Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies

Abstract

Acrolein (Acr) is a ubiquitous environmental pollutant found in cigarette smoke and automobile exhaust. It can also be produced endogenously by oxidation of polyunsaturated fatty acids. The Acr-derived 1,N(2)-propanodeoxyguanosine (Acr-dG) adducts in DNA are mutagenic lesions that are potentially involved in human cancers. In this study, monoclonal antibodies were raised against Acr-dG adducts and characterized using ELISA. They showed strong reactivity and specificity toward Acr-dG, weaker reactivity toward crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N(2)-propanodeoxyguanosines, and weak or no reactivity toward 1,N(6)-ethenodeoxyadenosine and 8-oxo-deoxyguanosine. Using these antibodies, we developed assays to detect Acr-dG in vivo: first, a simple and quick FACS-based assay for detecting these adducts directly in cells; second, a highly sensitive direct ELISA assay for measuring Acr-dG in cells and tissues using only 1 μg of DNA without DNA digestion and sample enrichment; and third, a competitive ELISA for better quantitative measurement of Acr-dG levels in DNA samples. The assays were validated using Acr-treated HT29 cell DNA samples or calf thymus DNA, and the results were confirmed by LC-MS/MS-MRM. An immunohistochemical assay was also developed to detect and visualize Acr-dG in HT29 cells as well as in human oral cells. These antibody-based methods provide useful tools for the studies of Acr-dG as a cancer biomarker and of the molecular mechanisms by which cells respond to Acr-dG as a ubiquitous DNA lesion.

Figure 1. Structures of 1,N²-propanodeoxyguanosine adducts of Acr as regio-isomers

Acr-dG1/2 (α-OHPdG) and Acr-dG3 (γ-OHPdG).

Figure 2. Reactivity of mAbs against immunogens by ELISA

The mAbs were incubated with varying concentrations of immunogens, Acr-Guo1/2- or Acr-Guo3-conjugated BSA. All four A3-mAbs and three A1/2-mAbs showed strong binding to their respective immunogens, while A1/2-mAb3 displayed only low reactivity.

Figure 3. Specificity of mAbs by competitive ELISA and Slot-Blotting

(A) Different amounts of dG, dA, dC and T were used to determine their effects on the binding to mAbs to immunogens. Of all the mAbs examined, A3-mAb4 did not bind to normal nucleosides, as did A3-mAb1 (not shown). (B) All other mAbs, represented by A3-mAb3, showed cross-reactivity to dG at the highest concentration. (C) The A3-mAb4 and A3-mAb1 were further studied in a competitive ELISA assay coated with Acr-Guo3-conjugated BSA for their reactivity towards Acr-dG1/2, Acr-dG3, Acr-Guo1/2, Acr-Guo3, Cro-dG, HNE-dG, 8-oxo-dG, edA. No stereospecificity was observed; A3-mAb4 bound equally well to Acr-dG3 and Acr-dG1/2 as to Acr-Guo1/2 and Acr-Guo3. A3-mAb4 displayed significant cross-reactivity towards Cro-dG, but less for HNE-dG. It did not at all recognize 8-oxo-dG and showed minimal reactivity towards edA only at the highest concentration. (D) Acr modified CTDNA was coated on plates and Acr-dG3 and ring-opened form of AcrdG3 (Acr-dG3 RO) were added in competitive ELISA. A3-mAb4 showed binding to Acr-dG3 and no reactivity towards ring-opened form of Acr-dG3. The coated antigens were different in (C) and (D), so the competitive binding curves for Acr-dG3 were different. Data were obtained from duplicate experiments. (E) Slot-blot assay further demonstrated the specificity of these antibodies towards Acr modified plasmid DNA with no reactivity towards BPDE, H₂O₂, MDA modified DNA. The left panel of blot image shows the binding of antibodies to the modified DNA samples and binding only occurred with Acr-Modified DNA in a dose-dependent manner, the middle panel shows the DNA loading markers and the right panel is the information for the corresponding modified DNA samples.

