Polydatin attenuates cadmium-induced oxidative stress via stimulating SOD activity and regulating mitochondrial function in Musca domestica larvae

Chemosphere 248 (2020) 126009

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Polydatin attenuates cadmium-induced oxidative stress via stimulating SOD activity and regulating mitochondrial function in Musca domestica larvae Yuming Zhang a, b, Yajing Li b, Qin Feng b, Menghua Shao b, Fengyu Yuan b, Fengsong Liu a, b, * a b

The International Centre for Precision Environmental Health and Governance, College of Life Sciences, Hebei University, Baoding, 071002, China Key Laboratory of Zoological Systematics and Application of Hebei Province, College of Life Sciences, Hebei University, Baoding, 071002, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Cd induces oxidative stress and mitochondria dysfunction in housefly.
PD improves SOD activity and scavenges ROS in Cd-exposed larvae.
PD reduces generation of 8-oxoG by Cd exposure.
PD attenuates Cd toxicity via regulating mitochondria activity.
PD relieves Cd-induced growth inhibition in housefly larvae.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 November 2019 Received in revised form 20 January 2020 Accepted 21 January 2020 Available online 22 January 2020

Cadmium (Cd) is a widespread environment contaminant due to the development of electroplating and metallurgical industry. Cd can be enriched by organisms via food chain, causing the enlarged environmental problems and posing threats to the health of humans. Polydatin (PD), a natural stilbenoid compound derived from Polygonum cuspidatum, shows pronouncedly curative effect on oxidative damage. In this work, the protective effects of PD on oxidative damage induced by Cd in Musca domestica (housefly) larvae were evaluated. The larvae were exposed to Cd and/or PD, subsequently, the oxidative stress status, mitochondria activity, oxidative phosphorylation efficiency, and survival rate were assessed. Cd exposure generated significant increases of malondialdehyde (MDA), reactive oxygen species (ROS) and 8-hydroxy-2-deoxyguanosine (8-oxoG) in the housefly larvae, causing mitochondrial dysfunction and survival rate decline. Interestingly, pretreatment with PD exhibited obviously mitochondrial protective effects in the Cd-exposed larvae, as evidenced by reduced MDA, ROS and 8-oxoG levels, and increased activities of superoxide dismutase (SOD), mitochondrial electron transfer chain, and mitochondrial membrane potential, as well as respiratory control ratio. These results suggested that PD could attenuate Cd-induced damage via maintaining redox balance, stimulating SOD activity, and regulating mitochondria activity in housefly larvae. As a natural polyphenolic chemical, PD can act as a potential candidate compounds to relieve Cd injury. © 2020 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Polydatin Cadmium Oxidative stress Musca domestica Mitochondria

* Corresponding author. College of Life Sciences, Hebei University, Baoding, 071002, China. E-mail address: [email protected] (F. Liu). https://doi.org/10.1016/j.chemosphere.2020.126009 0045-6535/© 2020 Elsevier Ltd. All rights reserved.

