Nasal Carcinogen Research Articles

Updating the biologically based dose-response model for the nasal carcinogenicity of inhaled formaldehyde in the F344 rat.

Chronic inhalation of formaldehyde by F344 rats causes nasal squamous cell carcinoma (SCC). This outcome is well-characterized: including dose-response and time course data for SCC, mechanistic endpoints, and nasal dosimetry. Conolly et al. (Toxicol. Sci. 75, 432-447, 2003) used these resources to develop a biologically based dose-response (BBDR) model for SCC in F344 rats. This model, scaled up to humans, has informed dose-response conclusions reached by several international regulatory agencies. However, USEPA concluded that uncertainties precluded its use for cancer risk assessment. Here, we describe an updated BBDR model that addresses uncertainties through refined dosimetry modeling, revised analysis of labeling index data, and an extended dataset where both inhaled (exogenous) and endogenous formaldehyde (exogF, endoF) form DNA adducts. Further, since Conolly et al. (ibid) was published, it has become clear that, when controls from all F344 inhalation bioassays are considered, accounting for over 4000 rats, at most one nasal SCC occurred. This low spontaneous incidence constrains possible contribution of endoF to the formation of nasal SCC via DNA reactivity. Further, since both exogF and endoF form DNA adducts, this constraint also applies to exogF. The revised BBDR model therefore drives SCC formation through the cytotoxicity of high concentration exogF. An option for direct mutagenicity associated with DNA adducts is retained to allow estimation of an upper bound on adduct mutagenicity consistent with the lack of a spontaneous SCC incidence. These updates represent an iterative refinement of the 2003 model, incorporating new data and insights to reduce identified model uncertainties.

Relative contributions of endogenous and exogenous formaldehyde to formation of deoxyguanosine monoadducts and DNA-protein crosslink adducts of DNA in rat nasal mucosa.

Understanding the dose-response for formaldehyde-induced nasal cancer in rats is complicated by (1) the uneven distribution of inhaled formaldehyde across the interior surface of the nasal cavity and, (2) the presence of endogenous formaldehyde (endoF) in the nasal mucosa. In this work, we used computational fluid dynamics (CFD) modeling to predict flux of inhaled (exogenous) formaldehyde (exogF) from air into tissue at the specific locations where DNA adducts were measured. Experimental work has identified DNA-protein crosslink (DPX) adducts due to exogF and deoxyguanosine (DG) adducts due to both exogF and endoF. These adducts can be considered biomarkers of exposure for effects of endoF and exogF on DNA that may be part of the mechanism of tumor formation. We describe a computational model linking CFD-predicted flux of formaldehyde from air into tissue, and the intracellular production of endoF, with the formation of DPX and DG adducts. We assumed that, like exogF, endoF can produce DPX. The model accurately reproduces exogDPX, exogDG, and endoDG data after inhalation from 0.7 to 15 ppm. The dose-dependent concentrations of exogDPX and exogDG are predicted to exceed the concentrations of their endogenous counterparts at about 2 and 6 ppm exogF, respectively. At all concentrations examined, the concentrations of endoDPX and exogDPX were predicted to be at least 10-fold higher than that of their DG counterparts. The modeled dose-dependent concentrations of these adducts are suitable to be used together with data on the dose-dependence of cell proliferation to conduct quantitative modeling of formaldehyde-induced rat nasal carcinogenicity.

Sex Differences in the Acute Nasal Antioxidant/Antielectrophilic Response of the Rat to Inhaled Naphthalene

Naphthalene is a nasal carcinogen, inducing respiratory adenomas in male and olfactory neuroblastomas in female rats, respectively. The reasons for the site and sex-specific tumorigenic response are unknown. Naphthalene is bioactivated to electrophilic metabolites; cytotoxicity followed by regenerative cell proliferation is likely involved in the tumorigenic response. To examine sex differences in the acute nasal response to naphthalene, male and female F344 rats were nose-only exposed to 0, 1, 3, 10, or 30 ppm naphthalene vapor for 4 or 6 h. Following exposure, respiratory/transitional mucosa (RTM) and olfactory mucosa (OM) were isolated and analyzed for markers of oxidant/electrophilic stress and/or toxicity, including reduced/oxidized glutathione levels (GSH/GSSG), mRNA levels of electrophile-responsive genes, and epithelial cytoxicity (as measured by membrane permeability to ethidium homodimer-1). Naphthalene caused significant depletion of GSH in RTM and OM with no increase in GSSG. Cytotoxicity was apparent at concentrations of 15 and 30 ppm. No consistent sex differences were observed in these responses. Sex differences were observed in the induction of antielectrophilic genes in OM: glutamyl cysteine ligase (catalytic subunit) (Gclc), NADPH quinone oxidase 1 (Nqo1), and heme oxygenase 1 (Hmox1) were all induced to a greater extent in the male OM compared with the female. No consistent sex differences were observed in the RTM. Although the mechanism of the sex difference in the RTM adenoma response remains enigmatic, sex differences in the induction of antioxidant/electrophile-responsive genes may contribute to the heightened sensitivity of the female OM to the carcinogenic effects of naphthalene.

