Effect of lead and cadmium exposure on antioxidant defences in rock lizard Psammophilus blanfordanus

Effect of lead and cadmium exposure on antioxidant defences in rock lizard Psammophilus blanfordanus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of lead and cadmium exposure on antioxidant defences in rock lizard Psammophilus blanfordanus Bijayeeta Deb, Sibaprasad Parida, Puspanjali Parida This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7512613/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Heavy metals cause environmental pollution, and profoundly impact biological systems. This study investigates the toxic effects of cadmium (Cd) and lead (Pb) on the antioxidant defense system in Rock lizard, Psammophilus blanfordanus . The P. blanfordanus has a common occurrence in rocky and mining rich areas of Chota-Nagpur and Eastern India, exposed to heavy metals that are released as a process of weathering. Lizards were orally gavaged with CdCl₂ or Pb (NO₃)₂, to mimic the natural exposure condition and evaluated at 24, 48, and 72-hour intervals with respect to control. Results indicated that Cd-exposure significantly decreased liver protein content and catalase activity while increasing lipid peroxidation. Similarly, Pb-exposure led to significant reductions in catalase and ascorbic acid levels, with an initial increase followed by a decrease in superoxide dismutase and glutathione (GSH) levels over time. These findings underscore the oxidative stress and cellular damage induced by Cd and Pb, reflecting their detrimental impacts on reptilian physiology. The study also highlights the potential of Psammophilus blanfordanus as an indicator for monitoring environmental/heavy metal pollution in areas with mineral mining. Our study also emphasizes the need for stringent controls on heavy metal pollution in future to mitigate their ecological and health consequences. Antioxidant heavy metals lipid peroxidation catalase liver reptiles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION One of the major events of human history, the Industrial Revolution, came with the cost of environmental pollution due to the lack of awareness and increasing anthropogenic activity. Pollution of heavy metals and their accumulation in the biological system pose a great threat to biological species (Mitra et al. 2022 ). Numerous studies have highlighted the toxic effects of heavy metals on humans and other animals, like the disruption of cellular events, including growth, proliferation, differentiation, damage-repairing processes, nephrotoxicity, neurotoxicity, hepatotoxicity, skin toxicity, cardiovascular toxicity, and apoptosis (Jomova et al. 2025 ). Also, heavy metal-induced toxicity impacts ROS generation, weakening of the antioxidant defence, enzyme inactivation, and oxidative stress (Balali-Mood et al. 2021 ; Mitra et al. 2022 ). According to the Lancet Commission on Pollution and Health, one in every six deaths is due to environmental pollution (Fuller et al. 2022 ). Cadmium is a non-essential second-row transition metal and has no known biological function, and it harms almost all life forms (Suhani et al. 2021 ). Cadmium (Cd) disrupt the cellular homeostasis by interfering with the function of biologically essential metal ions such as copper (Cu), zinc (Zn) and calcium (Ca) (Noël et al. 2004 ). Similarly, Lead (Pb) is a heavy metal with no significant biological role (Abadin et al. 2007 ; U.S. Department of Health and Human Services. Centers for Disease Control and Prevention (U.S.). Agency for Toxic Substances and Disease Registry 2020). As it interferes with the function of essential metals (Zn, Fe) it hampers the function of metalloenzymes, in turn Inhibiting ribosomal function and protein translation (Bertin and Averbeck 2006 ). Lead damages the liver, kidneys, pulmonary, gastrointestinal, haematological, central nervous system and cardiovascular systems (Khan et al. 2008 ; Balali-Mood et al. 2021 ). Areas with mining activity has high levels of heavy metal concentration in soil and water due to weathering of the ores and minerals 11,12 . Further, open cast mining of mineral ores contributes to the weathering process and increase heavy metal pollution of the surrounding area (Dudka and Adriano 1997 ). The heavy metals affect the surrounding plant and animals. Odisha is one of the mineral rich states in India and heavy metal pollution is one of the major concerns in the mining areas of the state (Yadav et al. 2019 ; Nanda et al. 2022 ; Panda et al. 2024 ). The current study highlight the toxic effect of the Cd and Pb as it accumulates in the food chain and possess great threat to the flora and fauna of the surrounding areas (Bouida et al. 2022 ; Surenbaatar et al. 2023 ). Environmental pollutants like heavy metals impede huge oxidative stress on the exposed animals (Isaksson 2010 ) , (Sun et al. 2022 ). An increase in oxidative stress can damage cellular homeostasis; cause cell damage and cell death (Klein and Ackerman 2003 ). If the condition persists for a longer duration, it can manifest itself in multiple life-threatening conditions, including the death of the animal. The body has developed several endogenous antioxidant systems to deal with the production of reactive oxygen intermediates. Antioxidants may be enzymes or non-enzyme small molecules such as Vitamin C and Vitamin E. The important enzymatic antioxidants are Superoxide Dismutase (SOD), Catalase (CAT), and Glutathione System (Pamplona and Costantini 2011 ; Demirci-Çekiç et al. 2022 ). As anthropogenic pollutant Cd and Pb destabilize the antioxidant defence system by altering the function of antioxidant enzymes and inducing oxidative stress (the denaturation of proteins and lipid peroxidation) (Beyersmann and Hechtenberg 1997 ; Dailiah Roopha and Padmalatha 2012 ; Jiang et al. 2021 ). Besides its role in impairment of antioxidant defences, Cd and Pb-induced oxidative also activates both intrinsic (mitochondrial) and extrinsic apoptotic pathways (Ahmed et al. 2013 ; Yuan et al. 2018 ; Yi et al. 2022 ; Chlubek and Baranowska-Bosiacka 2024 ). Further, the liver is the primary organ associated with the metabolism of drugs and toxic substances. Along with the liver, muscle tissue is also known as the deposition organ for toxic and heavy metal substances (Squadrone et al. 2013 ; Tomaszewska et al. 2015 ; Chałabis-Mazurek et al. 2021 ). Hence, we choose liver and muscle as study organ. Recent studies have highlighted the importance of reptiles as biological indicators of heavy metal pollution (Oyekunle 2012 ). A study has shown heavy metal pollution strongly affects fossorial reptiles by altering antioxidant responses and inducing oxidative stress (Martín et al. 2021 ). The studies on reptiles can also indicate the potential threat of heavy metal pollutants on humans. Besides, the effect of pollution on reptiles and amphibians is widespread, it drastically affects the diversity and population of these two classes (Croteau et al. 2008 ). Current evaluation by IUCN suggests that 28% of accessed species are under threat of extinction yet 21% of the reptiles fall in this category 37 . Moreover, 61% of the lizards and uropeltids are under threat of extinction (Cox et al. 2022 ). Environmental pollution has been recognized as one of the main contributing factors to this extinction, directly or indirectly. Heavy metals, as one of the major sources of environmental pollution, must have a great effect on the reptile population, specifically fossorials (Fuentes et al. 2020 ; Martín et al. 2021 ). Psammophilus blanfordanus (Rock lizard) is a land-dwelling animal with distribution mostly overlapping with the mineral deposits of Eastern India. This makes it one of the most suited organisms to understand the toxic effect of heavy metal (Dudka and Adriano 1997 ; Parida et al. 2012 ; Kasturi 2022 ). Several ecotoxicological studies have been carried out to identify, characterize the modulation of antioxidant defences and other biochemical systems. This is improving our knowledge about how pollutants can interact with living organisms (Jan et al. 2015 ; Paithankar et al. 2021 ). In this regard, our study focuses on the assessment of the toxic effect of cadmium and lead on the antioxidant system of Psammophilus blanfordanus (Rock lizard). We determined the level of antioxidants (SOD, CAT, lipid peroxidation, reduced glutathione and Ascorbic acid) along with other biochemical parameters like protein and lipid to unzip the effect of lead and cadmium on the antioxidant system of Psammophilus blanfordanus . Our results indicate that lead and cadmium dampen the antioxidant response in the Rock lizard with an increase in time of exposure. MATERIALS AND METHODS Animal We use a list concern stable Agamidae Psammophilus blanfordanus 44 as the model organism. Animals weighing between 18g-30g were collected in the early morning or night from the locality of Baripada, Odisha. The study protocol was approved by the Institutional Animal Ethics Committee (IAEC) of the North Orissa University (Letter No-12/15/IAEC/NOU). Prior to exposure the animals were acclimatized for 48 hrs. The animals were segregated Group A- 0 hour/control, Group B- 24 hours, C- 48 hours and D- 72 hours. Treatment process and preparation of supernatant The animals were orally gavaged with 50µl of 1mg/ml cadmium chloride (CdCl 2 ) or lead nitrate (Pb(NO₃)₂) orally. The dose (sub-toxic) for the treatment was decided based on previous dose response study conducted in our lab involving the same animal for both CdCl 2 and Pb(NO₃)₂ (Data not shown). After treatment the animals were incubated for 24, 48 and 72 hours. Post completion of incubation these are sacrificed and samples were prepared for assessment of