Paradoxical Radiosensitizing Effect of Carnosic Acid on B16F10 Metastatic Melanoma Cells: A New Treatment Strategy.

Carnosic acid (CA) is a phenolic diterpene characterized by its high antioxidant activity; it is used in industrial, cosmetic, and nutritional applications. We evaluated the radioprotective capacity of CA on cells directly exposed to X-rays and non-irradiated cells that received signals from X-ray treated cells (radiation induced bystander effect, RIBE). The genoprotective capacity was studied by in vivo and in vitro micronucleus assays. Radioprotective capacity was evaluated by clonogenic cell survival, MTT, apoptosis and intracellular glutathione assays comparing radiosensitive cells (human prostate epithelium, PNT2) with radioresistant cells (murine metastatic melanoma, B16F10). CA was found to exhibit a genoprotective capacity in cells exposed to radiation (p < 0.001) and in RIBE (p < 0.01). In PNT2 cells, considered as normal cells in our study, CA achieved 97% cell survival after exposure to 20 Gy of X-rays, eliminating 67% of radiation-induced cell death (p < 0.001), decreasing apoptosis (p < 0.001), and increasing the GSH/GSSH ratio (p < 0.01). However, the administration of CA to B16F10 cells decreased cell survival by 32%, increased cell death by 200% (p < 0.001) compared to irradiated cells, and increased cell death by 100% (p < 0.001) in RIBE bystander cells (p < 0.01). Furthermore, it increased apoptosis (p < 0.001) and decreased the GSH/GSSG ratio (p < 0.01), expressing a paradoxical radiosensitizing effect in these cells. Knowing the potential mechanisms of action of substances such as CA could help to create new applications that would protect healthy cells and exclusively damage neoplastic cells, thus presenting a new desirable strategy for cancer patients in need of radiotherapy.

Radiation-Induced Bystander Effect: Loss of Radioprotective Capacity of Rosmarinic Acid In Vivo and In Vitro.

In radiation oncology, the modulation of the bystander effect is a target both for the destruction of tumor cells and to protect healthy cells. With this objective, we determine whether the radioprotective capacity of rosmarinic acid (RA) can affect the intensity of these effects. Genoprotective capacity was obtained by determining the micronuclei frequencies in in vivo and in vitro assays and the cell survival was determined by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) (MTT) assay in three cell lines (PNT2, TRAMPC1 and B16F10), both in direct exposure to X-rays and after the production of radiation-induced bystander effect. The administration of RA in irradiated cells produced a decrease in the frequency of micronuclei both in vivo and in vitro, and an increase in cell survival, as expression of its radioprotective effect (p < 0.001) attributable to its ability to scavenge radio-induced free radicals (ROS). However, RA does not achieve any modification in the animals receiving serum or in the cultures treated with the irradiated medium, which expresses an absence of radioprotective capacity. The results suggest that ROS participates in the formation of signals in directly irradiated cells, but only certain subtypes of ROS, the cytotoxic products of lipid peroxidation, participate in the creation of lesions in recipient cells.

Genoprotective Effect of Some Flavonoids against Genotoxic Damage Induced by X-rays In Vivo: Relationship between Structure and Activity.

Flavonoids constitute a group of polyphenolic compounds characterized by a common gamma-benzo- pyrone structure considered in numerous biological systems to possess antioxidant capacity. Among the different applications of flavonoids, its genoprotective capacity against damage induced by ionizing radiation stands out, which has been related to antioxidant activity and its chemical structure. In this study, we determined the frequency of appearance of micronucleus in vivo by means of the micronucleus assay. This was conducted in mice treated with different flavonoids before and after exposure to 470 mGy X-rays; thereafter, their bone marrow polychromatophilic erythrocytes were evaluated to establish the structural factors enhancing the observed genoprotective effect. Our results in vivo show that the presence of a monomeric flavan-3-ol type structure, with absence of carbonyl group in position C4 of ring C, absence of conjugation between the carbons bearing the C2 = C3 double bond and the said ring, presence of a catechol group in ring B and characteristic hydroxylation in positions 5 and 7 of ring A are the structural characteristics that determine the highest degree of genoprotection. Additionally, a certain degree of polymerization of this flavonoid monomer, but maintaining significant levels of monomers and dimers, contributes to increasing the degree of genoprotection in the animals studied at both times of their administration (before and after exposure to X-rays).

Effect of Rosmarinic Acid and Ionizing Radiation on Glutathione in Melanoma B16F10 Cells: A Translational Opportunity.

