Zebrafish Tissues Research Articles

Methylglyoxal-mediated Gpd1 activation restores the mitochondrial defects in a yeast model of mitochondrial DNA depletion syndrome

Human MPV17, an evolutionarily conserved mitochondrial inner-membrane channel protein, accounts for the tissue-specific mitochondrial DNA depletion syndrome. However, the precise molecular function of the MPV17 protein is still elusive. Previous studies showed that the mitochondrial morphology and cristae organization are severely disrupted in the MPV17 knockout cells from yeast, zebrafish, and mammalian tissues. As mitochondrial cristae morphology is strictly regulated by the membrane phospholipids composition, we measured mitochondrial membrane phospholipids (PLs) levels in yeast Saccharomyces cerevisiae MPV17 ortholog, SYM1 (Stress-inducible Yeast MPV17) deleted cells. We found that Sym1 knockout decreases the mitochondrial membrane PL, phosphatidyl ethanolamine (PE), and inhibits respiratory growth at 37 ̊C on rich media. Both the oxygen consumption rate and the steady state expressions of mitochondrial complex II and super-complexes are compromised. Apart from mitochondrial PE defect a significant depletion of mitochondrial phosphatidyl-choline (PC) was noticed in the sym1∆ cells grown on synthetic media at both 30 ̊C and 37 ̊C temperatures. Surprisingly, exogenous supplementation of methylglyoxal (MG), an intrinsic side product of glycolysis, rescues the respiratory growth of Sym1 deficient yeast cells. Using a combination of molecular biology and lipid biochemistry, we uncovered that MG simultaneously restores both the mitochondrial PE/PC levels and the respiration by enhancing cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase 1 (Gpd1) enzymatic activity. Further, MG is incapable to restore respiratory growth of the sym1∆gpd1∆ double knockout cells. Thus, our work provides Gpd1 activation as a novel strategy for combating Sym1 deficiency and PC/PE defects.

Cell-type-specific responses to the microbiota across all tissues of the larval zebrafish.

Animal development proceeds in the presence of intimate microbial associations, but the extent to which different host cells across the body respond to resident microbes remains to be fully explored. Using the vertebrate model organism, the larval zebrafish, we assessed transcriptional responses to the microbiota across the entire body at single-cell resolution. We find that cell types across the body, not limited to tissues at host-microbe interfaces, respond to the microbiota. Responses are cell-type-specific, but across many tissues the microbiota enhances cell proliferation, increases metabolism, and stimulates a diversity of cellular activities, revealing roles for the microbiota in promoting developmental plasticity. This work provides a resource for exploring transcriptional responses to the microbiota across all cell types of the vertebrate body and generating new hypotheses about the interactions between vertebrate hosts and their microbiota.

Using machine learning to parametrize large scale tumor simulations.

While clinical imaging of tissues focuses on macroscopic tumors, many experiments investigate only small clusters of cells. We aim on providing a scale-bridging link by performing large scale tissue simulations. We employ highly parallelized code in an HPC setting to simulate mm-sized virtual tissues such as embryogenetic zebrafish tissue or breastcancer tumors with more than a million µm-resolved individual cells. We deploy Cells in Silico (CiS), which combines a cellular potts model with an agent based layer and is thus capable of accurately representing many physical and biological properties, such as individual cell shapes, cell division, cell motility etc. Using a model with such a strong representational capacity poses the task of adjusting a large number of parameters to reproduce experimental findings. Prior work has attempted to characterize the similarity between experimental and simulated data by extracting different features and using statistical tests to establish a distance measure. This work highlights how this difficult task can be circumvented by training neural networks to distinguish between experimental and simulated data while simultaneously optimizing the model parameters to maximize the error rate of the network.

