Evaluation of phthalate migration potential in vacuum-packed.

In recent years, the presence and migration of PAEs in packaging materials and consumer products has become a serious concern. Based on this concern, the aim of our study is to determine the possible migration potential and speed of PAEs in benthic fish stored in vacuum packaging, as well as to monitor the storage time and type as well as polyethylene (PE) polymer detection.As a result of the analysis performed by µ-Raman spectroscopy, 1 microplastic (MP) of 6 µm in size was determined on the 30th day of storage in whiting fish muscle and the polymer type was found to be Polyethylene (PE) (low density polyethylene: LDPE). Depending on the storage time of the packaging used in the vacuum packaging process, it has been determined that its chemical composition is affected by temperature and different types of polymers are formed. 10 types of PAEs were identified in the packaging material and stored flesh fish: DIBP, DBP, DPENP, DHEXP, BBP, DEHP, DCHP, DNOP, DINP and DDP. While the most dominant PAEs in the packaging material were determined as DEHP, the most dominant PAEs in fish meat were recorded as BBP and the lowest as DMP. The findings provide a motivating model for monitoring the presence and migration of PAEs in foods, while filling an important gap in maintaining a safe food chain.

Whole-Mount Immunohistochemical and Immunofluorescence Assays in Zebrafish Embryos.

Zebrafish embryos are an important organism used as an in vivo model in a wide variety of disciplines from the past to the present. Immunohistochemistry analyses are an important method used to determine the localization of specific antigens in tissue sections with labeled antibodies depending on antigen-antibody interactions in zebrafish embryos. Immunofluorescence assays are an immunohistochemistry method that uses fluorophores to determine diverse cellular antigens. Zebrafish embryos and larvae, with their small size, are the most ideal model organisms for whole-mount immunohistochemical and immunofluorescent methods today. The small size of these organisms allows simultaneous evaluation of different tissues and organs, and results are obtained in a shorter time. In this section, whole-mount immunohistochemical and immunofluorescent analysis methods in zebrafish embryos, and larvae are summarized in detail, taking into account different studies and recent advances.

Mitigation potential of zingerone and rutin on toxicity mechanisms of nickel to zebrafish based on morphological, DNA damage and apoptosis outcome analysis.

Although nickel (Ni) is an important cofactor for various enzymes in biological systems, it can cause serious problems when insufficient or excessive in an organism. Therefore, it is very important to investigate Ni in biological systems, especially in cells with its related pathogenic mechanism. This study was carried out to demonstrate the effects of zingerone (ZO) and rutin (RN) administration against nickel chloride (NiCl2) toxicity on neurobehavioral performance and brain oxidative status in zebrafish (Danio rerio) embryos/larvae on histological perspective. The experimental design of the study, which included twenty groups of fish, each containing 10 embryos, was prepared as semi-static and the trial continued for 96 hpf. In the obtained findings, it was determined that ZO and RN had a mitigating effect in this toxicity table where Ni caused oxidative stress in zebrafish larvae, induced DNA damage and apoptosis. A similar picture is valid for malformation processes as well as survival and hatching rates. These results showed that nickel is toxic to developing embryos via acting different mechanisms. In conclusion, we observed that ZO and RN have a greater effect on physiology, DNA damage and apoptosis than gross morphology, with a significant ameliorative effect.

Has PdCu@GO effect on oxidant/antioxidant balance? Using zebrafish embryos and larvae as a model.

