecting intracellular redox status. Finally, OA also inhibits pro-inflammatory cytokines and triggers anti-inflammatory ones, thus possibly inhibiting the cascade pathway of apoptosis induced by Cd. Therefore, consumption of OA or, OA fortified diet or, diets containing nutritionally relevant olive oil or, aqueous bark extract of Terminalia arjuna [3] could be used as a promising protective measure against Cdinduced cardiac and hepatic tissue injuries which may have future therapeutic relevance. Acknowledgments: Bharati Bhattacharjee gratefully acknowledges the receipt of a Senior Research Fellowship (SRF) under INSPIRE (IF140691) program of Department of Science and Technology, Govt. of India. Dr. Palash Kumar Pal is a Dr. D. S. Kothari Post-Doctoral Fellow (BL/16-17/0502) of University Grant Commission (UGC), Govt. of India. Dr. Ritesh Tiwari (CRNN), Souvik Roy and Arijit Pal from DBT-IPLS scheme of University of Calcutta, Department of 21

Journal Pre-proof Biotechnology (DBT), Govt. of India are acknowledged. AC is supported by funds available to her from Department of Science and Technology, Govt. of West Bengal. This work is also partially supported from Major Research Project Grant to Prof. DB [F. No. 37-396/2009 (SR)]. Prof. DB also extends his grateful thanks to University Grants Commission, Govt. of India, for the award of a Research Project under Centre with Potential for Excellence in a Particular Area (CPEPA), at University of Calcutta. DB also gratefully acknowledges the support he received from DST-PURSE Program awarded to University of Calcutta, and Departmental Research Grant (BI 92).

of

Author’s contribution: Dr. DB and Dr. AC contributed to the conception, revised the manuscript critically and

ro

approved it. BB executed the experiment, analyzed the data, prepared figures, drafted the

-p

manuscript and edited it. Dr. PKP contributed to analyzing the data, prepared figures, drafted

re

and revised the manuscript and edited it. Conflict of interest:

lP

Authors declare that there are no conflicts of interest to declare.

na

References:

1. E. Waterman, B. Lockwood, Active components and clinical applications of olive oil,

Jo ur

Altern. Med. Rev. 12 (2007) 331–343. 2. F. Visioli, C. Galli, Biological properties of olive oil phytochemicals, Crit. Rev. Food Sci. Nutr. 42 (2002) 209–221. doi: 10.1080/10408690290825529. 3. B. Bhattacharjee, P.K. Pal, A.K. Ghosh, A. Mishra, A. Chattopadhyay, Aqueous bark extract of Terminalia arjuna protects against cadmium-induced hepatic and cardiac injuries in male Wistar rats through antioxidative mechanisms, Food Chem. Toxicol. 124 (2019) 249–264. https://doi.org/10.1016/j.fct.2018.12.008. 4. F. Visioli, A. Poli, C. Gall, Antioxidant and other biological activities of phenols from olives

and

olive

oil,

Med.

Res.

Rev.

22

(2002)

65–75.

https://doi.org/10.1002/med.1028. 5. J.A. Menendez, A. Papadimitropoulou, L. Vellon, R. Lupu, A genomic explanation connecting “Mediterranean diet,” olive oil and cancer: oleic acid, the main monounsaturated fatty acid of olive oil, inducesformation of inhibitory “PEA3 transcription factor PEA3 DNA binding site” complexes at the Her-2/neu (erbB-2) 22

Journal Pre-proof oncogene promoter in breast, ovarian and stomach cancer cells, Eur. J. Cancer. 42 (2006) 2425–2432. https://doi.org/10.1016/j.ejca.2005.10.016. 6. J.S. Perona, R. Cabello-Moruno, V. Ruiz-Gutierrez, The role of virgin olive oil components in the modulation of endothelial function, J. Nutr. Biochem. 17 (2006) 429–445. doi: 10.1016/j.jnutbio.2005.11.007. 7. C.I. Gill, A. Boyd, E. McDermott, M. McCann, M. Servili, Potential anti-cancer effects of virgin olive oil phenols on colorectal carcinogenesis models in vitro, Int. J. Cancer. 117 (2005) 1-7. doi: 10.1002/ijc.21083. 8. A.A. Berbert, C.R. Kondo, C.L. Almendra, T. Matsuo, I. Dichi, Supplementation of

of

fish oil and olive oil in patients with rheumatoid arthritis, Nutrition. 21 (2005) 131–

ro

136. doi: 10.1016/j.nut.2004.03.023.

9. S. Mishra, A. Chattopadhyay, S. Naaz, A.K. Ghosh, A.R. Das, Oleic acid ameliorates

-p

adrenaline induced dysfunction of rat heart mitochondria by binding with adrenaline: An isothermal titration calorimetry study, Life Sci. 218 (2018) 96-111. doi:

re

10.1016/j.lfs.2018.12.035.

