Beta-estradiol protects against copper-ascorbate induced oxidative damage in goat liver mitochondria in vitro by binding with ascorbic acid

Life Sciences 250 (2020) 117596

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Beta-estradiol protects against copper-ascorbate induced oxidative damage in goat liver mitochondria in vitro by binding with ascorbic acid

T

Arnab K. Ghosha,1,2, Bharati Bhattacharjeea,2, Sanatan Mishraa,b,2, Souvik Royc, ⁎ Aindrila Chattopadhyayb,2, Adrita Banerjeeb, Debasish Bandyopadhyaya, a

Oxidative Stress and Free Radical Biology Laboratory, Department of Physiology, University of Calcutta, 92, APC Road, Kolkata 700009, India Department of Physiology, Vidyasagar College, 39, Sankar Ghosh Lane, Kolkata 700006, India c DBT-IPLS section, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: β-Estradiol Cu2+-ascorbate Mitochondria JC-1 dye Fluctuation in heat change (ΔH) Oxidative stress Antioxidant

Aims: β-Estradiol (β-E), one of the chemical forms of female gonad hormone exhibited antioxidant efficacy in biochemical system, in vitro. The aim of the study was to investigate whether any other mechanism of protection by β-E to hepatic mitochondria in presence of stressor agent i.e.,a combination of Cu2+ and ascorbic acid is involved. Main methods: Freshly prepared goat liver mitochondria was incubated with stressors and 1 μM β-E and post incubated with the same concentration at 37 °C at pH 7.4. Mitochondrial viability, biomarkers of oxidative stress, activities of Krebs cycle enzymes, mitochondrial membrane potential, Ca2+ permeability were measured. Mitochondrial morphology and binding pattern of β-E with stressors were also studied. Key findings: Upon incubation of mitochondria with Cu, ascorbic acid and their combination there is a significant decline in activities of four of Krebs cycle enzymes in an uncompetitive manner with a concomitant increase in Ca2+ permeability and membrane potential of inner mitochondrial membrane, which is withdrawn during coincubation with β-E, but was not reversed during post incubation with the β-E. The final studies on mitochondrial membrane morphology using scanning electron microscope also exhibited damage. Isothermal titration calorimetry data also showed the negative heat change in the mixture of β-E with ascorbic acid and also its combination with Cu2+. Significance: Our results for the first time demonstrated that β-E protects againstCu2+-ascorbate induced oxidative stress by binding with ascorbic acid. The new mechanism of binding of β-E with stress agents may have a future therapeutic relevance.

1. Introduction Estra 1,3,5-triene 3,17 β-diol (β-E), is one form of estrogen, whose physiological impact on mammalian female reproductive system is well established [1].The antioxidant potential of this pure compound has been established through our recent work [2] using the Cu2+-ascorbate system, well known for generation of oxidative stress, in vitro [3]. Recent evidences have suggested that Cu2+in combination with ascorbic acid can exert uncompetitive inhibition on Krebs cycle enzymes and thereby affecting the pathway leading to ATP synthesis strategy [4,5]. So considering this fact, in our present work, an attempt has been

made to elucidate whether the β-E exhibits any protective effect in isolated goat liver mitochondria against copper-ascorbate induced oxidative damage, in vitro. To the best of our knowledge and belief this is the first report on the mechanism of protection of β-E against Cu2+ascorbic acid induced oxidative stress. 2. Materials and methods 2.1. Preparation of mitochondria from liver tissue Isolation of mitochondria was done according to the method of Hare

⁎ Corresponding author at: Oxidative Stress and Free Radical Biology Laboratory, Department of Physiology, University Colleges of Science and Technology, 92, APC Road, Kolkata 700009, India. E-mail address: [email protected] (D. Bandyopadhyay). 1 Presently, Department of Biochemistry, School of Life Science and Biotechnology, Adamas University, Adamas Knowledge City, P.O. Jagannathpur, BarasatBarrackpore Road, Kolkata-700,126. 2 The authors have equal contribution.

https://doi.org/10.1016/j.lfs.2020.117596 Received 31 December 2019; Received in revised form 23 March 2020; Accepted 27 March 2020 Available online 30 March 2020 0024-3205/ © 2020 Elsevier Inc. All rights reserved.