Figure 3. Specificity of mAbs by competitive ELISA and Slot-Blotting

(A) Different amounts of dG, dA, dC and T were used to determine their effects on the binding to mAbs to immunogens. Of all the mAbs examined, A3-mAb4 did not bind to normal nucleosides, as did A3-mAb1 (not shown). (B) All other mAbs, represented by A3-mAb3, showed cross-reactivity to dG at the highest concentration. (C) The A3-mAb4 and A3-mAb1 were further studied in a competitive ELISA assay coated with Acr-Guo3-conjugated BSA for their reactivity towards Acr-dG1/2, Acr-dG3, Acr-Guo1/2, Acr-Guo3, Cro-dG, HNE-dG, 8-oxo-dG, edA. No stereospecificity was observed; A3-mAb4 bound equally well to Acr-dG3 and Acr-dG1/2 as to Acr-Guo1/2 and Acr-Guo3. A3-mAb4 displayed significant cross-reactivity towards Cro-dG, but less for HNE-dG. It did not at all recognize 8-oxo-dG and showed minimal reactivity towards edA only at the highest concentration. (D) Acr modified CTDNA was coated on plates and Acr-dG3 and ring-opened form of AcrdG3 (Acr-dG3 RO) were added in competitive ELISA. A3-mAb4 showed binding to Acr-dG3 and no reactivity towards ring-opened form of Acr-dG3. The coated antigens were different in (C) and (D), so the competitive binding curves for Acr-dG3 were different. Data were obtained from duplicate experiments. (E) Slot-blot assay further demonstrated the specificity of these antibodies towards Acr modified plasmid DNA with no reactivity towards BPDE, H₂O₂, MDA modified DNA. The left panel of blot image shows the binding of antibodies to the modified DNA samples and binding only occurred with Acr-Modified DNA in a dose-dependent manner, the middle panel shows the DNA loading markers and the right panel is the information for the corresponding modified DNA samples.

Figure 4. Acr-dG levels in HT29 cells measured with A3-mAb4 by FACS analysis, sensitive ELISA assay and LC-MS/MS-MRM

(A) In the FACS assay, the left panel shows that the average fluorescence intensity (F mean) indicates the antibodies bound in a concentration-dependent manner to the cellular DNA containing Acr-dG. The right panel shows the original histograms with X-axis as the fluorescence (F) and Y axis as the number of cells (N). The p values from t-test for 0 vs 50, 50 vs 100, 100 vs 150 and 150 vs 200 μM comparisons are 0.4, 0.14, 0.14 and 0.08, respectively. (B) A highly sensitive ELISA assay shows the Acr-dG levels in DNA extracted from HT29 cells. Only one μg DNA from each sample was used, and the chemiluminescence signal (CL), representing the binding of antibodies to DNA, showed that the Acr-dG levels increased in a concentration-dependent manner. The corresponding p values as (A) are 0.26, 0.3, 0.3 and 0.02, respectively. (C) To validate this, Acr-dG levels in the same DNA samples were quantified with by LC-MS/MS-MRM. The corresponding p values as (A) are 0.2, 0.3, 0.08 and 0.06, respectively. All results were obtained from duplicate experiments. (D) Quantification of Acr-dG by a competitive ELISA assay. To the ELISA plates were coated with different levels of modified CTDNA, different amount of Acr-dG standards were added together with the anti-Acr-dG antibodies. The approximate detection ranges that can be used to quantify the Acr-dG adducts are: 100pmol to more than 1000pmol for the 20% Acr-CTDNA coated plates, 10pmol to 1000pmol for the 2% Acr-CTDNA coated plates and <100fmol to 100pmol for and the un-modified CTDNA coated plates.

Figure 5. Immunohistochemical staining of Acr-dG in HT29 cells and human oral cells using A3-mAb4

HT29 cells treated with 200 μM Acr showed increased fluorescent intensity in nuclei (Alexa 488 labeled green) as compared to untreated controls. Human oral cells were subjected to FFPE-based staining with A3-mAb4. The positive cells show red fluorescence in nuclei stained with Cy3.