rGO outperforms GO in generating oxidative stress and DNA strand breaks in zebrafish liver cells.

Graphene oxide (GO) and reduced graphene oxide (rGO) are both widely applicable and there is a massive production throughout the world which imply in inevitable contamination in the aquatic environment by their wastes. Nevertheless, information about their interaction at the cellular level in fish is still scarce. We investigated the metabolic activity, reactive oxygen species (ROS) production, responses of antioxidant defenses, and total antioxidant capacity (TAC) as well as oxidative stress and DNA integrity in zebrafish liver cells (ZFL) exposed to (0.001, 0.01, 0.1 and 1 µg mL-1) of GO and rGO after two exposure period (24 and 72 h). Higher ROS production and no significant changes in the antioxidant defenses resulted in lipid peroxidation in cells exposed to rGO. Cells exposed to GO increased the activity of antioxidant defenses sustaining the TAC and avoiding lipid peroxidation. Comet assay showed that both, GO and rGO, caused DNA strand breaks after 24 h of exposure; however, only rGO caused DNA damage after 72 h of exposure. The exposure to rGO was significantly more harmful to ZFL cells than GO, even at very low concentrations. The cells showed a high capacity to neutralize ROS induced by GO preventing genotoxic effects and metabolic activity, thus sustaining cell viability. The time of exposure had different impacts for both nanomaterials, GO caused more changes in 24 h showing recovery after 72 h, while cells exposed to rGO were jeopardized at both exposure times. These results indicate that the reduction of GO by removal of the oxygen functional groups (rGO) increased toxicity leading to adverse effects in the cells, even at very low concentrations.

Liver dysfunction and energy storage mobilization in traíra, Hoplias malabaricus (Teleostei, Erythrinidae) induced by subchronic exposure to toxic cyanobacterial crude extract.

Microcystins (MC) are hepatotoxic for organisms. Liver MC accumulation and structural change are intensely studied, but the functional hepatic enzymes and energy metabolism have received little attention. This study investigated the liver and hepatocyte structures and the activity of key hepatic functional enzymes with emphasis on energetic metabolism changes after subchronic fish exposure to cyanobacterial crude extract (CE) containing MC. The Neotropical erythrinid fish, Hoplias malabaricus, were exposed intraperitoneally to CE containing 100 µg MC-LR eq kg-1 for 30 days and, thereafter, the plasma, liver, and white muscle was sampled for analyses. Liver tissue lost cellular structure organization showing round hepatocytes, hyperemia, and biliary duct obstruction. At the ultrastructural level, the mitochondria and the endoplasmic reticulum exhibited disorganization. Direct and total bilirubin increased in plasma. In the liver, the activity of acid phosphatase (ACP) increased, and the aspartate aminotransferase (AST) decreased; AST increased in plasma. Alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were unchanged in the liver, muscle, and plasma. Glycogen stores and the energetic metabolites as glucose, lactate, and pyruvate decrease in the liver; pyruvate decreased in plasma and lactate decreased in muscle. Ammonia levels increased and protein concentration decreased in plasma. CE alters liver morphology by causing hepatocyte intracellular disorder, obstructive cholestasis, and dysfunction in the activity of key liver enzymes. The increasing energy demand implies glucose mobilization and metabolic adjustments maintaining protein preservation and lipid recruitment to supply the needs for detoxification allowing fish survival.

Curcumin/d-mannitol as photolarvicide: induced delay in larval development time, changes in sex ratio and reduced longevity of Aedes aegypti.

BACKGROUND: Resistant populations of Ae. aegypti have been a major problem in arboviruses epidemic areas, generating a strong demand for novel methods of vector control. In this regard, our group has demonstrated the use of curcumin as an efficient photoactive larvicide to eliminate Ae. aegypti larvae. This work was aimed to evaluate the Ae. aegypti (Rockefeller) development under sublethal conditions, using a curcumin/d-mannitol (DMC) formulation. The photolarvicidal efficacy under semi-field and field conditions (wild populations) was also analyzed, as well as the photobleaching and residual activity of DMC. RESULTS: A delay in development time when larvae were exposed to sublethal concentrations of DMC was observed, followed by significant changes in sex ratio and reduction in longevity. DMC also presented a low residual activity when compared to usual larvicides, and had a substantial photolarvicidal activity against wild populations in field trials, achieving 71.3% mortality after 48 h. CONCLUSIONS: Overall, these findings are of great biological importance for the process of enabling the implementation of DMC as a new product in the control of Ae. aegypti larvae, and contributes to the improvement of new plant-based larvicides. © 2021 Society of Chemical Industry.

Curcumin in formulations against Aedes aegypti: Mode of action, photolarvicidal and ovicidal activity.

Combating the Aedes aegypti vector is still the key to control the transmission of many arboviruses, such as Dengue, Zika, and Chikungunya. As few products are efficient for Aedes aegypti control, the search for new strategies have become pivotal., t Substances with photodynamic activity, such as curcumin and their formulations, are strongly encouraged, due to their multi-target mechanism of action. In this study, we evaluated the photolarvicidal and ovicidal activity of curcumin in the presence of sucrose (named SC) and d-mannitol (named DMC). To support the understanding of the larvicidal action of these formulations, Raman micro-spectroscopy was employed. We also studied the morphological changes in Danio rerio (Zebrafish) gills, a non-target organism, and demonstrate that this is an environmentally friendly approach. Both SC and DMC presented a high photo-larvicidal potential. DMC showed the highest larval mortality, with LC50-24h values between 0.01 and 0.02 mg.L-1. DMC also significantly decreased egg hatchability, reaching a hatching rate of 10 % at 100 mg.L-1. The analysis of molecular mechanisms via Raman micro-spectroscopy showed that DMC is highly permeable to the peritrophic membrane of the larva, causing irreversible damage to the simple columnar epithelium of the digestive tube. Histological changes found in the D. rerio gills were of minimal or moderate pathological importance, indicating an adaptive trait rather than detrimental characteristics. These findings indicate that curcumin in sugar formulations is highly efficient, especially DMC, proving it to be a promising and safe alternative to control Aedes mosquitoes. Moreover, Raman micro-spectroscopy demonstrated high potential as an analytical technique to understand the mechanism of action of larvicides.

