Potential chemopreventive effects of Broccoli extract supplementation against 7, 12 dimethyl Benz(a)anthracene (DMBA) -induced toxicity in female rats.

Dietary components have recently received rapidly expanding attention for their potential to halt or reverse the development of many oxidative stress-mediated diseases after exposure to environmental toxicants. 7, 12 dimethylbenz(a)anthracene (DMBA) is one of the most common environmental pollutants. The present study aimed to evaluate the chemo-preventive effects of broccoli as a nutritional component against DMBA intoxication in rats. A daily dose of aqueous (1 ml/rat) and methanolic (150 mg/kg) broccoli extracts, respectively, was given to 50-day-old female rats for 26 successive weeks after carcinogen intoxication with a single dose of 20 mg/ml of DMBA. DMBA intoxication resulted in a redox imbalance (a decreased GSH level and an increased MDA level) and increased DNA fragmentation in the liver, kidney, and brain. Besides, it affected the level of expression of the bcl2 gene in the liver, kidney, and brain tissue but didn't affect cfos gene expression accompanied by histopathological changes. The aqueous and methanolic broccoli extract supplements ameliorated the adverse effects by increasing the level of GSH, decreasing the MDA level, and reducing DNA fragmentation. Besides, broccoli extracts decreased the expression of bcl2 in the liver and brain and up-regulated bcl2 expression in the kidney, accompanied by lowering NF-κß 65 expression in the liver and brain and γ-catenin expression in the liver and kidney. In conclusion, broccoli as a dietary component had a strong chemoprotective effect against oxidative stress, DNA damage, and genotoxicity induced by DMBA intoxication in rats.

Circulating miR-146b and miR-27b are efficient biomarkers for early diagnosis of Equidae osteoarthritis.

One of the most orthopedic problems seen in the equine is osteoarthritis (OA). The present study tracks some biochemical, epigenetic, and transcriptomic factors along different stages of monoiodoacetate (MIA) induced OA in donkeys in serum and synovial fluid. The aim of the study was the detection of sensitive noninvasive early biomarkers. OA was induced by a single intra-articular injection of 25 mg of MIA into the left radiocarpal joint of nine donkeys. Serum and synovial samples were taken at zero-day and different intervals for assessment of total GAGs and CS levels as well as miR-146b, miR-27b, TRAF-6, and COL10A1 gene expression. The results showed that the total GAGs and CS levels increased in different stages of OA. The level of expression of both miR-146b and miR-27b were upregulated as OA progressed and then downregulated at late stages. TRAF-6 gene was upregulated at the late stage while synovial fluid COL10A1 was over-expressed at the early stage of OA and then decreased at the late stages (P < 0.05). In conclusion, both miR-146b and miR-27b together with COL10A1 could be used as promising noninvasive biomarkers for the very early diagnosis of OA.

COL2A1 and Caspase-3 as Promising Biomarkers for Osteoarthritis Prognosis in an Equus asinus Model.

Osteoarthritis (OA) is one of the most degenerative joint diseases in both human and veterinary medicine. The objective of the present study was the early diagnosis of OA in donkeys using a reliable grading of the disease based on clinical, chemical, and molecular alterations. OA was induced by intra-articular injection of 25 mg monoiodoacetate (MIA) as a single dose into the left radiocarpal joint of nine donkeys. Animals were clinically evaluated through the assessment of lameness score, radiographic, and ultrasonographic findings for seven months. Synovial fluid and cartilage samples were collected from both normal and diseased joints for the assessment of matrix metalloproteinases (MMPs) activity, COL2A1 protein expression level, and histopathological and immunohistochemical analysis of Caspase-3. Animals showed the highest lameness score post-induction after one week then decreased gradually with the progression of radiographical and ultrasonographic changes. MMP activity and COL2A1 and Caspase-3 expression increased, accompanied by articular cartilage degeneration and loss of proteoglycan. OA was successfully graded in Egyptian donkeys, with the promising use of COL2A1and Caspase-3 for prognosis. However, MMPs failed to discriminate between early and late grades of OA.

Oxidative Stress and DNA methylation in male rat pups provoked by the transplacental and translactational exposure to bisphenol A.

