2002 Progress Report: Molecular Characterization of a Biological Threshold in Developmental Toxicity

EPA Grant Number: R827445
Title: Molecular Characterization of a Biological Threshold in Developmental Toxicity
Investigators: Knudsen, Thomas B. , Charlap, Jeffrey H. , Craig, Robert C. , Nibbio, B. J.
Current Investigators: Knudsen, Thomas B.
Institution: Jefferson Medical College
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1999 through September 30, 2002 (Extended to September 30, 2003)
Project Period Covered by this Report: October 1, 2001 through September 30, 2002
Project Amount: $207,170
RFA: Children's Vulnerability to Toxic Substances in the Environment (1999) RFA Text |  Recipients Lists
Research Category: Children's Health , Health Effects , Human Health , Health


The objective of this research project is to characterize, in molecular terms, the biological basis of a threshold in developmental toxicity and place this into the context of a quantitative dose-response model for risk assessment. In our research, we have undertaken a large-scale analysis of gene expression in the early mouse embryo using cDNA microarrays, with the goal of uncovering functional relationships between gene expression and developmental toxicity. The research prototype is 2-chloro-2'-deoxyadenosine (2CdA), an ocular teratogen in mouse embryos exposed on day 8 of gestation. Susceptibility to 2CdA-induced microphthalmia is dependent on the p53 tumor suppressor genotype of the embryo. Microarray analysis was used to address changes in gene expression under three experimental contexts: (1) dosages across the threshold for p53 protein induction and developmental toxicity; (2) trajectories across the critical period of p53 protein induction; and (3) effects sensitive to mitochondrial benzodiazepine receptor (Bzrp) ligands that alter the physiological state of the embryo to suppress the p53 response.

Progress Summary:

To date, this project has 42 microarray hybridizations of RNA from headfold-stage mouse embryos collected under 21 different experimental conditions. Ten to 20 headfolds were pooled for each sample. Quality assurance of RNA was determined by an absorbance A260/280 ratio of >1.7, 1 percent agarose gel electrophoresis, and assessment was determined by RNA 6000 Nano microchip electrophoresis on the Agilent 2100 Bioanalyzer. RNA (1 microgram) was converted to cDNA with incorporation of biotin-11-dCTP, or fluorescein-11-dCTP, and approximately 15 ng labeled cDNA from each sample was hybridized competitively to the MPS621 microarray probe (PerkinElmer Life Sciences). Histochemical detection used peroxidase-conjugated anti-fluorescein and cyanine-3-tyramide (FL-Cy3) reaction followed by peroxidase-conjugated streptavidin and cyanine-5-tyramide (BN-Cy5) reaction. Each sample was repeated in the reversed labeling scenario. The log-intensity ratio was normalized with locally weighted regression, standardized using absolute deviation of data points from their mean, and equalized between paired swaps for each gene in the matrix. Data reduction and visualization used GeneSpring software (v 5.2, Silicon Genetics, Redwood City, CA).

Biochip Performance. Of the 2,400 genes (4,800 elements) spotted on the MPS621 biochip, 2,338 genes yielded signals above the detection threshold (500 median absolute signal). When reverse transcription-polymerase chain reaction (RT-PCR) was applied to a panel of 50 target genes that were positive by microarray, it amplified the correct size PCR band in 42 cases (84 percent success rate). The success rate in end-point PCR varied with intensity of the microarray signal reflecting relative transcript abundance, sequence homology of cDNA elements, cellular heterogeneity, and labeling efficiencies. This end-point PCR panel reproduced the qualitative effect of the microarray in 32 of 50 target genes (64 percent success rate). A representative sample of 232 elements from the DNA biochip produced a 79.8 percent average sequence homology between human-mouse (CI = 77.0-82.7 percent). Therefore, genes representing the lower quartile (190-1123 median absolute signal) were deemed unreliable and were filtered from the microarray data set.

Dose Response. Samples collected during Year 1 of the project focused on the dose response characteristics at 3.0 hours post-exposure. Benchmark Dose Software (BMDS) predicts the threshold for microphthalmia occurring at 2.0 mg/kg 2CdA. This dose represents the lower 95 percent confidence limit for modeled dose associated with an increased 5 percent risk for the defect (BMD5), and is intermediate between the no observable adverse effect level (1.5 mg/kg) and the BMD5 (2.5 mg/kg) modeled from the dose response curve. We identified 182 genes changing by at least 1.5-fold in the 2CdA dose response (0.625 to 5.0 mg/kg) and partitioned them by K-means clustering (see Figure 1). This showed a segmental response of the transcriptome, which seemed to flicker at intermediate levels and burn above the threshold dose for microphthalmia; however, somewhat unexpectantly, the transcripts could be grouped to those spiking at the threshold dose for developmental toxicity (2.5 mg/kg) versus those altered only at the overtly teratogenic dose (5.0 mg/kg). This implies a novel cellular regulation invoked as the toxicant exposure crosses the threshold for developmental toxicity.

