|Year : 2017 | Volume
| Issue : 1 | Page : 27-38
Modulatory role of Saccharomyces cerevisiae against cadmium-induced genotoxicity in mice
Omaima M Abd-El-Moneim1, Heba A.M Abd El-Kader1, Abeer H Abd El-Rahim1, Hasnaa A Radwan1, M Fadel2, Ibrahim M Farag1
1 Department of Cell Biology, National Research Centre, Dokki, Cairo, Egypt
2 Department of Microbial Chemistry, National Research Centre, Dokki, Cairo, Egypt
|Date of Submission||23-Feb-2017|
|Date of Acceptance||21-Mar-2017|
|Date of Web Publication||22-Jun-2017|
Ibrahim M Farag
Department of Cell Biology, National Research Centre, Dokki, Giza, 12311
Source of Support: None, Conflict of Interest: None
Background/aim Cadmium (Cd) was found to be a major heavy metal utilized in different agricultural and industrial processes. However, exposure to this element was found to cause serious environmental pollution and induce deleterious effects on human and animal health. Therefore, this study was conducted to estimate the protective and therapeutic role of Saccharomyces cerevisiae against Cd genotoxicity.
Materials and methods Forty-eight male mice were used in this study by dividing them into six groups (eight animals each): the control group, which was fed a basal diet; a group fed a basal diet contaminated with Cd (0.03 g/kg diet) for 3 weeks; two protection groups fed a basal diet supplemented with S. cerevisiae at low (5%) or high (7.5%) levels along with contamination with Cd; and two therapeutic groups fed a basal diet supplemented with S. cerevisiae at the same levels for 15 days after cessation of contamination with Cd. At the end of the experimental period, genetic and sperm parameters of the mice were evaluated. Genetic parameters included the DNA comet assay, random amplified polymorphic DNA-PCR analysis, micronucleus test, and chromosomal examinations. Sperm parameters involved sperm-shape abnormalities and sperm count.
Results The results showed that feeding on a basal diet contaminated with Cd caused significant increases in abnormal genetic parameters and sperm-shape abnormalities as well as significant decrease in sperm count. The utilization of S. cerevisiae yeast as a protective or therapeutic agent significantly improved the genetic and sperm parameters as compared with Cd treatment alone. The best findings were revealed when S. cerevisiae was used as a protective agent, especially treatment at high levels (7.5%).
Conclusion This work showed that S. cerevisiae yeast is a strong probiotic agent against Cd detoxification in foods, where the addition of this probiotic to animal diets significantly minimized the deleterious effects caused by Cd on the genetic and sperm parameters of mice.
Keywords: Cadmium, comet assay, cytogenetics, mice, random amplified polymorphic dna-pcr, saccharomyces cerevisiae, sperm
|How to cite this article:|
Abd-El-Moneim OM, Abd El-Kader HA, Abd El-Rahim AH, Radwan HA, Fadel M, Farag IM. Modulatory role of Saccharomyces cerevisiae against cadmium-induced genotoxicity in mice. J Arab Soc Med Res 2017;12:27-38
|How to cite this URL:|
Abd-El-Moneim OM, Abd El-Kader HA, Abd El-Rahim AH, Radwan HA, Fadel M, Farag IM. Modulatory role of Saccharomyces cerevisiae against cadmium-induced genotoxicity in mice. J Arab Soc Med Res [serial online] 2017 [cited 2020 Oct 25];12:27-38. Available from: http://www.new.asmr.eg.net/text.asp?2017/12/1/27/208863
| Introduction|| |
Cadmium (Cd) is considered a major heavy metal that is extensively used in agriculture and industry: for example, in the application of pesticides and phosphate fertilizers, in plastic and pigment production, in paints, electroplating, soldering, and in the manufacture of alloys and batteries ,. Cd was found to be an extremely toxic metal causing serious environmental pollution ,,.
Humans and animals might be exposed daily to Cd through fumes, dust, smoking, and polluted food and water ,,. This metal was revealed to accumulate mostly in the liver and kidney, although its toxicity can extend to other organs such the lungs, the bladder, the brain, bone, and the reproductive system, causing deleterious effects and a variety of diseases such as cancer, genotoxicity, reproductive disorders and infertility, arteriosclerosis, immunotoxicity, and anemia ,.
The toxicity of Cd might be due to its metabolites in the liver leading to the generation of reactive oxygen species (ROS) that include hydrogen peroxide and superoxide anion ,,,. ROS can attack cellular biomolecules in several organisms resulting in deleterious oxidation of protein, formation of lipid peroxidation, reduction of antioxidant parameters, DNA damage, chromosomal aberrations, alterations of gene expression, and apoptosis ,. Cd ions were found to have the ability to pass through the blood–testes barrier, inducing testis toxicity ,. Testis toxicity includes spermatogenesis aberrations, inhibition of chromatin condensation of sperm, fragmentation of sperm DNA, decrease in sperm motility and viability, and abnormalities in sperm shape ,. Also, ROS generated through exposure to Cd can affect the sperm structure and function by inducing peroxidation of membrane lipids, as well as by decreasing each cell membrane phosphatide and levels of ATP . Thus, contamination with Cd was found to have multiple deleterious effects on human and animal health. Therefore, control and prevention of toxic effects of this metal has become essential. Interventions focusing on probiotics that lead to inactivation or elimination of the bioavailability of Cd ions in contaminated products show promise in controlling the impact of diet-polluted mutagens.
The yeast of Saccharomyces cerevisiae is considered one of the major probiotics (microorganisms that are believed to provide health benefits when consumed). This yeast was for many centuries used in food production, and recently it has been utilized in pharmaceutical industries as well as in the production of certain substances that have beneficial properties in humans and animals, such selenium, zinc, glutathione, and cysteine ,,. Glutathione, cysteine, zinc, and selenium were reported to have antioxidant substances that could scavenge free radicals and peroxynitrite; they were also found to be strong hepatoprotective agents against many toxicants . Probiotics were found to stimulate the detoxification process by removing or eliminating toxin traces without leaving deleterious residues and without impairing the quality of nutrition of the commodity ,. Moreover, S. cerevisiae was found to be a potent protective agent against genotoxicity induced by exposure to aflatoxin (AF) in somatic and germ cells of mice, where the rate of cytogenetic changes and sperm abnormalities was significantly minimized in the animals exposed to AF and treated with S. cerevisiae as compared with those exposed only to AF .
