Poly(allylamine)-tripolyphosphate Ionic Assemblies as Nanocarriers: Friend or Foe?

Designing effective drug nanocarriers that are easy to synthesize, robust, and nontoxic is a significant challenge in nanomedicine. Polyamine-multivalent molecule nanocomplexes are promising drug carriers due to their simple and all-aqueous manufacturing process. However, these systems can present issues of colloidal instability over time and cellular toxicity due to the cationic polymer. In this study, we finely modulate the formation parameters of poly(allylamine-tripolyphosphate) complexes to jointly optimize the robustness and safety. Polyallylamine was ionically assembled with tripolyphosphate anions to form liquid-like nanocomplexes with a size of around 200 nm and a zeta potential of -30 mV. We found that nanocomplexes exhibit tremendous long-term stability (9 months of storage) in colloidal dispersion and that they are suitable as protein-loading agents. Moreover, the formation of nanocomplexes induced by tripolyphosphate anions produces a switch-off in the toxicity of the system by altering the overall charge from positive to negative. In addition, we demonstrate that nanocomplexes can be internalized by bone-marrow-derived macrophage cells. Altogether, these nanocomplexes have attractive and promising properties as delivery nanoplatforms for potential therapies based on the immune system activation.

Cytotoxic Screening and Enhanced Anticancer Activity of Lippia alba and Clinopodium nepeta Essential Oils-Loaded Biocompatible Lipid Nanoparticles against Lung and Colon Cancer Cells.

Plant and herbal essential oils (EOs) offer a wide range of pharmacological actions that include anticancer effects. Here, we evaluated the cytotoxic activity of EO from Lippia alba (chemotype linalool), L. alba (chemotype dihydrocarvone, LaDEO), Clinopodium nepeta (L.) Kuntze (CnEO), Eucalyptus globulus, Origanum × paniculatum, Mentha × piperita, Mentha arvensis L., and Rosmarinus officinalis L. against human lung (A549) and colon (HCT-116) cancer cells. The cells were treated with increasing EO concentrations (0-500 µL/L) for 24 h, and cytotoxic activity was assessed. LaDEO and CnEO were the most potent EOs evaluated (IC50 range, 145-275 µL/L). The gas chromatography-mass spectrometry method was used to determine their composition. Considering EO limitations as therapeutic agents (poor water solubility, volatilization, and oxidation), we evaluated whether LaDEO and CnEO encapsulation into solid lipid nanoparticles (SLN/EO) enhanced their anticancer activity. Highly stable spherical SLN/LaDEO and SLN/CnEO SLN/EO were obtained, with a mean diameter of 140-150 nm, narrow size dispersion, and Z potential around -5mV. EO encapsulation strongly increased their anticancer activity, particularly in A549 cells exposed to SLN/CnEO (IC50 = 66 µL/L CnEO). The physicochemical characterization, biosafety, and anticancer mechanisms of SLN/CnEO were also evaluated in A549 cells. SLN/CnEO containing 97 ± 1% CnEO was highly stable for up to 6 months. An increased in vitro CnEO release from SLN at an acidic pH (endolysosomal compartment) was observed. SLN/CnEO proved to be safe against blood components and non-toxic for normal WI-38 cells at therapeutic concentrations. SLN/CnEO substantially enhanced A549 cell death and cell migration inhibition compared with free CnEO.

Nanostructured lipid carriers containing benznidazole: physicochemical, biopharmaceutical and cellular in vitro studies.

