DuPont Haskell Laboratory, E. I. du Pont de Nemours and Co., Inc., P.O. Box 50, Newark, Delaware 19714
Received September 21, 2001; accepted December 21, 2001
ABSTRACT
Cancerbioassays have demonstrated the carcinogenic activity of vinyl acetate in rodents. Tumors appear only at the site of contact and mechanistic data suggest that the carcinogenic mechanism involves carboxylesterase-mediated metabolism of vinyl acetate to acetic acid. It has been hypothesized that intracellular formation of acetate causes a reduction of intracellular pH (pHi) at noncytotoxic levels, but that prolonged exposure to reduced pHi is cytotoxic and/or mitogenic and drives proliferative responses. Coupled with exposure to metabolically formed acetaldehyde at high administered concentrations, nonlinear dose-response curves for epithelial tumors are produced. Freshly isolated rat hepatocytes were used as a model system to test the concept that exposure of cells to vinyl acetate causes a reduction in pHi. Quantitative fluorescence imaging ratio microscopy showed that exposure of hepatocytes to vinyl acetate concentrations ranging from 10 to 1000 µM caused rapid and sustained reductions of approximately 0.03 to 0.65 pH units. Cellular acidification was rapidly reversed to control pHi upon removal of vinyl acetate. There was minimal accumulation of protons during the exposure period, as suggested by minor differences in pHi of cells with or without prior exposure to vinyl acetate. The effect of vinyl acetate on pHi was attenuated by prior exposure to the carboxylesterase inhibitor bis(p-nitrophenyl)phosphate. These results support the concept that intracellular acidification is a sentinel pharmacodynamic response of cells to vinyl acetate exposure and that pHi is an appropriate metric dose for use in quantitative risk assessments of cancer and noncancer human health risk assessment.
Key Words: vinyl acetate; intracellular acidification; mode of action; cytotoxicity; mitogenicity.
Vinyl acetate is a synthetic organic ester with a wide range of uses including polyvinyl acetate emulsions in latex paints and in paper and paperboard coatings. By the inhalation route of exposure, vinyl acetate is carcinogenic in rats (600 ppm) but not mice (Bogdanffy et al., 1994a). When administered in drinking water, vinyl acetate is carcinogenic in rats and mice (
2000 ppm) (Bogdanffy et al., 1994b
; Japan Bioassay Research Center reported in U.S. EPA, 1997
). Vinyl acetate is only carcinogenic at the portal of entry, inducing nasal tumors by the inhalation route and oral cavity and esophageal tumors by the oral route. Vinyl acetate is metabolized via carboxylesterase to acetic acid and acetaldehyde (Bogdanffy and Taylor, 1993
; Simon et al., 1985
), and mechanistic studies have shown that formation of these metabolites is a key step in the pathogenesis of nonneoplastic and neoplastic responses (Kuykendall et al., 1993
). Previous efforts to develop a pharmacokinetic/pharmacodynamic model for nasal and oral cavity effects of vinyl acetate indicated that three protons should be produced per mole of vinyl acetate, leading to acidification of exposed tissues (Bogdanffy et al., 1999b
; Sarangapani et al., submitted). The ability of cellular metabolism of vinyl acetate to cause tissue acidification has not yet been verified.
Maintenance of pHi within a critical range is of paramount importance to the normal function of cellular machinery. Since enzyme systems have critical pH optima, perturbations in normal pHi can adversely affect physiology and reproduction of cells (Goldsmith and Hilton, 1992). 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). Thus addition of protons to the intracellular environment through metabolic formation of acetic acid would be offset by these mechanisms, although it might be expected that normal pHi could be altered beyond homeostatic bounds at high vinyl acetate concentrations. 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 could result in disproportionate risk reduction.
Presently, it is difficult to validate these predictions using nasal epithelial cells or to conduct the study in nasal tissues in vivo. A surrogate system was therefore developed by exposing hepatocytes to vinyl acetate and observing transient alterations in pHi with the use of a pH-sensitive fluorescent marker that is held within the cytoplasm of the isolated hepatocytes. Freshly isolated rat hepatocytes have the advantage of being readily available, can be isolated in large numbers, have the metabolic machinery necessary to metabolize vinyl acetate, and have carboxylesterase activity similar to nasal epithelium (Bogdanffy and Taylor, 1993; Simon et al., 1985
). Thus, the objectives of the current project were (1) to demonstrate "proof of concept" that pHi is reduced upon exposure of freshly isolated hepatocytes to vinyl acetate, and (2) to demonstrate that changes in pHi following vinyl acetate exposure are related to carboxylesterase-mediated metabolism.
