Dosimetry modeling of inhaled formaldehyde: comparisons of local flux predictions in the rat, monkey, and human nasal passages.

Formaldehyde-induced nasal squamous cell carcinomas in rats and squamous metaplasia in rats and rhesus monkeys occur in specific regions of the nose with species-specific distribution patterns. Experimental approaches addressing local differences in formaldehyde uptake patterns and dose are limited by the resolution of dissection techniques used to obtain tissue samples and the rapid metabolism of absorbed formaldehyde in the nasal mucosa. Anatomically accurate, 3-dimensional computational fluid dynamics models of F344 rat, rhesus monkey, and human nasal passages were used to estimate and compare regional inhaled formaldehyde uptake patterns predicted among these species. Maximum flux values, averaged over a breath, in nonsquamous epithelium were estimated to be 2620, 4492, and 2082 pmol/(mm(2)-h-ppm) in the rat, monkey, and human respectively. Flux values predicted in sites where cell proliferation rates were measured as similar in rats and monkeys were also similar, as were fluxes predicted in a region of high tumor incidence in the rat nose and the anterior portion of the human nose. Regional formaldehyde flux estimates are directly applicable to clonal growth modeling of formaldehyde carcinogenesis to help reduce uncertainty in human cancer risk estimates.

Respiratory tract toxicity in rats exposed to Mexico City air.

The rat has been used extensively as a health sentinel, indicator, or monitor of environmental health hazards, but this model has not been directly validated against human exposures. Humans in Mexico City show upper respiratory tract lesions and evidence of pulmonary damage related to their environmental inhalation exposure. In this study, male and female F344 rats were exposed (23 hr/day) in Mexico City to local Mexico City air (MCA)* for up to seven weeks. Controls were maintained at the same location under filtered air. Prior to these exposures, several steps were taken. First, the nasal passages of normal male rats shipped from the United States and housed in Mexico City were examined for mycoplasma infection; no evidence of infection was found. In addition, a mobile exposure and monitoring system was assembled and, with an ozone (O3) exposure atmosphere, was tested along with supporting histopathology techniques and analysis of rat nasal and lung tissues. Last, the entire exposure model (equipment and animals) was transported to Mexico City and validated for a three-week period. During the seven-week study there were 18 one-hour intervals during which the average O3 concentration of MCA in the exposure chamber exceeded the US National Ambient Air Quality Standard (NAAQS) of 0.120 ppm 03 (hourly average, not to be exceeded more than once per year). This prolonged exposure of healthy F344 rats to MCA containing episodically low to moderate concentrations of 03 (as well as other urban air pollutants) did not induce inflammatory or epithelial lesions in the nasal airways or lung as measured by qualitative histologic techniques or quantitative morphometric techniques. These findings agree with those of previous controlled O3 inhalation studies, but they are in contrast to reports indicating that O3-polluted MCA causes significant nasal mucosal injury in adults and children living in southwestern Mexico City. Taken together, these findings may suggest that human airways are markedly more susceptible to the toxic effects of MCA than are the airways of the F344 rat.

Psychological factors in the determination of suicide in self-inflicted gunshot head wounds.

Self-inflicted contact gunshot wounds to the head have usually been considered presumptive of suicide. This study evaluates whether sufficient psychological data are gathered in such cases to support a manner of death determination of suicide. We suggest that law enforcement agencies and coroner's departments do not fully explore the decedent's state of mind at the time of death. We studied the first 50 consecutive deaths in 1993 in a major metropolitan county due to self-inflicted gunshot wounds to the head. The sample consists primarily of unmarried, white males, with a median age of 35 years, who displayed psychiatric disturbance, primarily depression, before their death. Younger individuals were often under the influence of alcohol and/or drugs at the time of death. Stressors, such as the loss of a loved one, are common among young and middle-aged persons, while serious health problems are found among the majority of middle-aged and elderly individuals. Many of the findings of this study are consistent with the literature regarding individuals who commit suicide. Although data on many important psychological risk factors are missing in most cases, sufficient psychological material is gathered about the decedent's mental condition at the time of death to support a suicide determination.

Olfactory neuron loss in adult male CD rats following subchronic inhalation exposure to hydrogen sulfide.