Industrial products containing PdCu@GO can gain access to the aquaculture environment, causing dangerous effects on living biota. In this study, the developmental toxicity of zebrafish treated with different concentrations (50, 100, 250, 500 and 1000 µg/L) of PdCu@GO was investigated. The findings showed that PdCu@GO administration decreased the hatchability and survival rate, caused dose-dependent cardiac malformation. Reactive oxygen species (ROS) and apoptosis were also inhibited in a dose-dependent manner, with acetylcholinesterase (AChE) activity affected by nano-Pd exposure. As evidence for oxidative stress, malondialdehyde (MDA) level increased and superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (GPx) activities and glutathione (GSH) level decreased due to the increase in PdCu@GO concentration. Our research, it was determined that the oxidative stress stimulated by the increase in the concentration of PdCu@GO in zebrafish caused apoptosis (Caspase-3) and DNA damage (8-OHdG). Stimulation of ROS, inflammatory cytokines, tumor Necrosis Factor Alfa (TNF-α) and interleukin - 6 (IL-6), which act as signaling molecules to trigger proinflammatory cytokine production, induced zebrafish immunotoxicity. However, it was determined that the increase of ROS induced teratogenicity through the induction of nuclear factor erythroid 2 level (Nrf-2), NF-κB and apoptotic signaling pathways triggered by oxidative stress. Taken together with the research findings, the study contributed to a comprehensive assessment of the toxicological profile of PdCu@GO by investigating the effects on zebrafish embryonic development and potential molecular mechanisms.

Microplastic-induced oxidative stress response in turbot and potential intake by humans.

Microplastic (MP) pollution has become a health concern subject in recent years. Althoughann increasing number of studies about the ingestion of microplastics by fish, research on the oxidative stress response to MPs in natural environments is quite limited. In this study, the identification and characterization of MPs in gill (G), muscle tissues (M), and gastrointestinal tract (GI) of turbot (Scophthalmus maximus) were evaluated. Oxidative damage of MPs on the brain (B), liver (L), gill (G), and muscle (M) tissues as well as their effect on superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), paraoxonase (PON), arylesterase (AR) myeloperoxidase (MPO), and malondialdehyde (MDA) biomarkers were evaluated. The potential transmission of MPs from muscle tissues to humans was examined. Results showed that gills contain the highest amounts of MPs, ethylene propylene is the most dominant polymer type, black and blue are the most common MP color, fiber is the most common shape, and 50-200 µm is the most common MP size. Results showed that MPs cause oxidative stress of tissues with inhibiting effect on enzyme activities and promoting impact on lipid peroxidation. The oxidative damage mostly affected the liver (detoxification organ) followed by gill tissue. The intake of MPS in the European Union was estimated by EFSA as 119 items/year, while in Turkey it is 47.88 items/year. This study shows that more research is needed in terms of ecosystem health and food chain safety. The risk assessment of MPs in living organisms and environmental matrices including food safety and human health should be considered a public health issue.

Thermal processing implications on microplastics in rainbow trout fillet.

Heat treatment is an inevitable step in making meat and meat products ready for human consumption. Researches on ready-to-eat foods had shown that foods can also contain microplastics (MPs). The source of the presence of MPs in foods is: air, raw materials, food production stages, or plastics used in packaging. This study was carried out to evaluate the possible effects of the sous-vide (So-Vc) technique applied in rainbow trout (Oncorhynchus mykiss) fillets at different temperatures and time intervals on MPs degradation or migration mechanisms and the level of uptake by humans. For this purpose, 7 treatment temperature × 3 various cooking times and So-Vc technique were applied on rainbow trout fillets. Then, in these fillets, MP presence, size, and shape were researched, as well as polymer types and possible levels of MP uptake by humans were determined. In the analyses, 1.27 ± 0.54 MP/g was found in 1 g of fish tissue. Dimensionally, 67% of MPs was detected as <50 µm and 8% of 500-1000 µm. The dominant shape was determined as a fragment, and the color was black. Six polymer types were determined. The results showed that high temperature (> 65°C) applications promoted polymer degradation. MP migration from packaging material to fillets was not detected. By calculations made on these findings, the lowest intake level by a human was estimated as 6140 MPs units/year. The obtained data provided the initial data to explore and optimize the current understanding of thermally processed products in terms of MPs. This study proved that the sous vide method causes polymer degradation at high temperatures and longer time periods.

In vivo toxicity assessment of Remazol Gelb-GR (RG-GR) textile dye in zebrafish embryos/larvae (Danio rerio): Teratogenic effects, biochemical changes, immunohistochemical changes.

