In the 1970s exposure to vinyl chloride (VC) was shown to cause liver angiosarcoma in VC workers. We have developed a new LC-MS/MS method for analyzing the promutagenic DNA adduct N2,3-ethenoguanine (εG) and have applied this to DNA from tissues of both adult and weanling rats exposed to 1100 ppm [13C2]-VC for 5 days or 1100 ppm VC for 1 day. This assay utilizes neutral thermal hydrolysis and an HPLC clean-up prior to quantitation by LC-MS/MS. The number of endogenous and exogenous εG adducts in DNA from tissues of adult rats exposed to [13C2]-VC for 5 days was 4.1±2.8 adducts/108 guanine of endogenous and 19.0±4.9 adducts/108 guanine of exogenous εG in liver, 8.4±2.8 adducts/108 guanine of endogenous and 7.4±0.5 adducts/108 guanine of exogenous εG in lung and 5.9±3.3 adducts/108 guanine of endogenous and 5.7±2.1 adducts/108 guanine of exogenous εG in kidney (n=4). Additionally, the data from weanling rats demonstrated higher numbers of exogenous εG, with ~4 fold higher amounts in liver DNA of weanlings (75.9±17.9 adducts/108 guanine) in comparison to adult rats and ~2 fold higher amounts in lung (15.8±3.6 adducts/108 guanine) and kidney (12.9±0.4 adducts/108 guanine) (n=8). The use of stable isotope labeled VC permitted accurate estimates of the half life of εG for the first time by comparing [13C2]-εG in adult rats with identically exposed animals killed 2, 4 or 8 weeks later. The half life of εG was found to be 150 days in liver and lung and 75 days in kidney, suggesting little or no active repair of this promutagenic adduct.
We have generated a photoactivatable form of sonic hedgehog protein by modifying the N-terminal cysteine with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (Bzm). The Bzm modification on ShhN imparted a significant increase in activity as assessed in the C3H10T1/2 functional assay with potency comparable to that of the endogenous dual-lipidated form of ShhN (ShhNp). Reversed-phase HPLC analysis indicated that the increase in activity compared to unmodified ShhN may be due in part to the hydrophobic nature of the benzophenone group. In contrast to the fully processed ShhNp, Bzm-ShhN is monomeric as assessed by analytical SEC and does not require detergent to be soluble. Further, we demonstrated that the Bzm-ShhN was able to crosslink in vitro in the presence of a known binding partner, heparin. We suggest that Bzm-ShhN can serve as a relatively facile and preferred source of ShhNp for in vitro assays and as a probe to identify novel Hh protein interactions.
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