ROS and ERK Pathway Mechanistic Approach on Hepatic Insulin Resistance After Chronic Oral Exposure to Cadmium NOAEL Dose.

Cadmium is a critical toxic agent in occupational and non-occupational settings and acute and chronic environmental exposure situations that have recently been associated with metabolic disease development. Until now, the no observed adverse effect level (NOAEL) of cadmium has not been studied regarding insulin resistance development. Therefore, we aimed to monitor whether chronic oral exposure to cadmium NOAEL dose induces insulin resistance in Wistar rats and investigate if oxidative stress and/or inflammation are related. Male Wistar rats were separated into control (standard normocalorie diet + water free of cadmium) and cadmium groups (standard normocalorie diet + drinking water with 15 ppm CdCl2). At 15, 30, and 60 days, oral glucose tolerance, insulin response, and insulin resistance were analyzed using mathematical models. In the liver glycogen, triglyceride, pro- and anti-inflammatory cytokines, cadmium, zinc, metallothioneins, and redox balance were quantified. Immunoreactivity analysis of proteins involved in metabolic and mitogenic insulin signaling was performed. The results showed that a cadmium NOAEL dose after 15 days of exposure causes ROS and mitogenic arm of insulin signaling to increase while hepatic glycogen diminishes. At 30 days, Cd accumulation accentuated ROS production, hepatic triglyceride overaccumulation, and mitogenic signals that develop insulin resistance. Finally, inflammation and lipid peroxidation appear after 60 days of Cd exposure, while lipids and carbohydrate homeostasis deteriorate. In conclusion, environmental exposure to cadmium NAOEL dose causes hepatic Cd accumulation and ROS overproduction that chronically declines the antioxidant defense, deteriorates metabolic homeostasis associated with the mitogenic pathway of insulin signaling, and induces insulin resistance.

Acrylamide induces a thyroid allostasis-adaptive response in prepubertal exposed rats.

Some endocrine-disrupting chemicals (EDCs) can affect the endocrine system through covalent interactions with specific sites, leading to deregulation of physiological homeostasis. The acrylamide (AA) present in some fried or baked foods is an example of an electrophile molecule that is able to form adducts with nucleophilic regions of nervous system proteins leading to neurological defects. A positive correlation between increased urinary AA metabolite concentration and reduced levels of thyroid hormones (TH) was described in adolescents and young adults. Thus, this study aimed to evaluate whether AA affects the physiology of the hypothalamus-pituitary-thyroid (HPT) axis and the possible repercussions in peripheral TH-target systems. For this, male Wistar rats were exposed to doses of 2.5 or 5.0 mg AA/Kg/day, based on the LOAEL (Lowest Observed Adverse Effect Level) during prepubertal development. The expression of molecular markers of HPT functionality was investigated in the hypothalamus, pituitary, thyroid, heart and liver, as well as the hormonal and lipid profiles in blood samples. Herein, we showed that AA acts as EDCs for thyroid gland function, increasing the transcript expression of several proteins related to TH synthesis and altering hypothalamus-pituitary-thyroid axis homeostasis, an effect evidenced by the higher levels of THs in the serum. Compensatory mechanisms were observed in TH-target tissues, such as an increase in Dio3 mRNA expression in the liver and a reduction in Mct8 transcript content in the hearts of AA-treated rats. Together, these results pointed out an allostatic regulation of the HPT axis induced by AA and suggest that chronic exposure to it, mainly associated with food consumption, might be related to the higher prevalence of thyroid dysfunctions.