Dyes, which are very important for various industries, have very adverse effects on the aquatic environment and aquatic life. However, there are limited studies on the toxic properties of dyes on living things. This research elucidated the sublethal toxicity of acute exposure of the textile dye remazol gelb-GR (RG-GR) using zebrafish embryos and larvae for 96 h. The 96 h-LC50 for RG-GR in zebrafish embryos/larvae was determined to be 151.92 mg/L. Sublethal 96 hpf exposure was performed in RG-GR concentrations (0.5; 1.0; 10.0; 100.0 mg/L) to determine the development of toxicity in zebrafish embryos/larvae. RG-GR dye affected morphological development, and decreased heart rate, hatching, blood flow, and survival rates in zebrafish embryos/larvae. The immunopositivity of 8-hydroxy 2 deoxyguanosine (8-OHdG) in larvae exposed to RG-GR at high concentrations was found to be intense. Depending on the RG-GR dose increase, some biochemical parameters such as glutathione peroxidase (GSH) level, acetylcholinesterase (AChE) activity, catalase (CAT) activities, superoxide dismutase (SOD), and nuclear factor erythroid 2 (Nrf-2) levels were detected to be decreased in larvae, while malondialdehyde (MDA) content, nuclear factor kappa (NF-kB), tumor necrosis factor-α (TNF-α), DNA damage (8-OHdG level), interleukin-6 (IL-6) and apoptosis (Caspase-3) levels were found to be increased. The experimental results revealed that RG-GR dye has high acute toxicity on zebrafish embryo/larvae.

Investigation of the Oxidative Stress Response of a Green Synthesis Nanoparticle (RP-Ag/ACNPs) in Zebrafish.

Silver nanoparticles (AgNPs) are prominent nanomaterials that are efficiently used in different industries including medical products, water treatment, and cosmetics. However, AgNPs are known to cause adverse effects on the ecosystem and human health. In this study, aqueous extract of Rumex patientia (RP) was used as a reducing and stabilizing agent in AgNP biosynthesis. The obtained activated carbon (AC) from Chenopodium album (CA) plant was combined with RP-AgNPs to synthesize RP-Ag/AC NPs. Next, the effects of these green synthesis RP-Ag/AC NPs on zebrafish (Danio rerio) embryos and larvae were investigated. First, we characterized the RP-Ag/AC NPs by using X-ray diffraction (XRD) and transmission electron microscopy (TEM) and determined LC50 value as 217.23 mg/L at 96 h. Next, the alterations in survival rate, hatching rate, and morphology of the larvae at 96 h were monitored. The survival rates decreased in a dose-dependent manner. Morphological defects such as yolk sac edema, pericardial edema, spinal curvature, and tail malformation in the NP-treated larvae were observed. RP-Ag/AC NPs stimulated the production of neuronal NOS (nNOS) and 8-OHdG in zebrafish brain tissues in a dose-dependent manner and enhanced neutrophil degeneration and necrosis at concentrations of 50 and 100 mg/L. Thus, the obtained data suggest that the green synthesis process is not sufficient to reduce the effect of oxidative stress caused by AgNPs on oxidative signaling.

Ecotoxicological Effects of Bimetallic PdNi/MWCNT and PdCu/MWCNT Nanoparticles onto DNA Damage and Oxidative Stress in Earthworms.