1. Introduction Environmental pollution, especially heavy metal, endangers

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human health. The problem has received increasing attention. Cadmium (Cd), an industrial and environmental pollutant, arises mainly from electroplating, plastic and metallurgical industry. In the polluted soil and water, Cd can be taken up by organisms, then, it is accumulated and transferred to humans through food chain. Cd is persistent in the environment, causing serious injuries, such as renal dysfunction, hepatic toxicity, genotoxicity and apoptotic effects (Muller, 1986; Lopez et al., 2003; Jarup and Akesson, 2009; Turner, 2019; Wilczek et al., 2019). Cd accumulation in living systems varies not only by location but also within and among various species. For instance, gastropods from Korea contain more Cd than bivalves from the same region, and Boops boops have Cd concentration 3e4 times higher than Merluccius merluccius (Zhang and Reynolds, 2019). For occupational exposure, automotive batterymanufacturing workers have a mean blood Cd level of around 0.03 g/L (Zhang and Reynolds, 2019). A recent study indicates that Cd concentration in rice samples is 0.05e0.12 mg/kg across China (Wang et al., 2019c). Multiple studies suggest that oxidative stress is an important way of Cd toxicity (Kumar and Sharma, 2019; Wallace et al., 2019). Cd can indirectly induce ROS via Fenton chemistry and HabereWeiss reaction (Moriwaki et al., 2008). Cd also reduces the activity of anti-oxidative defense system enzymes by interfering with the proteins’ sulfhydryl groups (Kumar and Sharma, 2019). The main part of ROS in living organisms is produced by mitochondrial electron transport chain (ETC), endoplasmic reticulum, plasmatic and nuclear membranes (Zorov et al., 2014). In eukaryocyte, mitochondria play vital roles in processes related to energy production, cell growth and cell autophagy (West, 2017). The predominant physiological function of mitochondria is the generation of ATP, and the additional functions involve the participation in metabolism and regulation of immunity (Brand and Nicholls, 2011). Excess ROS can trigger mitochondrial permeability transition pore and attack enzymes in mitochondrial membrane, leading to mitochondrial destruction (Kowaltowski et al., 2001; Briston et al., 2017). As many studies states, Cd-induced ROS can trigger cellular oxidative stress through inhibiting and damaging ETC (Nemmiche, 2017; Tarnawska et al., 2019), resulting in loss of redox balance. Thus, therapies targeting basic mitochondrial processes, such as energy metabolism or free-radical generation, or specific interactions of disease-related proteins with mitochondria, hold great promise (Li et al., 2019b; Wallace et al., 2019). The terms mitochondrial ‘function’ and ‘dysfunction’ are widely employed in bioenergetics and cell biology. Mitochondrial membrane potential (MMP), ETC activity and OXPHOS (oxidative phosphorylation) efficiency are considered as common parameters for monitoring mitochondrial function in cells and tissues (Kuznetsov et al., 2008; de Carvalho et al., 2017; Kake-Guena et al., 2017). Amelioration of Cd-induced toxicity with rebalancing the adverse prooxidant/antioxidant ratio through supplementation of antioxidant attracts wide attentions (Patra et al., 2011; Gong et al., 2019; Wang et al., 2019b). Polydatin (PD), a natural stilbenoid compound derived from Polygonum cuspidatum, shows pronouncedly chemo-preventive activity (Yuan et al., 2019), and is used for treating cystic duct and hepatic diseases in traditional Chinese medicine (Fang et al., 2019). PD is a precursor and metabolite of resveratrol (Zhang et al., 2012), and it possesses a higher solubility in an aqueous environment compared with resveratrol (Sohretoglu et al., 2018). The curative effects of PD on damage induced from oxidative, reperfusion injury, and human tumor have been reported (Ma et al., 2016; Li et al., 2019b), however, the underlying mechanism of PD function remains to be fully understood. Musca domestica (housefly) shows a world-wide distribution, which is highly adapted to severe environmental pollutants, and seldom exhibits ill effects (Tang et al., 2011). Drosophila