Chagas disease is a neglected endemic disease prevalent in Latin American countries, affecting around 8 million people. The first-line treatment, benznidazole (BNZ), is effective in the acute stage of the disease but has limited efficacy in the chronic stage, possibly because current treatment regimens do not eradicate transiently dormant Trypanosoma cruzi amastigotes. Nanostructured lipid carriers (NLC) appear to be a promising approach for delivering pharmaceutical active ingredients as they can have a positive impact on bioavailability by modifying the absorption, distribution, and elimination of the drug. In this study, BNZ was successfully loaded into nanocarriers composed of myristyl myristate/Crodamol oil/poloxamer 188 prepared by ultrasonication. A stable NLC formulation was obtained, with ≈80% encapsulation efficiency (%EE) and a biphasic drug release profile with an initial burst release followed by a prolonged phase. The hydrodynamic average diameter and zeta potential of NLC obtained by dynamic light scattering were approximately 150 nm and -13 mV, respectively, while spherical and well-distributed nanoparticles were observed by transmission electron microscopy. Fourier-transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, and small-angle X-ray scattering analyses of the nanoparticles indicated that BNZ might be dispersed in the nanoparticle matrix in an amorphous state. The mean size, zeta potential, polydispersity index, and %EE of the formulation remained stable for at least six months. The hemolytic effect of the nanoparticles was insignificant compared to that of the positive lysis control. The nanoparticle formulation exhibited similar performance in vitro against T. cruzi compared to free BNZ. No formulation-related cytotoxic effects were observed on either Vero or CHO cells. Moreover, BNZ showed a 50% reduction in CHO cell viability at 125 µg/mL, whereas NLC-BNZ and non-loaded NLC did not exert a significant effect on cell viability at the same concentration. These results show potential for the development of new nanomedicines against T. cruzi.

Efectividad de la administración de hierro en forma semanal y diaria para la prevención de anemia por deficiencia de hierro en lactantes / Effectiveness of weekly and daily iron administration for the prevention of iron deficiency anemia in infants

Introducción. La deficiencia de hierro (DH) es la carencia nutricional más prevalente y la principal causa de anemia en lactantes. Existe consenso en la suplementación diaria con hierro como estrategia de prevención; también se demostró que la suplementación semanal es eficaz, pero la evidencia en lactantes es escasa. El objetivo fue comparar la efectividad de la administración diaria de hierro frente a la semanal para la prevención de la anemia por DH del lactante. Población y métodos. Ensayo clínico controlado y aleatorizado. Lactantes atendidos en un centro de salud público, sin anemia a los 3 meses de edad, aleatorizados en tres grupos: suplementación diaria (1 mg/kg/día), semanal (4 mg/kg/semana) o sin suplementación (grupo control con lactancia materna exclusiva [LME]). Se evaluó anemia y DH a los 3 y 6 meses. Se registró grado de adherencia y efectos adversos. Los datos se analizaron con el software R versión 4.0.3. Resultados. Participaron 227 lactantes. A los 6 meses el grupo de lactantes con LME sin suplementación (control) presentó prevalencias de DH y anemia por DH (ADH) mayores que los grupos intervenidos (diario y semanal). DH: 40,5 % vs. 13,5 % y 16,7 % (p = 0,002); ADH: 33,3 % vs. 7,8 % y 10 % (p < 0,001). No hubo diferencias entre los grupos diario y semanal. Tampoco hubo diferencias en el porcentaje de alta adherencia a la suplementación (50,6 % diaria vs. 57,1 % semanal), ni en los efectos adversos. Conclusiones. No se hallaron diferencias significativas en la efectividad entre la administración diaria y semanal para la prevención de ADH del lactante.

Introduction. Iron deficiency (ID) is the most prevalent nutritional deficiency and the main cause of anemia in infants. There is consensus on daily iron supplementation as a preventive strategy; and weekly iron supplementation has also been shown to be effective, but evidence in infants is scarce. The objective of this study was to compare the effectiveness of daily versus weekly iron administration for the prevention of ID anemia (IDA) in infants. Population and methods. Randomized, controlled clinical trial. Infants seen at a public health center, without anemia at 3 months of age, were randomized into 3 groups: daily supplementation (1 mg/kg/ day), weekly supplementation (4 mg/kg/week), or no supplementation (control group with exclusive breastfeeding [EB]). Anemia and ID were assessed at 3 and 6 months old. Adherence and adverse events were recorded. Data were analyzed using the R software, version 4.0.3. Results. A total of 227 infants participated. At 6 months, the group of infants with EB without supplementation(control) had a higher prevalence of ID and IDA than the intervention groups (daily and weekly). ID: 40.5% versus 13.5% and 16.7% (p = 0.002); IDA: 33.3% versus 7.8% and 10% (p < 0.001). There were no differences between the daily and weekly supplementation groups. There were also no differences in the percentage of high adherence to supplementation (50.6% daily versus 57.1% weekly) or adverse events. Conclusions. No significant differences in effectiveness were observed between daily and weekly administration for the prevention of infant IDA.

