key: cord-1052836-7v8wi994
authors: Datta, Amit; Alam, Md. Jibran; Khaleda, Laila; Al-Forkan, Mohammad
title: Protective effects of Corchorus olitorius and Butea monosperma against Arsenic induced aberrant methylation and mitochondrial DNA damage in wistar rat model
date: 2020-12-19
journal: Toxicol Rep
DOI: 10.1016/j.toxrep.2020.12.017
sha: 55884bc34b12530e94220879adedcb928f0d8b1f
doc_id: 1052836
cord_uid: 7v8wi994

Millions of people around the world are chronically exposed to Arsenic (As) through food and drinking water. Studies revealed that Arsenic is genotoxic and causes damage to DNA. In this study, we evaluated Corchorus olitorius and Butea monosperma for their alleviative properties against Arsenic induced genotoxicity in vivo using Wistar Rat model. Arsenic exposed rats were given C. olitorius leaf powder and B. monosperma flower powder as supplementation with normal food. Methylation status of p53 promoter was measured using Methylation Sensitive Restriction Endonuclease PCR (MSRE-PCR) assay and mitochondrial DNA (mtDNA) copy number as well as occurrence of a common deletion in mtDNA in liver and kidney tissue was determined through quantitative realtime PCR (qPCR). Arsenic exposed rats after supplementation showed relatively less severe effects of toxicity evident by significantly higher amount of (p<0.05) mtDNA copy number and reduced occurrence of deletion containing mtDNA as well as lower levels of methylation in p53 gene promoter. Histopathological analysis revealed less severe histopathological changes of liver and kidney and normal liver and kidney function parameters in supplemented rats. So, the protective properties of B. monosperma and C. olitorius against Arsenic toxicity is evident in molecular level.

Arsenic (As) is a ubiquitous toxic metalloid present in the environment all over the world in organic and inorganic form [1] . It is one of the most epidemiologically important toxicant affecting millions of people around the world [2] . Beside severe skin pigmentation and keratosis, Arsenic causes respiratory disease, peripheral neuropathy, liver fibrosis, edema of legs, anemia and cancers. Chronic exposure to Arsenic especially through drinking water and food is associated with a number of different cancers (e.g., skin, bladder, liver and urinary tract) [3, 4] .

Arsenic is a proven carcinogen [5] . Though the exact mechanism is still not apparent it is understood that As is a potent genotoxic substance. It has the ability to directly affect the genetic material in numerous ways [6] . The genotoxic effects of Arsenic include induction of oxidative stress and DNA damage, inhibition of DNA repair enzymes, tumor promotion, cell proliferation, chromosomal aberrations (CA), signal transduction, [7] inhibition of DNA ligase [8] , interference with tubulin polymerization in the mitotic spindle [9] and influencing telomere length through the stimulation of telomerase reverse transcriptase [10] . Also, exposure to heavy metal pollutants can influence neuro-developmental disorders such as Autism spectrum disorder (ASD) through various mechanisms [11] . Reports found association between the levels of heavy metals such as Arsenic, lead etc. in hair and severity of ASD symptoms [12] . In addition, prolonged inhalation of heavy metal polluted particulate matter (PM 2.5) can cause respiratory dysfunction and aggravated symptoms in respiratory viral infections such as influenza and COVID-19 [13] . Recent reports also suggest that Arsenic is an epi-mutagen [14] . That is Arsenic is capable of changing the methylation pattern of a single gene to even whole genome [15] . Epigenetic changes are closely related with various diseases such as cancer [16] , neurodegenerative diseases [17] and cardiovascular diseases [18] .

Despite being a very concerning problem worldwide there is no dependable and safe treatment for arsenic toxicity [19] . Currently, Arsenic toxicity is treated with Sulfhydryl containing chelating agents like 2,3-dimercaptopropane-1-sulfonate, Meso-2,3-dimercaptosuccinic acid etc. [20] . These are prescribed in combination with natural antioxidants like vitamin C and E. But most of these chelating agents have severe side effects and are toxic in nature [21] . At this context, medicinal plants attracted much attention lately; largely due to the antioxidative properties of many plant derivatives. The free radical scavenging property of plant derivatives can reduce the oxidative damage induced by Arsenic [22] .