Bimetallic nanoparticles are synthesized using two different metal elements and used recently in many fields. However, limited studies related to the ecotoxic effects of nanoparticles available in the literature. The purpose of this study is to synthesize and characterize bimetallic PdCu/MWCNT and PdNi/MWCNT NPs and investigate their ecotoxic effects on earthworms. For this purpose, we injected approximately 20 µL of various concentrations of bimetallic PdCu/MWCNT and PdNi/MWCNT NPs (1, 10, 100, 1000, and 2000 mg/L) into the coelomic space of earthworms. We evaluated survival rate, malformations, reactive oxygen species (ROS) level, 8-OHdG content, and histopathological changes in earthworms at the 48th hour after exposure. PdCu/MWCNT and PdNi/MWCNT NPs were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD) pattern, and Raman-scattering spectroscopy. Toxicological examinations showed that PdCu/MWCNT NPs reduced the survival rate of earthworms (2000 mg/L, 84%) and caused various malformations (various lesions, thinning, swelling, and rupture), but nonsignificant effects of survival rate and malformations were observed in earthworms using PdNi/MWCNT NPs. The histopathological examinations of earthworm tissues exposed with PdNi/MWCNT determined that tissues in all treatment groups had a normal histological appearance. However, at a concentration of 2000 mg/L of PdCu/MWCNT NPs, atrophy in the longitudinal muscle layer and less degenerative cells in the epidermis layer were observed in earthworm tissues. It was determined that PdNi/MWCNT and PdCu/MWCNT NPs caused significant increases in ROS levels and 8-OHdG activity in earthworm tissues after 48 h. Finally, our results demonstrated that the toxicity of PdNi/MWCNT NPs was detected to be lower than PdCu/MWCNT NPs. However, both nanoparticles may pose a toxicological risk at high concentrations (1000 and 2000 mg/L). These findings will provide valuable information to studies on the use of PdNi/MWCNT NPs in wastewater treatment systems, industrial and medical fields, which have been determined to have less ecotoxicological risk.

Evaluation of the hydrogen-rich water alleviation potential on mercury toxicity in earthworms using ATR-FTIR and LC-ESI-MS/MS spectroscopy.

The toxic effects of mercury in earthworms and the potential alleviation effect of hydrogen-rich water (HRW) using ATR-FTIR and LC-MS analysis methods were investigated. Different concentrations of mercury chloride (H1: 5 µg/mL, H2: 10 µg/mL, H3: 20 µg/mL, H4: 40 µg/mL, and C1: control) and mercury chloride prepared in hydrogen-rich water (H5: 5 µg/mL, H6: 10 µg/mL, H7: 20 µg/mL, H8: 40 µg/mL, and C2: control) were injected into earthworms. The changes and reductions in some bands representing proteins, lipids, and polysaccharides (3280 cm-1, 2922 cm-1, 2855 cm-1, 1170 cm-1, and 1047 cm-1) showed that protective effects could occur in groups prepared with hydrogen-rich water. In the FTIR results, it was found that these bands in the H3 group were more affected and decreased by the influence of mercury on earthworms than the H7 group prepared with hydrogen. LC-MS analysis showed that the changes in some ions of the highest dose groups (H4 and H8) were different, and mercury caused oxidative DNA damage in earthworms. When the high-level application groups of mercury, i.e., H4 and H8 were compared with the controls, the ion exchange ([M + H] + ; m/z 283.1) representing the 8-Oxo-dG level in earthworms was higher in the H4 group than the H8 group. This reveals that HRW exhibited the potential ability to alleviate the toxic effects of mercury; however, a longer period of HRW treatment may be necessary to distinguish an obvious effect. The ATR-FTIR spectroscopy provided a rapid and precise method for monitoring the changes in biological tissues caused by a toxic compound at the molecular level.

A new record for the presence of microplastics in dominant fish species of the Karasu River Erzurum, Turkey.

It is known that microplastics (MPs) are increasingly detected in aquatic environments (sea and fresh water), and the presence of these pollutants have worrying potential effects on the biota. This study is the first research to measure and characterize MPs in freshwater ecosystems (inland waters) in Turkey. Accordingly, the identification and characterization of MPs in the gastrointestinal systems of fish by making samples of three species [chub (Squalius cephalus), common carp (Cyprinus carpio), and mossul bleak (Alburnus mossulensis)] of the carp family living in Karasu River in Erzurum. Hydrogen peroxide application and Fourier transform infrared-attenuated total reflectance (ATR-FTIR) analyses were done for this purpose. In the obtained results, 232 microplastics were found in all three fish gastrointestinal systems. While the highest determined color was black (39-58%), the most common shape was fiber (88%), fragment (6%, and pellet (6%); MPs in the range of maximum 1001-2000 mm were detected in size. Plastics are defined as polyethylene, polyester, poly (vinyl stearate), polyethylene terephthalate, polypropylene, and cellulose. Among the studied species, the most common type of plastic pollutants was found in S. cephalus. The findings indicated the presence of microplastics in dominant species. However, these findings will be basic information for future studies on living things and microplastics in inland waters.