Preliminary transcriptomic analysis of peripheral blood from German Shepherd dogs with degenerative joint disease for the identification of diagnostic biomarkers.

OBJECTIVE: Degenerative joint disease (DJD) includes a group of disorders characterised by the deterioration of the articular cartilage. In this study, we investigated the transcriptomic profile of peripheral blood in German Shepherd dogs with DJD to identify putative diagnostic biomarkers. METHODS: Differential gene expression (DGE) and gene ontology (GO) analyses of the bulk RNA-seq experiment were performed in a cohort of 12 adult dogs (five cases and seven controls, classified by clinical and radiographic analyses). RESULTS: Radiographs of cases revealed severe signs of progressive DJD. Two up-regulated (LOC106559672 and THBS4) and one down-regulated (LOC106559235) differentially expressed genes (adjusted p value < 0.05) were identified. The DGE with log2 fold change < -1.5 and > 1.5 and non-adjusted p < 0.01 were selected for GO analysis. No significant enrichment terms were observed in the selected threshold. CONCLUSION: The gene-encoding protein THBS4 is correlated with DJD severity and long noncoding RNA LOC106559235 is probably involved in the DJD process. The THBS4 gene should be considered a good biomarker for DJD in dogs. Future studies using independent cohorts will be necessary to validate the present results.

Effectiveness of weekly and daily iron administration for the prevention of iron deficiency anemia in infants. / Efectividad de la administración de hierro en forma semanal y diaria para la prevención de anemia por deficiencia de hierro en lactantes.

Introduction. Iron deficiency (ID) is the most prevalent nutritional deficiency and the main cause of anemia in infants. There is consensus on daily iron supplementation as a preventive strategy; and weekly iron supplementation has also been shown to be effective, but evidence in infants is scarce. The objective of this study was to compare the effectiveness of daily versus weekly iron administration for the prevention of ID anemia (IDA) in infants. Population and methods. Randomized, controlled clinical trial. Infants seen at a public health center, without anemia at 3 months of age, were randomized into 3 groups: daily supplementation (1 mg/kg/day), weekly supplementation (4 mg/kg/week), or no supplementation (control group with exclusive breastfeeding [EB]). Anemia and ID were assessed at 3 and 6 months old. Adherence and adverse events were recorded. Data were analyzed using the R software, version 4.0.3. Results. A total of 227 infants participated. At 6 months, the group of infants with EB without supplementation (control) had a higher prevalence of ID and IDA than the intervention groups (daily and weekly). ID: 40.5% versus 13.5% and 16.7% (p = 0.002); IDA: 33.3% versus 7.8% and 10% (p < 0.001). There were no differences between the daily and weekly supplementation groups. There were also no differences in the percentage of high adherence to supplementation (50.6% daily versus 57.1% weekly) or adverse events. Conclusions. No significant differences in effectiveness were observed between daily and weekly administration for the prevention of infant IDA.

Introducción. La deficiencia de hierro (DH) es la carencia nutricional más prevalente y la principal causa de anemia en lactantes. Existe consenso en la suplementación diaria con hierro como estrategia de prevención; también se demostró que la suplementación semanal es eficaz, pero la evidencia en lactantes es escasa. El objetivo fue comparar la efectividad de la administración diaria de hierro frente a la semanal para la prevención de la anemia por DH del lactante. Población y métodos. Ensayo clínico controlado y aleatorizado. Lactantes atendidos en un centro de salud público, sin anemia a los 3 meses de edad, aleatorizados en tres grupos: suplementación diaria (1 mg/kg/día), semanal (4 mg/kg/semana) o sin suplementación (grupo control con lactancia materna exclusiva [LME]). Se evaluó anemia y DH a los 3 y 6 meses. Se registró grado de adherencia y efectos adversos. Los datos se analizaron con el software R versión 4.0.3. Resultados. Participaron 227 lactantes. A los 6 meses el grupo de lactantes con LME sin suplementación (control) presentó prevalencias de DH y anemia por DH (ADH) mayores que los grupos intervenidos (diario y semanal). DH: 40,5 % vs. 13,5 % y 16,7 % (p = 0,002); ADH: 33,3 % vs. 7,8 % y 10 % (p < 0,001). No hubo diferencias entre los grupos diario y semanal. Tampoco hubo diferencias en el porcentaje de alta adherencia a la suplementación (50,6 % diaria vs. 57,1 % semanal), ni en los efectos adversos. Conclusiones. No se hallaron diferencias significativas en la efectividad entre la administración diaria y semanal para la prevención de ADH del lactante.

