Chemicals. Sodium arsenite, sodium salt (99% pure), was purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure phosphoric acid was obtained from J.T. Baker (Phillipsburg, NJ). Sodium arsenate (iAs
V), sodium salt (96%; Sigma-Aldrich); methylarsonate (MAs
V), disodium salt (98%; Chem Service, West Chester, PA); and dimethylarsinic acid (DMAs
V; 98%; Strem Chemicals, Inc., Newburyport, MA) were used as standards for speciation analysis of As in mouse tissues. All other chemicals were of the highest grade commercially available.
Mice. Four-week-old male weanling C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the University of North Carolina–Chapel Hill (UNC-CH) Animal Facility, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. Mice were housed five per cage in polycarbonate cages with corncob bedding in controlled conditions (12-hr light/dark cycle, 22 ± 1°C, and 50 ± 10% humidity). Mice were fed a low-fat diet (LFD; 11% fat) or a high-fat diet (HFD; 58% fat; both from Research Diets, Inc., Brunswick, NJ) and drank either diH
2O or diH
2O plus iAs
III (25 or 50 ppm As). Water containing iAs
III was freshly prepared every 3–4 days to minimize oxidation of iAs
III to iAs
V. Water and food consumption and body mass were monitored in all exposure groups every week. Body composition was measured biweekly at the UNC-CH Nutrition Obesity Research Core, using EchoMRI-100 (EchoMRI, Houston, TX). The animals were treated humanely and with regard for alleviation of suffering. All procedures involving mice were approved by the UNC-CH Institutional Animal Care and Use Committee.
Oral glucose tolerance test (OGTT) and analyses of blood glucose and insulin. After 20 weeks, both control and iAs-treated LFD and HFD mice were fasted overnight before administration of OGTT.
d-Glucose (Sigma) was dissolved in diH
2O and orally administered to the fasted mice (2 g/kg of body weight) using a 20-gauge stainless steel gavage feeding needle (Fisher Scientific, Waltham, MA). Samples of whole blood (2–3 μL each) were collected from a tail-clip bleed immediately before and 15, 30, 60, 90, and 120 min after glucose administration. Blood glucose levels were measured using a Freestyle Glucose Monitoring System (Abbott Laboratories, Abbott Park, IL). Additional samples of whole venous blood (100 μL) were collected from tails immediately before and 15 min after glucose administration for determination of serum insulin levels. The blood was allowed to clot on ice for 15 min and then centrifuged (200 ×
g) at 4°C for 10 min.
Serum was analyzed using the Rat/Mouse Insulin ELISA kit according to the manufacturer’s protocol (Millipore, Billerica, MA). We used the FBG and FSI concentrations to calculate the homeostasis model assessment–insulin resistance (HOMA-IR) value:
HOMA-IR =
[FSI (in microunits per milliliter)
× FBG (in millimoles per liter)]
÷ 22.5.
The blood glucose and serum insulin levels recorded during OGTT were used to evaluate glucose tolerance and insulin response to glucose challenge, respectively. After OGTT, mice were returned to their cages and treatment continued for 7–10 days before necropsy.
Blood and tissue collection at necropsy. Whole blood samples were collected by submandibular bleeds, and mice were sacrificed by cervical dislocation. Hematocrit (a marker of dehydration) was determined in samples of fresh blood (~ 100 μL) using 40-mm heparin-coated capillary tubes. Capillary tubes were centrifuged at 12,000 rpm in a microhematocrit centrifuge (Unico, Dayton NJ) for 15 min, and the percentage of red blood cells was recorded for each mouse. Serum was isolated from submandibular blood as described above. Liver, inguinal adipose tissue, quadriceps, and pancreas were collected during necropsy and snap frozen in liquid nitrogen. Serum and the tissue samples were stored at –80°C until analysis.
Analyses of hepatic and serum triacylglycerol. We measured triacylglycerol (TAG) contents in serum and in liver homogenates spectrophotometrically (at 490 nm) after a two-step extraction in a chloroform/methanol (2:1) mixture and in pure chloroform, using a Stanbio Enzymatic Triglyceride Kit (Stanbio, Boerne, TX), following the manufacturer’s instructions.
Speciation analysis of As in tissues. For analysis of iAs metabolites, 10% (wt/vol) tissue homogenates were prepared in diH
2O and digested in ultrapure phosphoric acid. Concentrations of As species were determined in digested homogenates by hydride generation–cryotrapping–atomic absorption spectrometry (HG-CT-AAS), following previously described procedures (Hernández-Zavala et al. 2008 (
link)). This method detects and quantifies total iAs (iAs
III + iAs
V), total MAs (MAs
III + MAs
V), and total DMAs (DMAs
III + DMAs
V).
Statistical analysis. Results of the analyses are expressed as mean ± SE for each treatment group (
n = 6–10). Effects of diet and iAs exposure, as well as interactions between diet and iAs exposure, were analyzed by analysis of covariance. Differences between the treatment groups were evaluated by one-way analysis of variance with Tukey or Bonferroni multiple-comparison posttests. All statistical analyses were performed using Graphpad Prism (version 5.0; GraphPad Software, San Diego, CA). Differences among means with
p < 0.05 were considered statistically significant.
Paul D.S., Walton F.S., Saunders R.J, & Stýblo M. (2011). Characterization of the Impaired Glucose Homeostasis Produced in C57BL/6 Mice by Chronic Exposure to Arsenic and High-Fat Diet. Environmental Health Perspectives, 119(8), 1104-1109.