Protective effect of fisetin against subchronic chlorpyrifos-induced toxicity on oxidative stress biomarkers and neurobehavioral parameters in adult male albino mice

Abstract

Chlorpyrifos (CPF), a chlorinated organophosphate insecticide that is widely used in agriculture and public health, has neurotoxic effects in animals. In addition to acetylcholinesterase inhibition, CPF has been shown to induce alterations such as oxidative stress and lipid peroxidation. Fisetin is a dietary flavonol that protects the brain tissue against oxidative stress by modulating the activity of antioxidant enzymes. This study was designed to investigate the protective role of fisetin against brain oxidative damages and neurobehavioral parameters induced by subchronic oral exposure to CPF in albino mice. Adult albino mice (males, n = 32, weighing 20 ~ 25 g) were assigned randomly into 4 groups and treated accordingly for 7 weeks as follows: Group 1(S/OIL): served as the control group and were given 2 ml/kg of soya oil; Group 2 (CPF): received CPF (6.6 mg/kg; 1/5th of the LD₅₀); Group 3 (FIS): fisetin (15 mg/kg) and Group 4 (FIS + CPF): received fisetin at 15 mg/kg, followed by CPF (6.6 mg/kg) 30 min later. Co-treatment with FIS + CPF mitigated the increase in brain malondialdehyde concentration (0.28 ± 0.02 nmol/mg) and orchestrated the increase in the activities of catalase (81.35 ± 7.26 µ/mg), superoxide dismutase (93.03 ± 6.63 IU/mL), glutathione peroxidase (68.76 ± 3.554 nmol/mL) and acetylcholinesterase (11.59 ± 0.72 nmol/min/mL) when compared to the CPF group. The result showed that deficits in motor strength and excitability scores induced by subchronic CPF were mitigated by fisetin administration. It was concluded that fisetin has a protective potential in mitigating against oxidative stress and damages in the brain tissues, induced by subchronic exposure to CPF in adult male albino mice.

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Introduction

Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a naturally-occurring flavonoid, found in strawberry, persimmon, grape, onion, and cucumber [1–3]. This flavonoid is involved in the control of different aspects of the oxidative process, such as scavenging radical ions [4, 5] or as anti-lipoperoxidation compounds [6] in the biological system. It has been reported as a chemotherapeutic agent with neuroprotective function in mice and humans [7–10]. The ability of fisetin to scavenge free radicals contributes to its marked antioxidant activity and significant biological effects [11]. As a hydrophobic compound, it easily penetrates cell membranes, accumulating in cells to exert its anti-oxidative, neurotrophic and neuroprotective effects [12]. Fisetin has been reported to reduce the adverse effect of benzo(a)pyrene and aluminum chloride toxicity in Swiss albino mice by reducing lipoperoxidative damages and increasing the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [13]. It has also been found to exert neuroprotection by inhibiting reactive gliosis and inflammation during aluminum chloride-induced neurotoxicity [14].

Chlorpyrifos (CPF) is a broad-spectrum organophosphate insecticide that is toxic to mammals with widespread agricultural, domestic, and public health applications [15, 16]. It inhibits acetylcholinesterase (AChE) activity by binding to the active sites of the enzyme; thus, preventing the breakdown of acetylcholine (ACh) in the nervous system [17, 18]. This results in the accumulation of ACh in nerve endings, which causes continuous stimulation of cholinergic receptors [19] and eventual paralysis and death [20]. Chlorpyrifos (CPF) is a developmental neurotoxicant, affecting developing fetus [21], and at low doses disrupt brain development and cognitive function [22]. An important mechanism implicated in CPF toxicity is the induction of oxidative stress and excessive production of reactive oxygen species (ROS), currently identified as a major mechanism of CPF toxicity [23, 24]. Exposure to organophosphate in humans and animals is associated with neurologic symptoms, including those that affect cognitive, motor, sensory, and autonomic functions [25, 26].

Several studies have been conducted on the ameliorative effect of antioxidants induced by exposure to CPF [27–31]. However, there is a paucity of information on the protective effect of fisetin on oxidative stress biomarkers of the brain induced by exposure to CPF. Therefore, the present study was aimed at evaluating the effect of fisetin on some behavioral parameters and biomarkers of oxidative stress in the brain of adult male albino mice.