MATERIALS AND METHODS
Chemicals.
Vinyl acetate (>99% purity as supplied) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Buffers and media were obtained from Gibco Laboratories (Grand Island, NY). Liberase was from Roche Diagnostics Corporation (Indianapolis, IN). BCECF-AM and Pluronic F127 was from Molecular Probes, Inc. (Eugene, OR). All other reagents were from Sigma Chemical Co. (St. Louis, MO).
Animals.
Adult male CrlCD: BR rats (approximately 250350 g) were obtained from Charles River Breeding Laboratories (Raleigh, NC). Upon receipt, rats were placed in polycarbonate shoebox-style cages, housed individually, and provided with Purina Certified Rodent Chow (Ralston Purina, St. Louis, MO) and tap water ad libitum; they were quarantined for a one-week period. During the quarantine period, all rats were observed with respect to eating habits, weight gain, and any gross signs of disease or injury. All rats were clinically normal and free of antibody titers to pathogenic murine viruses and mycoplasma. They were also free of pathogenic endo- and ectoparasites and bacteria.
Isolation of rat hepatocytes.
Rats were deeply anesthetized by ip injection of approximately 80 mg/kg sodium pentobarbital. The hepatic portal vein was exposed and catheterized, the vena cava severed, and the liver perfused at a rate of 2030 ml/min. Perfusion was first with Hank's balanced salt solution (without calcium) containing HEPES buffer and EGTA and then for 812 min with Leibovitz's (L-15) medium containing HEPES, calcium chloride, and liberase. The liver was removed, the capsule broken, and the cells released and combed free. The cells were resuspended in cold L-15, washed twice by centrifugation (50 x g), and resuspended. Viable hepatocytes were then purified by Percoll separation and washed twice with L-15. Cell yield, viability, and density were then measured by Trypan blue dye exclusion. Viability was greater than 80%.
Approximately 100 µl of cell suspension was then dispensed over approximately a 15 x 30 cm2 area on a 24 x 40 cm, polylysine-coated glass cover slip. The cover slips were then incubated for approximately 2 h at 37°C to allow cell attachment.
Loading Cells with BCECF-AM and Fluorescence Imaging Microscopy
The method chosen for measuring pHi was quantitative fluorescence imaging ratio microscopy (QFIRM). QFIRM is the most common and widely accepted method for measuring pHI. The technique has the distinct advantage of measuring changes in pHi within a single cell, thereby allowing for the monitoring of selected cells within a heterogeneous population. The fluorescent dye 2`,7`-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluoroscein, acetoxymethyl ester (BCECF-AM) is loaded into test cells in buffered medium. BCECF-AM is a highly lipophilic compound that readily diffuses into cells. Esterases present in the cells hydrolyze the acetoxymethyl ester, leaving BCECF free acid trapped within the cell cytoplasm. BCECF is a dual-excitation indicator with excitation near the absorption maximum (495 nm), the intensity of which is pH-dependent, and a second excitation near the excitation isosbestic point (440 nm), which is insensitive to pH. The ratio of excitation intensity at 490 nm to that at 440 nm is pH-sensitive and signal errors are canceled in the ratio measurement. Emission ratios can be readily calibrated to yield quantitative measures of pHi. Nigericin-induced permeability of cells, and cell-specific measures of 495/440 emission ratio, allow for calibration curves to be generated for individual cells.
