Time- and Concentration-Dependent Increases in Cell Proliferation in Rats and Mice Administered Vinyl Acetate in Drinking Water

R. Valentine,1, J. R. Bamberger, B. Szostek, S. R. Frame, J. F. Hansen and M. S. Bogdanffy

E.I. du Pont de Nemours and Company, Haskell Laboratory for Toxicology and Environmental Sciences, Elkton Road, P.O. Box 50, Newark, Delaware 19714

Received October 17, 2001; accepted January 29, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic administration of vinyl acetate (VA) in drinking water to rats and mice has produced upper digestive tract neoplasms. These tumors were believed to arise from the intracellular metabolism of VA by carboxylesterases to cytotoxic and genotoxic compounds. We hypothesized that prolonged VA exposure at high concentrations would induce cytotoxicity and a restorative cell proliferation (CP). These endpoints were measured in F-344 rats and BDF1 mice administered drinking water containing 0, 1000, 5000, 10,000, or 24,000 ppm VA for 92 days. On test days, Days 1, 8, 29, and 92, upper digestive tract histopathology and oral cavity CP (pulsed 5-bromodeoxyuridine [BrdU] to measure S-phase DNA synthesis) were evaluated. Analysis of test solutions showed that VA spontaneously hydrolyzed, slowly releasing acetic acid and thereby lowering pH. Statistically significant, concentration-related increases in CP occurred in basal cells of the mandibular oral cavity mucosa of mice at 10,000 and 24,000 ppm but only after 92 days. CP increases were ~2.4- and 3.4-fold above controls and were considered to be toxicologically significant. Some statistically significant increases in CP were also measured in the oral cavity mucosa of rats; however, these changes were considered to be of equivocal biological relevance. No histopathological evidence of mucosal injury was seen in either species. The absence of cytotoxicity in the upper digestive tract mucosa suggests that the increased CP at high administered VA concentrations may be due to a mitogenic response, ostensibly from the loss of cell growth controls in oral cavity mucosa.

Key Words: vinyl acetate; drinking water; cell proliferation; basal cell; oral mucosa; mandible; subchronic; histopathology.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vinyl acetate (VA; CAS No. 108-05-4) is a volatile organic monomer used in polyvinyl acetate and vinyl acetate copolymer resin manufacture. Although the carcinogenic activity of VA via drinking water has previously been reported, the results of these studies were either inconclusive due to unconventional study design/practices, or negative or positive only at very high-administered doses. Lijinsky and Reuber (1983) administered commercial VA (purity unspecified) for 5 days/week for 100 weeks at 1000 or 2500 ppm to male and female rats. No upper digestive tract neoplasms were observed, although a low incidence of tumors was reported at 2 other sites. Maltoni et al. (1997) reported upper digestive tract tumors in male and female Swiss mice and their offspring administered 0, 1000, or 5000 ppm VA for 78 weeks. In 5000 ppm males and females found dead up to 168 weeks, VA produced oral cavity, tongue, esophagus, and forestomach squamous cell carcinomas. Carcinoma incidence decreased as the distance from the oral cavity increased (oral cavity and tongue > esophagus > forestomach). Bogdanffy et al. (1994) described the chronic toxicity of VA (>99.9% pure) following in utero and subsequent lifetime exposure in Crl:CD(SD) rats administered 0, 200, 1000, or 5000 ppm VA. Rats were exposed to VA for 10 weeks and mated; their offspring were divided into main and satellite groups and exposed for up to 104 weeks. No compound-related histopathology was found in satellite groups after 52 or 78 weeks. At 104 weeks, no significant increases in neoplastic lesions were observed. Although 2 oral cavity squamous carcinomas were seen in 5000-ppm males, they were within historical incidence and considered not treatment-related.

Most recently, an unpublished, 2-year drinking water carcinogenicity study in male and female BDF1 mice and F344 rats was described by the Japanese Bioassay Research Center (JBRC) (U.S. EPA, 1997Go). Groups of 50 male and 50 female rats and mice received 0, 400, 2000, or 10,000 ppm VA (98% pure) for 104 weeks; mean daily intake of VA was approximately 0, 36, 170, and 710 mg/kg/day for rats and 0, 72, 320, and 1500 mg/kg/day for mice, respectively. Statistically significant increases in VA-related preneoplastic changes (e.g., squamous cell hyperplasia, basal cell activation) and squamous cell neoplasms were observed at several sites in the upper digestive tract but only at 10,000 ppm. Tumors and pre-neoplastic lesions developed only along the upper digestive tract, with the highest incidence in the oral cavity, and decreased progressively towards the forestomach. For example, combined neoplastic and non-neoplastic lesion incidences in the oral cavity, esophagus, and forestomach among 10,000 ppm mice were 73/99, 37/99, and 20/99, respectively. Additionally, mice appeared to be the more susceptible species to neoplasia, since the incidence of oral cavity squamous cell carcinomas/papillomas at 10,000 ppm was 35/99 in mice and 10/100 in rats. Tumor location mapping revealed that the majority of the oral cavity carcinomas/papillomas was found in the mandible, as compared to the maxilla.

