RAPD-PCR Technique Research Articles

The Study of Genetic Affinity of Methicillin Resistance Staphylococcus Aureus Strains Isolated from Cream Pastries and Nasal Isolates at Shiraz Confectionaries

Background: Staphylococcus aureus (S. aureus) is responsible for most cases of food poisoning all around the world. These carriers and manipulated foodstuffs are the main sources of bacteria transmission to ready-to-eat food. This study aims to determine the genetic affinity of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from cream pastries and workers, nasal of Shiraz city confectioneries. Methods: 30 MRSA strains (7 nasal carriers, 23 food isolates) were selected from the bank of bacteria at Shiraz medical faculty. To determine the genetic affinity of the isolates, RAPD-PCR technique was performed using OLP6, OLP11, and OLP13 primers. RAPD-PCR patterns were analyzed using the software GelJ. Results: By using primer OLP6 only 5 RAPD-PCR patterns were produced from DNA ampliqons of creamy pastry isolates and were not enough to compare the genetic affinity of all the isolates. Based on 100% similarity, OLP13 primer produced 20 different patterns with some bands in the range of 1 to 11, and the OLP11 primer produced 22 patterns with some bands from 3 to 11 bands. At closely and possibly genetically related levels, the isolates are categorized into (13-15) and (1-5) clusters. In general, all the isolates are classified into human and food isolates. Conclusions: There was no genetic affinity of MDSA isolates regarding human and food samples; but, a high percentage of close genetic relationship between the isolates increases the possibility of bacteria transfer from humans to pastries and food poisoning.

Genetic relationship between local guinea fowl, quail and chicken using RAPD–PCR technique

Genetic relationship between local guinea fowl, quail and chicken using RAPD–PCR technique

Molecular Investigation Using RAPD-PCR Marker Field Populations of Pectinophora gossypiella Saunders (Lepidoptera: Gelechiidae) Exposed to Some Insecticide

Seasonally the cotton plant cultivated in Egyptian fields is suffering from the cotton Pink Bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) attacking fruits and buds. Insecticide applications reported control failure by the most recommended conventional classes. Monitoring pest DNA changes after sublethal exposure become the new technique to investigate. Then four insecticide treatments were screened by using the molecular marker polymerase chain reaction (PCR) inspection, using four Random amplified polymorphic deoxyribonucleic acid analysis (RAPD-PCR) primers partitioned based on band reproducibility. Result of LC50 was 0.55, 61.1, 69.3, and 0.23 for spinoteram, novaluron, metaflumezone, and dimeuron respectively. The insecticide treatments band of gel produced detected 47 loci ranging from 87 to 63 % polymorphism. The primer efficiency value of PIC =0.361, 0.34, 0.34, 0.355, and 0.262, RP = 5.30, H = 0.473, 0.434, 0.434, 0.462 and 0.31, and MI = 0.10, 0.1386, 0.138, 0.167 and 0.0582 for the same treated samples respectively. Distance and similarity were quantified based on Nei’s and genetic dissimilarity by the UPMGA method then a phylogenetic tree was constructed and grouped the entire genotypes into 2 major clusters and 6 subclusters. The RAPD primers revealed the number of alleles (Na = 0.324, 0.318, 0.318, 0.326, and 0.328), and the effective number of alleles (Ne = 0.571, 0.569, 0.558, 0.574, and 0.5784). The fixation-index (Fst) analysis narrated a very great genetic diversity (Fst = 0.626, 0.684, 0.684, 0.2, and 0.695) exists within the four treated samples respectively. The level of gene flow was (Nm = 0.239, 0.230, 0.230, 0.233, and 0.2187) respectively across the four genotypes studied. Results proved that the RAPD-PCR technique was suitable for distinguishing between insecticide treatments.