10. O. Benavente-Garcıa, J. Castillo, J. Lorente, A. Ortuno, J. Del-Rio, Antioxidant

lP

activity of phenolics extracted from Olea europaea L. leaves, Food Chem. 68 (2000) 457–462. https://doi.org/10.1016/S0308-8146(99)00221-6.

na

11. C.M. Bitler, T.M. Viale, B. Damaj, R. Crea, Hydrolyzed olive vegetation water in mice has anti-inflammatory activity, J. Nutri. 135 (2005) 1475–1479. doi:

Jo ur

10.1093/jn/135.6. 1475.

12. R.W. Owen, R. Haubner, G. Wurtele, W.E. Hull, B. Spiegelhalder, H. Bartsch, Olives and olive oil in cancer prevention, Eur. J. Cancer Prev. 13 (2004) 319–326. doi: 10.1097/01.cej.0000130221.19480.7e. 13. S. Satarug, J.R. Baker, S. Urbenjapol, M. Haswell-Elkins, P.E. Reilly, D.J. Williams, and M.R. Moore, A global perspective on cadmium pollution and toxicity in nonoccupationally

exposed

population,

Toxicol.

Lett.

137

(2003)

65–83.

https://doi.org/10.1016/S0378-4274(02)00381-8. 14. Agency for Toxic Substances and Disease Registry (ATSDR) 2005. Agency for Toxic Substances and Disease Registry, U.S. Toxicological profile for cadmium, Department of Health and Humans Service, Centers for Disease Control, Atlanta, GA. 15. S. Satarug, S.H. Garrett, M.A. Sens, D.A. Sens, Cadmium, environmental exposure and health outcomes, Environ. Health Perspect. 118 (2010) 182–190. doi: 10.1289/ehp.0901234. 23

Journal Pre-proof 16. WHO (World Health Organization) 1993. Evaluation of certain food additives and contaminants (Forty first Report of the Joint FAO/WHO Expert Committee on Food Additives) WHO Technical Report Series, No. 837. 17. A. Sauvageau, C. Jumarie, Different mechanisms for metal-induced adaptation to cadmium in the human lung cell lines A549 and H441, Cell Biol. Toxicol. 29 (2013) 159–173. doi: 10.1007/s10565-013-9243-4. 18. N. Ercal, H. Gurer-Orhan, N. Aykin-Burns, Toxic metals and oxidative stress part I: mechanisms involved in metal-induced oxidative damage, Curr. Top. Med. Chem. 1 (2001) 529-539. doi: 10.2174/1568026013394831.

of

19. J. Habeebu, J. Liu, C.D. Klaassen, Cadmium-induced apoptosis in mouse liver,

ro

Toxicol. Appl. Pharmacol. 149 (1998) 203–209.doi: 10.1006/taap.1997.8334. 20. G.H. El-Sokkary, A.A. Nafady, E.H. Shabash, Melatonin administration ameliorates

-p

cadmium-induced oxidative stress and morphological changes in the liver of rat, Ecotox. Environ. Safe. 73 (2010) 456– 463. doi:10.1016/j.ecoenv.2009.09.014.

re

21. F.M. El-Demerdash, M.I. Yousef, F.S. Kedwany, H.H. Baghdadi, Cadmium-induced changes in lipid peroxidation, blood hematology, biochemical parameters and semen

lP

quality of male rats: Protective role of vitamin E and beta-carotene, Food Chem. Toxicol. 42 (2004) 1563–1571. doi: 10.1016/j.fct.2004.05.001.

na

22. S.J. Stohs, D. Bagchi, Oxidative mechanisms in the toxicity of metal ions, Free Radic. Biol. Medic. 18 (1995) 321–336. https://doi.org/10.1016/0891-5849(94)00159-H.

Jo ur

23. S. Mitra, A. Deshmukh, R. Sachdeva, J. Lu, J.L. Mehta, Oxidized low-density lipoprotein and atherosclerosis implications in antioxidant therapy, Am. J. Med. Sci. 342 (2011) 135–142. doi: 10.1097/MAJ.0b013e318224a147. 24. J.L. Peters, T.S. Perlstein, M.J. Perry, E. McNeely, J. Weuve, Cadmium exposure in association with history of stroke and heart failure, Environ. Res. 110 (2010) 199– 206. doi: 10.1016/j.envres.2009.12.004. 25. M. Tellez-Plaza, E. Guallar, B.V. Howard, J.G. Umans, K.A. Francesconi, et al., Cadmium exposure and incident cardiovascular disease, Epidemiol. 24 (2013) 421– 429. doi: 10.1097/EDE. 0b013e31828b0631. 26. S.J. Kopp, H.M. Perry, E.F. Jr. Perry, M. Erlanger, Cardiac physiologic and tissue metabolic changes following chronic low-level cadmium and cadmium plus lead ingestion in the rat, Toxicol. Appl. Pharmacol. 69 (1983) 149–160.