The epigenetic changes induced by environmental contaminants play important roles in the inheritance of male reproductive dysfunction. The present study investigated DNA methylation changes and some oxidative stress biomarkers induced by bisphenol A (BPA) in male offspring. A total number of 48 female albino rats were administered orally with 50 µg/kg of BPA/day during gestation and/or lactation periods. At postnatal day 60, the samples were collected from the male pups to assess the serum testosterone, malondialdehyde (MDA) level, superoxide dismutase (SOD), glutathione S-transferase, and glutathione peroxidase (GSH-Px) activities in testicular tissue. DNA methylation in both DNA (cytosine-5)-methyltransferase 3A and estrogen receptor alpha genes was detected by methylation-specific PCR. BPA exposure resulted in significant decrease in the anogenital distance, testis and epididymis weights, serum testosterone level, SOD, GST, and GSH-Px levels with significant increase in weaning body weight and the MDA level. Additionally, BPA caused marked hypermethylation within Dnmt3A and ER- ∝ genes promoter regions in the testis of rat male pups. Graphical abstract.

Nano-sized selenium attenuates the developmental testicular toxicity induced by di-n-butyl phthalate in pre-pubertal male rats.

The current study was conducted to test the possible ameliorative role of selenium nanoparticles (Se-NPs) against oxidative damage of Leyding cells induced by di-n-butyl phthalate (DBP) in pre-pubertal male rat offspring. Forty-two pregnant female rats treated from gestation day (GD) 12 to postnatal day (PND) 14 day with two doses of Se-NPs (0.2 and 0.5 mg/kg/d) against developmental testicular toxicity induced by DBP (500 mg/kg/d). At PND 25 serum and testes of offspring were collected. Serum LH, the Leydig cells performance [total serum testosterone, LH and testosterone (LH/T) ratio, relative gene expression of insulin-like growth factor-3 (INSL3) and mineralocorticoid receptor (MR)], oxidative stress biomarker malondialdehyde (MDA) and antioxidant machinery [reduced glutathione (GSH), and the relative gene expression of antioxidant enzymes: superoxide dismutase (SOD), glutathione peroxidase (GPx)] were estimated in all groups. The obtained results revealed that maternal exposure to DBP significantly reduced total serum testosterone level, relative mRNA expression of INSL3 and MR genes with observed testicular damage revealed by increasing MDA and depressed levels of GSH and antioxidant enzymes. The histopathological changes include necrosis and desquamation of spermatogoneal cells. Co-administration of Se-NPs high dose along with DBP significantly increased serum testosterone, improved LH/T ratio and the relative mRNA expression of INSL3 and MR genes, decreased the level of MDA, and also improved all the antioxidant enzymes expression levels. In conclusion, Se-NPs could be a potent maternal prophylactic agent against the reduced total serum testosterone level and oxidative damage of Leydig cells induced by DBP via reducing the lipid peroxidation (LPO) and enhancing the antioxidant state in pre-pubertal male rat offspring.

Maternal exposure to di-n-butyl phthalate induces alterations of c-Myc gene, some apoptotic and growth related genes in pups' testes.

The aim of this study was to investigate the effects of maternal exposure to di-( n-butyl) phthalate (DBP) on testicular development and function in pre-pubertal and post-pubertal male rat offspring. Fourteen pregnant female rats were equally divided into two groups: a control group and a DBP-treated group. During gestation day (GD) 12 to postnatal day (PND) 14, the control group was administered 1 ml/day corn oil, and the DBP-treated group was administered DBP 500 mg/kg/day by oral gavage. On PND 25 (pre-puberty) and PND 60 (post-puberty), blood for serum and the testes were collected from five male offspring of each group. To determine the relationship between the methylation state of the c-Myc promoter and the expression of the c-Myc gene, some apoptotic-related genes, such as p53 and Bax, the anti-apoptotic Bcl-2 gene, and some growth arrest-related genes, such as BRD7 and GAS1, were examined. Compared with the control ( p < 0.05), at pre-puberty, DBP induces c-Myc hyper-methylation with significant downregulation for c-Myc, p53, Bax genes, and significant upregulation for Bcl-2, BRD7, and GAS1, while at post puberty, the methylation state and expression of c-Myc and apoptosis-related genes returned to control levels in the same sequence with the fold change in the expression of BRD7 and GAS1 genes. These findings suggest that DBP induced a transient pre-pubertal increase in c-Myc promoter methylation that may be associated with disruption of both apoptotic and growth mechanisms in the testes.