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Fig. 1. Effect of β-E on the Cu2+-ascorbic acid induced alteration of functional integrity and oxidative stress biomarkers of goat hepatic mitochondria.(A) Succinate dehydrogenase, (i) declining activity of this enzyme upon incubation with 0.2 mM CuCl2 only (Cu2+), upon incubation with 1 mM ascorbic acid (Asc) only and combination of CuCl2 with ascorbic acid (Cu-Asc) at the same concentration (as mentioned) with increasing time periods (15,30,45 and 60 min) (ii) shows the protective effect of β-E at 1 μM concentration in presence and absence of Cu2+-ascorbate, (Cu2+-Asc + E1 and E1, respectively) and effect of post incubation of the samples with β-E for 30 min (E30′) and 60 min (E60′) (i.e. after incubation with Cu2+-ascorbate for 1 h). Control (C) was only incubated with buffer.*p < .001 versus Control (C) and **p < .001 versus Cu2+-ascorbate (iii) Lineweaver-Burk double reciprocal plots of the activities of all above mentioned enzymes. Vmax and Km values in each case were calculated from the curve using straight line equation y = mx + c, where Vmax values of C, E1, Cu2+-Asc and Cu2+-Asc + E1 are 5.81, 3.48, 2.99, 7.14 units, respectively and Km values are 4.32, 2.35, 2.26, 4.56 mM respectively. (B) Lipid peroxidation level (C) protein carbonylation level and (D) reduced glutathione (GSH) in both co-incubated and post incubated mitochondria in presence and absence of Cu2+-ascorbate (with β-E). All values were expressed in terms of Mean ± SE, *p < .001 versus Control (C) and **p < .001 versus Cu2+-ascorbate (Cu-Asc).

In case of post-incubation experiments, highest concentration of βestradiol (1 μM) was added after one-hour incubation with Cu2+ (0.2 mM) and ascorbic acid (1 mM) [2,3,16] and incubated for 30 and 60 min respectively. Reactions in all incubated samples were terminated by addition of 0.04 ml of 35 mM EDTA [2]. In case of post-incubated samples, β-estradiol was added after one-hour incubation followed by addition of EDTA after 30 and 60 min post incubation with βestradiol.

et al. [6]. Goat liver was collected from Kolkata Municipal Corporation approved slaughter's house just after sacrifice, then washed with 0.9% NaCl, then cut into pieces and chopped at 4 °C, and homogenized with Potter-Elvehjem homogenizer in 50 mM phosphate buffer (pH 7.4). The homogenate was centrifuged at 600g for 10 min to sediment nuclear debri and supernatant was collected and again centrifuged at 16,000g for 45 min. The pellet was collected and suspended in 50 mM Trissucrose buffer (pH 7.8) and used as source of mitochondria. Since, the yield of mitochondria is abundant from goat liver and it is easily available, goat liver was chosen as the source of mitochondria.

2.3. Measurement of mitochondrial functional integrity and oxidative stress biomarkers 2.2. In vitro incubation of suspended mitochondria with copper chloride, ascorbic acid and β-estradiol

2.3.1. Mitochondrial viability determination Incubated mitochondrion was stained by Janus green B (SigmaAldrich) and its fluorescence intensity was measured at 540 nm using Olympus Fluorescence microscope, and this methodology was already described previously [2] Succinate dehydrogenase (SDH) activity of mitochondria was also measured, as described later.

A 50% mitochondrial suspension made with 50 mM potassium phosphate buffer(pH 7.4) were incubated at 37 °C in four different groups: (i) CuCl2 (0.2 mM), (ii) ascorbic acid (1 mM) (iii) ascorbic acid (1 mM) in combination with CuCl2(0.2 mM) (iv) β-estradiol (1 μM) coincubated with 0.2 mM CuCl2 and 1 mM ascorbic acid. The first three groups were incubated for 15, 30, 45 and 60 min. The fourth group was incubated for 60 min only. 2