24

Journal Pre-proof 27. D.A. Limaye, Z.A. Shaikh, Cytotoxicity of cadmium and characteristics of its transport in cardiomyocytes, Toxicol. Appl. Pharmacol. 154 (1999) 59–66. doi: 10.1006/taap.1998.8575. 28. J.N. Weiss, P. Korge, H.M. Honda, P. Ping, Role of the mitochondrial permeability transition in myocardial disease, Circ. Res. 93 (2003) 292–301. doi: 10.1161/01.RES. 0000087542. 26971.D4. 29. D. Bandyopadhyay, A.K. Chatterjee, A.G. Dutta, Effect of cadmium treatment on hepatic

flavin

metabolism,

J.

Nutr.

Biochem.

4

(1993)

510–514.

https://doi.org/10.1016/0955-2863(93)90086-C

of

30. E. Mitra, A.K. Ghosh, D. Ghosh, D. Mukherjee, A. Chattopadhyay, et al., Protective

ro

effect of aqueous Curry leaf (Murrayakoenigii) extract against cadmium-induced oxidative stress in rat heart, Food Chem. Toxicol. 50 (2012) 1340–1353. doi:

-p

10.1016/j.fct.2012.01.048.

31. B. Bhattacharjee, A.K. Ghosh, S. Mishra, J. Das, A. Chattopadhyay, D.

re

Bandyopadhyay, Terminalia arjuna aqueous bark extract protects against cadmium acetate-induced injury to rat liver and heart through antioxidant mechanisms: a dose

lP

response study, J. Pharmacy Res. 10 (2016) 771–792. 32. D. Mukherjee, S. Ghose-Roy, A. Bandyopadhyay, A. Chattopadhyay, A. Basu, E.

na

Mitra, A.K. Ghosh, R.J. Reiter, D. Bandyopadhyay, Melatonin protects against isoproterenol‐ induced myocardial injury in the rat: antioxidative mechanisms, J.

Jo ur

Pineal Res. 48 (2010) 251–262. doi:10.1111/j.1600-079X.2010.00749.x. 33. S. Reitman, S. Frankel, A colorimetric method for the determination of serum glutamic oxalacetic and glutamicpyruvic transaminases, Am. J. Clin. Pathol. 28 (1957) 56–63.https://doi.org/10.1093/ajcp/28.1.56. 34. C.F. Strittmatter, Studies on avian xanthine dehydrogenases, properties and patterns of appearance during development, J. Biol. Chem. 240 (1965) 2557–2564. 35. D. Mukherjee, A.K. Ghosh, A. Bandyopadhyay, A. Basu, S. Datta, Melatonin protects against

isoproterenol-induced

alterations

in

cardiac

mitochondrial

energy

metabolizing enzymes, apoptotic proteins, and assists in complete recovery from myocardial

injury

in

the

rat,

J.

Pineal

Res.

53

(2012)

166–179.

https://doi.org/10.1111/j.1600-079X.2012.00984.x. 36. L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, Analysis of nitrate, nitrite, and (15N) nitrate in biological fluids, Anal. Biochem. 126 (1982) 131– 138. https://doi.org/10.1016/0003-2697(82)90118-X. 25

Journal Pre-proof 37. Y. Suresh, U.N. Das, Differential effect of saturated, monounsaturated, and polyunsaturated fatty acids on alloxan-induced diabetes mellitus, Prostaglandins Leukot. Essent. Fatty Acids, 74 (2006) 199–213. doi: 10.1016/j.plefa.2005.11.006. 38. J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, Meth. Enzymol. 52 (1978) 302–310.https://doi.org/10.1016/S0076-6879(78)52032-6. 39. D. Bandyopadhyay, G. Ghosh, Bandyopadhyay A, Reiter RJ. Melatonin protects against piroxicam-induced gastric ulceration, J. Pineal Res. 36 (2004) 195–203. https://doi.org/10.1111/j.1600-079X.2004.00118.x. 40. R.L. Levine, J.A. Williams, E.R. Stadtman, E. Shacter, Carbonyl assays for

ro

https://doi.org/ 10.1016/S0076-6879(94)33040-9.

of

determination of oxidatively modified, Meth. Enzymol. 233 (1994) 346–357.