Effects of Endogenous Formaldehyde in Nasal Tissues on Inhaled Formaldehyde Dosimetry Predictions in the Rat, Monkey, and Human Nasal Passages

Formaldehyde is a nasal carcinogen in rodents at high doses and is an endogenous compound that is present in all living cells. Due to its high solubility and reactivity, quantitative risk estimates for inhaled formaldehyde have relied on internal dose estimates in the upper respiratory tract. Dosimetry calculations are complicated by the presence of endogenous formaldehyde concentrations in the respiratory mucosa. Anatomically accurate computational fluid dynamics (CFD) models of the rat, monkey, and human nasal passages were used to simulate uptake of inhaled formaldehyde. An epithelial structure was implemented in the nasal CFD models to estimate formaldehyde absorption from air:tissue partitioning, species-specific metabolism, first-order clearance, DNA binding, and endogenous formaldehyde production. At an exposure concentration of 1 ppm, predicted formaldehyde nasal uptake was 99.4, 86.5, and 85.3% in the rat, monkey, and human, respectively. Endogenous formaldehyde in nasal tissues did not significantly affect wall mass flux or nasal uptake predictions at exposure concentrations > 500 ppb; however, reduced nasal uptake was predicted at lower exposure concentrations. At an exposure concentration of 1 ppb, predicted nasal uptake was 17.5 and 42.8% in the rat and monkey; net desorption of formaldehyde was predicted in the human model. The nonlinear behavior of formaldehyde nasal absorption will affect the dose-response analysis and subsequent risk estimates at low exposure concentrations. Updated surface area partitioning of nonsquamous epithelium and average flux values in regions where DNA-protein cross-links and cell proliferation rates were measured in rats and monkeys are reported for use in formaldehyde risk models of carcinogenesis.

Nasal dosimetry of inspired naphthalene vapor in the male and female B6C3F1 mouse

Naphthalene vapor is a nasal cytotoxicant in the rat and mouse but is a nasal carcinogen in only the rat. Inhalation dosimetry is a critical aspect of the inhalation toxicology of inspired vapors and may contribute to the species differences in the nasal response. To define the nasal dosimetry of naphthalene in the B6C3F1 male and female mouse, uptake of naphthalene vapor was measured in the surgically isolated upper respiratory tract (URT) at inspiratory flow rates of 25 or 50ml/min. Uptake was measured at multiple concentrations (0.5, 3, 10, 30ppm) in controls and mice treated with the cytochrome P450 inhibitor 5-phenyl-1-pentyne. In both sexes, URT uptake efficiency was strongly concentration dependent averaging 90% at 0.5ppm compared to 50% at 30ppm (25ml/min flow rate), indicating saturable processes were involved. Both uptake efficiency and the concentration dependence of uptake were significantly diminished by 5-phenyl-1-pentyne indicating inspired naphthalene vapor is extensively metabolized in the mouse nose with saturation of metabolism occurring at the higher concentrations. A hybrid computational fluid dynamic physiologically based pharmacokinetic model was developed for nasal dosimetry. This model accurately predicted the observed URT uptake efficiencies. Overall, the high URT uptake efficiency of naphthalene in the mouse nose indicates the absence of a tumorigenic response is not attributable to low delivered dose rates in this species.

