* Department of Cell Biology and Anatomy, Southwest Environmental Health Science Center, The University of Arizona, P.O. Box 245044, Tucson, Arizona 85724, and
DuPont Haskell Laboratory for Health and Environmental Sciences, E. I. DuPont de Nemours and Co., Inc., P.O. Box 50, Newark, Delaware 19714
Received April 18, 2003; accepted July 9, 2003
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ABSTRACT |
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Key Words: vinyl acetate; nasal epithelial cells; acidification; dose-response; confocal microscopy.
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INTRODUCTION |
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Intracellular proton concentration is maintained by two basic mechanisms: intracellular buffers such as organic acids, bases, and proteins, and plasma membrane-bound transporters such as the Na+/H+ antiporter (Goldsmith and Hilton, 1992; LaPointe and Batlle, 1996
). At low vinyl acetate exposure levels, the addition of protons to the intracellular environment through the metabolic formation of acetic acid would be buffered by these mechanisms. However, at higher exposure levels, it might be expected that normal intracelluar pH (pHi) could be altered beyond homeostatic bounds. Since enzyme systems have critical pH optima, perturbations in normal pHi can adversely affect the physiology and replication of cells (Goldsmith and Hilton, 1992
). It is proposed that such a perturbation, if sustained, is an integral part of a sequence of events leading eventually to cellular proliferation, neoplastic transformation, and cancer. Conversely, controlling vinyl acetate exposure to levels below those that significantly reduce pHi should result in disproportionate risk reduction.
Recently published data indicate that exposure to vinyl acetate can lead to immediate decreases in pHi in isolated rat hepatocytes in a dose-dependent manner (Bogdanffy, 2002). However, vinyl acetateinduced decreases in pHi have not been directly observed in nasal epithelial cells. We have therefore developed an in vitro assay system for observing alterations in pHi by use of a pH-sensitive fluorescent marker that is held within the cytoplasm of isolated rat nasal epithelial cells. This system has the advantage that epithelial cells from both the respiratory and olfactory nasal regions can be independently isolated and tested. In addition, data can be collected on individual cells, thus allowing assessment of the cellular variability of responses. Thus, the objective of the current project was (1) to determine the pHi dose-response characteristics of nasal epithelial cells upon exposure to vinyl acetate, and (2) to demonstrate that changes in pHi following vinyl acetate exposure are related to carboxylesterase-mediated metabolism.
The method chosen for measuring pHi was confocal microscopy fluorescence image ratioing. This method has been used to investigate pH heterogeneity across the plasma membrane (Maouyo et al., 2000) and to examine the effect of oxygen deprivation on pHi (Yao et al., 2001
). This technique has the distinct advantage of measuring changes in pHi within large populations of single cells, thereby allowing for the monitoring of a heterogeneous population. The fluorescent dye carboxyseminaphthorhodafluor-1 (SNARF-1) is loaded into test cells in buffered medium using the acetoxymethyl ester form of SNARF-1 that readily diffuses into cells. Esterases present in the cells hydrolyze the acetoxymethyl ester, leaving the SNARF-1 trapped within the cell cytoplasm. SNARF-1 is a dual-emission indicator, which can be excited with the 488-nm line of an argon laser. The emission spectra vary as a function of pH. In the presence of more basic pH media, the SNARF-1 emission peak is above 600 nm. However, as the media pH become acidic, the peak shifts toward the blue. The ratio of emission intensity at greater than 665 nm to the emission at 600 can be used to determine the pH. The use of ratiometric measurements reduces errors due to cellular loading of dyes and to photobleaching. Emission ratios can be readily calibrated to yield quantitative measures of pHi. Nigericin-induced permeablization of cells, and cell-specific measures of 665/600 emission ratio, allow for calibration curves to be generated for individual cells.
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MATERIALS AND METHODS |
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Animals.
Adult male CrlCD: BR rats (approximately 250350 g) were obtained from Charles River Breeding Laboratories (Raleigh, NC). Upon receipt, the rats were placed in polycarbonate shoe-boxstyle cages, housed two per cage, provided with Purina Certified Rodent Chow (Ralston Purina, St. Louis, MO) and tap water ad libitum, and quarantined for a 1-week period. During the quarantine period, all of the rats were observed with respect to eating habits, weight gain, and any gross signs of disease or injury. All of the rats were clinically normal and free of antibody titers to pathogenic murine viruses and mycoplasma, and free of pathogenic endo- and ectoparasites and bacteria. All protocols were approved by the University Institutional Animal Care and Use Committee.
Isolation of cells.