Collectively, these data show that VA may produce oral cavity tumors, primarily above ~5000 ppm VA (corresponding to ingested doses above ~300 mg/kg/day) and exhibit a highly nonlinear dose-response (Fig. 1Go). This dose-response behavior pattern is consistent with the dose-response for nasal tumors following VA inhalation (Bogdanffy et al., 1999Go, 2001Go; Kuykendall et al., 1993Go). By analogy to the mode of action that these authors proposed for nasal tumors, the mode of action for orally administered, VA-induced upper digestive tract carcinogenicity is similarly hypothesized to involve a multistep process, beginning with the intracellular metabolism of VA by mucosal carboxylesterases to acetic acid and acetaldehyde. Genetic damage from acetaldehyde may occur at high doses through induction of DNA-protein cross links (Dellarco, 1988Go; Kuykendall and Bogdanffy, 1992aGo,bGo). The intracellular metabolism of VA and acetaldehyde also produces hydrogen ions, which lower pH. Once intracellular pH is reduced to a critical level, cytotoxicity resulting in cell death is thought to occur, triggering a restorative CP. Thus, it was hypothesized that tumor development occurs following prolonged CP in mucosa along portals of entry, allowing expression of acetaldehyde-induced genetic damage.



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 1. Composite incidence of oral cavity squamous cell carcinomas in rats and mice chronically exposed to vinyl acetate in the drinking water. (A) Incidence observed following administration of VA in drinking water at different concentrations. (B) Incidence observed based on ingested doses of VA. Data obtained from Bogdanffy et al., 1994Go, Maltoni et al., 1997Go, and U.S. EPA, 1997Go.

 
This study was conducted specifically to determine whether histopathological and CP alterations occur in the upper digestive tract mucosa following continuous exposure to VA. VA was administered under conditions similar to those used in the JBRC bioassay, except that the high dose was increased to 24,000 ppm (the water solubility limit) to maximize the likelihood of detecting changes. We hypothesized that prolonged VA exposure at high concentrations would induce cytotoxicity and a restorative CP in mucosa comparable to that observed in nasal olfactory and respiratory epithelium by VA inhalation (Bogdanffy et al., 1997Go). Increased CP is considered an essential step in the multistage process of carcinogenesis for both initiation and promotion of neoplasia (Butterworth, 1990Go; Cohen and Ellwein, 1990Go) and is central to the hypothesized mode of action for VA-induced neoplasia.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Test material.
Vinyl acetate (VA), a colorless flammable liquid with an ester-like odor at room temperature, was supplied by the DuPont Company (La Porte, TX). It was analyzed prior to study start and after study completion and had a purity of at least 99.98%. Hydroquinone (3.8 ppm) was present as a polymerization inhibitor. Analyses revealed: acetic acid (11 ppm), acetaldehyde (4.3 ppm), methyl acetate (26 ppm), ethyl acetate (31 ppm), and water (120 ppm).

Test species and animal husbandry.
Male CDF®(F-344)/CrlBr rats (F344) and male B6D2F1/CrlBr mice (BDF1) were obtained from Charles River Laboratories (Raleigh, NC) and were ~46 days old at study start. The F344 rat and BDF1 mouse were selected because they were the strains used in the JBRC chronic drinking water study with VA. Animal health was monitored during a 10-day quarantine period. Animals were divided by computerized, stratified randomization into 5 weight-matched groups, each consisting of 20 rats and 20 mice.

Rats and mice were housed individually in suspended stainless steel wire mesh cages and housed on separate cage racks. Animal rooms were artificially illuminated (fluorescent light) on a 12-h light/dark cycle. Animal rooms were targeted at a temperature of 22 ± 3°C and a relative humidity of 50 ± 10%. Throughout the study, all animals were provided test solutions and Certified Rodent LabDiet® 5002 (PMI Nutrition International, Inc.) ad libitum.

