Suppression of tumor growth and apoptosis induction by pomegranate seed nano-emulsion in mice bearing solid Ehrlich carcinoma cells.

Despite the high antioxidant and penetration ability of pomegranate seed oil (PSO), the in vivo antitumor activity of PSO nano-emulsion has not been well investigated. Therefore, this study was undertaken to estimate the antitumor activity and safety of PSO nano-emulsion in mice bearing Ehrlich solid carcinoma cells. For tumor inoculation, about 2 × 106 viable Ehrlich tumor cells (200 µl) were implanted intramuscularly in the left thigh of hind leg. Once a solid tumor appears on the 10th day of transplantation; the mice were randomly divided into five groups (5 animals/group). Characterization of the PSO nano-emulsion using a Zeta sizer Malvern instrument and transmission electron microscope (TEM) revealed that the PSO nano-droplets were well dispersed with an average particle size of 8.95 nm and a spherical shape. Treatment with PSO nano-emulsions caused a significant reduction in the tumor size and weight, in a dose dependent manner, compared to tumor control group. Marked dose dependent elevations in the DNA damage level together with significant increases in the tumor suppressor p53, Bax and Caspase genes and reductions in the anti-apoptotic Bcl2 gene were also observed in the tumor tissue of mice given PSO nano-emulsions. Histological examination also revealed apoptosis and necrosis of tumor cells and tumor infiltration with inflammatory cells after PSO nano-emulsion treatment. However, high DNA damage was noticed in the liver and kidney tissues of mice given the highest dose of PSO nano-emulsion (400 mg/kg). Therefore, we concluded that PSO nano-emulsion exhibited a potent antitumor activity through induction of DNA breaks that triggers apoptosis of tumor cells but the highest dose caused genotoxicity to liver and kidney tissues, thus it is recommended to use doses lower than 400 mg/kg of PSO nano-emulsion as an alternative drugs for chemotherapy.

Comparative analysis of Acomys cahirinus and Mus musculus responses to genotoxicity, oxidative stress, and inflammation.

The Egyptian spiny mouse, Acomys cahirinus, is a recently described model organism for regeneration studies. It has surprising powers of regeneration with relatively fast repairing mechanisms and reduced inflammation form compared to other mammals. Although several studies have documented the exceptional capabilities of Acomys to regenerate different tissues after injury, its response to different cellular and genetic stresses is not yet investigated. Therefore, the current study aimed to investigate Acomys abilities to resist genotoxicity, oxidative stress and inflammation induced by acute and subacute treatments with lead acetate. Responses of Acomys were compared with those of the lab mouse (Mus musculus), which displays signatures of the "typical" mammalian response to various stressors. Cellular and genetic stresses were induced by using acute and subacute doses of Lead acetate (400 mg/kg and 50 mg/kg for 5 days, respectively). The assessment of genotoxicity was carried out by using comet assay, while oxidative stress was evaluated by measuring the biomarkers; MDA, GSH and antioxidant enzymes CAT and SOD. Moreover, inflammation was assessed by analyzing the expression of some inflammatory-regeneration-related genes: CXCL1, IL1-ß, and Notch 2 and immunohistochemical staining of TNF-α protein in brain tissue, in addition to histopathological examination of brain, liver, and kidneys. The obtained results revealed a unique resistance potency of Acomys to genotoxicity, oxidative stress, and inflammation in certain tissues in comparison to Mus. Altogether, the results revealed an adaptive and protective response to cellular and genetic stresses in Acomys.

Association of new SNPs at DGAT1 gene with milk quality in Egyptian Zaraibi goat breed.

This study aimed to detect putative genomic loci in candidate genes associated with milk composition in Egyptian Zaraibi goats. A total number of 50 samples were tested to detect polymorphism in exons 15 and 16 of the diacylglycerol acyltransferase 1 (DGAT1) gene. The PCR products were sequenced and aligned. Sequence analysis showed three new genotypes in the studied samples: T1C1 (T12C SNP), T2C2 (T84C), and AG (G219A), then three groups were created: the first group was BB with C1T1 and AG genotypes, the second was DD which contains C2T2 and AG genotypes, and the third was AG with only AG genotype. GLM showed that the DD group with C84T and G219A SNPs had significantly the highest fat percent. Meanwhile, the BB group with C84T and G219A SNPs recorded significantly the highest total solids levels. On the other hand, the AG group which has G219A SNP showed a non-significant effect on milk components. Those new SNPs were submitted to GenBank and approved to be published. Moreover, translation of those sequences showed that the G219A SNP causes a substitution of Glycine to Serine in exon 16 at position 106. This SNP (G106S) was predicted to be tolerated by SIFT with a score of 0.48.

