Procedures

General Information for All Procedures: iAs # inorganic arsenic

Procedure 1: Treatment with sodium arsenite

Definitions & Abbreviations: DIW # deionized water

Reagents and solutions

• DIW containing sodium arsenite, NaAsO₂, 99% pure (Sigma-Aldrich, St. Louis, MO, USA)

Steps

1. Following the four-week acclimatization period, all mice are switched to DIW containing sodium arsenite at a final concentration of 100 µg As/L (100 ppb) for a period of 26 weeks.

Procedure 2: Food and water consumption

Equipment, software, and supplies

• Balance scale

Steps

1. Food consumption is measured either over a two or four weekly time period by recording the weight of food provided to test mice at the beginning of the time period, and comparing that to the food left at the end of the time period.
2. Water consumption is measured weekly is a similar manner.

Procedure 3: Body weight

Equipment, software, and supplies

• Balance scale

Steps

1. Body weight is measured from the time of arrival of the mice and then at selected time points, as described in the study design figure, until 26 weeks (sacrifice).

Procedure 4: Body composition and weight by MRI

Equipment, software, and supplies

• Body composition analyzer, Echo MRI Three-in-one Composition Analyzer and Labmaster (Echo Medical Systems, Three-in-one, Houston, TX, USA)

Steps

1. Whole body composition, including percentages of body mass represented by fat (% fat) and by lean mass (% lean mass) are measured by Echo-MRI. Measurements are carried out 2 weeks before the start of iAs exposure, as well as at 12 and 24 weeks respectively after the start of iAs exposure.
2. In addition to body weight recordings by balance scale (see above body weight procedure), body weights are also measured by the body composition analyzer. Measurements are carried out at the same time points as whole body composition assessments.

Procedure 5: Blood glucose and plasma insulin

Fasting (duration): 6h

Equipment, software, and supplies

• Glucometer (LifeScan Inc., One Touch Ultra glucometer, Milpitas, CA, USA)
• ELISA kit (Crystal Chem, Elk Grove Village, IL, USA)
• Ultracentrifuge

Reagents and solutions

• Glucose (Sigma-Aldrich, Burlington, MA, USA)

Steps

1. Blood glucose and plasma insulin levels are both measured after 6-hours of fasting, and 15 minutes after intraperitoneal (i.p.) injection of glucose (2 g/kg body weight) at 3-5 week intervals.
2. Plasma is isolated from blood by centrifugation at 1700xg for 15 minutes at 4°C and stored at -80°C. Fasting plasma insulin (FPI) and 15-minute plasma insulin (PI15) are measured using ELISA.
3. Fasting blood glucose (FBG) and 15-minute blood glucose (BG15) concentrations are measured by glucometer.
4. Sample preparations and glucose treatments are performed in the early afternoon and organized to facilitate the timing for each mouse and is consistent over the course of the study.

Procedure 6: Urine collection

Steps

1. Urine is collected, and its volume noted at set time points, specifically 2 weeks prior to iAs exposure and at 12 and 24 weeks after iAs exposure. Samples collected at 24 weeks are stored at -80°C for further analysis.

Procedure 7: Euthanasia

Steps

1. Mice are euthanized after 26 weeks of iAs exposure by cervical dislocation. Liver tissue is dissected, snap frozen, and stored -80°C for future analysis.

Procedure 8: iAs speciation analysis in liver and urine

Definitions & Abbreviations: MMA # monomethyl-arsenic acid; DMA # dimethyl-arsenic acid; As # Arsenic

Equipment, software, and supplies

• Hydride generation-atomic absorption spectrometer

Reagents and solutions

• 2% cysteine

Steps

1. Inorganic arsenic (iAs) and its metabolites are measured in liver, dissected at euthanasia; and in urine samples (50-100 µL), collected earlier following 24 weeks of iAs exposure.
2. Concentrations of iAs, MMAs and DMAs are measured by hydride generation-atomic absorption spectrometry coupled with a cyrotrap as described by Hernández-Zavala et al. (2008). This method is used to measure concentrations of arsenic species in 10% homogenates (^w/_v) of liver and urine. Both samples are treated with 2% cysteine for 1h prior to analysis, to convert all pentavalent arsenicals to trivalency. The instrumental limits of detections (LODs) for iAs, MMAs, and DMAs using this method are 14, 8, and 20 pg As, respectively (Hernández-Zavala et al. (2008). These instrumental LODs translate to 0.28, 0.16, and 0.40 ppb in urine and 0.5-5.6, 0.4-3.2, and 1.0-8 ppb in liver, respectively, given the injected sample volumes and dilutions used in this study.
3. A value of zero was imputed for each value below LOD. The relative contributions of each As species in each tissue are computed by dividing the concentration of each metabolite by the total arsenic concentration (tAs) in the tissue, which is calculated as the sum of the concentrations of iAs, MMAs, and DMAs. These proportions are then expressed as the percentage of tAs represented by iAs (%iAs), MMAs (%MMAs) or DMAs (%DMAs).