41. P.L. Wendell, Measurement of oxidized glutathione and total glutathione in the

-p

perfused rat heart, Biochem. J. 117 (1970) 661–665. doi: 10.1042/bj1170661. 42. J. Sedlak, R.H. Lindsay, Estimation of total protein bound and non-protein sulfhydryl

re

groups in tissue with Ellman’s reagent, Anal. Biochem. 25 (1968) 192–205. https://doi.org/ 10.1016/0003-2697(68)90092-4.

lP

43. S. Marklund, G. Marklund, Involvement of the superoxide anione radical in the autoxidation of pyragallol and a convenient assay for superoxide dismutase, Eur. J.

na

Biochem. 47 (1974) 469–474. https://doi.org/10.1111/j.1432-1033.1974.tb03714.x. 44. J.R.F. Beers, I.W. Sizer, A spectrophotometric method for measuring the breakdown

Jo ur

of hydrogen peroxide by catalase, J. Biol. Chem. 195 (1952) 133–140. 45. G. Krohne-Ehrich, R.H. Schirmer, R. Untucht-Grau, Glutathione reductase from human erythrocytes. Isolation of the enzyme and sequence analysis of the redoxactive peptide, Eur. J. Biochem. 80 (1977) 65–71. https://doi.org/10.1111/j.14321033.1977. tb11856.x.

46. D.E. Paglia, W.N. Valentine, Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase, J. Lab. Clin. Med. 70 (1967) 158–169. 47. A. Chattopadhyay, S. Biswas, D. Bandyopadhyay, C. Sarkar, A.G. Datta, Effect of isoproterenol on lipid peroxidation and antioxidant enzymes of myocardial tissue of mice and protection by quinidine, Mol. Cell. Biochem. 245 (2003) 43–49. 48. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature.27 (1970) 680–685.

26

Journal Pre-proof 49. N.J. Freehold, 1972. Worthington enzyme manual: enzymes, enzyme reagents, related biochemicals

Responsibility Worthington

Biochemical

Corporation,

Imprint,

Freehold, N. J., Worthington Biochemical Corporation. 50. D. Chretien, M. Pourrier, T. Bourgeron, M. Séné, A. Rötig, A. Munnich, P. Rustin, An improved spectrophotometric assay of pyruvate dehydrogenase in lactate dehydrogenase contaminated mitochondrial preparations from human skeletal muscles, Clin. Chim. Acta. 240 (1995) 129–136. https://doi.org/10.1016/00098981(95)06145-6. 51. P.A.

Srere,

Citrate

synthase.

Methods

Enzymol.

(1969)

3–5.

https:

of

//doi.org/10.1016/0076-6879(69)13005-0.

13

ro

52. M.J. Duncan, D.G. Fraenkel, Alpha-ketoglutarate dehydrogenase mutant of Rhizobium meliloti, J. Bacteriol. 137 (1979) 415– 419.

-p

53. C. Veeger, D.V. DerVartanian, W.P. Zeylemaker, Succinate dehydrogenase. Methods Enzymol, 13 (1969) 81–90. https://doi.org/10.1016/0076-6879(69)13020-7.

re

54. A.H. Mehler, A. Kornberg, S. Grisolia, S. Ochoa, The enzymatic mechanism of oxidation-reductions between malate or isocitrate and pyruvate, J. Biol. Chem. 174

lP

(1948) 961–977.

55. N. Goyal, V.M. Srivastava, Oxidation and reduction of cytochrome c by

na

mitochondrial enzymes of Setariacervi,J. Helminthol. 69 (1995) 13–17. doi: https://doi.org/10.1017/S0022149X00013778.

Jo ur

56. L. Greenlee, P. Handler, Xanthine oxidase. IV. Influence of pH on substrate specificity, J. Biol. Chem. 239 (1964) 1090-1095. 57. S.G. Roy, P. De, D. Mukherjee, V. Chander, A. Konar, D. Bandyopadhyay, A. Bandyopadhyay, Excess of glucocorticoid induces cardiac dysfunction via activating angiotensin

II

pathway,

Cell

Physiol.

Biochem.

24

(2009)

1–10.

doi:

10.1159/000227803. 58. I. Watanabe, K. Ogawa, E. Yamada, Taste buds of rabbits foliate papillae. A scanning electron microscopy study, Cienc. Cult. 40 (1988) 787–790. 59. D. Mukherjee, A.K. Ghosh, M. Dutta, E. Mitra, S. Mallick, B. Saha, R.J. Reiter, D. Bandyopadhyay, Mechanism of isoproterenol induced cardiac mitochondrial damages: protective action of melatonin, J. Pineal Res. 58 (2015) 275–290. doi: 10.1111/jpi.12213.