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Fig. 2. Cu -ascorbic acid induced changes on the activities of three NAD linked enzymes like (A) PDH, (B) ICDH and (C) α-KGDH in isolated goat hepatic mitochondria. A(i), B(i) and C(i) show the decline in the activity of the respective enzymes (as mentioned) upon incubation with 0.2 mM CuCl2 only (Cu2+), upon incubation with 1 mM ascorbic acid (Asc) only and combination of CuCl2 with ascorbic acid (Cu2+-Asc) at the same concentration (as mentioned) with increasing time periods (15, 30, 45 and 60 min). A(ii), B(ii) and C (ii) show the protective effect of β-E at 1 μM concentration in presence and absence of Cu2+-ascorbate, (Cu2+Asc + E1 and E1, respectively) and effect of post incubation of the samples with β-E for 30 min (E30′) and 60 min (E60′) (i.e. after incubation with Cu2+-ascorbate for 1 h). Control (C) was only incubated with buffer. All values were expressed in terms of Mean ± SE, *p < .001 versus Control (C) and **p < .001 versus Cu2+ascorbate (Cu2+-Asc) (iii) Lineweaver-Burk double reciprocal plots of the activities of all above mentioned enzymes. Vmax and Km values in each case were calculated from the curve using straight line equation y = mx + c, in case of PDH, Vmax values of C, E1, Cu-Asc and Cu-Asc + E1 are- 0.271, 0.26, 0.151, 0.225 units, respectively and Km values are 0.089, 0.083, 0.053,0.059 mM respectively. In case of ICDH, Vmax values of C, E1, Cu-Asc and Cu-Asc + E1 are- 0.27, 0.41, 0.086, 0.29 Units respectively and Km values are 1.011, 1.001, 0.129, 1.01 mM respectively, whereas in case of α-KGDH Vmax values of C, E1, Cu-Asc and CuAsc + E1 are- 1.2, 1.2, 0.091, 0.69 units, respectively and Km values are 0.553, 0.554, 0.086, 1.01 mM respectively. 2+

those samples was precipitated by 10% TCA, centrifuged at 2800 ×g for 10 min at room temperature to pellet down protein debris, supernatant discarded and pellets washed with ethanol-ethyl acetate (1:1) mixture to remove excess DNPH. To the pellet, 0.5 ml 6 M guanidine HCl and equal volume 0.5 M phosphate buffer (pH 2.5) were added, vortexed and absorbance was checked at 375 nm. All values are expressed in terms of nmoles protein carbonyl/mg protein.

2.4. Lipid peroxidation level Lipid peroxidation level of Cu2+-ascorbate treated and β-E co-incubated and post incubated mitochondria was measured by the method of Buege and Aust et.al [7]. Two ml TCA-TBA-HCl (containing 15% trichloroacetic acid, 0.375% thiobarbituric acid, 0.25 N HCl) was added to mitochondrial suspension containing 0.5 mg protein, the mixture heated to 80 °C for 20 min and centrifuged at 448 ×g for 10 min at 37 °C. The supernatant was collected and absorbance checked at 532 nm. Values are expressed in terms of nmoles TBARS/mg protein.

2.6. Reduced glutathione (GSH) level GSH content of Cu2+-ascorbate treated and β-E co-incubated and post incubated mitochondria was measured by the method of Sedlak and Lindsay et.al [9]. In this assay, mitochondrial protein was precipitated using equal volume of 10% trichloroacetic acid at 1:1 ratio, then centrifuged at 2800 ×g for 10 min at 4 °C to remove the protein precipitate, and then the supernatant was collected. To one volume of the supernatant, two volumes of 0.8(M) Tris-HCl (containing 2 mM EDTA), and 0.1 volume DTNB (5,5′-dithio-bisnitrobenzoic acid) was

2.5. Protein carbonylation level Protein carbonyl content of Cu2+-ascorbate treated and β-E co-incubated and post incubated mitochondria was measured by the method of Reznick et al. [8]. Mitochondrial suspension containing 0.6 mg protein containing mitochondria was incubated with 0.5 ml 10 mM 2,4dinitrophenylhydrazine (DNPH) in dark for 45 min. Then protein of 3

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Sample groups Fig. 3. Effect of Cu2+-ascorbate induced time dependent alteration in mitochondrial membrane potential (in terms of JC-1 fluorescence, where blue dots and red dots are indicating depolarized and polarized mitochondrial population respectively) and surface morphology (Scanning electron micrograph), where C = Control, CuAsc 15′ = 15 min incubation with Cu2+-Ascorbate, Cu-Asc 30′ = 30 min incubation with Cu2+-Ascorbate, Cu-Asc45′ = 45 min incubation with Cu2+-Ascorbate, CuAsc60′ = 60 min incubation with Cu2+-Ascorbate, [panel A(i) and (ii) respectively]. Protective effect of β-E; E1 = positive control of 1 μM β-E, Cu-Asc + E1 = Coincubation of Cu2+-ascorbate treated mitochondria with 1 μM β-E. E30′, E60′ = post incubated with identical concentration of β-E with Cu2+-ascorbate treated mitochondria for 30 and 60 min. [Panel B (i) and (ii) respectively]. *p < .001 versus Control(C) and **p < .001 versus Cu2+-ascorbate (Cu-Asc). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

added, and finally the absorbance was checked at 412 nm. All values are expressed in terms of nmoles GSH/mg protein.