Hydrogen-Rich Water Alleviates the Nickel-Induced Toxic Responses (Inflammatory Responses, Oxidative Stress, DNA Damage) and Ameliorates Cocoon Production in Earthworm.

In recent years, studies investigating the protective effect of hydrogen-rich water (HRW) against different diseases and the toxicity of some substances have attracted increasing attention. Here, we assessed the effects of hydrogen-rich water on different nickel-induced toxic responses (reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) of stress responses, histopathological changes) and cocoon production in earthworm model. Earthworms were randomly divided into two main groups: water (W) group including control (CW: ultrapure water), 10 (10W), 200 (200W), and 500 (500W), and hydrogen-rich ultrapure water (HRW) group including control (CHRW: hydrogen-rich ultrapure water), 10 (10HRW), 200 (200HRW), and 500 (500HRW) mg of nickel chloride kg-1 soil for 14 days. We found that cocoon production was less affected by the nickel exposure of earthworms in the 500HRW group compared to the 500W group. The ROS levels in 200HRW and 500HRW groups were less than that of 200W and 500W, respectively. The epithelial degeneration, epithelial necrosis, and necrosis in muscle fibers in tissues of earthworm were less damaged in 200HRW and 500HRW groups compared to 200W and 500W, respectively. HRW groups significantly reduced the expression of 8-OHdG induced by nickel exposure and inflammatory cytokine response including TNF-α. The study showed that hydrogen-rich water could alleviate the toxic effects of nickel-induced oxidative and inflammatory damages in earthworms. The HRW treatment known for its cheap and eco-friendly properties without any negative effects on the ecosystem can be used as a green method for alleviating the toxification effects of heavy metals in contaminated soil and increasing cocoon production of earthworms.

Microplastics in Tissues (Brain, Gill, Muscle and Gastrointestinal) of Mullus barbatus and Alosa immaculata.

The researches on MPs in commercial marine fish are very limited although in marine environments microplastic (MPs) pollution is a global problem. In this study, the presence, composition, and characterization of MPs in different tissues (brain, gill, muscle, and gastrointestinal tract) of commercial fish species [red mullet (Mullus barbatus) and pontic shad (Alosa immaculata Bennett 1835)] from the Black Sea were investigated. M. barbatus (demersal) and A. immaculata (pelagic) fish were preferred in the selection of fish species in order to represent demersal and pelagic environments. After dissected the fish, MPs were obtained from the tissues by extraction using the flotation method; then the MPs were counted and categorized according to shape, size, and color. The composition of the MPs was determined via ATR-FTIR spectroscopy. In terms of microplastic abundance in fish tissues, the gastrointestinal tract (40.0%) ranked first in both fish species, while the lowest MPs density was determined in brain tissues (7.0%). After the gastrointestinal tissue, gills were identified as the second tissue with the highest MPs density. Regardless of fish species, MPs characterization was mainly fibrous (51.0%), black colored (49.0%), and 50-200 µm in size (55.0%). Among the nine different polymers determined, polychloroprene (18.8%) and polyamide (15.0%) were found most frequently. This research provides data for tissue-based assessment of MPs in fish. The obtained data showed that MPs (one of the anthropogenic pollutants) are quite high in all tissues regardless of fish species. Moreover, it has emerged that these two fish species are suitable for monitoring microplastics in the study area.

Evaluation of different packaging methods and storage temperature on MPs abundance and fillet quality of rainbow trout.

There are many studies on microplastics (MPs) about the aquatic ecosystems and its components. However, there is limited study on the MPs abundance, identification and sources in processed seafood products which are manufactured for direct human consumption. In this study, rainbow trout (Oncorhynchus mykiss) fillets were packed with different packaging techniques and stored at two different temperatures (+4 and -20°C) for 21 days. The presence, shape, size and polymer type of MPs were determined by ATR-FTIR on certain days (7, 14 and 21 days) in fillets during storage. The chemical quality changes in fillets [with pH, thiobarbituric acid reactive substrate (TBARS), and total volatile basic nitrogen (TVB-N) data] were monitored and the effect of MPs presence was evaluated. At the last step, the estimated MPs intake level in humans was determined with considering the presence of MPs (determined in fillets). The presence of MPs was determined the most in the Polystyrene plate + wrapped film (S) group and the least in the Chitosan film + Polystyrene plate + wrapped film (C) group. When evaluated in terms of chemical parameters, although good results were obtained in all samples stored at - 20°C, the presence of MPs was determined at a high level in fillets which stored at this temperature. As a result of the study, it was determined that the packaging type and storage temperature have significant effects on the presence of MPs and fillet quality.