Dysosmia and anosmia are reported to occur following human exposure to hydrogen sulfide (H2S) gas. The clinical association between H2S exposure and olfactory dysfunction in humans necessitates evaluation of the nasal cavity and olfactory system in experimental animals used to study H2S toxicity. The purpose of this study was to subchronically expose 10-week-old male CD rats to relatively low concentrations of H2S and to histologically evaluate the nasal cavity for exposure-related lesions. Rats (n = 12/group) were exposed via inhalation to 0, 10, 30, or 80 ppm H2S 6 h/d and 7 d/wk for 10 weeks. Following exposure to 30 and 80 ppm H2S, a significant increase in nasal lesions limited to the olfactory mucosa was observed. The lesions, which consisted of olfactory neuron loss and basal cell hyperplasia, were multifocal, bilaterally symmetrical, and had a characteristic rostrocaudal distribution pattern. Regions of the nasal cavity affected included the dorsal medial meatus and the dorsal and medial portions of the ethmoid recess. The no observed adverse effect level for olfactory lesions in this study was 10 ppm. For perspective, the American Conference of Governmental Industrial Hygienists threshold limit value (TLV) recommendation for H2S is currently 10 ppm (proposed revision: 5 ppm), so the concentrations employed in the present study were 3 and 8 times the TLV. These findings suggest that subchronic inhalation exposure to a relatively low level of H2S (30 ppm) can result in olfactory toxicity in rats. However, because of differences in the breathing style and nasal anatomy of rats and humans, additional research is required to determine the significance of these results for human health risk assessment.

Prediction of structural and functional relationships of Repeat 1 of human interphotoreceptor retinoid-binding protein (IRBP) with other proteins.

PURPOSE: We compared the structure and function of interphotoreceptor retinoid-binding protein (IRBP) related proteins and predicted domain and secondary structure within each repeat of IRBP and its relatives. We tested whether tail specific protease (Tsp), which bears sequence similarity to IRBP Domain B, binds fatty acids or retinoids, and whether IRBP possessed protease activity resembling Tsp's catalytic function. These tests helped us to learn whether the primary sequence similarities of family members extended to higher order structural and functional levels. METHODS: Predictions derived from multiple sequence alignments among IRBP and Tsp family members and secondary structure computer programs were carried out. The first repeat of human IRBP (EcR1) and Tsp were expressed, purified, and tested for binding properties. Tsp was examined for fluorescence enhancement of retinol or 16-anthroyloxy-palmitic acid (16-AP) to test for ligand binding. IRBP was tested for protease activity. RESULTS: Tsp did not exhibit fluorescence enhancement with retinol or 16-AP. IRBP did not exhibit protease activity. The positions of critical residues needed for the ligand binding properties of retinol were predicted. Primary sequence and three-dimensional similarity was found between Domain A of IRBP Repeat 3 and eglin c. CONCLUSIONS: The sequence similarity of Tsp and IRBP raised the possibility that each might share the function of the other protein: IRBP might possess protease activity or Tsp might possess retinoid or fatty acid binding activity. Our studies do not support such a shared function hypothesis, and suggest that the sequence similarity is the result of maintenance of structure. The finding of similarity to eglin c in Domain A suggests the possibility of a tight interaction between Domain A and Domain B, possibly implying the need for Domain A in retinoid-binding, and suggesting that both Domains should be present in testing mutations. The positions of predicted critical amino acids suggest models in which a large binding pocket holds the retinoid or fatty acid ligand. These predictions are tested in a companion paper.

Effects of dispersed point substitutions in Repeat 1 of human interphotoreceptor retinoid binding protein (IRBP).

PURPOSE: The purpose of this study was to measure the effects of mutations on the retinol binding capability of human Repeat 1 of interphotoreceptor retinoid-binding protein (IRBP). First, we predicted important functional amino acids by several computer programs. We also noted the lack of shared functions between Tail-specific protease (Tsp) and IRBP, which bear sequence similarity, and this aided in predicting functional residues. We analyzed the effects of point substitutions on the retinol and fatty acid binding properties of Repeat 1 of human IRBP at 25 and 50 degrees C. METHODS: To find residues critical to retinol binding that might affect function, a series of thirteen mutations were created by site-specific mutagenesis between positions 140 and 280 in Repeat 1 of human IRBP. These mutants were expressed, purified, and tested for binding properties. The conformations of the proteins were examined by circular dichroism (CD) scans. RESULTS: Seven of the mutations exhibited reduced binding capacity, and five were not expressed at high enough levels to assess binding activity. Four of the mutants were purified, and their CD scans were very similar to those of Repeat 1. Only one of the mutations did not affect binding, folding, or expression when compare to wild type Repeat 1. CONCLUSIONS: Several IRBP mutants containing point mutations retained native structure but lost retinol binding function. The data suggest that retinol binding is affected by many different amino acid substitutions in or near a binding pocket. That even a single point substitution can profoundly affect binding without affecting overall conformation suggests that much of Domain B (from amino acid positions 80 to 300) is involved with ligand binding. This excludes three previously proposed IRBP-retinol binding mechanisms: (1) retinol binds to a small portion of the protein repeat, (2) retinol can bind to any hydrophobic patch in the protein, and (3) native conformation is not required for retinol binding to the repeat.