For the isolation of hepatocytes, the rats were anesthetized with sodium pentobarbitol. The livers were perfused via the hepatic portal vein with a Ca2+-free buffer followed by a collagenase buffer. The liver was then removed and placed in Waymouths media, and the cells were gently dissociated into suspension. The cell suspension was then filtered through gauze and centrifuged twice for 1 min at 400 rpm. The cells were then pre-incubated in Waymouths media for 2 h on Bioptechs confocal culture plates coated with Cell Tak. Each plate was coated with a solution containing 5-µl Cell Tak, 2.5-µl NaOH, and 67.5-µl sodium bicarbonate buffer, pH 8.0. The plates were air-dried for 20 min, rinsed with water, air-dried, and stored at 4°C until used.
For nasal epithelial cells, the ethmoturbinates (olfactory) and maxilloturbinates (respiratory) were removed from the rats as described in Uriah and Maronpot (1990). For the analysis of explants, the turbinates were cut into smaller pieces, loaded with dye, and observed directly with the confocal microscope. For cell isolation, the turbinates were placed separately in 5-ml digestion solution [0.5% Type XIV Protease, 0.1% collagenase (type IV), 0.1% hyaluronidase (type IV-S), as described by Steele and Arnold (1985)
] on a rotating shaker. After 30 min, the digestion solution was collected and the turbinates were placed in fresh 5 ml of digestion solution. The collected digestion solution was kept on ice until centrifuged. Following another 30 min, the digestion solution was again collected and pooled with the original digestion solution. The solutions were spun for 15 min at 300g in a Sorvall RT6000B refrigerated centrifuge and the supernatant was removed. The cell pellet was resuspended in 10 ml of incubation media [F-12 media, 0.1-µg/ml hydrocortisone, 5-µg/ml transferrin, 10-µg/ml insulin, 25-ng/ml EGF, 50-µg/ml bovine pituitary extract, 28-ng/ml retinol, 25-µg/ml gentamycin, 100-U/ml penicillin, and 100-µg/ml streptomycin, as described by Fanucchi et al. (1999)
]. The cells were respun and resuspended in 1-ml incubation media containing 20-ng/ml cholera toxin and were plated on to Bioptechs confocal culture plates. The cells were allowed to adhere for at least 1 h prior to analysis. The plates were coated with Cell Tak to increase cell adhesion.
Loading cells with SNARF-1 and confocal fluorescence imaging microscopy.
Explants or cells adhered to Bioptechs plates were washed three times in phosphate-buffered saline with glucose (PBSG) and incubated for 30 min at room temperature in 20-µM SNARF-1 in solution containing 0.004% Pluronic F127 (final concentration). The cells were then washed three times with PBS and kept at room temperature in the dark. The plates were then mounted onto a stage holder on either a Leica TCS or Zeiss 510 confocal microscope. Similar results were found with both microscopes. Fields were located that contained at least five cells. The intracellular pH was tracked using the argon excitation line of 488 nm. Emission was recorded using selected filters. For the Leica, the images were collected using an inverted microscope and x20 objective, NA 0.6. Emission was recorded using 600-nm bandpass, 30-nm width at half-max (green channel) and 665-nm long-pass (red channel) filters. For the Zeiss, the images were collected using an upright microscope and a x40 water immersion objective, NA 0.8. Emission spectra were recorded using the META system, with emissions collected at 578621 nm (green channel) and 621685 nm (red channel). The images were averaged over eight frames. Eight bit images were collected using the photomultiplier tubes (PMT) of the confocal microscopes. The laser power and PMT settings were adjusted to produce a full range of intensities without saturation of the PMTs. One to five fields per experimental manipulation were used to estimate the changes in intensity. The captured images were further processed using Compix Simple PCI (Version 4.06.1605) (Pittsburgh, PA) software. A threshold intensity level was used to separate the cells from the background. Cells in close proximity were isolated using the watershed algorithm. Size exclusion was used to further identify cells from small background fluorescent particles. All image manipulation was validated by operator observation. Background intensity levels were determined for both the green and red images. Data were collected as average intensity for each individually identified cell. Following background subtraction, the ratio of red intensity to green intensity was calculated as a measure of pH.
Cell treatments and pHi calibration.
