Genotoxicity Assessment of Quinoin, a Ribosome Inactivating Protein from Quinoa Seeds, in the Teleost Danio rerio.

BACKGROUND: Ribosome inactivating proteins (RIPs) are N-glycosylases found in various plants that are able to specifically and irreversibly inhibit protein translation, thereby leading to cell death. Their cytotoxic properties have attracted attention in the medical field in the context of developing new anticancer therapies. Quinoin is a novel toxic enzyme obtained from quinoa seeds and classified as a type 1 RIP (Chenopodium quinoa Willd.). Recently, quinoin was found to be cytotoxic to normal fibroblasts and keratinocytes in vitro, as well as to several tumor cell lines. METHODS: The aim of this study was to evaluate the in vitro and in vivo genotoxicity of quinoin in a zebrafish model. We evaluated its ability to induce DNA fragmentation, genomic instability, and reactive oxygen species (ROS) generation by means of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction, randomly amplified polymorphic DNA (RAPD) Polymerase Chain Reaction (PCR) technique, and dichlorofluorescine (DCF) assay, respectively. RESULTS: Quinoin was found to cause genomic damage in zebrafish, as shown by DNA fragmentation, polymorphic variations leading to genomic instability, and oxidative stress. Interestingly, longer quinoin treatment caused less damage than shorter treatments. CONCLUSIONS: This study demonstrated ROS-mediated genotoxicity of quinoin toward the zebrafish genome. The reduced damage observed after longer quinoin treatment could indicate the activation of detoxification mechanisms, activation of repair mechanisms, or the loss of protein activity due to enzymatic digestion. In order to clarify the genotoxic actions of quinoin, further investigations of the response pathways to DNA damage are needed. Overall, the ability of quinoin to cause breaks and instability in DNA, together with its clear cytotoxicity, make it an interesting candidate for the development of new drugs for cancer treatment.

Histopathological effects of long-term exposure to realistic concentrations of cadmium in the hepatopancreas of Sparus aurata juveniles.

In recent decades, cadmium has emerged as an environmental stressor in aquatic ecosystems due to its persistence and toxicity. It can enter water bodies from various natural and anthropogenic sources and, once introduced into aquatic systems, can accumulate in sediments and biota, leading to bioaccumulation and biomagnification in the food chain. For this reason, the effects of cadmium on aquatic life remain an area of ongoing research and concern. In this paper, a multidisciplinary approach was used to assess the effects of long-term exposure to an environmental concentration on the hepatopancreas of farmed juveniles of sea bream, Sparus aurata. After determining metal uptake, metallothionein production was assessed to gain insight into the organism's defence response. The effects were also assessed by histological and ultrastructural analyses. The results indicate that cadmium accumulates in the hepatopancreas at significant concentrations, inducing structural and functional damage. Despite the parallel increase in metallothioneins, fibrosis, alterations in carbohydrate distribution and endocrine disruption were also observed. These effects would decrease animal fitness although it did not translate into high mortality or reduced growth. This could depend on the fact that the animals were farmed, protected from the pressure deriving from having to search for food or escape from predators. Not to be underestimated is the return to humans, as this species is edible. Understanding the behaviour of cadmium in aquatic systems, its effects at different trophic levels and the potential risks to human health from the consumption of contaminated seafood would therefore be essential for informed environmental management and policy decisions.

Could Exposure to Glyphosate Pose a Risk to the Survival of Wild Animals? A Case Study on the Field Lizard Podarcis siculus.

Soil contaminants (herbicides, pesticides, and heavy metals) are among the main causes of change in terrestrial ecosystems. These substances lead to a general loss of biodiversity, both of flora and fauna and being able to biomagnify and pass through the food chain, they can endanger the survival of terrestrial vertebrates at the top of this chain. This review analyzes the risks associated with exposure to glyphosate, the active principle of many herbicide products, for the reproductive health of the field lizard (Podarcis siculus) potentially exposed to the substance in its natural habitat; therefore, introducing it as a possible model organism. Data demonstrate that glyphosate is toxic for this animal, affecting the health of the reproductive organs, both in males and females, and of the liver, the main detoxifying organ and closely involved in the female reproductive process. Sharing structural and functional characteristics of these organs with many other vertebrates, the information obtained with this reptile represents a wake-up call to consider when analyzing the cost/benefit ratio of glyphosate-based substances. The data clearly demonstrate that the P. siculus lizard can be considered a good target organism to study the reproductive risk assessment and hazards of exposure to soil contaminants on wild terrestrial vertebrates.