Teratogenic and Neurotoxic Effects of n-Butanol on Zebrafish Development.

In recent years, n-butanol, a type of alcohol, has been widely used from the chemical industry to the food industry. In this study, toxic effects of n-butanol's different concentrations (10, 50, 250, 500, 750, 1,000, and 1,250 mg/L) in Zebrafish Danio rerio embryos and larvae were investigated. For this purpose, Zebrafish embryos were exposed to n-butanol in acute semistatic applications. Teratogenic effects such as cardiac edema, scoliosis, lordosis, head development abnormality, yolk sac edema, and tail abnormality were determined at different time intervals (24, 48, 72, 96, and 120 h). Additionally, histopathological abnormalities such as vacuole formation in brain tissue and necrosis in liver tissue were observed at high doses (500, 750, and 1,000 mg/L) in all treatment groups at 96 h. It was determined that heart rate decreased at 48, 72, and 96 h due to an increase in concentration. In addition, alcohol-induced eye size reduction (microphthalmia) and single eye formation (cyclopia) are also among the effects observed in our research findings. In conclusion, n-butanol has been observed to cause intense neurotoxic, teratogenic, and cardiotoxic effects in Zebrafish embryos and larvae.

Textile dyes Maxilon blue 5G and Reactive blue 203 induce acute toxicity and DNA damage during embryonic development of Danio rerio.

Common textile dyes used in various industrial sectors are organic compounds and considered for the aquatic environment as pollutants. The textile dye industry is one of the main sectors that have serious impacts on the environment due to a large amount of wastewater released into the ecosystem. Maxilon blue 5G (MB-5G) and Reactive Blue 203 (RB-203) are widely used textile dyes. However, their potential toxicity on living organisms remains to be elucidated. Here, we investigate the acute toxicity and genotoxicity of MB-5G and RB-203 dyes using the zebrafish embryos/larvae. Embryos treated with each dye for 96 h revealed LC50 values of acute toxicity as 166.04 mg L-1 and 278.32 mg L-1 for MB-5G and RB 203, respectively. When exposed to MB-5G and RB-203 at different concentrations (1, 10, and 100 mg L-1) for 96 h, the expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, significantly increased in brain tissues as compared to control. MB-5G and RB-203 resulted in common developmental abnormalities including tail malformation, microphthalmia, pericardial edema, curved body axis, and yolk sac/pericardial edemas. Moreover, at its highest dose (100 mg L-1), RB-203 caused premature hatching after 48 h, while MG-5G did not. Our results collectively reveal that the textile dyes MB-5G and RB-203 cause genotoxicity and teratogenicity during embryonic and larval development of zebrafish. Thus, it is necessary to eliminate these compounds from wastewater or reduce their concentrations to safe levels before discharging the textile industry wastewater into the environment.

The effects of n-butanol on oxidative stress and apoptosis in zebra fish (Danio rerio) larvae.

In recent years, n-butanol has growing use in many areas, including the food industry. In this study, acute toxic effects of n-butanol to zebrafish (Danio rerio) larvae by applying different concentrations (10, 50, 250, 500, 750, 1000 and 1250 mg/L) to embryos were evaluated. For this purpose the data of oxidative stress, antioxidant - acetyl cholinesterase enzyme activities, malondialdehyde level and apoptosis were taken into consideration. At the end of the 96 h, antioxidant (Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)) and acetylcholinesterase (AChE) enzyme activities were decreased, however lipid peroxidation level, apoptotic cells, and reactive oxygen species increased (p < .05). As a result, it has been observed that high concentrations of n-butanol with its amphiphilic structure causes quite intense toxic effects in zebrafish embryos.