Genetic polymorphism in melatonin receptor 1A and arylalkylamine N-acetyltransferase and its impact on seasonal reproduction in Egyptian sheep breeds.

This study was carried out to detect polymorphisms in the melatonin receptor 1A (MTNR1A) and arylalkylamine N-acetyltransferase (AA-NAT) genes and their association with reproductive traits. Blood samples of 126 animals from three Egyptian sheep breeds were collected. DNA was extracted and subjected to PCR restriction fragment length polymorphism (RFLP) analysis using the RsaI and SmaI enzymes. Two alleles (C and T) and three genotypes (CC, CT and TT) for MTNR1A and for AA-NAT (A and G; GG, GA and AA) were detected. The alleles C and A and the genotypes CT and GA showed the highest frequencies for the MTNR1A and AA-NAT genes, respectively. Association analysis of the MTNR1A single nucleotide polymorphism (SNP) with ewe reproductive traits revealed significant associations in the Ossimi and Rahmani breeds with age at first lambing, and the C allele seemed to be the favorable allele. The results for the AA-NAT SNP demonstrated significant correlations in Ossimi with age at first lambing and litter size and in Rahmani with lambing interval; the G allele seemed to be the desirable allele. In the first conception season, ewes carrying CT exhibited a significantly lower age of first lambing in the unfavorable season. Additionally, GG ewes exhibited a significantly lower age of first lambing in the early favorable season, followed by the unfavorable season. To the best of our knowledge, this is the first study of these associations in Egyptian sheep breeds. In conclusion, the polymorphisms revealed in this study could be used as genetic markers to improve reproductive efficiency during the unfavorable season, and the obtained desirable genotypes could be considered in new genetic selection schemes.

Impact of seminal plasma superoxide dismutase and glutathione peroxidase on cryopreserved buffalo spermatozoa.

Fifty semen samples were collected from sixteen buffalo-bulls (4-10 years old) and evaluated before cryopreservation. The activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) as well as the levels of glutathione (GSH) and malondialdehyde (MDA) were assayed in the seminal plasma before freezing. Aspartate aminotransferase (AST) activity and cholesterol content were assayed in seminal plasma before freezing and after thawing. Results revealed the presence of SOD and GPx activities (0.07 ± 0.01 U/ml and 14.59 ± 0.50 nmol/min/ml, respectively) in buffaloes' seminal plasma. SOD activity was positively correlated with both of GSH level and GST activity in seminal plasma, and showed an inverse relationship with both cholesterol efflux and post-thaw abnormal tails of buffalo spermatozoa. A positive correlation was found between GPx activity in seminal plasma and abnormal tails and an inverse relationship with both post-thaw viability indices and increased motility in response to PTx. GST activity showed a positive correlation with the increased motility after addition of PTx and negative correlations with both of cholesterol level and AST activity. MDA levels were negatively correlated with motility after addition of PTx and positive correlations with both post-thaw abnormal acrosomes and tails. Buffalo seminal plasma contains high activities of SOD, GPx and GST enzymes and GSH levels that have an influence on the functional competence of cryopreserved spermatozoa.

Polymorphism of insulin-like growth factor I gene among chicken breeds in Egypt.

The insulin-like growth factor I (IGF-I) regulates growth, protein synthesis, and cell proliferation and differentiation in vertebrates. Polymorphisms of IGF-I gene transcripts of three breeds of chicken were assessed. The associations of these polymorphisms with the growth rate of the studied breeds were also evaluated. Total RNA was isolated from chicken livers, and the IGF-I gene was amplified from each breed RNA by RT-PCR using specific primers flanking a certain region of the gene. The amplified RT-PCR products were formed to identify the transcripts and to correlate them to the phenotype of growth, by performing single stranded conformation polymorphism (SSCP) analysis for genotype identification. In this report, we describe how SSCP analysis of RT-PCR products can be used to evaluate the transcript expression pattern of avian IGF-I polymorphism, and their effect on the growth traits of chickens.