Vitamin E protective effects on genomic and cellular damage caused by paediatric preventive supplementation for anaemia: an experimental model.

Iron deficiency is the leading cause of anaemia. In Argentina, the prevalence of anaemia and iron deficiency is very high; for that reason, the Argentine Society of Pediatrics recommends daily ferrous sulphate supplementation as a preventive treatment strategy. Alternatively, weekly ferrous sulphate supplementation has also been shown to be effective for anaemia prevention. Excess iron could be related to oxidative stress, which may in turn cause cytomolecular damage. Both can be prevented with vitamin E supplementation. We evaluated the effect of both daily and weekly ferrous sulphate supplementation combined with two doses of vitamin E on cell viability, oxidative stress and cytomolecular damage in peripheral blood cultured in vitro. The experimental design included the following groups: untreated negative control, two vitamin E controls (8·3 and 16·6 µg/ml), weekly ferrous sulphate supplementation (0·55 mg/ml) with each vitamin E dose, daily ferrous sulphate supplementation (0·14 mg/ml) with each vitamin E dose and a positive control. Daily ferrous sulphate supplementation decreased cell viability and increased the levels of reactive oxygen species, lipid peroxidation and cytomolecular damage (P < 0·5) compared with the weekly supplementation, probably due to the excess iron observed in the former. Vitamin E seemed to reduce ferrous sulphate-induced oxidative stress and genomic damage.

Colloidal delivery of vitamin E into solid lipid nanoparticles as a potential complement for the adverse effects of anemia treatment.

Vitamin E (VitE) is one of the most important antioxidants and plays a key role in decreasing the inflammatory effects of oxidative stress caused by recurrent doses of iron administration in anemia treatment. However, VitE is poorly soluble in aqueous environments. Here, VitE encapsulation into solid lipid nanoparticles (SLN) composed of myristil myristate to improve its bioavailability was proposed. A 99.9 ± 0.1% encapsulation efficiency with a drug/lipid ratio of 500 µg/mg and 478 higher VitE solubility was obtained. The antioxidant properties of VitE after encapsulation were maintained. SLN-VitE showed a 228.2 nm mean diameter with low polidispersitivity (0.335), and negative Z potential (ζ ≈ -9.0 mV). The SLN were well-dispersed, displayed spherical and homogeneous morphology by TEM. A controlled release of VitE from SLN was found. The XRD and FTIR analyses revealed the presence of a nanostructured architecture of SLN after VitE incorporation. We probed the safety of SLN-VitE after contact with three in vitro cell models: erythrocytes, lymphocytes and HepG2 cells. The cell viability in presence of SLN, SLN-VitE, and their combinations with iron was not affected. The comet assay demonstrated that the DNA damage caused by iron administration was decrease in presence of SLN-VitE.

Nanostructured Lipid Carriers Loaded with Dexamethasone Prevent Inflammatory Responses in Primary Non-Parenchymal Liver Cells.

Liver inflammation represents a major clinical problem in a wide range of pathologies. Among the strategies to prevent liver failure, dexamethasone (DXM) has been widely used to suppress inflammatory responses. The use of nanocarriers for encapsulation and sustained release of glucocorticoids to liver cells could provide a solution to prevent severe side effects associated with systemic delivery as the conventional treatment regime. Here we describe a nanostructured lipid carrier developed to efficiently encapsulate and release DXM. This nano-formulation proved to be stable over time, did not interact in vitro with plasma opsonins, and was well tolerated by primary non-parenchymal liver cells (NPCs). Released DXM preserved its pharmacological activity, as evidenced by inducing robust anti-inflammatory responses in NPCs. Taken together, nanostructured lipid carriers may constitute a reliable platform for the delivery of DXM to treat pathologies associated with chronic liver inflammation.

Binary Medical Nanofluids by Combination of Polymeric Eudragit Nanoparticles for Vehiculization of Tobramycin and Resveratrol: Antimicrobial, Hemotoxicity and Protein Corona Studies.

