Dimethyl sulfate (DMS) is an alkylating agent that is carcinogenic to the respiratory tract of rodents. DNA adducts, cell proliferation, and histopathology were assessed in rats to better understand the molecular dosimetry and tissue dynamics associated with repeated inhalation exposure to DMS. For DNA methylation, rats were exposed to DMS vapor 6 h/day for up to 10 days to 0.0, 0.1, 0.7 and 1.5 ppm. N7-Methylguanine and N3-methyladenine were detected in neutral thermal hydrolysates of DNA isolated from respiratory tract tissues by high-performance liquid chromatography (HPLC) using fluorescence and ultraviolet (UV) detection. DNA methylation was greatest in DNA isolated from nasal respiratory mucosa, less in olfactory, and little was found in lung. N7-Methylguanine levels in respiratory mucosa approached steady-state levels by day 5, and N7-methylguanine persistence following exposure for 5 consecutive days was also determined. Loss of N7-methylguanine from respiratory and olfactory mucosa appeared to follow first-order kinetics. N3-Methyladenine levels were at or below detection limits in all samples. The effect of DMS on histopathology and cell proliferation in the nasal epithelium was also investigated. Rats were exposed nose-only for 2 wk to DMS vapor at concentrations of 0, 0.1, 0.7, or 1.5 ppm. Inhalation exposure to DMS induced degenerative and inflammatory changes in nasal epithelium at ≥0.7 ppm. Cell proliferation evaluations showed a trend towards an increased response at 1.5 ppm. These experiments demonstrate that DMS can induce cytotoxic and proliferative effects and is a potent methylating agent of the nasal mucosa in vivo. These experiments will provide data for the development of dosimetry models useful for risk extrapolation.

The reactivity of guanines in an oligonucleotide containing mutational hot spots within thep53gene (codons 248 and 249), 5′-CCG1G2AG3G4CCCA-3′, toward dimethyl sulfate (DMS) and aflatoxin B1-8,9-epoxide (AFB1-8,9-epoxide) was investigated by a modified Maxam–Gilbert technique. 5-Methylcytosine in the CpG site of codon 248 did not appear to modulate the reactivity of target guanines G1, G2, G3, and G4toward either genotoxin when compared to the sequence containing a nonmethylated CpG site. A similar experiment was conducted in which a 0.5-kb fragment of the humanHPRTgene containing exon 1 and several CpG sites was treated with UV-activated aflatoxin B1. Results showed that guanine adduct formation was independent of the methylation status of the CpG site. These findings are discussed in relation to other studies that have shown that cytosine methylation has an inhibiting effect, an enhancing effect, or no effect on adduct formation with nearby guanine nucleotides.

The purpose of this review is to provide a compilation of work examining DNA damage in the nasal cavity. There are numerous methods to identify and quantify damage to DNA and the diversity of methods and toxicologic endpoints is illustrated by the range of studies presented here. There are a large number of independent studies measuring endpoints in the upper respiratory tract; however, with regard to toxicant induced DNA damage in the nasal passageway, the effects of two compounds, 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and formaldehyde (HCHO), appear to have been extensively characterized. The body of work on NNK and formaldehyde have provided insights into molecular mechanisms of DNA damage and repair and induced cell replication and its relationship to nasal cancer. With new technologies and molecular techniques, the sensitivity to enable evaluations of the minute quantities of nasal tissue available in test species and human biopsy impact the study of the nasal–toxicant interactions. As methods used to characterize DNA damage increase in sensitivity, the importance of both exogenous and endogenous sources of DNA damage, steady-state levels of cellular damage, repair, and resulting mutations, low-dose exposure assessments and inter-species extrapolation will become increasingly complex. Additional studies of DNA damage in the nasal passage will undoubtedly challenge future estimations of risk and impact what are perceived to be acceptable levels of exposure to known and predicted carcinogens. The aim of this paper is to provide to the interested scientist literature relevant to the effects of agents on nasal DNA, so that areas of insufficient information can be identified and used to further develop and expand the knowledge base for nasal DNA toxicant interactions.