Bioaccumulation, metabolism and toxicological effects of chiral insecticide malathion and its metabolites in zebrafish (Danio rerio)

The bioaccumulation, metabolism, tissue-specific distribution and toxicity of the widely used organophosphorous pesticide malathion to zebrafish were investigated on an enantiomeric level for evaluating the environmental risks. The metabolites were also monitored and evaluated. Malathion was metabolized by zebrafish very fast with the half-life of 0.12 d and showed a middle accumulation capacity in zebrafish with bioaccumulation factor (BCF) of 12.9 after a 15-d exposure. Brain could enrich higher concentration of malathion than other tissues. The metabolites malaoxon, malathion/malaoxon monocarboxylic acid (DMA), malathion/malaoxon dicarboxylic acid (DCA), dimethylthiophosphate (DMTP) and dimethyldithiophosphate (DMDTP) were found, in which DMTP and DCA were in higher level, indicating the metabolism was mainly induced by carboxylesterase degradation. The accumulation of malathion and malaoxon was stereoselective in zebrafish tissues, exhibiting S-enantiomer preferentially enriched. The acute toxicity test showed rac-malathion was low toxic to zebrafish, which was 1.2 and 1.6 folds more toxic than S-malathion and R-malathion respectively. Malaoxon was highly toxic to zebrafish and approximately 32 times more toxic than malathion. The toxicity of other metabolites was lower than malathion. Malathion could cause an apparent developmental toxicity to zebrafish embryo, including bradycardia, hatchability reduction and deformity, and abnormal movement patterns in zebrafish larva. Chronic toxicity indicated that malathion and malaoxon induced oxidative damage and neurotoxicity in the liver, brain and gill of zebrafish, and malaoxon exhibited a relatively high injury to the zebrafish brain. The results can provide information for the comprehensive assessment of the potential risk of malathion to aquatic organisms and highlight the necessity of consideration of stereoselectivity and metabolites when systemically evaluating pesticides.

Environmental fate of five brominated flame retardants co-exposure in a water-sediment-zebrafish microcosm system: Enrichment, removal, and metabolism mechanisms

Brominated flame retardants (BFRs) are widely used in electronic products. After electronics obsolescence, BFRs are gradually released and enriched in the environment, thus leading to frequent occurrence and detection. However, the environmental fate of multiple flame retardants in aquatic microcosms has not been explored, and it mainly focused on the environmental monitoring of BFRs and the toxicity of a single BFR to aquatic organisms. To fill the gap, based on actual investigation, a water-sediment-zebrafish microcosm system was co-exposed to five concentrations (0.2, 1, 5, 25, and 50 mg/kg) of pentabromotoluene (PBT), hexabromobenzene (HBB), 1,2-bis(2,4,6-tribromophenoxy) ethane (BTBPE), decabromodiphenyl ethane (DBDPE), and decabromodiphenyl ether (BDE-209) for the enrichment (35 days) and clearance (10 days) experiments. The results showed that the liver, intestine, and gill were the top three tissues enriched in BFRs, which were 7504.54, 6936.99, and 3532.97 ng/g, respectively. BFRs can also be transferred between a generation. The compositions of BFRs in the tissues of zebrafish were also different, and the average compositions of DBDPE and BDE-209 were 36.2% and 28.6%, respectively, indicating that DBDPE and BDE-209 were more easily absorbed by zebrafish. Additionally, the average scavenging half-lives of PBT, HBB, BTBPE, DBDPE, and BDE209 were 4.01, 4.09, 2.90, 3.72, and 3.75 days, respectively, suggesting that these chemicals barely lasted long in zebrafish. The metabolic pathways of the five BFRs were also probed, and gradual debromination occurred in the organisms. These observations would provide a basis for the study of the fate and toxicology of BFRs in the water environment.

A study to assess the health effects of an anticancer drug (cyclophosphamide) in zebrafish (Danio rerio): eco-toxicity of emerging contaminants.

Cyclophosphamide (CP) is widely used for treating various kinds of cancer. Because of its high intake, metabolism and excretion, these anticancer medications have been detected in the aquatic environment. There is very limited data on the toxicity and effects of CP on aquatic organisms. The present study aims to assess the toxic effect of CP on certain oxidative stress biomarkers (superoxide dismutase-SOD, catalase-CAT, glutathione peroxidase-GPx, glutathione-GSH, glutathione S-transferases-GST and lipid peroxidation-LPO), protein, glucose, metabolising enzymes (aspartate aminotransferase-AST, alanine aminotransferase-ALT), and ion-regulatory markers (sodium ions-Na+, potassium ions-K+, and chloride ions-Cl-), and histology in the gills and liver of Danio rerio at environmentally relevant concentrations (10, 100 and 1000 ng L-1). Exposure to CP for 42 days led to a significant decrease in SOD, CAT, GST, GPx and GSH levels in the gills and liver tissues of zebrafish. The level of lipid peroxidation in the gills and liver tissues of zebrafish was significantly increased compared to the control group. Chronic exposure significantly changes protein, glucose, AST, ALT, Na+, K+ and Cl- biomarkers. Fish exposed to different levels of CP showed necrosis, inflammation, degeneration and hemorrhage in the gills and hepatic tissues. The observed changes in the studied tissue biomarkers were proportional to both dose and time. In conclusion, CP at environmentally relevant concentrations causes oxidative stress, energy demand, homeostasis disturbances, and enzyme and histological alterations in the vital tissues of zebrafish. These alterations were similar to the toxic effects reported in mammalian models.

