Aging is characterized by loss of functional reserve, placing the elderly at increased risk of numerous diseases. Identifying the driving force behind this functional loss is essential for maintaining a healthy populace. Recent evidence from our lab and others implicates DNA damage as a cause of aging. This implies a strong environmental component to aging. The long term objective of this research is to understand the molecular mechanism by which DNA damage promotes aging. This coupled with identifying environmental causes of genotoxic stress will greatly facilitate prevention of age-associated diseases. ERCC1-XPF is an endonuclease required for repair of bulky monoadducts via nucleotide excision repair (NER) and DNA interstrand crosslinks (ICLs) via a distinct mechanism. Deletion of ERCC1-XPF in the mouse causes early onset aging. These mice therefore offer a unique, rapid and sensitive model system for discovering which genotoxins promote aging and how they do so. The phenotype of the Erccl mice cannot be attributed to loss of NER. Thus our working hypothesis is that rapid aging in ERCC1-XPF deficient mice is caused by their inability to repair ICLs and therefore a consequence of endogenous ICLs which are cytotoxic. To test this, the investigators engineered mice hypomorphic for ERCC1-XPF which age over the course of months, permitting interventional studies. These mice will be exposed to DNA crosslinking drugs and environmental agents that promote lipid peroxidation (LPO), a likely source of endogenous ICLs, to determine if these exposures exacerbate the progeroid symptoms of the mice. The investigators discovered a human progeria caused by mutation of XPF. Thus identifying the cause of rapid aging in ERCC1-XPF-deficient mice will have direct implications for human health.
The specific aims of this project are:
Aim I : To define the cellular response of ERCCl-XPF-deficient cells to DNA ICLs and LPO. ERCCl-XPF-deficient cells will be exposed to 8-MOP or angelicin, plant-derived psoralens. Photoactivation of 8-MOP induces ICLs and monoadducts, whereas angelicin produces only monoadducts. Cell survival, cellular senescence, apoptosis, mutation frequency and chromosomal aberrations will be measured. If our hypothesis is correct then, 8-MOP will be significantly more cytotoxic than angelicin under conditions where an equal number of DNA lesions are induced. ERCCl-XPF-deficient cells will also be exposed to cadmium, an environmental agent that promotes LPO, to determine if LPO elicits the same cellular response as ICLs.
Aim II : To directly test the hypothesis that unrepaired DNA ICLs promote aging. ERCC1-XPF hypomorphic mice will be chronically exposed to the crosslinking agent mechlorethamine. A second cohort will be exposed to 2-chloroethylamine (which induces structurally related monoadducts but not ICLs) using a dose that induces the same number of lesions as mechlorethamine. If our hypothesis is correct, mechlorethamine, but not 2-chloroethylamine, will exacerbate the progeria in these mice. Results will be confirmed by comparing skin aging in response to topical 8-MOP versus angelicin plus UV-A in mice genetically deleted for ERCC1-XPF in the skin only.
Aim III : To determine if lipid peroxidation (LPO) promotes aging in mice with defective ICL repair. LPO is caused by oxygen radical damage to membranes and yields products able to crosslink DNA. We hypothesize that LPO is a source of ICLs that contribute to the phenotype of the Erccl mice. LPO will be induced in ERCCl-XPF-deficient mice via exposure to CCL4 or cadmium. If our hypothesis is correct, Erccl mice will be hypersensitive to LPO compared to wild type mice and LPO will exacerbate their progeria. Results from these experiments will indicate if LPO promotes aging and if so, whether it does so by inducing DNA damage. These experiments will also reveal if two common industrial exposures promote aging.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Special Emphasis Panel (ZES1-JAB-C (R1))
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Mcallister, Kimberly A
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University of Pittsburgh
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