Formation of hydroxymethyl DNA adducts in rats orally exposed to stable isotope labeled methanol

Abstract

Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol's well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [¹³CD₄]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [¹³CD₄]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account.

FIG. 1.

Metabolism of methanol and two proposed MOAs for the carcinogenicity of methanol, adapted from Sweeting et al. (2010).

FIG. 2.

Schematic representation of experiment design and analytical approach for formaldehyde-DNA adducts from endogenous source and [¹³CD₄]-methanol.

FIG. 3.

Contribution of natural abundance of isotope peaks for 100 fmol N²-methyl-dG (A) and 40 fmol N⁶-methyl-dA analytical standards injected on column (B).

FIG. 4.

Typical LC-ESI-MS/MS SRM chromatograms of N²-Me-dG DNA adducts in spleen of rats exposed to 0 (A), 500 (B) or 2000 (C) mg/kg for 5 days.

FIG. 5.

Typical LC-ESI-MS/MS SRM chromatograms of N⁶-Me-dA DNA adducts in bone marrow of rats exposed to 0 (A), 500 (B) or 2000 (C) mg/kg for 5 days.

FIG. 6.

The number of exogenous N²-hydroxymethyl-dG adducts in different tissues (A) and the ratios of exogenous versus endogenous dG adducts (B).

FIG. 7.

Formation of exogenous hydroxymethyl-dG (A) and -dA adducts (B) in different regions of kidney.