Study of some Chemical, Anatomical and Molecular Indicators using RAPD-PCR Technique of Wheat Cultivars in Terms of Irrigation with Saline Water

This field experiment was conducted with the aim of studying the chemical, anatomical and molecular indicators of wheat cultivars under irrigation with levels of salinity stress. The results of the statistical analysis showed a significant difference in the effect of the high salinity concentration of irrigation water on the chemical traits such as the proline content and protein, the lowest average was (10.37 and 25.96)% respectively. It also affected the anatomical traits represented by the number of the stomatal of the upper and lower surfaces of the leaf with the lowest average of (8.11, 6.22) stomata mm-2 respectively, while the cultivar differed significantly, as the Russian cultivar showed superiority in the proline content and protein, while the Mawada cultivar was superior in the relative water content and stomata density on the upper and lower surfaces. The results of the molecular analysis by RAPD-PCR technology showed that the primer UBC1 gave the highest number of band, which amounted to 58 total band, and the primer OPD-02 scored 49 relevance band, including one unique band, while the primer OPS-19 showed the lowest band number, reached 30, including a unique band. It constitutes a genetic fingerprint for these two primers.

Analysis of the Genetic Distance of Several Generations of Barley (Hordeum valulgare L) by RAPD-PCR Technique

Random-amplified-polymorphic-DNA(RAPD) was assayed to detect the genetic variation of 6 barley generations from Iraq. Four primers generated a total of 17 scoreable bands in RAPD analysis) and resolving power, the three polymorphic primers differed (Rp). The use of RAPD marker systems to detect the genetic distance among barley generation was discovered to be beneficial. The RAPD dendrograms indicate a diverse grouping of 6 barely specimens, although we did see that certain groups were identical in several cases. As a result, the RAPD molecular markers reveal two genetic groups in the few specimens examined. Keywords. Barley, Genetic variation, RAPD-PCR.

Genotoxic potentials of some selected heavy metals exposure on Clarias gariepinus (Burchell, 1822) and Oreochromis niloticus (Linnaeus, 1758) using RAPD-PCR technique

Fish health and the aquatic ecosystem are strongly interwoven and interrelated. The aquatic ecosystem receives a range of anthropogenic chemicals which have toxicological or lethal health effects on the aquatic animals. The aim of this study was to determine the level of induced mutation and genomic stability of different sub-lethal doses (25%, 50% and 75% LC50) of heavy metal [Nickel (Ni), Mercury (Hg), Lead (Pb)and Zinc (Zn)] in Clarias gariepinus (Burchell, 1822) andOreochromis niloticus (Linnaeus, 1758) using RAPD-PCR technology over a period of 21 days. Four highly polymorphic RAPD markers were evaluated on isolated DNA from both heavy metal exposed fishes and control fishes after exposure. Observation of RAPD profiles in C. gariepinus revealed more damaging effect as sub lethal doses increased with zinc (a + b = 44 bands) mercury (a + b = 41 bands) nickel (a + b = 37 bands) lead (a + b = 35 bands) than with nickel (a + b = 37 bands) zinc (a + b = 34 bands) lead (a + b = 32 bands) mercury (a + b = 31 bands) for O. niloticus when compare to the control groups. Although, the genomic stability template decreased sub lethal heavy metal doses, higher stability was observed in O. niloticus (GTSPb = 26.32% GTSHg = 18.42% GTSZn = 10.53% GTSNi = 2.63%) than in C. gariepinus (GTSPb = 5.41% GTSNi = 0.0% GTSHg = -10.81% GTSZn = -18.92%). The results obtained showed differential variation in heavy metal induced genetic mutation and genomic stabilty in C. gariepinus and O. niloticus. These observable differences might be due to the physiological structure of the fish species evaluated. This study also confirms that RAPD–PCR technology is a useful tools in detecting the genotoxic effect of heavy metals in aquatic organisms but due to low reproducibility of RAPD results, it is recommended that this technology should be used along with other molecular techniques in fish genotoxicity studies.Keywords: Heavy metals, Induced, Genotoxicity, Fish, RAPD-PCR Technique

Genetic Variation in Peach Fruit Fly, Bactrocera zonata, Females Irradiated with Gamma Radiation as Revealed by RAPD-PCR Technique

Bactrocera zonata (Saunders) is a destructive polyphagous insect that infests various fruits and vegetables worldwide. Nowadays, radiation technology is widely used as a safe tool for controlling it. Therefore, this study was designed to explore the effect of gamma radiation on adult females of B. zonata. Full-grown pupae were irradiated with female sterilizing and substerilizing doses of gamma irradiation. Exposure to gamma radiation-induced alterations in DNA patterns of adult females, the appearance of some extra bands and the deficiency of others in the RAPD-PCR amplification patterns of irradiated females when compared with unirradiated females.