Chemical characterization and toxicological assessment of hydroethanolic extract of Mandevilla velame xylopodium

Abstract The present study was aimed to characterize the chemical profile and evaluate the cytotoxicity and sub-chronic toxicity of the hydroethanolic extract of the xylopodium Mandevilla velame (A.St.-Hil.) Pichon, Apocynaceae. Chemical profile was analyzed by high performance liquid chromatography. Cytotoxic potential of hydroethanolic extract of the xylopodium M. velame was evaluated using Chinese hamster ovary cells. The sub-chronic assessment was done on rats with hydroethanolic extract of the xylopodium M. velame (50, 200 and 800 mg/kg) was orally administered daily for 30 consecutive days. High performance liquid chromatography analysis confirmed the presence of gallic acid, ellagic acid, catechin, epigallocatechin gallate, naringin, myricetin, quercetin and naringenin. hydroethanolic extract of the xylopodium M. velame tested concentrations did not alter the viability of Chinese hamster ovary cells. In the sub-chronic test, 50 and 200 mg/kg were safe, but there were significant changes in relation to weight gain and water consumption by animals that received 800 mg/kg of hydroethanolic extract of the xylopodium M. velame. Among the haematological and biochemical parameters evaluated, only the number of neutrophils, lymphocytes, and creatinine concentration were changed at 800 mg/kg. Phytochemical profile of hydroethanolic extract of the xylopodium M. velame revealed the presence of phenolics and flavonoid compounds. The in vitro cytotoxicity assay result demonstrated that hydroethanolic extract of the xylopodium M. velame had no cytotoxic effects in Chinese hamster ovary cells. In the in vivo models, hydroethanolic extract of the xylopodium M. velame was shown to be relatively safe after sub-acute administration in rats which is relation to that the population daily takes a total dose of the plant xylopodium decoction or infusion about 23.29 times lower than the no-observed-adverse effect level dose in rats.

Acute and Repeated 28-Day Oral Dose Toxicity Studies of Thymus vulgaris L. Essential Oil in Rats.

Thymus vulgaris L. is widely used as an ingredient in cooking and in herbal medicine. However, there is little information about its toxicity. The present study was performed to evaluate the acute and repeated 28-day oral dose toxicity of thyme essential oil in rats. For the acute toxicity test, two groups of three rats were used. The rats received a single dose of essential oil: 300 or 2,000 mg/kg of body weight (bw). The rats were observed individually during the first four hours, and then daily until day 14. For the toxicity test with repeated doses, four groups of 10 rats were used. Doses of 100, 250, and 500 mg/kg/day were tested for 28 days. At the end of the experiment, blood was collected and the animals were sacrificed. Histopathological examination showed that in the lungs of rats given the 2,000 mg/kg bw dose, polymorph nuclear infiltrates, hemosiderin macrophages, and interstitial space thickening were present. In the repeated dose study, all rats survived the 28-day treatment period and apparently showed no signs of toxicity. The hematological and biochemical parameters were not altered. The histopathological study of the organs showed severe changes in the lung, with the dose of 500 mg/kg/day; in the other organs, no alterations were observed or the changes were slight. The body weight was only altered in male rats given the 500 mg/kg dose. The relative weight of the organs did not show any significant changes. Our studies revealed that the essential oil of Thymus vulgaris has moderate oral toxicity according to the results of the acute test, whereas the results of the 28-day oral toxicity test suggest that the no-observed-adverse effect level (NOAEL) is greater than 250 mg/kg/day.