Materials and methods

Experimental animals

A total of 32 adults (10 weeks’ old) male albino mice were used for the experiment. The mice were obtained from the animal house of Department of Veterinary Physiology, Ahmadu Bello University, Zaria, Nigeria. They were fed with standard mice pellets and water ad libitum. The animals were kept in metal cages at room temperature (25–26 °C) throughout the study period.

The study was approved by the Ahmadu Bello University Committee on Animal Welfare and Use (ABUCAUC/2016-17). All the mice were kept according to the guidelines and welfare regarding animal protection approved by National Research Council Local Ethical Review Committee and were conducted following the “Guide for the Care and Use of Laboratory Animals’’ [32].

Chemical acquisition and preparations

Commercial grade CPF 20% emulsifiable concentrate (Termifos^® by Alderelm Limited, UK) was obtained from a reputable chemical store. Soya oil (Grand Cereals and Oil Mills Ltd., Jos, Nigeria) was used as a vehicle to reconstitute the CPF into a 1% (w/v) working solution. The fisetin used in this study was of analytical grade and was obtained from Sigma Inc., St. Louis, MO, USA. Fisetin was administered at a dose of 15 mg/kg [14].

Subchronic toxicity studies

Thirty-two adult male albino mice, weighing between 20 and 25 g, used for the study were reared in the Animal House of the Department of Veterinary Pharmacology and Toxicology, Ahmadu Bello University (ABU), Zaria, Nigeria. The mice were allowed to acclimatize for 2 weeks in the laboratory before the commencement of the experiment.

Adult albino mice (males, n = 32, weighing 20 ~ 25 g) were assigned randomly into 4 groups and treated accordingly for 7 weeks as follows: Group 1(S/OIL): served as the control group and were given 2 ml/kg of soya oil; Group 2 (CPF): received CPF (6.6 mg/kg; 1/5th of the LD₅₀); Group 3 (FIS): fisetin (15 mg/kg) and Group 4 (FIS + CPF): received fisetin at 15 mg/kg, followed by CPF (6.6 mg/kg) 30 min later, respectively. During this period, the body weight, neurobehavioral and cognitive parameters were evaluated. There is a paucity of information regarding the protective effect of fisetin against CPF-induced neurotoxicity in mice.

Our justification for the use of the dosage was based on the study of Prakash et al. [14] that fisetin treatment at a concentration of 15 mg/kg of body weight significantly exerts a neuroprotective effect against aluminum chloride-induced neurotoxicity. Thus, 15 mg/kg of body weight was chosen as the dosage in this study. The median lethal dose (LD₅₀) for CPF using the method of Lorke (1983) [33] is 33 mg/kg. As such, 1/5th of the LD₅₀ was used in order to obtain a very low dose that would not have any acute or apparent effect for this subchronic study.

Assessment of the level of excitability scores

This neurobehavioral parameter was evaluated using the excitability scores as described by Ambali and Ayo [28]. Briefly, each mice was held by the tail upside down and held in that position for 30 s. The response of each mice was then rated using an ordinal scale of 0–5 as follows:

Grade 0—mice did not show any form of wriggling at all.

Grade 1—mice wriggling was low with feeble forepaw movement.

Grade 2—mice responded through a stronger wriggling and forepaw movement.

Grade 3—mice vigorously wriggled and a strong fore- and hind-limbs movement.

Grade 4—In addition to observation in grade 3 above, the mice made an unsuccessful attempt to climb on its tail.

Grade 5—In addition to observation in grade 3 above, the mice successfully climbed the tip of its tail. The excitability scores were assessed on day 0 and weeks 3 and 6.

Evaluation of motor strength using forepaw grip test

The forepaw grip time was used to evaluate the motor strength of the mice, as described by Abou-Donia et al. [34]. This was conducted by having mice hung down from a 5 mm diameter wood dowel gripped with both forepaws. The time spent by each mouse before releasing the grip was recorded in seconds. This parameter was evaluated on day 0, weeks 3 and, 6.