Genetic Analysis of (Oreochromis niloticus) Nirvana Tilapia Cultivated in Wanayasa and Galunggung, Lumajang, Bali, Minahasa Using Random Amplified Polymorphic DNA Method

This research aims to determine the kinship of nirvana tilapia (Oreochromis niloticus) cultivated in Wanayasa with Galunggung, Lumajang, Bali, and Minahasa by Random Amplified Polymorphic DNA method. All tilapia cultivated in these four places originated from Wanayasa. This research was conducted in the Biotechnology Laboratory, Laboratory of Microbiology and Molecular Biotechnology, FPIK UNPAD. The research method was an explorative experiment with the RAPD-PCR technique using primary OPA-3 and OPA-5. However, the results of DNA amplification with OPA-3 were better than with OPA-5. Therefore, further analysis was carried out using data from OPA-3. Kinship was analyzed based on the similarity index which was calculated by Numerical Taxonomy and Multivariate Analysis System (NTSYS) program. The results showed that the kinship between Nirvana tilapia cultivated in Wanayasa with Galunggung, Lumajang, Bali, and Minahasa, according to the similarity index was high, about 77-85%. The highest similarity index (85%) was obtained between nirvana tilapia cultivated in Wanayasa with Galunggung, and the lowest similarity index (77%) between tilapia cultivated in Bali with Minahasa. Genetic changes that occur because of differences in cultivation environments range from 15-23%.

Evaluation of Genetic Variability between Local White Chicken and Commercial Lines by RAPD-PCR and Sequencing of 18s rRNA Gene

The present study investigated and identified genetic diversity between native White chicken lines and two commercial Broiler (Rose) and Layer (Isa Brown) chicken breeds using RAPD markers and a sequencing technique. All primers applied produced 151 scorable bands with percentage polymorphic loci of 54.93 % within chicken populations, as per the results of the RAPD marker. The maximum amplified fragment by primer OPC-11 was 22 and the fewest by primer OPAA-03 was 7. For all loci analyzed, the effective number of alleles (ne), means the observed number of alleles (na), Shannon's information index (I), and gene diversity (h) was 1.4394, 1.5493, 0.3496, and 0.2441, respectively. The presence of a high number of polymorphisms and targeted (71) loci across all chicken populations indicates that RAPD-PCR techniques provide sufficient genetic distance and higher genetic variation among chicken populations. The highest identity of the blasted sequences of the 18srRNA gene of local white chicken is 90.41 % and 84.23 %. Likewise, a total of 46 and 27 nucleotides are altered with 27 and 10 gaps in both sequences for the first and second regions, respectively. According to both phylogenetic trees, the local white chicken had a stronger sense of individuality and was slightly closer to the commercial broiler breeds than the layer chicken breeds. As a result, it suggests that enhancing the local chicken line requires a broiler breeding program, as well as cross-breeding with other native chicken lines to obtain hereditarily significant new strains.

The probiotic properties and potential of vaginal Lactobacillus spp. isolated from healthy women against some vaginal pathogens.

During the last decade, probiotic research has progressed considerably and significant advances have been made in the selection and characterization of specific probiotic strains. The most studied probiotics belong to the genus Lactobacillus. In this study, 80 Lactobacillus spp. isolated from healthy women tolerated low pH and were able to grow in the presence of bile salts. RAPD PCR technique resulted in the identification of 38 different types. These isolates were then evaluated based on adhesion capacity, antibiotic susceptibility and tolerance in simulated gastrointestinal tract. Species-specific PCR and detection of bacteriocin-related genes were also surveyed. Among the isolates, five strains-Lacticaseibacillus rhamnosus NO21, Lacticaseibacillus casei NO1, Lactiplantibacillus plantarum NO4, Lactobacillus acidophilus NO7 and Lactobacillus gasseri NO38-presented acceptable antibiotic susceptibility pattern. Further analysis showed antimicrobial activity of Lacticaseibacillus culture against various bacterial pathogens and real-time PCR showed all five strains were able to prevent the colonization of bacterial pathogens. All five selected strains produced organic acids, hydrogen peroxide and were resistant to the spermicide. In addition, they lacked haemolytic activity with the ability of hydrophobicity, auto-aggregation and co-aggregation with pathogens. These results suggest that the vaginal microbiome could be a good source for the isolation of probiotics and the strains of this study may be considered as good probiotic candidates.