Corchorus olitorius otherwise known as 'Tossa' jute contains antioxidants such as carotenoids, flavonoids and vitamin C and antitumor agents such as phytol and monogalactosyl-diacylglycerol [23] . Studies also provided evidence of C. olitorius reducing oxidative damage in rat renal and hepatic tissue due to Arsenic [24] .

Butea monosperma is another medicinal plant used in folk medicine alike C. olitorius. According to Ayurvedic medicine and modern studies every part of B. monosperma contains medicinal properties. These include anti-oxidative, anti-inflammatory, anti-diarrheal, bactericidal and fungicidal properties [25] . Studies also reported that B. monosperma flower contains butein, butin, butrin, isobutrin, coreopsin etc., all of which have anti-oxidative attribute [26] . Therefore, evidences suggest that traditional herbal medicines such as B. monosperma and C. olitorius can potentially produce a protective effect against Arsenic and reduce its toxic effects, although this remains to be fully determined.

Drinking water is the most common route of exposure to Arsenic. But Arsenic exposure may also be through food, especially rice, via soil/ water-crop-food transfer [27] . Many studies confirmed presence of Arsenic in rice in concentration ranging from 0.03 to 1.83 mg per kilogram of rice. Indeed, one of the most disturbing facts is that the Arsenic content of soil in rice cultivating areas, where Arsenic may be found in the ground water, is significantly increasing. This poses a potential severe hazard for countries such as Bangladesh where rice is a staple component of the everyday diet [28] . Recent studies discovered a strong positive correlation between amount of As contaminated rice intake and increase in As induced toxicity [29] . Arsenic exposure may also occur from other dietary sources such as fish and sea foods [30] or even from indoor dusts [31] .

In this study, our objective was to ascertain the genotoxic and epigenetic effects of Arsenic contaminated rice induced toxicity in rat model alongside evaluating the potential protective effects of two medicinal plants Butea monosperma and Corchorus olitorius against the induced toxicity. This study will hopefully validate the deep laden health impacts of As contaminated rice and its prospective hazardous ability.

Rice grain of local BR-28 variety was collected and checked for background Arsenic contamination through flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS) according to Rahman et al. [32] . After confirming that no arsenic contamination is present, the rice grains were soaked in 20 mg/kg Sodium Arsenite solution for 36 h and subsequently tested for Arsenic concentration. Rice Arsenic content was 47.2 mg/kg. Then the grains were dried, grinded and mixed with pallet feed for the rats.

B. monosperma flowers were collected from University of Chittagong campus and C. olitorius leaves were collected from local plantations in the nearby Hathajari region. Both plant species were identified by Professor Dr. Sheikh Bakhtear Uddin, a taxonomist (Department of Botany, University of Chittagong, Bangladesh). Petals were separated from the flower and the leaves from the plant and washed thoroughly with distilled water. Then the petals and leaves were sundried and ground to powder. The powder was kept in sterile air tight containers and stored at a cool and dry place. As both the plant product is seasonal a single preparation was used through the study. After that B. monosperma flower powder and C. olitorius leaf powder were mixed with pellet feed at 4 % wt/wt ratio for supplementation.

For this study 20 Female Wistar albino rats (Rattus norvegicus), weighing 160-170 gm were collected from the animal breeding center of Bangladesh Council of Scientific and Industrial Research (BCSIR), Chittagong. Single sex of rats was used in this study to reduce variability. The average weight of the rats was 167.35gm and the rats were provided with sufficient laboratory rodent pellet diet and water. Then the rats were divided into four groups randomly placing five rats in each group. Group I, the control group received normal diet (pellet feed), Group II received normal diet mixed with Arsenic contaminated rice, Group III and IV was fed with Arsenic contaminated rice with C. olitorius leaf powder and B. monosperma flower powder mixed pellet feed supplementation in their diet respectively for 150 days for simulating subchronic exposure. All the experiments were carried out according the institutional and national guidelines.

The experimental animals were sacrificed on the 150th day after fasting them overnight. Blood was collected in K 2 EDTA tubes for each rat and centrifuged at 1500xg for 15 min. After centrifugation blood plasma was collected in pre labeled microcentrifuge tubes and stored at − 80 • C. Liver and kidney from sacrificed rats were removed carefully and after washing the organs with PBS a section was immersed in 10 % formalin solution for histology and the rest of the organs were immersed in absolute ethanol and preserved in an ultra-low temperature freezer (− 86 ℃) for molecular analysis and Arsenic measurement.