The development of smart nanoparticles (NPs) became a trend to enhance the delivery of drugs. In the present work, Tobramycin (TB), an aminoglycoside antibiotic that displays several undesirable side effects, has been encapsulated into cationic Eudragit®E100 (E100) NPs for the treatment of infections caused by Pseudomonas aeruginosa. Combination with neutral Eudragit®NE30D (NE30D) NPs containing resveratrol (RSV), a strong natural antioxidant, increased the antimicrobial activity of TB (75% higher than free TB). NPs were stabilized with 1.0% (w/v) poloxamer 188 (P188) or poloxamer 407 (P407) as surfactants. E100 NPs showed 83.3 ± 8.5%, and 70.1 ± 2.7 encapsulation efficiency (EE) of TB with P188 and P407 coatings, respectively. The presence of NPs was confirmed by DLS and TEM studies. TB was controlled released from NPs for 6 h. Hemotoxicity tests of NPs in the range of MIC values on human blood gave negative results. Analysis of Surface Plasmon Resonance verified that NE30D/P407/RSV does not interact with plasma proteins BSA, IgG or fibrinogen, besides E100/P188/TB interact with BSA, findings that are compatible with a negligible in vivo clearance of the nanovehicles. The obtained results show a potential binary fluid composed of two NPs to highly improve the effectiveness of conventional antibiotics.

Epidemiology of Cranial Cruciate Ligament Rupture and Patellar Luxation in Dogs from the Province of Buenos Aires, Argentina.

OBJECTIVE: The aim of this study was to investigate the prevalence of cranial cruciate ligament rupture and patellar luxation and the associated risk factors in dogs. MATERIALS AND METHODS: A total of 13,072 clinical records of dogs were reviewed from School Hospital (Faculty of Veterinary Sciences, National University of La Plata). Data of age, breed, sex, body weight, patellar luxation and cranial cruciate ligament rupture condition were registered. Chi-squared and Fisher's exact tests were used to compare the prevalence of cranial cruciate ligament rupture and patellar luxation with the variables and then univariable logistic regression was used to evaluate the risk of having cranial cruciate ligament rupture and patellar luxation. Multivariable logistic regression was used including all variables to assess the odds of having patellar luxation and cranial cruciate ligament rupture. RESULTS: Of 13,072 patients treated, 72 and 51 had cranial cruciate ligament rupture and patellar luxation respectively. Sex was not a major risk factor for either condition. Adult (odds ratio [OR] = 8.2) and senior (OR = 4.3) patients had increased risk of having cranial cruciate ligament rupture, while for patellar luxation age was not a risk factor. Groups 2, 3 and 8 were more likely to have cranial cruciate ligament rupture (OR = 5.5, OR = 9.1 and OR = 2.6), and group 11 had lower risk of having patellar luxation (OR = 0.08). Maxi (OR = 2.4) and giant (OR = 6.0) breeds had higher risk of having cranial cruciate ligament rupture, and medium and maxi breeds had higher risk of patellar luxation (OR = 0.05 and OR = 0.3). Multivariate OR test confirmed that age (adult), body size (giant and maxi) and breed group (Group 3) were significantly associated with having cranial cruciate ligament rupture, and age was associated with having patellar luxation. CLINICAL SIGNIFICANCE: This is the first epidemiological study of cranial cruciate ligament rupture and patellar luxation in dogs from School Hospital (Faculty of Veterinary Sciences, National University of La Plata). Giant and large adult dogs from the Molossoid and Terrier breeds were more likely to have cranial cruciate ligament rupture, while mixed and large dog breeds showed the lowest risk of having patellar luxation.

Doramectin induced cytotoxic and genotoxic effects on bovine peripheral lymphocytes and cumulus cells in vitro.

The effect of doramectin (DOR) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PL) and cumulus cells (CC). The cytotoxicity and genotoxicity of DOR were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, single cell gel electrophoresis assay (SCGE) and cytokinesis-block micronucleus cytome (CBMN Cyt) assay. Both cells were treated with three concentrations of DOR (20, 40, 60 ng mL-1) for 24 h. The results obtained from PL demonstrated that DOR was able to induce cytotoxic effect and DNA damage with all concentrations tested. Additionally, DOR increased micronuclei (MNi) frequency and nuclear buds (NBuds) with 20, 40, 60 ng mL-1, and nucleoplasmic bridges (NPBs) only with 40 ng mL-1. On the other hand, the three concentrations of DOR were not able to induce cytotoxic effect and DNA damage using SCGE in the bovine CC. Nevertheless, the two higher concentrations of DOR (20, 40 µg mL-1) significantly increased the frequency of micronucleus formation in bovine CC. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by DOR on bovine PL and CC.