melanogaster (fruit fly) is now gaining attention by environmental toxicologists as a suitable alternative model for toxicity testing (Peterson et al., 2017; Vimal et al., 2019). Analogous to fruit fly, housefly has similar advantages for toxicologic studies, including short generation times, ability to generate large sample sizes, and cost-efficient in vivo model for raising. In our previous studies, housefly has been developed as a model system to investigate the physiology, development, and immunity (Tang et al., 2011; Zhang et al., 2019d). Our previous studies also find that Cd exposure can enhance the expression of oxidative stress related genes in housefly such as SOD, heat shock protein 70 (HSP70), and HSP67B2-like rhodanese (RDH) (Tang et al., 2011, 2012, 2019). Given the remarkable anti-oxidative effects of PD, the present study aims to illustrate the toxicity of Cd in housefly, and to elucidate the protective effects and mechanism of PD in this process. Especially, we explore whether PD protects housefly against Cd injury via regulation redox balance and mitochondrial function. 2. Materials and methods 2.1. Housefly strain and treatment The housefly strain used in this study was provided by Prof. Fengqin He, Institute of Zoology, Chinese Academy of Sciences. Housefly larvae were raised at a temperature of 28  C in a medium composed of 35 g bran and 100 mL water. After eclosion, adult flies were fed on water, sugar and milk powder. For evaluating Cd toxicity to housefly larvae, Cd solution ranging from 2.5 mM to 20 mM was used to replace the water in the culture medium to feed the larvae according to the methods illustrated in our previous report (Tang et al., 2011). To investigate the protective effects of PD against the damages induced by Cd exposure in housefly larvae, four groups of the 3rd instar larvae were used in this work. (1) Control group: Control group was achieved by using normal culture medium. (2) Cd group: Cd solution of 20 mM was used to replace the water in the culture medium. (3) PD group: PD solution of 20 mM was used to replace the water in the culture medium. In this study, PD (C20H22O8, CAS no. 65914-14-2, MW 390.38 g/mol, purity 98%), with a 3,4,5-trihydroxystibene-3-bmono-D-glucoside molecular structure, was purchased from Zelang BioTech (Nanjing, China). (4) Combinations of Cd and PD group (Cd þ PD group): Solution of 20 mM Cd and 20 mM PD was used to replace the water in the culture medium. For each of the above groups, about 100 housefly larvae were selected. All the physiological and biochemical indicators in larvae were detected following a 24 h-treatment with Cd and/or PD. 2.2. Survival rate assay In the survival rate experiments, the 3rd instar larvae were treated continuously with Cd at different concentrations (2.5, 4, 6, 8, 10, 12, 14, 16, 18, and 20 mM) until the eclosion of adult flies. The newly emerged flies were counted to estimate their survivability. About 100 larvae were conducted in each experimental group, and the medium was changed daily. The survival rate of the houseflies was defined as [the number of emerged flies/the number of larvae subjected to experiment]  100%. The experiments of survival rate were repeated three times. 2.3. Determination of oxidative stress biomarkers Tissue homogenates of housefly larvae (10 larvae/dose; 3 replicates/group) were used to measure oxidative stress biomarkers of MDA, ROS and SOD. Protein concentration of each procedure was determined by using the Bradford method (Bradford, 1976). MDA

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level was measured by using an MDA detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following a previous method (Zhang et al., 2019c). ROS level was quantified by a fluorescence spectrophotometer (F-7000, Hitachi, Japan) using 20,70-dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular Probes) according to ROS assay kit instruction (Beyotime Biotechnology, Shanghai, China). The reaction was started by measuring fluorescence at an excitation/emission wave length of 485/525 nm, respectively, and the obtained fluorescence values were normalized by protein concentration. SOD activity was spectrophotometrically measured by using the nitro blue tetrazolium (NBT) method following a previous method (Tang et al., 2012). Here, one unit (U) of SOD was defined as the amount of protein that inhibited the rate of NBT reduction by 50%. SOD activity was expressed as U/mg tissue protein. 8-oxoG, a mutagenic guanine base lesion, is a major form of oxidative DNA damage. Level of 8-oxoG was measured by using a quantification direct kit (Yuduo Biotechnology, Shanghai, China) based on a method of ELISA (enzyme linked immunosorbent assay).

consumed up. Then, 2 mM ADP, 0.5 mM rotenone, 10 mM succinate, 8 mM cytochrome c and 6.25 mM antimycin A were added to measure CI and CII activities in the isolated mitochondria. All experiments were performed at 28  C with continuous stirring at 750 rpm, and the results were analyzed using DatLab 4.0 software (Oroboros Inc., Austria). The experiments were repeated three times.