Preparation of the brain sample

At the end of seven weeks, the mice were sacrificed using jugular venesection after initial light chloroform anesthesia. The whole brain was immediately (< 1 min) removed and washed in cold (4 °C) modified Krebs-Henseleit preincubation solution containing 120 mmol/L NaCl, 2 mmol/L KCl, 0.5 mmol/L CaCl₂, 26 mmol/L NaHCO₃, 10 mmol/L MgSO₄, 1.18 mmol/L KH₂PO₄, 11 mmol/L glucose, and 200 mmol/L sucrose [35]. The brain samples were stored in a freezer at − 80 °C. At the day of the oxidative stress assays, the samples were homogenized in physiological buffer saline at 10 g/100 mL (w/v) containing 140 mmol/L KCl, pH 7.4, and centrifuged at 750 g for 10 min at 4 °C. The supernatant was then collected for the assay of antioxidant enzyme activities.

Evaluation of serum malondialdehyde concentration

The malondialdehyde (MDA) concentration in the serum sample was determined using the double heating method of Draper and Hadley [36] and as modified by Yavuz et al. [37]. The MDA concentration in each serum sample was calculated by the absorbance coefficient of MDA-thiobarbituric acid complex at 1.56 × 10⁵ cm⁻¹ M⁻¹ and expressed in nmol/mg.

Glutathione peroxidase activity

Glutathione peroxidase (GPx) activity was measured using glutathione peroxidase assay kit protocol NWK-GPx01 (NWLSS™, the Northwest Life Science Specialist, Vancouver, Canada). Briefly, the standard procedure was used for microplate assay, required all reagents to be brought to room temperature. A diluted sample (50 µL) was added to the wells and then 50 µL of working nicotinamide adenine dinucleotide phosphate (NADPH) was added to each well. Working H₂O₂ (50 µL) was also added to each well. After waiting for 1 min, the microplate was placed in a plate reader and read at 340 nm wavelength [38].

Determination of brain superoxide dismutase activity

Superoxide dismutase (SOD) activity was analyzed using NWLSS™ superoxide dismutase activity assay kit (Northwest Life Science Specialities, Vancouver, WA, USA). The principle of the test was based on the monitoring of the autooxidation rate of hematoxylin as described by Martin et al. [39].

Determination of brain catalase activity

Catalase (CAT) was analyzed using NWLSS™ catalase activity assay kit (Northwest Life Science Specialities, LLC, 5131 Vancouver, WA 98662, USA). The principle of the test was based on the monitoring of the consumption of H₂O₂ substrate at 240 nm using the method described by Beers and Sizer [40].

Assessment of brain acetylcholinesterase activity

Brain samples collected were analyzed for AChE activity using the assay methods described by Ellman et al. [41]. The principle for the AChE assay was based on spectrophotometric measurement of the yellow color, developed following the reaction of thiocholine (break-down product of acetylcholine iodide and disthiobisnitrobenzoate).

Statistical analyses

Data obtained from the different groups were expressed as mean ± standard error of the mean (mean ± SEM) and subjected to one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test to compare mean values among the different groups. The excitability score and forepaw grip time performances were analyzed using the Kruskal–Wallis test followed by Dunn’s post-hoc test for comparison among the groups. Data were analyzed using GraphPad Prism, version 6.01 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Values of p < 0.05 were considered significant.

Results

Effect of treatment on body weight gain

There was a progressive gain in body weight in the S/OIL, FIS, and FIS + CPF groups during the study period. Although the FIS + CPF group had a decrease in body weight at weeks 1 and 2, a steady rise in weight gain at week 3 to 7 was observed. In the CPF group, there was a significant (p < 0.05) loss of body weight at weeks 4, 5, 6, and 7 (Table 1).

Table 1.

Effect of subchronic exposure of chlorpyrifos on body weight of adult male albino mice

Group	Week 0	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7
S/OIL	20.0 ± 0.3	20.4 ± 0.2	20.9 ± 0.2	22.4 ± 0.2	22.8 ± 0.28*	23.4 ± 0.2*	23.9 ± 0.3**	25.0 ± 0.2**
CPF	23.0 ± 0.2	22.9 ± 0.2	22.7 ± 0.2	22.1 ± 0.2	21.5 ± 0.2*	21.0 ± 0.1*	20.4 ± 0.2*	20.0 ± 0.2**
FIS	20.2 ± 0.1	20.5 ± 0.1	21.0 ± 0.2	21.5 ± 0.2	21.9 ± 0.1	22.5 ± 0.13*	23 ± 0.1*	24.0 ± 0.1**
FIS + CPF	21.0 ± 0.2	20.5 ± 0.2	20.0 ± 0.2	20.2 ± 0.2	20.8 ± 0.2	21.5 ± 0.2	22 ± 0.2*	22.5 ± 0.1*

