Testicular Toxicity of the Concurrent Administration of Cadmium And Arsenic Through The Food Chain

Theresa Ezedom^1* and Samuel Ogheneovo Asagba^{2 1}Department of Medical Biochemistry, Delta State University, PMB 1, Abraka, Nigeria
²Department of Biochemistry, Delta State University, PMB 1, Abraka, Nigeria
^*Correspondence: Theresa Ezedom, Department of Medical Biochemistry, Delta State University, PMB 1, Abraka, Nigeria, Email:

Received: Mar 08, 2022, Manuscript No. JEBMH-22-50711; Editor assigned: Mar 11, 2022, Pre QC No. JEBMH-22-50711 (PQ); Reviewed: Mar 25, 2022, QC No. JEBMH-22-50711; Revised: Mar 30, 2022, Manuscript No. JEBMH-22-50711 (R); Published: Apr 05, 2022, DOI: 10.18410/jebmh/2022/09.07.40

Citation: Theresa Ezedom, Samuel Ogheneovo Asagba. Testicular toxicity of the concurrent administration of cadmium and arsenic through the food chain. J Evid Based Med Healthc 2022;9(7):40.

Keywords

Bcl, Bax, Testes, Cadmium, Arsenic

Introduction

Several studies on the decline in male reproductive ability as a result of environmental toxicants have been reported. Of the different contaminants present in the environment, Cadmium (Cd) and Arsenic (As) are among the top list. Cd is an environmental contaminant gotten primarily from mining, plastics manufacturing, paint pigments, electroplating, alloy preparation, and batteries. For the non-smoking and non-occupationally exposed population, food is the most important source of exposure to Cd. It can exert both carcinogenic and non-carcinogenic effects on various tissues such as the lung, bone, liver, kidney etc. Cd has also been found to have adverse effects on the endocrine and reproductive systems and exerts its toxicity through several biochemical mechanisms associated with oxidative stress and cell death. Arsenic is a well - known environmental pollutant whose exposure could occur through drinking As-contaminated water. Other important sources of exposure to this chemical is through food and inhalation.^1-7 Arsenic is a carcinogen which is capable of causing tumors in a variety of tissues including the skin, liver, kidney, lung, prostate etc. It has also been shown to be capable of inducing oxidative stress and cell death. Cell death could occur through apoptosis, which is characterized by loss of plasma membrane phospholipids asymmetry, enzymatic cleavage of the DNA, condensation of nuclear chromatin and cell segmentation into apoptotic bodies. The main regulators of this process are the Bcl - 2 family proteins. These help in the suppression of cell death. Bax - like subgroup such as Bax, Bak, Bok and Bik has been reported to promote cell death. Although the exact mechanism through which Bcl - 2 family proteins regulate the apoptotic pathway has not been fully elucidated, it has been shown to be dependent on protein-protein interactions. This protein-protein interaction could be induced by mixtures of chemicals in the environment. According to a mixture is defined as the combination of two or more environmental agents. The toxicological actions of components in a mixture could be described using the concept of additively and interaction (which could be synergism or antagonism). In order to describe the combined action of the components in the mixture of metals, the most common approach utilized is to carry out experimental studies to compare the effects of the individual components to the effects of the mixture. However, a study on the possible effects of metals and mixture of metals when provided through the food-chain is scarce in literature. The importance of the food-chain as one of the major sources of human exposure to metals and the increasing rate of male infertility underscore the need for the present study. The aims of the study were to evaluate total protein concentration, antioxidant status as well as expression of Bax and Bcl - 2 genes of the testes of rats exposed to Cd and As (singly and in mixture) through a controlled food-chain and to evaluate the comparative effect of both metals when administered singly and in combination through a controlled food-chain.^8-15