Further, a previous study by Eshak et al.  revealed that the addition of S. cerevisiae yeast to quail feed that was contaminated with AF led to better growth performance and improvement in the expression levels of neural and gonadal genes as compared with AF diet alone.
Furthermore, the components of S. cerevisiae, such as β-glucan and mannan oligosaccharides, were considered to be strong antioxidant, antimutagenic, and anticarcinogenic agents, wherein treatment with these components led to a reduction in DNA damage caused by cancer development of human leukocytes , and minimized the proportion of micronuclei induced by cyclophosphamide in mice . Moreover, the β-glucan treatment was found to improve the quality of life of animals exposed to radiation, as β-glucan was observed to have bioaction and antioxidant activity due to its being capable of bioantimutagenic action, scavenging of free radicals, and exerting desmutagenic action . On the other hand, S. cerevisiae yeast and its components were observed to have the ability to adsorb and bind with toxicants and consequently protect the live organisms and their genetic constitutions from deleterious effects .
The present work aimed to assess the protective and therapeutic role of S. cerevisiae yeast against genotoxicity induced by Cd in mice. DNA comet assay, random amplified polymorphic DNA (RAPD)-PCR analysis, cytogenetic tests, and sperm examinations were performed to elucidate the study purpose.
| Materials and methods|| |
Cadmium chloride (CdCl2) in powder form was purchased from Sigma–Aldrich. CdCl2 was added and mixed well to the basal diet at 0.03 g/kg diet. This dose of CdCl2 has been considered an appropriate experimental dose that affects mouse organs ,. All experimental procedures involving animals were conducted in accordance to the ethical guidelines of the medical ethical committee of the National Research Centre in Egypt and approved by medical ethical committee of the National Research Centre in Egypt.
Yeast of Saccharomyces cerevisiae
The yeast of S. cerevisiae was obtained from Microbial Chemistry Department, National Research Centre, Egypt. Two levels, 5 and 7.5%/kg, of S. cerevisiae were added to the basal diet. These levels of S. cerevisiae were mixed well and fed according to the procedure detailed in the study by Parlat et al. . CdCl2 was incorporated into the basal diets before S. cerevisiae addition.
Albino mice of Swiss strain weighing 25–30 g were obtained from the Animal House, National Research Centre, Giza, Egypt. The animals were housed at an ambient temperature of 25±3.2°C under a light/dark cycle of 12/12 h. All mice were kept in clean polypropylene cages and administered food and water ad libitum.
The mice were divided into six equal groups of eight animals each. Group 1 was fed a normal basal diet (control group). Group 2 was fed a basal diet containing CdCl2 at 0.03 g/Kg diet for 3 weeks (cadmium group). The third and fourth groups were fed a basal diet containing S. cerevisiae at low (5%) (protection 1) or high (7.5%) (protection 2) levels along with treatment with CdCl2. The fifth and sixth groups were fed a basal diet containing S. cerevisiae at low (5%) (therapeutic 1) or high (7%) (therapeutic 2) levels for 2 weeks after cessation of CdCl2 treatment. Groups 3 and 4 were used to evaluate the protective role of S. cerevisiae against the potential mutagenic effects of CdCl2. Groups 5 and 6 were used to evaluate the therapeutic effect of S. cerevisiae against the potential mutagenic effects of CdCl2. At the end of the experiment, the mice were killed by cervical dislocation for studying their molecular genetics (using the DNA comet assay and RAPD-PCR), cytogenetics (micronuclei and chromosome aberrations), and sperm profile (sperm shape and count).
DNA comet assay (single-cell gel electrophoresis)
DNA damage was measured according to the method of Singh et al. . The DNA fragment migration patterns of 100 cells of liver tissue for each treatment, including those of controls, were evaluated with a fluorescence microscope [with excitation filter 420–490 nm (issue 510 nm)]. The comet tail lengths were measured from the middle of the nucleus to the end of the tail at ×40 magnification to determine the count and size of the comet. For visualization of DNA damage, EtBr-stained DNA was observed at ×40 magnification using a fluorescence microscope. Comet 5 image analysis software developed by Kinetic Imaging Ltd. (Liverpool, UK) linked to a charge-coupled device camera was used to assess the quantitative and qualitative extent of DNA damage in the cells by measuring the length of DNA migration and the percentage of migrated DNA. The program also calculates tail moment. Generally, 100 randomly selected cells were analyzed per sample.
Random amplified polymorphic DNA-PCR analysis
DNA extraction using the salting-out method
DNA was extracted from liver tissue of mice according to the method described by Miller et al.  and Sambrook et al. .
Random amplified polymorphic DNA-PCR amplification
Three commercial primers with size 10 bases and variable nucleotide proportions were used for the amplification process. The primers (supplied by Operon, Almeda, California, USA) were OPC05 (5′GATGACCGCC3′), OPA04 (5′AATCGGGCTG3′), and OPA06 (5′GGTCCCTGAC3′). The PCR protocol for RAPD analysis was as described by Williams et al. . Approximately 3 μl of the amplified DNA product and 2 μl of 1×loading dye were loaded on a 2% agarose gel and then subjected to electrophoresis in 1×tris/borate/EDTA buffer and stained with ethidium bromide (0.5 μl/ml) for verification. A Bio-Rad (USA) XR+molecular imager apparatus was used to visualize the PCR products. The ladder DNA (100 pb) (Fermentas; Thermo fisher scientific, USA) and LabImage were used for determination of the molecular size of the bands.
Random amplified polymorphic DNA profiles and data analysis
Quantitative estimation of DNA samples was done by a double-beam UV-spectrophotometer (UV-2450; Shimadzu, Japan). The DNA concentration was measured at 260 and 280 nm, respectively. The integrity of extracted genomic DNA was checked by electrophoresis in 0.8% agarose gel using a DNA molecular weight marker (Fermentas). The marked changes in RAPD profile such as disappearance and/or appearance of bands in comparison with untreated control treatments (b/a bands) were evaluated .