Solutions of vinyl acetate in PBS were prepared immediately prior to use. The pH of vinyl acetate solutions was checked prior to use and was 7.4. Following collection of the control images, the cells were exposed to vinyl acetate applied as a bolus to the plate. Doses applied to the cells were 100, 250, 500, and 1000 µM. Concentrations given are final concentrations in the plate. For washes, solutions were removed from the cells and the plates were washed with fresh saline solution. At the conclusion of the test interval, the cells were then washed with freshly prepared calibration solutions adjusted in 0.25 increments from pH 6.50 to pH 7.75. The buffer solutions contained 15-mM N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 15-mM 2-morpholinoethanesulfonic acid monohydrate (MES), 130-mM KCl, 1-mM MgCl2, and 10-µM nigericin and were calibrated against buffer standards (Beckman, Naguabu, Puerto Rico). The emission ratio was then monitored. At least three image readings were collected at each pH. These data were then averaged and a standard curve of emission ratio vs. pHi created. The emission ratio for each cell collected during the test interval was converted to pHi units. To account for differences between experiments, pH calibration was normalized to values at pH 7. The standard curve was drawn by linear lines between standards. Unknown pH values were calculated by linear interpolation between calibration points.
Statistics.
Average pHi values measured during the control (PBS alone) or test intervals were calculated on a per-cell basis. These values were then averaged for all of the cells monitored. Images were collected over one to five random fields, and data from each cell were used to calculate the overall pHi. The treatment-induced change in pHi (pHi) was calculated by subtracting the pHi value measured during the test phase from the pHi recorded during the treatment phase. For data obtained from respiratory epithelial cells, which are normally distributed, the data were analyzed by ANOVA (p < 0.05). Significant differences in distributions in data obtained from olfactory cells were analyzed using a chi-squared test (p < 0.05). Significant differences in
pHi were analyzed using ANOVA (p < 0.05).
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RESULTS |
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To validate the imaging measurements, rat hepatocytes were isolated and exposed in vitro to 1-mM vinyl acetate. Previous reports have demonstrated that the exposure of rat hepatocytes to vinyl acetate results in a rapid acidification (Bogdanffy, 2002). A total of 159 to 232 cells were analyzed in the control, vinyl acetate, and washed samples. The pHi levels measured in these experiments demonstrate a range of values from an apparent single population of cells. Upon addition of 1-mM vinyl acetate, the intracellular pH distribution shifted toward more acidic levels. Washing the cells resulted in a return to the control levels. The mean pHi values were 7.40 ± 0.01 for the control cells (N = 232) and 7.30 ± 0.01 following the wash (N = 185). After administration of vinyl acetate, the pHi decreased to 6.79 ± 0.02 (N = 159). These results are similar to those previously reported using different imaging techniques (Bogdanffy et al., 2002
), thus validating the use of SNARF-1 confocal microscopy image ratioing to determine pHi.
Response of Nasal Epithelial Cells
All cells analyzed were viable, since activation of the pH-sensitive dye requires the presence of active esterases in the cytoplasm. Damage of the cell membrane allows cytoplasmic esterases to leak from the cells. Damaged cells would not be able to trap and activate the dye and would therefore not fluoresce. While the absence of carboxylesterases has been shown in sensory cells in the olfactory epithelium (Bogdanffy et al., 1986), they will presumably contain other nonspecific cytoplasmic esterases capable of activation and trapping of SNARF-1. The cells appeared rounded and did not spread on the plates during the 1-h incubation. Cell viability was greater then 90% in all samples as measured using Live/Dead Assay from Molecular Probes. The cells isolated from the respiratory epithelium were slightly larger than the cells isolated from the olfactory epithelium (15 µm for respiratory versus 10 µm for olfactory cells). Cells isolated from the respiratory epithelium showed occasional beating cilia. The olfactory epithelial cells were much less adherent than the respiratory epithelial cells. Red blood cells isolated along with the epithelial cells were excluded from analysis based on size (approximately 5 µm).
The response of isolated respiratory and olfactory nasal epithelial cells is shown in Figure 1. The pHi values for the respiratory epithelial cells were distributed in a single modal distribution (Fig. 1A
). Upon exposure to 1-mM vinyl acetate, the pHi decreased. At this particular exposure and time (5 min after addition of vinyl acetate), the response suggests a bimodal distribution. This may be due to the activation of pH compensation mechanisms in the cells (Paradiso, 1997
), since lower concentrations and earlier time points did not show this effect. Subsequent washing of the cells restored the pHi to the control values.