To explain a paradoxical radiosensitizing effect of rosmarinic acid (RA) on the melanoma B16F10 cells, we analyzed the glutathione (GSH) intracellular production on this cell (traditionally considered radioresistant) in comparison with human prostate epithelial cells (PNT2) (considered to be radiosensitive). In PNT2 cells, the administration of RA increased the total GSH content during the first 3 h (p < 0.01) as well as increased the GSH/ oxidized glutathione (GSSG) ratio in all irradiated cultures during all periods studied (1h and 3h) (p < 0.001), portraying an increase in the radioprotective capacity. However, in B16F10 cells, administration of RA had no effect on the total intracellular GSH levels, decreasing the GSH/GSSG ratio (p < 0.01); in addition, it caused a significant reduction in the GSH/GSSG ratio in irradiated cells (p < 0.001), an expression of radioinduced cell damage. In B16F10 cells, the administration of RA possibly activates the metabolic pathway of eumelanin synthesis that would consume intracellular GSH, thereby reducing its possible use as a protector against oxidative stress. The administration of this type of substance during radiotherapy could potentially protect healthy cells for which RA is a powerful radioprotector, and at the same time, cause significant damage to melanoma cells for which it could act as a radiosensitive agent.

Radioprotective and Antimutagenic Effects of Pycnanthus angolensis Warb Seed Extract against Damage Induced by X Rays.

Although different studies have demonstrated different applications of Pycnanthus angolensis extracts in traditional African and Asian medicine, its possible antimutagenic or genoprotective capacities have never been explored. We studied these capabilities of Pycnanthus angolensis seed extract (PASE) by means of the two micronucleus assays, determining the frequency of micronucleus (MN) yield in mouse bone marrow (in vivo) and in human lymphocytes blocked by cytochalasin B (in vitro). PASE exhibited a significant genoprotective capacity (p < 0.001) against X-rays with a protection factor of 35% in both in vivo and in vitro assays. Further, its radioprotective effects were determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) cell viability test in two cell lines: one being radiosensitive (i.e., human prostate epithelium (PNT2) cells) and the other being radioresistant (i.e., B16F10 melanoma cells). In the radiosensitive cells, PASE showed a protection factor of 35.5%, thus eliminating 43.8% of X-ray-induced cell death (p < 0.001) and a dose reduction factor of 2.5. In the radioresistant cells, a protection factor of 29% (p < 0.001) with a dose reduction factor of 4 was realized. PASE elicited a greater radioprotective capacity than the substances currently used in radiation oncology and, thus, could be developed as a nutraceutical radioprotectant for workers and patients exposed to ionizing radiation.

Disminución de la toxicidad y del efecto terapéutico del ácido zoledrónico en el tratamiento combinado con diferentes extractos antioxidantes / Decrease in toxicity and therapeutic effect of zoledronic acid in combination therapy with different antioxidant extracts

Antecedentes. El ácido zoledrónico se utiliza en el tratamiento de diversas enfermedades, tumorales o no, aunque su uso se asocia con necrosis avascular ósea. Objetivo. Determinar un posible efecto protector de diferentes sustancias antioxidantes frente a la inhibición del crecimiento de células epiteliales de próstata humana (PNT2) y en células tumorales de adenocarcinoma transgénico de próstata murina (TRAMP-C1), en tratamientos combinados de ácido zoledrónico junto con radiación ionizante (IR). Material y métodos. Mediante un ensayo de viabilidad celular (MTT) se estudia la supervivencia celular de 2 líneas celulares en tratamientos aislados y combinados con ácido zoledrónico y con IR, así como, el efecto de la adición de diferentes sustancias antioxidantes. Resultados. El ácido zoledrónico muestra un efecto citotóxico significativo sobre las células PNT2 y TRAMP-C1 (p<0,001). La administración de diferentes sustancias antioxidantes en el tratamiento con ácido zoledrónico presenta un efecto protector sobre las células PNT2 (p<0,001), pero no sobre las células tumorales. Sin embargo, la administración de ácido rosmarínico y apigenina en el tratamiento combinado con ácido zoledrónico junto con IR presenta un efecto protector no solo sobre las células PNT2 (p<0,001), sino también sobre las células TRAMP-C1 (p<0,001). Conclusión. El uso de sustancias antioxidantes produce una disminución del efecto citotóxico del ácido zoledrónico sobre las células no tumorales, por lo que podrían ser utilizadas en enfermedades benignas no tumorales. Sin embargo, en un tratamiento combinado con IR, también pueden proporcionar protección a las células tumorales, y reducir de este modo el efecto terapéutico deseado (AU)

