Grantee Research Project Results

Final Report: Analysis of Genotoxic Biomarkers in Children Associated with a Pediatric Cancer Cluster and Exposure to Two Superfund Sites

EPA Grant Number: CR830757
Title: Analysis of Genotoxic Biomarkers in Children Associated with a Pediatric Cancer Cluster and Exposure to Two Superfund Sites
Investigators: Finette, Barry A.
Institution: University of Vermont
EPA Project Officer: Aja, Hayley
Project Period: March 1, 2003 through February 28, 2006 (Extended to December 31, 2007)
Project Amount: $775,141
RFA: Children's Vulnerability to Toxic Substances in the Environment (2002) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health

Objective:

The objective of this study was to evaluate the utility of specific biomarkers of effect and susceptibility for studying cancer risk in children following genotoxic exposures. We determined if children from an exposed population with elevated cancer incidence have an increase in chromosome aberrations or changes in HPRT mutational spectrum, such as: 1) an increase in frameshift mutations reflective of exposure to anthraquinone-based dyes and styrene-acrylonitrile trimers; 2) an increase in point mutations reflective of exposure to benzidine-based dyes, epichlorohydrin and trichloropropane; and 3) an increase in V(D)J recombinase mediated deletions reflective of exposure to aromatic hydrocarbons. We also determined if specific DNA polymorphisms in 18 carcinogen metabolizing and DNA repair enzymes were associated with increased mutagenic susceptibility to genotoxic exposure. Approach: We analyzed biomarkers of effect and susceptibility in exposed siblings of children in a CDC defined pediatric cancer cluster that has been linked to transplacental and childhood exposure to contaminated groundwater from two EPA designated superfund sites in Dover Township, NJ. Exposure studies focused on the siblings of children with cancer rather than the children with cancer because of the genotoxic effects of cancer treatment. However, biomarkers of susceptibility were also determined in children with cancer. Biomarkers of effect (chromosomal aberrations and HPRT mutations) in the exposed siblings were compared to measurements in unexposed children from neighboring communities. Biomarkers of susceptibility (DNA polymorphisms for carcinogen metabolizing enzymes) in the exposed siblings and unexposed children were compared to children with cancer to determine if the latter have a different prevalence of specific metabolic genotypes. In addition, the relationships between biomarkers of effect and susceptibility in exposed siblings and unexposed children were examined to see if the effects of exposure are modified by any of these metabolic polymorphisms. Exposures in all subjects will be evaluated from their residential and personal histories, using a computer model developed by the ATSDR to estimate exposure to different water sources over time.

Summary/Accomplishments (Outputs/Outcomes):

We completed and published our HPRT cloning assay analysis that measured the frequency of somatic mutations in 49 exposed siblings and 43 age/gender matched unexposed children (Vacek et. Al. Environ Mol Mutagen. 2005;45(4):339-45). These studies demonstrated that the frequency of somatic mutations (Mfs) in peripheral T cells from these children were not significantly different regardless of whether results were adjusted or unadjusted for age and CE (Residual lnMf). The observation that HPRT lnMf and residual lnMf were not significantly different does not preclude that significant genotoxic differences exist at the genomic level that would be reflected in a change in the mutational spectrum.

We performed a double-blinded mutation spectra analysis on 394 HPRT mutant T cells, 224 from exposed children and 170 from unexposed children. We identified at the cDNA and genomic level 338 (85.7%) unique mutation events, 192 from exposed children (85.7%) and 146 from unexposed children (85.8%). Our analysis revealed no significant difference in the classes of mutations from exposed and unexposed subjects. The analysis did reveal significant differences between two subclasses of mutations from exposed and unexposed subjects, specifically AT to GC transitions and GC to CG transversions (p=0.05). We also observed that genotypes for a number of polymorphisms were associated with specific types of mutations and that there were also several significant interactions between genotype and exposure. We also determined the incidence of mutant clone proliferation in these subjects by examining whether 2 or more mutant clones had identical HPRT mutations. The emergence of clonal proliferation is important since increased cell division/proliferation is itself a mutagenic mechanism and one of the phenotypic characteristics of malignant transformation. We observed that unexposed children had a significantly higher incidence of mutant clonal proliferation (30%) than exposed subjects (8%). The reasons for these observations are unclear but could be the cellular effects of chronic environmental exposures in the exposed subjects.

We also preformed double-blind FISH analysis for determining the frequency of chromosome aberrations (CA) on metaphase spreads generated from 81/92 (88%) of subjects, 44/49 (90%) from exposed subjects and 37/43 (86%) from unexposed subjects. In exposed subjects we analyzed an average of 2217 metaphase spreads (range 525-3890) and observed 230 aberrant cells (mean of 5.22 per subject) containing an average of 10.1 aberrations per subject. This corresponded to a mean aberration frequency of 0.47 and a mean genome aberration frequency of 1.11 in the exposed subjects. In the unexposed subjects we analyzed an average of 2818 metaphase spreads (range 471-5741) and observed 236 aberrant cells (mean of 6.38 per subject) containing an average of 13.3 aberrations per subject. This corresponds to a mean aberration frequency of 0.51 and a mean genome aberration frequency of 1.23. Chromosomal aberrations in the exposed and unexposed subjects were compared by T-test. The only significant difference was in the number of translocations per 1000 cells, which was lower in the exposed than in the unexposed subjects (0.38 vs. 0.84; p=0.024). There were no significant differences with respect to the other measures of chromosomal aberrations: percent of cells with aberrations, reciprocal translocations per 1000 cells, fragmentations per 1000 cells, insertions per 1000 cells, aberrations per 100 cells and genomic aberration frequency.