different parameters. Smear was prepared for the collected blood to understand the changes in the RBC structure and morphology. For preparation of 10% homogenate small amount of liver and muscle were collected from the freshly sacrificed animal, added to ice-cold PBS and ground using prechilled mortar and pestle. The homogenate was collected and subjected to centrifugation at 4000 rpm (1000Xg) for 10 minutes in cooling centrifuge (Remi) set at 4°C. The pellet was discarded after collecting the supernatant which is used for further biochemical analysis. Estimation of Protein For estimation of protein in the sample we have used the method of Lowry (Lowry et al. 1951 ). Briefly, 0.1 ml of homogenate was taken and adjusted to 0.5 ml by adding distilled water, to this 5 ml of biuret reagent was added and vortexed. The mixture was then incubated for 30 minutes at room temperature. Post incubation the absorbance of the mixture was recorded at 700nm by Systronics Visiscan 167 spectrophotometer with an appropriate blank. Amount of protein in each sample was calculated using standard curve and expresses as mg/g. Estimation of lipid peroxidation Formation of lipid peroxide is one of the important markers of stressed or nonfunctional antioxidant system. We have estimated the amount of lipid peroxides by Thiobarbituric acid test (TBA test) (Ohkawa et al. 1979 ). From the collected supernatant 0.1 ml was taken in a test tube and 1.9 ml. of TBA reagent was added. The mixture was incubated at 95°C for 1hr and appropriate measures were taken to avoid evaporation of the sample, after completion of the incubation period the samples were allowed to cooled to ambient temperature. The samples were then centrifuged at 4500 rpm (1000×g) for 10 minutes. The supernatant was transferred to a new tube and absorbance was estimated at 532nm. The TBA-RS concentration was calculated from the extinction coefficient of 156mM־1 cm־1(wills,1969) and expressed as n mole equivalent of MDA formed per mg protein. Reduced glutathione Reduced glutathione present in the collected tissue was estimated by Ellman's method with slight modification (Ellman 1959 ). Briefly, ice-cold Tri-carboxylic acid (10% concentration) was added to precipitate the supernatant followed by centrifugation. The resulting supernatant was used for GSH assay, 0.5ml. of supernatant was taken and 2.5 ml of DTNB (5, 5-Dithiobis 2 Nitro benzoic acid) was added to it after incubation the absorbance of the sample was measured at 412 nm against appropriate blank containing. The GSH content of the tissue was expressed as mg/g tissue. Ascorbic acid The ascorbic acid content of the tissue samples was determined using the method of Jagota and Dani (Jagota and Dani 1982 ). The tissue samples were homogenated in 5% metaphosphoric acid. Then diluted, the homogenate tissue 5 times with 10% ice-cold trichloroacetic acid and, with vigorous shaking, kept in an ice bowl for 5 minutes, then centrifuged at 3000 rpm for 5 minutes. 0.1 ml. of homogenate centrifuged tissue sample was added to 1.9 ml. of distilled water, then 0.2 ml. of Folin phenol reagent (10 times diluted) was added to it and incubated for 10 minutes. The absorbance was taken at 760 nm against an appropriate blank. The ascorbic acid content of the tissue was expressed as mg/g tissue. The above experiment was repeated for at least 5 times. Lipid estimation Lipid was estimated by Vanillin-phosphoric acid reagent according to the Folch method (Folch et al. 1957 ). The reagent was prepared by dissolving 300 mg of Vanillin in 50 ml of hot water, and 200 ml of 85% phosphoric acid was added to it accordingly. A cholesterol stock was prepared by dissolving 1 mg of cholesterol stock in 1 ml chloroform. In the standard test tube, 0.1 ml of cholesterol solution was added, whereas in the other tubes, 0.1 ml of tissue supernatant was added. In the blank tube, 0.1 ml of chloroform was added. Then the solution tubes were evaporated at 60ºc. After total evaporation, 0.2 ml of concentrated H2SO4. was added to each tube and incubated for 10 minutes at 60ºc. After cooling down the test tubes, 4.8 ml of Vanillin-phosphoric acid reagent was added to each. The solution was mixed thoroughly and was allowed to stand for 10 minutes at room temperature. The absorbance of the supernatant was taken at 525 nm against an appropriate blank and the lipid content of the tissue was expressed as mg/g tissue. The above experiment was repeated for at least 5 times. Estimation of super oxide dismutase (SOD) activity Superoxide dismutase (SOD; EC 1.15.1.1) activity was determined according to Das et al., method (Das et al. 2000 ). 0.5 ml of supernatant was passed through a 2 ml column of Sephadex G-25, and elute was used for the estimation of SOD activity. In this method, superoxide radicals are generated by photo reduction of riboflavin. Superoxide radicals were allowed to react with hydroxylamine hydrochloride to produce nitrite. The nitrite, in turn, reacts with sulphanilic acid to produce a diazonium compound, which subsequently reacts with N-1 Napthyl Ethylene Diamine Dihydrochloride (NED) to produce a red azo compound having absorption maxima at 543 nm. Superoxide dismutase scavenges superoxide radicals produced by photo reduction of riboflavin. Therefore, nitrite formation in the reaction is inversely proportional to the amount of SOD present in the sample. In this method, a 1.9 ml aliquot of the cocktail was taken in a test tube to which 0.1 ml test sample was added, followed by 25 µl of riboflavin. The tubes were exposed for 10 min to two 20 W Philips CFL lamps fitted in an aluminium foil-coated wooden box.Post processing and incubation 1 ml of freshly prepared Greiss reagent was added to the tube, the absorbance of the sample was estimated at 543 nm. The enzyme activity is calculated from the value (Vo/V)–1, where Vo is the absorbance of the control and V is the absorbance of the sample. Estimation of catalase (CAT) Catalase (CAT; EC 1.11.1.6) activity was estimated according to Beers and Sizer method (Rf and Iw 1952 ). Briefly, catalase activity was determined to be a decrease in absorbance in the following reaction. The reaction solution contained 50 mM phosphate buffer (pH 7.4) and 10 mM hydrogen peroxide (H2O2). 0.1 ml of supernatant was added to 1.9 ml reaction solution and mixed thoroughly. Absorbance was measured at 240 nm at 25°C using Systronics double beam spectrophotometer (model-2203). The decreases in absorbance at 240 nm were observed over one minute of the reaction. The activity of catalase was expressed as nkat/mg protein (1nkat = 1mole of substrate converted to product per sec, 1U = 16.67nkat). The above experiment was repeated for at least 5 times. Data presentation and statistical analysis The data was presented as a bar graph, showing the mean ± SD of five data points. The experiment includes 5 animals in each set. The graphs were plotted, and statics was applied using GraphPad prism 8. Unpaired t-tests were applied to determine the statistical significance. p < 0.05 was considered significant in all the cases. p-values of < 0.05, < 0.01, < 0.001 and < 0.0001 were represented with * , ** , *** and **** respectively non-significant was denoted as ns . The normality of the data set was assessed using The Kolmogorov-Smirnov Test of Normality, through an online available software 52 . RESULTS Cadmium and lead toxicity alters the morphology of liver and RBC in exposed Psammophilus blanfordanus Morphological alterations of body organs and blood cells may indicate the state of disease severity or toxicity. In order to access the toxic effect of CdCl 2 and Pb (NO₃)₂ extracted livers of P. blanfordanus were imaged at different time of exposure. Surprisingly, the liver colour changes and becomes darker with the increase in the time of exposure (Suppl. Figure 1a). 72 hours post exposure the liver colour becomes dark brown (Suppl. Figure 1a). Similarly, our investigation of RBC, post exposure revealed morphological deterioration (Suppl. Figure 1b). The RBCs becomes increasingly irregular in shape after exposure to cadmium and lead, though the effect was more profound in lead exposed group. Exposure to cadmium deregulates the antioxidant system in the liver and muscle To determine the effect of cadmium on the antioxidant defence of P. blanfordanus we treated the organisms with CdCl 2 . After completion of the incubation period, the animals were sacrificed, and an estimation of biochemical parameters was done in the liver and muscle. The total protein content of the liver decreases significantly 24 hours post-treatment (p < 0.05) (Fig. 1 a). Interestingly, the antioxidant enzyme catalase was significantly depleted in the Cd-treated group at all the time points (p < 0.01) (Fig. 1 b). Similar to catalase, the level of ascorbic acid was significantly downregulated in the Cd-exposed lizards’ livers (p < 0.001) (Fig. 1 d). Besides, the antioxidant enzyme SOD and GSH content increases significantly after exposure to CdCl 2 in the early time points (24 and 48 hours) (Fig. 1 c and e). Yet at a later time point, the level decreased in both SOD and GSH (Fig. 1 c and e). One of the important signatures of oxidative damage is the peroxidation of lipids, which results in the formation of lipid peroxides. In our quest for cadmium-mediated oxidative damage, we determined the level of lipid peroxides in the liver of untreated and CdCl2-treated P. blanfordanus. We found a significant increase in the lipid peroxide level with an increase in the time of treatment (p < 0.0001) (Fig. 1 g). We have also determined a significant decrease in the level of total lipid in the cadmium-exposed organisms compared to untreated ones at all the time points (p < 0.01) (Fig. 1 