27

Journal Pre-proof 60. S.J. Cooperstein, P.K. Dixit, A. Lazarow, Studies on the mechanism of Janus green B staining of mitochondria. IV. Reduction of Janus green B by isolated cell fractions, Anat. Rec. 138 (1960) 49–66. https://doi.org/10.1002/ar.1091380107. 61. A. Cossarizza, M. Baccarani-Contri, G. Kalashnikova, C. Franceschi, A new method for the cytofluorometric analysis of mitochondrial membrane potential using the Jaggregate forming lipophilic cation 5,5 ,6,6 -tetrachloro- 1,1,3,3 tetraethylbenzimidazolcarbocyanine iodide (JC-1), Biochem. Biophys. Res. Commun. 197 (1993) 40– 45. https://doi.org/10.1006/bbrc.1993.2438. 62. R.K. Bhogal, S.M. Curbishley, C.J. Weston, D.H. Adams, S.C. Afford, Reactive

of

oxygen species mediate human hepatocyte injury during hypoxia/reoxygenation,

ro

Liver Transplant. 16 (2010) 1303–1313. doi: 10.1002/lt.22157. 63. N. Dousset, G. Ferretti, M. Taus, P. Valdiguie, G. Curatola, Fluorescence analysis of peroxidation,

Meth.

Enzymol.

233

(1994)

459–469.

-p

lipoprotein

https://doi.org/10.1016/ S0076-6879(94)33052-2.

selective

re

64. C. Giulivi, K.J.A. Davies, Dityrosine: A marker for oxidatively modified proteins and proteolysis,

Meth.

Enzymol.

233

(1994)

363–371.

lP

https://doi.org/10.1016/S0076-6879(94)33042-5. 65. K.K. Minezaki, M.S. Suleiman, R.A. Chapman, Changes in mitochondrial function

na

induced in isolated guinea-pig ventricular myocytes by calcium overload, J. Physiol.476 (1994) 459–471. https://doi.org/10.1113/jphysiol.1994.sp020147.

Jo ur

66. S.G. Capar, J.T. Tanner, M.H. Friedman, K.W. Boyer, Multielement analysis of animal feed, animal wastes, and sewage sludge, Environ. Sci. Technol. 12 (1978) 785–790. doi: 10.1021/es60143a004. 67. J.F. Hare, E. Ching, G. Attardi, Isolation, subunit composition, and site of synthesis of human cytochrome c oxidase, Biochem. 19 (1980) 2023-2030. 68. C.F. Babbs, M.G. Steiner, Detection and quantitation of hydroxyl radical usingdimethylsulfoxide as a molecular probe, Meth. Enzymol. 186 (1990) 137-47. 69. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 19 (1951) 265-275. 70. A. Alghasham, T.A. Salem, A.R. Meki Effect of cadmium-polluted water on plasma levels of tumor necrosis factor-α, interleukin-6 and oxidative status biomarkers in rats: protective effect of curcumin, Food Chem. Toxicol. 59 (2013) 160-4. doi: 10.1016/j.fct.2013.05.059.

28

Journal Pre-proof 71. S. Li, C. Sun, Y. Gu, X. Gao, Y. Zhao, et al., Mutation of IDH1 aggravates the fatty acid induced oxidative stress in HCT116 cells by affecting the mitochondrial respiratory

chain,

Mol.

Med.

Rep.

19

(2019)

2509–2518.

doi:

10.3892/mmr.2019.9903. 72. S.V.S. Ranaa, R. Prakasha, A. Kumara, C.B. Sharma, A study of glycogen in the liver of metal-fed rats, Toxicol. Lett. 29 (1985) 1–4. https://doi.org/10.1016/03784274(85)90191-2. 73. P. Rajalekshmi, A. Mohandas, Amsterdam Effect of heavy metals on tissue glycogen levels in the freshwater mussel, Lamellidenscorrianus (Lea), Sci. Total Environ.

of

Supplement 1, 134 (1993) 617–630. https://doi.org/10.1016/S0048-9697(05)80064-6.

ro

74. P.C. Grimm, P. Nickerson, J. Gough, R. McKenna, E. Stern, Computerized image analysis of Sirius Red-stained renal allograft biopsies as a surrogate marker to predict

-p

long-term allograft function, J. Am. Soc. Nephrol. 14 (2003) 1662–1668. doi: https://doi.org/10.1097/01.ASN.0000066143.02832.5E.

re

75. M. Valko, H. Morris, M.T.D. Cronin, Metals, Toxicity and Oxidative Stress, Curr. Med. Chem. 12 (2005) 1161–1208. doi: 10.2174/0929867053764635.

of

cadmium

lP

76. M. Weisberg, P. Joseph, B. Hale, D. Beyersmann, Molecular and cellular mechanisms carcinogenesis:

a

review,

Toxicology,

192

(2003)

95–117.

na

https://doi.org/10. 1016/ S0300-483X(03)00305-6. 77. P. Gong, F.X. Chen, G.F. Ma, Y. Feng, Q. Zhao, Endomorphin 1 effectively protects