This study was conducted according to Good Laboratory Practice guidelines in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. The study protocol was reviewed and approved by the Du Pont Laboratory Animal Welfare Committee.

Test material administration.
During the test period, animals in each group were provided with drinking water solutions containing 0, 1000, 5000, 10,000, or 24,000 ppm (w/w) VA in opaque, glass water bottles equipped with stainless steel sipper tubes. VA was added to either tap or distilled water and thoroughly mixed using magnetic stirrers for ~5 min to ensure a homogeneous mixture. Test solutions were prepared twice weekly at 3- or 4-day intervals to mimic the protocol used by JBRC (U.S. EPA, 1997Go). Samples were analyzed to verify the concentration and stability of VA in drinking water as follows: The inherent stability of VA in water was determined at 0, 1000, and 24,000 ppm VA. Freshly made solutions of VA were poured into 250 ml opaque glass bottles fitted with a screw top closure and stored for up to 4 days at room temperature. On days 0 and 4, a sample was taken and analyzed for VA from each bottle.

Additionally, the stability of VA from the drinking water bottles provided to animals was evaluated. On Day 0, 5 water bottles (250 ml each) per dose level (e.g., 0, 1000, 5000, 10,000, and 24,000 ppm of VA) were picked randomly and marked. These selected water bottles were delivered to the animals together with other bottles so that the samples taken from these bottles represent the VA that animals actually received. On days 1, 2, 3, and 4, each marked bottle was sampled by removing approximately 5 ml of the solution through the metal sipper tube and processed to measure pH, and acetic acid and VA concentrations.

VA in drinking water was analyzed with a Hewlett Packard Model 1100 HPLC equipped with a Diode Array Detector (DAD; absorbance at 210 nm). Samples were chromatographed on a Zorbax RX-C18, 2.1 x 150 mm, 5 µm particle size column with isocratic mobile phase of 40% 3.1 mM phosphoric acid in water and 60% acetonitrile at a flow rate 0.3 ml/min. The retention time of VA was 1.6 min. Acetic acid in drinking water was analyzed with a Hewlett Packard Model 1100 HPLC equipped with a DAD (absorbance at 210 nm). Samples were chromatographed on a Zorbax SB-C18, 4.6 x 150 mm, 3.5 µm particle size column. Gradient elution conditions were used with the mobile phase composed of 3.1 mM phosphoric acid in water (solvent A) and acetonitrile (solvent B) and a flow rate of 1 ml/min. The concentrations of VA or acetic acid were determined by comparing the detector response of the samples to calibration curves derived from calibration standards.

In vivo parameters.
All animals were weighed twice during the first week, then once a week for the remainder of the study. The amount of food consumed by each animal was determined once per week and the amount of water twice per week. From the water consumption and body weight data, the daily intake of VA was calculated. Each animal was individually handled and examined for abnormal behavior and appearance during each weighing. Animals were observed daily to detect moribund or dead animals and abnormal behavior/appearance throughout the study.

Pathological evaluations.
On test days 1, 8, 29, and 92, 5 rats and 5 mice from each group were prepared for determination of oral cavity histopathology and CP using pulsed 5-bromodeoxyuridine (BrdU) as a measure of S-phase DNA synthesis. Animals were euthanized by carbon dioxide anesthesia and exsanguination. Each animal was given a complete gross examination, and the head and mandible, esophagus, and forestomach were saved at necropsy for CP evaluation and histopathology. Tissues were placed in 10% neutral buffered formalin for approximately 24 h and then transferred to 70% ethanol. After formalin fixation (48 h), the head and mandible were decalcified and transferred to 70% ethanol. After paraffin embedding, tissues were sectioned at 5 µm, stained with hematoxylin and eosin, and examined by light microscopy.

Evaluations of the oral cavity for CP and histopathology were limited to the maxilla, corresponding to sectioning levels III (both species), and the mandible, sectioning levels VI (rat), and V (mouse) initially from high-dose and control animals from all sacrifice periods (Fig. 2Go). These specific areas corresponded to the highest tumor incidence areas in the JBRC drinking water bioassay (U.S. EPA, 1997Go). Subsequently, only mandibular sections (level V) from the mouse intermediate-dose groups sacrificed at 92 days were evaluated to determine a no-observable-effect level (NOEL) for CP and histopathology. Maxillary sections included the buccal fold, and gingival and palatial mucosa. Mandibular sections included the buccal fold, and gingival and sublingual mucosa. Histopathologic examinations were also conducted on 4 transverse sections of the esophagus (from the pharynx to just above the carina, to include the pharynx, thyroid, mid-esophagus, and mediastinal lymph nodes), and 4 sections of the stomach (2 midline and 2 lateral sections covering both the glandular and nonglandular stomach) from the control and 24,000-ppm groups of rats and mice at each sacrifice interval.



