Evaluation of gene expression changes in human primary uroepithelial cells following 24‐Hr exposures to inorganic arsenic and its methylated metabolites

Abstract

Gene expression changes in primary human uroepithelial cells exposed to arsenite and its methylated metabolites were evaluated to identify cell signaling pathway perturbations potentially associated with bladder carcinogenicity. Cells were treated with mixtures of inorganic arsenic and its pentavalent or trivalent metabolites for 24 hr at total arsenic concentrations ranging from 0.06 lM to 18 lM. One series (five samples) was conducted with arsenite and pentavalent metabolites and a second (10 samples) with arsenite and trivalent metabolites. Similar gene expression responses were obtained for pentavalent or trivalent metabolites. A suite of eight gene changes was consistently identified across individuals that reflect effects on key signaling pathways: oxidative stress, protein folding, growth regulation, metallothionine regulation, DNA damage sensing, thioredoxin regulation, and immune response. No statistical significance of trend (NOSTASOT) analysis of these common genes identified lowest observed effect levels (LOELs) from 0.6 to 6.0 lM total arsenic and no observed effect levels (NOELs) from 0.18 to 1.8 lM total arsenic. For the trivalent arsenical mixture, benchmark doses (BMDs) ranged from 0.13 to 0.92 lM total arsenic; benchmark dose lower 95% confidence limits (BMDLs) ranged from 0.09 to 0.58 lM total arsenic. BMDs ranged from 0.53 to 2.7 lM and BMDLs from 0.35 to 1.7 lM for the pentavalent arsenical mixture. Both endpoints varied by a factor of 3 across individuals. This study is the first to examine gene expression response in primary uroepithelial cells from multiple individuals and to identify no effect levels for arsenical-induced cell signaling perturbations in normal human cells exposed to a biologically plausible concentration range.Environ. Mol. Mutagen. 54:82-98, 2013. V V C 2012 Wiley Periodicals, Inc.

Evaluation of gene expression changes in human primary lung epithelial cells following 24‐hr exposures to inorganic arsenic and its methylated metabolites and to arsenic trioxide

“…It is important to note, however, that in our earlier study, in which we ran whole genome expression microarrays following in vitro treatment with five doses of a mixture of iAs and its methylated metabolites on primary uroepithelial cells from 15 individuals [Yager et al, ], we conducted BMD analyses on the genes that were expressed most in common among at least eight out of 10 individuals for the trivalent arsenical mixture and three out of five for the pentavalent mixture. To conduct a relatively straightforward comparison of the BMD results in this current experiment utilizing primary lung epithelial cells from three different individuals, we chose to run the BMD analysis on those genes expressed most in common among all three individual's cells.…”

“…Two approaches were used for BMD analysis. Population analysisFor comparison to the results from the previous bladder cell study [Yager et al, ], which did not have replicates, the median of the replicates from each donor was used. The median of the normalized gene expression signal from the three replicates for each donor was matched with the other two donor's medians for a population analysis.…”

“…The median of the normalized gene expression signal from the three replicates for each donor was matched with the other two donor's medians for a population analysis. The analysis of the previous study's data [Yager et al, ] used three donors to create a comparable population. These three donors were selected using PCA by removing any donor that was an outlier until three remained. Individual analysisAn individual analysis was performed for each donor and arsenic treatment separately using the individual replicates from each donor.…”

“…A series of studies conducted in bladder tissue by the authors provided elucidation of important aspects of arsenic's MoA at the gene transcription level following administration of relatively low concentrations [Clewell et al, ; Yager et al, ,b]. In the most recent in vitro studies, differentiated primary human uroepithelial cells from multiple normal individuals were treated for 24 hr [Yager et al, ] and up to 60 days [Yager et al, ] with mixtures of iAs and its pentavalent or trivalent methylated metabolites representative of urinary concentrations and proportions reported in the epidemiological literature (total arsenic concentrations ranging from 0.06 to 18 µM). Benchmark dose (BMD) modeling of gene expression results in the 24‐hr study showed BMD lower confidence limits (BMDLs) or equivalents of a no observed effect level (NOEL) of 0.09–0.58 µM arsenic for trivalent arsenical mixtures; using an alternative statistical method (NOSTASOT), NOELs for any change in gene expression were identified ranging from 0.18 to 1.8 µM arsenic.…”

“…Given the results of the foregoing studies as well as other published work in mammalian species, a growing body of evidence from a number of disciplines indicates a transition in arsenic dose–response such that cellular effects related to frank toxicity and progression that are observed at high concentrations transition to adaptive effects at lower concentrations with the inflection in exposure dose or POD somewhere in the vicinity of 0.1 µM iAs [Gentry et al, ; Clewell et al, ; Cohen et al, ; Yager et al, ,b].…”