Environmental safety and mode of action of a novel curcumin-based photolarvicide.

Aedes aegypti is the vector of important diseases like dengue, zika, chikungunya, and yellow fever. Vector control is pivotal in combating the spread of these mosquito-borne illnesses. Photoactivable larvicide curcumin obtained from Curcuma longa Linnaeus has shown high potential for Ae. aegypti larvae control. However, the toxicity of this photosensitizer (PS) might jeopardize non-target aquatic organisms. The aim of this study was to evaluate the toxicity of this PS to Daphnia magna and Danio rerio, besides assessing its mode of action through larvae biochemical and histological studies. Three PS formulations were tested: PS in ethanol+DMSO, PS in sucrose, and PS in D-mannitol. The LC50 of PS in ethanol+DMSO to D. rerio was 5.9 mg L-1, while in D. magna the solvents were extremely toxic, and LC50 was not estimated. The PS formulations in sugars were not toxic to neither of the organisms. Reactive oxygen species (ROS) were generated in D. magna exposed to 50 mg L-1 of PS in D-mannitol, and D. rerio did not elicit this kind of response. D. magna feeding rates were not affected by the PS in D-mannitol. Concerning Ae. aegypti larvae, there were changes in reduced glutathione and protein levels, while catalase activity remained stable after exposure to PS in D-mannitol and sunlight. Histological changes were observed in larvae exposed to PS in sucrose and D-mannitol, most of them irreversible and deleterious. Our results show the feasibility of this photolarvicide use in Ae. aegypti larvae control and its safety to non-target organisms. These data are crucial to this original vector control approach implementation in public health policies.

Assessment of biomarkers in the neotropical fish Brycon amazonicus exposed to cypermethrin-based insecticide.

The effects of cypermethrin-based insecticide (CBI), commonly used in aquaculture and agriculture, were evaluated in matrinxa (Brycon amazonicus) exposed to sub-lethal concentration (20% of LC50) for 96 h. Physiological and biochemical effects were studied through biomarkers: lipid peroxidation (LPO), glutathione (GSH), and ascorbic acid concentrations; superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glucose-6-phosphate dehydrogenase (G6PDH) assays in the liver and gills. Besides, ions Na+, Cl-, and K+; protein and glucose concentrations were measured in the plasma. Red blood cells count (RBC), hemoglobin concentration (Hb), hematocrit (Ht), and hematimetric parameters were evaluated in the total blood. The NKA (Na+ /K+ ATPase) activity was assayed in the gills. The histopathological effects of CBI were also investigated in the gills. The liver and gill LPO increased 62 and 100%, respectively. The tripeptide GSH concentration reduced in the liver and increased in the gill of exposed fish. The SOD and CAT activities increased in the liver, whereas CAT reduced in the gill. Liver also presented an increase in G6PDH activity. Plasma Na+ and Cl- and glucose concentrations increased in the exposed fish. Levels of Ht, Hb and RBC were significantly increased. The gill NKA activity also increased. Exposed fish exhibited histological alterations in the gills such as hypertrophy and proliferation of chloride cells, blood vessels dilation, aneurysms and hemorrhage of the lamella. The histological index indicated moderate to heavy damage to the gills. CBI provokes liver and gills oxidative stress, gill structural damages, and ionic imbalance. A multi-biomarker approach allows us to see that B. amazonicus was unable to cope with CBI exposure.

Hepatotoxicity and metabolic effects of cellular extract of cyanobacterium Radiocystis fernandoi containing microcystins RR and YR on neotropical fish (Hoplias malabaricus).

The toxicological effect of cellular extract of cyanobacterium Radiocystis fernandoi strain R28 containing RR and YR microcystins was analyzed in the fish Hoplias malabaricus with emphasis on the liver structure and energetic metabolism, after short-term exposure. Fish were intraperitoneally (i.p.) injected with 100 µg of equivalent MC-LR kg-1 body mass containing in the cellular extract of R. fernandoi strain R28. Twelve and 96 h post-injection, the plasma, liver and white muscle were sampled for biochemical analyses and liver was also sampled for morphological analyses. After i.p. injection, the activity of acid phosphatase (ACP), alanine aminotransferase (ALT) and direct bilirubin increased in the plasma, while ALT and aspartate aminotransferase (AST) decreased in the liver. Glucose, lactate and pyruvate increased while protein decreased in the plasma; glycogen, pyruvate and lactate decreased in the liver; and glycogen and glucose increased in the muscle. Ammonia increased in the plasma, liver and muscle. The hepatocyte cell shape changed from polyhedral to round after cellular extract injection; there was loss of biliary canaliculus organization, but the biliary duct morphology was conserved in the liver parenchyma. In conclusion, microcystins present in the cellular extract of R. fernandoi strain R28 affect the liver structure of H. malabaricus, but the liver was able to continuously produce energy by adjusting its intermediate metabolism; glycogenolysis and gluconeogenesis maintained glucose homeostasis and energy supply.