View larger version (32K):
[in this window]
[in a new window]
 
FIG. 2. Sectioning locations for cell proliferation and histopathological evaluations in the oral cavity. Lines denote positions of transverse sectioning.

 
Cell proliferation evaluation.
Incorporation of the thymidine analog, BrdU, into newly synthesized DNA during the S-phase of cell division coupled with anti-BrdU immunohistochemical detection techniques is considered a simple, reliable measure of cell division kinetics (Gratzner, 1982Go). Approximately 1 h prior to sacrifice, each animal was injected with 100 mg/kg BrdU, ip, in phosphate-buffered saline (pH 7.4). Approximately 1 h after injection, animals were sacrificed by CO2 anesthesia and exsanguination. Tissue samples were collected from the oral mucosa and duodenum, and prepared as described above. Tissues were processed to paraffin blocks for immunohistochemical staining using the anti-BrdU antibody and avidin-biotin complex (Vectastain Elite, Burlingame, CA) techniques, adopted from Sano et al. (1992), to measure BrdU incorporation into DNA. The labeling indices of all groups were evaluated by tissue site (e.g., oral cavity levels III, V, or VI). CP was measured as the cell-labeling index, defined as the number of labeled cell nuclei per 1000 total basal cells that were counted along the basement membrane of the palatial, gingival, buccal, or sublingual mucosa. The duodenum was used as an immunohistochemical control. Since the majority of tumors from the chronic drinking water studies occurred in the oral cavity, CP evaluations were not conduced in tissues from the esophagus or forestomach.

Statistical analyses.
Body weight, and food and water consumption data were initially evaluated for lack of trend, and if not significant, sequential analysis by Jonckheere-Terpstra (Jonckheere, 1954Go) trend test was used. CP for each concentration group/time interval was evaluated for normality and homogeneity using Shapiro-Wilk (Shapiro and Wilk, 1965Go) and Levene's (Levene, 1960Go) tests, respectively. If the individual data were normally distributed and homogeneous, data were further evaluated by a repeated-measure analysis of variance (Snedecor and Cochran, 1965Go) followed by Dunnett's (Dunnett, 1955Go) test. The Mann-Whitney U test was used for CP data that were nonhomogeneous and nonnormal (Lehman, 1975Go). Statistical differences were declared at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Analytical
Analysis of the 1000- and 24,000-ppm VA solutions stored at room temperature showed that the percent VA remaining after 4 days was 94.5% and 103%, respectively, of initial concentrations. Stability assessments conducted on VA solutions from the drinking water bottles actually used by rats during the study indicated that the percentage of VA remaining in the animal bottles from Day 4 relative to Day 0 to was 112, 105, 102, and 90% for the 1000, 5000, 10,000 and 24,000 ppm levels, respectively; no significant loss with time was observed. Since these values were within 12% of nominal for at least 4 days, the test solutions were considered to be stable over the time period they were presented to animals.

Although VA concentrations were relatively constant, the composition of the test solutions slowly changed during the 3–4-day intervals that test solutions were presented to animals. Acetic acid concentrations in the drinking water rose approximately 10-fold over 4 days for each group. In the 24,000-ppm solution, for example, acetic acid levels increased from 55 ppm immediately after mixing to 650 ppm by Day 4. Overall, the formation rate of acetic acid averaged approximately 0.7% of the nominal VA concentration/day over a 4-day interval. The pH of each test solution also decreased as a function of the initial VA concentration and time. Notably, the pH of the 24,000-ppm VA solution decreased from 5.21 on Day 0 to 3.74 by Day 4; pH reductions of similar magnitude were also seen in the 5000- and 10,000-ppm groups.

In-Life Toxicology
All rats and mice survived to their scheduled termination. No clinical signs of toxicity attributable to VA administration were observed in rats or mice during the study.

Rats.
Rats in the 5000-, 10,000-, and 24,000-ppm groups had slight (up to 8%) but statistically significantly lower mean body weights and mean body weight gains than controls during the study. Body weight effects were seen within 4 days in the 10,000- and 24,000-ppm groups and within 67 days in the 5000-ppm group, and persisted throughout the remainder of the study (Fig. 3Go). Mean daily food consumption was significantly lower in rats from the 10,000- and 24,000-ppm groups compared to controls. Food consumption was lower but not significantly less than controls in the 5000-ppm group.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 3. Body weights in rats.

