Development and application of an LC-MS/MS method for the detection of the vinyl chloride-induced DNA adduct N(2),3-ethenoguanine in tissues of adult and weanling rats following exposure to [(13)C(2)]-VC

Abstract

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 N(2),3-ethenoguanine (εG) and have applied this to DNA from tissues of both adult and weanling rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days or 1100 ppm VC for 1 day. This assay utilizes neutral thermal hydrolysis and an HPLC cleanup prior to quantitation by LC-MS/MS. The number of endogenous and exogenous εG adducts in DNA from tissues of adult rats exposed to [(13)C(2)]-VC for 5 days was 4.1 ± 2.8 adducts/10(8) guanine of endogenous and 19.0 ± 4.9 adducts/10(8) guanine of exogenous εG in the liver, 8.4 ± 2.8 adducts/10(8) guanine of endogenous and 7.4 ± 0.5 adducts/10(8) guanine of exogenous εG in the lung, and 5.9 ± 3.3 adducts/10(8) guanine of endogenous and 5.7 ± 2.1 adducts/10(8) guanine of exogenous εG in the kidney (n = 4). Additionally, the data from weanling rats demonstrated higher numbers of exogenous εG, with ∼4-fold higher amounts in the liver DNA of weanlings (75.9 ± 17.9 adducts/10(8) guanine) in comparison to adult rats and ∼2-fold higher amounts in the lung (15.8 ± 3.6 adducts/10(8) guanine) and kidney (12.9 ± 0.4 adducts/10(8) 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 [(13)C(2)]-εG in adult rats with identically exposed animals euthanized 2, 4, or 8 weeks later. The half-life of εG was found to be 150 days in the liver and lung and 75 days in the kidney, suggesting little or no active repair of this promutagenic adduct.

Figure 1

Calibration curve for εG obtained under pseudo-SRM conditions using the present LC-MS/MS method. The plot shows the peak area ratio of εG to internal standard versus increasing fmol of εG. Data points correspond to standard mixtures that give 4.6, 18.5, 46, 200, and 1000 fmol εG and 144 fmol [¹³C₄ ¹⁵N₂]-εG per injection.

Figure 2

The product ion spectrum of εG (precursor ion m/z 176) shows low abundances of major products. The spectrum was obtained by injecting 200 fmol of εG on the LC system as described in the text, selecting m/z 176 in Q1, and scanning Q3 from m/z 50 to 200. Argon collision gas pressure was 1.5 mTorr, and collision energy was 20 eV.

Figure 3

These chromatograms prove the utility of the pseudo-SRM method for quantitation of low amounts of εG. A standard mixture giving 3 fmol εG and 37.5 fmol [¹³C₄ ¹⁵N₂]-εG on column was analyzed using SIM and pseudo-SRM. Chromatograms are plotted as absolute ion intensity versus time. Panel A) SIM results show only internal standard is visible above baseline. Panel B) Pseudo-SRM decreases background noise to reveal the low-level εG peak. A portion of the baseline is magnified 3-fold to illustrate improved S/N over SIM. Note the y axis scale and baseline level in Panel A are about 10 times higher than in Panel B for each ion monitored.

Figure 4

Chromatograms of A) liver DNA from an unexposed adult rat and B) liver DNA from an adult rat exposed to 1100 ppm [¹³C₂]-VC, 5 days, 6 hours per day.

Figure 5

The plot of log (adducts) versus days was used to determine the half life of N²,3εG in liver, lung and kidney of adult rats exposed to [¹³C₂]-VC (1100 ppm, 5 days, 6 hours per day) and killed at the end of exposure, or 2, 4 or 8 weeks later. The half life of N²,3εG in liver and lung is 150 days and in kidney is 75 days based on the equations.

Scheme 1

Formation of vinyl chloride induced DNA adducts, 7-OEG and εG. In the case of [¹³C₂]-VC exposures, * indicates positions of labeled atoms.