Association of PRLR, IGF1, and LEP genes polymorphism with milk production and litter size in Egyptian Zaraibi goat.

Studying variation in genes responsible for physiological characters is important to enhance goat productive and reproductive efficiency. This study aimed to detect specific nucleotide polymorphisms in prolactin receptor (PRLR), insulin-like growth factor (IGF1), and leptin (LEP) genes and their correlation with milk production (MP) and litter size (LS) traits in Zaraibi goat. PCR-SSCP products of different patterns of each gene were sequenced and aligned to reveal two mutations (T > C) and (G > A) in 3'UTR of PRLR gene and registered on NCBI with accession numbers OM418863 for TT and OM418864 for CT, while (G > A) variation was registered as OM418861 for GG and OM418862 for AG in exon 10. TT, CT, AG, and GG genotypes were distributed in the studied animals with frequencies 0.43, 0.57, 0.65, and 0.35, respectively. While alleles C, T, A, and G frequencies were 0.28, 0.72, 0.32, and 0.68, respectively. CT and AG genotypes associated significantly (P < 0.05) with higher MP and LS, respectively. By studying the haplotypes of PRLR, C-A and T-A were associated with the highest and the lowest level of MP, respectively. For LS, T-A and C-G showed significant correlation with the highest and the lowest rate, respectively. Regarding IGF1 gene, two polymorphisms were detected; T74C at exon 4 which registered on NCBI as OM418860, and combined mutations as ins. G470, A531G, and T534C (PP genotype) at 5' flanking region that registered as OM418859. For LEP, only one polymorphism was found in intron 2 (G281A) which submitted to NCBI as OM418855. All detected polymorphisms have shown to be involved in regulating the MP or LS as reproductive traits in goat.

Tissue Distribution, Histopathological and Genotoxic Effects of Magnetite Nanoparticles on Ehrlich Solid Carcinoma.

Nanoparticles can potentially cause adverse effects on cellular and molecular level. The present study aimed to investigate the histopathological changes and DNA damage effects of magnetite nanoparticles (MNPs) on female albino mice model with Ehrlich solid carcinoma (ESC). Magnetite nanoparticles coated with L-ascorbic acid (size ~ 25.0 nm) were synthesized and characterized. Mice were treated with MNPs day by day, intraperitoneally (IP), intramuscularly (IM), or intratumorally (IT). Autopsy samples were taken from the solid tumor, thigh muscle, liver, kidney, lung, spleen, and brain for assessment of iron content, histopathological examination, and genotoxicity using comet assay. The liver, spleen, lung, and heart had significantly higher iron content in groups treated IP. On the other hand, tumor, muscles, and the liver had significantly higher iron content in groups treated IT. MNPs induced a significant DNA damage in IT treated ESC. While a significant DNA damage was detected in the liver of the IP treated group, but no significant DNA damage could be detected in the brain. Histopathological findings in ESC revealed a marked tumor necrosis, 50% in group injected IT but 40% in group injected IP and 20% only in untreated tumors. Other findings include inflammatory cell infiltration, dilatation, and congestion of blood vessels of different organs of treated groups in addition to appearance of metastatic cancer cells in the liver of non-treated tumor group. MNPs could have an antitumor effect but it is recommended to be injected intratumorally to be directed to the tumor tissues and reduce its adverse effects on healthy tissues.

Induction of fetal abnormalities and genotoxicity by molybdenum nanoparticles in pregnant female mice and fetuses.