2.4. Mitochondrial activity and respiration measurement

3. Results

2.4.1. Mitochondria isolation procedures Housefly larvae (10 larvae; 3 replicates/group) were taken and put into ice-cold MIB (mitochondrial isolation buffer) solution. MIB contains 70 mM sucrose, 1 mM EDTA (ethylenediaminetetraacetic acid), 210 mM mannitol, and 10 mM HEPES (N-2hydroxyethylpiperazine-N-2-ethane sulfonic acid). The larvae were homogenized using a Potter homogenizer. The mitochondrial fractions were isolated by differential centrifugation as described in a previous study (Rodrigues et al., 2018). The final pellet enriched in mitochondria was resuspended in MIB. All the solutions used in mitochondria isolation were cooled to 4  C, and the above procedures were performed on ice. Mitochondrial protein concentration was assayed by using a Bradford method (Bradford, 1976).

3.1. Cd exposure induced oxidative damage in housefly larvae

2.4.2. Measurement of mitochondrial membrane potential To investigate the potential roles of PD and Cd in the MMP regulation, mitochondria of housefly larvae (10 larvae; 3 replicates/ group) were isolated and then the value of MMP was detected according to a mitochondrial membrane potential assay kit (Beyotime Biotechnology, Shanghai, China) by using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). The experiments were repeated three times. 2.4.3. Mitochondrial respiration assay Respiration assay was performed at 28  C using the isolated mitochondria of housefly larvae. According to the method proposed in the literatures (de Carvalho et al., 2017; Kake-Guena et al., 2017), an Oxygraph-2k (O2k, Oroboros Instruments, Innsbruck, Austria) apparatus was employed to measure the activities of complex I (CI) and complex II (CII), as well as the mitochondrial OXPHOS efficiency. The specific procedures were described as follows. The isolated mitochondria were added to each chamber containing 2 mL mitochondrial respiratory solution (0.5 mM EGTA, 3 mM MgCl2$6H2O, 60 mM K-lactobionate, 20 mM Taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose and 0.1% BSA). Then, 10 mM glutamate and 2 mM malate were added. In the case of stable breathing, the value of respiratory state 3 (state 3) could be obtained by adding the unsaturated ADP (adenosine diphosphate), and the respiratory state 4 (state 4) could be reached when ADP was exhausted. According to the ratio of state 3 to state 4, the respiratory control ratio (RCR) could be calculated and the molecular number of ADP consumed by each oxygen atom, that was, the ADP/O value, could be obtained based on the concentration difference of O2 at two points of adding ADP and waiting for it to be

2.5. Statistical analysis All data were given as means ± SD (standard deviation). After ensuring gaussian distribution of the data, the data were subjected to one-way analysis of variance (one-way ANOVA) followed by a multiple comparison among the means of three or more independent groups. In experiments of survival rate assay, student’s ttest was used for comparison between the treatment and control groups. Differences were considered significant at P < 0.05, and the data were analyzed using IBM SPSS Statistics 20.0.

When the housefly larvae were exposed to Cd of different concentrations, the intracellular levels of ROS, SOD and MDA in the host were shown in Fig. 1. With the concentration of Cd ranged from 2.5 to 20 mM in the medium, the induced MDA levels in the larvae increased. ROS production increased and reached the highest value at 20 mM Cd. A slight decline profile of SOD activity was observed during Cd exposure. Accumulations of ROS and MDA indicated that oxidative damage occurred in the larvae. To further characterize the damage effects of Cd on housefly larvae DNA, 8oxoG contents were detected. As shown in Fig. 1D, 8-oxoG production significantly increased due to 10 mM or 20 mM Cd exposure. 3.2. Protective effects of PD on Cd exposure As shown in Fig. 2, the levels of oxidative stress biomarkers (MDA, ROS and 8-oxoG) in the Cd þ PD group were significantly reduced compared with that of the Cd group (20 mM Cd exposure). SOD activity was stimulated by 20 mM PD treatment, and SOD activity in the Cd þ PD group was significantly improved compared with that in the 20 mM Cd exposure group (Fig. 2C). Taken together, the Cd-induced oxidative stress was attenuated by PD supplement. 3.3. Protective effects of PD against Cd-induced mitochondrial damage 3.3.1. Mitochondrial membrane potential As shown in Fig. 3, the larvae exposed to 20 mM Cd showed a decreased MMP level compared with that of the control group, indicating a significant damage occurred. In the experiments, no significant difference was observed between the control group and the PD group. It was worth pointing out that the MMP level of the larvae exposed to 20 mM Cd was attenuated significantly by 20 mM PD supplement. 3.3.2. PD supplement attenuated Cd-induced mitochondrial dysfunction The mitochondrial CI&II activities and OXPHOS efficiency were measured, the operation profiles and the results were illustrated in Fig. 4. Mitochondrial function between the control group and the PD group showed almost no difference. Compared with the control group, the mitochondrial CI&II activity in the Cd-exposed larvae

