The Protective Role of Coenzyme Q10 in Metallothionein-3 Expression in Liver and Kidney Upon Rats’ Exposure to Lead Acetate

Metallothionein-3 (MT3) is an antioxidant protein that alters after exposure to heavy metals. In this study, we investigated the hepatic and renal expression of MT3 gene following exposure to lead acetate (PbAc) alone and PbAc plus CoQ10 as an adjuvant antioxidant. Twenty-four rats were allocated into three groups, including control, PbAc (free access to drinking water contaminated with PbAc at 1g/100ml), and PbAc plus CoQ10 (10 mg/kg/day Oral). After 28 consecutive days of treatment, the mRNA expression of MT3 and Cyt-c genes and MT3 protein levels were assessed using real-time PCR and immunosorbent assay. The serum lipid prole was also monitored in the three groups. PbAc exposure signicantly reduced the hepatic and renal MT3 mRNA and protein expression compared to the control group. This reduction was signicantly increased with addition of CoQ10 to levels near those of the control group. The hepatic and renal expression of Cyt-c mRNA increased after treatment with PbAc, while such effect was reversed after addition of CoQ10. Alteration in lipid prole including increased cholesterol and low-density lipoprotein levels were observed after PbAc exposure which were counteracted by CoQ10. Our results conrm the cytotoxic effects of acute lead exposure manifested as changes in the serum lipid prole and cellular levels of Cyt-c mRNA. These cytotoxic effects may have been caused by decreased MT3 gene expression and be reduced by the protective role of CoQ10.


Introduction
Lead (Pb) is a heavy metal and environmental contaminant that induces toxicity in various body organs, including the liver, kidney, brain, and testis (Bokara et al., 2008;Fahim et al., 2013;Togao et al., 2020). Pbinduced intoxication is mainly caused by contaminated food, occupational conditions, inhalation of contaminated dust and activity in industries (Ericson et al., 2016). Pb is known as an oxidative stress factor and Pb exposure increases Reactive Oxygen Species (ROS) levels, decreases cellular antioxidant levels and consequently damages the proteins, DNA, mitochondria and biological membranes (Hsu and Guo, 2002;Sabath and Robles-Osorio, 2012). Some studies have investigated the lipid pro le after lead exposure. The long-term effects of exposure to low-dose lead acetate on the liver and kidney of rats show a signi cant increase in maleic dialdehyde levels, which is a characteristic of increased lipid peroxidation (Dai et al., 2013). Furthermore, previous studies have reported a signi cant increase in liver enzyme activities and serum cholesterol and triglyceride and a reduction in serum HDL in rats following lead poisoning (Abdelhamid et al., 2020;Liu et al., 2011).
Metallothioneins (MTs) are a family of cysteine-rich, low-molecular-weight proteins that bind to Pb in the liver and kidney (Fowler, 1998;Thirumoorthy et al., 2007;Vallee, 1995;Vašák, 2005). MTs are four isoforms of the murine MT (MT1 to MT4) and a metallothionein-like gene (MTL5) (Binz and Kägi, 1999; Moleirinho et al., 2011;Rahman et al., 2018). Among the four isoforms of MTs, MT1 and MT2 are largely expressed in mammalian cell types, with the highest expression being in the liver. MT3 was mainly expressed in the brain, and also in small amounts in the liver, kidney, heart, pancreas, and intestine MT4 is expressed exclusively in the skin and tongue (Sabolić et al., 2010;Thirumoorthy et al., 2011).
Nonetheless, the expression of MT3 in the liver and kidney is not well unclear.
Several studies have investigated the protective role of MTs against heavy metal toxicity (Sandbichler and Höckner, 2016;Siscar et al., 2014;Thirumoorthy et al., 2007). MT1 and MT2 have a role in the detoxi cation of heavy metals (Shen et al., 2019). They are mainly expressed in the liver and kidney of mice exposed to cadmium and lead (Dai et al., 2013;Shen et al., 2019). The high cysteine content of MT1 and MT2 makes them capable of binding to heavy metal ions (cadmium, silver, and lead) in vivo and in vitro (Shen et al., 2019). Unlike MT1 and MT2, MT3 isoform has been very little studied. MT3 has been shown to play a role in the development and cell apoptosis in mammalian cells, protection from DNA damage, diagnosis of tumorigenesis and cancer, and pathogenesis of some neurodegenerative diseases

Animals
Twenty-four male adult Wistar rats weighing 200-250 g were procured from Pasteur Institute of Iran. All the rats were housed in a room with a 12/12-hour dark-light cycle with controlled temperature (22± 3 °C). The rats had access to fresh water and food and were acclimatized to laboratory conditions for one week prior to the experiments. All protocols for animal experiments were approved by the institutional animal Ethical Committee, Parand branch, Islamic Azad University, Parand, Iran (IR.IAU.PIAU.REC.1399.003).