Materials and Methods

Catfish (first trophic level) were obtained from a local fish pond located in Imoje-Orogun, Delta State, Nigeria. Exposure of the fishes to the metals was done using plastic troughs for a period of 1 month. The fishes were divided into four groups. Group A which served as the control had fish kept in fresh water. In Groups B, C and D, fishes were kept in water contaminated with a nominal concentration of 0.4 mg of metal / 100 mL of water using Cd, as and Cd + As, respectively. The concentration of metal to which the fishes were exposed to is in accordance with previous studies. The water, in which the fishes were kept, was changed and re-contaminated every 24 hours. During the 1 month exposure, fishes were provided with normal commercial feed before being sacrificed, dried and used as a source of protein in compounding the experimental diet (Table 1 and 2). The Cd and as contents of these diets were determined by atomic absorption spectrophotometry.^16-21

Treatment of animals

The sixty-four (64) adult male albino rats with average weight of 126.25 ± 3.59 g which were used for the study were gotten from the Animal house of College of Health Sciences, Delta State University, Abraka, Nigeria. The rats were acclimatized for 1 week, given a 12 h light and dark cycle with free access to normal rat chow pellet and water. Thereafter, the animals were categorized into 4 experimental groups with 16 rats per group. Groups A served as the control. Rats in Group B were exposed to the Cd contaminated diet while rats in Groups C and D were exposed to the As and Cd + As compounded diets, respectively. After exposure to the experimental diet for 1 month, half the number of rats in each group were sacrificed and the other half after 3 months of exposure. The animals were sacrificed under chloroform anesthesia and their testes were excised and used immediately for biochemical analysis. The use of rat models in the present study was subject to approval by the ethical committee of the College of Health Sciences, Delta State University, Abraka, Nigeria. All sections of this report adhere to the arrive Guidelines for reporting animal research.^22-25

Metal Analysis

Weighed samples of the feed and testes of experimental rats of each group were digested separately in beakers with 20 ml of concentrated acid mixture (98 % w/ v HNO₃ / HClO₄, 4:1 v / v) at 100 °C. After digestion of samples, the Cd and as concentrations in the tissues and feeds were measured using a Varian AA 1475 spectrophotometer. An International Atomic Energy Agency (IAEA) reference biological sample was used for the evaluation of the accuracy and precision of the analysis.²⁶

Biochemical assays

The testes of the rats were weighed, homogenized, centrifuged and the supernatants obtained were used for the determination of the activities of superoxide dismutase (SOD), Catalase (CAT), Glutathione Transferase (GST) based on the methods the levels of reduced glutathione (GSH), total protein and lipid peroxidation were determined using the methods respectively.²⁷

Preparation of guanidine isothiocyanate (GITC) lysate

Weighed samples of testes from rats in each group and for both durations of study were pounded aseptically using mortar and pestle under cold conditions and then transferred into 2 mL eppendorf tube. GITC solution was activated and the GITC lysate prepared based on the method. The lystate were stored at – 16 °C prior to use.²⁸

Isolation and purification of RNA from GITC lysate

Total RNA of the GITC lysate of testes were extracted using Reliaprep RNA Kit, a product of Promega Corporation Madison, Wisconsin, USA. The integrity and purity of RNA obtained was electrophoretically verified by formaldehyde agarose gel stained with ethidium bromide based on the method of Lehrach (1977).²⁹

cDNA Synthesis Protocol

In a reverse transcription reaction mixture containing 1 x PCR buffer, 0.5 mMdeoxy-nucleoside triphosphates (dNTPs), 1 unit of RNase inhibitor, 2.5 μM of olio d (T) 16, and 2.5 units of MuLV reverse transcriptase (Perkin-Elmer, Roche Molecular Systems, Branchburg, New Jersey, USA). One microgram 1 μg of RNA was reverse transcribed into cDNA. This was incubated for 10 mins at room temperature to allow primer annealing, then, the reaction mixture was incubated at 42 °C for 15 min, heated to 95 °C for 5 min, and chilled at 4 °C for 5 min in a GeneAmp thermal cycler (Applied Biosystems, Foster City, CA, USA). Two microliters 2 μL of the resultant cDNA products was used for PCR amplification.³⁰