Liver and kidney function tests were performed by measuring liver enzymes (AST, ALT) and urea level in serum respectively. These tests were performed with commercially available kits from Human GmbH using CHEM-5v3 analyzer (Erba, Mannheim, Germany) following manufacturer's instruction.

Preserved liver and kidney tissues were cut in longitudinal and transverse pieces and passed through ascending series of ethanol washes. Then the samples were cleared with toluene and embedded in paraffin. 4μM sections of the paraffin embedded tissues were fixed on a glass slide after staining with hematoxylene and eosin and observed under light microscope.

The concentration of As in different organs (liver, kidney) was determined using FI-HG-AAS method. From each organ, 0.25gm sample was weighed and taken in beaker. The samples were digested with a mixture of HClO 4 -HNO 3 solution (ratio 1:3 v/v) at 130 • C. After removal of HNO 3 by evaporation, the digested samples were diluted with deionized water up to 100 mL. The concentrations of As in digested samples were measured at 193.7 nm wave length and 10 mA current using Atomic Absorption Spectrophotometer equipped with As lamp. Vapor generation accessory (VGA) was used to produce hydride vapors using 0.6 % sodium borohydride and 10 mM HCl [33] .

DNA from cryopreserved rat liver and kidney tissues were extracted following standard phenol-chloroform method [34] . After ethanol precipitation DNA was resuspended in nuclease free water. DNA was quantified and checked for purity in a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.) and stored in − 20 • C freezer.

Methylation Sensitive Restriction Endonuclease PCR (MSRE-PCR) technique was used to quantify the methylation level in the promoter region of the tumor suppressor gene TP53 following a previously described technique [35] . The extracted DNA from liver and kidney tissue of the rats from each group were digested with a methylation sensitive restriction endonuclease BstUI. BstUI enzyme recognizes a CpG site 5 ′ -CGCG-3 ′ within the 85bp basal promoter region of TP53. Methylation of the internal cytosine blocks cleavage. Thus the number of available template for amplification is dependent on the number of methylated and unmethylated copies of the template. After the restriction digestion only the unmethylated copies will be amplified.

Approximately 200 ng of genomic DNA was digested with 7 units of enzyme overnight (16 h) at 37 • C. Following the MSRE digestion the digested products were used as a template to amplify a 505bp region of the TP53 promoter which includes the 85bp basal promoter with the following primers, forward: 5 ′ -TCT GTT TCA AAA AGC CAA AAA GAT G-3 ′ and reverse: 5 ′ -CAG TCT TCA GGG GAG CGT G-3 ′ . PCR reactions were carried out in 25 μl volume and each reaction mixture contained 2x

GoTaq Reaction Buffer (Promega Corp.), 500 nM of each forward and reverse primer, 200 mM of each dNTPs and 1 unit of GoTaq DNA Polymerase (Promega Corp.). The cycling conditions were an initial denaturation of 5 min at 95 • C followed by 35 cycles of denaturation at 95 • C for 1 min, annealing at 56 • C for 1 min, elongation at 72 • C for 1 min and a final elongation at 72 • C for 5 min. The reactions were carried out in a Q-Cycler (HAIN Life Science, UK). The amplified region of p53 promoter region was detected by electrophoresis in 1.5 % agarose gel stained with ethidium bromide and visualized in a WiseDoc gel documentation apparatus (Daihan Scientific, Korea). After that, ImageJ software (National Institute of Health) was used to measure the relative intensity of each band for calculating the relative percentage of methylation in each group (Fig. 1 ).

mtDNA copy number and frequency of deletion in rat liver and kidney tissue was carried out following a method reported by Nicklas et al. [36] . For the quantification of mtDNA copy number and frequency of deletion, separate reactions were set up with 2x AddProbe qPCR cycles of denaturation at 95 • C for 15 s and hybridization and elongation at 60 • C for 30 s. Fluorescence was detected in the last step in a QuantStudio 3 system (Applied Biosystems, USA). Rat β-actin gene (Fwd 5 ′ -GGG ATG TTT GCT CCA ACC AA-3 ′ , Rev 5 ′ -GCG CTT TTG ACT CAA GGA TTT AA-3 ′ ) was used as the internal control to calculate the relative copy number of mtDNA and relative frequency of deletion in mtDNA using the 2 − ΔΔCT method.