Biocontrol potential of Chitosan extracted from Procambarus clarkii (Crustacea: Cambaridae) against Eobania vermiculata snails (Muller 1774) in Egypt

BackgroundLand snails, especially the chocolate banded snails, Eobania vermiculata (Muller 1774) are destructive pests of a wide range of field and vegetable crops, and biological treatment appears to be better alternative to the chemical snail control. Therefore, the goal of this work was to assess the molluscicidal activity of chitosan extracted from the crawfish Procambarus clarkii against E. vermiculata using oxidative stress, histopathological and genotoxic biomarkers.ResultsExposure of snails to LC50 (222.4 mg/l) chitosan for 1, 3 and 7 days induced a significant increase in glutathione S-transferase and catalase levels then decline in reduced glutathione content after 1 and 3 days as well as a slight decrease in CAT levels, GSH content and GST of the treated snails after 7 days exposure. Histologically, the stress induced by chitosan exposure leads to deformation of cells, dilatation of the intertubular spaces, and destruction of tubules with increase in lumen size, necrosis of digestive cells with rise in vacuoles number and increase in calcium cells number. Considerably, a great damage was observed with increasing time of exposure. Furthermore, genotoxicity was assessed using RAPD-PCR technique and the results revealed that change in RAPD profiles of E. vermiculata following chitosan treatment included loss of normal DNA bands and appearance of new one compared to control snails. The genomic template stability was 63.6, 36.4 and 18.2% 1, 3 and 7 days of exposure, respectively. The apparent of new bands increased as time of exposure decreased, while GTS values decreased confirming the effect of chitosan-induced DNA damage.ConclusionChitosan may be an ecofriendly acceptable alternative pesticide for snail control.

Identification of Some Breast Cancer Related Genes by RAPD Technique in Maysan Province, Iraq

Breast cancer is a heterogeneous disease regarding its morphology, invasive behavior, metastatic capacity, hormone receptor expression and clinical outcome. Many risk factors for breast cancer, including genetic factors, account for 25-30% of the incidence. About 15-30% of breast cancer is heritable due to known familiar highly penetrates genes and the others are sporadic; It is worthy to state that this study was the first in the world to include amplified genes as a PCR template to determine the relationship between their polymorphism and breast cancer incidence using, RAPD of amplified genes. The study was designed first to evaluate the association of ABCG2 gene polymorphism beside miRNA-152 and ER-a using the RAPD technique with breast cancer incidence in Maysan province women, and second to use those genes as indicators for breast cancer prediction and diagnosis. The study included 100 patients with breast cancer and 30 control healthy women, and then all samples were amplified by conventional PCR by specific F and R primer for (ABCG2, ER-α, miRNA-152) genes and then the best (20 PCR product) from which was chosen as the template for PCR RAPD PCR technique. The results revealed there are significant differences (P < 0.05) in the unique band of ABCG2 at marker OPAA 11, OPU 15, OPAA 17, significant differences (P < 0.05) in the total band of ER- α at marker OPAA11, significant differences in the polymorphic band of ER- α at marker OPU 15, significant differences in the unique band of ER- α at marker OPAA11, OPU 15, and significant differences (P < 0.05) in the bands that had been size (50-60) bp, (140 - 150) bp, (170-180 ) bp of miRNA-152 at marker OPAA 17, OPD 18 between breast cancer patients and control. Our study proved the relationship between genetic polymorphism of breast cancer-related genes (ABCG2, ER-α, miRNA-152) and a higher incidence of cancer; The current study recommends employing these results for future prediction and diagnosis of breast cancers.