Real time quantitative RT-PCR

Light cycler 2.0 system (Roche Applied Systems, USA) was used for the Real time quantitative RT-PCR to analysis the expression levels of Bax and Bcl-2 gene relative to glyceraldehyde - 3- phosphate dehydrogenase (GAPDH). The real-time quantitative PCR probe design software (Roche Applied Systems, Branchburg, New Jersey, USA) were used to design primer sets for GAPDH, Bax and Bcl-2. PCR reactions for these primers were first optimised using conventional PCR.³¹

For the quantitative Real-Time PCR, 20 μL amplification mixtures (Light Cycler Fast start DNA MasterPLUS SYBR Green Reaction Mix; Roche Applied Science) were prepared as per manufacturer’s instructions, containing cDNA (equivalent to 100 mg reverse transcribed RNA) and 0.5 μM of each primer. The cycling conditions were: 10 min polymerase activation at 95 °C and 40 cycles at 95 °C for 15 s, 58 °C for 15 s, and 72 °C for 15 s. After thermal cycling, the gel was run with 3 % agarose gel (that is, 3 g of agarose powder in 100 mL of TBE buffer). On cooling, 20 μL EtBr was added. Electrophoresis was run at 100 V for 30 min, watching the controls.³²

Statistical Analysis

All the data are expressed as mean ± standard deviation (SD). Statistical comparisons were performed by one way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) comparison when ANOVA results indicates statistically significant difference between groups. The SPSS software (version 20) was used in the statistical analysis using multiple comparison tests. A p - value of less than 0.05 (p < 0.05) was considered significant.³³

Results

The results of metal analyses carried out on the feed and testes of rats are shown in (Table 3). There were trace amount of the metals in the control most especially for as the level of Cd was below detection limit. The test groups also had slight contamination of these metals as evident by the mole ratio obtained. The results obtained further showed that the metals accumulated in the testes of rats after both period of exposure.

Results are expressed as Mean ± SD. Values not sharing same superscript in same column differs significantly at (P < 0.05). A, Control; B, Cd - contaminated diet; C, as contaminated diet, Cd + As contaminated diet (Table 4). Presents the changes in the body weight and testes / body weight ratio of rats used in the present study for the experimental period of 1 and 3 months. There was a significant (p < 0.05) reduction in the body weights of rats in all test groups relative to controls after both periods of study. The weight loss in the 3 months exposure was higher than that of the 1 month exposure and this effect was more severe in rats fed a combination of both metals in diet. There was a significant (p < 0.05) decrease in the testes/body weight ratio of rats fed with Cd-contaminated diet and Cd + As - contaminated diet relative to control after 1 month exposure. Similarly, the testes/body weight ratio of rats exposed to Cd and as singly or in combined form in diet was significantly decreased relative to control. Again, in both durations of exposure, the effect of the metals on testes / body weight ratio was more severe when combined in diet. The effects of the treatment on total protein and antioxidant status of the testes are shown in (Table 5).

The levels of total protein and malondialdehyde (MDA) (an index of lipid peroxidation) were significantly (p < 0.05) increased when compared to the control for both periods of exposure. After 1 Month exposure, there was a significant (p < 0.05) increase in the level of GSH and activities of SOD, CAT and GST but after 3 months exposure, these were significantly (p < 0.05) decreased.

Results are expressed as Mean ± SD. n = 16. Values not sharing same superscript in same column differ at (P < 0.05).

