Identification of tyrosine brominated extracellular matrix proteins in normal and fibrotic lung tissues.

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin ß1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/ß1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor ß1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.

Uric Acid Reacts with Peroxidasin, Decreases Collagen IV Crosslink, Impairs Human Endothelial Cell Migration and Adhesion.

Uric acid is considered the main substrate for peroxidases in plasma. The oxidation of uric acid by human peroxidases generates urate free radical and urate hydroperoxide, which might affect endothelial function and explain, at least in part, the harmful effects of uric acid on the vascular system. Peroxidasin (PXDN), the most recent heme-peroxidase described in humans, catalyzes the formation of hypobromous acid, which mediates collagen IV crosslinks in the extracellular matrix. This enzyme has gained increasing scientific interest since it is associated with cardiovascular disease, cancer, and renal fibrosis. The main objective here was to investigate whether uric acid would react with PXDN and compromise the function of the enzyme in human endothelial cells. Urate decreased Amplex Red oxidation and brominating activity in the extracellular matrix (ECM) from HEK293/PXDN overexpressing cells and in the secretome of HUVECs. Parallelly, urate was oxidized to 5-hydroxyisourate. It also decreased collagen IV crosslink in isolated ECM from PFHR9 cells. Urate, the PXDN inhibitor phloroglucinol, and the PXDN knockdown impaired migration and adhesion of HUVECs. These results demonstrated that uric acid can affect extracellular matrix formation by competing for PXDN. The oxidation of uric acid by PXDN is likely a relevant mechanism in the endothelial dysfunction related to this metabolite.

Anacardium microcarpum extract and fractions protect against paraquat-induced toxicity in Drosophila melanogaster.

Anacardium microcarpum Ducke (Anacardiaceae) is a native species of Brazil used in folk medicine for the treatment of several illnesses although its antioxidant activity has been reported in vitro, there is no evidence of this effect in an in vivo model. Here, we investigated the potential protective effect of hydroalcoholic extract (AMHE), methanol (AMMF) and acetate (AMAF) fraction of A. microcarpum against paraquat toxicity on survivorship, locomotor performance, antioxidant enzymes activity and reactive species using Drosophila melanogaster. Flies were exposed to the extract or fractions (1 and 10 mg/ml) in the presence or absence of paraquat (5 mM) in sucrose solution for 72 h. In addition, total phenolic content of extract and fractions was evaluated as well as ABTS radical scavenging capacity. Our results demonstrated that AMAF presented higher content of phenols and ABTS chelating potential. Treatment of flies with the extract or fractions did not alter the survivorship, locomotor ability, and acetylcholinesterase (AchE) activity per se. Paraquat caused 85 % mortality of flies and 30 % increase in reactive species generation, which were significantly attenuated by AMHE and AMMF. AAMF increased catalase activity (from 66.77 ± 6.64 to 223.94 ± 25.92 mU/mg of protein), while AMAF increased GST activity (from 477.76 ± 92 to 770.19 ± 147.92 mU/mg of protein) and catalase activity (from 66.77 ± 6.64 to 220.54 ± 26.63 mU/mg of protein). AMHE and AMMF were more effective in protecting against paraquat toxicity. Taken together, the data indicate the potential of this plant in acting as a protective and antioxidant agent in vivo.

Senecio brasiliensis impairs eclosion rate and induces apoptotic cell death in larvae of Drosophila melanogaster.