The use of internal transcribe spacer (ITS) as a molecular diagnostic tool for understanding variation among population of Metarhizium spp.

Direct analysis of DNA polymorphisms now is a general approach to identify and compare fungi at intraspecific, species, genus, or higher level. In this research using molecular genetic techniques with RAPD-PCR technique with ITS1 and ITS2 as promers were used. The samples are Metrahizium from Indonesia (M4 and M5) and Philippines (M6) using RAPD-PCR amplification, DNA sequencing and Analysis of ITS DNA sequences and ITS DNA sequence homologous alignment by search similarities used Blast Biological Software and multi-aligned with CLUSTALW Biological Software. It confirmed three isolates are: M. anisopliae and by multiple sequence alignment revealed transmission translation and transversion and deletion among them. The phylogram showed biogeographic area and their virulence.

Genetic Variability of Klebsiella Variicola by RAPD-PCR Technique and Bioremoval of Pb2+ and Cd2+ from Simulated Contaminated Soils

ABSTRACT Microbial bioremediation techniques continued to generate increasing interest due to its cost-effectiveness and being environmentally friendly. In the present study, Klebsiella variicola Wt, K. variicola MutAc and K.variicola MutEb were used to bioremediate Cd2+ and Pb2+ simulated contaminated soil in a pot experiment and their genetic variability were determined. The genetic variability among the Klebsiella variicola wild type and the mutant strains were investigated using 10 RAPD primers which revealed that 89.7% were polymorphic while 10.3% were monomorphic. The polymorphic information content (PIC) ranged from 0.52–0.86 while the diversity of the gene ranged from 0.56–0.88, this indicated an enormous heterozygosis associated with a high degree of polymorphism and diversity which make Klebsiella variicola mutant strains unique. The initial Cd2+ and Pb2+ in the simulated soil was 500 and 545 mg/Kg respectively while the Pb2+ concentration in the unsimulated soil was 45 mg/Kg. it was observed that K.variicola MutEb removed 14.6% of Cd2+ from the unsterilized soils while K.variicola MutAc removed 15.6% of Cd2+from the sterilized contaminated soils. However, K.variicola MutAc removed 27.8% and 11.6% of Pb2+ from the unsterilized and sterilized metal contaminated soils respectively. The results indicated that the autochthonous bacteria in the soil assisted in the remediation of the contaminated soil, most importantly, it was observed that the mutant strains performed better than the K.variicola wild type in terms of bioremediation potential, therefore, the strains could be suitable for the remediation of Cd2+ and Pb2+ contaminated soil.

Susceptibility of Fluconazole-Resistant Candida albicans to Thyme Essential Oil.

Candida spp. is the most common microbial pathogen in fungal infections. There has been a tremendous increase in cases of candidiasis, especially among critically ill non-neutropenic patients. Candida albicans’ isolates were procured from the Prince Sultan Military Hospital, Riyadh, KSA. The isolates were characterized for their identification using CHROMagar, carbohydrate metabolism, germ tube formation, and RAPD-PCR techniques. The essential oil of Thymus vulgaris was obtained by hydro-distillation and characterized to decipher the major bioactive phytoconstituents. The antifungal activity of the thyme essential oil (TEO) was evaluated against fluconazole-resistant C. albicans isolates. The major phytocomponents identified by GC/MS were thymol (68.1%) followed by γ-terpinene (8.9%), cymol (7.7%), caryophyllene (1.1%), linalool (1.4%). The TEO successfully reduced the growth of C. albicans isolates. At very low doses, the TEO proved to be fungi static and fungicidal. TEO also effectively inhibited the germ tube formation and budging of fungal pathogens. The time kill assays have shown that TEO was more effective against drug resistant clinical isolates than fluconazole. This study provides an array of experimental evidence regarding the therapeutic efficacy of TEO against the drug-resistant clinical isolates of C. albicans. The findings may be used in the development of a new antifungal agent accordingly.