Statistical analysis was performed with SPSS for Windows V.24. All data were analyzed by using one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMART) with a p-val-ue<0.05 considered to be statistically significant. All the values are expressed as mean ± SEM.

All the activities were found normal in both control and arsenictreated groups. No mortality was observed in any of the groups during the period of study. The albino rats of Group II developed 'Chromodacryorrhea' around their eyes after 80 days of Arsenic exposure. The food and water intake and body weight of the animals were monitored regularly although the study period. Change in body weight is used as an indicator for checking the animal health status. We observed significant (P < 0.05) changes in food and water intake after arsenic exposure. After treatment with C. olitorius and B. monosperma food and water intake both improved significantly (P < 0.05) ( Table 1 ). The mean initial body weight of Group I (Normal diet for 150 days), Group-II (As cont. rice for 150 days), Group-III (As cont. rice plus 4 % C. olitorius for 150 days), Group-IV (As cont. rice plus 4 % B. monosperma for 150 days) were 166.1 ± 2.46 gm, 163.78 ± 3.08 gm, 160.56 ± 3.12 gm, 163.18 ± 3.66 respectively. After 150 days study, the mean final body weight was 205.7 ± 3.60 gm, 183.9 ± 2.61 gm, 197.14 ± 3.49 gm, 205.48 ± 3.47 gm respectively. The mean final body weight of Group-II significantly (P < 0.05) decreased compared to Group-I. On the other hand, the mean final body weight of Group-III and Group-IV significantly (p < 0.05) increased compared to Group-II. 

Effect of C. olitorius and B. monosperma on daily food and water consumption of albino rats. Group II rats consumed significantly (p < 0.05) less food and water compared to control group. Whereas, in Group III and IV food and water consumption increased significantly (p < 0.05) after C. olitorius and B. monosperma treatment compared to arsenic exposed rats of group II. Here, values are expressed as MEAN ± SEM;

Arsenic accumulation in the rat organ samples were determined through FI-HG-AAS analysis. Fig. 2 is showing the mean ± SEM values of arsenic deposition in liver and kidney tissues of the three experimental groups exposed to arsenic. In control rats (Group I) mean Arsenic accumulation in liver and kidney was 0.44 ± 0.15 μg/gm and 0.46 ± 0.04 μg/gm respectively. After Arsenic exposure overall arsenic accumulation was significantly higher in kidney than liver in all groups.

Arsenic contaminated rice fed rats (Group II) had 33.872 μg/gm accumulated arsenic in kidney and 12.97 μg/gm accumulated arsenic in liver on average. However, rats of group III (C. olitorius leaf powder) and IV (B. monosperma flower powder) which received supplementation with arsenic contaminated rice had significantly less arsenic accumulation in both liver (5.27 μg/gm in GIII and 5.44 μg/gm in GIV) and kidney (18.34 μg/gm in GIII and 21.89 μg/gm in GIV) tissue compared to group II (no supplementation).

To further investigate the extent of protective capabilities of Corchorus olitorius and Butea monosperma histopathological analysis of liver and kidney tissue was done. The tissue samples from group II (only arsenic contaminated rice) exhibited severe changes in the histostructure (Fig. 3b) . We observed necrosis and degenerative changes of varied severity in hepatocytes and central veins including sinusoidal dilation, venous congestion, increased lymphatic cell population, free nuclei and fatty degeneration. Similar degenerative changes were also seen in kidney tissue samples (Fig. 3f) . Moderate to severe glomerulonephritis, coagulative and liquefactive necrosis along with epithelial damage and loss of nuclei was seen. Minor alterations in histostructure with mild degenerative changes in liver and kidney was observed in groups III and IV which received Corchorus olitorius (Fig. 3c, g) and Butea monosperma (Fig. 3d, h) supplementation respectively. The organ weight to body weight ratio was calculated to determine hypertrophy of organs in study animals. We observed mild liver hypertrophy in group II animals (Arsenic exposed) when compared to control rats (Group I) where the liver weight to body weight ratio was 3.13 ± 0.03 and 3.82 ± 0.09 in Group I and Group II respectively. In treatment groups the liver weight to body weight ratio was 2.97 ± 0.07 (Group IV-As + C. olitorius) and 2.89 ± 0.09 (Group III-As + B. monosperma). Kidney weight to body weight ratio was 0.29 ± 0.01, 0.37 ± 0.01, 0.31 ± 0.01, 0.29 ± 0.01 respectively in Group I, II, III and IV. However, none of these changes were statistically significant (p>0.05).