Cells that adhered to cover slips were washed three times in phosphate-buffered saline with glucose (PBSG) and then incubated for 30 min at room temperature in 0.5 µM BCECF-AM. Stock solutions of BCECF-AM were prepared to 1 mM strength in anhydrous DMSO. Pluronic F127 (10% in DMSO) was added such that the final concentration in PBSG was 0.002%. Cells were then washed three times with PBS and kept at room temperature in the dark. Cover slips were then mounted onto a perfusion well, and the well was mounted onto an inverted fluorescence microscope. The well is an RC-21 600 µl closed chamber system with no headspace (Warner Instruments, Hamden, CT). The cells were then perfused at a rate of 2 ml/min with PBS with or without varied concentrations of vinyl acetate. Cells were located such that 412 cells per field were visualized under bright field. Intracellular pH was tracked using the QFIRM system.
QFIRM imaging studies were conducted using an inverted Nikon Diaphot microscope with a 40 x Fluor oil immersion objective (NA 1.3). Approximately every 5 s, cells were excited for 33 ms with wavelength pairs of 495 and 440 nm using interference filters, with fluorescence emission monitored at 530 nm. Emission intensity data was averaged over 16 frames/s. Illumination was controlled using a filter wheel (Lambda-10; Sutter Instruments, Novato, CA), and images were captured using an intensified charge-coupled device (C240097; Hamamatsu Photonics, Hamamatsu City, Japan). Control of image acquisition and subsequent image processing were performed using the MetaFluorTM imaging system from Universal Imaging Corporation (Downingtown, PA). Vinyl acetate and/or bis(p-nitrophenyl)phosphate (BNPP) were delivered by bath application using a peristaltic pump (IPS; Ismatec SA, Glattbrugg-Zurich, Switzerland) and switching valve (MV-8; Amersham Pharmacia Biotech, Inc., Piscataway, NJ), enabling rapid changes of solutions through the experimental chamber. Cells adhering to polylysine-coated slides formed the base of the chamber. The emission intensity ratio of 495/440 was then calculated.
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 stable readings of the 495:440 ratio, cells were perfused with test solutions at 2 ml/min. At the conclusion of the test interval, cells were then perfused with freshly prepared buffer solutions, adjusted to near pH 6.20 and pH 7.50 using a Sentron Titan X pH meter (Sentron, Inc., Gig Harbor, WA). 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). Emission ratio 495:440 was then monitored for each cell. Readings were stabilized using the high pH standard before switching the perfusion medium to the low pH standard. At least six readings were collected at each pH. These data were then averaged and a standard curve of 495:440 emission ratio vs. pHi created for each cell. The 495:440 emission ratio for each cell collected during the test interval was converted to pHi units.
Statistics
Average pHi values measured during the control (PBS alone) or test interval were calculated on a per-cell basis. These values were then averaged for all of the cells monitored. At least four cells were monitored per experiment (i.e., n = 412). The treatment-induced change in pHi (pHi) was calculated by subtracting the pHi value measured during the test phase from the average pHi recorded during the control treatment phase. Treatment-related effects were analyzed by Student's t-test (p < 0.05).
RESULTS
The pHi of control hepatocytes was approximately 6.9 (range of approximately 6.87.1) and gave adequate fluorescence emission for experimentation (Fig. 1). Intracellular pH of hepatocytes exposed to vinyl acetate decreased immediately upon exposure. Steady-state pHi was achieved within approximately 70 s. The minimal concentration of vinyl acetate inducing a statistically significant change in pHi was 10 µM, which caused a drop in pHi of approximately 0.03 units (Fig. 2A
). Vinyl acetate-induced
pHi was dose-dependent, with a clear break in the dose-response curve occurring at 500 µM (Fig 2B
). Hepatocytes appeared normal and remained attached to the cover slips at concentrations as high as 1000 µM. There was no microscopic evidence of cytotoxicity.
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In the present research, we have implemented a method that enables testing of the hypothesis that exposure of cells to organic esters results in cellular acidification. Such a mechanism is believed to be central to the toxicity of inhaled organic esters and also, potentially, to orally ingested organic esters such as vinyl acetate. These results confirm that exposure of hepatocytes to vinyl acetate results in intracellular acidification, that the response is concentration-dependent, and that the response is attenuated by carboxylesterase inhibition.