Senecio brasilienis (Spreng) Less., is a species native from Brazil, popularly known as "Maria mole", and known to induce hepatotoxicity due to its high content of Pyrrolizidine alkaloids. Despite its toxicity, this plant is widely used in Brazilian folk medicine. Considering the antagonizing effects described for S. brasiliensis, we describe here molecular markers involved in the toxicity of hydroalcoholic extract from leaves of S. brasiliensis (HESB) in Drosophila melanogaster. Phytochemical analysis of HESB revealed the presence of phenolic acids and flavonoids. A significant antioxidant potential against ABTS+ and DPPH radical was found in parallel. Ingestion of extract did not alter the survival and locomotor activity of adult flies. However when ingested along the larval developmental phase, the eclosion rate of flies was interrupted at higher concentration of extract. To comprehend this phenomenon several analysis were conducted in larvae. HESB stimulated activity of antioxidant enzymes SOD and GST, and increased GSH/GSSG ratio and ROS production. Additionally, HESB caused a significant decrease of cell viability. The mRNA expression of Nrf2, TrxR, CAT, Drice and Dilp6 were also significantly up-regulated. HESB caused significant decrease on the phosphorylation of MAPKs and AKT. In parallel, PARP cleavage and caspases 3/7 activity were stimulated. In addition, glucose, glycogen and triglycerides levels were decreased. Taken together our study depicts a disruption in the eclosion of D. melanogaster possibly attributed to the inhibition of kinases implied in developmental process, energetic demand and induction of apoptotic cell death process.

Oxidant effects and toxicity of Croton campestris in Drosophila melanogaster.

CONTEXT: Croton campestris A.St.-Hil. (Euphorbiaceae) is a species native to Northeast Brazil used by traditional communities for the treatment of a variety of health problems. However, potential toxicological effects of this plant are unknown. OBJECTIVE: The potential toxicity of the hydroalcoholic extract of C. campestris leaves on Drosophila melanogaster insect model, additionally with phytochemical constitution and cellular mechanisms mediating the action of extract were analysed in this study. MATERIALS AND METHODS: Constituents of the extract were evaluated by HPLC. In vitro antioxidant potential of extract was analysed by DPPH, ABTS and FRAP. Flies injected culture medium mixed with extract (0.1-50 mg/mL) for 72 h. After, ROS production was evaluated by DCF-DA oxidation. Phosphorylation of MAPK signalling pathway was investigated by Western blotting method. Activity of antioxidant enzymes was analysed in homogenates. RESULTS: Major components of the extract include quercetin (38.11 ± 0.06 mg/g), caffeic acid (20.06 ± 0.17 mg/g) and kaempferol (15.45 ± 0.05 mg/g). Consumption of the extract impaired locomotor performance and induced fly death of flies (LC50 of 26.51 mg/mL). Augmented ROS formation and SOD, CAT and GST activity were observed from 0.1 mg/mL. JNK and p38 kinases phosphorylation was modulated and Paraquat-induced toxicity was augmented by extract. DISCUSSION AND CONCLUSION: Our data show important toxicological effects of C. campestris leading to increased mortality and impaired locomotor performance accompanied by induction of cell stress markers in flies. The study draws attention to the indiscriminate use of plant extracts.

Phytochemical Composition, Antifungal and Antioxidant Activity of Duguetia furfuracea A. St.-Hill.

UNLABELLED: Background. Duguetia furfuracea is popular plant used in popular medicine. Hypothesis/Purpose. This claim evaluated the phytochemical composition of the hydroethanolic extract (HEDF), fractions of Duguetia furfuracea, and antioxidant and antifungal activity. Methods. The chemical profile was carried out by HPLC-DAD. The total phenolic contents and flavonoid components were determined by Folin-Ciocalteu and aluminium chloride reaction. The antioxidant activity was measured by scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical and ferric reducing ability of plasma (FRAP) methods. The antifungal activity was determined by microdilution assay. RESULTS: HPLC analysis revealed caffeic acid and rutin as major compounds (HEDF), caffeic acid and quercitrin (Mt-OH fraction), and quercitrin and isoquercitrin (Ac-OEt fraction). The highest levels of phenols and total flavonoids were found for Ac-OEt fraction, and the crude extract showed higher in vitro antioxidant potential. The antifungal activity showed synergic effect with fluconazole and EHDF against C. krusei, fluconazole and Mt-OH against C. krusei and C. tropicalis, and Ac-OE and fluconazole against C. albicans. Conclusion. The highest levels of phenols and total flavonoids were marked with antioxidant effect. This is the first report of bioactivity of the synergic effect of HEDF and fractions. More studies would be required to better clarify its mechanism of synergic action.