Background. Zoledronic acid is used in the treatment of cancer-related diseases, although its use has been associated with avascular osteonecrosis. Aims. To determine the possible protective effect of a range of antioxidant substances against the inhibition of human prostate epithelial cell growth (PNT2) and transgenic adenocarcinoma mouse prostate tumour cells (TRAMP-C1), in treatments combining zoledronic acid and ionising radiation (IR). Material and method. Cell survival is studied via cell viability assays (MTT) for 2 cell lines in isolated and combined treatments with zoledronic acid and/or IR, as well as the effect of adding 3 antioxidant substances. Results. Zoledronic acid displays a significant cytotoxic effect over PNT2 and TRAMP-C1 cells (P<.001). The administration of antioxidants together with the zoledronic acid shows a protective effect for normal prostate cells, yet not so for prostate tumour cells. However, the administration of rosmarinic acid and apigenin in treatments combined with zoledronic acid provides a protective effect from the harmful effects of applying ionizing radiation, not only for normal PNT2 cells, but also for tumour cells. Conclusion. The use of antioxidant substances decreases the cytotoxic effect of zoledronic acid over non-tumour cells, and as such could be used in benign diseases. Furthermore, in the combined treatment using ionising radiation, these antioxidants also produced a protective effect in tumour cells, thus reducing the therapeutic effect sought by combining the treatment with radiation (AU)

[Decrease in toxicity and therapeutic effect of zoledronic acid in combination therapy with different antioxidant extracts]. / Disminución de la toxicidad y del efecto terapéutico del ácido zoledrónico en el tratamiento combinado con diferentes extractos antioxidantes.

BACKGROUND: Zoledronic acid is used in the treatment of cancer-related diseases, although its use has been associated with avascular osteonecrosis. AIMS: To determine the possible protective effect of a range of antioxidant substances against the inhibition of human prostate epithelial cell growth (PNT2) and transgenic adenocarcinoma mouse prostate tumour cells (TRAMP-C1), in treatments combining zoledronic acid and ionising radiation (IR). MATERIAL AND METHOD: Cell survival is studied via cell viability assays (MTT) for 2 cell lines in isolated and combined treatments with zoledronic acid and/or IR, as well as the effect of adding 3 antioxidant substances. RESULTS: Zoledronic acid displays a significant cytotoxic effect over PNT2 and TRAMP-C1 cells (P<.001). The administration of antioxidants together with the zoledronic acid shows a protective effect for normal prostate cells, yet not so for prostate tumour cells. However, the administration of rosmarinic acid and apigenin in treatments combined with zoledronic acid provides a protective effect from the harmful effects of applying ionizing radiation, not only for normal PNT2 cells, but also for tumour cells. CONCLUSION: The use of antioxidant substances decreases the cytotoxic effect of zoledronic acid over non-tumour cells, and as such could be used in benign diseases. Furthermore, in the combined treatment using ionising radiation, these antioxidants also produced a protective effect in tumour cells, thus reducing the therapeutic effect sought by combining the treatment with radiation.

Toxicity of a dental adhesive compared with ionizing radiation and zoledronic acid.

BACKGROUND: To determine the toxicity of aqueous dilutions of a universal self-priming dental adhesive (DA) and comparing these with those elicited by exposure to ionizing radiation (IR), Zoledronic acid (Z) treatment and the synergic effects of the combined treatment with IR+Z. MATERIAL AND METHODS: The genotoxic effect of DA was determined by the increase in the frequency of micronuclei in cytokinesis-blocked in cultured human lymphocytes before and after exposure to 2Gy of X-rays. The cytotoxic effect was studied by using the MTT cell viability test in normal prostate cell lines (PNT2) after exposure to different X-ray doses (0Gy-20Gy). The cell lines divided into different groups and treated with different test substances: DA in presence of O2, DA in absence of O2, Z-treated and control. RESULTS: An in vitro dose-dependent and time-dependent cytotoxic effect of DA, Z and IR on PNT2 cells (p>0.001) was demonstrated. DA without-O2, following the recommendations of manufacturers, had a more pronounced effect of increasing cell death than DA with-O2 (p<0.001). In the genotoxicity assay, DA at 25% of its original concentration significantly increased chromosome damage (p<0.001). The samples studied were found to be toxic, and the samples photo-polymerized in absence of O2 showed a bigger cytotoxic effect comparable to the additive toxic effect showed by the combined treatment of IR+Z. CONCLUSIONS: Additional effort should be carried out to develop adhesives, which would reduce the release of hazardous substances; since toxic effects are similar to that reported by other agents whose clinical use is controlled by the health authorities.