performed in 0.5 ml reaction mixture that contained 0.1 M phosphate buffer pH 7.5,0.5 mM α-ketoglutarate and 20 μl of 50% mitochondrial suspension as the source of the enzyme, and 0.35 mMNAD. The increase in absorbance at 340 nm was monitored for 90 s at interval of 10 s. Specific activity was expressed in terms of units/mg protein. Succinate dehydrogenase (SDH) activity of incubated mitochondria was measured through monitoring the reduction of potassium ferricyanide spectrophotometrically at 420 nm for 2 min according to the method of Veeger et al. [12]. The 0.5 ml reaction mixture contained 0.1 M phosphate buffer pH 7.5, 2% BSA, 2.5 mM potassium ferricyanide, 4 mM succinate and 20 μl of 50% mitochondrial suspension as the source of the enzyme. The specific activity was expressed in terms of units/mg protein. Kinetic behaviour of all of those enzymes was studied by increasing concentrations of their respective substrates.

2.7. Determination of activities of Krebs cycle enzymes Pyruvate dehydrogenase (PDH) activity of incubated mitochondria was measured following the reduction of NAD to NADH at 340 nm for 90 s according to the method of Chreiten et al. [10] using an UV/Vis Bio-Rad spectrophotometer. The 0.5 ml reaction mixture contained 0.1 M phosphate buffer pH 7.5,0.5 mM sodium pyruvate,0.5 mM NAD and 20 μl of 50% mitochondrial suspension as the source of the enzyme. Specific activity was expressed in terms of units/mg protein. Isocitrate dehydrogenase (ICDH) activity of incubated samples was measured according to the method of Duncan et al. [11]. The 0.5 ml reaction mixture contained 0.1 M phosphate buffer pH 7.5,10 mM isocitrate, 2.5 mM MnSO4 and 20 μl of 50% mitochondrial suspension. To start the reaction 5 mM NAD was added and the reaction was monitored following the increase in absorbance at 340 nm for 90 s at an interval of 10 s. Specific activity was expressed in terms of units/mg protein. α-Ketoglutarate dehydrogenase (α-KGDH) activity was determined according to the method of Duncan et al. [11]. This assay was

2.8. Measurement of mitochondrial membrane potential by JC-1 dye Mitochondrial membrane potential was measured according to the method of Joshi et al. [13]. Control and all treated mitochondrial suspension (12.5% in the incubated medium) were incubated with JC-1 dye (at 2 nM 4

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Fig. 4. Effect of β-E on Ca2+ ion permeability across the inner mitochondrial membrane (assayed by calcein fluorescence). C = Control, Cu-Asc 15′ = 15 min incubation with Cu2+-Ascorbate, Cu-Asc 30′ = 30 min incubation with Cu2+-Ascorbate, Cu-Asc45′ = 45 min incubation with Cu2+-Ascorbate, Cu-Asc60′ = 60 min incubation with Cu2+-Ascorbate, E1 = positive control of 1 μM β-E, Cu-Asc + E1 = Co-incubation of Cu2+-ascorbate treated mitochondria with 1 μM β-E. E30′, E60′ = post incubated with identical concentration of β-E with Cu2+-ascorbate treated mitochondria for 30 and 60 min. *p < .001 versus Control (C) and **p < .001 versus Cu2+-ascorbate (Cu-Asc).

concentration) and incubated at 37 °C for 30 min. Afterwards flow cytometry was done at 529 nm wavelength (excitation at 514 nm) using FACS Aria III. Data were analyzed using FACS Diva software and plotted on a dual percentage of colours (red and blue, where blue is indicating the percentage of depolarized mitochondria) and gates were assigned on depolarized mitochondria. The percentage of depolarized mitochondria in each gate was plotted as bar diagram.

CuCl2, 1 mM ascorbic acid and Cu2+-ascorbic acid combination. For a single run, titration was conducted with twenty injections of each ligand (2 μl each) with 150 s spacing between two successive injections for approximately 1 h at 37 °C. 3. Estimation of protein Protein concentration of mitochondria was determined by the method of Lowry et al. [17].

2.9. Assessment of mitochondrial surface morphology Surface morphology of mitochondria was studied according to the method of Ghosh et al. [14]. The 0.5 ml of incubated mitochondrial suspension (12.5% in the incubated medium) was mixed with an equal volume 2% glutaraldehyde and kept at 4 °C for two nights for fixation. Then those samples was dehydrated with gradual washing with increasing concentration of ethanol and n-amyl alcohol, dried and observed under Zeiss scanning electron microscope at 40× magnification. Ultimately morphology of mitochondrial surface was presented by histogram.