 
Mean daily water consumption was significantly lower among rats administered test solutions containing 5000, 10,000, and 24,000 ppm VA than controls (Table 1Go). Based on overall water consumption and the nominal VA concentrations, the mean daily intake of VA in rats ranged from 81 to 1400 mg VA/kg body weight/day for the 1000 and 24,000 ppm groups, respectively, during the study (Table 1Go).


View this table:
[in this window]
[in a new window]
 
TABLE 1 Overall Mean Daily Drinking Water Consumption and Mean Daily VA Intake in Rats and Mice
 
Mice.
In mice, no compound-related effects on mean body weight (Fig. 4Go), body weight gain, or food consumption were observed in any exposure group.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 4. Body weights in mice.

 
Compared to controls, the mean daily water consumption was significantly lower in mice from all test groups during the study (Table 1Go). Based on overall water consumption, the mean daily intake of VA (on a mg VA/kg body weight/day basis) in mice from each exposure group were approximately 3–4-fold greater than rats (Table 1Go).

Cell Proliferation
Rats.
In the 24,000-ppm group, statistically significant increases in mean CP relative to controls occurred in the oral cavity maxillary mucosa (level III) on Days 29 and 92, while increased CP occurred in the oral cavity mandibular mucosa (level VI) on Days 1 and 29. These increases were small (less than 2-fold). Although statistically significant, these increases were considered to be of equivocal biological significance, so evaluation of CP in rats from the intermediate groups was not conducted (Fig. 5Go).



View larger version (25K):
[in this window]
[in a new window]
 
FIG. 5. Cell proliferation in rat oral mucosa. Data are expressed as labeled cells/1000 mucosal basal cells. Note that only high dose and control groups were evaluated at each time interval. Values shown represent mean and standard deviation; * p < 0.05 by Mann Whitney U test; # p < 0.05 by Dunnett's test.

 
Mice.
In mice, statistically significant and dose-related increases in mean CP occurred in the oral cavity mandibular mucosa (level V) in the 10,000- and 24,000-ppm groups at 92 days. The increases in means were approximately 2.4- and 3.4-fold above the control group mean for the 10,000- and 24,000-ppm groups, respectively. Based on the magnitude of the increases and the dose-related nature of the response, these increases were considered to be compound-related and toxicologically significant. There were no other statistically significant increases in cell proliferation among the 24,000-ppm group mice compared to controls, at any of the time points evaluated (Fig. 6Go).



View larger version (26K):
[in this window]
[in a new window]
 
FIG. 6. Cell proliferation in mouse oral mucosa. Data are expressed as labeled cells/1000 mucosal basal cells. With the exception of day 92, only high-dose and control groups were evaluated at each time interval. Values shown represent mean and standard deviation; * p < 0.05 by Mann Whitney U test; # p < 0.05 by Dunnett's test.

 
Pathological Evaluations
In comparison to controls, there were no compound-related gross or microscopic lesions in the oral cavity, esophagus, or forestomach of rats or mice at 24,000 ppm VA at any time interval. No discernable cytotoxicity, inflammation, hyperplasia, hypertrophy, or apoptotic bodies were observed.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, presentation of VA-containing drinking water was limited to 4 days to mimic the JBRC study design. Over this period, the pH and acetic acid measurements indicated that spontaneous hydrolysis had changed the composition of the drinking water solutions. Consequently, acetic acid and H+ concentrations for each group increased about 10-fold over 4 days. Although not specifically measured, similar levels of acetaldehyde were probably formed, since this is the other hydrolysis product resulting from abiotic VA degradation. At higher VA dosing concentrations, the elevated acetaldehyde and acetic acid concentrations and lower test solution pH from spontaneous hydrolysis of VA may have complicated study interpretation, but was necessary in order to mimic the dosing conditions in the JBRC study.