Increasing the uses of molybdenum (Mo) nanoparticles in a wide range of applications including food, industry, and medicine, resulted in increased human exposure and necessitated the study of their toxic effects. However, almost no studies are available on their genotoxic effects, especially on pregnant females and their fetuses. Therefore, this study was undertaken to estimate the possible induction of genotoxicity and fetal abnormalities, especially fetal malformations and skeletal abnormalities by Mo nanoparticle administration in mice. Oral administration of Mo nanoparticles resulted in significant decreases in the maternal body weight, the number and length of fetuses as well as skeletal abnormalities mainly less ossification and less chondrification. Administration of Mo nanoparticles also caused DNA damage induction which elevated the expression levels of p53, the vital gene in maintaining the genomic stability and cell differentiation in both maternal and fetus tissues. Similarly, the expression levels of E-Cad and N-Cad genes that control skeleton development have also been increased in the tissues of female mice administered Mo nanoparticles and their fetuses. Thus, we concluded that oral administration of Mo nanoparticles induced genotoxic effects and fetal abnormalities that necessitated further studies on the possible toxic effects of Mo nanoparticles.

Estimation of genomic instability and mutation induction by graphene oxide nanoparticles in mice liver and brain tissues.

The rapidly growing interest in using graphene-based nanoparticles in a wide range of applications increases human exposure and risk. However, very few studies have investigated the genotoxicity and mutagenicity of the widely used graphene oxide (GO) nanoparticles in vivo. Consequently, this study estimated the possible genotoxicity and mutagenicity of GO nanoparticles as well as possible oxidative stress induction in the mice liver and brain tissues. Nano-GO particles administration at the dose levels of 10, 20, or 40 mg/kg for one or five consecutive days significantly increased the DNA breakages in a dose-dependent manner that disrupts the genetic material and causes genomic instability. GO nanoparticles also induced mutations in the p53 (exons 6&7) and presenilin (exon 5) genes as well as increasing the expression of p53 protein. Positive p53 reaction in the liver (hepatic parenchyma) and brain (cerebrum, cerebellum, and hippocampus) sections showed significant increase of p53 immunostaining. Additionally, induction of oxidative stress was proven by the significant dose-dependent increases in the malondialdehyde level and reductions in both the level of reduced glutathione and activity of glutathione peroxidase observed in GO nanoparticles administered groups. Acute and subacute oral administration of GO nanoparticles induced genomic instability and mutagenicity by induction of oxidative stress in the mice liver and brain tissues.

Apoptosis and oxidative stress as relevant mechanisms of antitumor activity and genotoxicity of ZnO-NPs alone and in combination with N-acetyl cysteine in tumor-bearing mice.

Background: Several in vitro studies have revealed that zinc oxide nanoparticles (ZnO-NPs) were able to target cancerous cells selectively with minimal damage to healthy cells. Purpose: In the current study, we aimed to evaluate the antitumor activity of ZnO-NPs in Ehrlich solid carcinoma (ESC) bearing mice by measuring their effect on the expression levels of P53, Bax and Bcl2 genes as indicators of apoptotic induction in tumor tissues. Also, we assessed the potential ameliorative or potentiation effect of 100 mg/kg N-acetyl cysteine (NAC) in combination with ZnO-NPs. Materials and methods: ESC bearing mice were gavaged with three different doses of ZnO-NPs (50, 300 and 500 mg/kg body weight) alone or in combination with NAC for seven consecutive days. In addition to measuring the tumor size, pathological changes, zinc content, oxidative stress biomarkers and DNA damage in ESC, normal muscle, liver and kidney tissues were assessed. Results: Data revealed a significant reduction in tumor size with a significant increase in p53 and Bax and decrease in Bcl2 expression levels in the tissues of ZnO-NPs treated ESC bearing mice. Moreover, a significant elevation of MDA accompanied with a significant reduction of CAT and GST. Also, a marked increase in all comet assay parameters was detected in ZnO-NPs treated groups. On the other hand, the combined treatment with ZnO-NPs and NAC significantly reduced reactive oxygen species production and DNA damage in liver and kidney tissues in all ZnO-NPs treated groups. Conclusion: ZnO-NPs exhibited a promising anticancer efficacy in ESC, this could serve as a foundation for developing new cancer therapeutics. Meanwhile, the combined treatment with ZnO-NPs and NAC could act as a protective method for the healthy normal tissue against ZnO-NPs toxicity, without affecting its antitumor activity.

Bone marrow derived-mesenchymal stem cells downregulate IL17A dependent IL6/STAT3 signaling pathway in CCl4-induced rat liver fibrosis.