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In contrast to the respiratory epithelial cells, the olfactory epithelial responses were not normally distributed (Fig. 2 shows data from one of nine dose-response experiments). For the data shown in Figure 2
, peak pHi values of responding cells in the olfactory population are 7.32 for controls (N = 69 cells), 7.29 for 100 µM (N = 87), 7.18 for 250 µM (N = 71), 7.10 for 500 µM (N = 69), and 7.00 for 1000 µM (N = 71). Examination of Figure 2
shows that, as the dose of vinyl acetate is increased, cells isolated from the olfactory region undergo acidification. Peak values shift toward a lower pHi at each subsequent dose. Chi-squared analysis of the distributions showed that all groups were significantly different from each other, with the exception of the 100- and 250-µM exposure groups, which were not significantly different from each other in this experiment (p = 0.056).
Data from all dose-response experiments are summarized in Figure 4. Data from each experiment were used to calculate the
pHi means and standard deviations. (For respiratory epithelial cells, N = 9 experiments for 100, 250, and 500 µM and 16 experiments for 1000 µM. For olfactory epithelial cells, N = 9 experiments for all doses). For respiratory epithelial cells,
pHi was calculated as the difference between the average cellular pHi for all control cells for that experiment and the average cellular pHi of each test field. For the olfactory epithelial cells, values for all cells and for responding cells alone were used to calculate
pHi. Responding cells were determined by evaluating the shifts in the distribution curves between the controls and each of the treatment doses.
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Role of Carboxyesterase
The role of carboxylesterase in the acidification was tested by application of bis(p-nitrophenyl)phosphate (BNPP) to inhibit carboxylesterase-mediated hydrolysis of vinyl acetate to acetic acid. Figure 5 shows a response for olfactory epithelial cells. Application of 100-µM vinyl acetate lead to an acidification. Following a wash, 100-µM BNPP was added to the cells. The addition of BNPP itself led to a slight acidification. However, the addition of 100-µM vinyl acetate in the presence of BNPP did not cause further acidification.
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DISCUSSION |
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Validation of the confocal technique was achieved using rat hepatocytes. Exposure of the hepatocytes to 1-mM vinyl acetate lead to an acidification of approximately 0.50.6 pH units, similar to the effect reported by Bogdanffy (2002). The use of confocal microscopy in conjunction with image analysis has allowed us to collect data on a large number of individual cells. Individual cellular pHi values were distributed over a broad range. This variability appears to be real differences in pH and not an artifact of the measurement system. Differences in dye loading and photobleaching cannot explain the dispersion. These differences are eliminated by use of the ratio method.
Differences in the pHi distributions were seen between the respiratory and olfactory nasal epithelium. The respiratory cells showed a normal distribution, suggesting a single population of cells. However, the olfactory epithelium showed two apparent populations with different basal pHi values (Fig.1B). Of these two populations, only one responded to vinyl acetate exposure. The nonresponding cells did change their pHi in response to nigericin calibration solutions, suggesting normal rather than artifactual behavior under the conditions of the assay. The calibration solutions also abolished the bimodal distribution.
The acidification response of the nasal epithelial cells was not as large as that of the hepatocytes. The maximal response in the respiratory nasal epithelial cells was only 0.3 unit. Smaller changes were observed among the entire olfactory cell population (maximum of 0.1 pH unit). However, a more pronounced response to vinyl acetate treatment was observed among a subset of olfactory cells. Previous reports have shown that in the olfactory epithelium, only sustentacular cells contain carboxylesterase, which is required for vinyl acetate metabolism to acetic acid (Bogdanffy et al., 1986; Lewis et al., 1994
). These cells constitute approximately 30% of the cells of the olfactory epithelium. If this same percentage is present in the cell isolates, then the actual change in the sustentacular cells alone would be three times that seen in the whole population.
The changes in pHi that were observed in individual cells are consistent with the changes seen in situ. Analysis of cells in the explants of the olfactory epithelium demonstrated a bimodal response following exposure to vinyl acetate (Fig. 7B), while cells analyzed in the respiratory epithelium showed a single distribution (Fig. 7A
). In addition, the changes in pHi seen in the olfactory explants is much larger than that seen in the respiratory expants.
In vivo, sustentacular cells are arranged in the epithelial surface of the olfactory mucosa adjacent to sensory cells and provide support much like glial cells support neurons of the central nervous system. Because these cells are joined by tight junctions, it is likely that acidification-induced cytotoxicity of sustentacular cells in vivo leads to secondary acidification and cytotoxicity of the sensory cells. Sustentacular cells are the primary olfactory target cells of inhaled esters, and the susceptibility of sensory cells to acidification-induced cytotoxicity has been established (Nedergaard et al., 1991, Trela et al., 1992
). In vitro, where these tight junctions are lost in single-cell suspensions, sensory cells would not be expected to acidify upon exposure to vinyl acetate.