Bone marrow slides were prepared and stained according to the method described by Krishna and Hayashi .
For chromosomal analysis both treated and control animals were killed, femurs were removed, and bone marrow cells were aspirated using saline solution. Metaphase spreads were prepared using the method of Preston et al. .
For sperm-shape analysis, epididymal sperms were prepared and stained with Eosin Y (aqueous), as per the methods described by Wyrobek and Bruce  and Farag et al. . At least 3000 sperms per group were assessed for morphological abnormalities. The sperm abnormalities were evaluated according to the standard method described by Narayana . Epididymal sperm count was also determined by means of a hemocytometer as described by Pant and Srivastava .
Statistical analysis was performed with SPSS software. Data were analyzed using one-way analysis of variance followed by Duncan’s post-hoc test for comparison between different treatments. Results were reported as mean±SE. The polymorphism values were determined according to the method of Taşpınar et al. .
| Results|| |
The results of the DNA comet assay are shown in [Table 1] and [Figure 1]. These findings showed damaged parameters of genomic DNA that included DNA tailed percentage, DNA untailed percentage, DNA tail length, tail DNA percentage, and tail moment. The present results revealed that feeding on a basal diet polluted with Cd had induced significant increases in DNA tailed percentage, DNA tail length, tail DNA percentage, and tail moment, whereas there was significant decrease in DNA untailed percentage as compared with the control group. In contrast, the addition of S. cerevisiae yeast to the basal diet along with (as a protective agent) or after cessation (as a therapeutic agent) of Cd treatment caused significant reduction in DNA tailed percentage, DNA tail length, tail DNA percentage, and tail moment, and significant increase in DNA untailed percentage compared with treatment with Cd alone. The results indicated that the use of a high level of S. cerevisiae yeast was more pronounced for improvement of DNA structure than the low level of yeast. Moreover, the best improvement in DNA structure was obtained when S. cerevisiae yeast was used as a protective agent than when it was used as a therapeutic agent.
|Table 1 Parameters of DNA damage in the comet assay in mice treated with Saccharomyces cerevisiae yeast as a protective or a therapeutic agent against cadmium toxicity|
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|Figure 1 Comet assay prepared of mice liver showing varying extent of DNA damage. These mice were treated with Saccharomyces cerevisiae yeast as a protective or a therapeutic agent against contamination with cadmium. [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6], represent control, Cd, protection 1, protection 2, therapeutic 1 and therapeutic 2, respectively.|
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Random amplified polymorphic DNA-PCR
The products of RAPD-PCR are recorded in [Table 2] and illustrated in [Figure 2]. The obtained results from three primers OPC05, OPA04, and OPA06 displayed a potent PCR amplification with distinct band profiles. In protection treatment, the total number of amplified bands was 97. These bands were observed in mice fed a basal diet, a basal diet contaminated with Cd, and a basal diet contaminated with Cd and supplemented with low or high levels of S. cerevisiae yeast. The molecular sizes of these bands ranged from 1664 to 208 (for OPC05 primer), from 2091 to 244 (for OPA04 primer), and from 1680 to 298 (for OPC06 primer). The present results showed that 56 bands (57%) out of 97 were polymorphic. The polymorphic bands in the RAPD profile occurred as a result of the appearance (gain) or disappearance (loss) of the amplified bands with respect to those found in the control RAPD profile. The OPC04 primer recorded the highest value (45 bands) of amplified bands including polymorphic ones. The high degree of genetic polymorphism (82.1) was observed in the mouse group fed the basal diet contaminated with Cd. In contrast, the rate of inducing the genetic polymorphism in genomic DNA was decreased to 60.7 or 57.1% in mice fed the basal diet contaminated with Cd and treated with low or high levels of S. cerevisiae yeast, respectively. The supplementation of Cd diet with high level of S. cerevisiae yeast caused more reduction in the degree of genetic polymorphism than did treatment with a low level of yeast.
|Table 2 Control DNA bands and total polymorphic bands of genomic DNA from random amplified polymorphic DNA-PCR of mice treated with Saccharomyces cerevisiae yeast as protective or a therapeutic agent against cadmium toxicity|
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|Figure 2 Control DNA bands and total polymorphic bands were shown in the random amplified polymorphic DNA-PCR profile. These bands were generated by OPA04 (a), OPC05 (b), and OPA06 (c) of genomic DNA extracted from mouse liver. M, ladder DNA, 100 pb. Lane 1: control; lane 2, cadmium; lane 3, protection 1; lane 4, protection 2; lane 5, therapeutic 1; lane 6, therapeutic 2.|
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In therapeutic treatment, the total number of amplified bands was 115. These bands were seen in mice fed the basal diet, the basal diet contaminated with Cd, and the basal diet supplemented with low or high levels of S. cerevisiae yeast after cessation of Cd treatment. The molecular sizes of amplified bands ranged from 1664 to 219 (for OPC05 primer), from 2091 to 244 (for OPA04 primer), and from 1707 to 298 (for OPA06 primer). The present findings revealed that 53 bands (61%) out of a total of 115 were polymorphic. The OPA06 gave the highest number (40 bands) of amplified bands including amplified polymorphic ones. The degree of genetic polymorphism in the Cd group was 81%; however, this genetic polymorphism decreased to 67.8% in mice fed the basal diet supplemented with low or high levels of S. cerevisiae yeast after cessation of Cd treatment. Thus, the present findings showed that using S. cerevisiae yeast as a protective agent gave the best results than when using it as a therapeutic agent against Cd toxicity, where the degrees of polymorphisms were lower in protection treatment (Cd+S. cerevisiae1 at 5% level and Cd+S. cerevisiae2 at 7.5% level) than in therapeutic treatments (Cd followed by S. cerevisiae1 at 5% level and Cd followed by S. cerevisiae2 at 7.5% level).