English

The application of bacteriophages biocontrol requires the formulation of genetically distinct bacteriophages in a phage cocktail. Random Amplified Polymorphic DNA (RAPD) - PCR is considered a cheap, reproducible, and readily applicable tool in detecting phage diversity compared to other molecular techniques such as whole-genome sequencing. We used in this study the RAPD-PCR technique to assess the genetic diversity of 28 bacteriophages infecting Pseudomonas aeruginosa and Staphylococcus aureus. According to their RAPD profiles, isolated phages were grouped into 2 main clusters which included phages from the same host. The typing by RAPD-PCR of newly isolated phages was useful to assess the genetic diversity bypassing previous whole-genome sequencing analysis. These genetically distinct phages lytic against P. aeruginosa and S. aureus could potentially be used in a phage cocktail for biocontrol against these clinically and industrially relevant bacteria.   Key words: Phages, genetic diversity, RAPD PCR, P. aeruginosa, S. aureus.

ANALYSIS OF GENETIC DIVERSITY OF 26 CASSAVA VARIETIES (Manihot esculenta CRANTZ) WITH DIFFERENT RESPONSES TO WATERLOGGING CONDITIONS BY USING RAPD MARKERS

RAPD (Randomly Amplified Polymorphic DNA) is an indicator for high and stable polymorphism, widely used in the study of the diversity of cassava. In this paper, the results of using 20 polymorphic primers OPK combined with the establishment of the phylogenetic tree to analyze the genetic diversity of 26 cassava varieties with different responses to waterlogging conditions by using the RAPD-PCR technique were presented. The purpose of this experiment was to show the genetic relevance of the studied cassava varieties. The results showed that the flood tolerance of cassava was not related to the polymorphism and branching characteristics of the stem. This information may be use as a basis for selecting flood-tolerant cassava varieties for cassava production, as well as the basis for selecting genetically different parents for breeding.

EVALUATION OF GENETIC VARIABILITY BETWEEN THREE LINES OF CHICKENS BASED ON RAPD-PCR AND 18S rRNA GENE SEQUENCING IN ERBIL (IRAQI KURDISTAN REGION)

The current study was carried out to evaluate and identify the genetic variation between Local Black chicken lines with two commercial Layer (Isa Brown) and Broiler (Rose) breeds using RAPD markers and sequencing approach of 18s rRNA gene. From the result of the RAPD marker, all primers used were produced 152 scorable bands ranging from 2 to 9 with the size ranging from 320 to 2990 bp with percentage polymorphic loci 64.86% among chicken populations. The highest amplified fragments by primer OPC-11 and lowest by OPAA-03 were 24 and 8, respectively. The mean of the observed number of alleles (na), effective number of alleles (ne), gene diversity (h), Shannon's information index (I) for all loci found to be 1.6486, 1.5189, 0.2883 and 0.4129, respectively. The existence of a high level of polymorphisms and targeted (74) loci throughout all chicken populations/primers indicate sufficient genetic distance and more genetic variability among chicken populations using RAPD-PCR techniques. Result of blasted sequences of 18srRNA gene of local chicken has GenBank accession number MT808178 and MT808179 by BLAST tool against Gallus gallus, it showed the highest identity 95.74% and 94.88% for data of first and second part, respectively. The overall dendrograms clustered showed that the local chicken was closer to the commercial layer than to the broiler chicken lines. Therefore, it suggests that improving the local Black chicken line according to the layer breeding program to collect genetically invaluable genetic resources

Anti-Genotoxicity Evaluation of Ellagic Acid and Curcumin—An In Vitro Study on Zebrafish Blood Cells

Genotoxicity is the ability of specific substances to cause DNA damage, affecting development, physiology, and reproduction. This is often mediated by induction of oxidative stress. This in vitro study aims to test the ability of two antioxidants, ellagic acid (EA, 100 µM) and curcumin (Cur, 40 µM) to protect zebrafish blood cells from the genotoxic action of benzene (10 µL/mL). Cells were treated for 30, 60, and 90 min with EA or Cur alone and in combination with benzene. The antigenotoxic role of antioxidants was evaluated in terms of cytotoxicity by trypan blue dye, genome stability by RAPD-PCR technique, DNA fragmentation and percentage of apoptotic cells using Comet and Diffusion assay, respectively. The results did not show statistical differences in terms of cell viability, genome stability, DNA damage and apoptosis between cells treated with antioxidants. When zebrafish blood cells were co-incubated with individual antioxidants and benzene, a significant improvement of these parameters was observed in comparison with cells incubated in benzene. Our results suggested that EA and Cur are able to protect zebrafish blood cells against DNA damage and apoptosis caused by mutagenic substance, and laid the foundation for future studies investigating their antigenotoxic potential in DNA oxidative damage therapy.