Inhalation carcinogenicity and toxicity of 1,2-dichloropropane in rats

The toxicity and carcinogenicity of 1,2-dichloropropane (DCP) were examined by inhalation exposure of male and female F344 rats to DCP for either 13 wk or 2 years. In the 13-wk study, the DCP concentrations used were 125, 250, 500, 1000, or 2000 ppm (v/v), and in the 2-year study the DCP concentrations were 80, 200, or 500 ppm (v/v). Thirteen-week exposure to DCP induced hyperplasia in the respiratory epithelium and atrophy of the olfactory epithelium at 125 ppm and above. At the higher levels of exposure, hemolytic anemia and lesions of liver and adrenal gland were observed. Two-year exposure to DCP significantly increased incidences of papilloma in the nasal cavity of male and female rats exposed to 500 ppm DCP. In addition, three cases of esthesioneuroepithelioma were observed in the DCP-exposed male rats. Total nasal tumors increased in a concentration-dependent manner. Hyperplasia of the transitional epithelium and squamous cell hyperplasia, both of which were morphologically different from the hyperplasia of the respiratory epithelium observed in the 13-wk exposure study, occurred in a concentration-dependent manner; these lesions are considered to be preneoplastic lesions. Atrophy of the olfactory epithelium, inflammation of the respiratory epithelium, and squamous cell metaplasia were also seen in the 2-year study. These results demonstrate that DCP is a nasal carcinogen in rats. Lifetime cancer risks for humans exposed to DCP in the ambient air and work environment were quantitatively estimated, using both nonthreshold and threshold approaches, with the data obtained from the 2-year study.

Formaldehyde: Integrating Dosimetry, Cytotoxicity, and Genomics to Understand Dose-Dependent Transitions for an Endogenous Compound

Formaldehyde (FA), an endogenous cellular aldehyde, is a rat nasal carcinogen. In this study, concentration and exposure duration transitions in FA mode of action (MOA) were examined with pharmacokinetic (PK) modeling for tissue formaldehyde acetal (FAcetal) and glutathione (GSH) and with histopathology and gene expression in nasal epithelium from rats exposed to 0, 0.7, 2, 6, 10, or 15 ppm FA 6 h/day for 1, 4, or 13 weeks. Patterns of gene expression varied with concentration and duration. At 2 ppm, sensitive response genes (SRGs)-associated with cellular stress, thiol transport/reduction, inflammation, and cell proliferation-were upregulated at all exposure durations. At 6 ppm and greater, gene expression changes showed enrichment of pathways involved in cell cycle, DNA repair, and apoptosis. ERBB, EGFR, WNT, TGF-β, Hedgehog, and Notch signaling were also enriched. Benchmark doses for significantly enriched pathways were lowest at 13 weeks. Transcriptional and histological changes at 6 ppm and greater corresponded to dose ranges in which the PK model predicted significant reductions in free GSH and increases in FAcetal. Genomic changes at 0.7-2 ppm likely represent changes in extracellular FAcetal and GSH. DNA replication stress, enhanced proliferation, squamous metaplasia, and stem cell niche activation appear to be associated with FA carcinogenesis. Dose dependencies in MOA, high background FAcetal, and nonlinear FAcetal/GSH tissue kinetics indicate that FA concentrations below 1 or 2 ppm would not increase risk of cancer in the nose or any other tissue or affect FA homeostasis within epithelial cells.

Distribution of DNA Adducts Caused by Inhaled Formaldehyde Is Consistent with Induction of Nasal Carcinoma but Not Leukemia

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.

Comparison of rat olfactory mucosal responses to carcinogenic and non-carcinogenic chloracetanilides

Alachlor and butachlor are chloracetanilide herbicides that induce olfactory tumors in rats, whereas propachlor does not. The mechanism by which alachlor induces tumors is distinct from many other nasal carcinogens, in that alachlor induces a gradual de-differentiation of the olfactory mucosa (OM) to a more respiratory-like epithelium, in contrast to other agents that induce cytotoxicity, followed by an aberrant regenerative response. We studied biochemical and genomic effects of these compounds to identify processes that occur in common between alachlor- and butachlor-treated rats. Because we have previously shown that matrix metalloproteinase-2 (MMP2) is activated in OM by alachlor, in the present studies we evaluated both MMP2 activation and changes in OM gene expression in response to carcinogenic and non-carcinogenic chloracetanilide treatments. All three chloracetanilides activated MMP2, and >300 genes were significantly up- or downregulated between control and alachlor-treated rats. The most significantly regulated gene was vomeromodulin, which was dramatically upregulated by alachlor and butachlor treatment (>60-fold), but not by propachlor treatment. Except for similar gene responses in alachlor- and butachlor-treated rats, we did not identify clear-cut differences that would predict OM carcinogenicity in this study.

