Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells.

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA differs in pregnancies with placental dysfunction from healthy pregnancies and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA from non-placental cells; yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 mM) or rotenone (0.2-50 mM) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane bound, non-membrane bound, and vesicular-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (p<0.0001), induced cell necrosis (p=0.0004) but not apoptosis (p=0.6471) and was positively associated with release of membrane-bound and non-membrane bound mtDNA (p<0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicular-bound form; p=0.0019) and reduced autophagy marker expression (LC3A/B, p=0.0002; p62, p<0.001). Rotenone treatment did not influence mtDNA release or cell death (p>0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes non-apoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress.

Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells.

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA differs in pregnancies with placental dysfunction from healthy pregnancies and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA from non-placental cells; yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 µM) or rotenone (0.2-50 µM) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane bound, non-membrane bound, and vesicular-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (p<0.0001), induced cell necrosis (p=0.0004) but not apoptosis (p=0.6471) and was positively associated with release of membrane-bound and non-membrane bound mtDNA (p<0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicular-bound form; p=0.0019) and reduced autophagy marker expression (LC3A/B, p=0.0002; p62, p<0.001). Rotenone treatment did not influence mtDNA release or cell death (p>0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes non-apoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress. NEW & NOTEWORTHY: This is the first study to test whether trophoblast cells release mitochondrial DNA in response to oxidative stress and to identify mechanisms of release and biological forms of mtDNA from this cellular type. This research identifies potential cellular mechanisms that can be used in future investigations to establish the source and biomarker potential of circulating mitochondrial DNA in preclinical experimental models and humans.

Prospección fitoquímica y actividad antiplasmódica del extracto hexánico de Montrichardia linifera (Arruda) Schott / Phytochemical prospection and antiplasmoidal activity of the hexane extract from Montrichardia linifera (Arruda) Schott

Introducción: los extractos de M. linifera presentaron alta toxicidad para Artemia salina, lo cual sugiere un alto potencial biológico para actividad antitumoral, antibacteriana, antifúngica y actividad contra el Trypanosoma cruzi. Objetivo: evaluar el potencial antimalárico del extracto hexánico obtenido de hojas de Montrichardia linifera (Arruda) Schott y conocer su perfil fitoquímico. Métodos: la prospección fitoquímica se realizó por el método de precipitación y la actividad antiplasmódica fue evaluada in vitro utilizando el clon W2 de Plasmodium falciparum. Resultados: la prospección fitoquímica sugirió solamente la presencia de esteroides. En relación con la actividad antiplasmódica, después de 24 h en la concentración de 100 µg/mL la inhibición del crecimiento parasitario fue de 50,5 por ciento y en las demás no fue observada una inhibición significativa. En 48 h, en la concentración de 100 µg/mL, la inhibición del crecimiento fue de 34,7 por ciento. Después de 72 h las concentraciones de 100 y 50 µg/mL presentaron hemólisis, que imposibilitó la determinación porcentual parasitada. Conclusiones: el extracto hexánico obtenido de las hojas de M. linifera tiene un bajo potencial antimalárico y mostró positivamente esteroides.

Introduction: Montrichardia linifera extracts showed high toxicity to Artemia salina, which indicated high biological potential for antitumoral, antibacterial, antifungal activities, and action against Trypanossoma cruzi. Objective: to evaluate the antimalarial potential of the hexane extract from the Montrichardia linifera (Arruda) Schott leaves and to discover its phytochemical profile. Methods: the phytochemical prospection was conducted by the precipitation method whereas the antiplasmodial activity was evaluated in vitro using the W2 Plasmodium falciparum clone. Results: the phytochemical prospection just suggested the presence of steroids. Regarding the antiplasmodial activity after 24 hours at 100 µg/mL concentration, the parasite growth inhibition was 50.5 percent but at other concentration ranges, significant inhibition was not observed. At 48 hours, the growth inhibition at 100 µg/mL concentrations was 34.7 percent. After 72 hours, hemolysis was observed at 100 µg/mL and 50 µg/mL concentrations, so the percentage determination of the parasites was not possible. Conclusions: the hexane extract obtained from the M. linifera leaves presented with low antimalarial potential and proved to be positive for steroids.