Cytotoxic and genotoxic effects induced by enrofloxacin-based antibiotic formulation Floxagen® in two experimental models of bovine cells in vitro: peripheral lymphocytes and cumulus cells.

The in vitro effect of enrofloxacin (EFZ) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PLs) and cumulus cells (CCs). The cytotoxicity and genotoxicity of this veterinary antibiotic were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, single-cell gel electrophoresis (SCGE) assay, and cytokinesis-block micronucleus cytome (CBMN cyt) assay. Cells were treated during 24 h, and three concentrations were tested (50 µg/mL, 100 µg/mL, 150 µg/mL). When EFZ was tested in PLs, the results demonstrated that the antibiotic was able to induce cell death and DNA damage with all concentrations. In addition, 50 µg/mL and 100 µg/mL EFZ increased frequencies of micronuclei (MNi). On the other hand, the highest EFZ concentration occasioned cellular cytotoxicity in CCs as evidenced by mitochondrial activity alterations. Nevertheless, EFZ was not able to induce DNA damage and MNi in CCs. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by EFZ in bovine PLs and CCs.

Oxidative Stress and Genomic Damage Induced In Vitro in Human Peripheral Blood by Two Preventive Treatments of Iron Deficiency Anemia.

Iron deficiency is the most prevalent nutritional deficiency and the main cause of anemia worldwide. Since children aged 6-24 months are among the most vulnerable groups at risk, daily supplementation with ferrous sulfate is recommended by the Argentine Society of Pediatrics as preventive treatment of anemia. However, a single weekly dose would have fewer adverse side effects and has been therefore proposed as an alternative treatment. Ferrous sulfate is known by its pro-oxidative properties, which may lead to increased oxidative stress as well as lipid, protein, and DNA damage. We analyzed the effect of daily and weekly preventive treatment of iron deficiency anemia (IDA) on cell viability, oxidative stress, chromosome, and cytomolecular damage in peripheral blood cultured in vitro. The study protocol included the following: untreated negative control; bleomycin, hydrogen peroxide, or ethanol-treated positive control; daily 0.14 mg ferrous sulfate-supplemented group; and weekly 0.55 mg ferrous sulfate-supplemented group. We assessed cell viability (methyl-thiazolyl-tetrazolium and neutral red assays), lipid peroxidation (thiobarbituric acid reactive substances assay), antioxidant response (superoxide dismutase and catalase enzyme analysis), chromosome damage (cytokinesis-blocked micronucleus cytome assay), and cytomolecular damage (comet assay). Lipid peroxidation, antioxidant response, and chromosome and cytomolecular damage decreased after weekly ferrous sulfate supplementation (p < 0.05), suggesting less oxygen free radical production and decreased oxidative stress and genomic damage. Such a decrease in oxidative stress and genomic damage in vitro positions weekly supplementation as a better alternative for IDA treatment. Further studies in vivo would be necessary to corroborate whether weekly supplementation could improve IDA preventive treatment compliance in children.

Evaluation of resveratrol radiomodifying potential for radiotherapy treatment.

Radiotherapy is widely used for cancer treatment. However, its adverse effects that may develop during the course of treatment have forced to search agents to protect biological systems against the deleterious effects of radiation. Resveratrol (3,4,5-trihydroxy-trans-stilbene; RSV) is a natural polyphenol currently promoted for its beneficial pleiotropic effects on health, which has been shown to exhibit antioxidant properties while inhibiting the growth of tumor cells. In radioresistant tumors, RSV could contribute to reduce recurrence and treatment failure. We evaluated the radiomodulatory and genotoxic effects of RSV in CHO-k1 and A549 cell lines and in peripheral human blood lymphocytes through both conventional and hypofractionated protocols, due to the widespread use of hypofractionation in recent years. RSV genotoxic and cytotoxic action was assessed at 15 and 60 µM concentrations with the comet and the MTT assay and in cell proliferation experiments. Our results show that RSV administration to tumor cells at a dose of 60 µM exerted a genotoxic effect and that this concentration also had the capacity of modulating the cytomolecular damage induced by 4 and 16 Gy. These doses are delivered in conventional and hypofractionated radiotherapy, respectively. In both treatments, a radiosensitizing effect was evidenced by the decrease in cell viability that was exacerbated over time. These effects were not found in peripheral blood, suggesting that RSV had a dual response. Although the results obtained in CHO-k1 transformed cells corroborated the genotoxic effect of the 60 µM dose of RSV observed in the tumor system, they also showed a radio-protective effect at the lowest dose (15 µM). While more studies are necessary, our results together with the good systemic tolerance of RSV and the lack of toxicity position the compound as a potential candidate for the prevention and treatment of cancer as well as for the optimization of the radiotherapeutic ratio.

