Investigating the role of DNA damage in tobacco smoking-induced spine degeneration

Abstract

Background context: Tobacco smoking is a key risk factor for spine degeneration. However, the underlying mechanism by which smoking induces degeneration is not known. Recent studies implicate DNA damage as a cause of spine and intervertebral disc degeneration. Because tobacco smoke contains many genotoxins, we hypothesized that tobacco smoking promotes spine degeneration by inducing cellular DNA damage.

Purpose: To determine if DNA damage plays a causal role in smoking-induced spine degeneration.

Study design: To compare the effect of chronic tobacco smoke inhalation on intervertebral disc and vertebral bone in normal and DNA repair-deficient mice to determine the contribution of DNA damage to degenerative changes.

Methods: Two-month-old wild-type (C57BL/6) and DNA repair-deficient Ercc1(-/Δ) mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 7 weeks) to model first-hand smoking in humans. Total disc proteoglycan (PG) content (1,9-dimethylmethylene blue assay), PG synthesis ((35)S-sulfate incorporation assay), aggrecan proteolysis (immunoblotting analysis), and vertebral bone morphology (microcomputed tomography) were measured.

Results: Exposure of wild-type mice to tobacco smoke led to a 19% increase in vertebral porosity and a 61% decrease in trabecular bone volume. Intervertebral discs of smoke-exposed animals also showed a 2.6-fold decrease in GAG content and an 8.1-fold decrease in new PG synthesis. These smoking-induced degenerative changes were similar but not worse in Ercc1(-/Δ) mice.

Conclusions: Short-term exposure to high levels of primary tobacco smoke inhalation promotes degeneration of vertebral bone and discs. Disc degeneration is primarily driven by reduced synthesis of proteoglycans needed for vertebral cushioning. Degeneration was not exacerbated in congenic DNA repair-deficient mice, indicating that DNA damage per se does not have a significant causal role in driving smoke-induced spine degeneration.

Keywords: Aggrecan; DNA damage repair; Intervertebral disc degeneration; Matrix metalloproteinases; Matrix proteoglycans; Tobacco smoking.

Fig 1. Cigarette smoke exposure decreased disc matrix proteoglycan

A, safranin O histological staining of disc for matrix proteoglycan in smoke-exposed mice and unexposed controls. Decreased safranin O staining of PG (red stain) in the NP was observed in smoke-exposed mice compared to unexposed mice. B, DMMB assay for total GAG content in NP tissue of smoke-exposed mice and untreated controls. NS, nonsmokers. S, smokers. * p<0.05. Average values from three mice each measured in duplicate are shown with one standard deviation.

Fig 2. Tobacco smoke exposure decreased new matrix protein synthesis in intervertebral disc

PG synthesis was measured by ³⁵S-sulfate incorporation. Average values from three mice each measured in duplicate are shown with one standard deviation. * p<0.05.

Fig. 3. Effects of cigarette smoke exposure on disc aggrecan proteolysis

(A) A schematic representation of mouse PG aggregate consisting of the core aggrecan protein bound to hyaluronan via a linker protein. The MMP-mediated cleavage site (yielding VDIPEN neo-epitope) and ADAMTS-mediated cleavage site (yielding NVTEGE neo-epitope) within the IGD residing between the G1 and G2 domain of aggrecan are indicated. (B) A representative immunoblot of G1 fragments bearing the NVTEGE and VDIPEN neo-epitopes. Protein size marker (M),unexposed controls (U), smoke-exposed (S). To control for loading, proteins extracted from 1 mg of disc tissue wet weight were loaded per well. The anti-NITEGE neoepitope antibody crossreacts with the NVTEGE neoepitope generated from mouse aggrecan.

Fig 4. Smoking exposure effects on vertebral bone

A, Representative 3D reconstruction of the micro-computed tomographical images of the spine. B, Smoking-induced changes in vertebral bone porosity and trabecular bone volume. Quantitative bone parameters (TV, BV). Average values from three mice each measured in duplicates are shown with one standard deviation. * p<0.05.