Jo ur

cadmium chloride-induced hepatic damage in mice, Toxicology,251 (2008) 35–44. doi: 10.1016/j.tox.2008.07.051. 78. R.A. Larson, The antioxidants of higher plants, Phytochemistry, 27 (1988) 969–978. https://doi.org/10.1016/0031-9422(88)80254-1. 79. E. Mitra, A.K. Ghosh, D. Ghosh, S.B. Firdaus, D. Mukherjee, Ameliorative effect of aqueous tulsi leaf (Ocimum sanctum) extract against cadmium-induced oxidative stress in rat liver, Int. J. Pharm. Pharm. Sci. 5 (2014) 557–568. 80. M.J. Stillman, A.J. Zelazowski, Domain specificity in metal binding to metallothionein. A circular dichroism and magnetic circular dichroism study of cadmium and zinc binding at temperature extremes, J. Biol. Chem. 263 (1988) 6128– 6133. 81. Y. Wang, J. Fang, S.S. Leonard, K.M. Rao, Cadmium inhibits the electron transfer chain and induces reactive oxygen species, Free Radic. Biol. Med. 36 (2004) 1434– 1443. doi: 10.1016/j.freeradbiomed.2004.03.010. 29

Journal Pre-proof 82. U.E. Obioha, S.M. Suru, K.K. Ola-Mudathir, T.Y. Faremi, Hepatoprotective potentials of onion and garlic extracts on cadmium-induced oxidative damage in rats, Biol. Trace Elem. Res. 129 (2009) 143–156. doi: 10.1007/s12011-008-8276-7. 83. S.K. Das, D.M. Vasudevan, Alcohol-induced oxidative stress, Life Sci. 81 (2007) 177187. doi: 10.1016/j.lfs.2007.05.005. 84. Z.A. Shaikh, T.T. Vu, K. Zaman, Oxidative stress as a mechanism of chronic cadmium-induced hepatotoxicity and renal toxicity and protection by antioxidants, Toxicol. Appl. Pharmacol. 154 (1999) 256–263. doi: 10.1006/taap.1998.8586. 85. J. Wang, H. Zhang, T. Zhang, R. Zhang, R. Liu, Y. Chen, Molecular mechanism on

of

cadmium-induced activity changes of catalase and superoxide dismutase, Int. J. Biol.

ro

Macromol. 77 (2015) 59-67. doi: 10.1016/j.ijbiomac.2015.02.037. 86. A. Cuypers, M. Plusquin, T. Remans, M. Jozefczak, E. Keunen, H. Gielen, K.

-p

Opdenakker, A.R. Nair, E. Munters, T.J. Artois, T. Nawrot, Cadmium stress: an oxidative challenge, Biometal, 23 (2010) 927–940. doi: 10.1007/s10534-010-9329-x.

re

87. R.J. Gerson, Z.A. Shaikh, Difference in the uptake of cadmium and mercury by the rat hepatocyte primary cultures. Role of sulfhydryl carrier, Biochem. Pharmacol. 33

lP

(1984) 199–203. https://doi.org/10.1016/0006-2952(84)90476-3. 88. E. Cadenas, K.J. Davies, Mitochondrial free radical generation, oxidative stress, and

5849(00) 00317-8.

na

aging, Free. Radic. Biol. Med. 29 (2000) 222–230. https://doi.org/10.1016/S0891-

Jo ur

89. S. Miccadei, A. Floridi, Sites of inhibition of mitochondrial electron transport by cadmium, Chem. Biol. Interact. 89 (1993) 159–167. https://doi.org/10.1016/00092797(93) 90006-K.

90. I. M. Sokolova, S. Evans, F. M. Hughes, Cadmium-induced apoptosis in oyster hemocytes involves disturbance of cellular energy balance but no mitochondrial permeability transition, J Exp. Biol. 207 (2004) 3369-3380. 91. A.A. Heikal, Intracellular coenzymes as natural biomarkers for metabolic activities and

mitochondrial

anomalies,

Biomark

Med.

4

(2010)

241–263.

doi:10.2217/bmm.10.1. 92. W. Qu, B.A. Diwan, J.M. Reece, C.D. Bortner, J. Pi, J. Liu, M.P. Waalkes, Cadmiuminduced malignant transformation in rat liver cells: Role of aberrant oncogene expression and minimal role of oxidative stress, Int. J. Cancer, 114 (2005) 346–355. https://doi.org /10.1002/ijc.20736.

30

Journal Pre-proof Legends to Figures Figure 1. Graphical representation of the changes in the levels of (A) LPO, (B) PCO content, and (C) GSH content in both heart and liver tissues of rats treated with different doses (5, 10, and 20 mg/kg BW; fed orally) of OA and/or, Cd (0.44 mg/kg BW, s.c.). (#p<0.001 vs. Control group; *p<0.001 vs. Cd-treated group; using one way ANOVA). Figure 2. Diagrammatic representation of the changes in the activities of (A) AST and ALT, (B) TLDH, LDH-5 and LDH-1 (C) NO level and levels of inflammatory markers (D) IL-1β, (E) IL-6, (F) TNF-α and (G) IL-10 in the serum of rats treated with Cd (0.44 mg/kg BW)

of

and/or, OA (10 mg/kg BW). (#p<0.001 vs. Control group; *p<0.001 vs. Cd-treated group;

ro

using one way ANOVA).

