Micronucleus test in reptiles: Current and future perspectives.

Micronucleus (MN) cell counting emerged in 1973-1975 as a valid alternative for characterizing chromosomal damage caused by different agents. It was first described in mammals, but its application was rapidly extended to other vertebrates, mainly fish. However, it was not until 28 years later that this test was implemented in studies on reptiles. Nowadays, reptiles are found to be excellent non-target species from environmental contamination exposure and MN test has become a fundamental tool for analyzing genotoxic effects induced by various xenobiotics. In this article we provide an updated review of the application of the MN test in reptile species, from an ecotoxicological perspective. Therefore, we present (I) a bibliometric analysis of the available research on genotoxic-induced MN formation in reptile species; (II) the use of reptiles as sentinel organisms in ecotoxicological studies; and (III) the strength and weakness of the application of the MN test in this group. With this review, we aim to provide a comprehensive view on the use of the MN test in ecotoxicology and to encourage further studies involving reptile species.

Evaluation of oxidative damage and genotoxicity in populations exposed to arsenic in drinking water from Santa Fe province, Argentina.

The presence of arsenic in the environment is a public health problem. Groundwater of certain regions of Argentina contains arsenic of natural origin in concentrations that exceed the guide level recommended by World Health Organization (WHO, 10 µg/L). Pathologies derived from chronic arsenic consumption justify the planning of human biomonitoring. Hence, the aim of this study was to evaluate oxidative damage and genotoxicity and its relationship with nutritional variables in populations exposed to arsenic through drinking water in Santa Fe province, Argentina. A total of 322 participants were analyzed for arsenic in urine together with biomarkers of genotoxicity (Comet assay in blood and frequency of Micronuclei and other Nuclear Abnormalities in exfoliated buccal cells) and oxidative stress (modified Comet assay with Endonuclease III, Lipid peroxidation and antioxidant enzyme activity), as well as nutritional and biochemical variables. Results showed that 45 % of participants excreted arsenic in the urine. Consumption of water with arsenic, whether currently or previously, was associated with statistically significant increase of oxidative DNA damage and lipid peroxidation. MN in exfoliated buccal cells serve as an early biomarker of genotoxicity and showed significant differences in the current exposed group. Biochemical results indicate dyslipidemias potentially linked to dietary choices, and insufficient intake of fruits and vegetables rich in antioxidants, was also noted. This study advocates risk communication to the population, educators, and health authorities, emphasizing the need for preventive health strategies and improved food education.

Acute exposure of zebrafish (Danio rerio) adults to psychotria carthagenensis leaf extracts: chemical profile, lack of genotoxicity and histological changes.

Psychotria carthagenensis is a shrubby plant, often consumed by traditional populations in religious rituals. Previous studies have shown that this plant's infusion can inhibit the activity of Acetylcholinesterase (AChE) in rats. Despite the therapeutic potential, there is a lack of research regarding its possible toxicological and genotoxic effects. Hence, this study aimed to analyze the chemical profile of the ethanol extract from P. carthagenensis leaves by LC-DAD-MS and assess its possible toxicity and genotoxicity in zebrafish (Danio rerio). Adult zebrafish (N = 9/group) were exposed at different concentrations and the LC50 was calculated. Frequencies of micronucleus (MN) and nuclear abnormalities (NA) were estimated for genotoxic effects, and degree of tissue changes (DTC) was used to assess the liver and gill histopathology. From the LC-DAD-MS analyses, the identified compounds included N-fructosyl valine, ethyl hexoside, 5-O-E-caffeoylquinic acid, N-feruloylagmatime, roseoside, di-O-deoxyhexoyl-hexosyl quercetin, loiolide, and oleamide. The calculated values of LC50 did not vary significantly during the time of exposure. At the concentrations of 1.25, 2.5, 3.75, 5, 7.5, 10 and 15 mg/L, there was no genotoxicity, and only low to moderate toxicity for the tissues was observed, despite mortality of 100% at doses of 20-100 mg/L of P. carthagenensis ethanolic leaf extract. There were changes in cytoplasm of hepatocytes at 1.25 mg/L, and karyorrhexis, karyolysis and megalocytosis at 10 mg/L. In the gills, the alterations were primary lamellar hyperplasia in all concentrations, and at 10 mg/L, secondary lamellar edema and vascular hyperemia were common. Additionally, the chemical composition of P. carthagenensis was expanded.

