1. Impaired Integrity of DNA After Recovery From Inflammation Causes Persistent Dysfunction of Colonic Smooth Muscle 
Kuicheon Choi, Jinghong Chen, Sankar Mitra, Sushil K. Sarna 
Gastroenterology 
Volume 141, Issue 4, Pages e3 (October 2011)
DOI: /j.gastro
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2. Figure 1
TNBS inflammation impairs integrity of DNA in the promoter of Cacna1c and suppresses the expression of the α1C subunit. (A) TNBS inflammation enhanced the mRNA expression of GDD45G, an indicator of DNA damage, from days 1 to 7. (B) LX-PCR showed DNA integrity of the entire Cacna1c promoter decreased until day 3 and then started to recover. The DNA integrity of the whole promoter was completely restored by day 56 after inflammation vs age-matched controls. (C) Inflammation did not affect integrity of the housekeeping β-actin gene. (D) We established the validity of LX-PCR by detecting impairment of mitochondrial DNA integrity owing to treatment of muscularis externae tissue with 200 μmol/L H2O2 for 30 minutes. (E and F) The mRNA and protein expressions of the Cacna1c bottomed out on day 3 after inflammation and recovered partially on day 7 after inflammation. The gene products remained suppressed on day 56 after inflammation, compared with age-matched controls. Mit sm, small amplicon of mitochondrial DNA; Mit lg, large amplicon of mitochondrial DNA. N = 4–6. *P < .05 vs controls. #P < .05 vs age-matched controls.
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3. Figure 2
TNBS inflammation induces strand breaks in specific sequences of the Cacna1c promoter. (A) The loci of 6 sequences in the Cacna1c promoter chosen for Long extension (LX)-PCR. (B) Inflammation did not affect integrity of DNA in segments 1, 2, 4, and 6 at any time during 56 days after induction of inflammation. (Data not shown for segments 2, 4, and 6.) (C and D) Inflammation impaired integrities of segments 3 and 5, which peaked on day 3 and recovered partially by day 7 after inflammation. The DNA strand breaks in segment 3 were completely repaired by day 56, whereas segment 5 was partially repaired. Mit sm, small amplicon of mitochondrial DNA. N = 4–10. *P < .05 vs controls.
Gastroenterology  , e3DOI: ( /j.gastro )
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4. Figure 3
The contractile response of (A) circular smooth muscle strips to acetylcholine (ACh) and to (B) membrane depolarization by KCl remains suppressed 56 days after inflammation. N = 4. *P < .05 vs controls.
Gastroenterology  , e3DOI: ( /j.gastro )
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5. Figure 4
TNBS inflammation significantly increased the markers of DNA damage on day 3 of inflammation. (A) The global level of apurinic/apyrimidinic (AP) sites, an indicator of base excision in genomic DNA, increased significantly on day 3 of inflammation in the muscularis externae tissues. (B and C) Immunofluorescence and Western blotting revealed significantly greater phosphorylated γ-H2AX, an indicator of a double-strand break, in smooth muscle cells. N = 4–6. *P < .05 vs controls.
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6. Figure 5
TNBS inflammation increased the markers of oxidative stress in the muscularis externae tissues, (A) myeloperoxidase (MPO), and (B) H2O2 from days 1 to 7 of inflammation. However, these markers returned to base level by day 56 after inflammation. (C) By contrast, inflammation suppressed the regulator of antioxidant proteins, which remained suppressed until at least day 56 after inflammation. N = 4–6. *P < .05 vs controls. #P < .05 vs age-matched controls.
Gastroenterology  , e3DOI: ( /j.gastro )
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7. Figure 6
Administration of sulforaphane in naive rats had no effect on (A) the expression of Nrf2, (B) integrity of segment 5 in the Cacna1c promoter, and (C and D) mRNA and protein expressions of Cacna1c. Sulforaphane treatment starting 3 days before inflammatory insult reverses (A) the suppression of Nrf2, (B) DNA damage in segment 5, and (C and D) mRNA and protein expressions of Cacna1c on day 3 after inflammation. Mit sm, small amplicon of mitochondrial DNA; Sul, sulforaphane. N = 4–6. *P < .05 vs controls.
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8. Figure 7
Daily sulforaphane treatment starting 3 days before TNBS insult and continuing until day 7 after inflammation prevented the suppression of smooth muscle reactivity to acetylcholine (ACh). Sulforaphane treatment in naive rats had no effect on reactivity of smooth muscle to ACh. Sul, sulforaphane. N = 4 rats in each group. *P < .05 vs controls (Ctr); #P < .05 vs TNBS inflammation.
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9. Supplementary Figure 1
LX-PCR showed TBS inflammation caused no DNA damage in promoters of genes encoding CPI-17 and MLCK, indicating specific damage to the Cacna1C gene. N = 4 rats each.
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10. Supplementary Figure 2
Time course of expressions of (A) genes involved in the base excision repair mechanism and (B) genes involved in the double-strand break repair mechanism.
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