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Annual Report for Fiscal Year 2017

Annual Report for Fiscal Year 2017
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https://ntp.niehs.nih.gov/go/838037

Toxicogenomic Studies

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 provide an opportunity to elucidate how chemicals affect metabolism within cells relative to changes in gene expression.

Several FY 2017 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.

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 (HTT) 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 2017 are listed below.

Toxicogenomic Studies Planned or Ongoing in FY 2017

Chemical (CASRN*) Species/ Cell Line Route Duration Test Type (Platform) Study Scientist

Anthraquinone (84-65-1)
Oxazepam (604-75-1)
DE-71 (32534-81-9)
Dibutyl phthalate (84-74-2)
PCTFT (98-56-6)
TCPP (115-96-8)
Bromodichloroacetic acid (5589-96-8)
Ginkgo biloba extract (90045-36-6)
Primaclone (125-33-7)Bromochloroacetic acid (5589-96-8)

Mouse

Gavage

2 years

Infinium DNA methylation bead array

Arun Pandiri

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)2,3-Hexanedione (3848-24-6)

Human airway epithelium cell line
 
In vitro
  
4 days
 
High-throughput transcriptomic screening William Gwinn
 

Bisphenol A and analogs (80-05-7)

Human hepatocyte cell line In vitro 2 days High-throughput transcriptomic screening Mike DeVito

BPAF (1478-61-1)
TBBPA (79-94-7)

Rat

Gavage 5 days

S1500+ NextGen sequencing

Sue Fenton

Bromodichloroacetic acid (5589-96-8)
Methyleugenol (93-15-2)***
  

Mouse
  
Gavage
 
2 years
 
NextGen sequencing
Exome-Seq (Illumina)
 
Arun Pandiri
 

Dieldrin (60-57-1)
Ethinyl estradiol (57-63-6)
Methyl mercury (115-09-3)
Rotenone (83-79-4)
Phenol (68937-41-7)
Isopropylated phosphate (3:1) (60348-60-9)
Pentabromodiphenyl ether (60348-60-9)

Mouse

In vitro

1 day

S1500+, NextGen sequencing

Alison Harrill

DE-71 (32534-81-9)
PCB-126 (57465-28-2)
Phenobarbital (50-06-6)***  

Rat Gavage   GD** to PND 22   Microarray (Affymetrix)

June Dunnick  

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)
Metabolomics

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

N/A Rat N/A 2 years Targeted resequencing

Arun Pandiri, Ramesh Kovi

2-Hydroxy-4- methoxybenzophenone (131-57-7)

Rat

Oral (food)  

90 days Microarray (Affymetrix) Scott Auerbach
 

Methyleugenol extract (93-15-2)
Ginkgo biloba extract (90045-36-6)***

Mouse  Gavage 2 years NextGen sequencing
Exome-Seq
  RNA-Seq (Illumina)

Arun Pandiri, Scott Auerbach, Alex Merrick

PCB-11 (2050-67-1)

Rat
Human hepatocyte cell line

In vitro 1 day

S1500+ NextGen sequencing

Mike DeVito

2,3-Pentanedione (600-14-6)

Rat Inhalation 14 and 28 days microRNA Microarray (Affymetrix)  Dan Morgan
 

PBDE-47 (32534-81-9)

Rat Gavage

21 days

Microarray

June Dunnick

Polycyclic aromatic compounds:
Acenaphthenequinone (82-86-0)
Benzo[b]fluoranthene (205-99-2)
Benzo(a)pyrene (50-32-8)
Dibenz[a,h]anthracene (53-70-3)
9-Methylanthracene (779-02-2)
1-Methylfluorene (1730-37-6)
Perinaphthenone (548-39-0)
Phenanthrene (85-01-8)
Pyrene (129-00-0)
Human hepatocyte cell line In vitro 2 days Cytotoxicity
Gene expression by quantitative polymerase chain reaction  

Cynthia Rider, Erik Tokar

2,2’,4,4’,5-Pentabromodiphenyl ethers (5436-43-1)
Pentabromodiphenyl oxide (technical) (DE-71) (32534-81-9)
3,3,4,4,5- Pentachlorobiphenyl (57465-28-8)
2,2'4,4'- Tetrabromodiphenyl ether (DE-47) (5436-43-1)

Rat
Mouse
Gavage GD** 6 through 3 weeks Microarray (Affymetrix

June Dunnick

Tetrabromobisphenol A (79-94-7)
BDE-47 (5436-43-1)
Pentabromodiphenyl oxide-technical (DE-71) (32534-81-9)

Rat Gavage GD** 6 through 3 weeks Microarray (Affymetrix)

Arun Pandiri, Ramesh Kovi

Tetrabromobisphenol A (79-94-7)Pentabromodiphenyl oxide-technical (DE-71) (32534-81-9)
Triclosan (3380-34-5)
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

Time-restricted feeding
Shift work-surrogate study

Rat In vivo

4 months

Microarray

Gopi Gadupudi, Arun Pandiri

*Chemical Abstracts Service Registry Number
**GD: gestational day
***This study will compare toxicogenomic effects among the chemicals listed together.

Related Links:
Disposition, Metabolism, and Toxicokinetic Studies
Genetic Toxicity Studies 
Organ System Toxicity Studies 
Toxicology and Carcinogenicity Studies
Modified One-Generation Reproduction Studies
Toxicogenomic Studies
Project Review Committee Approved