G239T mutation in Repeat 1 of human IRBP: possible implications for more than one binding site in a single repeat.

PURPOSE: Interphotoreceptor retinoid-binding protein(IRBP) is a four-repeat protein found in the interphotoreceptor space. Each repeat can bind retinoids and fatty acids. The purpose of this study was to examine the effects of the single amino acid substitution, G239T, versus the wild type sequence of human IRBP Repeat 1, on ligand binding at equilibrium, ligand off rates, and protection of retinol from degradation. METHODS: G239T was created by site-specific mutagenesis, expressed in E. coli, and purified. E. coli expressed wild type Repeat 1 (EcR1) and G239T were subjected to thermal denaturation and analyzed by circular dichroism spectroscopy. We compared the ligand binding properties by fluorescence enhancement of retinol and 16-anthroyloxy-palmitate, tryptophan quenching of the proteins by different ligands, binding competition assays, protection of retinol from degradation, and stopped-flow kinetics to measure transfer of ligands to and from model membranes. RESULTS: Circular dichroism, fluorescence, and absorbance spectroscopy of G239T and EcR1 showed similar wavelength scans. G239T exhibited about three-fold less fluorescence of bound all-trans-retinol or 13-cis-retinol versus EcR1. Retinol quenching of intrinsic protein fluorescence was reduced by 37% in G239T versus EcR1. Other retinoids used as quenchers produced no difference between intrinsic protein fluorescence of either G239T or EcR1; all exhibited saturable high affinity binding to each protein. Docosahexaenoic acid (DHA) served as a competitive inhibitor of retinol fluorescence enhancement with EcR1. However, DHA did not alter retinol fluorescence with G239T. 16-anthroyloxy-palmitate (16-AP) exhibited about 30% higher levels of fluorescence enhancement when bound to G239T versus EcR1. EcR1 prevented oxidative damage of all-trans-retinol, whereas G239T provided much less protection. Each protein could accept 9-cis-retinal from small unilamellar vesicles (SUVs) as measured by stopped flow kinetics. Off rates were the same in comparing G239T and EcR1 as acceptors. CONCLUSIONS: Despite the general similarity in shape between G239T and EcR1 and the nearly identical binding behavior with some ligands, distinct differences exist in the ligand binding properties of G239T and EcR1. Fluorescence enhancement/quenching and retinol protection experiments suggest that retinol binding is reduced by about 50% in G239T versus EcR1. The data suggest that either: (1) EcR1 contains two binding sites for retinol and G239T has lost one site or (2) EcR1 has a single binding site that is altered in G239T to reduce retinol binding. Results of all the experiments were consistent with the first model while some of the data were not consistent with the second model. Thus, it is possible that position 239, found in Domain B2 of IRBP Repeat 1, is located in or near one of two retinol binding sites.

Direct olfactory transport of inhaled manganese ((54)MnCl(2)) to the rat brain: toxicokinetic investigations in a unilateral nasal occlusion model.

Inhalation exposure of humans to high concentrations of manganese (Mn) is associated with elevated Mn levels in the basal ganglia and an extrapyramidal movement disorder. In the rat, direct olfactory transport of Mn from the nose to the brain has been demonstrated following intranasal instillation of (54)MnCl(2). However, the contribution this route makes to brain Mn delivery following inhalation is unknown and was the subject of our study. Male 8-week old CD rats underwent a single 90-min nose-only exposure to a (54)MnCl(2) aerosol (0.54 mg Mn/m(3); MMAD 2.51 microm). The left and right sides of the nose and brain, including the olfactory pathway and striatum, were sampled at 0, 1, 2, 4, and 8 days postexposure. Control rats were exposed to (54)MnCl(2) with both nostrils patent to evaluate the symmetry of Mn delivery. Another group of rats had the right nostril plugged to prevent nasal deposition of (54)MnCl(2) on the occluded side. Gamma spectrometry (n = 6 rats/group/time point) and autoradiography (n = 1 rat/group/time point) were used to compare the levels of (54)Mn found on the left and right sides of the nose and brain to determine the contribution of olfactory uptake to brain (54)Mn levels. Brain and nose samples from the side with the occluded nostril had negligible levels of (54)Mn activity, validating the nasal occlusion procedure. High levels of (54)Mn were observed in the olfactory bulb and tract/tubercle on the side or sides with an open nostril within 1-2 days following inhalation exposure. These results demonstrated, for the first time, that the olfactory route contributes the majority (up to >90%) of the (54)Mn found in the olfactory pathway, but not in the striatum, of the rat brain up to 8 days following a single inhalation exposure. These findings suggest that the olfactory route may make a significant contribution to brain Mn levels following inhalation exposure in the rat.