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Fig. 1. Determination of oxidative stress biomarkers in housefly larvae during a 24 h exposure to 0, 2.5, 5, 10, and 20 mM Cd. A: Malondialdehyde (MDA); B: Reactive oxygen species (ROS); C: superoxide dismutase (SOD); D: 8-oxoG; Results were presented as the mean ± standard deviation (SD) from three independent experiments, and the different letters (a, b) depict statistical significance (P < 0.05).

decreased significantly (Fig. 4A&B), indicating 20 mM Cd exposure inhibited mitochondrial ETC efficiency. Moreover, 20 mM Cd exposure decreased values of RCR and ADP/O, suggesting Cd exposure reduced OXPHOS efficiency and damaged the coupling between respiratory chain and ATP synthase. The results indicated that the ability of ATP synthesis in the larvae was obviously affected by Cd exposure. Interestingly, the larvae supplemented with 20 mM PD showed resistant ability to the damage caused by Cd exposure. As shown in Fig. 4, compared with the Cd group, the levels of CI&CII activities and OXPHOS efficiency (RCR and ADP/O) were improved in the Cd þ PD group larvae. In other words, PD could attenuate Cdinduced mitochondrial damages in the housefly larvae.

3.4. PD supplement increased the larval survival under Cd exposure As shown in Fig. 5, the survival rates of the larvae decreased gradually with the increased concentration of Cd, suggesting the bio-toxicity effects occurred to the larvae’s growth during longtime exposure. To further investigate the protective effects of PD on Cd exposure, 20 mM PD was added to the Cd-exposed larvae group individually. As a result, PD supplement improved the survival rates of the Cd-exposed larvae in the groups of Cd concentration lower than 10 mM.

4. Discussion Insects or soil invertebrates accumulate metal either by ingesting contaminated food, soil, and organic matter, or dermally adsorbing metal ions in soil solution through their cuticle (Wilczek et al., 2019; Fajana et al., 2020). Thus, low dose of Cd can reduce the reproduction and survival of populations of insects or soil invertebrates. For instance, 52 mg/L Cd (equal to about 0.46 mM Cd) exposure can negatively affect fecundity ability of Drosophila melanogaster and trigger the expression of genes associated with defense (Hu et al., 2019b). For Caenorhabditis elegans, 0.25 mM of Cd exposure can induce serotonergic neuron injury and reproduction damage (Wang et al., 2018). Housefly widely distributes in the field such as refuse landfill, slaughterhouses, food waste, and livestock manure (Zhang et al., 2019d). Cd can be easily accumulated in the habitats to a high dose compared to other sites (Ramos-Ruiz et al., 2017; Turner, 2019). Thus, in this study, Cd doses ranging from 2.5 to 20 mM are selected to evaluate its toxic effects on housefly larvae. Increased oxidative stress represents an imbalance between intracellular production of free radicals and the cellular defense mechanisms. One of the major toxic effects of Cd exposure is its attribution of inducing oxidative stress (Kumar and Sharma, 2019). Many lines of evidences suggest that ROS plays a central role in triggering oxidative stress (Aitken, 2017; Zhang et al., 2019b). The sources of ROS are both extracellular (pollutants, drugs or radiation) and intracellular (mitochondria or endoplasmic reticulum). A

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Fig. 2. Effects of 20 mM Cd exposure and/or 20 mM PD supplement on the oxidative stress levels in housefly larvae. A: Malondialdehyde (MDA); B: Reactive oxygen species (ROS); C: superoxide dismutase (SOD); D: 8-oxoG. Results were presented as the mean ± standard deviation (SD) from three independent experiments, and the different letters (a, b, c) depict statistical significance (P < 0.05).