4. Statistical evaluation Each experiment was repeated at least three times with different groups. Data are presented as means ± S.E. Significance of mean values of different parameters between the treatment groups were analyzed using one way post hoc tests (Tukey's HSD test) of analysis of variances (ANOVA) after ascertaining the homogeneity of variances between the treatments. Pairwise comparisons were done by calculating the least significance. Statistical tests were performed using Microcal Origin version 7.0 for Windows.

2.10. Measurement of Ca2+ ion permeability of mitochondrial membrane 5. Results Calcium ion permeability across the inner mitochondrial membrane was done following the method of Bratosin et al. [15]. Control and all treated mitochondrial suspension (12.5% in the incubated medium) were incubated with Calcein-AM dye (at 5 μM concentration) and incubated at 37 °C for 30 min. Then flow cytometric analysis was done at 515 nm wavelength (Excitation wavelength 495 nm) using BD FACS Verse. Data were analyzed by FACSuite software and histogram overlays of Ca2+ fluorescence were done and mean fluorescence intensity was plotted as a bar diagram.

5.1. Effect of β-E on Cu2+-ascorbate mediated alteration in functional integrity of mitochondria and oxidative stress biomarkers Fig. 1A depicts that incubation of mitochondria only with copper chloride at different durations (15, 30, 45 and 60 min) caused a slight but significant decrease in the activity of succinate dehydrogenase compared to control. Incubation of liver mitochondria with 1 mM ascorbic acid alone resulted in a more significant decline in the activities of the same enzymes compared to those samples incubated only with 0.2 mM CuCl2. But when the same mitochondria was treated with 1 mM ascorbic acid along with 0.2 mM CuCl2 in an identical manner, then there was a drastic fall in the activity of the SDH in a time dependent manner in comparison to control and other groups of mitochondria (incubated with only CuCl2 and only with ascorbic acid). However, when the mitochondrial suspension was co-incubated with 1 μM β-E in presence of Cu2+ and ascorbic acid, a significant protection was

2.11. Isothermal titration calorimetry (ITC) The binding pattern of β-estradiol with copper chloride, ascorbic acid, copper-ascorbate combination was analyzed by isothermal titration calorimetry using Microcal ITC-200, Malvern, UK following the method of Ghosh et al. [16]. For this assay, in the sample cell, 0.35 ml of 1 μM β-estradiol was separately titrated with 0.06 ml of 0.2 mM 5

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Fig. 5. Isothermal titration calorimetric data of β-E to evaluate its interaction with copper chloride and ascorbic acid, where the heat change versus time titration curve, each peak designates an injection of ligand into the sample cell that contains β-E, where the ligand used is (A) copper chloride (B) ascorbic acid and (C) copper chloride and ascorbic acid. The amount of heat change per second (ΔH) is represented by the area under the curve (top curve) and the heat change in terms of kcal mol−1 of injectant against molar ratio is shown at the bottom of the curve of each figure respectively. Values of ΔH, ΔS and no. of sites were expressed in terms of mean ± S.E.

chloride for different time intervals (15,30,45 and 60 min), caused a slight but significant decrease in the activities of NAD linked enzymes of Krebs cycle (PDH, ICDH, α-KGDH) compared to control. Upon incubation of mitochondria with 1 mM ascorbic acid in the same way resulted in more decline in the activities of the enzymes compared to those samples incubated only with 0.2 mM CuCl2. But when the same mitochondria was treated with 1 mM ascorbic acid along with 0.2 mM CuCl2 in an identical manner, then there was drastic fall in the activities of Krebs cycle enzymes in a time dependent manner in comparison to control and other groups of mitochondria (incubated with only CuCl2 and only with ascorbic acid). But when the mitochondrial suspension was co-incubated with 1 μM β-E in presence of Cu2+ and ascorbic acid, a significant protection was observed. Studying the activities of the same enzymes at increasing substrate concentrations, also showed the Cu2+-ascorbate mediated decrease (that is evident from decreased Vmax and Km value in Cu2+-ascorbate incubated mitochondria compared to control) which was removed upon co-incubation with β-E (also evident from Vmax and Km value compared to Cu2+-ascorbic acid incubated mitochondria) as designated in Fig. 2. But when the same concentration of β-E was added to the incubation mixture for 1 h after 1 h incubation with Cu2+-ascorbic acid, there was no protection in the activities of the enzymes.

