Formaldehyde-induced DNA Adducts Research Articles

Is high resolution a strict requirement for mass spectrometry-based cellular DNA adductomics?

Exposure to endogenous and exogenous factors can result in the formation of a wide variety of DNA adducts, and these may lead to gene mutations, thereby contributing to the development of cancer. DNA adductomics, a novel tool for exposomics, aims to detect the totality of DNA adducts. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) is the state-of-the-art method for DNA adductomic analysis, although its high cost has limited widespread use. In this study, we compared the analytical performance between HRMS and the more popular/accessible triple-quadrupole MS (QqQ-MS). We initially developed and optimized a hybrid quadrupole-linear ion trap-orbitrap MS (Q-LIT-OT-MS) method, considering the detection of both purine and pyrimidine adducts. LC-Q-LIT-OT-MS and LC-QqQ-MS methods were compared by non-targeted screening of formaldehyde-induced DNA adducts. Using the results from Q-LIT-OT-MS as the gold standard, QqQ-MS successfully detected 12 out of 18 formaldehyde-induced DNA adducts/inter-strand crosslinks (ICLs). QqQ-MS however also produced nine false-positive results owing to the inherent instrumental mass resolution limits. To discriminate the false-positive results from the accurate ones, we firstly introduced a statistical analysis, partial least squares-discriminant analysis, which efficiently excluded the false signals. Six DNA adducts/ICLs were not detected by QqQ-MS, due to insufficient sensitivity. This could be overcome by employing a selected reaction monitoring scan mode with multiple injections. Overall, this study demonstrated that high resolution may not be a strict requirement for MS-based DNA adductomics. LC-QqQ-MS with statistical analysis, could also provide a comparable performance as HRMS for pre-screening purposes.

Evaluation of inhaled low-dose formaldehyde-induced DNA adducts and DNA-protein cross-links by liquid chromatography-tandem mass spectrometry.

As a widespread industrial chemical, formaldehyde carcinogenicity has been highly controversial. Meanwhile, formaldehyde is an essential metabolite in all living cells. Previously, we have demonstrated exogenous formaldehyde causes DNA adducts in a nonlinear manner between 0.7 and 15.2ppm using [13CD2]-formaldehyde for exposure coupled with the use of sensitive mass spectrometry. However, the responses from exposure to low doses of formaldehyde are still unknown. In this study, rats were exposed to 1, 30, and 300ppb [13CD2]-formaldehyde for 28days (6h/day) by nose-only inhalation, followed by measuring DNA mono-adduct (N2-HOMe-dG) and DNA-protein crosslinks (dG-Me-Cys) as formaldehyde specific biomarkers. Both exogenous and endogenous DNA mono-adducts and dG-Me-Cys were examined with ultrasensitive nano-liquid chromatography-tandem mass spectrometry. Our data clearly show that endogenous adducts are present in all tissues analyzed, but exogenous adducts were not detectable in any tissue samples, including the most susceptible nasal epithelium. Moreover, formaldehyde exposure at 1, 30 and 300ppb did not alter the levels of endogenous formaldehyde-induced DNA adducts or DNA-protein crosslinks. The novel findings from this study provide new data for risk assessment of exposure to low doses of formaldehyde.

Dosimetry ofN6-Formyllysine Adducts Following [13C2H2]-Formaldehyde Exposures in Rats

With formaldehyde as the major source of endogenous N6-formyllysine protein adducts, we quantified endogenous and exogenous N6-formyllysine in the nasal epithelium of rats exposed by inhalation to 0.7, 2, 5.8, and 9.1 ppm [13C2H2]-formaldehyde using liquid chromatography-coupled tandem mass spectrometry. Exogenous N6-formyllysine was detected in the nasal epithelium, with concentration-dependent formation in total as well as fractionated (cytoplasmic, membrane, nuclear) proteins, but was not detected in the lung, liver, or bone marrow. Endogenous adducts dominated at all exposure conditions, with a 6 h 9.1 ppm formaldehyde exposure resulting in one-third of the total load of N6-formyllysine being derived from exogenous sources. The results parallel previous studies of formaldehyde-induced DNA adducts.

Formation of Hydroxymethyl DNA Adducts in Rats Orally Exposed to Stable Isotope Labeled Methanol

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.

Formaldehyde and leukemia: epidemiology, potential mechanisms, and implications for risk assessment.

Formaldehyde is widely used in the United States and other countries. Occupational and environmental exposures to formaldehyde may be associated with an increased risk of leukemia in exposed individuals. However, risk assessment of formaldehyde and leukemia has been challenging due to inconsistencies in human and animal studies and the lack of a known mechanism for leukemia induction. Here, we provide a summary of the symposium at the Environmental Mutagen Society Meeting in 2008, which focused on the epidemiology of formaldehyde and leukemia, potential mechanisms, and implication for risk assessment, with emphasis on future directions in multidisciplinary formaldehyde research. Updated results of two of the three largest industrial cohort studies of formaldehyde-exposed workers have shown positive associations with leukemia, particularly myeloid leukemia, and a recent meta-analysis of studies to date supports this association. Recent mechanistic studies have shown the formation of formaldehyde-induced DNA adducts and characterized the essential DNA repair pathways that mitigate formaldehyde toxicity. The implications of the updated findings for the design of future studies to more effectively assess the risk of leukemia arising from formaldehyde exposure were discussed and specific recommendations were made. A toxicogenomic approach in experimental models and human exposure studies, together with the measurement of biomarkers of internal exposure, such as formaldehyde-DNA and protein adducts, should prove fruitful. It was recognized that increased communication among scientists who perform epidemiology, toxicology, biology, and risk assessment could enhance the design of future studies, which could ultimately reduce uncertainty in the risk assessment of formaldehyde and leukemia.