Immunofluorescence/fluorescence assessment of brain-derived neurotrophic factor, c-Fos activation, and apoptosis in the brain of zebrafish (Danio rerio) larvae exposed to glufosinate.

In this study, we investigated the potential neuro-toxicological mechanism of the glufosinate in the brain of zebrafish larvae in terms of BDNF and c-Fos proteins by evaluating apoptosis, immunofluorescence BDNF, and c-FOS activation. We also measured survival rate, hatching rate, and body malformations during 96 h exposure time. For this purpose, zebrafish embryos were treated with graded concentrations of dosing solutions (0.5, 1, 3, and 5 ppm) of glufosinate. End of the treatment, acridine orange staining was used to detect apoptotic cells in the brain of zebrafish larvae at 96 hpf. Texas Red and FITC/GFP labeled protein-specific antibodies were used in immunofluorescence assay for BDNF and c-FOS, respectively. The results have indicated that exposure to glufosinate caused to embryonic death, hatching delay, induction of apoptosis, increasing of c-FOS activity and the level of BDNF in a dose-dependent manner. As a conclusion, we suggested that c-Fos might play a role in the regulation of BDNF which responses to prevent the cell from apoptosis even in case of unsuccessful in zebrafish larvae exposed to glufosinate.

Determination of developmental toxicity of zebrafish exposed to propyl gallate dosed lower than ADI (Acceptable Daily Intake).

Propyl gallate (PG) is an antioxidant substance widely used in cosmetics, pharmaceutical and food industries. The aim of this study was to evaluate the potential toxic effect of PG injected to zebrafish embryos. To this end, zebrafish embryos were exposed to PG with 0, 1, 10 and 50 ppm concentrations which are lower than ADI and were monitored at 24, 48, 72 and 96 hpf. Survival rate, hatching rate and malformations were evaluated during this period. Moreover, it has been detected the accumulation of fluorescence signal of ROS and apoptotic cell in whole body at the end of 96 hpf. According to results, survival rate slightly decreased in highest concentration, and PG accelerated hatching in 1 and 10 ppm concentrations whereas delayed in 50 ppm concentration. In addition, it has been detected accumulation of fluorescence signal of ROS and apoptotic cells in a dose dependent-manner. Consequently, it has been considered that increased embryonic or larval malformation in this study may have been caused by ROS-induced apoptosis. The obtained data suggested that the developmental toxicity caused by PG and/or multiple hydroxyl groups arose when PG hydrolyze to gallic acid is probably triggered by the induction of ROS formation and consequent apoptosis.

Immunofluorescence evaluation of 4-hydroxynonenal and 8-hydroxy-2-deoxyguanosine activation in zebrafish (Daino rerio) larvae brain exposed (microinjected) to propyl gallate.

Propyl gallate (PG) is a chemical compound obtained by esterification of propanol with gallic acid. Due to its antioxidative properties, it is widely used in cosmetics and pharmaceutical industries as well as to protect the oils in foods such as butter, milk-based desserts, chewing gum, mayonnaise, meat, soups, cereals, spices and seasonings from rancidity. This study has been designed to assessment 8-OHdG and 4-HNE activity, and histopathological changes in the brain tissues of zebrafish larvae, which is a lecithotrophic organism, after 96 h of PG exposure via microinjecting to yolk sac of embryo. To this end, approximately 5 nL of various concentrations of PG (1, 10, and 50 ppm) has been injected into yolk sac of fertilized embryo (final exposure concentrations are 5, 50, 250 pg/egg) with micro manipulator system. After 96 h exposure time, propyl gallate caused immunofluorescence positivity of 8-OHdG and 4-HNE in the brain tissues of zebrafish larvae. PG was not effect brain tissue histopathological in low concentrations (1 and 10 ppm) but highest concentration (50 ppm) caused degenerative changes in brain. These results suggests that PG treatment could lead oxidative DNA damage by causing an increase 8-OHdG and 4-HNE activities. This strategy will enable us to better understand the mechanisms of propyl gallate in brain tissues of zebrafish larvae.