Iodoacetic Acid

Participants and procedures. Participants in the current study, the Folate and Oxidative Stress (FOX) Study, were recruited between February and July of 2008 in Araihazar, Bangladesh. Potential participants were identified on the basis of well water As (wAs) concentrations obtained from a well survey in the year 2000 (van Geen et al. 2002 (link)) in order to ensure a wide range of As exposures for the examination of dose-dependent relationships. A new water sample was collected at the time of enrollment for analysis of wAs concentration. Individuals were eligible to participate in FOX if they a) were between 30 and 65 years of age; b) were not pregnant; c) were not taking nutritional supplements; d) did not have known diabetes, cardiovascular or renal disease, chronic obstructive pulmonary disease, or cancer; and e) had been drinking water from their current well for at least 3 months. Trained recruiters identified eligible participants, explained the nature of the study, obtained informed consent, and scheduled a field clinic visit. Because GSH is unstable and therefore requires that blood samples be processed immediately, all visits were conducted in the laboratory at our field clinic in Araihazar. During the field clinic visit, a trained interviewer administered a detailed questionnaire to each participant, and a physician collected a venous blood sample. Urine samples were collected in 50-mL acid-washed polypropylene tubes and frozen at –20°C.
The primary aim of this study was to examine the dose–response relationship between As exposure and measures of oxidative stress. We aimed to recruit 75 participants each from five wAs concentration categories: < 10, 10–100, 101–200, 201–300, and > 300 µg/L. However, the final sample included more particpants with lower exposures because many households switched to lower-As wells after wells in the region were surveyed for As in 1999–2000 (Chen et al. 2007 (link)). Therefore, the final distribution among well wAs exposure categories was < 10 µg/L (n = 76), 10–100 µg/L (n = 104), 101–200 µg/L (n = 86), 201–300 µg/L (n = 67), and > 300 µg/L (n = 45).
Oral informed consent was obtained by our Bangladeshi field staff physicians, who read an approved assent form to the study participants. This study was approved by the Bangladesh Medical Research Council and the institutional review board of Columbia University Medical Center.
Sample collection and handling. After the initial processing of blood samples in the field clinic, the blood and plasma aliquots were immediately frozen at –80°C. Samples on dry ice were transported in batches to Dhaka, Bangladesh, by car and again stored at –80°C (blood and plasma) or –20°C (urine). Samples were then packed on dry ice in coolers and transported by air to Columbia University.
wAs. Field sample collection and laboratory analysis procedures have been previously described in detail (Cheng et al. 2004 (link); van Geen et al. 2005 (link)). Water samples were collected in 20-mL polyethylene scintillation vials. The samples were acidified to 1% with high-purity Optima HCl (Fisher Scientific, Pittsburg, PA, USA) at least 48 hr before analysis (van Geen et al. 2007 ). Water samples were analyzed by high-resolution inductively coupled plasma mass spectrometry (ICP-MS) after 1:10 dilution and addition of a germanium spike to correct fluctuations in instrument sensitivity. The detection limit of the method is typically < 0.2 μg/L (Cheng et al. 2004 (link)). A standard with an As concentration of 51 µg/L was run multiple times in each batch. The intraassay and interassay coefficients of variation (CVs) for this standard were 6.01% and 3.76%, respectively.
Total urinary As. All urine samples were analyzed for total urinary As (uAs) in the Columbia University Trace Metals Core Laboratory by graphite furnace atomic absorption spectrometry (Nixon et al. 1991 (link)) using the AAnalyst 600 graphite furnace system (PerkinElmer, Shelton, CT, USA). A method based on the Jaffe reaction was used to measure urinary creatinine concentrations (Slot 1965 (link)). Method and instrument precision was checked by running four different urine samples with known concentrations (to cover the whole linearity range of the standard curve) every day immediately after the instrument calibration with aqueous standards. A urine sample with an As concentration in the middle of the linearity range was run after every 10 study samples. The intraassay and interassay CVs based on this quality control sample were 3.9% and 5.6%, respectively. For duplicate study samples, the intraassay and interassay CVs were 3.8% and 5.1%, respectively.