Anatomical, physiological, biochemical and molecular factors that contribute to chemical-induced nasal carcinogenesis are either largely divergent between test species and humans, or we know very little of them. These factors, let alone the uncertainty associated with our knowledge gap, present a risk assessor with the formidable task of making judgments about risks to human health from exposure to chemicals that have been identified in rodent studies to be nasal carcinogens. This paper summarizes some of the critical attributes of the hazard identification and dose-response aspects of risk assessments for nasal carcinogens that must be accounted for by risk assessors in order to make informed decisions. Data on two example compounds, dimethyl sulfate and hexamethylphosphoramide, are discussed to illustrate the diversity of information that can be used to develop informed hypotheses about mode of action and decisions on appropriate dosimeters for interspecies extrapolation. Default approaches to interspecies dosimetry extrapolation are described briefly and are followed by a discussion of a generalized physiologically based pharmacokinetic model that, unlike default approaches, is flexible and capable of incorporating many of the critical species-specific factors. Recent advancements in interspecies nasal dosimetry modeling are remarkable. However, it is concluded that without the development of research programs aimed at understanding carcinogenic susceptibility factors in human and rodent nasal tissues, development of plausible modes of action will lag behind the advancements made in dosimetry modeling.

Adult male CrlCD:BR rats were exposed nose-only to several concentrations of dimethyl sulfate (DMS) vapors to determine the relationships between vapor uptake and DNA methylation. Following DMS exposure, nasal respiratory and olfactory mucosa and lung tissue were removed and DNA was isolated for the analysis of methylated purines. DMS vapor uptake was complex and related to exposure concentration; clearance appeared to increase with increasing DMS concentrations between 0.5 and 8 ppm. Plethysmorgraphic measurements correlated with the time-dependent disappearance of dimethyl sulfate from a closed exposure apparatus. Above an initial DMS concentration of 8 ppm, sensory irritancy apparently altered normal respiratory parameters, clearance, and regional DNA methylation. DMS-dependent N7-methylguanine formation in DNA isolated from nasal respiratory mucosa was detectable 30 min following a 20-min exposure to an initial DMS concentration of 1 ppm. DMS-dependent methylation of DNA, as evidenced by N7-methylguanine and N3-methyladenine formation, showed concentration-response relationships in all tissues examined and was correlated with vapor uptake. DNA adduct formation showed regional differences characteristic of the absorption of a water-soluble vapor; methylation was greatest in DNA isolated from respiratory mucosa, less in olfactory, and little in lung. Repair of N7-methylguanine did not appear to be significantly different between nasal respiratory and olfactory tissues. These experiments demonstrate that DMS is a potent methylating agent of nasal mucosa in vivo.

Previous work has demonstrated that hydrazine after formylation to its corresponding hydrazone may be activated both in vivo and in vitro to a methylating intermediate resulting in the formation of O6-methyl- and N7-methylguanines in DNA. Incubation of calf thymus DNA with the hydrazine derivative, hydralazine, and formaldehyde resulted in the production of N7-methylguanine and two aberrant bases in DNA. These bases were separated by strong cation-exchange high-performance liquid chromatographic fractionation of neutral thermal hydrolysates. Administration of hydralazine to rats resulted in the formation of N7-methylguanine in liver DNA, but the two unknown bases observed in the in vitro experiment could not be demonstrated in vivo. In contrast to hydrazine, administration of hydralazine resulted in the methylation of DNA only at doses approaching the LD50, suggesting that formylation does not represent a significant mechanism for hydralazine toxicity in the system described. Hydralazine in combination with formaldehyde resulted in the formation of triazolophthalazine, a metabolite which has been characterized in man. The ability of 17 other hydrazine derivatives to alkylate liver DNA was determined after single administration to young adult male Sprague-Dawley rats or C57BL6 mice. Quantifiable amounts of N7-methylguanine were measured in liver DNA from animals treated with 10 of the 17 compounds. In 3 of the 10 cases quantifiable amounts of O6-methylguanine were also measured. Methylation of liver DNA guanine was obtained with hydrazine, hydralazine, procarbazine, isoniazid, phenylhydrazine, nialamide, nitrofurazone, maleic hydrazide, sulfomethoxypyridazine, and sulfamethiazole and two hydrazine-formaldehyde polymerization products, formalazine and tetraformyltrisazine.

Effects on N-methyl-N-nitrosourea (MNU) mediated methylation of the N7 position of guanine were compared in defined sequences of DNA containing cytosine or 5-methylcytosine (5mC) using a Maxam-Gilbert sequencing technique. Cytosine methylation in 5'-CpG-3' pairs within a subcloned fragment of the 5' region of the human HPRT gene was generated with SssI methylase and S-adenosylmethionine. Cytosine methylation was demonstrated by both the inhibition of DNA restriction by methylation sensitive endonucleases and the lack of cleavage at 5-methylcytosines by hydrazine. MNU-dependent methylation of the N7 position of guanine was inhibited up to 18% when 5mC was a 5' neighboring base to guanine and was inhibited up to 36% in an alternating CpG region in which both 5' and 3' neighboring bases of guanine were enzymatically altered to 5mC. It can be concluded that 5-methylcytosine has discernible effects on MNU methylation of the N7 position of specific guanine bases in DNA.