Figure 3. Graphical representation of the changes in the levels of (A) LPO, (B) PCO content,

-p

(C) GSH content, (D) GSSG and (E) GSH:GSSG ratio in both heart and liver tissues of Cd

re

treated (0.44 mg/kg BW, s.c.) rats and/or, OA (10 mg/kg BW, fed orally). (#p<0.001 vs. Control group; *p<0.001 vs. Cd-treated group; using one way ANOVA).

lP

Figure 4. Representation of the changes in the activities of (A) SOD 1, (B) SOD 2, (C) CAT, (D) GPx and (E) GR in both heart and liver tissues of Cd treated (0.44 mg/kg BW, s.c.) rats

na

and/or, OA (10 mg/kg BW, fed orally). (#p<0.001 vs. Control group; *p<0.001 vs. Cd-treated

Jo ur

group; using one way ANOVA).

Figure 5. Diagrammatic representation of the immunoblots [I: A-L] and relative densities of SOD 1, SOD 2, CAT, GPx, GR and NFκB in heart [II: A-F] and liver [III: G-L] tissues of rat treated with Cd (0.44 mg/kg BW) and/or, OA (10 mg/kg BW, fed orally). The values of densitometric analyisis was performed through ImageJ software (NIH), and values are expressed as mean ± SEM. (#p<0.001versus Control group; *p<0.001 versus Cd-treated group; using one way ANOVA) Figure 6. Diagrammatic representation of the changes in the activities of (A) Hexokinase, (B) PDH, (C) CS, (D) ICDH, (E) α-KGDH (F) SDH, (G) MDH, (H) Cyt-c oxidase and (I) Cyt-c oxidoreductase in both heart and liver tissues of Cd treated (0.44 mg/kg BW, s.c.) rats and/or, OA (10 mg/kg BW, fed orally). (#p<0.001 vs. Control group; *p<0.001 vs. Cd-treated group; using one way ANOVA).

31

Journal Pre-proof Figure 7. Diagrammatic representation of the immunoblots [I: A-F] and relative densities of Cytoplasmic Cyt c, mitochondrial Cyt c and HSP70 in heart [II: A-C] and liver [III: D-F] tissues of rat treated with Cd (0.44 mg/kg BW) and/or, OA (10 mg/kg BW, fed orally). The values of densitometric analyisis was performed through ImageJ software (NIH), and values are expressed as mean ± SEM. (#p<0.001versus Control group; *p<0.001 versus Cd-treated group; using one way ANOVA) Figure 8. Representation of the changes in the activities of (A) XO, (B) XDH, (C) total enzyme activity (XO+XDH) and (D) XO/XDH ratio in both heart and liver tissues of Cd treated (0.44 mg/kg BW, s.c.) rats and/or, OA (10 mg/kg BW, fed orally).Protective effect of

of

OA against Cd-induced increasein hydroxyl radical generation in vitro in rat heart and liver

ro

mitochondrial fractions (E) of control (Con), OA (1µM), Cd (100 µM) and Cd (100 µM) +OA (1 µM) groups. (#p<0.001 vs. Control group; *p<0.001 vs. Cd-treated group; using one

-p

way ANOVA).

re

Figure 9. Representative images (40X magnification) of HE stained heart (panel A) and liver (panel B) tissue sections, and also PAS stained liver (panel C) tissue sections of control

lP

(CON), OA only (OA), Cd treated (Cd) and OA protected (Cd+OA) groups. Blue colour arrows in HE stained tissue sections indicate Cd induced infiltration of inflammatory cell,

na

capillary dilatation (marked by green arrows), and irregular myofibrils in cardiac tissue (marked by black arrow) along with dilated central vein (marked by brown arrow) in hepatic

Jo ur

sections. PAS stained Cd-treated hepatic sections indicated (blue arrow) pale and slightly stained hepatocytes and decreased glycogen content along with vacuolated cytoplasm (blue arrow).