observed. Studying the activity of the same enzyme at increasing substrate concentrations, also exhibited the Cu2+-ascorbate mediated decrease (that is evident from decreased Vmax and Km value in Cu2+ascorbate incubated mitochondria compared to control) which was removed upon co-incubation with β-E (also evident from Vmax and Km value compared to Cu2+-ascorbic acid incubated mitochondria). But when the same concentration of β-E was added to the incubation mixture for 1 h after 1 h incubation with Cu2+-ascorbic acid, there was no protection in the activity of succinate dehydrogenase. There were significant elevations in both lipid peroxidation and protein carbonyl level in Cu2+-ascorbate incubated mitochondria (73% and 3.35 fold compared to control respectively) with a significant decline in GSH level (35% compared to control) as designated from Fig. 1(B), (C) and (D), respectively. But upon co-incubation with 1 μM β-E both of those parameters were protected from being altered whereas post incubation of Cu2+-ascorbate treated mitochondria with β-E did not restore lipid peroxidation and protein carbonylation to the basal level.

5.2. Effect of β-E on Cu2+-ascorbate mediated inhibition on Krebs cycle enzymes Figs. 2A,B,C show that incubation of mitochondria only with copper 6

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5.3. Effect of β-E on mitochondrial membrane potential and Ca2+ permeability across the inner mitochondrial membrane

5.5. Measurement of interaction of β-E with CuCl2 and ascorbic acid by isothermal titration calorimetry

Fig. 3 [panels A (i) and B (i)] depicts that there is a gradual, timedependent and significant elevation in the percentage of depolarized mitochondria upon treatment with Cu2+-ascorbate up to 30 min compared to control. But then there was a steep fall in the percentage of depolarized mitochondria at 45 min and 60 min. Co-incubation of mitochondria with β-E at 1 μM concentration showed a significant and as well as dose dependent protection of membrane potential from being increased. Mitochondrial suspension which was post incubated with same concentration of β-E for 30 to 60 min did not show any restoration of this parameter to the basal level. Calcium ion concentration of Cu2+-ascorbate incubated mitochondria increased significantly in comparison to control whereas co-incubation of 1 μM β-E with mitochondria in the same system, showed a restoration in Ca2+ ion permeability to the control level. But post incubation of mitochondria in presence of Cu2+-ascorbate with β-E increased Ca2+ ion permeability and did not show its restoration to control level as designated in Fig. 4.

Upon injection of ascorbic acid in the sample cell containing β-E, a constant heat change was observed leading to gradual saturation. But when only Cu2+ was in contact with β-E, an abrupt fluctuation in heat change (ΔH) was observed. When Cu2+ and ascorbic acid both were present together in the ligand cell and came in contact with β-E, then there was a significant increase in heat change (compared to Cu2+ and ascorbic acid alone in ligand cell), where at initial stage an exponential decay in heat change followed by an exponential elevation in heat change at later stage and then a gradual inclination towards saturation was evident as depicted in Fig. 5A, B, C. 6. Discussion Ascorbic acid (Vitamin C) is a well-known antioxidant [18]. To scavenge reactive oxygen species (ROS) it donates its hydrogen to shield the charge of extra electron at their antibonding orbitals thereby converting it into dehydroascorbate [19]. But above a certain concentration it acts as a pro-oxidant. Its pro-oxidant activity is positively modulated in presence of Cu2+ that results in increased protein carbonylation, lipid peroxidation, thiol group depletion as well as DNA damage in cell and mitochondria mainly due to hydroxyl radical [20,21]. On the other hand, it can also modulate the activities of cellular antioxidant enzymes like Cu-Zn-SOD, catalase and Krebs cycle enzymes [4,5,16]. Decrease in activities of Krebs cycle enzymes can also affect the activities in electron transport chain (ETC) associated enzymes affecting thereby the mitochondrial membrane potential, which can cause disturbance in ATP synthesis [22]. In this situation only molecular oxygen can act as electron sink, because of its high electron affinity [23]. So the leaked free electrons due to reduced activity of ETC linked enzymes are accepted by molecular oxygen that can generate superoxide anion free radical, hydrogen peroxide and ultimately hydroxyl radicals, that can cause oxidative damages of

5.4. Effect of β-E on Cu2+-ascorbate mediated time dependent alteration of mitochondrial surface morphology Cu2+-ascorbate treatment of isolated goat hepatic mitochondria resulted in a gradual increase in surface roughness in a time dependent manner as evident from Fig. 3 [panels A (ii) and B (ii)]. But on coincubation of hepatic mitochondria with β-E in presence of Cu2+-ascorbate, the change in the roughness of surface was protected from being increased whereas post incubation of hepatic mitochondria with β-E showed increased surface roughness just as Cu2+-ascorbate incubated sample.