Metal accumulation in female green sea turtles (Chelonia mydas) from Eastern Atlantic affects their egg quality with potential implications for embryonic development.

Sea turtles, with their global distribution and complex life cycle, often accumulate pollutants such as metals and metalloids due to their extended lifespan and feeding habits. However, there are limited studies exploring the impact of metal pollution on the reproductive health of female sea turtles, specifically focusing on the quality of their eggs, which has significant implications for the future generations of these charismatic animals. São Tomé Island, a crucial nesting and feeding habitat for green sea turtles, underscores the urgent need for comprehensive research in this ecologically significant area. This study aimed to investigate whether metals and metalloids in the blood of nesting female green sea turtles induce genotoxic effects in their erythrocytes and affect their egg morphometric characteristics and the composition of related compartments. Additionally, this study aimed to evaluate whether the quality of energetic reserves for embryo development (fatty acids in yolk's polar and neutral lipids) is influenced by the contamination status of their predecessors. Results revealed correlations between Cu and Hg levels and increased "lobed" erythrocytes, while As and Cu negatively influenced shell thickness. In terms of energy reserves, both polar and neutral lipid fractions contained primarily saturated and monounsaturated fatty acids, with prevalent 18:1n-9, 18:0, 16:0, 14:0, and 12:0 fatty acids in yolk samples. The yolk polar fraction was more susceptible to contaminant levels in female sea turtles, showing consistent negative correlations between pollution load index and essential n3 fatty acids, including linolenic, eicosatrienoic, eicosapentaenoic, and docosapentaenoic acids, crucial for embryonic development. These metals accumulation, coupled with the reduced availability of these key fatty acids, may disrupt the eicosanoid and other important pathways, affecting reproductive development. This study reveals a negative correlation between metal contamination in female sea turtles' blood and egg lipid reserves, raising concerns about embryonic development and the species' future generations.

Nuclear Abnormalities in LMNA p.(Glu2Lys) Variant Segregating with LMNA-Associated Cardiocutaneous Progeria Syndrome.

The LMNA gene encodes lamin A and lamin C, which play important roles in nuclear organization. Pathogenic variants in LMNA cause laminopathies, a group of disorders with diverse phenotypes. There are two main groups of disease-causing variants: missense variants affecting dimerization and intermolecular interactions, and heterozygous substitutions activating cryptic splice sites. These variants lead to different disorders, such as dilated cardiomyopathy and Hutchinson-Gilford progeria (HGP). Among these, the phenotypic terms for LMNA-associated cardiocutaneous progeria syndrome (LCPS), which does not alter lamin A processing and has an older age of onset, have been described. Here, we present the workup of an LMNA variant of uncertain significance, NM_170707.2 c. 4G>A, p.(Glu2Lys), in a 36-year-old female with severe calcific aortic stenosis, a calcified mitral valve, premature aging, and a family history of similar symptoms. Due to the uncertainty of in silico predictions for this variant, an assessment of nuclear morphology was performed using the immunocytochemistry of stable cell lines to indicate whether the p.(Glu2Lys) had a similar pathogenic mechanism as a previously described pathogenic variant associated with LCPS, p.Asp300Gly. Indirect immunofluorescence analysis of nuclei from stable cell lines showed abnormal morphology, including lobulation and occasional ringed nuclei. Relative to the controls, p.Glu2Lys and p.Asp300Gly nuclei had significantly (p < 0.001) smaller average nuclear areas than controls (mean = 0.10 units, SD = 0.06 for p.Glu2Lys; and mean = 0.09 units, SD = 0.05 for p.Asp300Gly versus mean = 0.12, SD = 0.05 for WT). After functional studies and segregation studies, this variant was upgraded to likely pathogenic. In summary, our findings suggest that p.Glu2Lys impacts nuclear morphology in a manner comparable to what was observed in p.Asp300Gly cells, indicating that the variant is the likely cause of the LCPS segregating within this family.

Study of the frequencies of erythrocyte abnormalities as in situ biomarkers of genotoxic risk of chemicals in special fish stock in water supply reservoirs.