Chemical characterisation and toxicity assessment in vitro and in vivo of the hydroethanolic extract of Terminalia argentea Mart. leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia argentea Mart. (Combretaceae), known mainly as "capitão", is a native tree, not endemic, that occurs in the Amazon, Caatinga, Cerrado and Atlantic Forest in Brazil. Leaf infusion is popularly mentioned by riverine communities that inhabit the microregion of Northern Araguaia (Mato Grosso, Brazil) for the treatment of gastric ulcer, bronchitis and haemorrhage. Considering the wide medicinal use, lack of studies that evaluate the safety of use and the scarcity of phytochemical studies of T. argentea leaves, this work was carried out with the objective of evaluating the toxicity of the hydroethanolic extract of the leaves of T. argentea Mart. (HETa) in experimental models in vivo and in vitro, as well as to advance the phytochemical analysis of HETa. MATERIALS AND METHODS: HETa was prepared by macerating the leaf powder in hydroethanolic solution. Phytochemical characterisation was carried out by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and mass spectrometry through direct flow infusion coupled with electrospray ionization and ion-trap analyzer (DFI-ESI-IT-MS analyses) The contents of phenols, flavonoids and phytosterols were analysed by colorimetric methods. Cytotoxicity was assessed by the Alamar blue assay on Chinese hamster ovary epithelial cells (CHO-K1) and human gastric adenocarcinoma cells (AGS). In vitro genotoxicity of HETa (10, 30 or 100 µg/mL) was assessed by micronucleus (MN) and comet tests using CHO-K1 cells. The acute toxicity assessment was performed by oral administration of HETa in single dose Swiss mice (males and females) up to 2000 mg/kg and sub-chronic toxicity by daily oral administration of HETa (50, 200 and 800 mg/kg) in Wistar rats for 30 days. The parameters related to the clinical and toxicological observations were determined every 6 days and at the end of the treatment the blood was collected for biochemical and haematological analysis, and some organs were removed for macroscopic and histopathological analysis. RESULTS: Preliminary phytochemistry and TLC analysis of HETa revealed the presence of phenolic compounds (18.8%), flavonoids (10.8%), saponins, tannins and phytosterols (19%). The HPLC data revealed the presence of gallic acid, rutin, ellagic acid, catechin, quercetin and kaempferol. In the analysis by DFI-ESI-IT-MS, the presence of gallic acid, rutin, ellagic acid and quercetin was confirmed and identified caffeic acid, quinic acid, galloylmucic acid, quercetin xyloside, quercetin rhamnoside, quercetin glucoside, caffeoyl ellagic acid, quercetin galloyl xyloside, terminalin, quercetin galloyl glucose, corilagin, quercetin digalloyl xyloside, quercetin digalloyl glucoside, punicalin and punicalagin. HETa showed no cytotoxic effect on CHO-K1 and AGS cells. In the MN assay, HETa increased the number of MNs and nuclear buds (NBUDs) in binucleate cells at the three concentrations tested and the nucleoplasmic bridges (NPBs) number at 30 µg/mL. In the comet test, HETa (10 and 100 µg/mL) alone showed a genotoxic effect on CHO-K1 cells. In pre-treatment, HETa at all concentrations tested prevented DNA damage induced by H2O2. In co-treatment with H2O2, HETa showed genotoxic effects at the three concentrations, and post-treatment DNA damage in exposed CHO-K1 cells to H2O2 was repaired in 22.5% with 10 µg/mL HETa. In the acute toxicity test, the HETa did not cause death in the mice, being verified only by piloerection and reversible in 2 h in males and in 4 days in females. No macroscopic changes were observed in the analysed organs. In the sub-chronic toxicity test, the HETa did not cause death in the rats after 30 days and the few changes were: absolute (103/mm3) and relative (%) values of basophils increased by 477.8% and 423% (p < 0.001), respectively, with 50 mg/kg; reduction in feed intake (23.6%, p < 0.01) only on day 18; total cholesterol concentration (13.1%, p < 0.05) and relative heart weight (13.2% %, p < 0.05) at a dose of 800 mg/kg. These effects were not dose-dependent nor followed by clinical signs and symptoms of intoxication, nor of macroscopic and histopathological changes in the organs of animals treated with HETa. CONCLUSIONS: The results demonstrated that HETa had no cytotoxic in vitro effects for CHO-K1 and AGS cells. In in vitro genotoxicity assays, the HETa induced different responses, according to concentration and experimental condition. In the MN test the HETa presented genotoxic potential by increasing the number of MNs, NBUDs and NPBs. In the comet assay, HETa was genotoxic by itself and in the co-treatment protocol with H2O2. In pre-treatment or post-treatment protocols with H2O2, HETa presented an antigenotoxic effect by preventing or repairing, respectively, the genotoxicity induced by H2O2. In the in vivo models, HETa was shown to be relatively safe after acute administration in mice [no-observed-adverse effect level (NOAEL) of 2000 mg/kg] and sub-chronic in rats (NOAEL of 800 mg/kg), confirming the riverine information that it is non-toxic in the dosage used. Phytochemical analysis of HETa revealed the presence of phenolic compounds, flavonoids, saponins, tannins and phytosterols. Among the flavonoids and tannins, we highlight gallic acid, rutin, ellagic acid, quercetin, caffeic acid, quinic acid, corilagin, punicalin and punicalagin. Thus, it can be stated that HETa has a good safety margin for therapeutic use.