Genomic instability related to zinc deficiency and excess in an in vitro model: is the upper estimate of the physiological requirements recommended for children safe?

Micronutrients are important for the prevention of degenerative diseases due to their role in maintaining genomic stability. Therefore, there is international concern about the need to redefine the optimal mineral and vitamin requirements to prevent DNA damage. We analyzed the cytostatic, cytotoxic, and genotoxic effect of in vitro zinc supplementation to determine the effects of zinc deficiency and excess and whether the upper estimate of the physiological requirement recommended for children is safe. To achieve zinc deficiency, DMEM/Ham's F12 medium (HF12) was chelated (HF12Q). Lymphocytes were isolated from healthy female donors (age range, 5-10 yr) and cultured for 7 d as follows: negative control (HF12, 60 µg/dl ZnSO4); deficient (HF12Q, 12 µg/dl ZnSO4); lower level (HF12Q + 80 µg/dl ZnSO4); average level (HF12Q + 180 µg/dl ZnSO4); upper limit (HF12Q + 280 µg/dl ZnSO4); and excess (HF12Q + 380 µg/dl ZnSO4). The comet (quantitative analysis) and cytokinesis-block micronucleus cytome assays were used. Differences were evaluated with Kruskal-Wallis and ANOVA (p < 0.05). Olive tail moment, tail length, micronuclei frequency, and apoptotic and necrotic percentages were significantly higher in the deficient, upper limit, and excess cultures compared with the negative control, lower, and average limit ones. In vitro zinc supplementation at the lower and average limit (80 and 180 µg/dl ZnSO4) of the physiological requirement recommended for children proved to be the most beneficial in avoiding genomic instability, whereas the deficient, upper limit, and excess (12, 280, and 380 µg/dl) cultures increased DNA and chromosomal damage and apoptotic and necrotic frequencies.

Possible radioprotective effect of folic acid supplementation on low dose ionizing radiation-induced genomic instability in vitro.

Ionizing radiation (IR) induces DNA damage through production of single and double-strand breaks and reactive oxygen species (ROS). Folic acid (FA) prevents radiation-induced DNA damage by modification of DNA synthesis and/or repair and as a radical scavenger. We hypothesized that in vitro supplementation with FA will decrease the sensitivity of cells to genetic damage induced by low dose of ionizing radiation. Annexin V, comet and micronucleus assays were performed in cultured CHO cells. After 7 days of pre-treatment with 0, 100, 200 or 300 nM FA, cultures were exposed to radiation (100 mSv). Two un-irradiated controls were executed (0 and 100 nM FA). Data were statistically analyzed with X2-test and linear regression analysis (P 0.05). We observed a significantly decreased frequency of apoptotic cells with the increasing FA concentration (P <0.05). The same trend was observed when analyzing DNA damage and chromosomal instability (P <0.05 for 300 nM). Only micronuclei frequencies showed significant differences for linear regression analysis (R2=94.04; P <0.01). Our results have demonstrated the radioprotective effect of folic acid supplementation on low dose ionizing radiation-induced genomic instability in vitro; folate status should be taken into account when studying the effect of low dose radiation in environmental or occupational exposure.

Protein energy-malnutrition: does the in vitro zinc sulfate supplementation improve chromosomal damage repair?