In three water-supply reservoirs in the catchment area of the Odra River (Czech Republic), a special fish stock was monitored for control of health to estimate the mutagenic effect of chemicals. The results contribute to obtaining initial information about the morphology of erythrocyte abnormalities classified in 21 categories in 16 fish species in reservoirs with abundant salmonids (the Morávka Reservoir) or with the prevalence of cyprinids (the Kruzberk and Sance Reservoirs), not directly exposed to the adverse environmental effects such as industrial, urban, agricultural and intensive farming activities. The different intensities and prevalence of nuclear abnormalities (NA) and cytoplasmic abnormalities (CA) in fish from the same reservoir habitat show that to be able to obtain an objective view of the genotoxic risk of chemicals, it is necessary to respect the different requirements of the fish for the exploitation of the food available in the biotope and to subject all representatives of piscivorous, omnivorous and benthophagous fishes in the reservoir to cytogenetic analysis. The occurrence of certain categories of erythrocyte abnormalities in diseased fish draws attention to the need to know the state of health of the fish and to employ this knowledge to exclude parasitological, viral and other infectious agents. These results are the first report of the frequencies of erythrocyte abnormalities in native fish. They should serve to check which of the categories examined could be of use in assessing the genotoxic risk in other stagnant and running aquatic ecosystems affected by anthropogenic activities.

Cytogenotoxic potential and toxicity in adult Danio rerio (zebrafish) exposed to chloramine T.

Background: Chloramine-T (CL-T) is a synthetic sodium salt used as a disinfectant in fish farms to combat bacterial infections in fish gills and skin. While its efficacy in pathogen control is well-established, its reactivity with various functional groups has raised concerns. However, limited research exists on the toxicity of disinfection by-products to aquatic organisms. Therefore, this study aims to assess the sublethal effects of CL-T on adult zebrafish by examining biomarkers of nucleus cytotoxicity and genotoxicity, acetylcholinesterase (AChE) inhibition, and histopathological changes. Methods: Male and female adult zebrafish (wildtype AB lineage) specimens were exposed to 70, 140, and 200 mg/L of CL-T and evaluated after 96 h. Cytotoxic and genotoxic effects were evaluated by estimating the frequencies of nuclear abnormalities (NA), micronuclei (MN), and integrated optical density (IOD) of nuclear erythrocytes. Histopathological changes in the gills and liver were assessed using the degree of tissue changes (DTC). AChE activity was measured in brain samples. Results and conclusions: At a concentration of 200 mg/L, NA increased, indicating the cytogenotoxic potential of CL-T in adult zebrafish. Morphological alterations in the nuclei were observed at both 70 and 200 mg/L concentrations. Distinct IOD profiles were identified across the three concentrations. There were no changes in AChE activity in adult zebrafish. The DTC scores were high in all concentrations, and histological alterations suggested low to moderate toxicity of CL-T for adult zebrafish.

Toxicological effects of aqueous extract of Genipa americana L. leaves on adult zebrafish (Danio rerio): Chemical profile, histopathological effects and lack of genotoxicity.

Genipa americana is a native plant of Brazil with potential applications in folk medicine. Whereas most of the phytochemical and pharmacological studies on this plant have focused on its fruits, the crude extracts of its leaves contain chemical metabolites that may have toxicity to organisms, which have yet to be investigated. This study aimed to determine the main groups of secondary metabolites in the aqueous extract of the leaves of G. americana by phytochemistry and qualitative HPLC, and to evaluate the possible toxicological effects and histopathological changes caused by this extract in zebrafish (Danio rerio) adults, through micronucleus test, nuclear abnormalities and histopathological analyses of gills and liver. While three metabolites of high intensity (phenolic compounds, flavonoids and triterpenes) were found in the phytochemical evaluation, the HPLC showed results compatible with flavonoids and iridoids, all belonging to common classes for this species and the Rubiaceae family. The acute toxicity test did not induce mortality or genotoxicity in zebrafish, but after exposure for 96 h, it was possible to observe injuries to the fish gill tissue, such as lamellar fusion, vasodilation and telangiectasia; in the liver, necrosis was visualized at 40 mg/L, and at higher concentrations (80 and 100 mg/L) induced sinusoidal widening was identified. In conclusion, the results demonstrated the toxic potential of this plant for aquatic species.