A side-by-side comparison of carbon dioxide resurfacing lasers for the treatment of rhytides.

BACKGROUND: The use of cutaneous resurfacing lasers to treat rhytides is widely accepted. Several carbon dioxide lasers, many using fundamentally different technologies, are available. OBJECTIVE: The purpose of this study was to compare the results obtained and side effects after treating rhytides with 3 different carbon dioxide resurfacing lasers. METHODS: We performed a randomized, blinded, prospective study wherein 16 subjects had either periorbital or perioral rhytides resurfaced with 1 of 3 carbon dioxide lasers on either side of the face. RESULTS: We were unable to demonstrate any significant differences in improvement in rhytides, patient satisfaction, posttreatment erythema, or other side effects. CONCLUSION: Our results suggest that operator technique and patient selection are more important factors than laser type with respect to outcome.

Structure-function relationships in the four repeats of human interphotoreceptor retinoid-binding protein (IRBP).

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) binds hydrophobic ligands in the interphotoreceptor space. Human IRBP consists of 1230 amino acids in four 300 amino acid long repeats. We asked: 1. Whether each of the four repeats can bind retinoids or fatty acids, 2. Whether each repeat can prevent retinol degradation in aqueous solutions, 3. Whether a ligand can stabilize the protein from thermal denaturation, 4. Whether the four repeats can be further classified into two groups. Our rationale was to make each repeat from the human cDNA and then examine structural and functional characteristics. METHODS: Individual repeats were produced in E. coli and the whole protein was expressed in baculovirus. Binding properties with all-trans-retinol were characterized by ligand fluorescence enhancement. The quenching of protein fluorescence by retinol, 9-cis-retinal, all-trans-retinoic acid, beta-ionine, alpha-ionine, trans-parinaric acid, and DHA was also examined. Binding curves were analyzed by nonlinear regression. Prevention of retinol decomposition was measured by absorption spectroscopy. Circular dichroism was examined in the far UV range to study protein secondary structure and the near UV range to study ligand binding effects on the tryptophan environment. RESULTS: Temperature dependent denaturation suggests that EcR1 is the most stable of the four repeats. Each repeat possesses the capability of binding 9-cis-retinal, all-trans-retinol, all-trans retinoic acid, docosahexaenoic acid, alpha- and beta-ionine, and trans-parinaric acid. Protein fluorescence quenching by retinol and retinol fluorescence enhancement assays yielded similar binding parameters for each repeat. Each expressed repeat prevents the degradation of retinol in aqueous solutions. CONCLUSIONS: The data contrast with the idea that two or more repeats are needed to bind one molecule of ligand. Each repeat binds both retinoids and analogs, suggesting that each has multiple ligand binding sites or one binding site with affinity for different ligands. Together, the results suggest that each repeat retains all functions of the whole protein. However, there are distinguishing characteristics among the repeats in their ligand binding properties, though the four repeats cannot be classified into just two distinctive groups. Last, these data fit well with the current model of multiple binding sites in IRBP derived from quadruplication of an ancestral monomeric binding protein.

Correlation of regional formaldehyde flux predictions with the distribution of formaldehyde-induced squamous metaplasia in F344 rat nasal passages.