The evidence of Arsenic induced damage was also clear from the biochemical assays. As the data on Table 2 represents the Arsenic exposed rats had abnormal liver function due to significantly increased level of liver enzymes (AST and ALT) compared to control group of rats.

These enzymes work as markers for liver abnormalities. Arsenic exposed rats also suffered from kidney abnormalities having significantly higher levels of serum urea. However, all the markers were maintained nearly within the normal levels with C. olitorius leaf powder and B. monosperma flower powder supplementation showing significant difference from arsenic exposed group of rats. In our study we did not find any significant difference in serum urea level between the treatment groups.

Methylation status of a CpG site within the 85bp basal promoter region of rat TP53 gene was analyzed in liver and kidney tissue of rats from experimental groups. The semiquantitative analysis revealed control group of rats had about 10 % methylation at the CpG site (both in liver and kidney) but arsenic exposed rats had significantly higher percentage of methylated CpG (25.5 % in liver and 17 % in kidney) at the specific site of the p53 promoter (Fig. 4) . However, after B. monosperma flower powder and C. olitorious leaf powder supplementation despite arsenic exposure rats showed significantly lower site-specific methylation both in liver (12.55 % in GIII and 17.8 % in GIV) and kidney (12.6 % in GIII and 13.4 % in GIV) tissue. On the other hand, the mean percentage of methylation was higher in kidney than liver of arsenic exposed rats. Although DNA hypermethylation may precede histological changes but in this study no significant correlation was seen between hypermethylation of p53 promoter and degree of histological damage both in liver and kidney tissue.

The basic mechanism of arsenic induced toxicity is oxidative stress [37] . Therefore, mitochondria are the key target of arsenic induced oxidative damage. To ascertain the mitochondrial damage after arsenic exposure in liver and kidney tissue mtDNA number were measured through qPCR. The relative number of mtDNA in liver and kidney tissue of rats from experimental groups are shown in Fig. 5 . The relative number of mtDNA was significantly (p < 0.05) lower in group II (72 % and 57 % of normal value in liver and kidney respectively) when compared to group I. Conversely, B. monosperma and C. olitorious helped to retain higher level of mtDNA despite arsenic exposure both in liver (79 % and 86 % of normal value in GIII and GIV respectively) and kidney (76 % and 70 % of normal value GIII and GIV respectively). Both the plants showed good nephroprotective activity but showed lesser potential in retaining hepatic mtDNA after arsenic exposure. C. olitorius supplementation in GIII did not have significant effect on liver tissue.

Similar results (Fig. 6) were also found in case of deletion in mtDNAs. Deletions in mtDNA is seen as a marker of excessive oxidative stress and may cause mitochondrial dysfunction [38] . Arsenic exposure caused drastic increase in deletion containing mtDNA population in liver tissue (4.2-fold) compared to control rats. In kidney tissue however this increase in deleted mtDNA population was also significant (2.2-fold) but not as marked as in liver tissue. The nephroprotective potential of B. monosperma and C. olitorious was also evident in preserving mtDNA as the group of rats receiving supplementation retained near normal level (1.46-fold and 1.3-fold more than normal in GIII and GIV respectively) of deleted mtDNA population. This difference was significant (p<0.05) compared to the arsenic exposed group. However, we observed contrasting result in liver tissue as neither plant supplementation produced significant difference between the arsenic exposed rats and treated rats.

In our previous studies we have shown that C. olitorius [39] and B. monosperma [40] can alleviate the altered histological and Fig. 2 . Effects of C. olitorius and B. monosperma on As deposition pattern. Group III (Arsenic + Corchorus) and Group IV (Arsenic + Butea) both had significantly reduced (p < 0.05) Arsenic accumulation both in liver and kidney tissue than Arsenic exposed rats (Group II). Overall Arsenic accumulation was higher in kidney than liver tissue in experimental animals. Each bar represents the mean ± S.E.M.

hepato-biochemical parameters caused by rice induced arsenic toxicity. In this study, we aimed to investigate the genotoxic effects of arsenic contaminated rice in rat model and assess the protective effects of Butea monosperma and Corchorus olitorius two plant species used in traditional herbal medicine, against arsenic induced toxicity.