Fig. 3. Alterations of mitochondrial membrane potential (MMP) in housefly larvae after a 24 h exposure to 20 mM Cd and/or 20 mM PD. Results were presented as the mean ± standard deviation (SD) from three independent experiments, and the different letters (a, b) depict statistical significance (P < 0.05).

high level of ROS or MDA is considered as a phenomenon of oxidative stress. Meanwhile, guanine is the main target for ROS in DNA, with 8-oxoG being the most frequent base lesion. In organisms, cells evolve the following two strategies to maintain redox balance (Pamplona and Costantini, 2011). Nonenzymatic

antioxidants, such as glutathione, tocopherol, flavonoids, alkaloids, and carotenoids, are employed to eliminate oxidative stress (Ahmad et al., 2012). On the other hand, enzymatic ROS scavenging mechanisms include SOD, ascorbate peroxidase, and glutathione peroxidase. Most impressively, SOD acts as the first line of defense against excess ROS (Kim et al., 2015). In the present study, the levels of ROS, MDA and 8-oxoG in the housefly larvae increased in a concentration-dependent manner after Cd exposure, and the decrease of SOD activity was observed (Fig. 1), indicating that oxidative damage occurs in the Cd-exposed larvae. Effects of Cd on expression and enzymatic activity of SOD have been extensively studied. Gong et al. (2019) reveal that Cd exposure can significantly increase the SOD protein level, however, SOD activity was down-regulated by Cd exposure. Huang et al. (2006) find that Zn2þ plays an important role in maintaining the structure of SOD protein, and Cd exposure can decrease the content of Zn2þ, change the conformation of SOD protein to lower its enzyme activity. More studies focus on the protective effects of natural bioactive materials, such as melatonin (Zhang et al., 2019a), isoliquiritigenin (Wang et al., 2019a), blueberry anthocyanin (Zhang et al., 2019c) and ectoine (Bownik, 2019). Since one of major toxic effects of Cd-exposure is oxidative damage (Kumar and Sharma, 2019; Wallace et al., 2019), ROS scavengers may have positive functions to rescue the stimuli. In our previous work, SOD has been identified as an important member in housefly defense system to eliminate oxidative stress (Tang et al., 2012). In the present study, 20 mM PD supplement could stimulate the larvae’s SOD activity, and the Cd-induced oxidative stress was attenuated (Fig. 2). PD is

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Fig. 4. Effects of 20 mM Cd exposure and/or 20 mM PD supplement on the mitochondrial function in housefly larvae. A: Relative activity of CI; B: Relative activity of CII; C: RCR; D: ADP/O; E: Representative trace of evaluation of mitochondrial function in isolated mitochondria from housefly larvae with a multiple substrate-inhibitors titration protocol (the trace represents the oxygen flux as a function of time). Results were presented as the mean ± standard deviation (SD) from three independent experiments, and the different letters (a, b, c) depict statistical significance (P < 0.05).

reported to show high biological activity at low dose, for instance, equal or less than 20 mM PD can promote Nrf2-ARE anti-oxidative pathway in rat glomerular messangial cells (Huang et al., 2015). PD of 10 mM is also reported to reduce the production of ROS and

inhibit adipogenesis in orbital fibroblasts (Li et al., 2019a). Recently, direct interactions between PD and SOD are observed by using native mass spectrometry (Zhuang et al., 2019). It is deduced to be the reason that PD can show high biological activity even at low