The instability of (GpT)n and (ApC)n microsatellites induced by formaldehyde in Escherichia coli

Formaldehyde, a potential human nasal carcinogen, has been reported to induce DNA lesions. However, the effect of formaldehyde on microsatellite instability has not previously been reported. Plasmids containing different lengths of complementary (ApC)(n) or (GpT)(n) dinucleotide repeats on the leading strand were constructed to investigate whether the mutagenesis by formaldehyde can contribute to microsatellite instability. We observed that exposure of Escherichia coli to 2.5 mM formaldehyde increased the frequency of expansions and deletions of the dinucleotide repetitive sequences. After being induced by formaldehyde, the microsatellite mutation frequencies of (GpT)(n) and (ApC)(n) were 2- to 24-fold higher than those in the control. Although complementary to each other, (ApC)(n) and (GpT)(n) had different mutation frequencies when they were on the leading strand: mutation frequencies of (GpT)(n) were 13- to 24-fold higher than the control group, whereas frequencies of (ApC)(n) were only 2- to 3-fold higher the control group. Sequencing of the repetitive and flanking sequences in mutant clones showed that all mutants displayed expansions or deletions of dinucleotide repeats. These results clearly suggest that formaldehyde can increase microsatellite instability by affecting the fidelity of microsatellite maintenance. We presumed that a mutagenic mechanism of formaldehyde and the temporal formation of left-handed helix Z-DNA might be related to the microsatellite instability.

Nasal Cytotoxic and Carcinogenic Activities of Systemically Distributed Organic Chemicals

Toxicity and carcinogenicity in the mucosa of the nasal passages in rodents has been produced by a variety of organic chemicals which are systemically distributed. In this review, 14 such chemicals or classes were identified that produced rodent nasal cytotoxicity, but not carcinogenicity, and 11 were identified that produced nasal carcinogenicity. Most chemicals that affect the nasal mucosa were either concentrated in that tissue or readily activated there, or both. All chemicals with effects in the nasal mucosa that were DNA-reactive, were also carcinogenic, if adequately tested. None of the rodent nasal cytotoxins has been identified as a human systemic nasal toxin. This may reflect the lesser biotransformation activity of human nasal mucosa compared to rodent and the much lower levels of human exposures. None of the rodent carcinogens lacking DNA reactivity has been identified as a nasal carcinogen or other cancer hazard to humans. Some DNA-reactive rodent carcinogens that affect the nasal mucosa, as well as other tissues, have been associated with cancer at various sites in humans, but not the nasal cavity. Thus, findings in only the rodent nasal mucosa do not necessarily predict either a toxic or carcinogenic hazard to that tissue in humans.

Cytotoxicity of chloroacetanilide herbicide alachlor in HepG2 cells independent of CYP3A4 and CYP3A7

Alachlor is cytotoxic to human hepatoblastoma HepG2s, a cell line that expresses constitutive CYP3A7 and dexamethasone (DEX)-inducible CYP3A4 and CYP3A7. CYP3A4 catalyzes alachlor N-dealkylation to 2-chloro- N-(2,6-diethylphenyl)acetamide (CDEPA), precursor of 2,6-diethylbenzoquinoneimine, putative reactive metabolite for rat nasal carcinogenicity. We hypothesized that HepG2 alachlor cytotoxicity would be mediated by CYP3A4/7 and increased with DEX. Here, we report time-dependent alachlor cytotoxicity (EC 50 ∼ 500 μM and 264 ± 17 μM at 6 and 24 h, respectively) as assessed by lactate dehydrogenase leakage. DEX pretreatment (25 μM, 48 h) significantly increased CYP3A7-catalyzed luciferin 6′ benzylether O-debenzylation, but had no effect on alachlor toxicity. Further, CYP3A4/7 inhibitor triacetyloleandomycin did not prevent, but rather potentiated, alachlor cytotoxicity. In agreement, CDEPA was less toxic than parent alachlor. HepG2 CYP3A4 activity was unaffected by 48 h DEX pretreatment; therefore, studies were done in DPX-2 cells, a HepG2 derivative engineered to overexpress pregnane-X receptor (PXR) that exhibits rifampicin (RIF)-inducible endogenous CYP3A4. Alachlor cytotoxicity in DPX-2 cells occurred over a concentration range equivalent to that in HepG2. CYP3A4 activity of DPX-2 cells treated with RIF (10 μM, 48 h) was twice that of untreated cells, but RIF did not increase alachlor toxicity. These results demonstrate that neither CYP3A4 nor CYP3A7 initiate a pathway leading to a toxic alachlor metabolite.