Therapeutic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) has been reported in several animal models of liver fibrosis. Interleukin (IL) 17A, IL6 and Stat3 have been described to play crucial roles in chronic liver injury. However, the modulatory effect of MSCs on these markers was controversial in different diseases. BM-MSCs might activate the IL6/STAT3 signaling pathway and promote cell invasion in hepatocellular carcinoma, but the immunomodulatory role of BM-MSCs on IL17A/IL6/STAT3 was not fully elucidated in liver fibrosis. In the present study, we evaluated the capacity of the BM-MSCs in the modulation of cytokines milieu and signal transducers, based on unique inflammatory genes Il17a and Il17f and their receptors Il17rc and their effect on the IL6/STAT3 pathway in CCl4-induced liver fibrosis in rats. A single dose of BM-MSCs was administered to the group with induced liver fibrosis, and the genes and proteins of interest were evaluated along six weeks after treatment. Our results showed a significant downregulation of Il17a, Il17ra, il17f and Il17rc genes. In accordance, BM-MSCs administration declined IL17, IL2 and IL6 serum proteins and downregulated IL17A and IL17RA proteins in liver tissue. Interestingly, BM-MSCs downregulated both Stat3 mRNA expression and p-STAT3, while Stat5a gene was downregulated and p-STAT5 protein was elevated. Also P-SMAD3 and TGFßR2 proteins were downregulated in response to BM-MSCs treatment. Collectively, we suggest that BM-MSCs might play an immunomodulatory role in the treatment of liver fibrosis through downregulation of IL17A affecting IL6/STAT3 signaling pathway.

Fibulin-2 is required for basement membrane integrity of mammary epithelium.

Fibulin-2 (FBLN2) is a secreted extracellular matrix glycoprotein which has been associated with tissue development and remodelling. In the mouse mammary gland, FBLN2 can be detected during ductal morphogenesis in cap cells and myoepithelial cells at puberty and early pregnancy, respectively. In an attempt to assign its function, we knocked down Fbln2 in the mouse mammary epithelial cell line EpH4. FBLN2 reduction led to an increase in the size of spheroidal structures when compared to scrambled control shRNA-transduced cells plated on Matrigel matrix. This phenotype was associated with a disruption of the collagen IV sheath around the epithelial spheroids and downregulation of integrin ß1, suggesting a role for FBLN2 in stabilizing the basement membrane (BM). In contrast to mice, in normal adult human breast tissue, FBLN2 was detected in ductal stroma, and in the interlobular stroma, but was not detectable within the lobular regions. In tissue sections of 65 breast cancers FBLN2 staining was lost around malignant cells with retained staining in the neighbouring histologically normal tissue margins. These results are consistent with a role of FBLN2 in mammary epithelial BM stability, and that its down-regulation in breast cancer is associated with loss of the BM and early invasion.

Magnetite nanoparticles inhibit tumor growth and upregulate the expression of p53/p16 in Ehrlich solid carcinoma bearing mice.

BACKGROUND: Magnetite nanoparticles (MNPs) have been widely used as contrast agents and have promising approaches in cancer treatment. In the present study we used Ehrlich solid carcinoma (ESC) bearing mice as a model to investigate MNPs antitumor activity, their effect on expression of p53 and p16 genes as an indicator for apoptotic induction in tumor tissues. METHOD: MNPs coated with ascorbic acid (size: 25.0±5.0 nm) were synthesized by co-precipitation method and characterized. Ehrlich mice model were treated with MNPs using 60 mg/Kg day by day for 14 injections; intratumorally (IT) or intraperitoneally (IP). Tumor size, pathological changes and iron content in tumor and normal muscle tissues were assessed. We also assessed changes in expression levels of p53 and p16 genes in addition to p53 protein level by immunohistochemistry. RESULTS: Our results revealed that tumor growth was significantly reduced by IT and IP MNPs injection compared to untreated tumor. A significant increase in p53 and p16 mRNA expression was detected in Ehrlich solid tumors of IT and IP treated groups compared to untreated Ehrlich solid tumor. This increase was accompanied with increase in p53 protein expression. It is worth mentioning that no significant difference in expression of p53 and p16 could be detected between IT ESC and control group. CONCLUSION: MNPs might be more effective in breast cancer treatment if injected intratumorally to be directed to the tumor tissues.