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integrity as evident from mitochondrial membrane potential and surface topology, thereby protecting the hepatic mitochondria from oxidative damage.

mitochondria [24]. Hence, scavenging of stress mediating agents may be a good initiative to prevent oxidative stress induced alterations in mitochondria. In our experiment according to the results as displayed in Figs. 1 and 2 the NAD linked and FAD linked enzymes were inhibited in an uncompetitive manner, which was confirmed by the pattern of double reciprocal plot, Vmax and Km values, that is indicating towards the binding of Cu2+-ascorbate with enzymes that is removed in presence of β-E which was also assured from Vmax and Km value. But in case of post incubation there was no restoration of the activities of enzymes to the basal level. The results indicate that the inhibition of those enzymes was due to binding of Cu2+-ascorbate and also ROS generation that is also confirmed by elevated levels of lipid peroxidation and protein carbonylation in mitochondria (Fig. 1). Isothermal titration calorimetry (ITC) measures the heat change upon injection of specific ligand in the sample cells thereby determining the possibility of binding between the ligand and sample [25]. Here in our experiment, abrupt heat change fluctuation indicate towards a weak binding of β-E with CuCl2 with gradual but very slowly proceeding towards saturation, whereas a constant negative heat change is indicating towards an exothermic reaction and a possibility of single site binding of ascorbic acid with β-E. But in case of the combination of ascorbic acid and CuCl2 and β-E, there is an initial significant increase in heat change for subsequent injections, gradually showing inclination towards saturation which indicates multi-site and sequential binding of β-E with Cu2+-ascorbate. JC-1, a novel cationic carbocyanine dye that accumulates in mitochondrial matrix, exists as a monomer at low concentrations and yields green fluorescence. At higher concentrations, the dye forms Jaggregates that exhibit a broad excitation spectrum and an emission maximum at ~590 nm [26]. In our experiment, gradual elevation in JC1 fluorescence accumulation in matrix of Cu2+-ascorbate incubated mitochondria up to 30 min indicate towards time dependent depolarization of inner mitochondrial membrane which possibly indicate porous nature of mitochondrial membrane. But, in mitochondria, incubated for 45 min or more, gradual decrease in fluorescence of JC-1 indicated mitochondrial exhaustion leading towards death. But in presence of β-E, the change in membrane potential was prevented as designated in Fig. 3 (Panel A). On the other hand, calcein is a dye used as a Ca2+ indicator because its fluorescence is directly sensitive to Ca2+ ion only at strongly alkaline pH. This fluorescence quenching response has been exploited for detecting the opening of the mitochondrial permeability transition pore (mPTP) and for measuring cell volume changes [27]. In our experiment, Ca2+ ion permeability was increased in Cu2+-ascorbate treated mitochondria (in comparison to control), as Ca2+ transporters are present on inner mitochondrial membrane [28,29], leading to easy entry of Ca2+ within mitochondrial matrix to make it positively charged which is an indicator of mitochondrial depolarization, and this was prevented in presence of β-E as evident from Fig. 4. The schematic diagram (Fig. 6) represents the mechanism of protection offered by β-E against oxidative damage induced by Cu2+ and ascorbate in hepatic mitochondria as is evidenced through change in mitochondrial membrane potential. Furthermore, Scanning electron microscopy (SEM), a determinant of surface morphology of cell and mitochondria [30], showed time dependent increase in the surface roughness (as shown in inset histograms of Fig. 3, Panel-B) upon incubation with Cu2+-ascorbate which justified the cause of membrane depolarization and Ca2+ permeability, which was protected, but not reversed in presence of β-E.