The implausibility of leukemia induction by formaldehyde: a critical review of the biological evidence on distant-site toxicity

Formaldehyde is a naturally occurring biological compound that is present in tissues, cells, and bodily fluids. It is also a potent nasal irritant, a cytotoxicant at high doses, and a nasal carcinogen in rats exposed to high airborne concentrations. The normal endogenous concentration of formaldehyde in the blood is approximately 0.1 mM in rats, monkeys, and humans, and it is 2- to 4-fold higher in the liver and nasal mucosa of the rat. Inhaled formaldehyde enters the one-carbon pool, and the carbon atom is rapidly incorporated into macromolecules throughout the body. Oxidation to formate catalyzed by glutathione-dependent and -independent dehydrogenases in nasal tissues is a major route of detoxication and generally precedes incorporation. The possibility that inhaled formaldehyde might induce various forms of distant-site toxicity has been proposed, but no convincing evidence for such toxicity has been obtained in experimental studies. This review summarizes the biological evidence that pertains to the issue of leukemia induction by formaldehyde, which includes: (1) the failure of inhaled formaldehyde to increase the formaldehyde concentration in the blood of rats, monkeys, or humans exposed to concentrations of 14.4, 6, or 1.9 ppm, respectively; (2) the lack of detectable protein adducts or DNA–protein cross-links (DPX) in the bone marrow of normal rats exposed to [ 3H]- and [ 14C]formaldehyde at concentrations as high as 15 ppm; (3) the lack of detectable protein adducts or DPX in the bone marrow of glutathione-depleted (metabolically inhibited) rats exposed to [ 3H]- and [ 14C]formaldehyde at concentrations as high as 10 ppm; (4) the lack of detectable DPX in the bone marrow of Rhesus monkeys exposed to [ 14C]formaldehyde at concentrations as high as 6 ppm; (5) the failure of formaldehyde to induce leukemia in any of seven long-term inhalation bioassays in rats, mice, or hamsters; and (6) the failure of formaldehyde to induce chromosomal aberrations in the bone marrow of rats exposed to airborne concentrations as high as 15 ppm or of mice injected intraperitoneally with formaldehyde at doses as high as 25 mg/kg. Biological evidence that might be regarded as supporting the possibility of leukemia induction by formaldehyde includes: (1) the detection of cytogenetic abnormalities in circulating lymphocytes in seven studies of human subjects exposed to ambient concentrations in the workplace (but not in seven other studies of human subjects or in rats exposed to 15 ppm); (2) the induction of leukemia in rats in a single questionable drinking water study with formaldehyde concentrations as high as 1.5 g/L (but not in three other drinking water studies with concentrations as high as 1.9 or 5 g/L); (3) the detection of chromosomal aberrations in the bone marrow of rats exposed to very low concentrations of formaldehyde (0.4 or 1.2 ppm) (but not in another study at concentrations as high as 15 ppm); and (4) an apparent increase in the fraction of protein-associated DNA (assumed to be due to DPX) in circulating lymphocytes of humans exposed to ambient concentrations in the workplace (1–3 ppm). This evidence is regarded as inconsequential for several reasons, including lack of reproducibility, inadequate reporting of experimental methods, inconsistency with other data, or insufficient analytical sensitivity, and therefore, it provides little justification for or against the possibility that inhaled formaldehyde may be a leukemogen. In contrast to these inconclusive findings, the abundance of negative evidence mentioned above is undisputed and strongly suggests that there is no delivery of inhaled formaldehyde to distant sites. Combined with the fact that formaldehyde naturally occurs throughout the body, and that multiple inhalation bioassays have not induced leukemia in animals, the negative findings provide convincing evidence that formaldehyde is not leukemogenic.

Preferential metabolic activation of N-nitrosopiperidine as compared to its structural homologue N-nitrosopyrrolidine by rat nasal mucosal microsomes.