Normalization of nano-sized TiO2-induced clastogenicity, genotoxicity and mutagenicity by chlorophyllin administration in mice brain, liver, and bone marrow cells.

The intensive uses of titanium dioxide (TiO2) nanoparticles in sunscreens, toothpaste, sweats, medications, etc. making humans exposed to it daily by not little amounts and also increased its risks including genotoxicity. Thus, the present study was designed as one way to reduce nano-titanium-induced clastogenicity, genotoxicity, and mutagenicity in mice by co-administration of the free radical scavenger chlorophyllin (CHL). In addition, markers of oxidative stress were detected to shed more light on mechanism(s) underlying nano-sized TiO2 genotoxicity. Male mice were exposed to multiple injection into the abdominal cavity for five consecutive days with either CHL (40 mg/kg bw/day), or each of three dose levels of nano-sized TiO2 (500, 1000, or 2000 mg/kg bw/day) alone, or both simultaneously and sacrificed by cervical dislocation 24 h after the last treatment. After CHL co-administration, the observed dose-dependent genotoxicity of TiO2 nanoparticles indicated by the significant elevations in frequencies of both micronuclei and DNA damage induction was significantly decreased and returned to the negative control level. The observed induced mutations in p53 exons 5, 7, & 8 and 5 & 8 in the liver and brain, respectively, were declined in most cases. Moreover, CHL significantly decreased hepatic malondialdehyde level and significantly increased glutathione level and superoxide dismutase, catalase, and glutathione peroxidase activities that were significantly disrupted in animal groups treated with nano-TiO2 alone. In conclusion, the evidenced in vivo genotoxicity of nano-TiO2 in the present study was normalized after CHL co-administration which supports the previously suggested oxidative stress as the possible mechanism for titanium toxicity.

Evaluation of the role of the antioxidant silymarin in modulating the in vivo genotoxicity of the antiviral drug ribavirin in mice.

Ribavirin (1-ß-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a widely used broad-spectrum antiviral drug. Recently, several reports revealed genotoxic effects of ribavirin in vivo and in vitro, which were correlated with the production of reactive oxygen species (ROS). This study aimed to evaluate the genotoxicity of ribavirin and to investigate the role of the natural antioxidant silymarin to modulate this genotoxicity. Male albino mice (age, 8-10 weeks) were injected intraperitoneally (i.p.) with ribavirin at three dose levels (20, 75 and 130 mg/kg bw) either in a single injection (acute treatment) or in multiple injections on five consecutive days (sub-acute treatment). Other comparable groups were treated with silymarin (70 mg/kg bw) 1h before the injection with ribavirin. Mice were sacrificed at different sampling times (24, 48 and 72 h) after the last ribavirin treatment. Micronucleus (MN) and single-strand conformation polymorphism (SSCP) assays were used to assess genotoxic and cytotoxic effects of ribavirin and to evaluate the protective effect of the pre-treatment with silymarin. Our results reveal genotoxic and cytotoxic effects of ribavirin in the MN assay. Pre-treatment with silymarin reduced the toxicity of ribavirin. In the SSCP assay, ribavirin treatment did not induce any mutations in the two selected sites in the D-loop of mitochondrial DNA (mtDNA).

Genotoxic evaluation for the tricyclic antidepressant drug, amitriptyline.

Tryptizol(®) [amitriptyline HCl (AT); El-Kahira Pharmacological and Chemical Co., Cairo, Egypt], a widely used antidepressant drug in Egypt, was evaluated for its genotoxicity. The evaluation was performed in somatic (bone marrow) and germ (spermatocytes) cells, as well as well as the sperm morphology (i.e., head and tail) and count of the resulting sperm. Three doses were tested (low, medium, and high); they were chosen according to the drug manufacturer. The low-dose group received orally 1 mg/kg body weight (b.w.) daily for a total period of 1 month; the medium-dose group received 1 mg/kg b.w. daily for 15 days and 2 mg/kg b.w. daily for another 15 days; and the high-dose group received 1 mg/kg b.w. daily for 10 days, then 2 mg/kg b.w. daily for another 10 days and, finally, 4 mg/kg b.w. daily for 10 more days. The results showed that AT treatment induced structural and numerical chromosome abnormalities in somatic cells (bone marrow) and germ cells (spermatocytes). Moreover, AT significantly reduced both the mitotic index and meiotic activity after the different treatments used. AT was found to increase significantly the incidence of sperm-cell head and tail abnormalities. The sperm-cell count was also significantly decreased with the 3 doses tested. In general, results of chromosome abnormalities in both somatic and germ cells as well as sperm morphology and count showed that the effect of AT was dose dependent. The results of the current study showed that AT is a genotoxic agent for both somatic and germ cells and should be taken under special precautions and medical supervision.