Glyphosate Interference in Follicular Organization in the Wall Lizard Podarcis siculus.

Glyphosate (Gly) is a broad-spectrum herbicide widely used thanks to its high efficiency and low toxicity. However, evidence exists of its toxic effects on non-target organisms. Among these, the animals inhabiting agricultural fields are particularly threatened. Recent studies demonstrated that exposure to Gly markedly affected the morphophysiology of the liver and testis of the Italian field lizard Podarcis siculus. The present study aimed to investigate the effects of the herbicide on the female reproductive system of this lizard in order to have a full picture of Gly-induced reproductive impairment. The animals were exposed to 0.05 and 0.5 µg/kg of pure Gly by gavage for 3 weeks. The results demonstrated that Gly, at both doses tested, profoundly interfered with ovarian function. It induced germ cells' recruitment and altered follicular anatomy by anticipating apoptotic regression of the pyriform cells. It also induced thecal fibrosis and affected oocyte cytoplasm and zona pellucida organizations. At the functional levels, Gly stimulated the synthesis of estrogen receptors, suggesting a serious endocrine-disrupting effect. Overall, the follicular alterations, combined with those found at the level of the seminiferous tubules in males, suggest serious damage to the reproductive fitness of these non-target organisms, which over time could lead to a decline in survival.

Molecular Research on Reproductive Toxicity.

Fertility rates in animals have shown a progressive decrease in recent decades, and reproductive toxicity is considered an important regulatory endpoint in health hazard assessment [...].

The Italian Wall Lizard Podarcis siculus as a Biological Model for Research in Male Reproductive Toxicology.

Spermatogenesis is a genetically driven differentiation process that occurs in the testis and leads to the formation of spermatozoa. This process is extensively studied in several experimental models, particularly in vertebrates that share the morphological structure and functionality of the mammalian testis. Although reptiles are not generally considered biological models, the lizard Podarcis siculus has represented a suitable organism for the study of spermatogenesis over the years. In this lizard, the process of spermatogenesis is regulated by the interaction between systemic factors such as gonadotropins and local factors, i.e., molecules produced by the somatic and germinal cells of the testis. Many exogenous substances are able to alter the production of these regulative factors, thus altering the course of spermatogenesis, and P. siculus has proven to be an excellent model for studying the effects of various endogenous or exogenous substances on mechanisms underlying spermatogenesis. This review summarizes the available data on the effects of different substances on the control of spermatogenesis, highlighting the induced morphological and molecular alterations. Overall, the data show that sex hormone levels as well as the final stages of spermatogenesis are most affected by an imbalance of endogenous compounds or contamination by environmental pollutants. This is helpful for the male individual, since the damage, not affecting the spermatogonial stem cells, can be considered transient and not irreversible.

Vanadium Modulates Proteolytic Activities and MMP-14-Like Levels during Paracentrotus lividus Embryogenesis.

The increasing industrial use of vanadium (V), as well as its recent medical use in various pathologies has intensified its environmental release, making it an emerging pollutant. The sea urchin embryo has long been used to study the effects induced by metals, including V. In this study we used an integrated approach that correlates the biological effects on embryo development with proteolytic activities of gelatinases that could better reflect any metal-induced imbalances. V-exposure caused morphological/morphometric aberrations, mainly concerning the correct distribution of embryonic cells, the development of the skeleton, and the embryo volume. Moreover, V induced a concentration change in all the gelatinases expressed during embryo development and a reduction in their total proteolytic activity. The presence of three MMP-like gelatinases (MMP-2, -9, and -14) was also demonstrated and their levels depended on V-concentration. In particular, the MMP-14-like protein modified its expression level during embryo development in a time- and dose-dependent manner. This enzyme also showed a specific localization on filopodia, suggesting that primary mesenchyme cells (PMCs) could be responsible for its synthesis. In conclusion, these results indicate that an integrated study among morphology/morphometry, proteolytic activity, and MMP-14 expression constitutes an important response profile to V-action.

Metallothionein gene expression in rat tissues: response to dietary restriction after orally dichlorodiphenyldichloroethylene (DDE) exposure and high-fat feeding.