N-Nitrosopiperidine (NPIP) is a potent rat nasal carcinogen whereas N-nitrosopyrrolidine (NPYR), a hepatic carcinogen, is weakly carcinogenic in the nose. NPIP and NPYR may be causative agents in human cancer. P450-catalyzed alpha-hydroxylation is the key activation pathway by which these nitrosamines elicit their carcinogenic effects. We hypothesize that the differences in NPIP and NPYR metabolic activation in the nasal cavity contribute to their differing carcinogenic activities. In this study, the kinetics of tritium-labeled NPIP or NPYR alpha-hydroxylation mediated by Sprague-Dawley rat nasal olfactory or respiratory microsomes were investigated. To compare alpha-hydroxylation rates of the two nitrosamines, tritiated 2-hydroxytetrahydro-2H-pyran and 2-hydroxy-5-methyltetrahydrofuran, the major NPIP alpha-hydroxylation products, and tritiated 2-hydroxytetrahydrofuran, the major NPYR alpha-hydroxylation product, were quantitated by HPLC with UV absorbance and radioflow detection. These microsomes catalyzed the alpha-hydroxylation of NPIP more efficiently than that of NPYR. K(M) values for NPIP were lower as compared to those for NPYR (13.9-34.7 vs 484-7660 muM). Furthermore, catalytic efficiencies (V(max)/K(M)) of NPIP were 20-37-fold higher than those of NPYR. Previous studies showed that P450 2A3, present in the rat nose, also exhibited this difference in catalytic efficiency. For both types of nasal microsomes, coumarin (100 muM), a P450 2A inhibitor, inhibited NPIP and NPYR alpha-hydroxylation from 63.8 to 98.5%. Furthermore, antibodies toward P450 2A6 inhibited nitrosamine alpha-hydroxylation in these microsomes from 68.8 to 78.4% whereas antibodies toward P450 2E1 did not inhibit these reactions. Further immunoinhibition studies suggest some role for P450 2G1 in NPIP metabolism by olfactory microsomes. In conclusion, olfactory and respiratory microsomes from rat nasal mucosa preferentially activate NPIP over NPYR with P450 2A3 likely playing a key role. These results are consistent with local metabolic activation of nitrosamines as a contributing factor in their tissue-specific carcinogenicity.

The effect of deuterium and fluorine substitution upon the mutagenicity of N-hydroxy-2,6-dimethylaniline

2,6-Dimethylaniline (2,6-DMA) is an intermediate in the manufacture of several products, including pesticides, dyestuffs, and synthetic resins. It is also present in nanogram amounts in tobacco smoke, and is a major metabolite of the potent anesthetic and antiarrhythmic drug lidocaine, as well as a nasal carcinogen in rats. As with other aromatic amines, 2,6-DMA can undergo metabolic activation through cytochrome P450-mediated N-hydroxylation, followed by O-esterification to a reactive derivative capable of forming DNA adducts. We have recently characterized four DNA adducts resulting from this metabolic pathway. Three of the adducts arose from reaction of the exocyclic heteroatoms of deoxyadenosine and deoxyguanosine with the carbon para to the arylamine nitrogen. The fourth adduct resulted from reaction of the 2,6-DMA nitrogen with the C8 atom of deoxyguanosine. In order to investigate the relative contribution of the exocyclic heteroatom adducts as compared to the C8-deoxyguanosine adduct to the toxicities elicited by 2,6-DMA, we synthesized and compared the mutagenicity of N-hydroxy-2,6-DMA, N-hydroxy-4-deutero-2,6-DMA, 2,6-dimethylnitrosobenzene, 4-deutero-2,6-dimethylnitrosobenzene, and N-hydroxy-4-fluoro-2,6-DMA. In Salmonella typhimurium TA100, the two deuterated compounds and their non-deuterated analogues gave similar mutagenic responses (∼25 revertants/nmol). Likewise in S. typhimurium TA98, a similar mutant frequency (∼0.7 revertants/nmol) was obtained with the four compounds. With N-hydroxy-4-fluoro-2,6-DMA, the mutant frequency was reduced by ∼90% in S. typhimurium TA100 and ∼50% in S. typhimurium TA98. The results suggest that multiple adducts contribute to base substitution mutations detected by S. typhimurium TA100 while the C8-deoxyguanosine adduct is primarily responsible for the frameshift mutations detected by S. typhimurium TA98.

LACK OF DNA BINDING IN THE RAT NASAL MUCOSA AND OTHER TISSUES OF THE NASAL TOXICANTS ROFLUMILAST, A PHOSPHODIESTERASE 4 INHIBITOR, AND A METABOLITE, 4-AMINO-3,5-DICHLOROPYRIDINE, IN CONTRAST TO THE NASAL CARCINOGEN 2,6-DIMETHYLANILINE*