h). Besides, the liver muscle is another important organ affected by heavy metal toxicity (Khan et al., 2020). To understand the effect of cadmium on the antioxidant system of the muscle of P. blanfordanus . We determined the level of antioxidant enzymes and lipid peroxides along with level of total lipid and protein (Fig. 2 ). The protein content of the muscle decreased significantly in the CdCl2-exposed organism compared to the untreated group at all the time points (p < 0.05) (Fig. 2 a). The level of catalase enzyme and ascorbic acid was also significantly reduced in the treated group at all the time points compared to control (p < 0.0001) (Fig. 2 b and d). Interestingly the SOD and GSH levels were elevated at the 24-hour time point (p < 0.001), and later, at 48- and 72-hours post-exposure, the level of these antioxidant enzymes depleted, suggesting a significant oxidative burden and toxicity with an increase in time of exposure (Fig. 2 c and e). The eluted protein used for SOD level estimation remains more or less similar in all the samples except for the 48-hour time point, where it was reduced significantly (Fig. 2 f). The lipid peroxides increased, and the total lipid decreased significantly in the muscle of CdCl2-exposed animals compared to the untreated group (p < 0.01) (Fig. 2 g and h) Lead toxicity impairs the antioxidant system in Psammophilus blanfordanus liver and muscle Lead is one of the most abundant heavy metals in the world, with no known biological role. Lead toxicity affects almost all creatures in the world (Rahman and Singh 2019 ). We have determined the effect of lead toxicity on the antioxidant system of the P. blanfordanus by evaluating the status of antioxidant enzymes and lipid peroxides in the liver and muscle of the Pb(NO₃)₂ exposed organisms (Figs. 3 and 4 ). Post-lead exposure, the total protein content of the liver and muscle change significantly only in 72-hour exposure (Fig. 3 a and 4 a). In 72 hours, post-exposure, there was a significant decrease (p < 0.05) in the protein level of the liver in the lead-exposed group compared to the control (Fig. 3 a). Moreover, the level of catalase enzyme was significantly lowered in the Pb exposed group in both muscle and liver of the P blanfordanus (Fig. 3 b and 4 b). The exposure to lead increases the level of Glutathione in the liver of P blanfordanus significantly (p < 0.001) (Fig. 3 c). While in the case of muscle, the GSH level increased significantly (p < 0.01) in the 24 hour and 48-hour exposure group, yet at 72 hours, it decreased (Fig. 4 c). Similar to the cadmium, the level of Ascorbic acid in the lead exposed animal’s muscle and liver diminished significantly (p < 0.001) (Fig. 3 d and 4 d). Surprisingly, in case of lead exposed P. blanfordanus muscle the ascorbic acid level was 11-21-fold lower in the treated groups compared to untreated control (Fig. 4 d). In case of SOD the level increased significantly (p < 0.001) at the initial time point (24 hours) in both liver and muscle. In the later time points it started depleting in both muscle and liver, and depleted significantly after 72 hours exposure in case of liver (p < 0.0001) (Fig. 3 e and 4 e). The lipid peroxidation increased and total lipid decreased significantly post Pb (NO₃)₂ treatment in P. blanfordanus liver and muscle (Fig. 3 g-h and 4 g-h). DISCUSSION Cadmium and lead are toxic heavy metals with no known biological role (Faroon et al. 2012 ; U.S. Department of Health and Human Services. Centers for Disease Control and Prevention (U.S.). Agency for Toxic Substances and Disease Registry 2020; Suhani et al. 2021 ). However, the accumulation of these heavy metals in the surroundings possess great threat to all the creatures and imped toxic effect on the animal body (Wu et al. 2016 ; Briffa et al. 2020 ). Lizards and other reptiles are considered indicators of pollution due to their extended exposure to environmental factors (Oyekunle 2012 ; Silva et al. 2020 ). In the current study, we evaluated the toxic effect of cadmium and lead on the antioxidant defence of Rock lizard ( Psammophilus blanfordanus ). Psammophilus blanfordanus is a land-dwelling animal and is exposed to different anthropogenic activities, specifically soil pollutants a lot 44 . Further the distribution of the Psammophilus blanfordanus matches widely with the mineral rich areas of India, with extensive mining activity (Parida et al. 2012 ; Kasturi 2022 ). Further, open cast mining of mineral ores contributes to the weathering process and increase heavy metal pollution of the surrounding area (Dudka and Adriano 1997 ). Considering all these we have used Rock lizard as a model organism for our study. To understand the toxic effect of cadmium and lead on Psammophilus blanfordanus we imaged the liver of the organism post exposure with CdCl 2 and Pb (NO₃)₂ at 24, 48 and 72 hours. Similar to the findings of Ansari et al we have also found darkening of liver colour after exposure to high dose to toxic substances (Ansari et al. 2023 ). Further evaluation of RBC morphology also revealed structural deformities in the RBC. Previous study by Medina et al has shown erythrocyte deformation upon exposure to Cadmium in amphibian ( Rhinella arenarum ) (Medina et al. 2016 ). Further we determined the toxic effect of these heavy metals though determination of biochemical parameters and antioxidant system alteration. The results from the evaluation of toxicological effect on these animals shows that the total protein in the liver and muscle was decreased after exposure to cadmium or lead. A previous studies by Rajeshkumar et al. have suggested that the total protein in the liver and muscle decreases significantly in the heavy metal-exposed common carp (Rajeshkumar et al. 2017 ). Catalase is one of the most important antioxidant enzymes in the animal body. Similar to the previous findings, the exposure to cadmium and lead decreases the level of catalase in the liver and muscle of Psammophilus blanfordanus in our study (Elarabany and Bahnasawy 2019 ; Massányi et al. 2020 ). Reduced Glutathione (GSH) is an essential non-enzymatic component of the antioxidant system (Averill-Bates 2023 ). We have seen an increase in the GSH levels at the early time point of cadmium or lead exposure, and later, the level starts to deplete with an increase in the time of exposure. A similar pattern was also observed in the level of enzymatic antioxidant SOD. This biphasic trend in SOD and GSH levels might be due to an initial antioxidant response, followed by depletion due to sustained ROS burden. This unique pattern in GSH and SOD levels may indicate a similar function that differs from that of catalase. Ascorbic acid is an important component of antioxidant defence that neutralizes the adverse effects of free radicals and toxins (Adebiyi et al. 2022 ). Previous studies have suggested that treatment of ascorbic acid decreases cadmium and lead toxicity in animals (Simon 1999 ; Wang et al. 2007 ; Adebiyi et al. 2022 ). We have found a significant decrease in the level of ascorbic acid in both lead and cadmium treatment. As the level of all the antioxidant system components got deregulated after exposure to cadmium and lead, the level of oxidative radicals might have increased in the Psammophilus blanfordanus body. To understand the level of oxidative damage caused by the deregulation of the antioxidant system, we determined the level of lipid peroxides, an important marker of oxidative damage (Poletta et al. 2016 ; Yan et al. 2023 ). Similar to previous reports, cadmium and lead significantly induced lipid peroxidation in both the liver and muscle of Psammophilus blanfordanus (Pillai and Gupta 2005 ; Bertin and Averbeck 2006 ; Romero-Calderón et al. 2022 ; Ravikumar et al. 2023 ). Conclusively, our study highlights the damaging antioxidant effect of cadmium and lead in Psammophilus blanfordanus. Our findings suggest that cadmium has a higher toxic potential than lead, and it damages the antioxidant system of the organism earlier. Besides, the response of catalase and ascorbic acid to cadmium and lead toxicity is similar in nature. While that of SOD and GSH and similar. This shows the variation within the antioxidant defence response and how it responds to cadmium and lead toxicity in Psammophilus blanfordanus. The toxicity of these heavy metals increases with the increase in the time of exposure, which is evident from the deteriorating biochemical and antioxidant parameters. Our findings and its correlation with previous works suggest that Cd and Pb toxicity extends beyond oxidative damage and may initiate intrinsic and extrinsic path of apoptosis. Our study is one of the earliest to report the antioxidant response of Psammophilus blanfordanus in response to cadmium and lead toxicity (Fig. 5 ). This will help to plan a comprehensive conservation plan for Agamidae in awake of the increasing environmental pollution. The study may also aid in formulating human centric strategies in light of the toxic effect of cadmium and lead in Agamidae. Declarations ACKNOWLEDGMENTS The authors would like to thank MSCBDU, Baripada and (Department of Science and Technology, Government of Odisha-Letter no- 2805/ST) for funding. We would like to thank Budhadev Baral and Abhilipsa Patra for their kind help in collecting the animals. We are also thankful to Rajashree Biswal, Nibedita Mohapatra, Jyotipragnaya Majhi, Harapriya Mangaraj, Pragati Priyambada Pattanaik, Soumya Ranjan Sahu, Puspita Behera and Laxmipriya Mohanta for their help in performing some of the experiments. AUTHOR CONTRIBUTION PP : Study design and administration, fund acquisition, data analysis and validation, manuscript editing and review. BD : Study design, investigation, data acquisition and processing, manuscript writing, and preparation of figures. SPP : Manuscript writing and reviewing, data validation CONFLICT OF INTERESTS Authors have no conflicts of interests to declare. DATA AVAILABILITY All the data used in this manuscript are included in the manuscript itself. 