Genotoxicity and cytotoxicity of sevoflurane in two human cell lines in vitro with ionizing radiation.

OBJECTIVE: To determine the in vitro toxicity of different concentrations of sevoflurane in cells exposed to X-ray. METHODS: The genotoxic effects of sevofluorane were studied by means of the micronucleus test in cytokinesis-blocked cells of irradiated human lymphocytes. Subsequently, its cytotoxic effects on PNT2 (normal prostate) cells was determined using the cell viability test (MTT) and compared with those induced by different doses of X-rays. RESULTS: A dose- and time-dependent cytotoxic effect of sevofluorane on PNT2 cells was determined (p >0.001) and a dose-dependent genotoxic effect of sevofluorane was established (p >0.001). However, at volumes lower than 30 µL of sevofluorane at 100%, a non-toxic effect on PNT2 cells was shown. CONCLUSION: Sevofluorane demonstrates a genotoxic capacity as determined in vitro by micronucleus test in cytokinesis-blocked cells of irradiated human lymphocytes.

OBJETIVO: Determinar la capacidad genotóxica del anestésico sevofluorano en en células expuestas a radiación ionizante. MÉTODOS: La genotoxicidad del sevofluorane se determinó mediante el test del bloqueo citocinético de linfocitos humanos irradiados bloqueados con citochalasina. La capacidad citotóxica se determino mediante el test de viabilidad celular e inhibición del crecimiento celular (MTT) en células PNT2 (epiteliales de próstata), comparando sus resultados con los inducidos por diferentes dosis de rayos X. RESULTADOS: Se ha determinado un efecto citotóxico del sevofluorane sobre las células PNT2 que presenta correlación con la dosis administrada y el tiempo de estudio utilizado (p >0.001), así como un efecto genotóxico con características dosis-dependientes (p >0.001). Sin embargo, con volúmenes de sevofluorane puro inferiores a 30 µL no encontramos efecto citotóxico sobre las células PNT2. CONCLUSIÓN: Sevofluorane muestra una significativa capacidad genotóxica in vitro determinada mediante el test de micronúcleos en linfocitos humanos irradiados con bloqueados citocinético mediante citochalsina.

Radiosensitizing effect of rosmarinic acid in metastatic melanoma B16F10 cells.

BACKGROUND: Rosmarinic acid is an ester of caffeic acid with interesting biological activities including antioxidant effects and scavenging of oxygen-free radicals. AIM: To determine the potentially paradoxical effect of rosmarinic acid, typically being radioprotective when applied to non-tumorous cells, yet conversely displaying a sensitizing action when applied to metastatic B16F10 melanoma cells. MATERIALS AND METHODS: The genoprotective effect was studied by means of micronucleus tests for anti-mutagenic activity in which the reduction in the frequency of micronuclei was evaluated using cytokinesis-blocked human lymphocytes. The radioprotective effect was studied via a cell viability test in PNT2 (human prostate epithelium) and B16F10 melanoma cells. RESULTS: Rosmarinic acid exhibits a significant genoprotective capacity (p<0.001) against X-rays with a protection factor of 58%, and a dose reduction factor of 7.2. Cell survival obtained after exposure to 10 Gy of X-rays showed a protection factor of 47.5%, thus eliminating 29.1% of radiation-induced cell death in normal prostate epithelial cells (p<0.001). However, in metastatic B16F10 melanoma cells, rosmarinic acid acted not as a radioprotector, but as a sensitizing agent, increasing cellular death by 42% (p<0.001), with an enhancement ratio of 2.36. CONCLUSION: Rosmarinic acid has an increased capacity for producing radio-induced damage, and thus a paradoxical damaging effect in melanoma cells. Potentially, research into substances such as rosmarinic acid could help clarify mechanisms that provide protection on healthy normal cells, while exclusively damaging neoplastic cells, thus presenting a new strategy for patients undergoing radiotherapy for cancer.

Effects of bisphosphonates in combination with ionizing radiation and antioxidants on the growth of prostate and melanoma cells lines.

