Development of methods for measuring biological markers of formaldehyde exposure.
Formaldehyde, a widely used industrial chemical that is also present in automobile exhaust, causes nasal tumors in rats and mice after prolonged inhalation exposure to high concentrations. The induction of squamous cell carcinomas in rats by formaldehyde displayed a highly nonlinear dose response with a disproportionately large number of tumors at higher exposure concentrations. A sufficient amount of formaldehyde reaching target cells, and the saturation of formaldehyde metabolism to formate can increase the covalent binding of formaldehyde to DNA. The carcinogenicity of formaldehyde may result from its ability to induce DNA-protein cross-links and/or hydroxymethyl adducts in DNA. Measuring these products can indicate the dose of this carcinogen at a critical target site, and such assessment has been conducted for formaldehyde by measuring DNA-protein cross-links. The objective of this study was to develop methods for measuring hydroxymethyl adducts in DNA that do not require the use of radiolabeled formaldehyde. The detection of N6-hydroxymethyldeoxyadenosine and N2-hydroxymethyldeoxyguanosine, the major adducts formed by the reaction of formaldehyde with DNA in vitro, is complicated by their instability. The stabilization of hydroxymethyl adducts by reaction with sodium bisulfite in aqueous solution at 4 degrees C before isolating DNA from homogenates was investigated. On treatment of calf thymus DNA or isolated rat liver nuclei with [14C]formaldehyde, followed by reaction with bisulfite and isolation of DNA, radioactive peaks corresponding in retention time to N6-sulfomethyldeoxyadenosine and N2-sulfomethyldeoxy-guanosine were detected by high-performance liquid chromatography of nucleoside digests. However, on treatment of cultured lymphoblasts with [14C]formaldehyde, extensive metabolic incorporation of radioactivity into normal nucleosides precluded the detection of the derivatives. Methods for detecting these derivatives that do not involve the use of radiolabeled formaldehyde, such as 32P-postlabeling and electrophore postlabeling, were investigated. For electrophore postlabeling, several reactions for preparing a derivative suitable for analysis by gas chromatography with mass spectrometry were investigated unsuccessfully. For 32P-postlabeling, a method was developed for detecting sulfomethyldeoxyadenosine 3',5'-diphosphate that involved separating sulfomethyldeoxyadenosine 3'-monophosphate from normal nucleotides by reverse-phase high-performance liquid chromatography using two columns with column switching. The purified adduct fractions were subjected to 32P-postlabeling, and the labeled adduct was separated by two-dimensional thin-layer chromatography on polyethyleneimine-cellulose plates. The adduct spots were quantitated by comparing them with standards labeled directly or mixed with normal nucleotide 3'-monophosphates and separated by high-performance liquid chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)
Fennell
T R
TR
Chemical Industry Institute of Toxicology, Research Triangle Park, NC 27709.
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D002849:Chromatography, Gas; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis; D005557:Formaldehyde / Q000032:analysis* / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D009705:Nucleosides / Q000032:analysis / Q000302:isolation & purification; D009711:Nucleotides / Q000032:analysis / Q000302:isolation & purification; D051381:Rats; D011916:Rats, Inbred F344; D012680:Sensitivity and Specificity
D015415:Biomarkers; D018736:DNA Adducts; D009705:Nucleosides; D009711:Nucleotides; D005557:Formaldehyde; D006147:Guanine; D004247:DNA; D000225:Adenine