Micronuclei and nuclear buds in amniotic tissue of rats treated with cyclophosphamide.

Fetal development can be altered by DNA damage caused by maternal exposure to chemical, physical, or biological agents during gestation. One method of assessing genotoxicity is to detect micronuclei (MNs) and/or nuclear abnormalities. This can be performed in vivo and requires only frequently dividing tissues, such as amniotic tissue (AT), which is in contact with the fetal environment and is composed of very thin layers of cells. This study evaluated the presence of MNs, nucleoplasmic bridges, and nuclear buds (NBs) in the fetal AT following maternal exposure to cyclophosphamide (CP) during pregnancy. Pregnant Wistar rats were divided into a negative control group and an experimental group that was orally administered CP (10 mg/kg). Daily blood smears were obtained from pregnant rats on days 14-19 of gestation. The rats were dissected, and fetal ATs were obtained on the 19th day of gestation. The MN and NB frequencies in AT cells were analyzed using a fluorescence microscope (100 ×). Micronucleated erythrocytes in the peripheral blood of the control rats were also assessed. Micronucleated polychromatic erythrocyte frequencies were significantly higher than those in the controls. Polychromatic erythrocyte frequencies were lower in CP-treated rats than in controls at 48-120 h. Fetuses in the CP-treated group also showed a significant increase in MNs and NBs in AT cells. In conclusion, AT could be used for analyzing MNs and NBs in rats following maternal exposure to a genotoxic agent and as a viable alternative for analyzing the integrity of fetal DNA during gestation.

Occupational exposure to pesticides: DNA damage in horticulturist from Nativitas, Tlaxcala in Mexico.

Mexico is a country where agricultural activity is of great importance, but biomonitoring data are still scarce. With more intensive pesticides use per unit area/surface in horticultural productivity, there is a higher impact on environmental contamination and workers' health. Considering that exposure to various pesticide and pesticide mixtures represents an additional genotoxic risk, the appropriate characterization of exposure, confounding factors and the risk itself are very much needed. We compared genetic damage in 42 horticulturists and 46 unexposed controls (Nativitas, Tlaxcala) using alkaline comet (whole blood) and micronucleus (MN) test with nuclear abnormalities (NA) (buccal epithelial cells). Workers demonstrated significantly higher levels of damage (TI%=14.02 ± 2.49 vs. 5.37 ± 0.46; MN=10.14 ± 5.15 vs. 2.40 ± 0.20), with more than 90% of them not using protective clothing nor gloves during application. Combined DNA damage techniques and periodic monitoring together with educational programs for safe pesticide application is the best strategy to assess and prevent workers' health risks.

Ecotoxicological effects of conventional herbicides and a natural herbicide on freshwater fish (Danio rerio).

The contamination of aquatic environments has increased over time, affecting environmental integrity and human health. Herbicides represent a source of this contamination, and among the most commercialized are the triazines and glyphosate. In contrast, there are natural herbicides, which are less harmful to the environment. The aim of this study was to verify the ecotoxicological effects of the conventional herbicides (Atrazine and Glyphosate) and a natural herbicide on Zebrafish (Danio rerio). Fish were exposed for 72 h to different concentrations of conventional herbicides (1, 65, and 5,000 µg L-1) and the natural herbicide (0.62, 1.25, and 2.5 µg L-1) in aquaria (20 L). At end of the experiment, blood was collected from the fish, and slides were prepared to verify the frequency of nuclear abnormalities and micronuclei. A greater number of morphological nuclear alterations were recorded in the conventional herbicide treatments. In addition, significant differences were observed in the composition of abnormalities between treatments. Atrazine and Glyphosate can cause greater damage in D. rerio, demonstrating potential toxicity at all concentrations applied. The natural herbicide showed less mutagenic potential and was less harmful to fish. Thus, natural herbicides can be a better option for the preservation of the environment.

In vivo assessment of genotoxic effects in Cyprinus carpio L., 1758 (Teleostei: Cyprinidae) exposed to selected metal(oid)s.