Total blood As. We used a Perkin-Elmer Elan DRC II ICP-MS equipped with an AS 93+ autosampler to analyze whole blood samples for total blood As (bAs) concentration, as described previously (Hall et al. 2006 (link)). The intraassay and interassay CVs were 3.2% and 5.7%, respectively, for quality control samples. For study samples, the intraassay and interassay CVs were 2.1% and 4.9%, respectively.
Blood GSH and GSSG and plasma Cys and CySS. Whole blood GSH and GSSG and plasma Cys and CySS were assayed essentially as described by Jones et al. (1998) (link). Blood was collected with a butterfly needle and syringe and then immediately transferred into Eppendorf tubes. For whole blood measurements, the Eppendorf tubes contained 5% perchloric acid, 0.1 M boric acid, and γ-glutamyl glutamate as an internal standard. For plasma measurements, the tubes contained 0.53 g l-serine, 25 mg heparin, 50 mg bathophenanthrolene, 300 mg iodoacetic acid, and 10 mL borate buffer stock (12.4 g boric acid, 19 g sodium tetraborate decahydrate, and 500 mL distilled water). The samples for plasma measurements were centrifuged for 1 min, and 200 µL of supernatant was transferred into Eppendorf tubes containing an equal volume of 10% perchloric acid and 0.2 M boric acid. For derivatization, plasma samples were centrifuged at 13,000 rpm for 2 min, 300 µL of supernatant was transferred to a fresh tube, and the pH was adjusted to 9.0. After incubating for 20 min at room temperature, dansyl chloride was added, and samples were incubated at room temperature in the dark for 24 hr. The derivatized samples were then stored at –80°C until delivered to Columbia University for analysis. Free dansyl chloride was extracted from thawed samples with 500 µL chloroform, and then 20 µL of the sample was injected onto the HPLC. Separation was achieved using a Supelcosil LC-NH₂ column (catalog no. 58338; Sigma Chemical Co., St. Louis, MO, USA). Initial solvent conditions were 60% A (80% methanol, 20% water), 40% B (acetate-buffered methanol, pH 4.6) run at 1 mL/min for 10 min. A linear gradient to 20% A, 80% B was run during the 10- to 50-min period. From 50 to 52 min, the conditions were returned to 60% A, 40% B. Metabolites were detected using a Waters 474 scanning fluorescence detector (Waters Corp., Milford, MA, USA), with 335 nm excitation and 515 nm emission. Within-assay CVs were all between 0.05 and 0.10, and interassay CVs were between 0.11 and 0.18.
Plasma folate. Plasma folate was analyzed by radio protein-binding assay (SimulTRAC-S; MP Biomedicals, Orangeburg, NY, USA). To determine folate concentrations, we used folic acid as pteroylglutamic acid for calibration, and its ¹²⁵I-labeled analog as the tracer. The intraassay and interassay CVs were 0.06 and 0.14, respectively.
Calculation of the reduction potential. The reduction potential (E_h) of the thiol/disulfide GSH/GSSG and Cys/CySS redox pairs (blood GSH E_h and plasma Cys E_h, respectively) were calculated using the Nernst equation:
E_h = E_o + RT/nF ln(acceptor/(donor)²,
where E_o is the standard potential for the redox couple, R is the gas constant, T is the absolute temperature, n = 2 for the number of electrons transferred, and F is Faraday’s constant (Jones et al. 2002 (link)). For GSH and GSSG, the equation simplifies to
E_h (mV) = −264 + 30 log[(GSSG)/(GSH²)],
where (GSH) and (GSSG) are molar concentrations, and the E_o value assumes a physiologic pH of 7.4. A more positive E_h value indicates a more oxidized redox state.
Statistical methods. We calculated descriptive statistics for characteristics of the study sample, As exposure variables (wAs, uAs, and bAs), and outcome variables (blood GSH and GSSG, plasma Cys and CySS), both for the total sample and by sex. Bivariate associations were examined using scatter plots and Spearman’s correlation coefficients. To examine the bivariate associations between dichotomous covariates and As exposure variables or continuous outcome variables we used t-tests or the nonparametric Wilcoxon rank sum test.