We determined the prevalence of 37 polymorphisms in 18 genes associated with susceptibility to cancer and with a potential role in metabolizing known contaminants found in the Toms River aquifer. Genotype analysis was performed on 32 subjects with cancer and 49 siblings all of which were exposed to contaminated ground water. In addition, we also performed genotype analysis on 43 control subjects from neighboring regions of Dover Township and 32 subjects with cancer from a previously studied cohort from Vermont for which there was no evidence of exposure to contaminated ground water. We initially examined a subset of nineteen polymorphisms in the four study groups: The genotype distributions differed among the groups for the following polymorphisms ABCB1 (P = 0.041) and CYP3A4B (P = 0.024). The “t/t” genotype of ABCB1 was higher in the exposed siblings (30.6%) compared to the other groups (12.5%-16.3%). The “a/a” genotype of CYP3A4B was lower in the unexposed controls (81.4%) compared to the other groups (90.6%-100%). Logistic regressions to assess associations between genotype and cancer after adjustment for exposure, as well as potential gene-exposure interactions revealed an increasing prevalence of the “c” allele for the EPHX genotype, the “c” allele for the ABCB1 genotype, “a” allele of the EPHX1B genotype. A significant genotype by exposure interaction was also observed for ABCB1, ABCB1B, GSTP1B, and APEX1. For ABCB1 the “t” allele was associated with cancer in the unexposed subjects, while the “c” allele was associated with cancer in the exposed subjects. For ABCB1B cancer was associated with the “t” allele in the unexposed subjects and the “g” allele in exposed subjects. For GSTP1B cancer was associated with the “t” allele in the unexposed and the “c” allele in the exposed subjects. For APEX1 cancer was associated with the “g” allele in the unexposed and the “c” allele in the exposed subjects.

An additional two-way analysis of variance was preformed to assess the effect of genotype on HPRT Mf in exposed siblings and unexposed subjects, which revealed a significant group by genotype interaction for GSTT1. In the exposed siblings there were no differences in lnMf between GSTT1 genotypes but in the controls lnMf was higher in subjects with the +/+ genotype than in those with the -/+ and -/+ genotypes. We also performed a two-way analysis of variance to assess the effect of genotype on chromosomal aberrations in exposed siblings and unexposed subjects, as well as to test for interactions between the effects of genotype and exposure. For GSTP1A, the “g/g” genotype was significantly associated with more translocations per 1000 than the “a/a” and “g/g” genotypes, but the effect was more pronounced in the control subjects (p = 0.014 for the gene-exposure interaction). No overall effect of genotype was observed for any the other genes that were examined, but there were some significant interactions between genotype and exposure.

Conclusions:

Future activities will include completion and submission of 3 additional manuscripts on the mutation spectra/clonal proliferation studies; the chromosomal aberrations analysis and the genotype analysis for metabolic, DNA repair and transport proteins. We will also be submitting abstracts to present these data to national meetings over the next year. In addition we are still planning to compare these results with the ATSDR Historical Reconstruction of the Water-Distribution System Serving the Dover Township Area, New Jersey.

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

Publications Views
Other project views:	All 12 publications	1 publications in selected types	All 1 journal articles

Publications
Type	Citation	Project	Document Sources
Journal Article	Vacek PM, Messier T, Rivers J, Sullivan L, O'Neill JP, Finette BA. Somatic mutant frequency at the HPRT locus in children associated with a pediatric cancer cluster linked to exposure to two Superfund sites. Environmental and Molecular Mutagenesis 2005;45(4):339-345.	CR830757 (2004) CR830757 (2005) CR830757 (2006) CR830757 (Final)	Abstract from PubMed

Supplemental Keywords:

biomarkers, drinking water, risk assessment, human health, infants, children, genetic pre-disposition, hydro-geology, northeast, industry, vulnerability, sensitive population, genetics, Health, RFA, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, Susceptibility/Sensitive Population/Genetic Susceptibility, Health Risk Assessment, Risk Assessments, genetic susceptability, Children's Health, Risk Assessment, Human Health Risk Assessment, computer models, environmental hazard exposures, sensitive populations, biological markers, neurotoxic, neurodevelopmental toxicity, genotoxic biomarkers, human exposure, childhood cancer, contaminated groundwater, developmental effects, neurobehavioral effects, residential populations, children, children's vulnerablity, toxics, assessment of exposure, developmental neurotoxicology, biomarkers

Progress and Final Reports:

Original Abstract

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.