Lead acetate and arsenic trioxide induce instability of microsatellites at three different fragile sites (6q21, 9q32-9q33 and 15p14) within the genome of the rat.

Human exposure to metals is of increasing concern due to the well-documented toxic and carcinogenic effects of metals and metal compounds, and the rising environmental levels due to industrial processes and pollution. It has been reported that metals can be genotoxic by several modes of action including generation of reactive oxygen species and inhibition of DNA repair. The aim of this study was to evaluate microsatellite instability (MSI) in three microsatellite loci (D6mit3, D9mit2 and D15Mgh1) located within three common fragile sites in the genome of the laboratory rat (6q21, 9q32-9q33 and 15p14) exposed to acute and chronic doses of a metal salt (lead acetate trihydrate) and a metalloid oxide (arsenic trioxide). In the acute and sub-chronic studies with the two compounds, MSI was observed in the three loci as deletions or additions of microsatellite repeats. The percentages of MSI were 36.4% and 42.1% for lead acetate and arsenic trioxide, respectively. Results of the present work indicate that the microsatellites located within fragile sites provide a convenient assay system to detect changes in DNA sequences resulting from exposure to genotoxic agents.

Microsatellite instability at three microsatellite loci (D6mit3, D9mit2 and D15Mgh1) located in different common fragile sites of rats exposed to cadmium.

Cadmium (Cd) is a non-essential element and is a widespread environmental pollutant. Exposure to cadmium can result in cytotoxic, carcinogenic and mutagenic effects. Mutagenesis is an indicative of genetic instability and can be assayed using microsatellites instability. The aim of the present study is to investigate; based on the rat model, the effects of oral acute (single 8.8mg/kg BW, 1/10 LD(50)) and sub-chronic (2.93mg/kg BW, 1/30 LD(50), for 4 weeks) doses of cadmium chloride on microsatellite instability at D6mit3, D9mit2 and D15Mgh1 loci, which are located in three different common fragile sites (6q21, 9q32-q33 and 15p14, respectively), within rat genome. In the acute study, no microsatellite instability (MSI) was observed in all the three tested loci (D6mit3, D9mit2 and D15Mgh1). In the sub-chronic study, the MSI were observed in the three studied loci and was in the form of deletion of 2-3bp or addition of 3-6bp. These finding may indicate the sensitivity of microsatellite sequences located at the fragile sites and the sensitivity of the simple sequence repeats (SSRs) assay for the detection of small variations in DNA sequence. However, additional chronic toxicological trials are needed in order to assess genotoxic effects of chronic exposure to Cd.

Beta-glucan inhibits the genotoxicity of cyclophosphamide, adriamycin and cisplatin.

The inhibitory effects of beta-glucan (betaG), one of the biological response modifiers, on the induction of chromosomal aberrations in the bone marrow and spermatogonial cells of mice treated with various anti-neoplastic drugs were investigated. beta-Glucan (100 mg/kg bw, i.p.) pre-treatment reduced the total number of cells with structural chromosomal aberrations scored after the treatment with cyclophosphamide (CP) (2.5 mg/kg bw, i.p.) adriamycin (ADR) (12 mg/kg bw, i.p.) and cis-diamminedichloroplatinum-II (cisplatin) (5 mg/kg bw, i.p.) by about 41.1, 26.9 and 57.7% in bone marrow and 44.4, 55 and 57.1% in spermatogonial cells, respectively. This protective effect of beta-glucan could be attributed to its scavenging ability to trap free-radicals produced during the biotransformation of these anti-neoplastic drugs. Beta-glucan also markedly restored the mitotic activity of bone marrow cells that had been suppressed by the anti-neoplastic drugs. These results indicate that in addition to the known immunopotentiating activity of beta-glucan, it plays a role in reducing genotoxicity induced by anti-neoplastic drugs during cancer chemotherapy.