Fagr Kh. Abdel-Gawad

Roles of reactive oxygen species in the spermatogenesis regulation.

Spermatogenesis is a complex process of male germ cells proliferation and maturation from diploid spermatogonia, through meiosis, to mature haploid spermatozoa. The process involves dynamic interactions between the developing germ cells and their supporting Sertoli cells. The gonadal tissue, with abundance of highly unsaturated fatty acids, high rates of cell division, and variety of testis enzymes results very vulnerable to the overexpression of reactive oxygen species (ROS). In order to address this risk, testis has developed a sophisticated array of antioxidant systems comprising both enzymes and free radical scavengers. This chapter sets out the major pathways of testis generation, the metabolism of ROS, and highlights the transcriptional regulation by steroid receptors of antioxidant stress enzymes and their functional implications. It also deals with of the advantages of the system biology for an antioxidant under steroid control, the major selenoprotein expressed by germ cells in the testis, the phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4) having multiple functions and representing the pivotal link between selenium, sperm quality, and species preservation.

Electron Spin Resonance (ESR) for the study of Reactive Oxygen Species (ROS) on the isolated frog skin (Pelophylax bergeri): A non-invasive method for environmental monitoring

Background Reactive oxygen species (ROS) in biological tissues of elected biosentinels represent an optimal biomarker for eco‐monitoring of polluted areas. Electron spin resonance (ESR) is the most definitive method for detecting, quantifying and possibly identifying radicals in complex systems. Objective A non‐invasive method for monitoring polluted areas by the quantitative determination of ROS in frog skin biopsy is presented. Methods We assessed by ESR spectroscopy the ROS level in adult male of Pelophylax bergeri, specie not a risk of extinction, collected from the polluted Sarno River (SA, Italy) basin. The spin‐trap ESR method was validated by immunohistochemical analysis of the well‐assessed pollution biomarkers cytochrome P450 aromatase 1A (CYP1A) and glutathione S‐transferase (GST), and by determining the poly(ADPribose) polymerase (PARP) and GST enzymatic activity. Results ROS concentration in skin samples from frogs collected in the polluted area is significantly higher than that determined for the unpolluted reference area. Immunohistochemical analysis of CYP1A and GST supported the reliability of our approach, even in the absence of evident morphological and ultrastructural differences. PARP activity assay, connected to possible oxidative DNA damage, and the detoxification index by GST enzymatic assay give statistically significant evidence that higher levels of ROS are associated to alterations of the different biomarkers. Conclusions ROS concentration, measured by ESR on isolated frog skin, through the presented non‐lethal method, is a reliable biomarker for toxicity screening and represents a useful basic datum for future modelling studies on environmental monitoring and biodiversity loss prevention. HighlightsROS levels in frog skin biopsy were determined by spin‐trapping ESR spectroscopy.Skin of frogs collected in polluted areas contains higher ROS concentration.ROS levels determined in skin biopsy follow the same trend of those from liver samples.Immunohistochemical analyses and enzymatic activity tests validate the method.ROS concentration determined by ESR in biosentinel biopsy is an effective biomarker.

Seawater Polluted with Highly Concentrated Polycyclic Aromatic Hydrocarbons Suppresses Osteoblastic Activity in the Scales of Goldfish, Carassius auratus

We have developed an original in vitro bioassay using teleost scale, that has osteoclasts, osteoblasts, and bone matrix as each marker: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Using this scale in vitro bioassay, we examined the effects of seawater polluted with highly concentrated polycyclic aromatic hydrocarbons (PAHs) and nitro-polycyclic aromatic hydrocarbons (NPAHs) on osteoblastic and osteoclastic activities in the present study. Polluted seawater was collected from two sites (the Alexandria site on the Mediterranean Sea and the Suez Canal site on the Red Sea). Total levels of PAHs in the seawater from the Alexandria and Suez Canal sites were 1364.59 and 992.56 ng/l, respectively. We were able to detect NPAHs in both seawater samples. Total levels of NPAHs were detected in the seawater of the Alexandria site (12.749 ng/l) and the Suez Canal site (3.914 ng/l). Each sample of polluted seawater was added to culture medium at dilution rates of 50, 100, and 500, and incubated with the goldfish scales for 6 hrs. Thereafter, ALP and TRAP activities were measured. ALP activity was significantly suppressed by both polluted seawater samples diluted at least 500 times, but TRAP activity did not change. In addition, mRNA expressions of osteoblastic markers (ALP, osteocalcin, and the receptor activator of the NF-κB ligand) decreased significantly, as did the ALP enzyme activity. In fact, ALP activity decreased on treatment with PAHs and NPAHs. We conclude that seawater polluted with highly concentrated PAHs and NPAHs influences bone metabolism in teleosts.