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Toxicology 469:153136. https://doi.org/10.1016/j.tox.2022.153136 Surenbaatar U, Lee S, Kwon J-Y, et al (2023) Bioaccumulation of Lead, Cadmium, and Arsenic in a Mining Area and Its Associated Health Effects. Toxics 11:519. https://doi.org/10.3390/toxics11060519 Tomaszewska E, Winiarska-Mieczan A, Dobrowolski P (2015) Hematological and serum biochemical parameters of blood in adolescent rats and histomorphological changes in the jejunal epithelium and liver after chronic exposure to cadmium and lead in the case of supplementation with green tea vs black, red or white tea. Experimental and Toxicologic Pathology 67:331–339. https://doi.org/10.1016/j.etp.2015.02.005 U.S. Department of Health and Human Services. Centers for Disease Control and Prevention (U.S.). Agency for Toxic Substances and Disease Registry (2020) Toxicological profile for lead. Agency for Toxic Substances and Disease Registry Wang C, Liang J, Zhang C, et al (2007) Effect of ascorbic Acid and thiamine supplementation at different concentrations on lead toxicity in liver. Ann Occup Hyg 51:563–569. https://doi.org/10.1093/annhyg/mem036 Wu X, Cobbina SJ, Mao G, et al (2016) A review of toxicity and mechanisms of individual and mixtures of heavy metals in the environment. Environ Sci Pollut Res 23:8244–8259. https://doi.org/10.1007/s11356-016-6333-x Yadav AK, Sahoo SK, Dubey JS, et al (2019) Assessment of particulate matter, metals of toxicological concentration, and health risk around a mining area, Odisha, India. Air Qual Atmos Health 12:775–783. https://doi.org/10.1007/s11869-019-00688-7 Yan Z, Chen Q, Xia Y (2023) Oxidative Stress Contributes to Inflammatory and Cellular Damage in Systemic Lupus Erythematosus: Cellular Markers and Molecular Mechanism. JIR Volume 16:453–465. https://doi.org/10.2147/JIR.S399284 Yi L, Shang X-J, Lv L, et al (2022) Cadmium-induced apoptosis of Leydig cells is mediated by excessive mitochondrial fission and inhibition of mitophagy. Cell Death Dis 13:928. https://doi.org/10.1038/s41419-022-05364-w Yuan Y, Zhang Y, Zhao S, et al (2018) Cadmium-induced apoptosis in neuronal cells is mediated by Fas/FasL-mediated mitochondrial apoptotic signaling pathway. Sci Rep 8:8837. https://doi.org/10.1038/s41598-018-27106-9 soil pollution in odisha - https://magazines.odisha.gov.in/Orissareview/Sept2004/englishPdf/soil.pdf. Accessed 24 Mar 2025a The IUCN Red List of Threatened Species. In: IUCN Red List of Threatened Species. https://www.iucnredlist.org/en. Accessed 19 Jun 2024b Psammophilus blanfordanus. The Reptile Database. https://reptile-database.reptarium.cz/species.php?genus=Psammophilus&species=blanfordanus. Accessed 19 Jun 2024c Kolmogorov-Smirnov Calculator (Test of Normality). https://www.socscistatistics.com/tests/kolmogorov/default.aspx. Accessed 25 Mar 2025d Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7512613","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509433348,"identity":"3f65d831-cee3-4571-b8c2-4e6b71ba803e","order_by":0,"name":"Bijayeeta Deb","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0000-6627-300X","institution":"Dharanidhar University","correspondingAuthor":true,"prefix":"","firstName":"Bijayeeta","middleName":"","lastName":"Deb","suffix":""},{"id":509433349,"identity":"e6d3c777-084c-4a09-9b4a-65813ec9b7d4","order_by":1,"name":"Sibaprasad Parida","email":"","orcid":"","institution":"Centurion University of Technology and Management","correspondingAuthor":false,"prefix":"","firstName":"Sibaprasad","middleName":"","lastName":"Parida","suffix":""},{"id":509433350,"identity":"cb27db56-ae9a-4cd4-9658-2b3b4a0ed7a5","order_by":2,"name":"Puspanjali Parida","email":"","orcid":"","institution":"Maharaja Sriram Chandra Bhanja Deo University","correspondingAuthor":false,"prefix":"","firstName":"Puspanjali","middleName":"","lastName":"Parida","suffix":""}],"badges":[],"createdAt":"2025-09-02 02:51:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7512613/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7512613/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90928621,"identity":"d716ec44-cd3e-467b-bc8f-2d7ffbd1aae6","added_by":"auto","created_at":"2025-09-09 15:57:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":848995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of cadmium toxicity on liver of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePsammophilus blanfordanus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e The protein content (a), Catalase activity (b), GSH content (c), Ascorbic acid content (d), SOD activity (e), Protein content in elute (f), Lipid peroxide content (g) and Lipid content (h) was estimated in the animals exposed to 50μL of CdCl2 (stock concentration 4mg/ml) for different time points (24, 48 and 72 hours). The experiment includes 5 animals in each set, and the results are shown as the mean ± SD of five data points. Unpaired t-tests were applied to determine the statistical significance. p\u0026lt;0.05 was considered significant in all the cases. p-values of \u0026lt;0.05, \u0026lt;0.01, \u0026lt;0.001 and \u0026lt;0.0001 were represented with *, **, *** and **** respectively non-significant was denoted as \u003cstrong\u003ens\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7512613/v1/4c9150a0537a39f1e1259f27.png"},{"id":90930807,"identity":"d869a0d6-e496-4726-ad67-7228a909a1a7","added_by":"auto","created_at":"2025-09-09 16:13:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":782795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStatus of antioxidant system in cadmium exposed \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePsammophilus blanfordanus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emuscle\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eThe protein content (a), Catalase activity (b), GSH content (c), Ascorbic acid content (d), SOD activity (e), Protein content in elute (f), Lipid peroxide content (g) and Lipid content (h) was estimated in the animals exposed to 50μL of CdCl\u003csub\u003e2\u003c/sub\u003e (stock concentration 4mg/ml) for different time points (24, 48 and 72 hours). The experiment includes 5 animals in each set, and the results are shown as the mean ± SD of five data points. Unpaired t-tests were applied to determine the statistical significance. p\u0026lt;0.05 was considered significant in all the cases. p-values of \u0026lt;0.05, \u0026lt;0.01, \u0026lt;0.001 and \u0026lt;0.0001 were represented with *, **, *** and **** respectively non-significant was denoted as \u003cstrong\u003ens\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7512613/v1/eb96cd31f3cef48855993d7d.png"},{"id":90929616,"identity":"15d69346-0ac4-47a9-aeb6-aeb9566ca7fc","added_by":"auto","created_at":"2025-09-09 16:05:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":802778,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Lead toxicity on liver of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePsammophilus blanfordanus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003eThe protein content (a), Catalase activity (b), GSH content (c), Ascorbic acid content (d), SOD activity (e), Protein content in elute (f), Lipid peroxide content (g) and Lipid content (h) was estimated in the animals exposed to 50μL of Pb(NO₃)₂ (stock concentration 4mg/ml) for different time points (24, 48 and 72 hours). The experiment includes 5 animals in each set, and the results are shown as the mean ± SD of five data points. Unpaired t-tests were applied to determine the statistical significance. p\u0026lt;0.05 was considered significant in all the cases. p-values of \u0026lt;0.05, \u0026lt;0.01, \u0026lt;0.001 and \u0026lt;0.0001 were represented with *, **, *** and **** respectively non-significant was denoted as \u003cstrong\u003ens\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7512613/v1/09dd662b8242a4370d0ab167.png"},{"id":90928622,"identity":"173858fe-22ce-4814-a701-f3ad2434f73d","added_by":"auto","created_at":"2025-09-09 15:57:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":83431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStatus of antioxidant system in Lead exposed \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePsammophilus blanfordanus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e muscle.