The present results obtained in [Table 3] revealed that feeding of a basal diet contaminated with Cd induced highly significant rates of micronuclei as compared with feeding of on a normal basal diet. However, the supplementation of S. cerevisiae yeast to the basal diet along (as a protective agent) or after cessation (as a therapeutic agent) of Cd treatment led to significant reduction in micronuclei compared with the Cd diet alone. The use of a high level of S. cerevisiae yeast was more effective for decreasing micronuclei than the use of a low level of yeast. Better results were observed by using S. cerevisiae yeast as a protective agent than by using S. cerevisiae yeast as a therapeutic agent, as the rate of inducing micronuclei in protection treatment was lower than that in therapeutic treatment.
|Table 3 The proportions of micronuclei in mice treated with Saccharomyces cerevisiae yeast as a protective or therapeutic agent against pollution with cadmium|
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The results presented in [Table 4] show that mice fed the basal diet contaminated with Cd had highly significant chromosomal aberrations in comparison with mice fed the normal basal diet. In contrast, chromosomal aberrations were significantly decreased in mice fed basal diets that were supplemented with S. cerevisiae yeast along with (as a protective agent) or after cessation (as a therapeutic agent) of Cd contamination as compared with those found in mice fed the Cd diet alone. Supplementation with a high level of S. cerevisiae yeast in the Cd diet resulted in the lowest rate of chromosomal aberrations compared with addition of a low level of yeast. Further, the frequencies of chromosomal aberrations were more minimized in mice fed a diet supplemented with S. cerevisiae yeast along with Cd treatment than those observed in mice fed a diet supplemented with S. cerevisiae yeast after cessation of Cd treatment.
|Table 4 Rates of chromosome aberrations in mice treated with Saccharomyces cerevisiae yeast as a protective or therapeutic agent against pollution with cadmium|
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Sperm abnormalities and count
The sperm examination in the present study revealed head and tail abnormalities. The sperm head abnormalities included amorphous, no hock, small head, big head, and banana types. The tail aberrations involved coiled and divided ones. The present findings found that sperm abnormalities were more frequent in mice fed the basal diet contaminated with Cd compared with those found in the control. Further, these results revealed that Cd treatment significantly decreased the sperm count as compared with that in control. The addition of S. cerevisiae yeast to the Cd diet as a protective or a therapeutic agent significantly decreased the sperm abnormalities and significantly increased the sperm count compared with feeding a basal diet contaminated with Cd alone. The use of a high level of S. cerevisiae yeast resulted in minimization of the sperm-shape abnormalities and improvement in the sperm count as compared with low level of yeast. In all cases, the supplementation of S. cerevisiae yeast as a protective agent gave much better results by reducing the sperm abnormalities and improving the sperm count compared with addition of S. cerevisiae yeast as a therapeutic agent ([Table 5]).
|Table 5 Sperm shape abnormalities and sperm count in mice treated with Saccharomyces cerevisiae yeast as protective or therapeutic agent against pollution with cadmium|
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| Discussion|| |
In the present study, the rates of induced DNA damage significantly increased in mice fed a diet contaminated with Cd as compared with those found in controls. These observations were detected by means of the DNA comet assay and RAPD-PCR analysis. The results of the comet assay showed significant elevation of DNA tailed percentage, DNA tail length, tail DNA percentage, and tail moment and significant decrease in DNA untailed percentage. Our findings were similar to those reported by Karimi et al. , who, by using the comet assay, observed significant alterations in DNA (including DNA in the head and DNA in the tail) in mouse kidney cells exposed to Cd as compared with control mice. Also, in an in-vitro study, Skipper et al.  found by using the comet assay significant elevation of tail lengths of DNA comets in liver carcinoma cells of humans treated with Cd compared with those in controls.
The RAPD-PCR analysis revealed that treatment with Cd caused significant elevation of RAPD profile changes, where the rate of induced genetic polymorphisms (the appearance of new bands and absence or loss of existing bands) of DNA amplified bands was higher than that of controls. Also, similar results were observed in fish  and plants ,. Mahrous et al.  reported higher genetic polymorphisms in the genomic DNA of the liver of Tilapia fish treated with Cd compared with those found in the control. Moreover, high modifications of genomic DNA were observed in each of viciafaba  and Arabidopsis plantlet shoots  that were treated with Cd as compared with the controls.
The obtained results of DNA comet assay showed distinct variation in the size of the nuclear DNA lesion induced by Cd treatment. This damage to DNA reveals the specific genotoxic effect of Cd on the nuclear DNA leading to increase in DNA fragmentation migration (comet tail) from the nucleus (comet head) ,,. The genotoxicity induced by Cd might be because this metal is metabolized inside liver cells, causing decreased capacity of antioxidant agents and generation of ROS that interact with DNA molecules resulting in oxidative DNA damage ,. Also, the increase in ROS rates due to genotoxic stress leads to many types of DNA damages, such as breaks in single-stranded or double-stranded DNA, insertion or deletion of nitrogenous base pairs, alkylation or oxidation of nitrogenous base pairs, and inhibition of mismatch repair of DNA adduct ,,. Moreover, the presence of DNA damage in the comet assay might be due to inducing forks of DNA replication during the S-phase under stress of genotoxicity of toxicants leading to high loss of DNA molecules (as comet tails) .
The RAPD profile of Cd-treated mice showed characteristic changes in the banding patterns of DNA inducing high rates of genetic polymorphism as compared with control. These genetic DNA polymorphisms might be caused by DNA damage due to Cd genotoxicity especially during DNA replication or during the process of gene expression ,. Thus, the gain (or new appearance) or loss (or disappearance) of the RAPD profile bands of DNA by Cd treatment might be related to changes in sequences, such as inducing point mutation, deletion or addition (insertion) of nitrogenous base sequences, or complex genomic rearrangements that influence the sites of primer binding ,,. The gain or appearance of new RAPD profile bands might be related to structural changes in the DNA, such as large addition (insertion) of nitrogenous base sequences or transpositions or point mutation that makes available some new sites of nucleotide sequences to bind or interact with primers ,. In contrast, the loss or disappearance of some DNA bands of RAPD profile might be due to DNA polymerase interaction with changes in nucleotide sequences (DNA damage), nucleotide sequence alterations at the sites of primer binding, single-strand and double-strand breaks of DNA, and complex genomic rearrangements ,. Therefore, this study proves that gain or disappearance of DNA bands of the RAPD profile is considered a good biomarker for identification of genotoxicity caused by contamination with Cd.