Evaluation of genotoxicity and subchronic toxicity of the standardized leaves infusion extract of Copaifera malmei Harms in experimental models.

ETHNOPHARMACOLOGICAL RELEVANCE: Copaifera malmei Harms (Fabaceae), known mainly as óleo-mirim, is a native and endemic plant found in the states of Mato Grosso and Goiás of Brazil. The plant's leaves infusion is popularly used by riverine communities of the northern Araguaia microregion, Mato Grosso, Brazil, for the treatment of gastric ulcers and inflammatory diseases of the respiratory tract. The gastric antiulcer activity of the standardized leaves infusion extract of Copaifera malmei (SIECm) in rodents has been reported. The objective of this study was to advance the investigation of the safety profile of SIECm by evaluating the genotoxicity and subchronic toxicity using in vitro and in vivo experimental models. MATERIALS AND METHODS: SIECm was prepared by infusion, by incubating the powdered dried leaves material in boiled water for 15min. In vitro genotoxicity of SIECm (10, 30 or 100µg/mL) was assessed by micronucleus and comet tests using Chinese hamster ovary (CHO-k1) epithelial cells. The evaluation of subchronic toxicity profile was performed by daily oral administration of SIECm (100, 400 or 1000mg/kg) to Wistar rats for 30 days. Clinical observations of toxicological related parameters were done every 6 days. After the treatment period, blood was collected for hematological and biochemical analysis, and some organs were removed for macroscopic and histopathological analysis. RESULTS: In the micronucleus assay, SIECm demonstrated anti-mutagenic activity. In the comet assay, SIECm presented anti-genotoxic effect preventing DNA damage at all the three concentrations tested with pre-treatment, while the same effect was only observed in the co-treatment at the lowest concentration. Post-treatment with SIECm increased the genetic damage induced by hydrogen peroxide (H2O2) at the highest concentration. In the subchronic toxicity test, few changes were observed, such as increase in feed consumption in the group of animals treated with 100mg/kg of the SIECm, which reversed after 6 days. There were no macroscopic, histological and relative weights changes in the organs of animals treated with SIECm. No toxicologically relevant changes were observed in the hematological analysis. Subchronic administration of SIECm reduced levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in animals treated with 100mg/kg and serum triglyceride levels at 400 and 1000mg/kg. However, the hematological and biochemical changes observed are within the physiological ranges for this animal species. CONCLUSION: The results demonstrate that SIECm is not genotoxic, and does not present toxicity when used orally for up to 30 days. In addition, it showed protection to the genetic damage induced by H2O2. The SIECm therefore has a high safety margin for therapeutic use.

Assessment of the potential health risks associated with the aluminium, arsenic, cadmium and lead content in selected fruits and vegetables grown in Jamaica.

