Evaluation of a toxicogenomic approach to the local lymph node assay (LLNA).
Genomic technologies have the potential to enhance and complement existing toxicology endpoints; however, assessment of these approaches requires a systematic evaluation including a robust experimental design with genomic endpoints anchored to traditional toxicology endpoints. The present study was conducted to assess the sensitivity of genomic responses when compared with the traditional local lymph node assay (LLNA) endpoint of lymph node cell proliferation and to evaluate the responses for their ability to provide insights into mode of action. Female BALB/c mice were treated with the sensitizer trimellitic anhydride (TMA), following the standard LLNA dosing regimen, at doses of 0.1, 1, or 10% and traditional tritiated thymidine ((3)HTdR) incorporation and gene expression responses were monitored in the auricular lymph nodes. Additional mice dosed with either vehicle or 10% TMA and sacrificed on day 4 or 10, were also included to examine temporal effects on gene expression. Analysis of (3)HTdR incorporation revealed TMA-induced stimulation indices of 2.8, 22.9, and 61.0 relative to vehicle with an EC(3) of 0.11%. Examination of the dose-response gene expression responses identified 9, 833, and 2122 differentially expressed genes relative to vehicle for the 0.1, 1, and 10% TMA dose groups, respectively. Calculation of EC(3) values for differentially expressed genes did not identify a response that was more sensitive than the (3)HTdR value, although a number of genes displayed comparable sensitivity. Examination of temporal responses revealed 1760, 1870, and 953 differentially expressed genes at the 4-, 6-, and 10-day time points respectively. Functional analysis revealed many responses displayed dose- and time-specific induction patterns within the functional categories of cellular proliferation and immune response, including numerous immunoglobin genes which were highly induced at the day 10 time point. Overall, these experiments have systematically illustrated the potential utility of genomic endpoints to enhance the LLNA and support further exploration of this approach through examination of a more diverse array of chemicals.
Boverhof
Darrell R
DR
Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674, USA. RBoverhof@dow.com
Gollapudi
B Bhaskar
BB
Hotchkiss
Jon A
JA
Osterloh-Quiroz
Mandy
M
Woolhiser
Michael R
MR
D000465:Algorithms; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D016000:Cluster Analysis; D003627:Data Interpretation, Statistical; D004305:Dose-Response Relationship, Drug; D023381:Endpoint Determination; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D007111:Immunity, Cellular / Q000187:drug effects; D023441:Local Lymph Node Assay; D051379:Mice; D008807:Mice, Inbred BALB C; D020411:Oligonucleotide Array Sequence Analysis; D010796:Phthalic Anhydrides / Q000633:toxicity; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013936:Thymidine / Q000378:metabolism; D043922:Toxicogenetics / Q000379:methods*
D010796:Phthalic Anhydrides; D012313:RNA; C015559:trimellitic anhydride; D013936:Thymidine