We used linear regression models to further examine the associations between As exposure variables, as continuous variables, and the outcome variables, with and without adjustment for potential confounders. Age and sex were included in all covariate-adjusted regression models. Other covariates considered for inclusion in the regression models were variables reported to be associated with the exposures or outcomes based on previous publications and/or variables associated with the exposure and outcome variables in the present study population. These variables included television ownership (as a surrogate for socioeconomic status), cigarette smoking, body mass index (BMI), urinary creatinine, and plasma folate. We adjusted for GSH laboratory batch (as a categorical variable) in order to reduce extraneous variation in the outcome variables. We also calculated the change in R² between models for each outcome that included covariates only and corresponding models that included both the covariates and As exposure.
To facilitate comparisons among the different measures of exposure (wAs, uAs, and bAs), we report the estimated change in the mean value of blood GSH, blood GSH E_h, plasma CySS, and plasma Cys E_h associated with an interquartile range (IQR) increase in each exposure. For outcome variables that were natural log-transformed (blood GSSG and plasma Cys) we report the ratio of estimated geometric means for an IQR change in As exposure.
To examine possible nonlinear relationships, we also created quintiles of As exposure variables and computed covariate-adjusted mean values of the outcome variables for categories of As exposure; plots of quintile-specific adjusted mean values were examined to determine if the association was approximately linear.
We ran separate linear regression models to examine the covariate-adjusted associations between As exposure and the outcome variables stratified by sex or by folate status. We then used a Wald test to detect differences in the covariate-adjusted associations between As exposure and outcome variables by sex or by folate status. All analyses were performed using SAS (version 9.2; SAS Institute Inc., Cary, NC, USA); all statistical tests were two-sided with a significance level of 0.05.

We prepared 39.44 mM sodium sulfite (Fujifilm Wako Pure Chemical Corporation), as an oxygen scavenger, in PBS (oxygen concentration of 0%). We also prepared an oxygen-saturated PBS solution containing sodium sulfite solution (oxygen concentration of 21%, matching that of air) by leaving it for a day. The oxygen concentrations in the solutions were verified using a TX3 trace oxygen microoptode. We also prepared 10 µM antimycin (Enzo Life Sciences), oligomycin (Abcam), and FCCP (Cayman chemical) in PBS as inhibitors of the mitochondrial respiratory pathway. In addition, as glycolytic pathway inhibitors, we prepared 50 µM heptelidic acid (Abcam) and 1 mM iodoacetic acid (Fujifilm Wako Pure Chemical Corporation) in PBS. To investigate the effect of pH on sperm motility, 150 mM NaCl with pH 7.1 and 8.6 by adding NaOH and HCl, respectively were also prepared. To prevent change in pH, the NaCl solution was used within 5 min of pH adjustment for each experiment.
A 0.5-mm deep silicone rubber spacer was placed on a slide to keep space between the slide and coverslip. Sperm cells from the spermathecae of C. osakensis queens were exposed to 20 µL of test solution on the slide and covered with a coverslip, and sperm motility was observed and recorded under a differential interference contrast microscope (Olympus BX53) with a 3CCD digital camera (Olympus DP74).
Sperm motility levels were scored based on the frequency of motile sperm cells as follows: 0, no motile sperm cells; 1, some sperm cells exhibiting motility; 2, intermediate motility, between scores 1 and 3; 3, 80–90% motility; and 4, maximum sperm cells motility. Scoring was performed by a trained observer within 2 min of dissection. In the experiments using an oxygen scavenger and NaCl, the motility scores of all test samples (with one exceptional sample in the PBS control) were either score 0 (immotile) or 4 (motile) (Figs. 2 and 6c). In the experiments using glycolysis and respiration inhibitors, intermediate motility was observed (Fig. 3). To ensure consistent scoring of sperm motility when testing the inhibitors, we performed all observations within 11 days of the experiments.