Results are expressed as Mean ± SD. Values not sharing same superscript in same column differs significantly at (P < 0.05). A, Control; B, Cd-contaminated diet; C, As-contaminated diet; D, Cd + As contaminated diet. The changes in the expressions of Bax And Bcl - 2 genes mRNA in the testes of rats exposed to Cd and As contaminated diet for both periods of exposure are shown in (Figure 1). There was a significant (p < 0.05) increase in the level of Bax gene mRNA expression for the Cd and Cd + As contaminated group when compared to the control after 1 month of exposure. However, there was a significant (p < 0.05) increase in the mRNA expression of Bax gene in the testes for all test groups when compared to the control after 3 months exposure. The expression of Bcl - 2 genes mRNA was found to be down regulated when compared to the control for all groups. In the group exposed to As contaminated diet, this reduction was not significant (p < 0.05) after 1 month exposure when compared to the control. However, after 3 months exposure, there was a significant (p < 0.05) decrease in the level of Bcl - 2 mRNA for all test group.

Discussion

Humans are exposed to Cadmium (Cd) and Arsenic (As) at low concentrations either voluntarily through supplementation or involuntarily through intake of contaminated food and water or contact with contaminated soil, dust, or air. These metals could have adverse impacts on male reproductive health. Metal analysis on the compounded experimental feed showed trace contamination of the control and the test groups. The mole ratio clearly indicated the trace presence of as in the Cd-contaminated diet and the trace presence of Cd in the As-contaminated diet. It was observed in the present study, that the control testes also had some very minimal level of contamination of the metals. This could be attributed to the pervasiveness of Cd and as in the general environment. The present study revealed a reduction in the body weight and testes/body weight ratio of rats exposed to the experimental diets for both periods of exposure. The severe effect on body weight gain and testes / body weight ratio of rats observed after 3 months exposure could be attributed to toxicity of the metals due to their increased accumulation relative to 1 month exposure. Again, the findings also indicated that combination of the metals in Group D had a more pronounced effect on the weight gain of rats. This is in accordance with the report of who showed that both As and Cd decreased body weight gain and food utilization with a more pronounced effects for the mixture. It has been shown that testicular germ cells are susceptible to oxidative damage by free radicals due to the presence of polyunsaturated fatty acids in the plasma membrane. This leads to the excessive generation of ROS which could outweigh the cell's antioxidant defense system. Thus, markers of oxidative stress and antioxidant enzymes were analyzed in the testes of experimental rats. After 1 month exposure, the activities of the antioxidant enzymes (SOD and CAT), and GST, a phase 1 drug metabolizing enzyme were all increased in the testes by the Cd and as (singly and in mixture) contaminated die. This is apparently traceable to the low level of oral dose of the metals in the diet as well as the short duration of exposure. This is in consonance with the study of where an induction of SOD and CAT activities were recorded following exposure to Cd and Cd and As, respectively at low dose. Increase in the activities of the antioxidant enzymes could also be attributed to the ability of the testes to tolerate the metal stress have also reported increase in oxidative enzymes in testes of rats after 1 month exposure of rats to Cd and As contaminated diets. The reduction in the activities of these enzymes after 3 months exposure may imply enzyme inactivation caused by excess reactive oxygen species (ROS) formation, displacement of essential cofactor such as Zn or Cu or binding to thiol groups of the enzyme. The current study also showed that exposure to Cd and As caused a significant (p < 0.05) increase in membrane lipid peroxidation (LPO) in the testes of exposed rats for both durations of study which is an indication of oxidative stress and could lead to intra and inter-molecular cross-links in proteins and nucleic acids occasioned by LPO products such as MDA. In the present study, significant (p < 0.05) increase in GSH levels in the testes was observed after 1 month exposure however, at the end of 3 months exposure, there was a significant (p < 0.05) decrease in the level of GSH. The increase in tissue GSH content after 1 month exposure might be attributed to the system trying to mop up free radicals generated by the metals in these organs. High level of GSH as observed after 1 month exposure could protect against cell death. The decreased GSH level observed after 3 months exposure could be due to the oxidation of GSH by free radicals or as a result of depletion of the sulfhydryl group of cysteine moiety in GSH due to its high affinity for Cd and As forming Cd / As-GSH complex or its electron donor ability. A reduction in the level of GSH, might initiate the onset of cytotoxicity. After 1 month exposure through the food-chain, non-significant (p < 0.05) increase in the level of Bax gene mRNA in the testes of rats was recorded but down regulated for Bcl - 2. After 3 months exposure, Bcl - 2 was significantly (p < 0.05) down regulated in all test groups while Bax was significantly (p < 0.05) up-regulated in all test groups. Interactions between Bcl - 2 and Bax regulate cytochrome c release from mitochondria and establish baseline sensitivity to apoptotic stimuli. Induction of apoptosis has been utilized for the treatment of cancer, but cancer cells have developed different strategies to resist death by apoptosis. One of such strategy is an increase in the expression of anti-apoptotic Bcl - 2 family proteins. Upon binding to Bax, Bcl - 2 can prevent pore formation and cytochrome c release. On the other hand, increase in the expression of Bax can induce cell death and is utilized in eliminating tumor cells. Based on different reports showing a reduction in the expression of Bax and a corresponding increase in the expression of Bcl - 2 in different drug-resistant tumor cells, it could be hypothesized that induction of mitochondrial apoptosis pathway by Cd and As in the testes of rats through the food chain might be mediated through the Bcl - 2 and Bax proteins. The disruption of the blood-testis barrier (BTB) could be responsible for the sensitivity of the testes to Cd and as toxicity. Rodent testes under normal conditions have higher levels of metallothionein (MT) than other organs (liver and kidney excluded), therefore, the high susceptibility of the testes to Cd toxicity could possibly be related to genetic background. MT is a metal binding protein that protects cells from toxicity of metals by binding to them. Environmentally, humans and animals are exposed to the combinations of various risk elements. The present study thus took account of the effects of long-term exposure to low levels of these elements through the food chain. Very few information is available on the interactive effects of as and Cd upon ingestion have shown using sub-chronic dietary studies in rats that neither metal significantly affected accumulation of the other in kidney, liver or brain tissue found that both As and Cd increased RBC count and decreased haematocrit; responses to the mixture were less than additive. One of the most striking findings from this study was the pronounced effect of the diet containing a combination of Cd and As compared to diets containing the individual metals on most of the parameters studied which is evident in the percentage changes from the controls. These findings may have arisen from the additive effect and/or synergism of the two metals on the parameters studied. Thus, the result of the present study is in consonance with the observation of that showed that As (as arsenite) and Cd were more lethal to rats when administered as a mixture than when injected alone. In the studies of each metal lowered the LD50 for the other, and the two metals at single fixed doses caused greater than additive lethality as compared to either alone.³⁵