The phosphodiesterase 4 inhibitor Roflumilast (B9302-107) (RF) and its metabolite 4-amino-3,5-dichloropyridine (ADCP) produced nasal toxicity in preclinical safety studies with rats. The purpose of this study was to assess the possible formation of DNA adducts, by RF and ADCP, in the nasal mucosa, liver and testes of male rats using the 32P-postlabeling assay. For comparison, rats were exposed to the DNA-reactive carcinogens 2,6-dimethylaniline (DMA), also known as 2,6-xylidine, a nasal carcinogen, and the aromatic amine carcinogens 4,4′-methylene-bis(2-chloroaniline) (MOCA), which yields monocyclic DNA adducts, and 2-acetylaminofluorene (2-AAF). In the case of RF, possible sources of DNA adducts include the parent molecule and its ADCP moiety by enzymatic N-hydroxylation and sulfation, reactions typical of carcinogenic aromatic amines. 4-Acetoxylamino-3,5-dichloropyridine (N-acetoxy-ADCP), a chemically activated derivative of ADCP, was prepared and used to modify DNA which was then used to establish the chromatographic conditions with which to reliably detect whether or not such adducts were formed metabolically from RF and ADCP. Similarly, a standard N-hydroxy-DMA was prepared, but the corresponding N-acetoxy derivative was unstable and decomposed during synthesis. Both N-hydroxy-DMA and N-acetoxy-ADCP were mutagenic in the Salmonella typhimurium Ames assay using strain TA100 without an exogenous bioactivation system, with the former being more potent. N-hydroxy-ADCP was essentially inactive in this assay. For the 32P-postlabeling assay, male Wistar rats were exposed to the test substances and carrier control compounds by intragastric instillation at the selected dose levels for 7 days. Subsequently, the nasal mucosa, liver, and testes of the rats exposed to the test or control compounds were extirpated, the DNA extracted and the samples postlabeled. The patterns of adducts formed with the test compounds were compared to those formed in N-acetoxy-ADCP- and N-hydroxy-DMA-adducted DNA, which were assayed by both nuclease P1 and butanol enhancement methods. Based upon the similarity of results from the two enhancement methods, only the former was used for the in vivo studies. No evidence was obtained for the formation of DNA adducts from RF or its metabolites, specifically ADCP, under the conditions of these assays despite the ability to detect adducts from DNA modified chemically with N-acetoxy-ADCP and DNA adducts from the other compounds in their target organs. In the absence of a pattern of compound-related spots, we conclude that RF does not form DNA adducts having the potential to initiate neoplasia in these three tissues.

Analysis of the lidocaine metabolite 2,6-dimethylaniline in bovine and human milk.

2,6-Dimethylaniline (2,6-xylidine; 2,6-DMA) is a nasal carcinogen in rats. Humans may be exposed to this compound via several routes: 2,6-DMA is found in cigarette smoke; it is a pharmacologically inactive metabolite of some drugs (e.g., the local anesthetic lidocaine) and pesticides (e.g., metalaxyl); and it is an impurity in technical grade metalaxyl. The potential transfer of 2,6-DMA from mother to nursing infant via milk is of toxicological concern. Solid-phase microextraction with separation and detection using gas chromatography-mass spectrometry was optimized and used for the analysis of 2,6-DMA in milk. 2,6-DMA-d9 was synthesized and used for quantitation by the isotope ratio method. At a concentration of 5 ppb 2,6-DMA, the method detection limit was 0.20 ppb, and the relative standard deviation was 3.6%. Samples of milk were obtained from bovines administered lidocaine (2.9-3.9 mg/kg) during surgery. A breast milk sample was also obtained from a human donor who received 36 mg lidocaine during dental work. 2,6-DMA was present at levels ranging from 14.5 to 66.0 ppb in bovine milk and was detected at 1.6 ppb in the human milk sample. Our results demonstrate that 2,6-DMA, formed by the metabolism of lidocaine, is transferable to bovine and human milk.

Dosimetry modeling of inhaled formaldehyde: the human respiratory tract.

Formaldehyde (HCHO), which has been shown to be a nasal carcinogen in rats and mice, is used widely and extensively in various manufacturing processes. Studies in rhesus monkeys suggest that the lower respiratory tract may be at risk and some epidemiologic studies have reported an increase in lung cancer associated with HCHO; other studies have not. Thus, an assessment of possible human risk to HCHO exposure based on dosimetry information throughout the respiratory tract (RT) is desirable. To obtain dosimetry estimates for a risk assessment, two types of models were used. The first model (which is the subject of another investigation) used computational fluid dynamics (CFD) to estimate local fluxes in a 3-dimensional model of the nasal region. The subject of the present investigation (the second model) applied a 1-dimensional equation of mass transport to each generation of an adult human symmetric, bifurcating Weibel-type RT anatomical model, augmented by an upper respiratory tract. The two types of modeling approaches were made consistent by requiring that the 1-dimensional version of the nasal passages have the same inspiratory air-flow rate and uptake during inspiration as the CFD simulations for 4 daily human activity levels. Results obtained include the following: (1) More than 95% of the inhaled HCHO is predicted to be retained by the RT. (2) The CFD predictions for inspiration, modified to account for the difference in inspiration and complete breath times, are a good approximation to uptake in the nasal airways during a single breath. (3) In the lower respiratory tract, flux is predicted to increase for several generations and then decrease rapidly. (4) Compared to first pulmonary region generation fluxes, the first few tracheobronchial generations fluxes are over 1000 times larger. Further, there is essentially no flux in the alveolar sacs. (5) Predicted fluxes based on the 1-dimensional model are presented that can be used in a biologically based dose-response model for human carcinogenesis. Use of these fluxes will reduce uncertainty in a risk assessment for formaldehyde carcinogenicity.