Heavy metals and metalloids originating from industrial, agricultural, and urban wastes and increasing in aquatic ecosystems cause genotoxic damage to fish species. This study aimed to determine the potential genotoxic effects of mixtures of aluminum, arsenic, and manganese in Cyprinus carpio. The effects of the mixtures on erythrocyte cells of C. carpio were examined using the comet assay, micronucleus test, and erythrocytic nuclear abnormalities in two groups after exposure to three doses of the mixtures (Group A; Dose 1: 0.3 + 0.1 + 0.02 mg/L, Dose 2: 0.6 + 0.2 + 0.04 mg/L, Dose 3: 0.9 + 0.3 + 0.06 mg/L and Group B; Dose 1: 1 + 3 +0.1 mg/L, Dose 2: 2 + 6 + 0.2 mg/L, Dose 3: 3 + 9 + 0.3 mg/L). Experimental groups were formed according to the permissible limits specified in the Turkish Surface Water Quality Regulation (TSWQR). The results of comet assay parameters such as tail DNA %, tail moment, and olive tail moment confirmed the genotoxic effect of metal(oid)s mixtures on erythrocyte cells compared with control groups and showed that DNA damage increased with increasing the concentrations. The micronucleus and other nuclear abnormalities such as blebbed nuclei, notched nuclei, eightshaped nuclei, lobed nuclei, and binucleated cells were detected in the erythrocyte cells exposed to the mixtures. Consequently, it was found that the frequency of micronucleus and erythrocytic nuclear abnormalities significantly increased in the erythrocyte cells exposed to metal(oid) concentrations compared to control groups. These results show the existence of potential genotoxicity in C. carpio even at the minimum values specified in the TSWQR after exposure to the mixtures.

How can environmental conditions influence dicofol genotoxicity on the edible Asiatic clam, Meretrix meretrix?

Genotoxic effects of dicofol on the edible clam Meretrix meretrix were investigated through a mesocosm experiment. Individuals of M. meretrix, were exposed to environmental concentration (D1 = 50 ng/L) and supra-environmental concentration (D2 = 500 ng/L) of dicofol for 15 days, followed by the same depuration period. DNA damage (i.e., strand breaks and alkali-labile sites) was evaluated at day 1, 7 and 15, during uptake and depuration, using Comet assay (alkaline version) and nuclear abnormalities (NAs) as genotoxicity biomarkers. The protective effects of dicofol against DNA damage induced by ex vivo hydrogen peroxide (H2O2) exposure were also assessed. Comet assay results revealed no significant DNA damages under dicofol exposure, indicating 1) apparent lack of genotoxicity of dicofol to the tested conditions and/or 2) resistance of the animals due to optimal adaptation to stress conditions. Moreover, ex vivo H2O2 exposure showed an increase in the DNA damage in all the treatments without significant differences between them. However, considering only the DNA damage induced by H2O2 during uptake phase, D1 animals had significantly lower DNA damage than those from other treatments, revealing higher protection against a second stressor. NAs data showed a decrease in the % of cells with polymorphic, kidney shape, notched or lobbed nucleus, along the experiment. The combination of these results supports the idea that the clams used in the experiment were probably collected from a stressful environment (in this case Pearl River Delta region) which could have triggered some degree of adaptation to those environmental conditions, explaining the lack of DNA damages and highlighting the importance of organisms' origin and the conditions that they were exposed during their lives.

Genotoxic and mutagenic effects of chlorothalonil on the estuarine fish Micropogonias furnieri (Desmarest, 1823).

Chlorothalonil is a fungicide widely used in agriculture as well as an active ingredient in antifouling paints. Although it causes toxic effects on non-target organisms and can accumulate in fish tissues, little is known about its sublethal effects. Thus, genotoxic and mutagenic effects of intraperitoneal injected chlorothalonil in Micropogonias furnieri, an estuarine fish of frequent human consumption and a promising test-organism for ecotoxicological assays, were assessed. Chlorothalonil showed to be genotoxic (DNA damage by comet assay) and mutagenic (micronuclei, nuclear buds, apoptotic fragments, and bilobed cells) even at the lowest dose tested (0.35 µg g-1) and in a dose-dependent manner (0.35 and 3.5 µg g-1) for micronuclei, apoptotic fragments, and bilobed cells. As genomic instability may lead to carcinogenesis, the present evidence can assist decision-makers in banning this compound since any benefit toward food production is outweighed by the hazard to aquatic ecosystems and human health.