Antimutagenic efficacy of some natural compounds on cyclophosphamide-induced p53 alterations.

Mutations in the p53 tumour suppressor gene have been associated with chemical carcinogens. Natural antimutagens are promising modulators for reducing the cancer risk. The present study was carried out to assess the protective efficacy of some natural antimutagens against p53 alterations. We investigated the ability of curcumin (100 mg/kg BW) and chlorophyllin (3 mg/kg BW) pretreatment, for three times per week for three successive weeks, to inhibit mutations induced by intraperitoneal injection of a single dose of 40 mg/kg BW of cyclophosphamide (CP). Forty male albino rats were assigned into four groups: control nontreated group, CP-treated group, curcumin-CP-treated group, and chlorophyllin-CP-treated group. Liver samples were collected for DNA isolation two days after CP injection. The isolated DNA was used in single-strand conformational polymorphism (SSCP) analysis of polymerase chain reaction (PCR)-amplified products of four regions: two in exon 5, one in exon 6, and one in exon 7. The amplified products of p53 different regions were found to be in the expected molecular size of the designed primers. SSCP analysis of these amplified products showed that CP-induced mutation in the p53 gene was found only in exon 7 shifting its electrophoretic mobility. Chlorophyllin treatment prior to CP injection had a more potent protective efficacy (80%) than that with curcumin (33.3%).

Conserved and non-conserved loci of the glucagon gene in old world ruminating ungulates.

The homology and diversification of genomic sequence encoding glucagon gene among native Egyptian buffalos, camel and sheep were tested using cattle as model. Oligodeoxynucleotide primers designed from the available GenBank data were used for PCR probing of the glucagon gene encoding sequence at different loci. The DNA oligomer probes were constructed to flank either the whole gene encoding sequence or different intra-gene encoding sequences. The PCR products were visualized using agarose gel electrophoresis. All species showed a same size band of prepro-glucagon when PCR was used to amplify the whole gene encoding sequence. In contrary, amplifications of different intra-gene loci failed to give the same results. The results indicated variable degrees of diversity among old world ruminating ungulates in the glucagon gene encoding sequence. Compared with other ruminants, the variation appears predominantly in camel. Surprisingly, the similarity in size between both amplification products of whole gene encoding sequence and the proposed size of glucagon cDNA definitely excludes the possibility of large intervening introns spanning the genomic sequence of the glucagon gene in these species. This indicates that, in contrast to other tested mammals, the glucagon gene includes an essentially full-length copy of glucagon mRNA. The study revealed a possible new aspect of glucagon gene evolution in order to correlate its corresponding protein function among different ruminant species.

Isolation and characterization of raw heparin from dromedary intestine: evaluation of a new source of pharmaceutical heparin.

Heparin, a heterogeneous anionic polysaccharide, is the glycosaminoglycan (GAG) used clinically an anticoagulant. This anticoagulant activity is primarily derived from its binding to the serine protease inhibitor antithrombin III, a potent inhibitor of thrombin (factor IIa) and factor Xa. Heparin is a complex natural product and its in vitro synthesis is not yet possible due to the difficulty of organizing the many biosynthetic enzymes required for its synthesis. The principle natural sources for heparin include porcine intestine and bovine lung. These two sources pose concerns for religious and health reasons, respectively. To circumvent these concerns, GAG from the intestinal tissue of one humped camel was isolated. Chemical characterization of this newly isolated GAG and spectroscopic analysis by 1D and 2D 1H-NMR were undertaken. Unsaturated disaccharide compositional analysis was performed on the enzymatically depolymerized GAG and the molecular weight of the isolated GAG was determined by gradient polyacrylamide gel electrophoresis. Anticoagulant activity of the newly isolated GAG was tested by using an anti-factor Xa assay. The results of these studies suggest that the GAG from one humped camel intestine is a mixture of heparin and heparan sulfate and represents an alternative source of heparin.