\u003c/strong\u003e The protein content (a), Catalase activity (b), GSH content (c), Ascorbic acid content (d), SOD activity (e), Protein content in elute (f), Lipid peroxide content (g) and Lipid content (h) was estimated in the animals exposed to 50μL of Pb(NO₃)₂ (stock concentration 4mg/ml) for different time points (24, 48 and 72 hours). The experiment includes 5 animals in each set, and the results are shown as the mean ± SD of five data points. Unpaired t-tests were applied to determine the statistical significance. p\u0026lt;0.05 was considered significant in all the cases. p-values of \u0026lt;0.05, \u0026lt;0.01, \u0026lt;0.001 and \u0026lt;0.0001 were represented with *, **, *** and **** respectively non-significant was denoted as \u003cstrong\u003ens\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7512613/v1/0a20ce721359ce81f70de1cb.png"},{"id":90931616,"identity":"0146c55e-367b-4ffa-8917-00875bc187f1","added_by":"auto","created_at":"2025-09-09 16:21:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":166391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExposure to Cadmium and Lead deteriorates antioxidant response in rock lizard (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePsammophilus blanfordanus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7512613/v1/55c1fd5b2585315736315530.png"},{"id":91356085,"identity":"80aca6d4-343c-45a2-b220-f9ad50a90427","added_by":"auto","created_at":"2025-09-15 15:28:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3606207,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7512613/v1/8f74afe5-4175-4811-bb77-8d14d3dd50c4.pdf"},{"id":90929619,"identity":"846cf85a-c6fe-4d15-9a36-eeb055d0639a","added_by":"auto","created_at":"2025-09-09 16:05:36","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1575708,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7512613/v1/0a9cd06fa092b16eef1c7b02.docx"}],"financialInterests":"","formattedTitle":"Effect of lead and cadmium exposure on antioxidant defences in rock lizard Psammophilus blanfordanus","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eOne of the major events of human history, the Industrial Revolution, came with the cost of environmental pollution due to the lack of awareness and increasing anthropogenic activity. Pollution of heavy metals and their accumulation in the biological system pose a great threat to biological species (Mitra et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Numerous studies have highlighted the toxic effects of heavy metals on humans and other animals, like the disruption of cellular events, including growth, proliferation, differentiation, damage-repairing processes, nephrotoxicity, neurotoxicity, hepatotoxicity, skin toxicity, cardiovascular toxicity, and apoptosis (Jomova et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Also, heavy metal-induced toxicity impacts ROS generation, weakening of the antioxidant defence, enzyme inactivation, and oxidative stress (Balali-Mood et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mitra et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). According to the Lancet Commission on Pollution and Health, one in every six deaths is due to environmental pollution (Fuller et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Cadmium is a non-essential second-row transition metal and has no known biological function, and it harms almost all life forms (Suhani et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cadmium (Cd) disrupt the cellular homeostasis by interfering with the function of biologically essential metal ions such as copper (Cu), zinc (Zn) and calcium (Ca) (No\u0026euml;l et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Similarly, Lead (Pb) is a heavy metal with no significant biological role (Abadin et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; U.S. Department of Health and Human Services. Centers for Disease Control and Prevention (U.S.). Agency for Toxic Substances and Disease Registry 2020). As it interferes with the function of essential metals (Zn, Fe) it hampers the function of metalloenzymes, in turn Inhibiting ribosomal function and protein translation (Bertin and Averbeck \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Lead damages the liver, kidneys, pulmonary, gastrointestinal, haematological, central nervous system and cardiovascular systems (Khan et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Balali-Mood et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Areas with mining activity has high levels of heavy metal concentration in soil and water due to weathering of the ores and minerals \u003csup\u003e11,12\u003c/sup\u003e. Further, open cast mining of mineral ores contributes to the weathering process and increase heavy metal pollution of the surrounding area (Dudka and Adriano \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The heavy metals affect the surrounding plant and animals. Odisha is one of the mineral rich states in India and heavy metal pollution is one of the major concerns in the mining areas of the state (Yadav et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nanda et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Panda et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The current study highlight the toxic effect of the Cd and Pb as it accumulates in the food chain and possess great threat to the flora and fauna of the surrounding areas (Bouida et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Surenbaatar et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEnvironmental pollutants like heavy metals impede huge oxidative stress on the exposed animals (Isaksson \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)\u003csup\u003e,\u003c/sup\u003e(Sun et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). An increase in oxidative stress can damage cellular homeostasis; cause cell damage and cell death (Klein and Ackerman \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). If the condition persists for a longer duration, it can manifest itself in multiple life-threatening conditions, including the death of the animal. The body has developed several endogenous antioxidant systems to deal with the production of reactive oxygen intermediates. Antioxidants may be enzymes or non-enzyme small molecules such as Vitamin C and Vitamin E. The important enzymatic antioxidants are Superoxide Dismutase (SOD), Catalase (CAT), and Glutathione System (Pamplona and Costantini \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Demirci-\u0026Ccedil;eki\u0026ccedil; et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As anthropogenic pollutant Cd and Pb destabilize the antioxidant defence system by altering the function of antioxidant enzymes and inducing oxidative stress (the denaturation of proteins and lipid peroxidation) (Beyersmann and Hechtenberg \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Dailiah Roopha and Padmalatha \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Besides its role in impairment of antioxidant defences, Cd and Pb-induced oxidative also activates both intrinsic (mitochondrial) and extrinsic apoptotic pathways (Ahmed et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yuan et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yi et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chlubek and Baranowska-Bosiacka \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Further, the liver is the primary organ associated with the metabolism of drugs and toxic substances. Along with the liver, muscle tissue is also known as the deposition organ for toxic and heavy metal substances (Squadrone et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tomaszewska et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chałabis-Mazurek et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hence, we choose liver and muscle as study organ.\u003c/p\u003e\u003cp\u003eRecent studies have highlighted the importance of reptiles as biological indicators of heavy metal pollution (Oyekunle \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A study has shown heavy metal pollution strongly affects fossorial reptiles by altering antioxidant responses and inducing oxidative stress (Mart\u0026iacute;n et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The studies on reptiles can also indicate the potential threat of heavy metal pollutants on humans. Besides, the effect of pollution on reptiles and amphibians is widespread, it drastically affects the diversity and population of these two classes (Croteau et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Current evaluation by IUCN suggests that 28% of accessed species are under threat of extinction yet 21% of the reptiles fall in this category \u003csup\u003e37\u003c/sup\u003e. Moreover, 61% of the lizards and uropeltids are under threat of extinction (Cox et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Environmental pollution has been recognized as one of the main contributing factors to this extinction, directly or indirectly. Heavy metals, as one of the major sources of environmental pollution, must have a great effect on the reptile population, specifically fossorials (Fuentes et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mart\u0026iacute;n et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e (Rock lizard) is a land-dwelling animal with distribution mostly overlapping with the mineral deposits of Eastern India. This makes it one of the most suited organisms to understand the toxic effect of heavy metal (Dudka and Adriano \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Parida et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kasturi \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral ecotoxicological studies have been carried out to identify, characterize the modulation of antioxidant defences and other biochemical systems. This is improving our knowledge about how pollutants can interact with living organisms (Jan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Paithankar et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this regard, our study focuses on the assessment of the toxic effect of cadmium and lead on the antioxidant system of \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e (Rock lizard). We determined the level of antioxidants (SOD, CAT, lipid peroxidation, reduced glutathione and Ascorbic acid) along with other biochemical parameters like protein and lipid to unzip the effect of lead and cadmium on the antioxidant system of \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e. Our results indicate that lead and cadmium dampen the antioxidant response in the Rock lizard with an increase in time of exposure.