Distribution of DNA adducts caused by inhaled formaldehyde is consistent with induction of nasal carcinoma but not leukemia

Abstract

Inhaled formaldehyde is classified as a known human and animal carcinogen, causing nasopharyngeal cancer. Additionally, limited epidemiological evidence for leukemia in humans is available; however, this is inconsistent across studies. Both genotoxicity and cytotoxicity are key events in formaldehyde nasal carcinogenicity in rats, but mechanistic data for leukemia are not well established. Formation of DNA adducts is a key event in initiating carcinogenesis. Formaldehyde can induce DNA monoadducts, DNA-DNA cross-links, and DNA protein cross-links. In this study, highly sensitive liquid chromatography-tandem mass spectrometry-selected reaction monitoringmethods were developed and [(13)CD(2)]-formaldehyde exposures utilized, allowing differentiation of DNA adducts and DNA-DNA cross-links originating from endogenous and inhalation-derived formaldehyde exposure. The results show that exogenous formaldehyde induced N(2)-hydroxymethyl-dG monoadducts and dG-dG cross-links in DNA from rat respiratory nasal mucosa but did not form [(13)CD(2)]-adducts in sites remote to the portal of entry, even when five times more DNA was analyzed. Furthermore, no N(6)-HO(13)CD(2)-dA adducts were detected in nasal DNA. In contrast, high amounts of endogenous formaldehyde dG and dA monoadducts were present in all tissues examined. The number of exogenous N(2)-HO(13)CD(2)-dG in 1- and 5-day nasal DNA samples from rats exposed to 10-ppm [(13)CD(2)]-formaldehyde was 1.28 +/- 0.49 and 2.43 +/- 0.78 adducts/10(7) dG, respectively, while 2.63 +/- 0.73 and 2.84 +/- 1.13 N(2)-HOCH(2)-dG adducts/10(7) dG and 3.95 +/- 0.26 and 3.61 +/- 0.95 N(6)-HOCH(2)-dA endogenous adducts/10(7) dA were present. This study provides strong evidence supporting a genotoxic and cytotoxic mode of action for the carcinogenesis of inhaled formaldehyde in respiratory nasal epithelium but does not support the biological plausibility that inhaled formaldehyde also causes leukemia.

FIG. 1.

The formation of N²-hydroxymethyl-dG (A) and dG-dG cross-links (B) originating from both endogenous and exogenous formaldehyde.

FIG. 2.

Typical LC-ESI-MS/MS-SRM chromatograms of 0.8 fmol of N²-CH₃-dG and 80 fmol of internal standard [¹³C₁₀¹⁵N₅]-N²-CH₃-dG loaded on the column (A). 0.15 fmol of N⁶-CH₃-dA and 37.5 fmol of internal standard [¹⁵N₅]-N⁶-CH₃-dA (B). Typical calibration curve used for the quantitation of N²-Me-dG adducts (C). Typical calibration curves used for the quantitation of N⁶-Me-dA adducts (D).

FIG. 3.

LC-ESI-MS/MS-SRM chromatograms of N²-Me-dG in typical tissues: 1-day exposed nasal respiratory epithelium (A), 5-day exposed nasal respiratory epithelium (B), bone marrow (C), and spleen (D).

FIG. 4.

LC-ESI-MS/MS-SRM chromatograms of N⁶-Me-dA of typical tissues of rats: nasal respiratory epithelium of a 1-day exposed rat (A), nasal respiratory epithelium of a 5-day exposed rat (B), bone marrow of a 5-day exposed rat (C), and spleen of a 5-day exposed rat (D).

FIG. 5.

The amount of exogenous N²-HO¹³CD₂-dG in nasal respiratory epithelial DNA of rats exposed to 10-ppm formaldehyde for 1 day or 5 days (A). The ratio of exogenous versus endogenous N²-hydroxymethyl-dG for 1-day and 5-day exposed nasal respiratory epithelial DNA (B).

FIG. 6.

LC-ESI-MS/MS-SRM chromatograms of dG-dG cross-links in typical tissues of rats: nasal respiratory epithelium of a 1-day exposed rat (A), nasal respiratory epithelium of a 5-day exposed rat (B), liver of a 5-day exposed rat (C), and spleen of a 5-day exposed rat (D). The exogenous dG-¹³CD₂-dG only could be detected in nasal respiratory epithelial DNA and not in DNA from any tissue remote from the portal of entry.

FIG. 7.

Formation of artifacts under the conditions used to analyze dG-dG cross-links in nasal DNA samples. The extent of artifacts (∼65%) was determined by the area ratio of peak_{562.5→152.1} (middle panel) versus peak_{547.5→152.1} (top panel) in parallel control experiments by adding amounts of [¹³C₁₀¹⁵N₅]-dG equal to the amount of dG in the sample during sample workup and storage.