Conclusion

In conclusion, this study has provided evidence that consumption of food contaminated with Cd and as through the food-chain could be a contributory factor to reproductive disorders. The results of the present study showed that Cd and As accumulated through the food chain can potentially affect negatively the weight gain and testes / body weight ratioof rats most especially as the duration of exposure increases with the mixture of metals having greater effect than either of the metals alone. Exposure to the metalsalso altered the activities of antioxidant enzymes leading to increased level of LPO by ROS, induced histological changes and altered the normal ratio of the mRNA expression of Bax and Bcl – 2 genes in the testes of exposed rats and these effects could lead to loss of reproductive functions. The up regulation of the mRNA expression of Bax and down regulation of Bcl – 2 gene expressions after both periods of exposure could be hypothesized as mediating induction of apoptotic pathway leading to cell death and consequently, loss of cell function. The findings of the study also revealed that the combined metals had a more pronounced biochemical and histological effects on the testes of the exposed rats than the individual metals especially in the three months exposed rats.

References

1. Anyanwu BO, Ezejiofor AN, Igweze ZN, et al. Heavy Metal Mixture Exposure and Effects in Developing Nations: An Update. Toxics 6(65).

   [Cross Ref][Google Scholar][Indexed At]

2. Beutler E, Duron O, Kelly BM. Improved method for determination of blood glutathione. J Lab Clin Med 1963;61:882-888.

   [Indexed At]

3. Casalino E, Calzaretti G, Sblano C et al. Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium. Toxicol 2002;179:37-50.