Declaration of competing interest Authors declare that there are no conflicts of interest. Acknowledgements Dr. AKG acknowledges the receipt of a Research Associate (RA) award from CSIR, Govt. of India, New Delhi. SM is supported by the funds from the “Teacher's Research Grant” available to Prof. DB from BI-92 of Department of Physiology University of Calcutta. BB acknowledges the receipt of a DST-Inspire fellowship from the Department of Science and Technology (DST), Govt. of India. SR acknowledges the financial support of DBT-IPLS scheme of University of Calcutta, which is supported by Department of Biotechnology (DBT), Govt. of India. Dr. AC is supported from the grants available to her from West Bengal Dept. of Science and Technology. This work is also partially supported from the funds available to Prof. DB from UGC Major Research Project under CPEPA Scheme of UGC awarded to University of Calcutta (Grant No: 8-2/2008(NS/PE) DATED 14.12.2011). Prof. DB further gratefully acknowledges the support that he has received from DST PURSE Grant awarded to Calcutta University. Dr. AKG also acknowledges Pratyush Sengupta of Centre for Nanoscience and Nanotechnology, University of Calcutta for providing assistance in using the facility of scanning electron microscopy. References [1] D. Pfaff, M. Keiner, Atlas of estradiol concentrating cells in the central nervous system of female rat, J. Comp. Neurol. 151 (1973) 121–157, https://doi.org/10. 1002/cne.901510204. [2] A.K. Ghosh, B. Bhattacharjee, S. Mishra, et al., Estra-1,3,5(10)- triene-3,17 β-diol protects mitochondria against cu-ascorbate induced oxidative damage in in vitro system: a novel therapeutic approach, J Pharma Res 10 (2016) 594–608. [3] A. Chattopadhayay, T.D. Choudhury, M.K. Basu, et al., Effect of Cu2+-ascorbic acid on lipid peroxidation, Mg2+-ATPase activity and spectrin of RBC membrane and reversal by erythropoietin, Mol. Cell. Biochem. 118 (1992) 23–30, https://doi.org/ 10.1007/BF00249691. [4] M. Dutta, A.K. Ghosh, V. Mohan, et al., Trigonelline [99%] protects against copperascorbate induced oxidative damage to mitochondria: an in vitro study, J Pharma Res 11 (2014) 1694–1718. [5] M. Dutta, A.K. Ghosh, A. Chattopadhyay, et al., Trigonelline [99%] protects against copper-ascorbate induced oxidative damage to aortic mitochondria in vitro: involvement of antioxidant mechanism(s), Int J PharmSci Rev Res 29 (2014) 312–323. [6] J.F. Hare, E. Ching, G. Attardi, et al., Isolation, subunit composition, and site of synthesis of human cytochrome c oxidase, Biochemistry 19 (1980) 2023–2030, https://doi.org/10.1021/bi00551a003. [7] J.A. Buege, S.G. Aust, Microsomal lipid peroxidation, MethEnzymol 52 (1978) 302–310, https://doi.org/10.1016/s0076-6879(78)52032-6. [8] A.J. Reznick, C.E. Cross, M.L. Hu, et al., Modifications of plasma protein by cigarette smoke as measured by protein carbonyl formation, Biochem. J. 286 (1992) 607–611, https://doi.org/10.1042/bj2860607. [9] J. Sedlak, R.H. Lindsay, Estimation of total, protein bound, and non protein sulfhydryl groups in tissue with Ellman’s reagent, Anal. Biochem. 25 (1968) 192–205, https://doi.org/10.1016/0003-2697(68)90092-4. [10] D. Chretien, M. Pourrier, T. Bourgeron, et al., 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/10.1016/0009-8981(95)06145-6. [11] M.J. Duncan, D.G. Fraenkel, Alpha-ketoglutarate dehydrogenase mutant of Rhizobium meliloti, J. Bacteriol. 137 (1979) 415–419. [12] C. Veeger, DerVartanian DV, W.P. Zeylemaker, et al., Succinate dehydrogenase, Meth Enzymol 13 (1969) 81–90. [13] D.C. Joshi, J.C. Bakowska, Determination of mitochondrial membrane potential and reactive oxygen species in rat cortical neuron, J VisExp 51 (2011) 1–4, https://doi. org/10.3791/2704. [14] A. Ghosh, M. Dutta, A.K. Ghosh, et al., Melatonin affords protection against myocardial ischaemia induced cerebral mitochondrial dysfunction: an in vivo study, J. Pharm. Res. 9 (2015) 105–118. [15] D. Bratosin, L. Mitrofan, C. Palii, et al., Novel fluorescence assay using Calcein-AM for the determination of human erythrocyte viability and aging, Cytometry Part A 66A (2005) 78–84, https://doi.org/10.1002/cyto.a.20152.

7. Conclusion Finally, it can be concluded that binding of β-E with Cu2+ and ascorbic acid restores the functional integrity of hepatic mitochondria as evident from the activities of Krebs cycle enzymes and structural 8

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