A Systematic Review of Studies on Genotoxicity and Related Biomarkers in Populations Exposed to Pesticides in Mexico.

In agricultural activities, pest control is essential, and the most effective method is the use of chemical agents that also represent an important source of exposure to potentially toxic compounds. Pesticides constitute a heterogeneous group of compounds designed specifically to control different pests. Besides measuring their levels or that of their metabolites in air, plasma, serum, blood, urine, etc., some studies reported increased DNA damage levels after occupational or environmental pesticides exposure, evidenced by several cytogenetic biomarkers such as chromosomal aberrations (CA), sister chromatid exchanges (SCE), micronuclei frequency (MN) together with other nuclear abnormalities (NA), alkaline comet assay, but also changes in oxidative stress parameters and miRNA levels. Single or combined, these techniques have also been used in genotoxic biomonitoring studies of workers occupationally exposed to pesticides in Mexico. Despite being a country with great agricultural activity and reported excessive pesticide use, genotoxic studies have been relatively few and, in some cases, contradictory. A review was made of the studies available (published until the end of 2020 on PubMed, Web of Science, Redalyc and Scielo, both in English and Spanish) in the scientific literature that evaluated occupational exposure of human samples to pesticides assessed with DNA damage and related biomarkers in Mexico.

Genotoxic effects of urban pollution in the Iguaçu River on two fish populations.

Environmental impacts on water resources and discharges of pollutants are some of the main problems of aquatic ecosystems. Currently, the Iguaçu River is considered the second most polluted river in Brazil, therefore, given the importance of understanding the effects of water pollution on living organisms, this study aimed to analyze the possible genotoxic effects in Astyanax bifasciatus (Characiformes; Characidae) and Geophagus brasiliensis (Perciformes: Cichlidae) in response to the water quality of the Iguaçu River. Four sampling points were determined on the Iguaçu River, with different levels of environmental impact. The micronuclei and nuclear abnormalities were quantified by counting 2000 erythrocytes from each specimen. There was a statistical difference in the frequency of nuclear abnormalities both for A. bifasciatus (Kruskal-Wallis 3;30 = 10.9; p = 0.01) and for G. brasiliensis (Kruskal-Wallis 3;26 = 12.27; p = 0.006), with point 1 (critically degraded) showing a higher frequency of nuclear abnormalities and point 4 (moderately degraded) showing a lower frequency of nuclear abnormalities for both species, showing genotoxic potential in fish erythrocytes. This result demonstrates the need for further discussion on the conservation of the Iguaçu River as well as broadening the discussion on its potability.

Habitat differences affect the nuclear morphology of the erythrocytes and the hepatic melanin in Leptodactylus fuscus (Anura) in the Brazilian Cerrado savanna.

The sensitivity of anuran to the effects of habitat destruction and contamination has led to a preoccupying global decline in their populations. Morphological biomarkers such as micronuclei and other erythrocyte nuclear abnormalities (ENAs), as well as the occurrence of hepatic melanin, can be used to evaluate the effects of habitat impacts. In the present study, these two parameters were combined for the in situ assessment of the effects of soybean cultivation on the grassfrog, Leptodactylus fuscus. Specimens were also collected from a protected area to provide a reference site (non-agricultural environment). The frequency of some of the nuclear abnormalities in the animals from the soybean plantation was much higher than that recorded at the reference site, in particular micronuclei, which were 3.6 times more frequent in the plantation, lobulated nuclei (3.4 times more frequent), and reniform nuclei, which were four times more common than at the reference site. The combined analysis of all the ENAs together also revealed a frequency approximately 1.4 times higher in the animals from the soybean plantation, in comparison with the protected area. Smaller areas of hepatic melanin were observed in the specimens from the soybean plantation. These results provide further evidence of the sensitivity of anurans to habitat impacts and indicate that animals found in soybean plantations are susceptible to systematic alterations of their cells.

Biological effects of multimetal (Ni, Cd, Pb, Cu, Cr, Zn) mixture in rainbow trout Oncorhynchus mykiss: Laboratory exposure and recovery study.

