Grantee Research Project Results

Final Report: Liver and the Metabolic Syndrome

EPA Grant Number: R834594C001
Subproject: this is subproject number 001 , established and managed by the Center Director under grant R834594
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Formative Center for the Evaluation of Environmental Impacts on Fetal Development
Center Director: Boekelheide, Kim
Title: Liver and the Metabolic Syndrome
Investigators: Gruppuso, Phillip
Institution: Rhode Island Hospital
EPA Project Officer: Hahn, Intaek
Project Period: December 1, 2009 through November 30, 2012
RFA: Children's Environmental Health and Disease Prevention Research Centers: Formative Centers (with NIEHS) (2009) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health

Objective:

The original goals of this project were to develop a model in which human fetal liver is transplanted into nude rats. We intended to demonstrate that manipulation of the host environment in the adult rat xenograft recipients will induce changes in fetal liver, then go on to characterize the effect of host arsenic exposure, rapamycin administration and dietary restriction on the xenograft epigenome. However, the goals changed significantly by the end of the second year of the project, by which time we had concluded that the fetal liver transplantation model had not succeeded. At that point, we transferred our efforts to the xenotransplantation of human fetal white adipose tissue (WAT).

Summary/Accomplishments (Outputs/Outcomes):

In focusing on WAT, we have made progress on the original overall goal of the application, to assess the effect of environmental factors on the fetus with an emphasis on fetal origins of metabolic syndrome in the offspring. Initial feasibility studies involved the transplantation of WAT derived from perigonadal fat of the rat into SCID beige mice, and WAT from mice into Rowett Nude (RNU) rats. At time points up to 7 days after transplantation, there was a marked and progressive loss of adipocytes. This was associated with the appearance of extracellular fat droplets and hemorrhage. However, at periods from 14 to 28 days, there developed a healthy appearing, growing fat pad. Given that mature adipocytes lack the ability to proliferate, the only potential explanation for this observation was de novo adipogenesis.

Having established the rodent-to-rodent xenotransplant model, we moved on to perform human to SCID-Beige mouse xenotransplantation. WAT tissue from spontaneous fetal loss at 18 to 22 weeks gestation yielded consistent results. At 2 and 8 days, the xenografts had the gross appearance of connective tissue with no obvious WAT at the transplant sites. Small foci of WAT development were present at 14 days. These progressed to obvious fat pads at the longer time points. Histology showed small WAT foci at 14 days with evidence for active adipogenesis. The markedly expanded WAT at 28, 35 and 60 days also contained adipocytes that were extremely heterogeneous in size. There was also evidence in all samples for numerous, small pericytes in close proximity to blood vessels. Immunohistochemical staining using an antibody specific for the human endothelial cell adhesion molecule CD31 demonstrated rich vascularity of the transplanted tissue. At later time points (28 and 35 days), there were xenograft-derived blood vessels in the host renal parenchyma, indicating extensive angiogenesis.

We have gone on to perform extensive characterization of gene expression and immunostaining for markers of preadipocytes, mature adipocytes and angiogenesis. In the aggregate, our studies have established human fetal WAT xenografts as a model for the long term (up to 3 months) modulation of fetal WAT tissue development by manipulation of the host environment. The work establishing and validating this model is in preparation for publication.

We pursued the one aspect of the liver project that was available to us, a comparison of gene expression and DNA methylation in mid-gestation human fetal liver versus adult liver. Triplicate samples of each were used to prepare total RNA and DNA, which were analyzed for gene expression (Affymetrix GeneChip Human Gene 1.0 ST Arrays) and DNA methylation (Illumina Infinium HumanMethylation450 BeadChips). Genes overexpressed in fetal liver were ordered based on fold-differences in expression. We selected 94 genes that were overexpressed by > 3-fold in fetal relative to adult liver and 46 genes that were overexpressed by > 3-fold in adult relative to fetal liver. These genes were submitted for analysis of methylation state by gene region (within 200 or 1500 bp of the transcription start site; within the 5 and 3 UTRs; within the 1st exon and gene body). Results were generated using R software for statistical computing (http://www.r- project.org/). More specifically, we examined the frequency (density) of methylation rates associated with the selected genes. The fetal overexpressed genes showed minimal differences from their adult counterparts in methylation adjacent to the transcription start site and within the 5UTR. However, there were marked differences in 1st exon methylation, with fetal overexpressed genes showing lower methylation in this region. This differed from results with the genes overexpressed in adult liver. For these genes, there appeared to be a reduction in methylation in the transcription start site, 5UTR and 3UTR regions. These findings support the novel observation that 1st exon methylation is functionally associated with fetal, but not adult, hepatic gene expression. This work will soon be in preparation for publication. We will extend this same approach to analysis of fetal and adult WAT and to the WAT xenografts at extended time points.

Conclusions:

Despite an extensive effort and multiple transplant trials in various immunodeficient rodent hosts, human fetal liver xenotransplants consistently led to a rapid loss of hepatocytes and the selective development within the xenografts of maturing biliary duct structures. Since the goal of Research Project 1 was to study toxicant- induced metabolic syndrome, institutional funds were used to explore xenotransplantation of human fetal adipose tissue as a model to study adipogenesis and obesogens. This approach has been successful, and has led Dr. Gruppuso to seek independent R01 funding to support a research program focused on human adipogenesis and the molecular response to the potential obesogens.

Supplemental Keywords:

In utero exposure, mammalian, metals, bisphenol A, perinatal programming, bioavailability, exposure assessment, biochemical research, intrauterine exposure, developmental effects, perinatal exposure, children's health, biological pathways, Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, Health Risk Assessment, Risk Assessments, Biology, Risk Assessment, bioavailability, fetal exposure, perinatal exposure, biological pathways, children's health, developmental effects, biochemical research

Progress and Final Reports:

Original Abstract

2010

2011

Main Center Abstract and Reports:

R834594 Formative Center for the Evaluation of Environmental Impacts on Fetal Development

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R834594C001 Liver and the Metabolic Syndrome
R834594C002 Prostate and Endocrine Disruption
R834594C003 Lung, Arsenic Exposure, and Tissue Remodeling

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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.