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Annual Report for Fiscal Year 2016

Annual Report for Fiscal Year 2016
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https://ntp.niehs.nih.gov/go/807118

Toxicogenomic Studies

NTP is incorporating the latest toxicogenomic technologies into its testing program to gain further insight regarding the toxicity of environmental substances. Toxicogenomics examines how the entire genetic structure, or genome, is involved in an organism's response to environmental toxicants. Microarray, next-generation (NextGen) sequencing, proteomics, and metabolomics are among the advanced technologies that NTP is using to study the way chemical exposures change the expression of genes, proteins, and metabolites in critical cells and tissues. Measuring genome-wide changes in affected tissues may be useful for identifying biomarkers of disease or exposure to toxic substances, and for understanding individual genetic susceptibilities. Once biomarkers are validated, they can be repeatedly sampled during long-term NTP studies to determine whether chemical exposures can be detected, or whether developing cancers provide a genetic signature.

NTP is researching whether gene expression pattern analysis can provide indicators of toxicity (1) at earlier time points and (2) at lower doses than is possible for traditional toxicology parameters. Evaluating patterns of gene expression may provide more than just a link between genetics and morphology. It is also expected to provide insights into the pathogenesis of the disease and how different rodent models respond to toxicants. In addition, metabolomics represents a promising area of study, as it can elucidate how chemicals affect metabolism within cells relative to changes in gene expression.

Several FY 2016 toxicogenomic studies used NextGen sequencing technologies, which provided improvements to gene expression analysis, including base-pair level resolution of accuracy and increased sensitivity compared to microarray platforms. While micorarrays are a stable and well understood technology for assaying gene expression, NextGen sequencing methods like RNA-Seq will likely become more common as sequencing costs drop and bioinformatic pipelines become standardized and integrated with genomic sequencing. A promising area of research is the use of exome sequencing (Exome-Seq) that can be applied 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 mutations; indels, which are insertions or deletions; and other genetic abnormalities associated with disease. Three NTP studies are using Exome-Seq as a means for mutation profiling at a genome-wide scale to understand differences between spontaneous and chemically induced tumors.

NTP is evaluating study conditions that may 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 have been made to optimize methods for DNA and RNA extraction from archival tissues for molecular analysis. FY 2016 planned or ongoing NTP toxicogenomic studies are listed in the table below.

Toxicogenomic Studies Planned or Ongoing in FY 2016

Chemical CASRN* Species/ Cell Line Route Length Test Type (Platform) Study Scientist
Aging cohort study, mouse strains:
  • 129S1/SvImJ
  • B6C3F1 (Jackson)
  • C3H/HeJ
  • C57BL/6J (Jackson)
  • CAST/EiJ (M. m. castaneus)
  • NZO/HiLtJ
  • PWK/PhJ
  • WSB/EiJ (M. m. domesticus)
  • A/J
  • NOD. B10Sn-H2(b)/J
N/A
 
Mice
 
N/A
 
2 years
 
NextGen sequencing
Exome-Seq (Illumina)
 
Dunnick
 
Arsenite
 
7784-46-5
 
Human prostate cell lines
 
In vitro exposure
 
30 weeks
 
NextGen sequencing
Exome-Seq (Illumina)
 
Merrick
 
2,3-butanedione (diacetyl)
2,3-hexanedione
 
431-03-8
3848-24-6
 
Human airway epithelium cell line
 
In vitro exposure
 
4 days
 
High throughput transcriptomics Gwinn
 
Bisphenol A and BPA Analogs 80-05-7 Human hepatocyte cell line In vitro exposure 2 days High throughput transcriptomics Devito
Bromodichloroacetic acid
Methyleugenol***
 
5589-96-8
93-15-2
 
Mice
 
Gavage
 
2 years
 
NextGen sequencing
Exome-Seq (Illumina)
 
Pandiri
 
DE-71
PCB-126
Phenobarbital***
 
32534-81-9
57465-28-2
50-06-6
 
Rats
 
Gavage
 
GD** to PND 22
 
Microarray (Affymetrix)
 