The obtained results showed that treatment with Cd induced significant increases in the frequencies of cytogenetic changes that included micronuclei and chromosomal aberrations as compared with controls. Similarly, Fahmy and colleagues ,, reported significant elevations in induced micronuclei and chromosomal aberrations in murine bone marrow cells exposed to Cd as compared with those found in the control. Also, Mahrous et al.  observed highly significant frequencies of micronuclei and different types of chromosomal aberrations in Nile tilapia fish treated with Cd compared with controls. Moreover, Singh and Sankhla  revealed significant reduction in mitotic activity and significant elevation in chromosomal aberrations in bone marrow cells of mice administered Cd in comparison with controls. El-Refaiy and Eissa  found marked chromosomal aberrations with highly significant frequencies in rats that received Cd, when compared with the untreated group. The high frequencies of cytogenetic changes that were observed by Cd treatment in the present study might be due to incorporation of Cd ions into the DNA structure causing random DNA fragmentations or DNA strand breaks that lead to the formation of micronuclei or induce chromosomal aberrations . Several studies reported that Cd ions have strong toxic properties and affect the formation of mitotic spindle fibers, causing anomalies in mitotic division and inducing high rates of micronuclei and different kinds of chromosomal aberrations ,.
Moreover, the exposure to various kinds of toxicants, including Cd, causes generation of ROS that interact with different biomolecule cellular components, especially nucleic acids, lipids, and proteins, leading to disturbance of homeostasis, oxidative stress, and consequently cytogenetic changes such as reducted mitotic  activity, increased micronuclei formation, and increased chromosomal aberrations. Furthermore, Cd ions were found to have toxic effect on the enzymes responsible for DNA repair, leading to DNA damage and resulting in micronuclei and chromosomal aberration ,. Thus, the Cd genotoxicity in the present work might be related to the incorporation of Cd ions into DNA or due to the toxic effect of such ions on mitotic spindle and enzymes responsible for DNA repair, or due to the formation of ROS that cause DNA lesions leading to increased constitution of micronuclei and chromosomal aberrations.
Concerning the sperm examination in the present study, the results clarified that Cd treatment caused significant increase in sperm-shape abnormalities and significant decrease in viable epididymides sperm count. These findings were in concordance with those of previous studies by Oliveira et al. , Kaur and Sharma , and Metwally and Hashem , who observed significant increase in sperm-shape abnormalities and significant decrease in sperm count in mice exposed to Cd as compared with those found in the control groups. Dietrich et al.  and Annabi et al.  revealed that treatment with Cd could affect calcium ions (which were found to be a necessary element for sperm activities), leading to sperm abnormalities. Hew et al.  and Dietrich et al.  reported that Cd ions can cross the blood–testes barrier, inducing suppression of spermatogenesis; further, these ions can bend with sperm enzymes, affecting sperm cell metabolism and leading to sperm deformation. Moreover, Hew et al.  detected that Cd ions could alter the adhesion of Sertoli cells More Details by disrupting cell junctions and subsequently cause degeneration of seminiferous tubules and lead to a reduction of epididymides sperm count. On the other hand, Acharya et al.  clarified that ROS generated by genotoxicity of Cd can peroxide the fatty acids in spermatozoa, causing lipid peroxidation and deleterious effects on cell membrane phosphatides as well as inducing sperm DNA oxidation and consequently sperm-shape abnormalities and decrease in viable sperm count.
The present findings identified that the use of S. cerevisiae yeast as a protective or a therapeutic agent could suppress the genotoxic and spermatotoxic effects caused by Cd in mouse tissues. The inducing of tail lengths of DNA comets, genetic DNA polymorphism of the RAPD-PCR profile, rate of micronuclei, frequencies of chromosomal aberrations, and proportion of sperm-shape abnormalities were significantly minimized as well as the sperm count significantly ameliorated in S. cerevisiae groups (Cd+S. cerevisiae or Cd then S. cerevisiae) as compared with the Cd group alone. Better results were obtained on using S. cerevisiae as a protective agent. Thus, our results revealed that the use of S. cerevisiae as a natural product might have protective effects on genetic materials and sperm parameters against Cd genotoxicity. Supporting this, Darwish et al.  and Abdel-Aziz et al.  observed that the supplementation of S. cerevisiae to mouse diets contaminated with mycotoxin [AF or Ochratoxin (OTA)] significantly decreased the chromosomal aberrations (in somatic and germ cells), micronuclei, and sperm-shape abnormalities as well as significantly increased mitotic and meiotic activities and sperm count in comparison with mycotoxin (AF of OTA) diet alone. Eshak et al.  found that the addition of S. cerevisiae yeast to quail diet contaminated with AFB1 (aflatoxins B1) significantly enhanced growth performances and expression level of neural and gonadal genes compared with AFB1 diet alone. Oliveira et al.  demonstrated that the utilization of β-glucan (which is a major constituent in the cell wall polysaccharide of S. cerevisiae yeast) in the culture of Chinese hamster ovary cell lines exposed to methylmethanesulfonate led to significant decreases in DNA damage (comet assay) and rate of micronuclei formation compared with methylmethanesulfonate culture alone. Also, Oliveira et al.  found that β-glucan (isolated from S. cerevisiae yeast) reduced the DNA damage (comet assay) and micronuclei caused by cyclophosphamide in mice. In a previous study, Silva et al.  observed that β-glucan had an anticlastogenic effect on cultured cells that were exposed to ultraviolet radiation.