   [Cross Ref][Google Scholar][Indexed At]

4. Chaudhari RT, Harshad RK, Thorat SR. Effect of cadmium chloride on the biochemical charecteristics of fresh water fish, Cyprinuscarpio. Int J Manag Soc Sci Res Rev 2015;3:85-89.

   [Google Scholar]

5. Chomczynski P, Sacchi N. single step method of RNA isolation by acid guanidiniumthiocyanate–phenol–chloroform extraction. Anal Biochem 1987;162:156-159.

   [Cross Ref][Google Scholar][Indexed At]

6. Choudhury H, Mudipalli A. Potential considerations and concerns in the risk characterization for the interaction profiles of metals. Ind J Med Res 2008;128:462-483.

   [Google Scholar][Indexed At]

7. Christian EI, Olatoye TR, Bamidele FS, et al. Cadmium-induced testicular toxicity, oxidative stress and histopathology in Wistar rats: Sustained effects of polyphenol-rich extract of Vernoniaamygdalina leaf J Interdiscip Histopathol 2016;4(3):54-62.

8. Ezedom T, Asagba S, Tonukari NJ. Toxicological effects of the concurrent administration of cadmium and arsenic through the food chain on the liver and kidney of rats. J Basic Appl Zool 2020;81:1-12.

   [Google Scholar]

9. Ezedom T, Asagba SO. Effect of a controlled food-chain mediated exposure to cadmium and arsenic on oxidative enzymes in the tissues of rats. Toxicol Rep.2016;3:08–715.

   [Cross Ref][Google Scholar][Indexed At]

10. Giaccio L, Cicchella D, De Vivo B, et al. Does heavy metals pollution affects semen quality in men? A case of study in the metropolitan area of Naples (Italy). J Geochem Explor 2012;112;218–225.

    [Cross Ref][Google Scholar]

11. Gornall A, Bardsmill C T, David M M. Determination of serum protein by means of biuret reaction. J Biol Chem 1949;177: 751-766.

    [Google Scholar][Indexed At]

12. Gutteridge JMC, Wilkins C. Copper dependent hydroxyl radical damage to ascorbic acid. Formation of thiobarbituric acid reactive products. FEBS Lett 1982;137:327-340.

    [Cross Ref][Google Scholar][Indexed At]

13. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974; 249:7130-7139.

    [Cross Ref][Google Scholar][Indexed At]

14. Horiguchi H, Sato M, Konno N, et al. Long-term cadmium exposure induces anemia in rats through hypoinduction of erythropoietin in the kidneys. Arch Toxicol 1996;71:11–9.

    [Cross Ref][Google Scholar][Indexed At]

15. Khan MA, Khan S, Khan A, et al. Soil contamination with cadmium, consequences and remediation using organic amendments. Sci Total Environ 2017;601–602.

    [Cross Ref][Google Scholar][Indexed At] 

16. Kiklugawa K, Kosugi H, Asakura T. Effect of MDA a product of lipid peroxidation on the function and stability of haemoglobin. Arch Biochem Biophys. 1984;7:229-232.

    [Cross Ref][Google Scholar][Indexed At]

17. Kilkenny C, Browne WJ, Cuthill IC, et al. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoSBiol 2010;8: e1000412.

    [Cross Ref][Google Scholar][Indexed At]

18. Kumar KMK, Kumar NM, Nagesh R, et al. Arsenic induces oxidative stress and apoptosis in human lung cancer cells. Int J of Life Sci 2017;5(4): 551-560.

19. Lehrach H, Diamond D, Wozney JM, et al. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical re-examination. Biochem 1977;16:4743–51.