Dichlorodiphenyldichloroethylene (DDE) is an environmental pollutant that accumulates in adipose tissue through the food chain. Hypercaloric, high-fat diet is considered to promote the accumulation of toxic lipophilic substances in tissues, whereas the loss of body fat through caloric restriction results in a recirculation of these substances. In rats, oral administration of DDE causes the onset of tissues damage; the concomitant intake of a high-fat diet ameliorates tissues status, probably because of the entrapment of the lipophilic substance in fat depots. Recent evidence demonstrates that DDE alters the expression of metallothioneins, proteins involved in cellular defense from oxidative stress, in a diet- and tissue-specific manner. This study is aimed to verify if 2 weeks of caloric restriction after the oral DDE treatment can modify metallothionein gene expression in tissues of high-fat fed rats. Real-time PCR analysis demonstrates that metallothionein gene expression after calorie restriction is tissue-specific and strongly influenced by both previous dietary conditions and DDE exposure. To avoid misleading conclusions on the interference of toxic xenobiotics on metallothionein gene expression is particularly important to consider the tissue, the cellular conditions, and the nutritional status of the animals, especially when the protein is used as an index of cells health.

Aluminium exposure leads to neurodegeneration and alters the expression of marker genes involved to parkinsonism in zebrafish brain.

Aluminium, despite being extremely widespread in the world, is a non-essential metal to human metabolism. This metal is known to have toxic effects on a variety of organs including the brain and is considered an etiological factor in neurodegenerative diseases. However, the molecular mechanisms by which aluminium exerts neurotoxic effects are not yet completely understood. Zebrafish is an animal model also used to study neurodegenerative diseases since the overall anatomical organization of the central nervous system is relatively conserved and similar to mammals. Adult zebrafish were exposed to 11 mg/L of Al for 10, 15, and 20 days and the neurotoxic effects of aluminium were analysed by histological, biochemical, and molecular evaluations. Histological stainings allowed to evaluation of the morphology of the brain parenchyma, the alteration of myelin and the activation of neurodegenerative processes. The expression of the Glial Fibrillary Acidic Protein, a marker of glial cells, was evaluated to observe the quantitative alteration of this important protein for the nervous system. In addition, the poly(ADP-ribose) polymerase activity was measured to verify a possible oxidative DNA damage caused by exposure to this metal. Finally, the evaluation of the markers involved in Parkinsonism was assessed by Real-Time PCR to better understand the role of aluminium in the regulation of genes related to Parkinson's neurodegenerative disease. Data showed that aluminium significantly affected the histology of cerebral tissue especially in the first periods of exposure, 10 and 15 days. This trend was also followed by the expression of GFAP. At longer exposure times, there was an improvement/stabilization of the overall neurological conditions and decrease in PARP activity. In addition, aluminium is involved in the deregulation of the expression of genes closely related to Parkinsonism. Overall, the data confirm the neurotoxicity induced by aluminium and shed a light on its involvement in neurodegenerative processes.

Toxicity of Vanadium during Development of Sea Urchin Embryos: Bioaccumulation, Calcium Depletion, ERK Modulation and Cell-Selective Apoptosis.

Vanadium toxicology is a topic of considerable importance as this metal is widely used in industrial and biomedical fields. However, it represents a potential emerging environmental pollutant because wastewater treatment plants do not adequately remove metal compounds that are subsequently released into the environment. Vanadium applications are limited due to its toxicity, so it is urgent to define this aspect. This metal is associated with sea urchin embryo toxicity as it perturbs embryogenesis and skeletogenesis, triggering several stress responses. Here we investigated its bioaccumulation and the correlation with cellular and molecular developmental pathways. We used cytotoxic concentrations of 1 mM and 500 µM to perform quantitative analyses, showing that vanadium accumulation interferes with calcium uptake during sea urchin development and provokes a disruption in the biomineralization process. At the end of the whole treatment, the accumulation of vanadium was about 14 and 8 µg for embryos treated respectively with 1 mM and 500 µM, showing a dose-dependent response. Then, we monitored the cell signaling perturbation, analyzing key molecular markers of cell survival/cell death mechanisms and the DNA fragmentation associated with apoptosis. This paper clarifies vanadium's trend to accumulate directly into embryonic cells, interfering with calcium uptake. In addition, our results indicate that vanadium can modulate the ERK pathway and activate a cell-selective apoptosis. These results endorse the sea urchin embryo as an adequate experimental model to study metal-related cellular/molecular responses.

Molecular and Histological Effects of Glyphosate on Testicular Tissue of the Lizard Podarcis siculus.