Drugs and experimental groups
Lead acetate (acetate trihydrate, Sigma-Aldrich, Merck, Germany) and CoQ10 (Sigma, St. Louis, MO, USA) were purchased from Kimia Pars Inc., Tehran, Iran. Lead acetate (PbAc) was dissolved in water and CoQ10 was dissolved in corn oil.
The rats were randomly divided into three equal groups as follows: Group 1 or control group was fed a normal diet for 28 consecutive days. Group 2 received (PbAc) (1 g/100 ml) by drinking water for 28 consecutive days. Group 3 received PbAc (1g/100 ml) by drinking water + CoQ10 (10 mg/kg/day) by gavage for 28 consecutive days. All the groups received oral corn oil as a vehicle of CoQ10. The PbAc solution was prepared daily. The dose of PbAc and CoQ10 was chosen according to previous research, respectively (Baranowska-Bosiacka et al., 2013; Rauscher et al., 2001). On the twenty-ninth day, the rats were rapidly euthanized with a combination of ketamine hydrochloride (100 mg/kg) and xylazine (10 mg/kg), their thoracic was opened, and blood samples were drawn from their left ventricle. Then, their liver and kidney were collected and washed using saline solution, and sections of the liver and kidney tissues were used for the enzyme-linked immunosorbent assay (Elisa) assays, and the remainder of the liver and kidney tissues was stored at −80 °C for mRNA isolation (Amanpour et al., 2020).

Blood biochemistry
To determine the serum concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL) and high-density lipoprotein cholesterol (HDL), the blood samples obtained from the heart were stored in test tubes. Serum TC, TG, LDL and HDL levels were measured using enzymatic kits (Wako, Osaka, Japan) according to the manufacturer's instructions.

Sample preparation for protein expression by Elisa
The level of MT3 protein was measured by a sandwich Elisa kit according to the manufacturer's protocol (BioSource, Inc.). In brief, one ml PBS was added to 0.5 g of the liver and kidney tissues of the rats and was homogenized and centrifuged at 4 °C, 600 X g, for 10 min. Then, their supernatant was centrifuged at 1000 X g, 4 °C, for 20 min. The resultant sediment was removed and diluted by 1 ml PBS, and then, 1 mmol Ethylenediaminetetraacetic acid (EDTA), 10 mmol tris at PH=7.4, 200 ml PBS, and 66 ml Sodium Dodecyle Sulfate (SDS) %16 were also added. This homogenate was centrifuged at 1100 X g for 20 min at room temperature. The supernatant was removed and stored at -80 °C and then used for the Elisa assays to determine the derived protein.

Results
Effect of PbAc and PbAc+CoQ10 on the lipid pro le of rats' blood serum Total cholesterol and LDL levels increased in the blood serum of the rats treated with PbAc (1 g/100 mL) compared to the control group (P<0.01 and P<0.05, respectively). This increase was countered by CoQ10 (10 mg/kg/day) (P<0.05 and P<0.01, respectively). HDL level also increased in the blood serum of the rats treated with CoQ10+PbAc compared to the control and PbAc groups. No signi cant differences in the rat's blood serum TG and HDL composition between PbAc and control group were found (P<0.05; Figure   1).
The effect of PbAc and PbAc+CoQ10 on MT3 gene mRNA expression in rats' liver and kidney using realtime PCR MT3 gene mRNA expression decreased in the liver and kidney of the rats treated with PbAc (1 g/100 ml) compared to the control group, (P<0.01 and P<0.001, respectively). MT3 gene mRNA expression increased in the liver and kidney of rats treated with PbAc+CoQ10 compared to the PbAc-treated group (P<0.05 and P<0.001, respectively). This level decreased in the PbAc+CoQ10-treated group compared to the control group (P<0.05; Figure 2).

The effect of PbAc and PbAc+CoQ10 on level of MT3 protein in rats' liver and kidney using Elisa assays
The level of MT3 protein decreased in the liver and kidney of the rats treated with PbAc compared to the control group (P<0.001). On the contrary, the level of MT3 protein increased in the rats treated with PbAc+CoQ10 compared to the rats treated with PbAc (P<0.01; Figure 3). MT3 expression was highly increased in the liver after exposure to cadmium (Al-Waeli et al., 2012), which shows that MT3 mediates the mechanism of cytotoxicity in different mammalian organs. Tsui et al.
(2019) have shown that MT3 is a tumorigenesis factor and increasing invasiveness and cell growth of bladder carcinoma, which is upregulated by hypoxia and arsenic in vivo. Also, the overexpression of the mRNA or protein of MT3 has been observed in many human bladder, prostate and breast cancers (Sens et al., 2001). Moreover, the MT3 gene is involved in cellular growth and heavy metals' metabolism during oxidative stress situations in the human brain (Bonaventura et al., 2018), and the MT3 gene expression also decreases with oxidative stress (Tahmasbpour et al., 2016). Thus, the present ndings suggest that MT3 expression can decrease after acute exposure to PbAc during oxidative stress conditions (Bonaventura et al., 2018).
The reduction of MT3 levels is indicated in metal-associated neurodegenerative diseases, and this function is due to the diminution of cellular capacity to neutralize ROS (Tsuji et al., 1992 International journal of environmental research and public health. 16