Qualitative and quantitative analysis of accumulation and biodistribution of polystyrene nanoplastics in zebrafish (Danio rerio) via artificial freshwater

PSN-spiked water exposure medium facilitated the spread of PSN in zebrafish tissues. New developed methods of MALDI-TOF-MS enabled an accurate analysis of PSN features in an aquatic organism.

Fast Adaptive Optics in optically sectioned fluorescence microscopes for functional neuroimaging

We demonstrate how a novel approach for closed-loop Adaptive Optics (AO) specifically adapted to microscopy enables straightforward integration in Light-Sheet and Multiphoton microscopes, as well as fast aberration correction. We present corresponding experimental setups as well as first demonstrations of the benefits of the correction of sample-induced aberrations in zebrafish and mouse brain tissue, with the ultimate goal to enable high-speed, high-sensitivity functional imaging at large depths.

Frozen Block Tissue Staining for Eye Structure of Zebrafish Embryo.

Histology is a technique used to study the morphology of cell structures by cutting samples into thin sections. Histological cross-section and staining are the techniques needed to visualize the morphology of cell tissues. A suitable tissue staining experiment was created to observe changes in the retinal layer in zebrafish embryos. Zebrafish have a human-like visual system, retina, and eye structures. Due to the small size of zebrafish and undeveloped bones in the embryonic stage, the resistance through cross-section is inevitably small. Here, we present optimized protocol changes in eye tissue of zebrafish using frozen blocks.

Detection of Zebrafish Retinal Proteins by Infrared Western Blotting.

The zebrafish retina is a canonical vertebrate retina. Since the past few years, with the continually growing genetic toolbox and imaging techniques, zebrafish plays a crucial role in retinal research. This protocol describes a method to quantitatively evaluate the expression of Arrestin3a (Arr3a) and G-protein receptor kinase7a (Grk7a) in the adult zebrafish retina at protein levels by infrared fluorescence western blot. Our protocol can be easily adapted to measure protein levels in additional zebrafish tissues.

Fluoride exposure causes behavioral, molecular and physiological changes in adult zebrafish (Danio rerio) and their offspring

Fluoride exposure through drinking water, foods, cosmetics, and drugs causes genotoxic effects, oxidative damage, and impaired cognitive abilities. In our study, the effects of fluoride on anxiety caused by the circadian clock and circadian clock changes in a zebrafish model were investigated at the molecular level on parents and the next generations. For this purpose, adult zebrafish were exposed to 1.5 ppm, 5 ppm, and 100 ppm fluoride for 6 weeks. At the end of exposure, anxiety-like behaviors and sleep/wake behaviors of the parent fish were evaluated with the circadian rhythm test and the novel tank test. In addition, antioxidant enzyme activities and melatonin levels in brain tissues were measured. In addition, morphological, physiological, molecular and behavioral analyzes of offspring taken from zebrafish exposed to fluoride were performed. In addition, histopathological analyzes were made in the brain tissues of both adult zebrafish and offspring, and the damage caused by fluoride was determined. The levels of BMAL1, CLOCK, PER2, GNAT2, BDNF and CRH proteins were measured by immunohistochemical analysis and significant changes in their levels were determined in the F- treated groups. The data obtained as a result of behavioral and molecular analyzes showed that parental fluoride exposure disrupts the circadian rhythm, causes anxiety-like behaviors, and decreases the levels of brain antioxidant enzymes and melatonin in parents. In addition, delay in hatching, increase in death and body malformations, and decrease in blood flow velocity, and locomotor activity was observed in parallel with dose increase in offspring. On the other hand, an increase in offspring apoptosis rate, ROS level, and lipid accumulation was detected. As a result, negative effects of fluoride exposure on both parents and next generations have been identified.