    [Cross Ref][Google Scholar][Indexed At]

20. Leibowitz B, Yu J. Mitochondrial signaling in cell death via the Bcl-2 family. Cancer BiolTher 2010;9(6):417–22.

    [Cross Ref][Google Scholar][Indexed At]

21. Mahaffey KR, Capar SG, Gladen BC, et al. Concurrent exposure to lead, cadmium, and arsenic. Effects on toxicity and tissue metal concentrations in the rat. J Lab Clin Med 1981;98:463-81.

    [Cross Ref][Google Scholar][Indexed At]

22. Mandal P. An insight of environmental contamination of arsenic on animal health. Emerging Contaminants 2017;3(10) 17-22.

    [Cross Ref][Google Scholar]

23. Misra HP, Fridovich I. The role of superoxide ion in auto-oxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972;247: 3170-3175.

    [Google Scholar][Indexed At]

24. Moazamian R, Polhemus A, Connaughton H, et al. Oxidative stress and human spermatozoa: Diagnostic and functional significance of aldehydes generated as a result of lipid peroxidation. MHR Basic Sci Reprod Med 2015;21: 502–515.

    [Cross Ref][Google Scholar][Indexed At]

25. Nachmias B, Ashhab Y, Ben Yehuda D. The inhibitor of apoptosis protein family (IAPs): an emerging therapeutic target in cancer. Semin Cancer Biol.2004;14(4):231–43.

    [Cross Ref][Google Scholar][Indexed At]

26. Naseri MH, Mahdavi M, Davoodi J, et al. Up regulation of Bax and down regulation of Bcl2 during 3-NC mediated apoptosis in human. Cancer Cell Int 2015;15:55.

    [Cross Ref][Google Scholar][Indexed At]DOI 10.1186/ s12935-015-0204-2.

27. Nna VU, Bakar ABA, Ahmad A, et al. Oxidative Stress, NF-κB-Mediated Inflammation and Apoptosis in the Testes of Streptozotocin–Induced Diabetic Rats: Combined Protective Effects of Malaysian Propolis and Metformin. Antioxidants 2019;8(10):465. 

    [Cross Ref][Google Scholar][Indexed At]  

28. Pavlova E, Atanassova N. Impact of Cadmium on Male Fertility. Acta morphologica et anthropologica 2018;25:1-2.

    [Google Scholar]

29. Radabaugh TR, Aposhian HV. Enzymatic reduction of arsenic compounds in mammalian systems: reduction of arsenate to arsenite by human liver arsenate reductase Chem Res Toxico;13:26-30.

    [Cross Ref][Google Scholar][Indexed At]

30. Rana SVS. Protection of Metal Toxicity by Melatonin-Recent Advances. EC Pharmacol Toxicol 2018;6(9): 851-864.

31. Schmolke G, Elsenhans B, Ehtechami C, et al. Arsenic-copper interaction in the kidney of the rat. Hum Exp Toxicol 1992;11:315-21.

    [Cross Ref][Google Scholar][Indexed At]

32. Sexton K, Hattis D. Assessing cumulative health risks from exposure to environmental mixtures—three fundamental questions. Environ Health Perspect 2007;115:825-832.

    [Cross Ref][Google Scholar][Indexed At]

33. Sinha AK. Colorimetric assay of catalase. Anal Biochem. 1972;47:389-394.

    [Cross Ref][Google Scholar][Indexed At]

34. Sukhn C, Awwad J, Ghantous A, et al. Associations of semen quality with non-essential heavy metals in blood and seminal fluid: data from the Environment and Male Infertility (EMI) study in Lebanon. J Assist Reprod Genet 2018;35:1691–1701.

    [Cross Ref][Google Scholar][Indexed At]

35. Zhang FQ, Wang YS, Lou ZP, et al. Effect of heavy metal stress on antioxidative enzymes and lipid peroxidation in leaves and roots of two mangrove plant seedlings (Kandeliacandel and Bruguieragymnorrhiza). Chemosphere 2007;67(1):44-50.

    [Cross Ref][Google Scholar][Indexed At]