Squamous epithelium lines the nasal vestibule of the rat, rhesus monkey, and human. Respiratory, transitional, and olfactory epithelia line most areas posterior to the nasal vestibule. Inhaled formaldehyde gas induces squamous metaplasia posterior to the nasal vestibule and does not induce lesions in the nasal vestibule in rats and rhesus monkeys, indicating that squamous epithelium is resistant to irritant effects of formaldehyde and that squamous metaplasia may be an adaptive response. If squamous metaplasia is determined by formaldehyde dosimetry rather than by tissue-specific factors, squamous epithelium may be protective by absorbing less formaldehyde than other epithelial types. In a previous study, a three-dimensional, anatomically accurate computational fluid dynamics (CFD) model of the anterior F344 rat nasal passages was used to simulate inspiratory airflow and inhaled formaldehyde transport. The present study consisted of two related parts. First, the rat CFD model was used to test the hypothesis that the distribution of formaldehyde-induced squamous metaplasia is related to the location of high-flux regions posterior to squamous epithelium. Regional formaldehyde flux into nonsquamous epithelium predicted by the CFD model correlated with regional incidence of formaldehyde-induced squamous metaplasia on the airway perimeter of one cross-sectional level of the noses of F344 rats exposed to 10 and 15 ppm formaldehyde gas for 6 months. Formaldehyde flux into nonsquamous epithelium was estimated to vary by an order of magnitude depending on the degree of formaldehyde absorption by squamous epithelium. These results indicate that the degree to which squamous epithelium absorbs formaldehyde strongly affects the rate and extent of the progression of squamous metaplasia with continued exposure to formaldehyde. In the second part of this study, the CFD model was used to predict squamous metaplasia progression. Data needs for verification of this model prediction are considered. These results indicate that information on the permeability of squamous epithelium in rats, monkeys, and humans is important for accurate prediction of uptake in regions posterior to the nasal vestibule.

Computer simulation of inspiratory airflow in all regions of the F344 rat nasal passages.

Data from laboratory animal experiments are often used in setting guidelines for safe levels of human exposure to inhaled materials. The F344 rat has been used extensively in laboratory experiments to determine effects of exposure to inhaled materials in the nasal passages. Many inhaled materials induce toxic responses in the olfactory (posterior) region of the rat nasal passages. The location of major airflow routes has been proposed as playing a dominant role in determining some olfactory lesion location patterns. Since nasal airflow patterns differ significantly among species, methods are needed to assess conditions under which these differences may significantly affect extrapolation of the effects of local dose in animals to potential disease outcome in humans. A computational fluid dynamics model of airflow and inhaled gas uptake has been used to predict dose to airway walls in the anterior F344 rat nasal passages (Kimbell et al., Toxicol. Appl. Pharmacol., 1993; 121, 253-263). To determine the role of nasal airflow patterns in affecting olfactory lesion distribution, this model was extended to include the olfactory region. Serial-step histological sections of the nasal passages of a F344 rat were used to construct the computer model. Simulations of inspiratory airflow throughout the rat nasal passages were consistent with previously reported experimental data. Four of the five major simulated flow streams present in the anterior nose (dorsal lateral, middle, ventral lateral, and ventral medial streams) flowed together to exit ventrally at the nasopharyngeal duct, bypassing the ethmoid recesses. The remaining dorsal medial stream split to flow both medially and laterally through the olfactory-epithelium-lined ethmoid recesses in a Z-shaped pattern when viewed sagitally. Simulated flow in the ethmoid recesses was more than an order of magnitude slower than flow in the anterior and ventral parts of the nasal passages. Somewhat higher volumes of flow were predicted in the dorsal medial stream when the nasal vestibule was reshaped to be upturned, and more flow was allocated to the dorsal medial stream with increased inspiratory airflow rate, suggesting that rats may be able to allocate more airflow to this stream by both modifying the shape of the nasal vestibule and increasing inhaled air velocity during sniffing. The present study provides the first description of flow in the complex olfactory region of the nose of the F344 rat. This model will be used to evaluate the role of airflow patterns in determining the distribution of xenobiotically induced olfactory mucosal lesions. This information, combined with models of disposition in the airway lining, will provide comprehensive dosimetry models for extrapolating animal response data to humans.

Upper respiratory tract surface areas and volumes of laboratory animals and humans: considerations for dosimetry models.