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimal\u003c/h2\u003e\u003cp\u003eWe use a list concern stable Agamidae \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e\u003csup\u003e44\u003c/sup\u003e as the model organism. Animals weighing between 18g-30g were collected in the early morning or night from the locality of Baripada, Odisha. The study protocol was approved by the Institutional Animal Ethics Committee (IAEC) of the North Orissa University (Letter No-12/15/IAEC/NOU). Prior to exposure the animals were acclimatized for 48 hrs. The animals were segregated Group A- 0 hour/control, Group B- 24 hours, C- 48 hours and D- 72 hours.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTreatment process and preparation of supernatant\u003c/h3\u003e\n\u003cp\u003eThe animals were orally gavaged with 50\u0026micro;l of 1mg/ml cadmium chloride (CdCl\u003csub\u003e2\u003c/sub\u003e) or lead nitrate (Pb(NO₃)₂) orally. The dose (sub-toxic) for the treatment was decided based on previous dose response study conducted in our lab involving the same animal for both CdCl\u003csub\u003e2\u003c/sub\u003e and Pb(NO₃)₂ (Data not shown). After treatment the animals were incubated for 24, 48 and 72 hours. Post completion of incubation these are sacrificed and samples were prepared for assessment of different parameters. Smear was prepared for the collected blood to understand the changes in the RBC structure and morphology. For preparation of 10% homogenate small amount of liver and muscle were collected from the freshly sacrificed animal, added to ice-cold PBS and ground using prechilled mortar and pestle. The homogenate was collected and subjected to centrifugation at 4000 rpm (1000Xg) for 10 minutes in cooling centrifuge (Remi) set at 4\u0026deg;C. The pellet was discarded after collecting the supernatant which is used for further biochemical analysis.\u003c/p\u003e\n\u003ch3\u003eEstimation of Protein\u003c/h3\u003e\n\u003cp\u003eFor estimation of protein in the sample we have used the method of Lowry (Lowry et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1951\u003c/span\u003e). Briefly, 0.1 ml of homogenate was taken and adjusted to 0.5 ml by adding distilled water, to this 5 ml of biuret reagent was added and vortexed. The mixture was then incubated for 30 minutes at room temperature. Post incubation the absorbance of the mixture was recorded at 700nm by Systronics Visiscan 167 spectrophotometer with an appropriate blank. Amount of protein in each sample was calculated using standard curve and expresses as mg/g.\u003c/p\u003e\n\u003ch3\u003eEstimation of lipid peroxidation\u003c/h3\u003e\n\u003cp\u003eFormation of lipid peroxide is one of the important markers of stressed or nonfunctional antioxidant system. We have estimated the amount of lipid peroxides by Thiobarbituric acid test (TBA test) (Ohkawa et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). From the collected supernatant 0.1 ml was taken in a test tube and 1.9 ml. of TBA reagent was added. The mixture was incubated at 95\u0026deg;C for 1hr and appropriate measures were taken to avoid evaporation of the sample, after completion of the incubation period the samples were allowed to cooled to ambient temperature. The samples were then centrifuged at 4500 rpm (1000\u0026times;g) for 10 minutes. The supernatant was transferred to a new tube and absorbance was estimated at 532nm. The TBA-RS concentration was calculated from the extinction coefficient of 156mM־1 cm־1(wills,1969) and expressed as n mole equivalent of MDA formed per mg protein.\u003c/p\u003e\n\u003ch3\u003eReduced glutathione\u003c/h3\u003e\n\u003cp\u003eReduced glutathione present in the collected tissue was estimated by Ellman's method with slight modification (Ellman \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1959\u003c/span\u003e). Briefly, ice-cold Tri-carboxylic acid (10% concentration) was added to precipitate the supernatant followed by centrifugation. The resulting supernatant was used for GSH assay, 0.5ml. of supernatant was taken and 2.5 ml of DTNB (5, 5-Dithiobis 2 Nitro benzoic acid) was added to it after incubation the absorbance of the sample was measured at 412 nm against appropriate blank containing. The GSH content of the tissue was expressed as mg/g tissue.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAscorbic acid\u003c/h2\u003e\u003cp\u003eThe ascorbic acid content of the tissue samples was determined using the method of Jagota and Dani (Jagota and Dani \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). The tissue samples were homogenated in 5% metaphosphoric acid. Then diluted, the homogenate tissue 5 times with 10% ice-cold trichloroacetic acid and, with vigorous shaking, kept in an ice bowl for 5 minutes, then centrifuged at 3000 rpm for 5 minutes. 0.1 ml. of homogenate centrifuged tissue sample was added to 1.9 ml. of distilled water, then 0.2 ml. of Folin phenol reagent (10 times diluted) was added to it and incubated for 10 minutes. The absorbance was taken at 760 nm against an appropriate blank. The ascorbic acid content of the tissue was expressed as mg/g tissue. The above experiment was repeated for at least 5 times.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLipid estimation\u003c/h3\u003e\n\u003cp\u003eLipid was estimated by Vanillin-phosphoric acid reagent according to the Folch method (Folch et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1957\u003c/span\u003e). The reagent was prepared by dissolving 300 mg of Vanillin in 50 ml of hot water, and 200 ml of 85% phosphoric acid was added to it accordingly. A cholesterol stock was prepared by dissolving 1 mg of cholesterol stock in 1 ml chloroform. In the standard test tube, 0.1 ml of cholesterol solution was added, whereas in the other tubes, 0.1 ml of tissue supernatant was added. In the blank tube, 0.1 ml of chloroform was added. Then the solution tubes were evaporated at 60\u0026ordm;c. After total evaporation, 0.2 ml of concentrated H2SO4. was added to each tube and incubated for 10 minutes at 60\u0026ordm;c. After cooling down the test tubes, 4.8 ml of Vanillin-phosphoric acid reagent was added to each. The solution was mixed thoroughly and was allowed to stand for 10 minutes at room temperature. The absorbance of the supernatant was taken at 525 nm against an appropriate blank and the lipid content of the tissue was expressed as mg/g tissue. The above experiment was repeated for at least 5 times.\u003c/p\u003e\n\u003ch3\u003eEstimation of super oxide dismutase (SOD) activity\u003c/h3\u003e\n\u003cp\u003eSuperoxide dismutase (SOD; EC 1.15.1.1) activity was determined according to Das et al., method (Das et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). 0.5 ml of supernatant was passed through a 2 ml column of Sephadex G-25, and elute was used for the estimation of SOD activity. In this method, superoxide radicals are generated by photo reduction of riboflavin. Superoxide radicals were allowed to react with hydroxylamine hydrochloride to produce nitrite. The nitrite, in turn, reacts with sulphanilic acid to produce a diazonium compound, which subsequently reacts with N-1 Napthyl Ethylene Diamine Dihydrochloride (NED) to produce a red azo compound having absorption maxima at 543 nm. Superoxide dismutase scavenges superoxide radicals produced by photo reduction of riboflavin. Therefore, nitrite formation in the reaction is inversely proportional to the amount of SOD present in the sample. In this method, a 1.9 ml aliquot of the cocktail was taken in a test tube to which 0.1 ml test sample was added, followed by 25 \u0026micro;l of riboflavin. The tubes were exposed for 10 min to two 20 W Philips CFL lamps fitted in an aluminium foil-coated wooden box.Post processing and incubation 1 ml of freshly prepared Greiss reagent was added to the tube, the absorbance of the sample was estimated at 543 nm. The enzyme activity is calculated from the value (Vo/V)\u0026ndash;1, where Vo is the absorbance of the control and V is the absorbance of the sample.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEstimation of catalase (CAT)\u003c/h2\u003e\u003cp\u003eCatalase (CAT; EC 1.11.1.6) activity was estimated according to Beers and Sizer method (Rf and Iw \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1952\u003c/span\u003e). Briefly, catalase activity was determined to be a decrease in absorbance in the following reaction. The reaction solution contained 50 mM phosphate buffer (pH 7.4) and 10 mM hydrogen peroxide (H2O2). 0.1 ml of supernatant was added to 1.9 ml reaction solution and mixed thoroughly. Absorbance was measured at 240 nm at 25\u0026deg;C using Systronics double beam spectrophotometer (model-2203). The decreases in absorbance at 240 nm were observed over one minute of the reaction. The activity of catalase was expressed as nkat/mg protein (1nkat\u0026thinsp;=\u0026thinsp;1mole of substrate converted to product per sec, 1U\u0026thinsp;=\u0026thinsp;16.67nkat). The above experiment was repeated for at least 5 times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eData presentation and statistical analysis\u003c/h2\u003e\u003cp\u003eThe data was presented as a bar graph, showing the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of five data points. The experiment includes 5 animals in each set. The graphs were plotted, and statics was applied using GraphPad prism 8. Unpaired t-tests were applied to determine the statistical significance. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant in all the cases. p-values of \u0026lt;\u0026thinsp;0.05, \u0026lt;\u0026thinsp;0.01, \u0026lt;\u0026thinsp;0.001 and \u0026lt;\u0026thinsp;0.0001 were represented with \u003cb\u003e*\u003c/b\u003e, \u003cb\u003e**\u003c/b\u003e, \u003cb\u003e***\u003c/b\u003e and \u003cb\u003e****\u003c/b\u003e respectively non-significant was denoted as \u003cb\u003ens\u003c/b\u003e. The normality of the data set was assessed using The Kolmogorov-Smirnov Test of Normality, through an online available software \u003csup\u003e52\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eCadmium and lead toxicity alters the morphology of liver and RBC in exposed\u003c/b\u003e \u003cb\u003ePsammophilus blanfordanus\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMorphological alterations of body organs and blood cells may indicate the state of disease severity or toxicity. In order to access the toxic effect of CdCl\u003csub\u003e2\u003c/sub\u003e and Pb (NO₃)₂ extracted livers of \u003cem\u003eP. blanfordanus\u003c/em\u003e were imaged at different time of exposure. Surprisingly, the liver colour changes and becomes darker with the increase in the time of exposure (Suppl. Figure\u0026nbsp;1a). 72 hours post exposure the liver colour becomes dark brown (Suppl. Figure\u0026nbsp;1a). Similarly, our investigation of RBC, post exposure revealed morphological deterioration (Suppl. Figure\u0026nbsp;1b). The RBCs becomes increasingly irregular in shape after exposure to cadmium and lead, though the effect was more profound in lead exposed group.\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eExposure to cadmium deregulates the antioxidant system in the liver and muscle\u003c/h2\u003e\u003cp\u003eTo determine the effect of cadmium on the antioxidant defence of \u003cem\u003eP. blanfordanus\u003c/em\u003e we treated the organisms with CdCl\u003csub\u003e2\u003c/sub\u003e. After completion of the incubation period, the animals were sacrificed, and an estimation of biochemical parameters was done in the liver and muscle. The total protein content of the liver decreases significantly 24 hours post-treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Interestingly, the antioxidant enzyme catalase was significantly depleted in the Cd-treated group at all the time points (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Similar to catalase, the level of ascorbic acid was significantly downregulated in the Cd-exposed lizards\u0026rsquo; livers (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Besides, the antioxidant enzyme SOD and GSH content increases significantly after exposure to CdCl\u003csub\u003e2\u003c/sub\u003e in the early time points (24 and 48 hours) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and e). Yet at a later time point, the level decreased in both SOD and GSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and e). One of the important signatures of oxidative damage is the peroxidation of lipids, which results in the formation of lipid peroxides. In our quest for cadmium-mediated oxidative damage, we determined the level of lipid peroxides in the liver of untreated and CdCl2-treated \u003cem\u003eP. blanfordanus.\u003c/em\u003e We found a significant increase in the lipid peroxide level with an increase in the time of treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). We have also determined a significant decrease in the level of total lipid in the cadmium-exposed organisms compared to untreated ones at all the time points (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh).\u003c/p\u003e\u003cp\u003eBesides, the liver muscle is another important organ affected by heavy metal toxicity (Khan et al., 2020). To understand the effect of cadmium on the antioxidant system of the muscle of \u003cem\u003eP. blanfordanus\u003c/em\u003e. We determined the level of antioxidant enzymes and lipid peroxides along with level of total lipid and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The protein content of the muscle decreased significantly in the CdCl2-exposed organism compared to the untreated group at all the time points (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The level of catalase enzyme and ascorbic acid was also significantly reduced in the treated group at all the time points compared to control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and d). Interestingly the SOD and GSH levels were elevated at the 24-hour time point (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and later, at 48- and 72-hours post-exposure, the level of these antioxidant enzymes depleted, suggesting a significant oxidative burden and toxicity with an increase in time of exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and e). The eluted protein used for SOD level estimation remains more or less similar in all the samples except for the 48-hour time point, where it was reduced significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The lipid peroxides increased, and the total lipid decreased significantly in the muscle of CdCl2-exposed animals compared to the untreated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg and h)\u003c/p\u003e\u003cp\u003e\u003cb\u003eLead toxicity impairs the antioxidant system in\u003c/b\u003e \u003cb\u003ePsammophilus blanfordanus\u003c/b\u003e \u003cb\u003eliver and muscle\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLead is one of the most abundant heavy metals in the world, with no known biological role. Lead toxicity affects almost all creatures in the world (Rahman and Singh \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We have determined the effect of lead toxicity on the antioxidant system of the \u003cem\u003eP. blanfordanus\u003c/em\u003e by evaluating the status of antioxidant enzymes and lipid peroxides in the liver and muscle of the Pb(NO₃)₂ exposed organisms (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePost-lead exposure, the total protein content of the liver and muscle change significantly only in 72-hour exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In 72 hours, post-exposure, there was a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the protein level of the liver in the lead-exposed group compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Moreover, the level of catalase enzyme was significantly lowered in the Pb exposed group in both muscle and liver of the \u003cem\u003eP blanfordanus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The exposure to lead increases the level of Glutathione in the liver of \u003cem\u003eP blanfordanus\u003c/em\u003e significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). While in the case of muscle, the GSH level increased significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the 24 hour and 48-hour exposure group, yet at 72 hours, it decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Similar to the cadmium, the level of Ascorbic acid in the lead exposed animal\u0026rsquo;s muscle and liver diminished significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Surprisingly, in case of lead exposed \u003cem\u003eP. blanfordanus\u003c/em\u003e muscle the ascorbic acid level was 11-21-fold lower in the treated groups compared to untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In case of SOD the level increased significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) at the initial time point (24 hours) in both liver and muscle. In the later time points it started depleting in both muscle and liver, and depleted significantly after 72 hours exposure in case of liver (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). The lipid peroxidation increased and total lipid decreased significantly post Pb (NO₃)₂ treatment in \u003cem\u003eP. blanfordanus\u003c/em\u003e liver and muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-h and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-h).\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCadmium and lead are toxic heavy metals with no known biological role (Faroon et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; U.S. Department of Health and Human Services. Centers for Disease Control and Prevention (U.S.). Agency for Toxic Substances and Disease Registry 2020; Suhani et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the accumulation of these heavy metals in the surroundings possess great threat to all the creatures and imped toxic effect on the animal body (Wu et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Briffa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Lizards and other reptiles are considered indicators of pollution due to their extended exposure to environmental factors (Oyekunle \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Silva et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the current study, we evaluated the toxic effect of cadmium and lead on the antioxidant defence of Rock lizard (\u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e). \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e is a land-dwelling animal and is exposed to different anthropogenic activities, specifically soil pollutants a lot \u003csup\u003e44\u003c/sup\u003e. Further the distribution of the \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e matches widely with the mineral rich areas of India, with extensive mining activity (Parida et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kasturi \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Further, open cast mining of mineral ores contributes to the weathering process and increase heavy metal pollution of the surrounding area (Dudka and Adriano \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Considering all these we have used Rock lizard as a model organism for our study.