Solid-State NMR-Based Metabolomics Imprinting Elucidation in Tissue Metabolites, Metabolites Inhibition, and Metabolic Hub in Zebrafish by Chitosan.

In this study, we demonstrated that chitosan-applied zebrafish (Danio rerio) tissue metabolite alteration, metabolic discrimination, and metabolic phenotypic expression occurred. The spectroscopy of solid-state 1H nuclear magnetic resonance (ss 1H-NMR) has been used. Chitosan has no, or low, toxicity and is a biocompatible biomaterial; however, the metabolite mechanisms underlying the biological effect of chitosan are poorly understood. The zebrafish is now one of the most popular ecotoxicology models. Zebrafish were exposed to chitosan concentrations of 0, 50, 100, 200, and 500 mg/L, and the body tissue was subjected to metabolites-targeted profiling. The zebrafish samples were measured via solvent-suppressed and T2-filtered methods with in vivo zebrafish metabolites. The metabolism of glutamate, glutamine, glutathione (GSH), taurine, trimethylamine (TMA), and its N-oxide (TMAO) is also significantly altered. Here, we report the quantification of metabolites and the biological application of chitosan. The metabolomics profile of chitosan in zebrafish has been detected, and the results indicated disturbed amino acid metabolism, the TCA cycle, and glycolysis. Our results demonstrate the potential of comparative metabolite profiling for discovering bioactive metabolites and they highlight the power of chitosan-applied chemical metabolomics to uncover new biological insights.

New insight on the mechanism of eating disorder in females based on metabolic differences of bisphenol S in female and male zebrafish

The different capacity of glucuronic acid metabolism might lead to the difference of bisphenol S (BPS) residual in tissues of male and female zebrafish. This may be the fundamental reason why BPS causes different effects in females and males. Here, adult zebrafish are exposed to 1, 10 and 100 μg/L BPS for 35 days to explore the main effect on females. After exposure, the liver of females showed stronger BPS metabolism ability than males, resulting in the accumulation of BPS in the gut of females. The results of neurotransmitters in gut of females revealed that the content of serotonin was decreased by BPS treatments. In addition, the mRNA expression levels of tryptophan 5-monooxygenase (Tph1) that regulated serotonin synthesis was reduced in gut of females in all BPS groups, and Tph1 protein has very high affinity with BPS molecule. Adult females treated with BPS exhibited symptoms including overeating, a decrease of serotonin in the gut, hypoglycemia and hyperlipidemia, a similar effect of Tph1 protein inhibitor LP533401 on adult females. This hypoglycemia stimulates brain agrp/pomc and orexin neurons to induce overfeeding behavior, causing intestinal homeostasis imbalance and hyperlipidemia. Our data elucidate a potential pathogenesis of eating disorder under pollutant stress.

Optical transparency and label-free vessel imaging of zebrafish larvae in shortwave infrared range as a tool for prolonged studying of cardiovascular system development

Optical techniques are utilized for the non-invasive analysis of the zebrafish cardiovascular system at early developmental stages. Being based mainly on conventional optical microscopy components and image sensors, the wavelength range of the collected and analyzed light is not out of the scope of 400–900 nm. In this paper, we compared the non-invasive optical approaches utilizing visible and near infrared range (VISNIR) 400–1000 and the shortwave infrared range (SWIR) 900–1700 nm. The transmittance spectra of zebrafish tissues were measured in these wavelength ranges, then vessel maps, heart rates, and blood flow velocities were calculated from data in VISNIR and SWIR. An increased pigment pattern transparency was registered in SWIR, while the heart and vessel detection quality in this range is not inferior to VISNIR. Obtained results indicate an increased efficiency of SWIR imaging for monitoring heart function and hemodynamic analysis of zebrafish embryos and larvae and suggest a prolonged registration period in this range compared to other optical techniques that are limited by pigment pattern development.

