Grantee Research Project Results

Final Report: Lung, Arsenic Exposure, and Tissue Remodeling

EPA Grant Number: R834594C003
Subproject: this is subproject number 003 , 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: Lung, Arsenic Exposure, and Tissue Remodeling
Investigators: Depaepe, Monique
Institution: Women & Infants Hospital of Rhode Island
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:

This project was guided by the hypothesis that arsenic exposure of human fetal lung xenotransplants results in disrupted postglandular lung remodeling mediated by molecular and epigenetic alterations.

The Specific Aims of the original application were:

To develop a human fetal lung xenograft model of in utero arsenic exposure.
To determine the effects of arsenic exposure on growth dynamics and gene expression in the developing human lung.
To determine arsenic-induced epigenetic changes in the developing human lung.

Summary/Accomplishments (Outputs/Outcomes):

Significant Results (Positive and Negative):

1. Optimization and validation of the human fetal lung xenograft model

We optimized and validated the human fetal lung xenograft model (Specific Aim 1). Human fetal lung tissues, derived from 12-22 weeks gestation stillbirths (n = 12), were implanted below the kidney capsule and/or subcutaneously in immune-suppressed mice or rats. The animals were sacrificed and grafts dissected at 2 and 4 weeks post-transplantation. Lung grafts were preserved in RNAlater for molecular genetic/epigenetic studies and formalin-fixed for morphologic studies.

Results

According to the original protocol, immune-suppressed nude rats were used as recipients for the first 11 human lung harvests (n = 38 recipient rats). Using this recipient model, varying degrees of inflammatory response was detected in the xenografts, in some cases replicating the allograft rejection reaction ("bronchiolitis obliterans") seen in human transplant lungs. This host-versus-graft immune reaction had variable effects on graft growth and vascularization, which might confound interpretation of the effects of arsenic exposure on such lungs. For this reason, we transitioned to the SCID-beige immune-suppressed mouse as recipient model from the ninth human lung harvest onward (n = 21 recipient mice). In contrast to the nude rat, the SCID-beige mouse does not contain natural killer cells, and is thus more profoundly immune suppressed. Following adoption of the SCID-beige mouse as model, host-derived inflammation in the grafts was virtually absent, and graft growth and vascularization were significantly enhanced.
In the original experiments, fetal lung tissues were implanted in renal subcapsular as well as subcutaneous sites. While the subcutaneous grafts showed higher levels of lung growth and proliferation, they displayed a non-physiologic type of lung growth, characterized by an abnormal dissociation of epithelial and vascular growth (epithelial overgrowth).

Conclusion:

Renal subcapsular grafts in SCID-beige mice provide the best model of normal postcanalicular human fetal lung development and were used in subsequent experiments.
Fetal lung tissues derived from stillbirths show unanticipated resilience and are capable of restoring prolifetaive activity even when obtained up to 48 hours after death (potential implications for organ transplantation/pulmonary regenerative medicine).

2. Analysis of effects of arsenic exposure on growth dynamics, gene expression and epigenetic changes in the developing lung

Following optimization and validation of the fetal lung xenograft model, studies were performed to determine the effects of arsenic exposure on growth dynamics and gene expression in the developing human lung (Specific Aim 2) as well as epigenetic changes (DNA methylation) (Specific Aim 3). Animals were exposed to 10 or 100 ppb inorganic arsenic as sodium arsenite, added to drinking water, for 2 or 4 weeks post-transplantation. Controls received arsenic-free water. Arsenic levels were determined in host tail at 4 weeks post-transplantation (Dartmouth). Lung grafts were preserved in RNAlater for gene expression (microarray and real-time RT-PCR) or DNA methylation studies and formalin-fixed for morphologic studies.

Results

Arsenic exposure was well tolerated and did not lead to weight loss or other systemic effects.
Exposure to 100 ppb sodium arsenite resulted in significantly increased tissue (tail) arsenic levels in host mice, indicating arsenic addition to drinking water at time of xenografts provides effective delivery.
Histopathologic analysis revealed architectural maturation in arsenic-exposed and control xenografts. Qualitatively, and based on the first arsenic experiments only, the airspaces of arsenic-exposed grafts appeared enlarged and simplified compared with those of controls. In addition, the microvasculature of arsenic-exposed lungs appeared more tortuous than that of controls. Morphometric studies of alveolar and microvascular development of the xenografts as well as proliferation analyses are pending.
Gene expression studies were initially performed by Affymetrix microarray analysis on three control and four arsenic-exposed xenografts (post-transplantation week 4). Various differentially regulated genes were identified, including the angiogenesis regulators ANGPT2, ESM1 (endocan) and LYVE1. Upregulation of ANGPT2 and ESM1 in arsenic-exposed lungs was confirmed by real-time RT-PCR analysis. Additional larger Affymetrix microarray analyses failed to confirm significant differential expression of these or other genes. Prominent batch effect was noted (inter-graft variability, likely attributable to clinical conditions). Pathway analyses are in progress. Similarly, DNA methylation studies failed to reveal significant differences in DNA methylation between grafts exposed to 100 ppb arsenic and controls.

Conclusion:

Exposure of human fetal lung xenografts to 100 ppb arsenic appears to result in morphologic alterations (including the microvasculature).
Arsenic exposure, at the doses studied and with the sample sizes available, did not have significant effects on gene expression or DNA methylation.
Of note, microvascular growth patterns noted in some grafts were reminiscent of the dysangiogenesis characteristic of new bronchopulmonary dysplasia (chronic lung disease of preterm newborn), suggesting the xenografts may serve as model of this disease.

Conclusions:

Pilot project 3 established the human fetal lung xenograft as an invaluable model system to study the effects of antenatal toxicant exposure on postcanalicular fetal lung development. In a first set of studies, it was determined that human fetal lung tissues retain the capacity to proliferate and differentiate into functional lung tissue, even when harvested and transplanted more than 24 hours after spontaneous miscarriage. This unanticipated degree of resilience of human fetal lung tissue supported the use of fetal lungs from stillbirths (as opposed to lungs obtained from induced abortions) for xenotransplant purposes. In a second set of experiments, it was demonstrated that the human fetal lung xenograft, especially in renal subcapsular position and in severely immune suppressed rodent hosts, faithfully replicates not only epithelial, but also microvascular postcanalicular development in situ. After optimization of the human fetal lung xenograft model in these preliminary studies, experiments involving exposure of human fetal lung xenografts to clinically relevant levels of arsenic (0-10-100 ppb) were initiated.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Publications Views
Other subproject views:	All 2 publications	2 publications in selected types	All 2 journal articles
Other center views:	All 13 publications	8 publications in selected types	All 8 journal articles

Publications
Type	Citation	Sub Project	Document Sources
Journal Article	De Paepe ME, Chu S, Hall S, Heger NE, Thanos C, Mao Q. The human fetal lung xenograft: validation as model of microvascular remodeling in the postglandular lung. Pediatric Pulmonology 2012;47(12):1192-1203.	R834594 (2012) R834594 (Final) R834594C003 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Abstract: Wiley-Abstract Exit
Journal Article	De Paepe ME, Chu S, Heger N, Hall S, Mao Q. Resilience of the human fetal lung following stillbirth: potential relevance for pulmonary regenerative medicine. Experimental Lung Research 2012;38(1):43-54.	R834594 (2012) R834594 (Final) R834594C003 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Abstract: Taylor & Francis-Abstract Exit

Supplemental Keywords:

In utero exposure, mammalian, metals, bisphenol A, developmental biology, 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, perinatal exposure, fetal exposure, children's health, biological pathways, developmental effects, exposure assessment

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.