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Fig. 5. Survival rates of the Cd-exposed and PD-supplied housefly larvae. The 3rd instar larvae were treated continuously with Cd at different concentrations (2.5, 4, 6, 8, 10, 12, 14, 16, 18, and 20 mM) until the eclosion of adult flies. PD was added to the culture medium to evaluate its protection effect. Significant differences between the Cd-exposed group and the Cd þ PD group are indicated by an asterisk (P < 0.05) or two asterisks (P < 0.01). ns, not significant. Results were presented as the mean ± standard deviation (SD) from three independent experiments.

dose. In the meanwhile, a recent study indicates that high dose of PD show toxicity to cell, for instance, PD with dose of 50e250 mM can inhibit osteosarcoma cell proliferation in a dose- and timedependent manner (Hu et al., 2019a). Moreover, the water solubility of PD is limited, and the high dose of PD solution is obtained usually by virtue of DMSO (dimethyl sulfoxide). Therefore, in this study, 20 mM PD is selected to evaluate its protective effects on Cdexposed housefly larvae. The mechanisms of PD’s antioxidant properties have been attracting wide attentions. PD could attenuate H2O2-induced oxidative stress in human umbilical vein endothelial cells via PKC (protein kinase C) pathway (Qiao et al., 2016). PD can inhibit the oxidative stress-induced proliferation of vascular smooth muscle cells by Sirt1 activation (Ma et al., 2016). It is well known that Sirt1, Nrf2-ARE (Nuclear factor-E2 related factor 2 anti-oxidant response element), and PKC pathway play important roles in regulating oxidation-reduction equilibrium (Thompson et al., 2013). Nrf2 is an important sensor protein of xenobiotic toxic substance and oxidants in cell, Huang et al. (2015) reveal that PD can promote Nrf2ARE pathway through activating Sirt1 in rat glomerular messangial cells, leading to increased expression of sod (one of target gene of Nrf2), with a result of quenching ROS overproduction. Similar research also reveals that PD supplement leads to reversal of oxidative stress and activities of SOD in cisplatin treated rats (Ince et al., 2014). Therefore, in the present study, the regulation of SOD activity is deduced to be one of the mechanisms for PD’s function of relieving Cd toxicity. DNA damage is another manifestation of Cd toxicity, and 8-oxoG is considered as a biomarker of DNA damage (Nemmiche, 2017; Kumar and Sharma, 2019). In the present study, PD supplement lowers the level of 8-oxoG in the Cd-exposed larvae, and the DNA injuries are alleviated. Thus, we speculate that PD reduces Cd damage in housefly larvae by improving SOD activity and scavenging Cd-generated ROS to maintain redox balance. PD has been reported as a promising agent for detoxification caused by many environmental pollutants, such as paraquat, propoxur, cypermethrin and fenvalerate (Arslan-Acaroz et al., 2018; Evcimen et al., 2018; Ma and Jia, 2018). Generally, environmental pollutants are inducers of oxidative stress, however, the exactly mechanism

underlying the effects of PD has not been clearly understood. Thus, in the present work, an in-depth investigation is undertaken to uncover the protective effects of PD on Cd-exposed housefly larvae by investigating mitochondrial function. Studies of mitochondrial function have drawn much attention to evaluate toxicity caused by environmental pollutants (Wang et al., 2004; Perdiz et al., 2019; Wallace et al., 2019). ETC activity is a direct parameter for evaluating mitochondrial function, and ADP/O ratio characterizes the mitochondrial activity for ATP synthesis. RCR is a parameter to reflect mitochondrial OXPHOS efficiency. In the present work, the Cd-mediated mitochondrial toxic effects are found in the housefly larvae, meanwhile, an ameliorative mitochondrial function is observed in the larvae treated with PD. The results give us a solid evidence that Cd exposure directly causes mitochondrial dysfunction in the larvae and the injury can be attenuated by PD supplement. To reveal whether PD supplement could cancel the effects of Cd exposure on survival rate, PD was added to the culture medium to evaluate its protective functions. As we expected, after PD supplement, the larvae’s survival rates increased, the reason thereof is deduced to be the declined oxidative stress and stimulated mitochondrial activity affected by PD treatment. It is worth noting that the survival rates of the larvae exposed to Cd with dose exceed than 10 mM are below 20% (Fig. 5), indicating a severe injury is generated by Cd during long-time exposure, thus, the protective effects of PD are limited. Our results reveal the PD’ protective effects against Cd-induced toxicity, thus, PD may be a new remedy for the treatment of disease connected with Cd-induced oxidative damage. Moreover, the role of PD in protecting mitochondria should deserve attention. It is well established that PD and resveratrol can be interconvertible after oral administration, and PD is the dominant ingredient (Wang et al., 2011, 2015). PD can prevent the induction of secondary brain injury after traumatic brain injury by protecting neuronal mitochondria (Li et al., 2019b), and plays a protective role on nicotinamideinduced mitochondrial dysfunction in early bovine embryo (Yuan et al., 2019). Multiple researches have explained modulatory effects of resveratrol on mitochondrial function and its contribution to organismal health. The mechanisms relate to increasing insulin