Figure 9. Representative images (40X magnification) of HE stained heart (panel A) and liver (panel B) tissue sections, and also PAS stained liver (panel C) tissue sections of control (CON), OA only (OA), Cd treated (Cd) and OA protected (Cd+OA) groups. Black colour arrows in HE stained tissue sections indicate Cd induced infiltration of inflammatory cell in both the tissues along with dilated central vein in hepatic section. PAS stained Cd-treated hepatic sections indicated (black arrow) pale and slightly stained hepatocytes and decreased glycogen content along with vacuolated cytoplasm. Figure 10. Representative confocal images (40X magnification) of picrisirius red stained heart (panel A) and liver (panel B) tissue sections of control (CON), OA only (OA), Cd treated (Cd) and OA protected (Cd+OA) groups. Yellow arrows denote the area of collagen. 32

Journal Pre-proof Histogram showing collagen volume (%) of Sirius red (C) stained hepatic and cardiac tissue sections. Values are expressed as means ± SEM. (#p<0.001 vs. Control group; *p<0.001 vs. Cd-treated group; using one way ANOVA). Figure 11. Representative SEM images of rat heart and liver tissue (panel A and B, respectively; magnification 5KX) as well as mitochondria from respective tissues (panel C and D, respectively; Magnification 20 KX) of control (CON), OA only (OA), Cd treated (Cd) and OA protected (Cd+OA) groups. Yellow arrow heads indicate distorsion induced furrowing in both tissues following Cd-treatment; while, Cd-induced blebbing of

of

mitochondrial surface is indicated in both the tissue sections by similar arrow. Figure 12. Representative images of Janus green B stained mitochondrial samples of heart

ro

(Panel A) and liver (Panel B) (20X magnifications) tissues of control (CON), OA only (OA),

-p

Cd treated (Cd) and OA protected (Cd+OA) groups. Similarly, Cd induced changes in heart (Panel D) and liver (Panel E) mitochondrial samples by FACS analysis following JC1

re

staining. Histogram showing fluorescent intensity (%) of (C) Janus green Band (F) JC1 (percent of mitochondrial membrane depolarization) stained heart and liver mitochondrial

lP

samples. The values are expressed as mean ± SEM. (#p<0.001 vs. Control group; *p<0.001

na

vs. Cd-treated group; using one way ANOVA). Figure 13. Representative images of Cd induced changes in heart and liver samples by FACS analysis after DCFDA (A and B, respectively) staining in control (CON), OA only (OA), Cd-

Jo ur

treated (Cd) and OA protected (Cd+OA) groups. Quantification of ROS is represented in terms of DCF fluorescence intensity in the heart (a) and liver (b) samples. The values are expressed as mean ± SEM. (#p<0.001 vs. Control group; *p<0.001 vs. Cd-treated group; using one way ANOVA).

Figure 14. Graphical representation of the changes in fluorescence intensities of (A) tryptophan (B) dityrosine and (C) NADH autofluorescence in both heart and liver mitochondrial samples. Similarly, the changes in (D) cadmium concentrations in both heart and liver tissues of control (CON), OA only (OA), Cd treated (Cd) and OA protected (Cd+OA) groups. The values are expressed as mean ± SEM. (#p<0.001 vs. Control group; *p<0.001 vs. Cd-treated group; using one way ANOVA). Figure 15. Diagrammatic representation of the binding study of cadmium with pure GSH and catalase through isothermal calorimetric profile (ITC). In the titration curve, each peak 33

Journal Pre-proof represents an interaction of pure (A) GSH and Cd, (B) GSH and OA and (C) GSH with Cd and OA as ligand (D) catalase and Cd, (E) catalase and OA and (F) catalase with Cd and OA as ligand. Heat change amount per second (ΔH) is represented by the area under the curve and the heat change in terms of kcal mol−1 of injectant. Values of ΔH, ΔS and number of sites were expressed in terms of mean ± SEM. Graphical Abstract. Schematic diagram showing possible protective mechanism(s) of oleic acid against cadmium

Jo ur

na

lP

re

-p

ro

of

induced oxidative damage in the heart and liver tissues of male Wistar rat.

34

Journal Pre-proof List of Ten (10) chemicals used in this present research work mentioned in the current manuscript

1. Cadmium acetate [(CH3COO)2Cd.2H2O] 2. Oleic acid [CH3(CH2)7CH=CH(CH2)7COOH] 3. JC-1(5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’tetraethylbenzimidazolcarbocyanine iodide) 4. Bovine serum albumin (BSA) 5. N-(1-naphthyl) ethylenediaminedihydrochloride (NED) 6. Sulfanilic acid

of

7. Sodium nitrite

ro

8. 2, 2-dithiobis-nitro benzoic acid (DTNB) 9. Bromophenol blue

Jo ur

na

lP

re

-p

10. Glycerol

35

Jo ur

na

lP

re

-p

ro

of

Journal Pre-proof

36

Journal Pre-proof

Highlights: Treatment of rats with cadmium caused injury to heart and liver tissues.



Pre-treatment of rats with oleic acid protected against cadmium induced injuries.



Oleic acid provided protection against tissue injury through multiple mechanisms.

Jo ur

na

lP

re

-p

ro

of



37

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15