The expansion of agriculture produces a steady increase in habitat fragmentation and degradation due to the increased use of pesticides and herbicides. Habitat loss and alteration associated with crop production play an important role in reptile decline, among which lizards are particularly endangered. In this study, we evaluated testicular structure, steroidogenesis, and estrogen receptor expression/localization after three weeks of oral exposure to glyphosate at 0.05 and 0.5 µg/kg body weight every other day in the field lizard Podarcis siculus. Our results show that glyphosate affected testicular morphology, reduced spermatogenesis, altered gap junctions and changed the localization of estrogen receptors in germ cells, increasing their expression; the effects were mostly dose-dependent. The result also demonstrates that glyphosate, at least at these concentrations, did not influence steroidogenesis. Overall, the data indicate that this herbicide can disturb the morphophysiology of the male lizard's reproductive system, with obviously detrimental effects on their reproductive fitness. The effects of glyphosate must be considered biologically relevant and could endanger the reproductive capacity not only of lizards but also of other vertebrates, including humans; a more controlled and less intensive use of glyphosate in areas devoted to crop production would therefore be advisable.

Toxicological Impact of Rare Earth Elements (REEs) on the Reproduction and Development of Aquatic Organisms Using Sea Urchins as Biological Models.

The growing presence of lanthanides in the environment has drawn the attention of the scientific community on their safety and toxicity. The sources of lanthanides in the environment include diagnostic medicine, electronic devices, permanent magnets, etc. Their exponential use and the poor management of waste disposal raise serious concerns about the quality and safety of the ecosystems at a global level. This review focused on the impact of lanthanides in marine organisms on reproductive fitness, fertilization and embryonic development, using the sea urchin as a biological model system. Scientific evidence shows that exposure to lanthanides triggers a wide variety of toxic insults, including reproductive performance, fertilization, redox metabolism, embryogenesis, and regulation of embryonic gene expression. This was thoroughly demonstrated for gadolinium, the most widely used lanthanide in diagnostic medicine, whose uptake in sea urchin embryos occurs in a time- and concentration-dependent manner, correlates with decreased calcium absorption and primarily affects skeletal growth, with incorrect regulation of the skeletal gene regulatory network. The results collected on sea urchin embryos demonstrate a variable sensitivity of the early life stages of different species, highlighting the importance of testing the effects of pollution in different species. The accumulation of lanthanides and their emerging negative effects make risk assessment and consequent legislative intervention on their disposal mandatory.

Effects of Cadmium Exposure on Gut Villi in Danio rerio.

In aquatic organisms, cadmium exposure occurs from ovum to death and the route of absorption is particularly wide, being represented by skin, gills and gastrointestinal tract, through which contaminated water and/or preys are ingested. It is known that cadmium interferes with the gut; however, less information is available on cadmium effects on an important component of the gut, namely goblet cells, specialized in mucus synthesis. In the present work, we studied the effects of two sublethal cadmium concentrations on the gut mucosa of Danio rerio. Particular attention was paid to changes in the distribution of glycan residues, and in metallothionein expression in intestinal cells. The results show that cadmium interferes with gut mucosa and goblet cells features. The effects are dose- and site-dependent, the anterior gut being more markedly affected than the midgut. Cadmium modifies the presence and/or distribution of glycans in the brush border and cytoplasm of enterocytes and in the goblet cells' cytoplasm and alters the metallothionein expression and localization. The results suggest a significant interference of cadmium with mucosal efficiency, representing a health risk for the organism in direct contact with contamination and indirectly for the trophic chain.

Heart Mitochondrial Metabolic Flexibility and Redox Status Are Improved by Donkey and Human Milk Intake.

The biological mechanisms linking nutrition and antioxidants content of the diet with cardiovascular protection are subject of intense investigation. It has been demonstrated that dietary supplementation with cow, donkey or human milk, characterized by distinct nutritional properties, triggers significant differences in the metabolic and inflammatory status through the modulation of hepatic and skeletal muscle mitochondrial functions. Cardiac mitochondria play a key role for energy-demanding heart functions, and their disfunctions is leading to pathologies. Indeed, an altered heart mitochondrial function and the consequent increased reactive oxygen species (ROS) production and inflammatory state, is linked to several cardiac diseases such as hypertension and heart failure. In this work it was investigated the impact of the milk consumption on heart mitochondrial functions, inflammation and oxidative stress. In addition, it was underlined the crosstalk between mitochondrial metabolic flexibility, lipid storage and redox status as control mechanisms for the maintenance of cardiovascular health.

Exposure to Dichlorodiphenyldichloroethylene (DDE) and Metallothionein Levels in Rats Fed with Normocaloric or High-Fat Diet: A Review.