After one week (day 0), the animals were allocated to 3 subgroups of 10 animals each. The mean weight of the rats was 210 g and did not differ significantly between the groups. The animals were anaesthetised with the use of a VMS MATRX inhalation anaesthesia machine in a chamber for anaesthesia of laboratory animals and Isoflurane AErrane (BAXTER, Lublin, Poland) at a concentration of 4%. General anaesthesia was maintained with 2% Isoflurane applied through a rodent face mask.
The animals of each of the three subgroups received monoiodoacetic acid (MIA—Sigma-Aldrich Co., St. Louis, MO, USA) at a concentration of 0.03 mg diluted in 50 μg of sterile saline. It was injected through the patellar ligament of the right knee joint with the use of a U-100 insulin needle. In turn, 50 μg of saline was administered through the patellar ligament in the left knee joint (control).
The rats in the first group were stimulated to exercise on a treadmill (RR—running rats). Three days after the MIA administration, a week-long training period began and the animals ran on a treadmill for 10 min/day at a speed of 10 m/min. After a week, the running time was prolonged to 30 min/day at a speed of 18 m/min. This training regimen was maintained for 3 weeks. The total training time was 28 days.
The animals in the second group were allowed to lead a natural lifestyle (no treadmill stimulation; WR—walking rats). In the third group, all parts of the right hind limb muscle were injected with Clostridium botulinum toxin type A (botox) supplied by Allergan (Irvine, CA, USA) at a dose of 0.8 units/kg b.w. to disable the movement of the right hind limb (Botox-injected rats, IR—immobilised rats). Experimental design is shown on Scheme 1.
The experiment was terminated on day 28 of the exercise imposed on the RR and WR rats and immobilisation of the IR rats. The animals were anaesthetised via intraperitoneal injection of 10 mg/kg b.w. xylazine (BIOWET, Puławy, Poland) and 80 mg/kg b.w. of ketamine (VET-AGRO, Lublin, Poland). Next, the animals were immobilised on the section table. After exposure of the heart, a 12-gauge needle was inserted into the right ventricle and blood was drained. In the next step, physiological saline was introduced gravitationally into the bloodstream to remove morphotic elements; next, 10% formalin was administered. After ca. 45–60 s, the needle was inserted into the right atrium to fill the vascular bed with formalin.

All viability assays were performed in 96-well black-walled, clear bottom plates. Growth media was removed following indicated treatments, and 100 µL of a room-temperature solution of Cell Titer-Glo and Opti-MEM was immediately added to each well (1:1 Cell Titer-Glo:Opti-MEM). Luminescence (Cell Titer-Glo) readings were taken after 10 min room-temperature incubation. Student’s t-test was used to determine the significance of specified gene knockdown values from non-targeting siRNA values. All cell number assays were CyQUANT measurements, and were performed in 96-well black-walled, clear bottom plates. Growth media was removed following indicated treatments and cells were processed according to the manufacturer’s protocol. Student’s t-test was used to determine the significance of specified gene knockdown values from non-targeting siRNA values. All ATP/cell assays were performed in 96-well black-walled, clear bottom plates. Growth media was removed following indicated treatments, and 100 µL of a room-temperature solution of Cell Titer-Glo, Opti-MEM, and CyQUANT was immediately added to each well (1:1:0.0058 Cell Titer-Glo:Opti-MEM:CyQUANT). Luminescence (Cell Titer-Glo) and fluorescence (CyQUANT) readings were consecutively taken after 10 min room-temperature incubation. Cell Titer-Glo values were divided by CyQUANT values to generate ATP/cell values. Inhibitors and drug treatments: Cells were treated with iodoacetic acid (IAA, 10 μM), oligomycin (500 ηM), or DMSO (vehicle, equivalent percent as drug) for four hours prior to ATP/cell measurements. Cells were treated with AZD8055 at the indicated concentrations for 18 h prior to cell viability measurements. Cells were treated with 6-aminonicotinamide (6-AN, 100 μM) or DMSO (equivalent percent as drug) for 24 h prior to cell number measurements.