P–033 In vitro protective effect of α -tocopherol and anthocyanin against TiO2-NPs induced genotoxicity on human spermatozoa

Abstract Study question Do α -tocopherol and anthocyanin counteract human sperm DNA damage provoked by titanium dioxide nanoparticles (TiO2-NPs)? Summary answer: ↑-tocopherol and anthocyanin are able to counteract TiO2-NPs genotoxicity on human sperm cells reducing oxidative stress. What is known already The environmental release and the extensive use of TiO2-NPs have been implicated in poor human sperm functionality.TiO2-NPs is genotoxic on human sperm cells causing a loss of sperm DNA integrity, an increase of apoptotic process and a reduction of genomic stability related to an over production of intracellular ROS.Antioxidants are the substances that can scavenge free radicals. α -tocopherol, present in vegetables, is the most important lipophilic antioxidant involved in restore sperm parameters in several experimental models. Anthocyanin, present in Aronia melanocarpaand belonging to the flavonoid family, is able to prevent damage caused by varicocele-induced ROS in rats. Study design, size, duration Semen samples from 132 men were obtained by masturbation following 3–5 days sexual abstinence and were examined for sperm concentration, viability, motility and morphology according to WHO 2010. The sperm cells, after purification with 45–90% double density gradient, were exposed in vitro to 1 µg/L of TiO2-NPs, 1 µg/L of TiO2-NPs whit 1 mg/L of anthocyanin and 1 µg/L of TiO2-NPs plus 1 mg/L of α -tocopherolfor 15,30,45 and 90 minutes at 37 °C. Participants/materials, setting, methods Sperm motility and concentration were analyzed with Makler camber while sperm viability and morphology were evaluated by Eosin-Nigrosin Test and by Testsimplets® prestained slides respectively. Antigenotoxicity was evaluated by Comet assay, TUNEL test and RAPD-PCR technique and Genomic Template Stability (GTS,%) calculation. The intracellular ROS level was assessed by DFC Assay. The data were analyzed using ANOVA test by GraphPad Prism 6 and considered significant if p-value ≤ 0.05. Main results and the role of chance Sperm analyses showed none statistically significant changes in sperm viability and motility (progressive and non-progressive) for each treatment. Anthocyanin and α -tocopherol counteracted sperm DNA damage induced in vitro by TiO2-NPs neutralizing ROS in a time-dependent way. Comet assay displayed that both antioxidants reduced sperm DNA strand breaks produced by TiO2-NPs, in particular the damage was no longer statistically significant starting from 30 and 90 minutes of anthocyanin-TiO2-NPs and α-tocopherol-TiO2-NPs co-exposure respectively. The antioxidant supplementation induced a statistically decrease of sperm DNA fragmentation provoked by TiO2-NPs after 45 co-treatment minutes.The RAPD-PCR technique evidenced variations of bands number in the TiO2-NPs treated sperm compared to the negative control and anthocyanin and α -tocopherol-TiO2-NPs co-treated samples. Human sperm genomic stability increased after anthocyanin and α -tocopherol TiO2-NPs co-exposure respect to the TiO2-NPs single treatment, until it almost reaches the negative control at 90 minutes. Intracellular ROS percentage was significantly lower both in anthocyanin and α -tocopherol TiO2-NPs co-treated compared to TiO2-NPs alone starting from 45 minutes. Limitations, reasons for caution In vitro study. Wider implications of the findings: Our results showed a protective effect of anthocyanin and α -tocopherol on human DNA by neutralizing intracellular ROS induced by TiO2NPs. We suggest anthocyanin and α -tocopherol as suitable molecules to defend human sperm DNA from oxidative stress, with a potentially role in treatmentof male infertility due to environmental factors. Trial registration number None