Project description:We analyzed the transcriptional response of the actinomycete Rhodococcus aetherivorans I24 to biphenyl and polychlorinated biphenyls (PCBs). This species has not been extensively exposed to PCBs, as it was first isolated from a toluene contaminated aquifer, rather than a site contaminated with polychlorinated hydrocarbons. Using a microarray targeting 3524 genes, we assessed gene expression in minimal medium supplemented with various substrates (e.g. PCBs) and in both PCB-contaminated and non-contaminated sediment slurries. Relative to the reference condition (minimal medium supplemented with glucose), 408 genes were up-regulated in the various treatments. In medium and in sediment, PCBs elicited the up-regulation of a common set of 100 genes, including chaperones (groEL), a superoxide dismutase (sodA), alkyl hydroperoxide reductase protein C (ahpC), and a catalase/peroxidase (katG). Analysis of the R. aetherivorans I24 genome sequence identified orthologs of many of the genes in the canonical biphenyl pathway, but very few of these genes were up-regulated in response to PCBs or biphenyl. This study is one of the first which utilizes microarrays to assess the transcriptional response of a soil bacterium to a pollutant under conditions which more closely resemble the natural environment. Our results indicate that the transcriptional response of R. aetherivorans I24 to PCBs, in both medium and sediment, is primarily directed towards reducing oxidative stress, rather than catabolism. In addition, the identification of numerous genes expressed in contaminated soil specifically may have implications for the development of biosensors. Finally, comparative genomic and transcriptomic analyses suggest that the mere presence of orthologs of the required enzymes may not be sufficient to confer a vigorous biphenyl/PCB metabolism. RNA was isolated from cells incubated in the following: sediment from a PCB-contaminated industrial site, uncontaminated sediment from a comparable site, and defined media supplemented with glucose (3 g/L), glucose and biphenyl (3 g/L, 4.5 Î¼M), or glucose and PCBs (3 g/L, 5 mg/L Aroclor 1254). In all cases, there were 3 biological replicates and 2 technical replicates (repeat hybridizations). A total of 3524 genes are represented on the arrays; of these, 41 and 176 are found on the plasmids pRA2 and pRA3, respectively. On average, there are 3 distinct 24nt probes per gene.

Project description:(Abstract) Toxicogenomics has great potential for enhancing our understanding of environmental chemical toxicity, hopefully leading to better-informed human health risk assessments. This study employed toxicogenomic technology to reveal species differences in response to two prototypical aryl hydrocarbon receptor (AHR) agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the polychlorinated biphenyl (PCB) congener PCB 126. Dose responses of primary cultures of rat and human hepatocytes were determined using species-specific microarrays sharing over 4,000 gene orthologs. Forty-seven human and 79 rat genes satisfied dose response criteria for both chemicals and were subjected to further analysis including the calculation of EC50 and the relative potency (REP) of PCB 126 for each gene. Only 5 responsive orthologous genes were shared between the two species, yet the geometric mean of the REPs for all rat and human modeled responsive genes were 0.06 (95% Confidence Interval (CI); 0.03-0.1) and 0.002 (95% CI; 0.001-0.005), respectively, suggesting broad species differences in the initial events that follow AHR activation but precede toxicity. This indicates that there are species differences in both the specific genes that responded and the agonist potency and relative potency for those genes. This observed insensitivity of human cells to PCB 126 is consistent with more traditional measurements of AHR activation (i.e., CYP1A1 enzyme activity) and suggests that the species difference in PCB 126 sensitivity is likely due to certain aspects of AHR function. That a species divergence also exists in this expanded AHR-regulated gene repertoire is a novel finding and should help when extrapolating animal data to humans. Experiment Overall Design: Primary hepatocyte cultures, separated into 2 pools of 3 female Sprague Dawley rats each, were treated with vehicle (0.5% DMSO), TCDD (-14 to -6.5 log10 M) , or PCB 126 (-12 to -5 log10 M) for 48h. Total RNA was extracted and screened with RG-U34A microarrays for dose response.

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Wastewater metagenome

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