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The absence of a routine continuous in vitro cultivation method for Plasmodium vivax, an important globally distributed parasite species causing malaria in humans, has restricted investigations to field and clinical sampling. Such a method has recently been developed for the Berok strain of P. cynomolgi, a parasite of macaques that has long been used as a model for P. vivax, as these two parasites are nearly indistinguishable biologically and are genetically closely related. The availability of the P. cynomolgi Berok in routine continuous culture provides for the first time an opportunity to conduct a plethora of functional studies. However, the initial cultivation protocol proved unsuited for investigations requiring extended cultivation times, such as reverse genetics and drug resistance. Here we have addressed some of the critical obstacles to this, and we propose a set of modifications that help overcome them.

Exposures in post-accidental situations are complex and include both external exposure and internal contamination with several radionuclides. However, in vivo and in vitro studies generally use simplified exposures, while a recent study suggested that combined external irradiation and internal contamination may induce more severe biological effects compared to single exposures. In an attempt to test the hypothesis of potential non-additive effects of multiple radiological exposures, we used a mouse model of combined external x-ray irradiation at 1 and 5 Gy and internal contamination with injection of 20 KBq 137Cs. The results showed differential kinetics of 137Cs elimination in irradiated animals compared to sham-irradiated, 137Cs injected animals. Moreover, changes in plasma potassium and in relative testis weight were observed 38 days after irradiation and injection in co-exposed animals compared to 137Cs injection alone. These results demonstrate that an external exposure combined with an internal contamination may lead to unexpected changes in biokinetics of radionuclides and biological effects compared to single exposures.

International audience

International audience; 1 Background Humans can be exposed at low concentrations to toxic agents with anthropogenic and natural origins such as uranium and fluoride. Because of its functions of filtration, transport and reabsorption the kidney is a primary target organ of toxicity to foreign compounds. Uranium and fluoride are both known to be nephrotoxic, nevertheless there is a lack of knowledge of their mechanisms of nephrotoxicity and of the underlying molecular pathways involved [1, 2]. This study aims to compare these agents to identify the cellular and molecular pathways of nephrotoxicity in mouse. 2 Methods and results C57Bl6 mice are exposed to uranyl nitrate (UN) (0, 2, 4, 5 mg/kg) or sodium fluoride (NaF) (0, 2, 5, 7.5, 10 mg/kg) by intraperitoneal injection and put into metabolism cages for 24 hours before and directly after injection. Collected urines are used to quantify uranium or fluoride, to measure specific and sensitive biomarkers of nephrotoxicity levels and to assess clinical bioassays. Finally, animals are euthanized 48h and 72h after exposure, chosen as the peak of nephrotoxicity is observed after intraperitoneal injection [3]. The protocol is shown in figure 1.Renal phenotypic aspects and biological mechanisms are evaluated by urinary biochemistry, gene and protein expressions, enzyme activity, and histological analyses. Exposure to UN and NaF induces nephrotoxicity in a dose-dependent manner. A 5 mg/kg injection of UN induces mild histopathological alterations and respectively 44 and 6-fold increase in gene expressions of nephrotoxicity markers KIM1 and osteopontin. In comparison, 10 mg/kg of NaF induces high nephrotoxicity with histopathological alterations scored as severe and late appearing parameters of toxicity whereas 7.5 mg/kg induces mild histopathological scoring and gene expressions of KIM1 and clusterin enhanced respectively by 70 and 4-fold compared to control. No signs of nephrotoxicity are observed below 5 mg/kg of NaF. Clusterin is respectively increased by 2.4 and 4.4-fold in urines after 7.5 and 10 mg/kg injection of NaF, whereas 4 and 5 mg/kg of UN induce respectively a 2.2 and 2.7-fold increase of clusterin in urines. Apoptosis is evaluated through caspases 3/7 activity which is increased by 210% after UN treatment (5mg/kg) whereas NaF does not induce apoptosis significantly. Inflammation is implied in UN and NaF acute nephrotoxicity as shown by gene and in situ overexpressions of ICAM and VCAM measurement by immunohistochemical staining.3 ConclusionsUN and NaF acute exposures resulted in dose and time-dependent nephrotoxicity with a higher nephrotoxicity after 72h. Inflammation and apoptosis are both involved in UN or NaF toxicity. These observations allow us to identify the mechanisms that will be studied in a low-dose exposure protocol after a chronic exposure.

