NTP is incorporating the latest toxicogenomic technologies into its testing program to gain further insights into the toxicity of environmental substances. Toxicogenomics examines how the entire genetic structure, or genome, influences an organism’s response to environmental toxicants. Microarray, proteomic, metabolomics analyses, and next-generation (NextGen) sequencing are among the advanced technologies that NTP is using to study how chemical exposures change the expression of genes, proteins, and metabolites in targeted cells and tissues.
Measuring genome-wide changes in affected tissues could be useful for identifying disease biomarkers, detecting exposure to toxic substances, and understanding individual genetic susceptibilities. Once validated, biomarkers can be repeatedly sampled during long-term NTP studies to determine if chemical exposures can be detected or if developing diseases (e.g., cancer) provide a genetic signature.
NTP is investigating whether pattern analysis of gene expression can provide toxicity indicators at (1) earlier time points and (2) lower doses than are possible using traditional toxicological parameters. Evaluating patterns of gene expression is expected to provide insight into the pathogenesis of disease and how different rodent models respond to toxicants. In addition, metabolomics provides an opportunity to elucidate how chemicals affect metabolism within cells relative to changes in gene expression. Expression signatures linked to chemical exposures and apical measures are intended to contribute to NTP’s efforts in predictive toxicology.
Several FY 2018 toxicogenomic studies used NextGen sequencing technologies, which improves gene expression analysis, including base pair-level resolution of accuracy and increased sensitivity compared to microarray platforms. Although microarray analysis is a stable and well-understood technology for assaying gene expression, NextGen sequencing methods like RNA-seq likely will become more common as sequencing costs decline and bioinformatic analyses become standardized and integrated with genomic sequencing.
One promising research area is the application of exome sequencing (Exome-Seq) to either frozen or formalin-fixed, paraffin-embedded tissues. DNA can be extracted from either frozen or archival tissues. Coding portions of DNA, or exons, are captured by libraries of hybridization-based probes targeting over 200,000 exons and transcriptionally active regions. Exon-enriched DNA can be sequenced by DNA-Seq and then genomically aligned to find mutation insertions or deletions and other genetic abnormalities associated with disease. An additional area of development is the isolation of circulating, cell-free DNA (ccfDNA). DNA released from apoptosis or cell turnover normally appears in plasma, but chemical exposure can increase levels of ccfDNA due to toxicity, inflammation, or tumor development. The NTP is investigating the use and sequencing of ccfDNA in toxicology studies to determine its use as a new marker of chemical exposure.
Several NTP studies are using Exome-Seq for profiling mutations on a genome-wide scale to understand differences between spontaneous and chemically induced tumors. Another promising NextGen sequencing-related area in toxicogenomics is the S1500+ platform. This platform provides a way to use high-throughput transcriptomic screening for thousands of genes per sample and can be applied to both in vitro chemical toxicity screening and in vivo screening of RNA extracted from animal tissues.
NTP is evaluating study conditions that could contribute to differential gene expression, such as animal and tissue variability, methods for tissue sampling, and standards for conducting toxicogenomic studies under laboratory conditions. Efforts are underway to optimize methods for DNA and RNA extraction from archival tissues for molecular analysis. Planned or ongoing NTP toxicogenomic studies from FY 2018 are listed below.
Toxicogenomic Studies Planned or Ongoing in FY 2018
|Chemical (CASRN*)||Species/ Cell Line||Route||Duration||Test Type (Platform)||Study Scientist|
|Arsenite (7784-46-5)||Human prostate cell line||In vitro||30 weeks||NextGen sequencing Exome-Seq (Illumina)||Alex Merrick|
|2,3-Butanedione (diacetyl) (431-03-8)
|Human airway epithelium cell line||In vitro||4 days||High-throughput transcriptomic screening||William Gwinn|
|Bisphenol A and analogues (80-05-7)||Human hepatocyte cell line||In vitro||2 days||High-throughput transcriptomic screening||Mike DeVito|
|Bisphenol AF (1478-61-1)
Tetrabromobisphenol A (79-94-7)
|Rat||Gavage||5 days||S1500+ NextGen sequencing||Sue Fenton|
|Bromodichloroacetic acid (5589-96-8)
|Mouse||Gavage||2 years||NextGen sequencing Exome-Seq (Illumina)||Arun Pandiri|
Ethinyl estradiol (57-63-6)
Methyl mercury (115-09-3)
Isopropylated phosphate (3:1) (60348-60-9)
Pentabromodiphenyl ether (60348-60-9)
|Mouse||In vitro||1 day||S1500+, NextGen sequencing||Alison Harrill|
|Phosphate flame retardants:
tert-Butylphenyl diphenyl phosphate (56803-37-3)
2-Ethylhexyl diphenyl phosphate (1241-94-7)
Isodecyl diphenyl phosphate (29761-21-5)
Isopropylated phenol phosphate (68937-41-7)
|Rat||Gavage||5 days||Microarray (Affymetrix) Metabolomics||Scott Auerbach|
|Ginkgo biloba extract (90045-36-6)||Rat||Gavage||5 days||Microarray (Affymetrix)||Cynthia Rider, Scott Auerbach|
|Induced pluripotent stem cells Embryoid bodies Embryonic stem cells||Human stem cell||N/A||N/A||High-throughput transcriptomic screening||Erik Tokar, Mike DeVito|
Human hepatocyte cell line
|In vitro||1 day||S1500+ NextGen sequencing||Mike DeVito|
|PBDE-47 (32534-81-9)||Rat||Gavage||21 days||Microarray||June Dunnick|
|Polycyclic aromatic compounds:
|Human hepatocyte cell line||In vitro||2 days||Cytotoxicity and gene expression by quantitative polymerase chain reaction||Cynthia Rider, Erik Tokar|
|Tetrabromobisphenol A (79-94-7)
Pentabromodiphenyl oxide-technical (DE-71) (32534-81-9)
alpha, beta-Thujone (76231-76-0)
|Rat||Gavage||5 days||High-throughput transcriptomic screening||Mike DeVito, William Gwinn|
|112-chemical compound test set (pharmaceuticals and environmental compounds)||Human hepatocyte cell line-HepaRG||In vitro||2 days||High-throughput transcriptomic screening||Stephen Ferguson, Sreenivasa Ramaiahgari|
|20-chemical compound test set (environmental compounds)||Primary rat hepatocytes Human HepaRG cells||In vitro||3 days||S1500+ NextGen sequencing||William Gwinn, Mike DeVito|
|N/A||Human mRNA Reference Isolation||In vitro||N/A||S1500+ NexGen Sequencing||Richard Paules|
|Carcinogens (HCC100)||Mouse||In vivo||2 years||HCC RNA-seq, CNV array||Miaofei Xu,
*Chemical Abstracts Service Registry Number
**This study will compare toxicogenomic effects among the chemicals listed together.