BACKGROUND: Bisphosphonates are used in cancer-related hypercalcaemia, in complications of bone metastasis and in postmenopausal osteoporosis, and have often been associated with adverse complications. AIM: To determine the protective effect of apigenin against growth inhibition of normal epithelial human prostatic (PNT2), transgenic adenocarcinoma of mouse prostate (TRAMP-C1) and metastatic melanoma cells (B16F10) in combined treatments with bisphosphonates and ionizing radiation (IR). MATERIALS AND METHODS: The growth inhibition on PNT2, TRAMP-C1 and B16F10 cells in the combined treatments with bisphosphonates (zoledronic acid, ibandronate and pamidronate) and IR in the presence and absence of apigenin was studied using a cell viability test. RESULTS: Zoledronic acid had a cytotoxic effect on PNT2, TRAMP-C1 and B16F10 cells (p<0.001). However, ibandronate and pamidronate had a cytotoxic effect only on PNT2 cells (p<0.001). The administration of apigenin in combined treatment with bisphosphonates and IR showed: a decrease in the cytotoxic effect on TRAMP-C1 and B16F10 cells in the treatment with ibandronate; a protective effect on normal PNT2 and melanoma cells, but not on TRAMP-C1 cells in the treatment with zoledronic acid; and provided protection only to PNT2 cells in the treatment with pamidronate. CONCLUSION: The use of the antioxidant produced a greater decrease in the cytotoxic effect on the non-tumor than in tumor cells when treated with bisphosphonates-alone and could be used in non-tumor pathologies. However, in a combined treatment with IR, it can also provide protection to tumor cells, thus reducing the intended effect of the IR.

Canting of the occlusal plane: Perceptions of dental professionals and laypersons

Objectives: To determine if canting of the occlusal plane influences esthetic evaluation of the smile among orthodontists, dentists and laypersons. Study Design: A frontal photo of a smile with 0º occlusal plane canting in relation to the bipupillary plane was modified using Adobe Photoshop C3 (Adobe Systems Inc, San José, California) to generate two images with occlusal plane inclinations of 2º and 4º. The three images were evaluated esthetically by orthodontists (n=40) general dentists (n=40) and laypersons (n=40). Each image was awarded a score as follows: 1=esthetically acceptable;2=moderately acceptable; 3=esthetically unacceptable. Evaluators also placed the three images in order in preference. Data were analyzed using the Kruskal-Wallis (p<0.05) and the Mann-Whitney tests, applying the Bonferroni Correction (p<0.016).Results: No significant differences (p> 0.05) were found between the three groups for 0º and 2º cants (median for orthodontists=1; general dentists=1; laypersons=1). Orthodontists (median score=3) made evaluations of the image with 4º occlusal plane that were significantly different from general dentists (median=2) and laypersons(median=2). All three groups put the 0º image in first place in order of esthetic acceptability, the 2º image in second place and the 4º image in third place. Orthodontists placed the 0º image in first place with significantly greater frequency (p<0.016) than laypersons. Conclusions: Occlusal plane canting of 0º and 2º were evaluated as esthetically acceptable by the three groups. The 4º occlusal plane cant was evaluated more negatively by orthodontists than by general dentists and laypersons. All three groups placed the 0º image in first place of esthetic acceptability, 2º in second place and 4º in third. Orthodontists put the 0º image in first place with significantly greater frequency than laypersons (AU)

Canting of the occlusal plane: perceptions of dental professionals and laypersons.

OBJECTIVES: To determine if canting of the occlusal plane influences esthetic evaluation of the smile among orthodontists, dentists and laypersons. STUDY DESIGN: A frontal photo of a smile with 0 occlusal plane canting in relation to the bipupillary plane was modified using Adobe Photoshop C3 (Adobe Systems Inc, San José, California) to generate two images with occlusal plane inclinations of 2 and 4 . The three images were evaluated esthetically by orthodontists (n=40) general dentists (n=40) and laypersons (n=40). Each image was awarded a score as follows: 1=esthetically acceptable; 2=moderately acceptable; 3=esthetically unacceptable. Evaluators also placed the three images in order in preference. Data were analyzed using the Kruskal-Wallis (p<0.05) and the Mann-Whitney tests, applying the Bonferroni Correction (p<0.016). RESULTS: No significant differences (p> 0.05) were found between the three groups for 0 and 2 cants (median for orthodontists=1; general dentists=1; laypersons=1). Orthodontists (median score=3) made evaluations of the image with 4 occlusal plane that were significantly different from general dentists (median=2) and laypersons (median=2). All three groups put the 0 image in first place in order of esthetic acceptability, the 2 image in second place and the 4 image in third place. Orthodontists placed the 0 image in first place with significantly greater frequency (p<0.016) than laypersons. CONCLUSIONS: Occlusal plane canting of 0 and 2 were evaluated as esthetically acceptable by the three groups. The 4 occlusal plane cant was evaluated more negatively by orthodontists than by general dentists and laypersons. All three groups placed the 0 image in first place of esthetic acceptability, 2 in second place and 4 in third. Orthodontists put the 0 image in first place with significantly greater frequency than laypersons.