Thirteen Jamaican-grown food crops - ackee (Blighia sapida), banana (Musa acuminate), cabbage (Brassica oleracea), carrot (Daucus carota), cassava (Manihot esculenta), coco (Xanthosoma sagittifolium), dasheen (Colocasia esculenta), Irish potato (Solanum tuberosum), pumpkin (Cucurbita pepo), sweet pepper (Capsicum annuum), sweet potato (Ipomoea batatas), tomato (Solanum lycopersicum) and turnip (Brassica rapa) - were analysed for aluminium, arsenic, cadmium and lead by atomic absorption spectrophotometry and instrumental neutron activation analysis. The fresh weight mean concentrations in these food crops (4.25-93.12 mg/kg for aluminium; 0.001-0.104 mg/kg for arsenic; 0.015-0.420 mg/kg for cadmium; 0.003-0.100 mg/kg for lead) were used to calculate the estimated daily intake (EDI), target hazard quotient (THQ), hazard index (HI) and target cancer risk (TCR) for arsenic, associated with dietary exposure to these potentially toxic elements. Each food type had a THQ and HI < 1 indicating no undue non-carcinogenic risk from exposure to a single or multiple potentially toxic elements from the same food. The TCR for arsenic in these foods were all below 1 × 10-4, the upper limit used for acceptable cancer risk. There is no significant health risk to the consumer associated with the consumption of these Jamaican-grown food crops.

Evaluation of the acute and subchronic oral toxicity of ethanol extract from Valeriana pavonii species in Wistar rats / Evaluación de la toxicidad oral aguda y sub-crónica del extracto etanólico de la especie Valeriana pavonii en ratas Wistar

Introduction: One of the most frequent problems found in medicinal plants is the absence of clinical, toxicological, and pharmacological studies. Valeriana pavonii is one of the species used in Colombia as an anxiolytic. Further study of this specie is rendered to add information in the toxicological area. Objective: The acute and subchronic oral toxicity of V. pavonii ethanolic extract was evaluated in Wistar rats of both sexes. Materials and methods: The rats were distributed into four groups: the control group received the vehicle (0.5 mL/100 g of corporal weight) and the other three groups received increasing levels of the dosage for 90 days to evaluate characteristics like physical exam, laboratory test (blood chemistry and haematology), and anatomopathological findings. Results: This study reveals that there were no signs of toxicity, mortality, or significant alterations attributable to the ethanolic extract of V. pavonii. Conclusions: The Not Observed Adverse Effect Levels (NOAEL) of V. pavonii ethanolic extract were 2000 and 1000 mg/kg of body weight for the acute and subchronic toxicity studies, respectively.

Introducción: Uno de los problemas más frecuentes asociados con el uso de plantas medicinales es la ausencia de evidencias farmacológicas, toxicológicas y clínicas. Valeriana pavonii es una de las especies más utilizadas popularmente en Colombia con fines ansiolíticos. Es necesario avanzar en el estudio de esta especie para aportar información en el campo toxicológico. Objetivos: Evaluar la toxicidad oral aguda y sub-crónica del extracto etanólico de V. pavonii en ratas Wistar de ambos sexos. Materiales y métodos: En cada uno de los estudios se distribuyeron ratas en cuatro grupos; un grupo control que recibió únicamente vehículo (0.5 ml/100 g de peso corporal) y tres grupos correspondientes a niveles crecientes de dosis, así: para el estudio de toxicidad aguda se administraron en dosis única 20, 200 y 2000 mg/kg con un período de observación de 14 días y para el de toxicidad sub-crónica, dosis diarias de 250, 500 y 1000 mg/kg durante 90 días. Se evaluaron los parámetros de examen físico, los exámenes de laboratorio (química sanguínea y hematología) y el estudio anatomopatológico. Resultados: No se presentaron signos de toxicidad, letalidad ni alteraciones significativas atribuibles al consumo del extracto etanólico de V. pavonii, según el examen físico, el examen anatomopatológico y el análisis de las pruebas de química sanguínea y hematología. Conclusiones: Los valores de nivel sin efectos adversos observados (NOAEL) del extracto etanólico de V. pavonii, fueron 2000 y 1000 mg/kg de peso corporal para los estudios de toxicidad aguda y sub-crónica, respectivamente. No se encontraron valores de nivel más bajo de efecto adverso observado (LOAEL).