Intracellular pH dropped proportionately with vinyl acetate concentration up to 500 µM concentration, beyond which there was no further decrease in pHi (Fig. 2B). Saturation of vinyl acetate metabolism is unlikely to be the cause of the plateau observed in the 500 µM to 1000 µM concentration range, since the Km for hydrolysis is approximately 730 µM in rat liver microsomes (Simon et al., 1985
). It is possible that allosteric activation of the Na+/H+ antiporter at approximately pHi 6.36 contributes to the nonlinear behavior. In rat leukocytes, intracellular buffer capacity (mM/pH) doubles in the pHi range of 6.26.4 compared to a pHi range of 6.46.6 (Saleh and Batlle, 1990
). H+-dependent activation of the antiporter is reflected in the kinetic constants (Vmax
75 mM H/30 s; p Km
6.0, nH
1.5) where the Hill cooperativity coefficient is >1. Another possible contributor to the apparent nonlinear response is the known departure from linearity of the relationship between the 495:440 nm ratio and pH when <6.2 (Saleh and Batlle, 1990
).
The vinyl acetate-induced decrease in pHi was rapidly reversible upon removal of vinyl acetate (Fig. 4). This observation reflects both the high first order rate of clearance of vinyl acetate and the efficiency of the mechanisms buffering cells against the intracellular proton load that results from vinyl acetate metabolism. The first order rate of conversion of vinyl acetate to acetic acid, on a per-cell basis, is approximately 3 x 1010 µmol/s/cell/µM.2 Assuming complete ionization of acetic acid provides an equivalent rate of proton production from vinyl acetate.3 The high rate of proton efflux is illustrated by the complete and rapid return to control pHi within 50 seconds of removal of 200 µM vinyl acetate (Fig. 4
) and by the minimal accumulation of protons (calculated as
pHi ) observed during the dose addition experiment when compared to single dose treatments (Table 1
). Assuming hepatocyte antiport kinetic constants are equal to that of rat leukocytes, the proton exchange capacity of hepatocytes is near 2 x 1010 µmol/s/cell/µM, suggesting that cells are able to export protons at nearly the same rate as they are produced.4
The vinyl acetate-induced decrease in pHi was metabolism-dependent. BNPP, a nonspecific irreversible esterase inhibitor has been shown to inhibit the metabolism, cytotoxicity, and DNA-protein crosslinks of vinyl acetate in isolated rat nasal explants (Kuykendall et al., 1993). In vivo, BNPP pretreatment also reduces the nasal cavity extraction of inhaled vinyl acetate and exhalation of acetaldehyde (Bogdanffy et al., 1999a
). Pretreatment of rat hepatocytes with BNPP attenuated the vinyl acetate-induced decrease in pHi. These results demonstrate the specificity of the response and lend further support to the overall mode of action proposed for the carcinogenic activity of vinyl acetate in the upper respiratory and alimentary tract tissues.
Intracellular acidification is proposed as the first pharmacodynamic step in a series of events that culminate in tumorigenesis of nasal and upper gastrointestinal tract epithelial cells exposed to vinyl acetate (Fig. 6). The present research was aimed at demonstrating proof of the concept that the pHi of nasal epithelial cells decreases when exposed to vinyl acetate and that the decrease is a result of carboxylesterase-mediated metabolism. Hepatocytes were used as a model cell system. Intracellular pH is regulated within homeostatic bounds by two primary mechanisms: cellular buffers (such as phosphate, bicarbonate, and zwitterionic proteins) and plasma membrane transporters. Plasma membrane transporters include proton transporters (e.g., Na+/H+ antiporter, H+-ATPase, Ca2+ATPase, and an H+/K+-ATPase) and HCO3/CO2-dependent transporters (e.g., the Na+-independent Cl-/HCO3 exchanger, the Na+-dependent Cl-/HCO3 exchanger and the Na+, HCO3 symporter (LaPointe and Batlle, 1996).
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Demonstration that vinyl acetate exposure causes a reduction in pHi supports an approach to vinyl acetate risk assessment that utilizes [H+] as a surrogate dosimeter for inhaled or ingested vinyl acetate concentration. Anatomical, physiological, and biochemical differences between species necessitate that more biologically-based approaches to assessing tissue dosimetry be used to extrapolate rodent toxicity data to humans (Andersen et al., 1992; Bogdanffy and Jarabek, 1995
). In the case of vinyl acetate, it has been proposed that risk management practices, which reduce exposures such that proton burden is within homeostatic bounds, would prevent all toxic responses, including cancer, that would follow (Bogdanffy et al., 1999b
; Sarangapani et al., manuscript submitted).