Arsenic is known to cause both hypo and hypermethylation either in the promotor region or in whole genome [15] . We observed significantly increased methylation in p53 promoter of the Arsenic exposed rats of Group II (As containing rice for 150 days) both in liver and kidney tissues (Fig. 4) . Similar results confirming hypermethylated p53 promoter were observed in other studies [41, 42] . In other experiments, it was suggested that promoter regions of tumor suppressor genes are target of oxidative stress induced aberrant methylations [43] . As methylated promoters of tumor suppressor genes are frequently reported in malignant tissues, the findings of our study suggest that prolonged Arsenic exposure may result into malignant transformation in liver and kidney tissues.

Arsenic toxicity can cause mitochondrial dysfunction, mtDNA depletion and occurrence of deletions in mtDNA [38] . In our study we observed significant decrease in relative mtDNA copy number in arsenic exposed rat liver and kidney tissues (Fig. 5 ). There are different factors which could affect the number of mitochondrial DNA such as cellular ROS level, mitochondrial dysfunction and reduced expression of DNA polymerase and repair enzymes [38] Recent reports suggesting that Arsenic decreases the expression of DNA polymerase γ -the enzyme responsible for replication and repair of mtDNA could be the potential reason behind depletion of mtDNA [44, 45] . The findings of our study certainly agree with these explanations. We also observed significantly increased number of deletion containing mtDNA (Fig. 6) . Truncated expression and activity of mtDNA replicating enzymes might cause gradual increase in deleted mtDNA percentage in arsenic exposed rat liver and kidney. In addition to that the deleted segment of DNA contains a subunit of COX enzyme and three subunits of NADH dehydrogenase enzyme [46] . As a result, arsenic exposed tissues exhibit diminished COX activity giving rise to ROS that further promotes Arsenic induced genotoxicity and carcinogenesis [47] .

In our study, we observed marked adverse liver and kidney changes associated with Arsenic accumulation and mitochondrial damage in Group II animals. Varied degree of necrosis was present throughout liver and kidney tissues along with other histostructural changes (Fig. 3) . A previous work of Santra et al. [48] also obtained similar results and attributed the cause of damage to mitochondrial dysfunction [48] . Several other studies also published similar reports that Arsenic causes mitochondrial induction of cellular apoptosis and necrosis through activating caspase cascade signaling and induction of mitochondrial membrane permeability transition (MMP) and inhibition of electron transport system [49, 50] . Besides, Arsenic induced free radicals can promote lipid peroxidation which causes loss of membrane integrity and results in the leakage of cytosolic enzymes into the circulation [51] . This explains the increased level of liver enzymes (AST and ALT) in the serum in arsenic exposed rats compared to normal rats. Almost similarly, Group-II rats had significantly elevated serum urea level, which also indicates cellular damage in kidney. All these correspond with the evidence from histological analysis too.

As the major genotoxic mechanism of Arsenic is through activities of ROS it is understandable that antioxidants can significantly help to decrease the toxic effects of Arsenic. Many in vitro studies reported the capability of natural and chemical antioxidants that exhibits protective effects against Arsenic [52] . In our study we observed remarkable protective effect of Corchorus olitorius against Arsenic induced toxicity.

We observed significant decrease in methylation in rat liver and kidney of Group III (C. olitorius leaf powder supplementation) (Fig. 4) . Arsenic induced hypermethylation of gene promoters originates from reactive oxygen species (ROS) induced stress [43] . Thus, the oxidative effects could be countered by administering antioxidants. In group III, mtDNA copy number increased and mtDNA deletion decreased compared to Arsenic exposed Group II rats. However, the changes in both mtDNA damage parameters were significant in kidney tissue. Group III also suffered less amount of Arsenic accumulation and less severe histological damage in both liver and kidney. Previous studies reported similar protective effects of C. olitorius in rat brain, liver and kidney against oxidative damage. [24] C. olitorius leaves are rich in polyphenolics and flavonoids which are active antioxidants [53] . The mechanism of protection against Arsenic toxicity might be through Table 2 Effect of C. olitorius and B. monoperma supplementation on biochemical markers. All three biochemical parameters were elevated significantly (p < 0.05) after arsenic exposure in group II compared to control rats. After treatment with C. olitorius and B. monosperma respectively in Group II and IV, biochemical parameters reduced significantly (p < 0.05) compared to arsenic exposed group II. Values are expressed as mean ± SEM. Fig. 4 . Effect of Corchorus olitorius and Butea monosperma on p53 promoter methylation in liver and kidney tissue of wistar albino rats. After arsenic exposure Arsenic exposed group is showing significantly increased methylation in liver and kidney tissue compared to control group. Both treatment groups, Group III (As + C. olitorius) and IV (As + B. monosperma) showing significantly (p < 0.05) reduced methylation compared to non-treatment arsenic exposed group II both in liver and kidney tissue. Each bar represents the mean ± S.E.M.