Data are reported as mean ± standard error

S/OIL Soya oil, CPF Chlorpyrifos, FIS Fisetin, FIS+CPF Fisetin + Chlorpyrifos

*p < 0.05, **p < 0.01, compared with week 0

Effect of treatment on antioxidant enzymes, malondialdehyde concentration, and acetylcholinesterase activity

The alterations in the levels of antioxidant enzymes, MDA concentration and AChE were observed in the brain of adult male albino mice, exposed to a subchronic dose of CPF (Figs. 1, 2). The brain CAT activity showed a significant (p < 0.05) decrease in the CPF group (40.81 ± 3.41 µ/mg) relative to the S/OIL (93.13 ± 6.27 µ/mg), FIS (124 ± 8.86 µ/mg) and FIS + CPF (81.35 ± 7.26 µ/mg) (Fig. 1a). Although there was no significant (p > 0.05) change in the brain CAT activity in the FIS + CPF group when compared to the S/OIL group (Fig. 1a), the fisetin group showed the highest activity in brain CAT.

Fig. 1.

a–c Effect of subchronic exposure to chlorpyrifos (CPF) on brain antioxidant enzymes of male adult albino mice treated with fisetin. S/OIL Soya oil, CPF Chlorpyrifos, FIS Fisetin and FIS + CPF Fisetin + Chlorpyrifos. *p < 0.05, **p < 0.01 compared with CPF. Data are reported as mean ± standard error of mean (SEM)

Fig. 2.

a, b Effect of subchronic exposure to chlorpyrifos (CPF) and fisetin on brain malondialdehyde concentration and acetylcholinesterase activity of male adult albino mice treated with fisetin. S/OIL Soya oil, CPF Chlorpyrifos, FIS Fisetin and FIS + CPF Fisetin + Chlorpyrifos. *p < 0.05, **p < 0.01 compared with CPF. Data are reported as mean ± standard error of mean (SEM)

There was a significant (p < 0.05) decrease in brain GPx activity in the CPF group (50.28 ± 3.44 nmol/mL), when compared to those recorded in the S/OIL (79.47 ± 4.09 nmol/mL), FIS (98.32 ± 8.30 nmol/mL) and FIS + CPF (80.76 ± 4.55 nmol/mL) (Fig. 1b). There was also no significant (p > 0.05) change in brain activity in the FIS + CPF group compared to the S/OIL group. The highest activity in brain GPx was recorded in the FIS group (Fig. 1b).

The brain SOD activity was significantly (p < 0.01) reduced in the CPF group (46.38 ± 1.709 IU/mL) when compared to the S/OIL (76.58 ± 2.63 IU/mL), FIS (124.20 ± 7.87 IU/mL) and FIS + CPF (93.03 ± 6.63 IU/mL) groups (Fig. 2c). There was no significant (p > 0.05) change in the brain SOD activity in the FIS + CPF group, compared to the S/OIL group. The fisetin group had the highest activity in brain SOD (Fig. 1c).

A significant (p < 0.05) increase in the brain MDA concentration was recorded in the CPF group (0.348 ± 0.02 nmol/mg) when compared to the S/OIL (0.18 ± 0.01 nmol/mg), FIS (0.11 ± 0.01 nmol/mg) and FIS + CPF (0.19 ± 0.02 nmol/mg). The lowest MDA concentration was recorded in the fisetin group (Fig. 2a).

There was a significant (p < 0.05) decrease in the AChE activity in the CPF (8.97 ± 0.36 nmol/mL) when compared to S/OIL (12.57 ± 0.36 nmol/mL), fisetin (11.59 ± 0.72 nmol/mL) and FIS + CPF (13.67 ± 1.23 nmol/mL) groups. No significant (p > 0.05) change was observed between the FIS + CPF and S/OIL groups (Fig. 2b).