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sensitivity, reducing IGF-I (insulin-like growth factor-1) levels, increasing AMPK (AMP-activated protein kinase) and PGC-1a (peroxisome proliferator-activated receptor g coactivator 1a) activity, and increasing mitochondrial number (Baur et al., 2006; Lagouge et al., 2006). Mitochondrial CI is identified as a direct target of resveratrol. The binding of the two molecules can stimulate the above key signaling pathways, and enhance the antioxidant defenses (Gueguen et al., 2015). In the present study, PD supplement significantly stimulates the SOD activity and lowers the oxidative stress level in the larvae. Thus, it is deduced that an interaction between PD and mitochondrial CI occurs, then, signaling pathways related to antioxidant defense are activated. Meanwhile, the PDimproved ETC and OXPHOS efficiency can help housefly to generate more energy to maintain metabolic balance, synthesis of antioxidant enzyme, repair Cd-induced damages, and participate in other biological processes that consume ATP. Similar to resveratrol, a consensus is yet to be reached about the mechanisms by which PD modulates (Sohretoglu et al., 2018), although may account with the regulation of transcription factors that activate or repress the expression of mitochondria-related genes, causing alterations in the mitochondrial physiology and mitochondria-related redox status (Ma et al., 2016; Qiao et al., 2016; Cao et al., 2017). The present work describes the effects of PD on improving SOD activity in housefly larvae, and the subsequent elimination of oxidative stress, resulting in maintaining redox balance in housefly larvae. Importantly, PD exerts promising effects on mitochondria, and it brings a new hope to relieve Cd-induced injury. Certainly, further study is necessary to investigate exactly how PD affects mitochondriarelated signaling pathways. 5. Conclusions Cd, a widespread heavy metal pollutant in soil and water, generates oxidative stress, mitochondrial dysfunction, and growth inhibition in housefly larvae. The present work implies that PD can mitigate Cd-induced injury via improving SOD activity, scavenging ROS, and regulating mitochondria activity. Thus, PD is revealed to play an important role in maintaining the redox balance and strengthen the function of mitochondria. This work provides valuable and reliable information to protect organism suffering from Cd toxicity. Declaration of competing interest The authors declare they have no actual or potential competing financial interests. CRediT authorship contribution statement Yuming Zhang: Formal analysis, Investigation, Methodology, Funding acquisition, Writing - original draft. Yajing Li: Investigation, Methodology. Qin Feng: Investigation, Data curation. Menghua Shao: Investigation, Methodology. Fengyu Yuan: Investigation, Methodology. Fengsong Liu: Project administration, Funding acquisition, Conceptualization, Investigation, Writing review & editing. Acknowledgments This work is supported by the National Natural Science Foundation of China (No. 31572327), the Natural Science Foundation of Hebei Province, China (No. C2019201188 and C2019201194), the Postdoctoral Science Foundation of Hebei Province, China (No. B2016005001), and the Innovation Project for Postgraduates of Hebei University, China (No. hbu2018ss41).

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