Relative number of mtDNA copy in rat liver kidney tissue. Relative mtDNA count increased significantly (p < 0.05) in liver and kidney tissue in Group III (Arsenic + Corchorus) and IV (Arsenic + Butea) except in liver tissue of group III. Arsenic exposed group II had significantly (p < 0.05) decreased mtDNA count both in liver and kidney compared to control rats. Each bar represents the mean ± S.E.M. prevention of lipid peroxidation by chelating Arsenic induced ROS. Heavy burden of ROS due to Arsenic can impair the activity of antioxidative enzymes such as super oxide dismutase (SOD) [54] . Thus C. olitorius may provide protection from the oxidative damage by inhibiting lipid peroxidation with phytophenolic compounds to prevent mitochondrial damage and preventing the ensuing damaging effects. Similarly, rats of Group IV (B. monosperma flower powder for 150 days) showed lower p53 promoter methylation along with significant retention of mtDNA in both liver and kidney after B. monosperma flower power supplementation in their feed. Even after Arsenic exposure the mtDNA number was almost similar to control animals and showed significant increase in mtDNA count from the Group II animals. Reduced mitochondrial damage in Group IV is also accompanied by low As accumulation and less severe histological damage. This protective effect against Arsenic induced toxicity might be due to presence of butein in B. monosperma flower. Previous studies reported about butein's antioxidant, free radical scavenging and anti-apoptotic properties [25] . Moreover, the presence of different flavonoids, alkaloids, butrin and isobutrin etc. attribute to the antioxidant property of B. monosperma. Butrin and isobutrin also have hepatoprotective and nephoprotective capability against oxidative damage [55] . Ethanolic extract of B. monosperma flower is a potent free radical scavenger and actively reduces super oxide anion in vivo [56] . Because creating oxidative stress is one of the prominent modes of action for Arsenic toxicity, presence of natural antioxidants and active ROS scavenger properties of B. monosperma can reduce the burden of ROS imposed by Arsenic biotransformation.

Overall, we found that protective effects of Corchorus olitorius and Butea monospema were more pronounced in kidney tissue than in liver. We have observed significant (p < 0.05) improvements in terms of both reduced methylation and mtDNA damage. This protective effect is exhibited through marked reduction in As accumulation, retention of normal level of mtDNA and lower amount of stress induced mtDNA deletion in both liver and kidney.

There are many plants that contain anti-oxidative properties like B. monosperma and C. olitorius which, as we have shown in this study, can alleviate the Arsenic induced toxicity in molecular level. But more knowledge is required to recommend a specific and safe use of these plant derived products. Potentially, this study can provide data that can initiate a pharmacological research to develop a remedy against Arsenic toxicity in Bangladesh as well as other Arsenic affected regions of the world.

Thank you again for the prompt review of the manuscript. In this revision text modifications have been done as per suggestion. We mainly changed the captions for all the tables and figures to eliminate the use of symbols describing statistical significance which seemed to lack cohesion across the literature. Now we reported the results in the captions of respective images and tables. 

The authors declare that there is no known conflict of interest.

Arsenic exposure and toxicology: a historical perspective

Clinical aspects of chronic arsenic toxicity

IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, Arsenic, metals, fibres, and dusts

Mechanism of arsenic carcinogenesis: an integrated approach

Methylated trivalent arsenicals as candidate ultimate genotoxic forms of arsenic: induction of chromosomal mutations but not gene mutations

Mechanism of comutagenesis of sodium arsenite withnmethyl-n-nitrosourea

Disruption of microtubule assembly and spindle formation as a mechanism for the induction of aneuploid cells by sodium arsenite and vanadium pentoxide

Arsenic exposure through drinking water is associated with longer telomeres in peripheral blood