Effect of treatment on neurobehavioural parameters

The neurobehavioral changes (excitability scores and forepaw grip time) observed in adult male albino mice exposed to a subchronic dose of CPF are shown in Fig. 3. There was a progressive decline in the excitability scores of mice in the CPF group (p < 0.05) relative to the S/OIL, FIS, and FIS + CPF groups. Specifically, there was a significant (p < 0.05) decrease in the excitability scores at week 3 (2.86 ± 0.14) and week 6 (2.57 ± 0.20), when compared to day 0 (4.44 ± 0.2) (Fig. 3a).

Fig. 3.

a, b Effects of subchronic exposure to chlorpyrifos (CPF) on brain excitability scores and foregrip test of adult male albino mice: S/OIL Soya oil, CPF Chlorpyrifos, FIS Fisetin and FIS + CPF Fisetin + Chlorpyrifos. *p < 0.05 compared with CPF

There was a significant decline (p < 0.05) in the forepaw grip time from 20.71 ± 1.81 s in the CPF group at day 0 to 12.71 ± 1.06 s and 10.14 ± 0.80 s at weeks 3 and 6, respectively (Fig. 3b). The grip time in the CPF group was significantly (p < 0.01) lower when compared to the S/OIL, FIS, and FIS + CPF groups (Fig. 3b).

Discussion

The study revealed a progressive decrease in body weight gain in the CPF group compared to the S/OIL, FIS, and FIS + CPF groups. This is consistent with the reports of Ambali et al. [42] and Ezzi et al. [43] in mice and rats exposed to CPF. The decrease in body weight recorded in the CPF may be due to the depletion of lipids and proteins as a result of the toxic effects of the OP [44]. The progressive weight loss in the CPF group may be due to oxidative and cholinergic stress caused by the inhibition of cholesterol ester hydrolase. The present study showed a significant elevation in brain MDA concentration in the CPF group when compared to the fisetin treated and S/OIL groups. The increased lipoperoxidative changes in the brain in the CPF group demonstrates the role of oxidative stress in CPF-induced toxicity. The findings of the present study are in agreement with the works of Shittu et al. [45], Tuzmen et al. [46] and Ambali et al. [47], who observed a similar increase in lipoperoxidative damage to the brain following CPF exposure in rats. Variations in MDA concentration have been used as an indicator of lipid peroxidative damage in tissues [48]. In the present study, an elevation in MDA concentrations in the brain may be due to the inability of the body’s internal antioxidant mechanism to eliminate the ROS produced by the reaction of CPF with the brain tissue, when it penetrates the blood–brain barrier [49]. The sources of ROS generation in cells are various metabolic reactions with the incomplete reduction of oxygen in the mitochondrial electron transport chain during cellular respiration [50].

In the present study, pretreatment with fisetin reduced MDA concentration and increased the activities of CAT, SOD and GPx. The result may be due to the ability of fisetin to scavenge deleterious and toxic ROS, which reduced the activities of the antioxidant enzymes. The decrease in brain SOD activity in the CPF group compared to other groups is an indication of the reduced level of oxidative damage. This is similar to the findings of Gultekin et al. [51] and Shittu et al. [45], who observed reduction in brain SOD activity in rats exposed to CPF toxicity. Previous studies revealed that CPF decreases significantly the SOD activity in rats exposed to CPF [52]. Treatment with fisetin was observed to restore the SOD activity to its normal level, through its potent antioxidant activity in rats exposed to aflatoxin [53, 54]. The low level of brain SOD activity in the CPF group showed that the organophosphate hastened the degradation or inactivation of SOD. Supplementation with fisetin, significantly increased the activity of brain SOD in the CPF + fisetin and fisetin group. The increase in SOD is an indication of the antioxidant properties of fisetin [55]. The increase may be due to the ability of fisetin to penetrate the blood–brain barrier and exerts its antioxidant effects [56], or its ability to reduce the production of ROS [12].