In human patients with advanced prostate cancer, treatment with β-glucan was found to be a strong protective agent against the development of DNA lesions . Moreover, polysaccharides of S. cerevisiae yeast were found to be a potent bioprotective factor in cucumber, tobacco, and bean plants against infection from viruses of necrosis and tobacco mosaic ,. Therefore, S. cerevisiae yeast is considered a strong antigenotoxic agent. This property might be related to yeast polysaccharides that include β-d glucans, glucomannans, and mannanoligo − saccharide , or some constituents that were found in S. cerevisiae cells, such as vitamins, carotenoids, essential amino acids, and minerals ,,,. These components were revealed to have the potent antioxidant activities that suppress the reaction of free radicals of the oxidative process or disable and scavenge ROS species, causing reduction of DNA oxidation and consequently minimization or prevention of genotoxicity ,. Furthermore, Tsa1 (CTPX1), which is a major constituent of peroxiredoxins in S. cerevisiae yeast, was observed to possess a strong ability to scavenge free radicals, leading to avoidance of several mutation processes and causing stability of genomic DNA . In contrast, Abousadi et al.  and Parlat et al.  reported that the cells of S. cerevisiae yeast can produce some biological enzymes that have the ability to detoxify by interacting with toxicant molecules and subsequently increasing the efficiency of gut microflora and enhancing the bioavailability of nutrients during the digestion process in the intestine, leading to prevention or minimization of genotoxicity. Eshak et al.  demonstrated that the action of S. cerevisiae yeast (or its components) may be related to its suppression of the activation of cytochrome enzymes, resulting in improvement in liver and kidney functions and leading to mitigation of genetic abnormalities.
Peteri et al.  observed that S. cerevisiae yeast or its components had the ability to biodegrade carcinogens such as OTA by cleaving the amide bond, releasing OTA, and leading to adsorption of such toxicants into viable or nonviable yeast cells. Also, Rahaie et al.  reported that because of the physical properties of S. cerevisiae yeast cells, these cells can bind and adsorb with high levels of carcinogens such as AFB1; the rate of such binding reached 70–95% (w/w) in animal diets.
In conclusion, this work adds evidence that S. cerevisiae yeast is a strong probiotic that can attenuate the toxic effects of Cd contamination in food. The addition of this probiotic to animal diets significantly minimized the deleterious effects induced by Cd on the genetic and sperm parameters of mice.
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Conflicts of interest
There are no conflict of interest.
| References|| |
Waisberg M, Joseph P, Hale B, Beyersmann D. Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 2003; 192:95–117.
Oliveira H, Spanò M, Santos C, Pereira MdL. Adverse effects of cadmium exposure on mouse sperm. Reprod Toxicol 2009; 28:550–555.
Topal A, Alak G, Atamanalp M, Oruç E, Ceyhun SB, Uçar A et al.
Effects of humic acid on liver and kidney toxicity induced by cadmium in Brown Trout (Salmotruttafario L
). Turk J Fish Aquat Sci 2013; 13:621–627.
Kaur S, Sharma S. Evaluation of toxic effect of cadmium on sperm count, sperm motility and sperm abnormality in albino mice. Int J Adv Res 2015; 3:335–343.
Skipper A, Sims JN, Yedjou CG, Tchounwou PB. Cadmium chloride induces DNA damage and apoptosis of human liver carcinoma cells via oxidative stress. International Journal of Environmental Research. Int J Environ Res Public Health 2016; 13:1–10.
Jarup L, Berglund M, Elinder C, Nordberg G, Vahteram M. Health effects of cadmium exposure − a review of the literature and a risk estimate. Scand J Work Environ Health 1998; 1:1–52.
Kasuya M, Teranishi H, Aoshima K, Katoh T, Horiguchi H, Morikawa Y. Water pollution by cadmium and the onset of Itai-itai disease. Water Sci Technol 2000; 25:149–156.
Amoruso MA, Goldstein BD. Enhancement of rat and human phagocyte superoxide anion radical production by cadmium in vitro. Toxicol Lett 1982; 10:133–138.
Ochi T, Otsuka F, Takahashi K, Shawa MO. Glutathione and metallothioneins as cellular defense against cadmium toxicity in culture Chinese hamster cells. Chem Biol Interact 1998; 65:1–14.
Karimi MM, Sani MJ, Mahmudabadi AZ, Sani AJ, Khatibi SR. Effect of acute toxicity of cadmium in mice kidney cells. Iran J Toxicol 2012; 6:691–698.
Singh P, Sankhla V. In situ protective effect of curcumin on cadmium chloride induced genotoxicity in bone marrow chromosomes of Swiss albino mice. J Cell Mol Biol 2010; 8:57–64.
Asadpour R, Shahbazfar D, Kianifard D, Azari M, Zaboli N. Comparison of the protective effects of garlic (Allium sativum L
) extract, vitamin E and N acetyl cystein on testis structure and sperm quality in rats treated with lead acetate. Rev Méd Vét 2013; 164:27–33.
Monsefi M, Alaee S, Moradshahi A, Rohani L. Cadmium-induced infertility in male mice. Environ Toxicol 2009; 25:94–102.
Wathes DC, Abayasekara DR, Aitken RJ. Polyunsaturated fatty acids in male and female reproduction. Biol Reprod 2007; 77:190–201.
Kim HS, Fay JC. Genetic variation in the cysteine biosynthesis pathway causes sensitivity to pharmacological compounds. Proc Natl Acad Sci USA 2007; 104:19387–19391.
Agbor GA, Vinson JA, Patel S, Patel K, Scarpati J, Shiner D et al.
Effect of selenium- and glutathione-enriched yeast supplementation on a combined atherosclerosis and diabetes hamster model. J Agric Food Chem 2007; 55:8731–8736.
Lai JT, Fang HL, Hsieh WT, Lin WC. Protective effect of a fermented substance from Saccharomyces cerevisiae
on liver injury in mice caused by acetaminophen. Biochem Biosci Biotechnol 2008; 72:2514–2520.
Abousadi AM, Rowghani E, Honarmand ME. The efficacy of various additives to reduce the toxicity of aflatoxin B1 in broiler chicks. Iran J Vet Res 2007; 8:144–150
Tovar-Ramirez D, Mazurais D, Gatesoupe JF, Quazuguel P, Cahu CL, Zambonino-Infante JL. Dietary probiotic live yeast modulates antioxidant enzyme activities and gene expression of sea bass (Dicentrachus labrax
) larvae. Aquaculture 2010; 300:142–147.