Effects of BPZ, BPC, BPF, and BPS Exposure on Adult Zebrafish (Danio rerio): Accumulation, Oxidative Stress, and Gene Expression

As substitutes for bisphenol A (BPA), bisphenol analogs (BPs) have been found to cause endocrine disorders and induce toxic effects. The objective of this study was to evaluate the bioaccumulation and subacute toxicity of bisphenol Z (BPZ), bisphenol C (BPC), bisphenol F (BPF), and bisphenol S (BPS) to zebrafish. Five-month-old zebrafish were exposed to 1/100 LC50, 1/50 LC50, and 1/10 LC50 of BPZ, BPC, BPF, and BPS for 13 days, respectively. Bioaccumulation, oxidative stress, and related mRNA expression in zebrafish tissues were measured on days 1, 7, and 13. After exposure, the four kinds of BPs all resulted in the accumulation of concentration and lipid peroxidation in zebrafish tissues to varying degrees. BPZ and BPC had the highest bioaccumulation level and had the greatest influence on malonic dialdehyde (MDA). In addition, the enzyme activities of superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GSH-PX), and the content of glutathione (GSH) in zebrafish tissues were also affected at different levels. However, the enzyme activities of SOD and POD were inactivated in zebrafish exposed to a high concentration of BPC. Further studies showed that BPs exposure down-regulated the transcription level of sod but up-regulated the relative expression levels of cat and gpx. The mRNA relative expression level of erα was not significantly changed, while the mRNA relative expression level of erβ1 was significantly down-regulated except under BPS exposure. These results indicate that BPZ, BPC, and BPF significantly affect the expression level of the estrogen receptor (ER) in zebrafish tissues. Overall, the results suggest that exposure to waterborne BPs can cause severe oxidative stress and tissue damage in adult zebrafish that is not sufficient to kill them after 13 days of waterborne exposure. The toxicity of BPs to organisms, therefore, should be further analyzed and evaluated.

Identification and expression analysis of zebrafish gnaq in the hypothalamic-Pituitary-Gonadal axis.

The G proteins have emerged as essential molecular switches in a wide variety of signal transduction pathways. Gαq, encoded by G protein subunit alpha q (gnaq), is a member of the G proteins and participates in regulating important biological activities in mammals; however, its function and regulatory mechanism in teleost remain largely unclear. In the current study, we cloned the cDNA of gnaq from zebrafish (Danio rerio) and investigated the expression characteristics of Gαq/gnaq in reproductive tissues. RT-PCR and WISH analyses showed that gnaq was widely expressed in zebrafish tissues, with high expression in the brain, olfactory brain, and hypothalamus. During the embryonic development stage, the gnaq was mainly distributed in the hypothalamus after 72h post-fertilization. In addition, immunohistochemistry analysis revealed that the Gαq protein was highly expressed in the diffuse nucleus of the inferior hypothalamic lobe (DIL), ventral zone of the periventricular hypothalamus (Hv), and caudal zone of the periventricular hypothalamus (Hc) in adult zebrafish. Furthermore, in the gonads, the Gαq protein was found in oocytes of all stages, except spermatids. Lastly, the gnaq mRNA exhibited relatively low expression in gonads on Day 4 during the reproductive cycle, while increasing drastically in the hypothalamus and pituitary afterward. Altogether, our results suggest that gnaq/Gαq might be important in fish reproduction.

Establishment and characterization of a new fibroblast-like cell line from the skin of a vertebrate model, zebrafish (Danio rerio).

The available fully sequenced genome and genetic similarities compared to humans make zebrafish a prominent in vitro vertebrate model for drug discovery & screening, toxicology, and radiation biology. Zebrafish also possess well developed immune systems which is ideal for studying infectious diseases. Fish skin confers immunity by serving as a physical barrier against the invading pathogens in the aquatic habitat. Therefore in vitro models from the skin tissue of zebrafish help to study the physiology, functional genes in vitro, wound healing, and pathogenicity of microbes. Hence the study aimed to develop and characterize a skin cell line from the wild-type zebrafish Danio rerio. A novel cell line designated as DRS (D. rerio skin) was established and characterized from the skin tissue of wild-type zebrafish, D. rerio, by the explant technique. The cells thrived well in the Leibovitz's -15 medium supplemented with 15% FBS and routinely passaged at regular intervals. The DRS cells mainly feature fibroblast-like morphology. The culture conditions of the cells were determined by incubating the cells at varying concentrations of FBS and temperature; the optimum was 15% FBS and 28°C, respectively. Cells were cryopreserved and revived with 70-75% viability at different passage levels. Two extracellular products from bacterial species Aeromonas hydrophila and Edwardsiella tarda were tested and found toxic to the DRS cells. Mitochondrial genes, namely COI and 16S rRNA PCR amplification and partial sequencing authenticated the species of origin of cells. The modal diploid (2n) chromosome number of the cells was 50. The cell line DRS was found to be free from mycoplasma. The cells were transfected with pMaxGFP plasmid and tested positive for green fluorescence at 24-48h post-transfection. The findings from this study thus confirm the usefulness of the developed cell line in bacterial susceptibility and transgene expression studies.

