Abstract
Summary Statement
DNA damage induced by systemic drugs or local γ-irradiation drives disc degeneration and DNA repair ability is extremely important to help prevent bad effects of genotoxins (DNA damage inducing agents) on disc.
Introduction
DNA damage (genotoxic stress) and deficiency of intracellular DNA repair mechanisms strongly contribute to biological aging. Moreover, aging is a primary risk factor for loss of disc matrix proteoglycan (PG) and intervertebral disc degeneration (IDD). Indeed, our previous evidences in DNA repair deficient Ercc1−/Δ mouse model strongly suggest that systemic aging and IDD correlate with nuclear DNA damage. Thus the aim of the current study was to test whether systemic or local (spine) genotoxic stress can induce disc degeneration and how DNA repair ability could help prevent negative effects of DNA damage on IDD. To test this hypothesis a total of twelve Ercc1−/Δ mice (DNA repair deficient) and twelve wild-type mice (DNA repair competent) were challenged with two separate genotoxins to induce DNA damage, i.e. chemotherapeutic crosslinking agent mechlorethamine (MEC) and whole-body gamma irradiation. Local effects of gamma irradiation were also tested in six wild-type mice.
Methods
Ercc1 −/Δ mice (n=6) and their wild-type littermates were chronically exposed to genotoxic stress beginning at 8 wks of age by subcutaneous administration of a subtoxic dose of MEC (8 μg/kg once per week for 6 weeks). Similarly, six Ercc1−/Δ mice and their wild-type littermates were exposed to genotoxic stress by whole-body administration of ∼10% radiotherapeutic dose of ionizing radiation (0.5 Gy 1x per week for 10 weeks). A third set of wild-type mice (n=6) were exposed to one shot local spine irradiation at 0, 6, and 10 Gy at 22 weeks old and sacrificed 10 weeks later. Histological staining for proteoglycan (Safranin O) and collagen (Masson's Trichrome), PG synthesis (35S-sulfate incorporation) and GAG content (DMMB assay), disc ADAMTS4, aggrecan and its fragments terminating in NITEGE-373 (immunohistochemistry (IHC)) were analyzed. Cellular senescence markers (p16) and apoptosis (TUNEL assay) were also measured.
Results
Histological staining revealed substantial reduction in matrix collagen, proteoglycan, and endplate cellularity in the discs of MEC-exposed and irradiated mice. IHC analysis showed decreased aggrecan and increased levels of ADAMTS4 and NITEGE-373 containing aggrecan proteolytic fragments. Disc PG synthesis was reduced 2–3 folds in MEC-treated mice and irradiated mice. Locally irradiated mice showed similar effects on disc matrix. Expression of p16 as well as apoptosis significantly increased in MEC-treated and irradiated mice. The overall effect of the treatments on disc matrix and endplate cartilage was more severe in Ercc1−/Δ mice than wild-type mice.
Discussion/Conclusion
MEC and IR treatment resulted in loss of disc matrix proteoglycan and collagen in adult wild-type and Ercc1−/Δ mice. The finding that loss of matrix proteoglycan was greater in the DNA repair deficient mice strongly supports the conclusion that DNA damage can drive disc degeneration and DNA repair ability is extremely important to help prevent these effects. Results of this work suggest that patients treated with genotoxic drugs (i.e. long-term cancer survivors) may be at increased risk of IDD.