Darwish HR, Abdel-Aziz KB, Farag IM, Nada SA, Amra H, Tawfek NS. Ameliorative effect of Saccharomyces cerevisiae
on aflatoxin-induced genotoxicity and spermatotoxicity in male albino mice. Researcher 2011; 3:38–45.
Eshak MG, Khalil WKB, Hegazy EM, Farag IM, Fadel M, Stino FKR. Effect of yeast (Saccharomyces cerevisiae
) on reduction of aflatoxicosis, enhancement of growth performance and expression of neural and gonadal genes in Japanese quail. J Am Sci 2010; 6:824–838.
Zimmerman JW, Lindermuth J, Fish PA, Palace GP, Stevenson TT, DeMong DE. A novel carbohydrate-glycosphingolipid interaction between a β-(1-3)-glucanimmunomodulator, PGG-glucan, and lactosylceramide of human leukocytes. J Biol Chem 1998; 273:22014–22020.
Turnbull JL, Patchen ML, Scadden DT. The polysaccharide, PGG-glucan, enhances human myelopoiesis by direct action independent of and additive to early-acting cytokines. Acta Haematol 1999; 102:66–71.
Chorvatovicova D, Navarova J. Suppressing effects of glucan on micronuclei induced by cyclophosphamide in mice. Mutat Res 1992; 282:147–150.
Silva AF, Oliveira RJ, Niwa AM, D’Epiro GFR, Ribeiro LR, Mantovani MS. Anticlastogenic effect of glucan, extracted from Saccharomyces cerevisiae
, on cultured cells exposed to ultraviolet radiation. Cytotechnology 2012; 65:41–48.
Rahaie S, Emam-Djomeh Z, Razavi1 S, Mazaheri M. Immobilized Saccharomyces cerevisiae
as a potential aflatoxin decontaminating agent in pistachio nuts. Braz J Microbiol 2010; 41:82–90.
OECD. Guideline for testing of chemicals, No. 420, acute oral toxicity–fixed dose method. Paris, France: Organization for Economic Co-operation and Development; 2001.
El-Refaiy AI, Eissa FI. Protective effects of ascorbic acid and zinc against cadmium-induced histopathological, histochemical and cytogenetic changes in rats. Comunicata Scientiae 2012; 3:162–180.
Parlat SS, Ozcan MH, Oguz H. Biological suppression of aflatoxicosis in Japanese quail (Coturnix coturnix japonica
) by dietary addition of yeast (Saccharomyces cerevisiae
). Res Vet Sci 2001; 71:207–211.
Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175:184–191.
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res 1988; 16:1215.
Sambrook L, Fritsch EF, Manitatis T. Molecular cloning: a laboratory manual cold spring. New York, USA: Harbor Press, Cold Spring Harbor; 1989.
Williams JKG, Kubelik AR, Livak KJ, Rafalsky JA, Tyngey SV. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 1990; 18:6531–6535.
El-Alfy NZ, Mahmoud MF, Abdullah AM. Cytogenetical and molecular studies to evaluate the cytotoxic effects of mitomycin C on male Albino mice. Glob J Pharmacol 2014; 8:532–541.
Krishna G, Hayashi M. In vivo rodent micronucleus assay: protocol, conduct and data interpretation. Mutat Res 2000; 455:155–166.
Preston RJ, Dean BJ, Galloway AF, Mcfee S. Mammalian in vivo cytogenetic assay − analysis of chromosomal aberration in bone marrow cells mutation. Mutat Res 1987; 189:157–165.
Wyrobek AJ, Bruce WR. The induction of sperm shape abnormalities in mice and humans. Chem Mutagens 1978; 5:237–285.
Farag IM, Abdou HSA, Ayesh AM, Osfr MMH. Chromosomal and sperm studies on the mutagenic effect of over heated meat and the protective role of green tea and gingeng on rats. Al-Azhar Bull Sci 2002; 13:105–120.
Narayana KJ. An aminoglycoside antibiotic gentamycin induces oxidative stress, reduces antioxidant reserve and impairs spermatogenesis in rats. J Toxicol Sci 2008; 33:85–96.
Pant N, Srivastava SP. Testicular and spermatotoxic effect of quinaphos in rats. J Appl Toxicol 2003; 23:271–274.
Taşpınar MS, Agar G, Yıldırım N, Sunar S, Aksakal O, Bozari S. Evaluation of selenium effect on cadmium genotoxicity in Viciafaba using RAPD. J Food Agric Environ 2009; 7:857–860.
Mahrous KF, Hassan AM, Radwan HA, Mahmoud MA. Inhibition of cadmium induced genotoxicity and histopathological changes in Nile tilapia fish by Egyptian and Tunisian montmorillonite clay. Ecotoxicol Environ Saf 2015; 119:140–147.
Liu W, Sun L, Zhong M, Zhou Q, Gong Z, Li P et al.
Cadmium-induced DNA damage and mutations in Arabidopsis plantlet shoots identified by DNA fingerprinting. Chemosphere 2012; 89:1048–1055.
Traore A, Ruiz S, Baudrimont I, Sanni A, Dano SD, Guarigues P et al.
Combined effects of okadaic acid and cadmium on lipid peroxidation and DNA bases modifications (m5dC and 8-(OH)-dG) in Caco-2 cells. Arch Toxicol 2000; 74:79–84.
Pourahmad J, O’Brien PJ, Jokar F, Daraei B. Carcinogenimetal induced sites of reactive oxygen species formation in hepatocytes. Toxicol In Vitro 2003; 17:803–810.
Casalino E, Casalino G, Calzaretti CS. Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium. Toxicology 2002; 179:37–50.
Abdelhalium E, Al-Huqail AA. Detection of protein and DNA damage induced by elevated carbon dioxide and ozone in Triticum aestivum L
., using biomarker and comet assay. Genet Mol Res 2016; 15:15028736.