Protein-energy malnutrition (PEM) is originated by a cellular imbalance between nutrient/energy supply and body's demand. Induction of genetic damage by PEM was reported. The purpose of this study was to determine the genetic effect of the in vitro zinc sulfate (ZnSO4) supplementation of cultured peripheral blood lymphocytes from children with PEM. Twenty-four samples from 12 children were analyzed. Anthropometric and biochemical diagnosis was made. For the anthropometric assessment, height-for-age index, weight-for-age index, and weight-for-height index were calculated (WHO, 2005). Micronutrient status was evaluated. A survey for assessed previous exposure to potentially genotoxic agents was applied. Results were statistically evaluated using paired sample t test and χ (2) test. Each sample was fractionated and cultured in two separate flasks to performed two treatments. One was added with 180 µg/dl of ZnSO4 (PEMs/ZnSO4) and the other remains non-supplemented (PEMs). Cytotoxic effects and chromosomal damage were assessed using the cytokinesis-block micronucleus assay (CBMN). All participants have at least one type of malnutrition and none have anemia, nor iron, folate, vitamin A, and zinc deficiency. All PEMs/ZnSO4 samples have a significant reduction in the micronucleus (MNi) frequency compared with PEMs (t = 6.25685; p < 0.001). Nuclear division index (NDI) increase in PEMs/ZnSO4 (t = -17.4226; p < 0.001). Nucleoplasmic bridge (NPBs) frequency was four times smaller in PEMs/ZnSO4 (χ (2) = 40.82; p < 0.001). No nuclear buds (NBuds) were observed. Cytotoxic effects and chromosomal damage observed in children suffering from PEM can be repaired in vitro with zinc sulfate supplementation.

Assessment of the adverse effects of the acaricide amitraz: in vitro evaluation of genotoxicity.

Amitraz is a formamidine widely used in Veterinary Medicine for the treatment of ectoparasites. It is a highly liposoluble compound that is quickly absorbed through the skin and mucous membranes, thus making exposure potentially dangerous for humans and animals. The aim of this study was to compare the genotoxic potential of the active constituent of the insecticide amitraz and a commercial product containing amitraz in vitro in hamster cells. The induction of primary DNA damage was evaluated by alkaline single-cell gel electrophoresis (comet assay) and the apoptotic ability was examined by the Annexin V/propidium iodide staining assay. The commercial formulation significantly increased the index of DNA damage at concentrations of 2.50-3.75 µg/mL compared to the control. The active constituent only induced significant DNA damage with the highest concentration (3.75 µg/mL). Although both tested products increased the frequency of cell death, neither of them induced significant differences. Genotoxic potential is a primary risk factor for long-term effects such as carcinogenic and reproductive toxicology. Results presented here highlight the need for further investigation of the potential health risk of this veterinary medicine.

Variations in estimates of underweight, stunting, wasting, overweight and obesity in children from Argentina comparing three growth charts.

OBJECTIVE: To compare estimates of underweight, stunting, wasting, overweight and obesity based on three growth charts. DESIGN: Cross-sectional study to estimate weight-for-age, length/height-for-age and weight-for-height comparing the 2006 WHO Child Growth Standards ('the WHO standards'), the 1977 National Center for Health Statistics (NCHS) international growth reference ('the NCHS reference') and the 1987 Argentine Pediatric Society Committee of Growth and Development reference ('the APS reference'). Cut-off points were defined as mean values ±2 s d. Epi-Info software version 6·0 (Centers for Disease Control and Prevention) was used for statistical evaluations (χ 2, P ≤ 0·05). SETTING: Greater La Plata conurbation, Buenos Aires, Argentina. SUBJECTS: A total of 2644 healthy, full-term children from 0 to 5 years of age. RESULTS: Prevalence of underweight was higher with the WHO standards than with the other references up to the first 6 months. For the rest of the ages, prevalence was lower with the WHO standards. Stunting prevalence was higher with the WHO standards at all ages. Prevalence of wasting was higher with the WHO standards compared with the NCHS reference up to the first 6 months and lower at 2-5 years of age. Overweight and obesity prevalences were higher with the WHO standards at all ages. CONCLUSIONS: The new WHO standards appear to be a solid and reliable tool for diagnosis and treatment of nutritional diseases, also being the only one built with infants fed according to WHO recommendations. Therefore, our results support the decision of the National Ministry of Health about the utilization of the new WHO standards to monitor the nutritional status of Argentinean children aged less than 5 years.