Blue Economy and Biodiversity Surveillance: Fish Caviar Substitute Rapid Discrimination

The species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in ecosystem and fisheries research and control, as well as processed fish products labelling in consumer protection. Here, we report a rapid blue biotechnological method applied for egg and fish species discrimination. The amplification of a region of the mitochondrial genome, the cytochrome b, by using the Polymerase Chain Reaction (PCR) permits seafood products species identification. The obtained PCR-products were cut with different restriction endonucleases resulting in species-specific Restriction Fragment Length Polymorphisms (RFLP), which allowed to discriminate, in one step, between different types of caviar substitute species. The following analysis of mitochondrial DNA to control the labelling between caviar substitute species of Cyclopterus lumpus, Mallotus villosus, Trisopterus minutus minutus and the caviar, Acipenser baerii and their monitoring and surveillance is suitable with only one restriction enzyme, MboII. This blue method, applied to a very significant number of samples, retrieved online, allowed a rapid and economic identification of the species, with high percentage of correct identification for fish caviar substitute.

Spectroscopic analyses and genotoxicity of dioxins in the aquatic environment of Alexandria

Dioxins have global concerns because of the bioaccumulation tendency and persistency in the environment. Water, seabream Pagrus auratus and seabass Dicentrarchus labrax samples were collected from Abu Qir, Alexandria to evaluate the concentration of dioxin. Fourier Transform Infrared Spectrometer (FTIR) and molecular modeling was applied for elucidating the molecular structure of fish samples. Furthermore, HPLC with UV detection was used to determine the concentration of dioxins (2,8-dichloro dibenzo-p-dioxin). RT-PCR assay was conducted to verify the expression of some immune genes in the fish species as a result of water pollution. The average detected concentrations varied from 0.2 to 1.3μg/l. Gene expression revealed that MHC class 1 and C3 were highly upregulated in liver and muscle of seabass and seabream while T2BP was highly regulated in seabass liver and seabream muscle and seabass muscle for transferrin, FTIR and molecular modeling indicate that dioxin finds its way to fish protein.

Fisheries and biodiversity along Mediterranean Sea: Italian and Egyptian coast overview

Mediterranean fish species living along Italian (Gaeta) and Egyptian (Alexandria) coasts were analyzed using DNA barcodes for molecular identification. Mitochondrial Cytochrome c Oxidase subunit 1 (COI) gene was sequenced from 31 different marine species to test whether the morphology-based assignment of individuals into 19 families, 6 orders was supported by DNA-based species delimitation and Neighbour Joining cladogram. All COI rRNA gene barcodes were matched with reference sequences of expected species, according to morphological identification. Neighbour joining tree was drawn based on COI rRNA gene and the majority of specimens clustered in agreement with their taxonomic classification. Our results updated Mediterranean edible fish knowledge providing graphical resources, taxonomical and bioinformatics references, improving the genetic fish database and the basic molecular information to strengthen the science–policy interface for biodiversity and ecosystem services as conservation, blue economy, and long-term human well-being.

Caves Biodiversity in the Marine Area of Riviera d'Ulisse Regional Park, Italy: Grotta del Maresciallo Overview

The caves have a significant economic importance in their role as tourist attraction and are of great importance for the presence of some rare species. The fauna identification in the “Grotta del Maresciallo” cave, situated in the Riviera di Ulisse Regional Park, started in march 2013 by a visual census and molecular approach. The studies allowed to detect 12 classes, 46 families, 47 species with their ecological niches, and the percentage contribution of each group to the Mediterranean marine cave diversity. Furthermore, we report the presence of two thermophilic species, as the star coral, Astroides calycularis (Pallas, 1766) and the goldblotch grouper, Epinephelus costae (Steindachner, 1878); the tropical species, the ringneck blenny Parablennius pilicornis (Cuvier, 1829); the endangered species, the dusky grouper Epinephelus marginatus (Lowe, 1834) included in the IUCN Red List; the rare species, the black brotula Grammonus ater (Risso, 1810), and some uncommon species such as the golden coral shrimp, Stenopus spinosus (Risso, 1826) and the spotted bumblebee shrimp, Gnatophillum elegans (Risso, 1816). Species of economic and medical interest were also recorded. In a DNA barcoding approach, Neighbour Joining (NJ) phylogenetic tree of 25 mitochondrial cytochrome oxidase subunit I species sequences, indicates that COI gene is suitable for an unambiguous identification. This first geological and biological attempt at the Marine Area of the “Grotta del Maresciallo” provides useful indications to focus future investigations, and may become a potential management tool for local administrations to protect these habitats.