Liu W, Yang YS, Li PJ, Zhou QX, Xie LJ, Han YP. Risk assessment of cadmium-contaminated soil on plant DNA damage using RAPD and physiological indices. J Hazard Mater 2009; 161:878–883.
Liu W, Zhou QX, Li PJ, Gao HR, Han YP, Li XJ. DNA mismatch repair related gene expression as potential biomarkers to assess cadmium exposure in Arabidopsis seedlings. J Hazard Mater 2009b; 167:1007–1013.
Atienzar FA, Jha AN. The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review. Mutat Res 2006; 613:76–102.
Cambier S, Gonzalez P, Durrieu G, Bourdineaud JP. Cadmium-induced genotoxicity in zebrafish at environmentally relevant doses. Ecotoxicol Environ Saf 2010; 73:312–319.
Fahmy MA, Aly FA. In vivo and in vitro studies on the genotoxicity of cadmium chloride in mice. J Appl Toxicol 2000; 20:231–238.
Celik A, Comelekoglu U, Yalin S. A study on the investigation of cadmium chloride genotoxicity in rat bone marrow using micronucleus test and chromosome aberration analysis. Toxicol Ind Health 2005; 21:243–248.
El-Habit OH, Abdel Moneim AE. Testing the genotoxicity, cytotoxicity and oxidative stress of cadmium and nickel and their additive effect in male mice. Biol Trace Elem Res 2014; 159:364–372.
Yang JM, Arnush M, Chenc QY, Wu XD, Pang B, Jiang XZ. Cadmium-induced damage to primary cultures of rat Leydig cells. Reprod Toxicol 2003; 17:553–560.
Natarajan A, Duivenvoorden W, Meijers M, Zaynesburg T. Induction of mitotic aneuploidy using Chinese hamster primary embryonic cells. Test results of 10 chemicals. Mutat Res 1993; 2874:7–56.
Theocharis S, Margeli A, Skopelitou A, Skaltsas S, Tsiakiri I, Mykoniatis M. Biochemical and histopathological changes after partial hepatectomy in rats. Iatrika Chronika IE 1992; 10:746–754.
Metwally MM, Hashem MA. Protective role of garlic against cadmium toxicity in rats clinico pathological and histopathological studies. Egypt J Comp Pathol Clin Pathol 2009; 22:114–140.
Dietrich GJ, Dietrich M, Kowalski RK, Dobosz S, Karol H, Demianowicz W, Glogowski J. Exposure of rainbow trout milt to mercury and cadmium alters sperm motility parameters and reproductive success. Aquat Toxicol 2010; 97:277–284.
Annabi A, Saïd K, Messaoudi I. Cadmium: toxic effects and physiological impairments in fishes. Int J Adv Res 2013; 1:372–382.
Hew KW, Heath GL, Jiwa AH, Welsh MJ. Cadmium in vivo causes disruption of tight junctions-associated microfilaments in rat Sertoli cells. Biol Reprod 1993; 49:840–849.
Dietrich MA, Żmijewski D, Karol H, Hejmej A, Bilińska B, Jurecka P et al.
Isolation and characterization of transferrin from common carp (Cyprinus carpio L
) seminal plasma. Fish Shelfish Immunol 2010; 29:66–74.
Acharya UR, Mishra M, Patro J, Panda MK. Effect of vitamins C and E on spermatogenesis in mice exposed to cadmium. Reprod Toxicol 2008; 25:84–88.
Abdel-Aziz KB, Farag IM, Tawfek NS, Nada SA, Amra HA, Darwish HR. Saccharomyces cereviciae
ameliorates oxidative stress, genotoxicity and spermatotoxic effects induced by Ochratoxin A in male Albino Mice. N Y Sci 2010; 3:177–190.
Oliveira RJ, Salles MJ, da Silva AF, Kanno TY, Lourenço AC, LeiteVda S et al.
In vivo evaluation of the antimutagenic and antigenotoxic effects of β-glucan extracted from Saccharomyces cerevisiae
in acute treatment with multiple doses. Genet Mol Biol 2013; 36:413–424.
Magnani M, Castro-Gomez RJH, Mori MP, Kyasne H, Gregório EP, Libos F Jr, Cólus IMS. Protective effect of carboxymethyl-glucan (CM-G) against DNA damage in patients with advanced prostate cancer. Genet Mol Biol 2011; 34:131–135.
Slovakova L, Subıkova V, Sandula J. Induction of antiviral resistance in plants with yeast polysaccharides. In: Blahutiak EŽ, editor. Works of the Institute of Experimental Phytopathology and Entomology. Bratislava: Vesna; 1993. pp. 69–77.
Kogan G, Machova E, Balisova A, Sandula J. Structure and biological activity of the cell wall polysaccharides of the technologically important yeasts. In: Suzuki S, SuzukiŽ M, editors. Fungal cells in biodefense mechanism. Tokyo, Japan: Saikon Publ; 1996. pp. 209–212.
Yiannikouris A, Andre G, Poughon L, Francois J, Dussap CG, Jeminet G et al.
Chemical and conformational study of the interactions involved in mycotoxincomplexation with D-glucans. Biomacromolecules 2006; 7:1147–1155.
Vetvicka V. B-glucans as immunomodulators. JANA 2001; 3:31–34.
Kogan G, Pajtinka M, Babincova M, Miadokova E, Rauko P, Slamenova D, Korolenko TA. Yeast cell wall polysaccharides as antioxidants and antimutagens: can they fight cancer? Neoplasma 2008; 55:387–393.
Vlckova V, Duhova V, Svidova S, Farkassova A, Kamasova S, Vlcek D et al.
Antigenotoxic potential of glucomannan on four model test systems. Cell Biol Toxicol 2004; 20:325–332.
Park SG, Cha MK, Jeong W, Kim IH. Distinct physiological functions of thiolperoxidase isoenzymes in Saccharomyces cerevisiae
. J Biol Chem 2000; 275:5723–5732.
Peteri Z, Teren J, Vagvolgyi C, Varga J. Ochratoxin degradation and adsorption caused by astaxanthin-producing yeasts. Food Microbiol 2007; 24:205–210.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]