The present study tested the biological consequences of exposure to a multimetal mixture as a multiple chemical stressor on Oncorhynchus mykiss at molecular, cellular, physiological and whole-organism levels and on biomarker responses of this fish during the depuration period. To represent environmentally relevant multiple chemical stressors, in our study, we used the mixture of Zn, Cu, Ni, Cr, Pb and Cd at the concentrations corresponding to Maximum-Permissible-Concentrations (MPCs) acceptable for the EU inland waters. This study was undertaken with a view to elucidate if changes in the MPC of the test mixture components (Ni, Pb, Cd) could cause significantly different biomarker responses in O. mykiss from those previously determined in the carnivorous and omnivorous fishes exposed to the mixture of the same metals but at different MPCs of Ni, Pb and Cd. This study has revealed that exposure to mixtures of metals at MPC produces genotoxic effects in fish blood erythrocytes and a lethargic effect on O. mykiss behaviour, and, also, significantly increases the levels of Cd, Cr and Ni accumulated in the gills tissue. O. mykiss successfully depurated Cr and Ni in less than 28 days, however, the level of Cd decreased by only approximately 40% over the same period. A significant capacity of O. mykiss to restore its DNA integrity (Comet assay) after exposure to metal mixtures was revealed. However, the 28-day recovery period proved to be insufficiently long for erythrocytes with nuclear abnormalities to recover to the unexposed level. In conclusion, changes in the MPCs of Ni, Pb and Cd in the test mixture produce biological effects similar to those previously determined in S. salar, R. rutilus and P. fluviatilis exposed to the mixture of the same metals but at lower MPCs of Ni and Pb and at higher MPC of Cd.

Genotoxic and melanic alterations in Lithobates catesbeianus (anura) tadpoles exposed to fipronil insecticide.

The aim of this study was to evaluate the genotoxic and morphological systemic effects of both an acute and a chronic exposure of bullfrog tadpoles to fipronil. Lithobates catesbeianus tadpoles had morphological biomarkers (skin, liver, and blood) analyzed at Gosner stages 36-38, when exposed to four different concentrations of Regent® 800 WG (80% fipronil): 0.00 (control), 0.04, 0.08, 0.4 mg/L, and four experimental times: 4, 8, 12, and 16 days. Body darkness responded directly to the treatment and exposure time. There was a treatment-dependent decrease in darkness of heads and tails. In relation to the biometric analysis, fipronil induced a decrease in the individual weight and liver mass at the end of the experiments, whereas the hepatosomatic index did not vary according to the treatment. For the exposed animals and for the control group, the area of hepatic melanin increased as exposure time increased. Fipronil has genotoxic effects on L. catesbeianus tadpoles even after short exposure times (e.g., 4 and 8 days), and the main nuclear abnormality is in the anucleate cells. A relevant correlation was observed between genotoxic biomarkers and cutaneous and internal melanin. The frequency of nuclear abnormalities is inversely correlated both with the hepatic melanin area and with the cutaneous melanin of animals. Fipronil has distinct systemic effects on tadpoles based on its concentration, as well as on its exposure time. Such alterations (pigmentation level and rate of erythrocyte abnormality) result in morphological and physiological effects, which may compromise the behavior and survival of the anurans.

Directly converted astrocytes retain the ageing features of the donor fibroblasts and elucidate the astrocytic contribution to human CNS health and disease.

Astrocytes are highly specialised cells, responsible for CNS homeostasis and neuronal activity. Lack of human in vitro systems able to recapitulate the functional changes affecting astrocytes during ageing represents a major limitation to studying mechanisms and potential therapies aiming to preserve neuronal health. Here, we show that induced astrocytes from fibroblasts donors in their childhood or adulthood display age-related transcriptional differences and functionally diverge in a spectrum of age-associated features, such as altered nuclear compartmentalisation, nucleocytoplasmic shuttling properties, oxidative stress response and DNA damage response. Remarkably, we also show an age-related differential response of induced neural progenitor cells derived astrocytes (iNPC-As) in their ability to support neurons in co-culture upon pro-inflammatory stimuli. These results show that iNPC-As are a renewable, readily available resource of human glia that retain the age-related features of the donor fibroblasts, making them a unique and valuable model to interrogate human astrocyte function over time in human CNS health and disease.