Formation of S-[1-(N2-deoxyguanosinyl)methyl]glutathione between glutathione and DNA induced by formaldehyde.

Formaldehyde is an essential metabolic intermediate in human cells and can also enter into the body through environmental exposures. It is classified as a human and animal carcinogen according to the International Agency for Research on Cancer (IARC). Previous research has demonstrated that formaldehyde is genotoxic, causing mutations in multiple genes. However, no exogenous formaldehyde-induced DNA adducts have been detected in animals after inhalation exposure, although formaldehyde can result in N(6)-deoxyadenosine, N(2)-deoxyguanosine, and N(4)-deoxycytidine adducts in vitro. This can be partially attributed to the rapid metabolism of formaldehyde by glutathione (GSH)-dependent enzyme systems. Among the intermediates in the pathway of formaldehyde detoxication, S-hydroxymethylglutathione is a reactive species and has the potential to further conjugate with DNA bases. Here, we have demonstrated the formation of S-[1-(N(2)-deoxyguanosinyl)methyl]glutathione between glutathione and DNA in the presence of formaldehyde. This adduct is unique because of the involvement of S-hydroxymethylglutathione which is a key player during the detoxication of formaldehyde.

Comparison of UV, Fluorescence, and Electrochemical Detectors for the Analysis of Formaldehyde-Induced DNA Adducts

A high-performance liquid chromatography (HPLC) method for normal and formaldehyde-modified deoxynucleosides after hydrolysis of exposed and unexposed human placental DNA was compared with ultraviolet (UV), fluorescence (FL), and electrochemical (EC) detectors. The lower quantifiable limits (LQL) for UV detection at 254 nm were 10-22 pmol for N(6)-hydroxymethyldeoxyadenosine (N(6)-dA), N(2)-hydroxymethyldeoxyguanosine (N(2)-dG), and N(4)-hydroxymethyldeoxycytidine (N(4)-dC), with N(4)-dC having the highest LQL and the 2 purines the lowest LQL. Similarly, LQLs using FL (excitation: 264 nm; emission: 340 nm) were 14-30 pmol, with N(2)-dG having the lowest LQL and N(6)-dA the highest. The LQL for N(2)-dG by EC detection at +1.10 V was 27 pmol, over 50-fold greater sensitivity than for the other hydroxymethyl deoxynucleosides; deoxyguanosine was similarly detected more sensitively than the other normal deoxynucleosides. Percent relative standard deviations ranged between 6 and 13% for both intra- and interrun assays for all detectors. HPLC-UV allows all the deoxynucleosides to be detected without the flow cell washing and use of fresh solutions necessary for the more selective FL detection, the latter not having enzyme blank interferences. EC allows only deoxyguanosine and N(2)-dG to be detected at pmol levels with no blank interferences. HPLC-UV allowed more sensitive detection of N(2)-dG and N(6)-dA than the other techniques and is recommended. The UV, FL, and EC properties of the hydroxymethyl deoxynucleosides of dA, dG, and dC are reported for the first time.

Formaldehyde-induced DNA adducts as biomarkers of in vitro human nasal epithelial cell exposure to formaldehyde

Formaldehyde (FA) is a mutagen that, at high concentrations and long durations, has been reported to cause nasal cancer in rats and in some humans. The level of FA-induced modified DNA in nasal cells should serve as a biomarker of FA exposure and effect. In the present study, a high-performance liquid chromatography (HPLC)-ultraviolet (UV) method at 254 nm was developed and optimized to detect and quantify hydroxymethyldeoxynucleosides after the isolated DNA in exposed human nasal epithelial cells (HNEC) was enzymically digested. Normal and modified deoxynucleosides were successfully resolved from one another and from tissue and enzyme blank interferences. The viability of HNEC exposed to FA in solution for 24 h decreased, and there was a linear dose response between % nonviability and FA dose from 10 to 500 μg/mL. Amounts of 18.0 ± 1.5 pmol N 6-dA and 12.0 ± 1.2 pmol N 2-dG derivatives were determined in a 10 μL injection after 1.4 × 10 7 HNEC (106 μg DNA) were exposed to 500 μg/mL in solution. The respective tissue concentrations in pmol hydroxymethyldeoxynucleoside/mg DNA were 170 ± 14 and 113 ± 11. The lower quantifiable limits were about 97 and 88 pmol/mg DNA, respectively. Diffusive exposure of HNEC to air FA up to 100 ppm (v/v) for 24 h did not produce quantifiable hydroxymethylnucleosides. FA-modified deoxynucleosides may be useful biomarkers for FA exposure in biological monitoring samples taken by nasal lavage or brush biopsy.