Mode-of-action-based dosimeters for interspecies extrapolation of vinyl acetate inhalation risk.

Vinyl acetate is used in the manufacture of many polymers. The Clean Air Act Amendments of 1990 require that an inhalation risk assessment be conducted to assess risks to human health from ambient exposures. Vinyl acetate is a nasal carcinogen in rats and induces olfactory degeneration in rats and mice. Because of the many unique aspects of the rodent nasal cavity compared to that of humans, conventional means for extrapolating dosimetry between species are not appropriate. Physiologically based pharmacokinetic (PBPK) and pharmacodynamic (PD) modeling can address many of these unique aspects. A PBPK/PD model has been developed for vinyl acetate, but the choice of appropriate dosimeter(s) to use for interspecies extrapolation depends on a hypothesis regarding mode of action. This article summarizes the key studies that formulate a mode of action hypothesis for vinyl acetate. Dose-response relationships for vinyl acetate-induced nonneoplastic and neoplastic responses are highly nonlinear, suggesting complex kinetic processes. Carboxylesterase-dependent metabolism of vinyl acetate forms acetic acid, a potent cytotoxicant, and acetaldehyde, a weak clastogen. Cell death, proposed to be the result of intracellular acidification, results in restorative cell proliferation. In conjunction with sufficient genetic damage, induced by spontaneous mutation and acetaldehyde-induced DNA-protein cross-links (DPX), olfactory degeneration progresses to a state of elevated proliferation and eventually, at high vinyl acetate concentrations, to neoplastic transformation. Thus, reduction in intracellular pH (pHi) is proposed as the dosimeter most closely linked to the earliest stages of vinyl acetate toxicity. Consequently, risk assessments that are based on protection of nasal epithelium from intracellular acidification will be protective of all subsequent pathological responses related to vinyl acetate exposure. Proposing a reasonable mode of action is an important step in any risk assessment and is critical to the choice of dosimeter(s) to be used for interspecies dosimetry extrapolation.

MODE-OF-ACTION?BASED DOSIMETERS FOR INTERSPECIES EXTRAPOLATION OF VINYL ACETATE INHALATION RISK

Vinyl acetate is used in the manufacture of many polymers. The Clean Air Act Amendments of 1990 require that an inhalation risk assessment be conducted to assess risks to human health from ambient exposures. Vinyl acetate is a nasal carcinogen in rats and induces olfactory degeneration in rats and mice. Because of the many unique aspects of the rodent nasal cavity compared to that of humans, conventional means for extrapolating dosimetry between species are not appropriate. Physiologically based pharmacokinetic (PBPK) and pharmacodynamic (PD) modeling can address many of these unique aspects. A PBPK/PD model has been developed for vinyl acetate, but the choice of appropriate dosimeter(s) to use for interspecies extrapolation depends on a hypothesis regarding mode of action. This article summarizes the key studies that formulate a mode of action hypothesis for vinyl acetate. Dose-response relationships for vinyl acetate-induced nonneoplastic and neoplastic responses are highly nonlinear, suggesting complex kinetic processes. Carboxylesterase-dependent metabolism of vinyl acetate forms acetic acid, a potent cytotoxicant, and acetaldehyde, a weak clastogen. Cell death, proposed to be the result of intracellular acidification, results in restorative cell proliferation. In conjunction with sufficient genetic damage, induced by spontaneous mutation and acetaldehyde-induced DNA?protein cross-links (DPX), olfactory degeneration progresses to a state of elevated proliferation and eventually, at high vinyl acetate concentrations, to neoplastic transformation. Thus, reduction in intracellular pH (pHi) is proposed as the dosimeter most closely linked to the earliest stages of vinyl acetate toxicity. Consequently, risk assessments that are based on protection of nasal epithelium from intracellular acidification will be protective of all subsequent pathological responses related to vinyl acetate exposure. Proposing a reasonable mode of action is an important step in any risk assessment and is critical to the choice of dosimeter(s) to be used for interspecies dosimetry extrapolation.