Flexible control of pulse intensity and repetition rate for multiphoton photostimulation

In deep tissue imaging, pulsed near-infrared lasers commonly provide high peak powers needed for nonlinear absorption, but average power and linear absorption can be limiting factors for tissue damage through heat. We implemented intra-cavity dumping within a mode-locked Ti:Sapphire laser used for two-photon computer generated holography stimulation. This system enables photostimulation in which pulse energy, average power, and repetition rate can each be varied and harnessed as degrees of freedom. We demonstrate how this system provides a new dimension of temporal control in photostimulation experiments to alter the ratio of nonlinear to linear light-tissue interactions, namely by tuning the laser repetition rate between single-shot and ≈ 3 MHz. Repetition rates below 1 MHz, yielding pulse energies over 60 nJ, facilitated holographic projections with more regions of interest than the highest repetition rate of 3 MHz. Even lower repetition rates ( ≈ 10 kHz) diminished thermal load on the sample, as characterized by quantification of heat shock protein expression in zebrafish tissue.

The role of the cd99l2 gene on leukocyte interstitial migration in zebrafish.

Leukocytes play an essential role in ontogeny, tissue regeneration, and innate and adaptive immunity. The migration of leukocytes to the infected or traumatized areas is necessary for their immune response function. As an adhesion molecule, CD99L2 is crucial in the extravasation of leukocytes, however, its role in the interstitial migration of leukocytes remains unclear. In this study, the cd99l2 gene was knock-out by TALEN (transcription activator-like effector nuclease) in zebrafish and discovered that the deletion had no effect on zebrafish development. The number of granulocytes and macrophages recruited to the wounded tissue was significantly reduced in the cd99l2 mutants following caudal fin damage. Further research revealed that the expression of mfap4 was drastically decreased in the cd99l2 mutants, which may be one of the reasons that affect the migration of macrophages to the wound site. Moreover,transgenic lines with labeled vasculature, neutrophils and macrophages demonstrated that neutrophils and macrophages migrate throughout the interstitial space to the wound tissue in both wild-type and mutant zebrafish at 60 hours post-fertilization, indicating that the cd99l2 gene is involved in the interstitial migration of leukocytes. Finally, RNA transcription, protein folding, and the P450 pathway were enriched in cd99l2 mutants by RNA-seq analysis. Previous research had demonstrated that the regulation of transcription and signal transduction could be affected by adhesion molecules, which may suggest that the cd99l2 gene is involved in the cascade signaling pathway of leukocyte migration as an adhesion molecule. In conclusion, this study uncovered a novel function of the cd99l2 gene in the process of leukocyte migration in zebrafish, which is expected to provide a theoretical foundation for inflammatory and immune-related diseases.

Antimony accumulation in zebrafish (Danio rerio) and its effect on genotoxicity, histopathology, and ultrastructure

Antimony (Sb) is a toxic metal in aquatic ecosystems. In this study, the accumulation of aqueous Sb in the liver, brain, gills and muscle of zebrafish (Danio rerio) and its effect on genotoxicity, histopathology and ultrastructure alterations were evaluated. The fishes were exposed to different concentrations (0, 8.29, 16.58, 33.16 mg L−1) of aqueous Sb for 18 days. The results showed that the order of Sb accumulation in different tissues was liver > gill > muscle > brain, and the accumulation increased with increasing Sb stress concentration. The mRNA expression levels of Nrf2, Cu/Zn-SOD, Mn-SOD, CAT and GPx genes showed different trends. In addition, significant histopathology and ultrastructure alterations were observed in the liver and gills exposed to Sb. Sb could accumulate in different tissues of zebrafish, inducing the expression of oxidative stress genes and activating antioxidant defense systems. Histological and ultrastructural changes could be used as valid biomarkers for the assessment of aqueous Sb contamination.