Fumigant activity of the Psidium guajava var. pomifera (Myrtaceae) essential oil in Drosophila melanogaster by means of oxidative stress.

The guava fruit, Psidium guajava var. pomifera (Myrtaceae family), is a native plant from South America. Its leaves and fruits are widely used in popular medicine in tropical and subtropical countries. Drosophila melanogaster has been used as one of the main model organisms in genetic studies since the 1900s. The extensive knowledge about this species makes it one of the most suitable organisms to study many aspects of toxic compound effects. Due to the lack of studies on the effects of the bioactive compounds present in the P. guajava var. pomifera essential oil, we performed a phytochemical characterization by CG-MS and evaluated the toxicity induced by the essential oil in the D. melanogaster insect model. In order to understand the biochemical mechanisms of toxicity, changes on the Nrf2 signaling as well as hallmarks of oxidative stress response were followed in the exposed flies. Our results showed that exposure of insects to the P. guajava oil increased mortality and locomotor deficits in parallel with an oxidative stress response signaling. Therefore, it suggested a bioinsecticidal activity for P. guajava volatile compounds by means of oxidative stress. Further studies are ongoing to identify which oil compounds are responsible for such effect.

Toxicity induced by Prasiola crispa to fruit fly Drosophila melanogaster and cockroach Nauphoeta cinerea: evidence for bioinsecticide action.

The adverse effects of the alga Prasiola crispa extract (PcE) were investigated in a fruit fly (Drosophila melanogaster) and cockroach (Nauphoeta cinerea) model. In flies, toxicity was assessed as mortality and biochemical alterations including acetylcholinesterase (AChE) activity and oxidative stress markers. The cardiotoxic action of PcE was also examined in a model of semi-isolated cockroach heart. The administration of PcE (2 mg/ml) to flies for 24 h resulted in a marked increase in mortality rate (7.6-fold rise compared to control). AChE activity, glutathione (GSH) levels, and hydroperoxide formation remained unchanged. Fly glutathione S-transferase (GST) and catalase (CAT) activity were significantly altered after PcE treatment. Fraction III (ethyl acetate) of PcE was significantly more toxic to flies compared to fractions I (methanol) and II (ethanol). A significant decrease was noted in cockroach semi-isolated heart function. The addition of 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB), an oxidizing agent, concomitant with the extract significantly blocked this effect, suggesting that reduced compounds may be involved in the cardiotoxic action produced by PcE. Our results show for the first time the adverse effects of PcE in two insect models, Drosophila melanogaster and Nauphoetacinerea. The insecticidal properties of PcE may be related to changes in important antioxidant/detoxifying systems, as well as to changes in insect cardiac function.

Drosophila melanogaster - an embryonic model for studying behavioral and biochemical effects of manganese exposure.

Embryonic animals are especially susceptible to metal exposure. Manganese (Mn) is an essential element, but in excess it can induce toxicity. In this study we used Drosophila melanogaster as an embryonic model to investigate biochemical and behavioral alterations due to Mn exposure. Flies were treated with standard medium supplemented with MnCl2 at 0.1 mM, 0.5 mM or 1 mM from the egg to the adult stage. At 0.5 mM and 1 mM Mn, newly ecloded flies showed significantly enhanced locomotor activity when assessed by negative geotaxis behavior. In addition, a significant increase in Mn levels (p < 0.0001) was observed, while Ca, Fe, Cu, Zn and S levels were significantly decreased. A significant drop in cell viability occurred in flies exposed to 1 mM Mn. There was also an induction of reactive oxygen species at 0.5 mM and 1 mM Mn (p < 0.05). At 1 mM, Mn increased Catalase (p < 0.005), Superoxide Dismutase (p < 0.005) and Hsp83 (p < 0.0001) mRNA expression, without altering Catalase or Superoxide Dismutase activity; the activity of Thioredoxin reductase and Glutatione-S-transferase enzymes was increased. Mn treatment did not alter ERK or JNK1/2 phosphorylation, but at 1 mM caused an inhibition of p38(MAPK) phosphorylation. Together these data suggest mechanisms of adaptation in the fly response to Mn exposure in embryonic life.