\u003c/p\u003e\u003cp\u003eTo understand the toxic effect of cadmium and lead on \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e we imaged the liver of the organism post exposure with CdCl\u003csub\u003e2\u003c/sub\u003e and Pb (NO₃)₂ at 24, 48 and 72 hours. Similar to the findings of Ansari \u003cem\u003eet al\u003c/em\u003e we have also found darkening of liver colour after exposure to high dose to toxic substances (Ansari et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Further evaluation of RBC morphology also revealed structural deformities in the RBC. Previous study by Medina \u003cem\u003eet al\u003c/em\u003e has shown erythrocyte deformation upon exposure to Cadmium in amphibian (\u003cem\u003eRhinella arenarum\u003c/em\u003e) (Medina et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurther we determined the toxic effect of these heavy metals though determination of biochemical parameters and antioxidant system alteration. The results from the evaluation of toxicological effect on these animals shows that the total protein in the liver and muscle was decreased after exposure to cadmium or lead. A previous studies by Rajeshkumar et al. have suggested that the total protein in the liver and muscle decreases significantly in the heavy metal-exposed common carp (Rajeshkumar et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Catalase is one of the most important antioxidant enzymes in the animal body. Similar to the previous findings, the exposure to cadmium and lead decreases the level of catalase in the liver and muscle of \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e in our study (Elarabany and Bahnasawy \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mass\u0026aacute;nyi et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Reduced Glutathione (GSH) is an essential non-enzymatic component of the antioxidant system (Averill-Bates \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We have seen an increase in the GSH levels at the early time point of cadmium or lead exposure, and later, the level starts to deplete with an increase in the time of exposure. A similar pattern was also observed in the level of enzymatic antioxidant SOD. This biphasic trend in SOD and GSH levels might be due to an initial antioxidant response, followed by depletion due to sustained ROS burden. This unique pattern in GSH and SOD levels may indicate a similar function that differs from that of catalase. Ascorbic acid is an important component of antioxidant defence that neutralizes the adverse effects of free radicals and toxins (Adebiyi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Previous studies have suggested that treatment of ascorbic acid decreases cadmium and lead toxicity in animals (Simon \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Adebiyi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We have found a significant decrease in the level of ascorbic acid in both lead and cadmium treatment. As the level of all the antioxidant system components got deregulated after exposure to cadmium and lead, the level of oxidative radicals might have increased in the \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e body. To understand the level of oxidative damage caused by the deregulation of the antioxidant system, we determined the level of lipid peroxides, an important marker of oxidative damage (Poletta et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similar to previous reports, cadmium and lead significantly induced lipid peroxidation in both the liver and muscle of \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e (Pillai and Gupta \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Bertin and Averbeck \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Romero-Calder\u0026oacute;n et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ravikumar et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eConclusively, our study highlights the damaging antioxidant effect of cadmium and lead in \u003cem\u003ePsammophilus blanfordanus.\u003c/em\u003e Our findings suggest that cadmium has a higher toxic potential than lead, and it damages the antioxidant system of the organism earlier. Besides, the response of catalase and ascorbic acid to cadmium and lead toxicity is similar in nature. While that of SOD and GSH and similar. This shows the variation within the antioxidant defence response and how it responds to cadmium and lead toxicity in \u003cem\u003ePsammophilus blanfordanus.\u003c/em\u003e The toxicity of these heavy metals increases with the increase in the time of exposure, which is evident from the deteriorating biochemical and antioxidant parameters. Our findings and its correlation with previous works suggest that Cd and Pb toxicity extends beyond oxidative damage and may initiate intrinsic and extrinsic path of apoptosis. Our study is one of the earliest to report the antioxidant response of \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e in response to cadmium and lead toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This will help to plan a comprehensive conservation plan for Agamidae in awake of the increasing environmental pollution. The study may also aid in formulating human centric strategies in light of the toxic effect of cadmium and lead in Agamidae.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank MSCBDU, Baripada and (Department of Science and Technology, Government of Odisha-Letter no- 2805/ST) for funding. We would like to thank Budhadev Baral and Abhilipsa Patra for their kind help in collecting the animals. We are also thankful to Rajashree Biswal, Nibedita Mohapatra, Jyotipragnaya Majhi, Harapriya Mangaraj, Pragati Priyambada Pattanaik, Soumya Ranjan Sahu, Puspita Behera and Laxmipriya Mohanta for their help in performing some of the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePP\u003c/strong\u003e: Study design and administration, fund acquisition, data analysis and validation, manuscript editing and review. \u003cstrong\u003eBD\u003c/strong\u003e: Study design, investigation, data acquisition and processing, manuscript writing, and preparation of figures. \u003cstrong\u003eSPP\u003c/strong\u003e: Manuscript writing and reviewing, data validation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors have no conflicts of interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data used in this manuscript are included in the manuscript itself. If the reader needs any specific information it can be availed from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbadin H, Ashizawa A, Stevens Y-W, et al (2007) Toxicological Profile for Lead. Agency for Toxic Substances and Disease Registry (US), Atlanta (GA)\u003c/li\u003e\n\u003cli\u003eAdebiyi O, Adigun K, David-Odewumi P, et al (2022) Gallic and ascorbic acids supplementation alleviate cognitive deficits and neuropathological damage exerted by cadmium chloride in Wistar rats. Sci Rep 12:14426. https://doi.org/10.1038/s41598-022-18432-0\u003c/li\u003e\n\u003cli\u003eAhmed MB, Ahmed MI, Meki A-R (2013) Neurotoxic Effect of Lead on Rats : Relationship to Apoptosis. 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Accessed 24 Mar 2025a\u003c/li\u003e\n\u003cli\u003eThe IUCN Red List of Threatened Species. In: IUCN Red List of Threatened Species. https://www.iucnredlist.org/en. Accessed 19 Jun 2024b\u003c/li\u003e\n\u003cli\u003ePsammophilus blanfordanus. The Reptile Database. https://reptile-database.reptarium.cz/species.php?genus=Psammophilus\u0026amp;species=blanfordanus. Accessed 19 Jun 2024c\u003c/li\u003e\n\u003cli\u003eKolmogorov-Smirnov Calculator (Test of Normality). https://www.socscistatistics.com/tests/kolmogorov/default.aspx. Accessed 25 Mar 2025d\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antioxidant, heavy metals, lipid peroxidation, catalase, liver, reptiles","lastPublishedDoi":"10.21203/rs.3.rs-7512613/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7512613/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeavy metals cause environmental pollution, and profoundly impact biological systems. This study investigates the toxic effects of cadmium (Cd) and lead (Pb) on the antioxidant defense system in Rock lizard, \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e. The \u003cem\u003eP. blanfordanus\u003c/em\u003e has a common occurrence in rocky and mining rich areas of Chota-Nagpur and Eastern India, exposed to heavy metals that are released as a process of weathering. Lizards were orally gavaged with CdCl₂ or Pb (NO₃)₂, to mimic the natural exposure condition and evaluated at 24, 48, and 72-hour intervals with respect to control. Results indicated that Cd-exposure significantly decreased liver protein content and catalase activity while increasing lipid peroxidation. Similarly, Pb-exposure led to significant reductions in catalase and ascorbic acid levels, with an initial increase followed by a decrease in superoxide dismutase and glutathione (GSH) levels over time. These findings underscore the oxidative stress and cellular damage induced by Cd and Pb, reflecting their detrimental impacts on reptilian physiology. The study also highlights the potential of \u003cem\u003ePsammophilus blanfordanus\u003c/em\u003e as an indicator for monitoring environmental/heavy metal pollution in areas with mineral mining. Our study also emphasizes the need for stringent controls on heavy metal pollution in future to mitigate their ecological and health consequences.\u003c/p\u003e","manuscriptTitle":"Effect of lead and cadmium exposure on antioxidant defences in rock lizard Psammophilus blanfordanus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 15:57:31","doi":"10.21203/rs.3.rs-7512613/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5a0fc17b-6f6c-462c-9bf2-89b815ae6fd1","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-15T15:20:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-09 15:57:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7512613","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7512613","identity":"rs-7512613","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}