Figure 1. K-means Clustering of 182 Genes That Changed 1.5-Fold in the Mouse Embryo at 3.0 Hours After Intrauterine Exposure to 2CdA on Day 8 of Gestation. Independent replicate analysis was performed on total RNA labeled with BN-dCTP or FL-dCTP and Cy5-tyramide or Cy3-tyramide signal amplification, respectively. Each segmental dose represents a true replicate sample with reversal of the label-dye assignments. Doses were 0.625, 1.25, 2.5, and 5.0 mg/kg. The threshold (BMDL) is modeled at 2.0 mg/kg (arrows).

Time Course. Samples collected during Year 2 of the project focused on the trajectories of gene expression at critical time points after exposure to 2.5 mg/kg 2CdA. Dams co-treated with 4.0 mg/kg PK11195 provided parallel samples of embryos, in which p53 protein induction is delayed or blocked by the Bzrp ligand. The test period was 3.0 to 6.0 hours post-exposure to encompass events on either side of p53 protein induction at 4.5 hours. We identified 180 genes sensitive to 2CdA and/or PK11195 co-treatment that fell into three principal behaviors by K-means clustering (see Figure 2). The 2CdA time course showed minor phasing of gene trajectories that was clearly promoted by PK11195 co-treatment. The effect of PK11195 on trajectories of gene expression was most pronounced at 3.0 hours post-exposure. Thus, PK11195 seemed to alter the regulation of the embryonic transcriptome during a period of time that ordinarily would signal p53 protein induction.

Figure 2. K-means Clustering of 180 Genes That Changed in the Mouse Embryonic Headfold After Intrauterine Exposure to 2.5 mg/kg 2CdA Alone or in Combination With 4.0 mg/kg PK11195 Co-Treatment on Day 8 of Gestation. Each time interval represents a true replicate sample with reversal of the label-dye assignments. Times were 3.0, 4.5, and 6.0 hours after treatment. The critical time based on p53 protein induction is 4.5 hours post-exposure (arrows).

Specificity of Intervention. Studies during Year 3 of the project provided microarray data comparing PK11195 with another well-studied Bzrp ligand, Ro5-4864. These ligands have different physiological activities at the Bzrp. RNA was collected from the headfold of early mouse embryos 4.5 hours after exposure to PK11195 (4.0 mg/kg), Ro5-4864 (4.0 mg/kg), or Ro5-4864 with 2CdA (2.5 mg/kg) on day 8 of gestation. Microarray hybridization and data analysis revealed that Ro5-4864 effected some genes; however, it did not substantially influence the patterns associated with the 180 genes in the 2CdA time course. Teratological evaluation showed Ro5-4864 and PK11195 differ substantially from one another with respect to three activities on the embryo: PK11195 blocked 2CdA-induced p53 protein accumulation, whereas Ro5-4864 did not; PK11195 suppressed 2CdA-induced microphthalmia, whereas Ro5-4864 did not; and Ro5-4864 had weak teratogenic activity at 4.0 mg/kg, whereas PK11195 did not. Microarray findings indicate that PK11195 and Ro5-4864 elicit different physiological responses by the embryonic transcriptome, consistent with the differential activities of these Bzrp ligands on teratological end-points.

An interesting finding from bioinformatic analysis pertained to the chromosomal linkage of many affected genes. Preliminary analysis mapped several syntenic hotspots with some ocular disorders at these regions. Many of the hotspots harbored reference to metabolic disorders of the biotin-coenzyme A cycle. Biotin is an essential water-soluble vitamin used as a prosthetic group in enzymes involved in coenzyme-A based carboxyl-group transfers. End-point PCR analysis revealed a specific effect of 2CdA exposure on genes coding for biotin-dependent carboxylation reactions in the mitochondrion (see Figure 3).

Figure 3. PATHWAY: Biotin-Dependent Carboxyl Group Transfers (Mitochondrial)

Future Activities:

We will continue to use bioinformatics and computational biology to identify potential linkages.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other project views: All 14 publications 2 publications in selected types All 1 journal articles
Type Citation Project Document Sources
Journal Article Charlap JH, Donahue RJ, Knudsen TB. Exposure-disease continuum for 2-chloro-2'-deoxyadenosine, a prototype ocular teratogen. 3. Intervention with PK11195. Birth Defects Research (Part A): Clinical and Molecular Teratology 2003;67(2):108-115. R827445 (2002)
R827445 (Final)
  • Abstract from PubMed
  • Full-text: InterScience Full Text
  • Other: InterScience PDF
  • Supplemental Keywords:

    risk assessment, health effects, teratogen, genetics, microarray., RFA, Health, Scientific Discipline, Toxicology, Health Risk Assessment, Susceptibility/Sensitive Population/Genetic Susceptibility, Children's Health, genetic susceptability, Molecular Biology/Genetics, biological threshold, birth defects, health effects, sensitive populations, developmental toxicity, dose response model, molecular characterization, exposure, human malformation, children, assessment of exposure, children's vulnerablity, dysmorphogenesis, functional assys, growth & development, biomedical research, developmental disorders, environmental hazard exposures

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    Progress and Final Reports:

    Original Abstract
  • 2000 Progress Report
  • 2001 Progress Report
  • Final Report