To facilitate the development of regional respiratory tract dosimetry comparisons between laboratory animal species and humans, published surface area (SA) and volume (VOL) data for the upper respiratory tract (URT) were reviewed. The review of the literature revealed that (1) different studies used different techniques to prepare specimens and make measurements, (2) different areas of the URT were measured, and (3) URT surface areas and volumes have been reported for a limited number of individual subjects within a species but for a relatively wide range of species. The published data are summarized in tables in this article. New measurements made in an F344 rat and in a female human subject are also presented. Despite the differences in experimental protocols, it was possible to fit allometric scaling equations to the data: In(SA, cm2) = -0.34 + 0.52 In(body weight, g) and In(VOL, cm3) = 1.70 + 0.78 In(body weight, g). Separate scaling equations were also fitted for rats alone. To illustrate the use of these scaling equations in quantitative human health risk assessment, two dose metrics (fractional absorption/cm2 URT SA and fractional absorption/g body weight) for predicted URT uptake in laboratory animals and humans were calculated for acrolein and epichlorohydrin. Expressed as an animal-to-human ratio, the 95% confidence interval for URT SA could change the predicted dose ratio by up to a factor of 2. Additional studies are needed to describe the entire URT (from the nares through the larynx) quantitatively and to decrease variability in scaling equation predictions as well as to develop additional species-specific scaling equations. Three-dimensional imaging techniques provide a noninvasive method to obtain URT surface areas and volumes in humans and the larger laboratory animals. Comparisons of magnetic resonance image (MRI) and computed tomography (CT) scans made as part of this study suggest that the greater clarity of the mucosal-air interface in the CT image provides better resolution for the study of anatomic features. Because there is no radiation exposure associated with MRI imaging, however, it is more safely used than CT scans in making repeated measurements in a subject to elucidate changes in URT geometry associated with normal nasal cycling or other physiological changes.

Structure-function relationships in interphotoreceptor retinoid-binding protein (IRBP).

PURPOSE: Interphotoreceptor retinoid binding protein (IRBP) binds hydrophobic ligands in the retina. The polypeptide consists of 1230 amino acids in four 300 amino acid long repeats. We asked whether each of the four repeats can bind one retinoid or fatty acid analog. Our rationale was to make protein variants from the human cDNA bearing one or more of the repeats and examine binding capacities and dissociation constants. METHODS: Proteins were characterized by SDS-PAGE, western blotting, N-terminal sequencing, and CD spectroscopy. Binding properties with all-trans-retinol and 16-anthryloxy-palmitic acid (16-AP) were characterized by ligand fluorescence enhancement and curve fitting. RESULTS: Binding capacities varied according to the length of each protein. Each repeat possesses the capability of binding retinol and 16-AP. CONCLUSIONS: The data contrast with the idea that two or more repeats are needed to bind one molecule of ligand. Each repeat binds a retinoid and fatty acid analog, suggesting that each has multiple ligand binding sites or one binding site with affinity for different ligands. Last, these data fit well with the current model of multiple binding sites in IRBP derived from quadruplication of an ancestral monomeric binding protein.

Correlation of regional and nonlinear formaldehyde-induced nasal cancer with proliferating populations of cells.

Formaldehyde induces nonlinear, concentration-related increases in nasal epithelial cell proliferation and squamous cell carcinomas (SCC) in rats. A formaldehyde carcinogenicity study was conducted in which a major end point was correlation of cell proliferation indices with sites of formaldehyde-induced SCC. A poor correlation in certain sites led to incorporation of the number of cells in each site into the correlation. Rats were exposed (6h/day, 5 days/week) to formaldehyde (0, 0.7, 2, 6, 10 or 15 ppm) for up to 24 months with interim sacrifice time points at 3, 6, 12, and 18 mo. A unit length labeling index (ULLI; S-phase nuclei/mm basement membrane) was determined for specific nasal regions in addition to a population-weighted ULLI (PWULLI). The PWULLI was defined as the product of regional ULLI and total number of nasal epithelial cells in the respective site. Nasal SCC sites of origin were mapped. Formaldehyde induced SCC in a highly nonlinear fashion, with no observed effect at the level of 2 ppm, a minimal response at 6 ppm, and a sharp increase at 10 and 15 ppm. The tumor incidence was 1, 22, and 47% at 6, 10 and 15 ppm, respectively. ULLI was significantly (P<0.05) increased at 10 and 15 ppm but not at the lower concentrations. There was a good correlation between PWULLI and regional tumor incidence (R(2) = 0.88), while the correlation of regional SCC with ULLI was relatively poor (R(2) = 0.46). We conclude that target cell population size and sustained increases of cell proliferation in these populations, determined by differences in regional airflow-driven formaldehyde binding to DNA dose to these sites, coupled with the known nonlinear kinetics of formaldehyde binding to DNA, can together account for the nonlinearity and site specificity of formaldehyde-induced nasal SCC in rats.