Toxic metal(loid)-based pollutants and their possible role in autism spectrum disorder

Metal and essential element levels in hair and association with autism severity

Toxic metal exposure as a possible risk factor for COVID-19 and other respiratory infectious diseases

Epigenetics and arsenic toxicity. Arsenic

Genetic and epigenetic changes induced by chronic low dose exposure to arsenic of mouse testicular Leydig cells

Cancer epigenomics: beyond genomics

Environmental-induced oxidative stress in neurodegenerative disorders and aging

Environmental exposures, epigenetics and cardiovascular disease

An overview of plantbased interventions to ameliorate arsenic toxicity

MESO-2,3-Dimercaptosuccinic acid: chemical, pharmacological and toxicological properties of an orally effective metal chelating agent

Oxidative mechanism of arsenic toxicity and carcinogenesis

Arsenic-induced oxidative myocardial injury: protective role of arjunolic acid

Antitumor Promoters in Leaves of Jute (Corchorus capsularis and Corchorus olitorius)

Protective effect of Corchorus olitorius leaves on sodium arseniteinduced toxicity in experimental rats

Butein imparts free radical scavenging, anti-oxidative and proapoptotic properties in the flower extracts of Butea monosperma

Antioxidant and antidiabetic activities of flower extract from Butea monosperma (Lam.) Taub, GSTF

Arsenic and heavy metal contamination of vegetables grown in Samta village

Spatial distribution and temporal variability of arsenic in irrigated rice fields in Bangladesh. 1. Irrigation water

Urinary and dietary analysis of 18,470 bangladeshis reveal a correlation of rice consumption with arsenic exposure and toxicity

Intake of arsenic and mercury from fish and seafood in a Northern Italy community

Bioaccessibility and health risk assessment of arsenic in soil and indoor dust in rural and urban areas of Hubei province, China

Arsenic accumulation in rice (Oryza sativa L.): human exposure through food chain

Quantitative Assay of Arsenic in Experimentally Intoxicated Guinea Pigs

Purification of nucleic acids by extraction with phenol: chloroform

HaCaT anchorage blockade leads to oxidative stress, DNA damage and DNA methylation changes

Development of a quantitative PCR (TaqMan) assay for relative mitochondrial DNA copy number and the common mitochondrial DNA deletion in the rat

Role of oxidative stress in arsenic-induced toxicity

Arsenic induced mitochondrial DNA damage and altered mitochondrial oxidative function: implications for genotoxic mechanisms in mammalian cells

Study of arsenic accumulation in rice and evaluation of protective effects of Chorchorus olitorius leaves against arsenic contaminated rice induced toxicities in Wistar albino rats

Protective effects of Butea monosperma against arsenic contaminated rice induced toxicity

DNA Hypermethylation of Promoter of Gene p53 and p16 in Arsenic-Exposed People with and without Malignancy

Effects of arsenic exposure on DNA methylation in cord blood samples from newborn babies and in a human lymphoblast cell line

Molecular mechanisms of oxidative stress-induced carcinogenesis: from epidemiology to oxygenomics

Decreased DNA repair gene expression among individuals exposed to arsenic in United States drinking water

The mitochondrial DNA polymerase as a target of oxidative damage

Contribution of common deletion to total deletion burden in mitochondrial DNA from inner ear of d-galactose-induced aging rats

Mitochondria and cancer: is there a morphological connection

Arsenic induces apoptosis in mouse liver is mitochondria dependent and is abrogated by Nacetylcysteine

Arsenic stimulates release of cytochrome from isolated mitochondria via induction of mitochondrial permeability transition

Mitochondrial damage mediates genotoxicity of arsenic in mammalian cells

Drug-induced liver injury: Is it somehow foreseeable?

Natural antioxidants against arsenic-induced genotoxicity

AAPS pharmSci: an advanced publication forum has matured

Cellular response to oxidative damage in lung induced by the administration of dimethylarsinic acid, a major metabolite of inorganic arsenics, in mice

Hepatoprotective evaluation of Butea monosperma against liver damage by paracetamol in rabbits

Evaluation of free radical scavenging activity of Butea monosperma Lam

Authors thank Mr. Minhazul Abedin for his critical contribution during the development of the study and Bangladesh Centre for Scientific and Industrial Research (BCSIR) for letting the group use their HG-AAS facility for Arsenic accumulation study.