Lipid Metabolism Alterations in a Rat Model of Chronic and Intergenerational Exposure to Arsenic

Chronic exposure to arsenic (As), whether directly through the consumption of contaminated drinking water or indirectly through the daily intake of As-contaminated food, is a health threat for more than 150 million people worldwide. Epidemiological studies found an association between chronic consumption of As and several pathologies, the most common being cancer-related disorders. However, As consumption has also been associated with metabolic disorders that could lead to diverse pathologies, suchas type 2 diabetes mellitus, nonalcoholic fatty liver disease, and obesity. Here, we used ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization/quadrupole time-of-flight mass spectrometry (ESI-QToF) to assess the effect of chronic intergenerational As exposure on the lipid metabolism profiles of serum from 4-month-old Wistar rats exposed to As prenatally and also during early life in drinking water (3 ppm). Significant differences in the levels of certain identified lyso-phospholipids, phosphatidylcholines, and triglycerides were found between the exposed rats and the control groups, as well as between the sexes. Significantly increased lipid oxidation determined by the malondialdehyde (MDA) method was found in exposed rats compared with controls. Chronic intergenerational As exposure alters the rat lipidome, increases lipid oxidation, and dysregulates metabolic pathways, the factors associated with the chronic inflammation present in different diseases associated with chronic exposure to As (i.e., keratosis, Bowen’s disease, and kidney, liver, bladder, and lung cancer)

Correction: Metformin Induces Cell Cycle Arrest, Reduced Proliferation, Wound Healing Impairment In Vivo and Is Associated to Clinical Outcomes in Diabetic Foot Ulcer Patients.

[This corrects the article DOI: 10.1371/journal.pone.0150900.]

Metformin inhibits HaCaT cell proliferation and colony formation.

<p>1000 cells/well human HaCaT cells were plated onto 6-well and incubated at 37°C with 5% CO₂. After 24h, the culture medium was replaced with fresh culture medium containing 0.0 mM, 10 mM or 20 mM every 3 days for 9 days. (A) At 9 days the cells colonies were stained and counted as described in the methods section. (B) Colony area was determined using Image J program. (C) Image is representative of the size of colonies observed for each of the treatments. Data are representative of six independent experiments.</p

Metformin inhibits wound healing <i>in vivo</i>.

<p>A rat model of wound healing was used to evaluate the effect of metformin on cell proliferation and wound closure. (A) The panel shows the effect of metformin (300mg/kg) in the healing of a standardized model of wound healing, PBS was used as a control and topical prednisolone (0.25 mg) has been used in this model as control of delayed healing. Representative photographs of each time point are shown. (B) Glucose concentrations were measured daily in the animals by means of a portable glucometer (mg/dl), to monitor the effect of metformin compared to PBS and prednisolone treatment. (C) Ulcer diameters were measured from the referenced images using Image J, the ruler in each photograph was used as a standard to set scale for each image. Groups of animals of N = 12 animals per treatment group were used for the experiments in two independent experiments. Analyses of glucose and area differences were made by means of a Two-way ANOVA. P<0.05 was considered statistically significant.</p

Metformin blocks cell cycle progression.

<p>Human HaCaT cells were grown in plate (5 x 105 cells). After 24h the culture medium was replaced with fresh culture medium containing 0 mM or 20 mM metformin for an additional 48h. Cell cycle progression in HaCaT cells was assessed by flow cytometry by analyzing PI fluorescence in slow acquisition and with doublet discrimination, 50000 events were acquired. Histogram showing cell cycle HaCaT with 0 mM and 20 mM of metformin, in the cells treated with 20 mM can be seen an increase in the proportion of cells which are in transition from G0/G1 to S phase. Histograms depicted above are representative of at least three independent experiments, which showed similar results.</p

Metformin Induces Cell Cycle Arrest, Reduced Proliferation, Wound Healing Impairment <i>In Vivo</i> and Is Associated to Clinical Outcomes in Diabetic Foot Ulcer Patients

<div><p>Background</p><p>Several epidemiological studies in diabetic patients have demonstrated a protective effect of metformin to the development of several types of cancer. The underlying mechanisms of such phenomenon is related to the effect of metformin on cell proliferation among which, mTOR, AMPK and other targets have been identified. However, little is known about the role that metformin treatment have on other cell types such as keratinocytes and whether exposure to metformin of these cells might have serious repercussions in wound healing delay and in the development of complications in diabetic patients with foot ulcers or in their exacerbation.</p><p>Material and Methods</p><p>HaCaT Cells were exposed to various concentrations of metformin and cell viability was evaluated by a Resazurin assay; Proliferation was also evaluated with a colony formation assay and with CFSE dilution assay by flow cytometry. Cell cycle was also evaluated by flow cytometry by PI staining. An animal model of wound healing was used to evaluate the effect of metformin in wound closure. Also, an analysis of patients receiving metformin treatment was performed to determine the effect of metformin treatment on the outcome and wound area. Statistical analysis was performed on SPSS v. 18 and GraphPad software v.5.</p><p>Results</p><p>Metformin treatment significantly reduces cell proliferation; colony formation and alterations of the cell cycle are observed also in the metformin treated cells, particularly in the S phase. There is a significant increase in the area of the wound of the metformin treated animals at different time points (P<0.05). There is also a significant increase in the size and wound area of the patients with diabetic foot ulcers at the time of hospitalization. A protective effect of metformin was observed for amputation, probably associated with the anti inflammatory effects reported of metformin.</p><p>Conclusions</p><p>Metformin treatment reduces cell proliferation and reduces wound healing in an animal model and affects clinical outcomes in diabetic foot ulcer patients. Chronic use of this drug should be further investigated to provide evidence of their security in association with DFU.</p></div

Clinical features of patients with foot ulcers.

<p>Clinical features of patients with foot ulcers.</p

Metformin induced inhibition of HaCaT cells proliferation.

<p>Human HaCaT cells were cultured 24 hours before CFSE labeling in FBS 1% to synchronize the culture cell cycle. Labeling was performed with CFSE as described in the methods section. After that the cells were cultured for 24 hours to adherence and then treated with 0 mM (PBS), 10 mM and 20 mM of metformin for 24 or 48 hours. The proliferation in HaCaT cells was assessed by flow cytometry. (A) Histograms show cell proliferation inhibition at 10 mM and 20 mM, the effect is observed after 24 hours of treatment. (B) The graph shows the mean fluorescence intensity of cells labeled with CFSE, it is clear that cells were treated with metformin have a higher intensity of fluorescence due to decreased cell proliferation. (C) Percentage of inhibition of proliferation is shown compared to control treated cells. Inhibition is observed in both the 10mM and 20 mM metformin treated cells and dependence on time is also observed.</p

Effect of metformin in history of diabetic foot ulcer.

<p>Data shown was taken from the clinical records of patients who had diabetic foot ulcer and stratified by treatment with metformin or others antidiabetic drugs (n = 72). (A) The graph shows patients at the time of the review of the record had already undergone some amputation. (B) The graph shows the number of previous amputations per treatment group. (C) The graph shows the area of ulceration reported by the treating physician, the size of ulcers is greater in the group of patients receiving metformin as part of their treatment for glycemic control (p = 0.029, Mann Whitney test). (D) The graph shows whether patients discharged from the hospital were amputated as a result of the severity of his ulcer, seen as the proportion of subjects amputees is higher in the group of patients treated with other antidiabetic drugs (p = 0.035 Fisher´s exact test). P<0.05 was considered statistically significant.</p