Literature reports suggest that reductions in pHi can be cytotoxic, or induce mitogenesis. For example, a reduction in pHi of neuronal cells below 0.15 pH units results in cytotoxicity (Nedergaard et al., 1991). Alterations in pHi are involved in stimulation of cell growth and transformation. For example, Syrian hamster embryo cells, cultured at pH 6.7, show a marked increase in lifespan compared to those cultured at pH 7.3, as measured by the number population doublings that occur before cellular senescence (Kerckaert et al., 1996
). The higher proton burden of the intracellular environment has been shown to displace Ca2+ from intracellular binding sites (Batlle et al., 1993
). Ca2+ displaced from the growth and differentiation factor (GDF) protein blocks the intracellular signaling that leads to differentiation (Isfort et al., 1993
). Blockage of the differentiation pathway could promote sustained proliferation, expansion of the undifferentiated cell population, and clonal expansion of spontaneous or chemical-induced mutants. Clearly, substantiation of the hypothesis that intracellular acidification is mitogenic in nasal or oral cavity mucosal cells will require further experimentation.
Physiologically based pharmacokinetic (PBPK) models, describing the uptake, metabolism, and intracellular proton burden following inhalation and oral exposure to vinyl acetate, have been developed for the rat and human (Bogdanffy et al., 1999b; Sarangapani et al., manuscript submitted). These models can be useful for providing context for the in vitro concentrations used here. For example, by the inhalation route, nasal olfactory epithelium is the target tissue most sensitive to vinyl acetate toxicity. Degeneration of the olfactory epithelium is significant at 200 and 600 ppm, while tumor incidence is significant only at 600 ppm. The no-observed-adverse-effect level (NOAEL) for all effects is 50 ppm. The inhalation model predicts that exposure to 50, 200, or 600 ppm levels of vinyl acetate reduces the pHi of rat olfactory epithelium by 0.08, 0.25, and 0.49 units, respectively. Under steady-state conditions, inhaled concentrations of 50, 200, or 600 ppm are predicted to yield 10, 50, or 178 µM vinyl acetate, respectively, in the nasal mucus overlying the olfactory epithelium. The isolated hepatocyte perfusion buffer can be analogized to the mucus overlying the nasal epithelium. Thus, the observed
pHi of 0.03 in hepatocytes exposed to 10 µM vinyl acetate is reasonably close to the model-predicted
pHi of 0.08. Furthermore, in vitro studies with nasal turbinates show that concentrations greater than 25 mM are required to induce cytotoxic damage (Kuykendall et al., 1993
). The changes in pHi presented here (over the concentration range 10 µM to 1 mM) precede cytotoxicity and would therefore be considered a sentinel pharmacodynamic response. In summary, to the extent that freshly isolated rat hepatocytes can serve as a model for nasal epithelial cells, these results support the proposed mode of action for vinyl acetate. These results also support the use of pHi as the dosimeter upon which vinyl acetate's health risk should be based (Bogdanffy et al., 2001
; Sarangapani et al., manuscript submitted).
In conclusion, these experiments show that pHi of rat hepatocytes is significantly reduced following exposure to concentrations of vinyl acetate 10 µM, that intracellular acidification is proportionate to dose up to 500 µM, and that the response is dependent on vinyl acetate metabolism. These changes preceded morphological evidence of cytotoxicity. To the extent that hepatocytes are a useful model for target epithelial cells, these results are consistent, both qualitatively and quantitatively, with the mode of action-based risk assessments developed for vinyl acetate. These data further support the use of tissue pH as the dosimeter most closely related to the sentinel pharmacodynamic steps of vinyl acetate carcinogenesis.
ACKNOWLEDGMENTS
The author thanks the following individuals for their technical help and advice: Mr. Benjamin Robinson, Dr. Raymond Kemper, and Mr. Daniel Cordova. This work was supported, in part, by the Vinyl Acetate Toxicology Group, Inc., Washington, DC.
NOTES
1 For correspondence via fax: (302) 366-5003. E-mail: matthew.s.bogdanffy{at}usa.dupont.com.
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