Dunnick
 
Phosphate flame retardants:
  • tert-Butylphenyl diphenyl phosphate
  • 2-Ethylhexyl diphenyl phosphate
  • Isodecyl diphenyl phosphate
  • Isopropylated phenol phosphate
56803-37-3
1241-94-7
29761-21-5
68937-41-7
 
Rats
 
Gavage
 
5 days
 
Microarray (Affymetrix)
Metabolomics
 
Auerbach
 
Ginkgo biloba extract
 
90045-36-6
 
Rats
 
Gavage
 
5 days
 
Microarray (Affymetrix)
 
Rider/ Auerbach
 
induced Pluripotent Stem Cells
Embryoid bodies
Embryonic stem cells
N/A Human N/A N/A High throughput transcriptomics Tokar/Devito
Harlan Sprague-Dawley rats N/A Rats N/A 2 years Targeted resequencing Pandiri/Kovi
2-Hydroxy-4-
methoxybenzophenone
 
131-57-7
 
Rats
 
Feed
 
90 days
 
Microarray (Affymetrix)
 
Auerbach
 
Methyleugenol extract
Ginkgo biloba extract***
 
93-15-2
90045-36-6
 
Mice
 
Gavage
 
2 years

 
NextGen sequencing
Exome-Seq

RNA-Seq (Illumina)
 
Pandiri/Auerbach/Merrick
 
2,3-Pentanedione
 
600-14-6
 
Rats
 
Inhalation
 
14 and 28 days
 
microRNA Microarray (Affymetrix)
 
Morgan
 
Polycyclic aromatic compounds:
  • Acenaphthenequinone
  • Benzo[b]fluoranthene
  • Benzo(a)pyrene
  • Dibenz[a,h]anthracene
  • 9-Methylanthracene
  • 1-Methylfluorene
  • Perinaphthenone
  • Phenanthrene
  • Pyrene


82-86-0
205-99-2
50-32-8
53-70-3
779-02-2 1730-37-6
548-39-0
85-01-8
129-00-0
 
Human hepatocyte cell line
 
In vitro exposure
 
48 hours
 
Cytotoxicity
Gene expression by quantitative polymerase chain reaction (qPCR)
 
Rider/ Tokar
 
BDE Toxicogenomics Studies:
  • 2,2’,4,4’,5-Pentabromodiphenyl ethers
  • Pentabromodiphenyl oxide (technical) (DE-71)
  • 3,3,4,4,5-Pentachlorobiphenyl
  • 2,2'4,4'-Tetrabromodiphenyl ether (DE-47)

5436-43-1

32534-81-9
57465-28-8

5436-43-1

Rats, Mice
 
Gavage
 
GD** 6 through 3 weeks
 
Microarray (Affymetrix)
 
Dunnick
 
Tetrabromobisphenol A
 
79-94-7
 
Rats
 
Gavage
 
90 days
 
Microarray (Affymetrix)
 
Dunnick/ Merrick
 
Tetrabromobisphenol A
BDE-47
Pentabromodiphenyl oxide-technical (DE-71)
79-94-7
5436-43-1
32534-81-9
Rats
 
Gavage GD** 6 through 3 weeks
 
Microarray (Affymetrix) Pandiri/Kovi
Tetrabromobisphenol A
Pentabromodiphenyl oxide-technical (DE-71)
Triclosan
alpha, beta-Thujone
79-94-7
32534-81-9
3380-34-5
76231-76-0
Rats Gavage 5 days High throughput transcriptomics Devito/Gwinn
112-Chemical compound test set; pharmaceuticals and environmental compounds N/A Human hepatocyte cell line-HEPARG In vitro exposure 2 days High throughput transcriptomics Ferguson/Ramaiahgari
* Chemical Abstracts Service Registry Number
**GD: gestational day
*** This study will compare toxicogenomic effects among the chemicals listed together.

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