The growing number of studies on metallothioneins (MTs), cysteine-rich metal-binding proteins, have been disclosing new functions of these proteins. Thanks to their inducibility, they were considered to play a pivotal role in regulating trace metals homeostasis and in detoxification from heavy metals; nowadays, it is known that they are involved in various physiological and pathological processes, such as regulation of apoptosis, elimination of free radicals, and protection of nucleic acids against toxic insults. MT induction has been demonstrated following stress factors other than heavy metals, such as endocrine-disrupting chemicals, insecticides, and herbicides. However, retrieved data are often controversial: in some cases, xenobiotics elicit MT expression and synthesis; under different conditions, they lead to a decrease in cellular MT content. This review describes the MT response to dichlorodiphenyltrichloroethane (DDT) contamination in mammalian tissues. In particular, attention focuses on changes in MT expression, synthesis, and localization in rat liver, kidneys, and testes following oral administration of dichlorodiphenyldichloroethylene (DDE), the main metabolite of DDT, under normal dietary conditions or in combination with a high fat diet potentially able to increase the cellular uptake of this lipophilic pesticide. The potential connection between MT expression and synthesis, lipophilic substances and trace metals availability is also discussed.

HSP70 localization in Podarcissiculus embryos under natural thermal regime and following a non-lethal cold shock.

The Heat Shock Proteins (HSPs) are a superfamily of molecular chaperones that maintain cellular homeostasis under stress. HSP70 represents the major stress-inducible family member, often activated in response to changes in thermal ranges of organisms, and therefore playing an important role enhancing thermal tolerance limits in ectothermic animals. The present study aimed to investigate the presence and the localization of HSP70 through the development of Podarcis siculus, an oviparous lizard inhabiting temperate Mediterranean regions, showing a limited potential to tolerate thermal changes during embryogenesis. Immunohistochemical analysis demonstrated that HSP70 protein is constitutively present in early embryonic stages, abundantly distributed in eye, in encephalic domains (predominantly in ventricular areas and in grey matter), in grey matter of spinal cord, in lung, gut mucosa, hepatic cords and kidney tubules. Interestingly, a severe drop in incubation temperature (5°C for 3 days) does not induce enhancements in HSP70 levels nor changes in tissues localization. These results suggest that the HSP70 found in P. siculus embryos represents a non-inducible, constitutive molecular chaperone that should be better called Heat Shock Cognate 70 (HSC70); the presence of stress-induced members of the HSP family in P. siculus has yet to be proven.

How Glyphosate Impairs Liver Condition in the Field Lizard Podarcis siculus (Rafinesque-Schmaltz, 1810): Histological and Molecular Evidence.

The potential toxicity of glyphosate, a widely used broad-spectrum herbicide, is currently a great matter of debate. As vertebrate insectivores, lizards protect plants from herbivorous insects increasing plant biomass via the trophic cascade and represent an important link between invertebrates and higher predators. A negative effect of glyphosate on lizards' survival could have major impacts at the ecological levels. In this study, we investigated the effects of the exposure to low doses of glyphosate on the liver of the wall lizard Podarcis siculus, a suitable bioindicator of soil pollution. Two different doses of pure glyphosate (0.05 and 0.5 µg/kg body weight) were orally administered every other day for 3 weeks to sexually mature males and females. The results demonstrated that both doses, despite being very low, are toxic for the liver that showed clear signs of suffering, regardless of sex. The histological analysis provided a scenario of severe hepatic condition, which degenerated until the appearance of fibrotic formations. The morphological observations were consistent with a loss of liver physiological functions. Immunocytochemical investigations allowed us to detect an involvement of antioxidant/cytoprotective proteins, such as superoxide dismutase 1 (Cu/Zn SOD, known as SOD1), glutathione peroxidase 1 (GPx1), metallothionein (MT), and tumor suppressor protein 53, (p53) suggesting that the liver was trying to react against stress signals and damage induced by glyphosate. Finally, in situ hybridization and Real-Time PCR analysis showed the upregulation of estrogen receptor α and vitellogenin gene expression, thus demonstrating the xenoestrogenic action of glyphosate. The imbalance of the hormonal homeostasis could threaten the lizards' reproductive fitness and survival, altering the trophic cascade.

Health status of the lizard Podarcis siculus (Rafinesque-Schmaltz, 1810) subject to different anthropogenic pressures.