In respiratory epithelial cells, the pHi dropped proportionately with vinyl acetate concentration up to 250 µM, beyond which there were only slight decreases in the pHi (Fig. 4). Exposure to 1 mM even caused a slight increase in the pHi. Saturation of vinyl acetate metabolism is unlikely to be the cause of the plateau (Bogdanffy, 2002
; Simon et al., 1985
). Potentially, acidification of the cells may lead to the activation of the Na+/H+ antiporter, although this usually occurs at lower pH values than was seen in these experiments. Another possible contributor is the response of SNARF-1 at more acidic levels.
The observation that the acidification of respiratory epithelial cells achieves a plateau while the responding olfactory epithelial cell population does not achieve a plateau (Fig. 4) has important implications for the mode of action of vinyl acetate in nasal tissue. Chronic inhalation exposure of rats causes degeneration of the olfactory epithelium while the respiratory epithelium is largely resistant to the cytotoxic effects of vinyl acetate. Resistance of the nasal respiratory epithelium and susceptibility of the olfactory epithelium to inhaled esters is a common observation (examples include propylene glycol monomethyl ether acetate, ethyl acrylate, methyl acrylate, n-butyl acrylate, ethyl acetate, and dibasic esters), but the reason for the resistance of the respiratory epithelium is not understood, especially since dosimetry models for vinyl acetate and other inhaled esters predict greater dosimetry to the regions lined by the respiratory epithelium (Andersen et al., 2002
). The results reported here, suggesting an increased capability of the respiratory epithelial cells to regulate pHi compared to the olfactory epithelium, provide such an explanation. While the presence and activity of the Na+/H+ exchanger have been reported in ciliated nasal epithelium (Paradiso, 1997
), no such data exist for olfactory nasal epithelium. This mechanism for tolerance of respiratory epithelial cells could be extended to other inhaled chemicals. Hydrogen sulfide, for example, has been proposed to damage selectively olfactory epithelium through the inhibition of cytochrome oxidase, an electron transport complex critical to mitochondrial respiration (Moulin et al., 2002
).
Intracellular acidification has been proposed as the first pharmacodynamic step in a series of events that culminate in the tumorigenesis of nasal and upper gastrointestinal tract epithelial cells exposed to vinyl acetate (Bogdanffy, 2002). Decreases in pHi may play a role in the stimulation of cell growth and transformation (Valentine et al., 2002
). Syrian hamster embryo cells, cultured at pH 6.7, show a marked increase in lifespan, compared to those cultured at pH 7.3 (Kerckaert et al., 1996
). A lower pHi has been shown to displace Ca2+ from intracellular binding sites (Batlle et al., 1993
). Displacement of Ca2+ from the growth and differentiation factor (GDF) protein blocks the intracellular signaling that leads to differentiation (Isfort et al., 1993
) and could promote sustained proliferation, expansion of the undifferentiated cell population, and clonal expansion of spontaneous- or chemical-induced mutants. A reduction in pHi of neuronal cells below 0.15 pH unit results in cytotoxicity (Nedergaard et al., 1991
). Describing the extent of acidification of cells in response to vinyl acetate exposure will help to define better the potential risks involved in exposure to vinyl acetate. In the case of vinyl acetate, it has been proposed that risk management practices that reduce exposures, such that the proton burden is maintained within homeostatic bounds, would prevent all toxic response, including cancer, that follow (Bogdanffy et al., 1999
; Sarangapani et al., in press).
In conclusion, these experiments have shown that the exposure of rat nasal epithelial cells to vinyl acetate results in a dose-dependent decrease in intracellular pH. Changes occurred at 100 µM but reached a plateau above 250 µM. Olfactory epithelial cells demonstrated a bimodal distribution, with only one population of cells responding to vinyl acetate treatment. BNPP, a carboxylesterase inhibitor, was able to attenuate the vinyl acetateinduced decrease in pHi. These results are consistent with the mode of action proposed and supply further data for developing appropriate risk assessment approaches for vinyl acetate exposure. For the inhalation route of exposure, intracellular acidification of target sites within the nasal epithelium appears to be a primary pharmacodynamic change; downstream responses such as mitogenesis, cytotoxicity, and cell proliferation are secondary. These secondary responses occur at high exposure levels and only under bioassay conditions might they eventually lead to tumorigenesis. It follows that decisions aimed at protecting the tissue from intracellular acidification will be protective of all the downstream effects of vinyl acetate inhalation exposure, including cancer. Work is currently underway to investigate this mode of action in oral cavity epithelium.
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ACKNOWLEDGMENTS |
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NOTES |
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