The low level of brain CAT activity recorded in the CPF group agrees with the findings of Altuntas et al. [57] and Mansour & Mossa [58]. The reduction of CAT activity may be related to CPF-induced decrease in the activities of SOD, which converts superoxide anion to hydrogen peroxide [59, 60]. Pretreatment with fisetin in the present study was able to increase the activity of CAT in the brain as a result of its ability to scavenge ROS, resulting in the reduction of superoxide anion. Fisetin, a natural polyphenolic compound, has been shown to upregulate glutathione function by modulating the activity or expression of glutathione reductase and transferase [61, 62]. Fisetin which is a flavonoid can scavenge free radicals as a result of its electron-donating capacity, due to the presence of two hydroxyl groups on one ring and one hydroxyl group on the other ring [4, 63]. The present study showed a significant decrease in brain GPx in mice exposed to CPF, when compared with the other groups. Previous studies have shown that CPF causes a decrease in the brain GPx [64], and fisetin modulates the activities of antioxidant enzymes, including GPx in rats exposed to aflatoxin B1 [53]. Fisetin has been shown to provide chemopreventive as well as chemotherapeutic effects in Swiss albino mice exposed to benzo(a)pyrene by its free-radical scavenging and antioxidant activities [13]. Prakash et al. [14] demonstrated that supplementation with fisetin significantly increases the activities of enzymatic antioxidants (SOD, CAT and GPx) in the brain tissue and enhances behavioral performance during aluminum chloride-induced neurotoxicity in adult Swiss albino mice [14]. Chen et al. [65] showed the neuroprotective function of fisetin in intracerebral hemorrhage induced-brain injury in mice by downregulation of proinflammatory markers.

The decrease in AChE activity by CPF results in the accumulation of ACh at the cholinergic receptors, leading to initial excitation followed by paralysis of muscular activity. This may be attributed to the impairment of motor strength recorded in the CPF group [66]. CPF being a lipophilic compound can cross the blood brain-barrier to exert its highly neurotoxic effect [67]. The neuroprotective effect of fisetin is due to its ability to cross the blood–brain barrier [68, 69]. The antioxidant, fisetin ameliorated the effect of CPF on the brain AChE activity by binding to the AChE active site forming multiple hydrogen bonds with the amino acids on the active site [70, 71]. Fisetin exhibits a competitive type of inhibition with CPF for the active site on AChE. Administration of fisetin resulted in the improvement of AchE activity that invariably enhances neuromuscular activity. This showed that fisetin which is a flavonoid has AChE restoration properties.

The progressive decrease in the excitability score of mice dosed with CPF only reflected the state of physical and mental alertness of the animals, indicating poor sensorimotor reflex and neuromuscular coordination. This deficit may be due to impairment in AChE activity, hence decreased neuronal activity in the nervous tissue [72]. Pretreatment with fisetin significantly improved the excitability scores. This may be due to improvement in AChE activity in the brain following its inhibition by CPF, thereby aiding in the restoration of neuromuscular function. Amelioration of CPF-induced lipoperoxidative damage to the brain by fisetin may have complemented the improvement in excitability scores. The significant reduction in forepaw grip time, reflecting a deficit in forepaw motor strength following subchronic CPF exposure, agreed with the finding obtained in an earlier study which showed a reduction in hindlimb grip strength following repeated CPF administration in rats [73]. Similarly, reduced hand strength, [74] altered peripheral nerve function [75], and loss of muscle strength [76] have been observed in humans following prolonged exposure to OPs. The reduced grip time following CPF exposure recorded in the present study is an indication of impaired motor strength. Fisetin administration increased the forepaw grip time in the CPF + fisetin and fisetin groups, which showed the protective effect of fisetin on motor strength. The increase in activities of antioxidant enzymes (SOD, CAT and GPx) and reduction of MDA in the S/OIL group showed that S/OIL acts as a free radical scavenger and radioprotectant [77].

The present study has shown the ability of subchronic CPF exposure to induce oxidative damage in the brain tissues of adult male albino mice. Thus, the low excitability scores, forepaw grip time, and CAT, SOD and GPx activities were as a result of the oxidative damage induced by CPF exposure. In this study, fisetin has been shown to ameliorate the oxidative damages in the brain tissues of adult male albino mice. It was concluded that fisetin has a protective potential in mitigating oxidative stress, induced by sub-chronic exposure to CPF in adult male albino mice. The administration of fisetin increased the excitability scores and forepaw grip time in adult albino mice, exposed to a sub-chronic dose of CPF.

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Conflict of interest

The authors declare that there is no conflict of interest in this work.