The terrestrial lizard Podarcis siculus is the most abundant reptile in Italy, where is considered a ubiquitous species. This lizard is widely distributed from the islands to the Apennines, from cultivated fields to anthropized areas such as gardens and city parks. For this reason, these animals are exposed to extensive physical and chemical stresses, as well as to the possibility of coming into contact with industrial pollutants and substances used in agricultural practices. Here, we review the health status of lizard specimens inhabiting natural non-anthropized areas and fields devoted to organic farming, considering the condition of (1) liver, representing the main detoxifying organ, directly influenced by feeding, and (2) gonads, essential for reproduction and, therefore, for the survival of the species. The morphological and biomolecular condition of these organs was then compared with those obtained from lizard specimens experimentally treated with nonylphenol, a co-formulant of many insecticides and plant protection products widely used in conventional farming, known to have harmful estrogenic effects. Taken together, data demonstrate that lizards inhabiting manured soil are in good health status and show a regular morphology of liver, testis, and ovary. Animals are found to be less exposed to the toxic heavy metals cadmium and lead if compared with specimens collected in areas not devoted to agriculture, but probably more exposed to vehicular traffic. However, manure, as well as nonylphenol, exerts a xeno-estrogenic effect, particularly evident in male specimens, more sensitive to estrogenic contamination.

Physiological Adaptation to Simultaneous Chronic Exposure to High-Fat Diet and Dichlorodipheniletylhene (DDE) in Wistar Rat Testis.

Environmental chemicals can be introduced by consuming contaminated foods. The environmental chemical dichlorodiphenyldichloroethylene (DDE), a persistent metabolite of dichlorodiphenyltrichloroethane (DDT), can affect spermatogenesis. Our study aims to evaluate, by using spectrophotometric analyses, western blot, and immunohistochemistry, the adaptive responses in testis of adult rats treated with a non-toxic dose of DDE, alone or in association with a high-fat diet (HFD). Four experimental groups were performed: N (normal diet); D (HFD); D + DDE (HFD + DDE); N + DDE (normal diet + DDE). D group showed a reduction in antioxidant capacity, and increases in lipid peroxidation, apoptosis, and proliferation associated with morphological impairment. A reduction in androgen receptor (AR) and serum testosterone levels were also found. DDE-treated groups exhibited higher lipid peroxidation levels compared to N and D, associated with pronounced defect in antioxidant capacity, apoptosis, cellular proliferation, as well as with tissue damage. Moreover, decreases in AR and serum testosterone levels were found in DDE-treated groups vs. N and D. In conclusion, HFD and DDE produced cellular stress leading to antioxidant impairment, apoptosis, and decreases in AR and serum testosterone levels associated with tissue damage. Cellular proliferation could be used as an adaptation to counterbalance the occurred damage, maintaining a pool of tubules that follow physiological maturation.

Oxidative stress and mitochondrial uncoupling protein 2 expression in hepatic steatosis induced by exposure to xenobiotic DDE and high fat diet in male Wistar rats.

Oxidative stress plays a key role in steatohepatitis induced by both xenobiotic agents and high fat diet (HFD). The present study aimed to evaluate hepatic oxidative stress and anti-oxidant systems response in rats exposed to HFD and/or non-toxic dose of dichlorodiphenyldichloroethylene (DDE), the first metabolite of dichlorodiphenyltrichloroethane. Groups of 8 rats were so treated for 4 weeks: 1- standard diet (N group); 2- standard diet plus DDE (10 mg/kg b.w.) (N+DDE group); 3- HFD (D group); 4- HFD plus DDE (D+DDE group). Oxidative stress was analyzed by determining malondialdehyde as lipid peroxidation product, while the anti-oxidant systems were evaluating by measuring the levels of the principal cytosolic and mitochondrial antioxidant proteins and enzymes, namely superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxidase 1 (GPx1) and uncoupling protein 2 (UCP2) involved in the control of hepatic reactive oxygens species (ROS) accumulation. The results showed malondialdehyde accumulation in livers of all groups, confirming the pro-oxidant effects of both HFD and DDE, but with a greater effect of DDE in absence of HFD. In addition, we found different levels of the analyzed anti-oxidant systems in the different groups. DDE mainly induced UCP2 and SOD2, while HFD mainly induced GPx1. Noteworthy, in the condition of simultaneous exposure to DDE and HFD, the anti-oxidant response was more similar to the one induced by HFD than to the response induced by DDE. Present findings confirmed that both HFD and xenobiotic exposure induced hepatic oxidative stress and showed that the anti-oxidant defense response was not the same in the diverse groups, suggesting that UCP2 induction could be an adaptive response to limit excessive ROS damage, mainly in condition of xenobiotic exposure.