Background Despite their differences in physicochemical properties, both uranium (U) and fluoride (F) are nephrotoxicants at high doses but their adverse effects at low doses are still the subject of debate. METHODS: This study aims to improve the knowledge of the biological mechanisms involved through an adaptive response model of C57BL/6 J mice chronically exposed to low priming doses of U (0, 10, 20 and 40 mg/L) or F (0, 15, 30 and 50 mg/L) and then challenged with acute exposure of 5 mg/kg U or 7.5 mg/kg NaF. Results We showed that an adaptive response occurred with priming exposures to 20 mg/L U and 50 mg/L F, with decreased levels of the biomarkers KIM-1 and CLU compared to those in animals that received the challenge dose only (positive control). The adaptive mechanisms involved a decrease in caspase 3/7 activities in animals exposed to 20 mg/L U and a decrease in in situ VCAM expression in mice exposed to 50 mg/L F. However, autophagy and the UPR were induced independently of priming exposure to U or F and could not be identified as adaptive mechanisms to U or F. Conclusion Taken together, these results allow us to identify renal adaptive responses to U and F at doses of 20 and 50 mg/L, probably through decrease apoptosis and inflammatory cell recruitment.

International audience; There is increasing evidence that environmental exposures early in fetal development influence phenotype and give rise to disease risk in the next generations. We previously found that lifelong exposure to uranium, an environmental contaminant, induced subtle testicular and hormonal defects; however, its impact on the reproductive system of multiple subsequent generations was unexplored. Herein, rats were exposed to a supra-environmental and non-nephrotoxic concentration of natural uranium (U, 40 mg·L−1 of drinking water) from postnatal life to adulthood (F0), during fetal life (F1), and only as the germ cells from the F1 generation (F2). General parameters (reproductive indices, epididymal weight) and sperm morphology were assessed in the three generations. In order to identify the epigenetic effects of U, we analyzed also the global DNA methylation profile and described for the first time the mRNA expression levels of markers involved in the (de)methylation system in rat epididymal spermatozoa. Our results showed that the F1 generation had a reduced pregnancy rate. Despite the sperm number being unmodified, sperm morphology was affected in the F0, F1 and F2 generations. Morphometric analysis for ten parameters was detailed for each generation. No common parameter was detected between the three generations, but the head and the middle-piece were always modified in the abnormal sperms. In the F1 U-exposed generation, the total number of abnormal sperm was significantly higher than in the F0 and F2 generations, suggesting that fetal exposure to uranium was more deleterious. This effect could be associated with the pregnancy rate to produce the F2 generation. Interestingly, global DNA methylation analysis showed also hypomethylation in the sperm DNA of the last F2 generation. In conclusion, our study demonstrates that uranium can induce morphological sperm defects and changes in the DNA methylation level after multigenerational exposure. The epigenetic transgenerational inheritance of U-induced reproductive defects should be assessed in further experiments.; Il existe de plus en plus de preuves de ce que les expositions environnementales auxquelles les individus sont soumis durant leur développement influencent le phénotype et le risque d’apparition de maladies chez les générations futures. Nous avons précédemment constaté que l’uranium, un contaminant environnemental, induisait après une exposition durant la vie entière de subtiles anomalies testiculaires et hormonales, mais son impact sur le système reproducteur des générations suivantes restait inexploré. Dans cette étude, des rats ont été exposés à une concentration supra-environnementale et non néphrotoxique d’uranium (U, 40 mg·L−1 dans l’eau de boisson) de la vie postnatale à l’âge adulte (F0), pendant la vie fœtale (F1), et seulement en tant que cellules germinales de la génération F1 (F2). Les paramètres généraux (indices reproductifs, poids) et la morphologie des spermatozoïdes ont été évalués sur les trois générations. Afin d’identifier les effets épigénétiques de l’uranium, nous avons également analysé le profil global de méthylation de l’ADN et décrit, pour la première fois, les niveaux d’expression d’ARNm des marqueurs impliqués dans le système de (dé)méthylation des spermatozoïdes épididymaires de rats. Nos résultats ont montré que la génération F1 avait un taux de naissance réduit. Bien que le nombre de spermatozoïdes n’ait pas été modifié, la morphologie des spermatozoïdes a été affectée chez les générations F0, F1 et F2. L’analyse morphométrique de dix paramètres a été détaillée pour chaque génération. Aucun paramètre commun n’a été détecté entre les trois générations, mais la tête et la pièce intermédiaire ont toujours été modifiées dans ces spermatozoïdes anormaux. Chez les générations F1 exposées à l’U, le nombre total de spermatozoïdes anormaux était significativement plus élevé que chez les générations F0 et F2, ce qui suggère que l’exposition fœtale à l’uranium était plus nocive et pouvait affecter le taux de naissance pour produire la génération F2. Il est intéressant de noter aussi que l’analyse globale de méthylation de l’ADN a montré une hypométhylation dans l’ADN des spermatozoïdes de la dernière génération F2. En conclusion, notre étude démontre que l’uranium peut induire des défauts morphologiques des spermatozoïdes et un changement de méthylation de l’ADN après une exposition multigénérationnelle. La transmission transgénérationnelle épigénétique des anomalies du système reproducteur induites par l’uranium devra être évaluée dans de futures expériences.