Statistically significant increases in mean CP were observed in the basal cells of the oral cavity maxillary mucosa (level III) of rats administered 24,000 ppm on Days 29 and 92 and in the mandibular mucosa (level VI) on Days 1 and 29. These increases were small (less than 2-fold) and were not consistent across time points. Moreover, from pilot studies, a 2-fold increase in CP was considered to be biologically significant and given method variability, statistically detectable using 5 animals/group. Therefore, the less than 2-fold CP increases observed in rats, while statistically different from controls, were considered to be of equivocal biological significance. Additionally, the equivocal CP response observed in rats may be a reflection of the weak oral cavity carcinogenic response (i.e., 0–6% incidence in rats vs. 0–22% in mice) noted among 10,000-ppm rats from the JBRC drinking water study (U.S. EPA, 1997Go). In contrast, mice exhibited statistically significant and dose-related increases in mean CP (~2.4- and 3.4-fold above controls for the 10,000- and 24,000-ppm groups, respectively) in the oral cavity mandibular mucosa (level V) but only after 92 days of exposure. The absolute magnitude and concentration-related nature of these CP increases signify the responses were compound-related. A 2-fold increase in CP may assume additional toxicological relevance, since CP increases of this magnitude occurred at a dose level (>=10,000 ppm), tissue site (mandibular mucosa), and species (mice) that produced oral cavity tumors in the JBRC study (U.S. EPA, 1997Go).

The increases in mandibular CP of mice qualitatively and quantitatively paralleled the JBRC tumor outcome data (U.S. EPA, 1997Go). For both species, the incidence of oral cavity neoplastic lesions was higher in the oral cavity mucosa of the mandible than the maxilla. For example, among 10,000-ppm mice, 28/99 carcinomas were found in mandibular mucosa vs. 8/99 in the maxilla. Also, for a given VA concentration, neoplastic lesion incidence was about 3-fold higher in mice compared to rats; the total number of mice with oral cavity squamous cell papillomas/carcinomas at 10,000 ppm was 35/99 vs. 10/100 for rats. Normalization of carcinoma incidence by amount of VA ingested, however, eliminated the apparent species difference in tumor response (Figs. 1A and 1BGo). The importance of ingested dose as the more relevant measure of response parallels the relative differences in CP among mice and rats. For example, the intake of VA per kg body weight was about 3.5-fold higher in mice compared to rats, and after 92 days, cell labeling indices were approximately 3-fold higher in mice than rats. At present, it is not possible to state whether the greater responsiveness of mice to VA-induced CP in the oral cavity in general or in the mandibular mucosa in particular, is due to an enhanced sensitivity or due to a higher tissue dose of VA or its metabolites, compared to rats.

The underlying hypothesis for the proposed mode of action of VA in the oral cavity is based on the effects of VA in the nasal cavity as outlined by Bogdanffy (Bogdanffy et al., 1999Go; Bogdanffy and Taylor, 1993Go; Plowchalk et al., 1997Go). In brief, oral cavity carboxylesterases (C. J. Reed, M. S. Bogdanffy, and A. Robinson, unpublished data) like those found in nasal respiratory and olfactory epithelium, hydrolyze VA-releasing acetaldehyde, a clastogen, and acetic acid, a cytotoxicant. Accordingly, concentration-dependent decreases in intracellular pH following the intracellular formation of acetic acid are thought to induce cytotoxicity and trigger restorative CP in basal cells. It has been hypothesized that sustained and increased CP in the presence of excess, nonphysiological levels of acetaldehyde and low pH induces mutations in genetic material, eventually resulting in increased tumor formation through accumulation of genetically altered cells (Bogdanffy et al., 1997Go). While this mode of action was developed initially for inhaled VA in the nasal cavity, these same features were also hypothesized to exist in the oral cavity with ingested VA (R. Sarangapani, unpublished data).

The dose-dependent, 2–3 fold increases in mandibular mucosal CP of mice after 92 days of exposure to 10,000 or 24,000 ppm VA provide some evidence supporting the proposed mode of action in the oral cavity. However, the increased CP was not accompanied by histopathological changes in the oral cavity, esophagus, or forestomach. As it relates to the proposed mode of action for VA, cytotoxicity was not discernable at the light microscopic level at any time point or exposure concentration. The cellular injury by VA or its metabolites may be either very subtle or obscured by the high level of mucosal cell turnover, requiring more sensitive methods for discernable cytotoxicity. In this regard, recent studies by Morris and colleagues show significantly increased epithelial permeability to Evans-blue dye in oral cavity rinses immediately after administration of 24,000 ppm VA in rats for 10–60 minutes (Morris et al., 2001Go). Also, the nature of the cellular response to VA or its hydrolysis products may simply not produce remarkable signs of cellular degeneration within the time frame of this study. Collectively, these findings indicate that CP in the oral cavity may be considerably delayed in onset and, in contrast to the nasal mucosa (Bogdanffy et al., 1997Go), occur in the absence of frank cytotoxic or inflammatory changes. Although the observed findings do not exclude the possible involvement of apoptosis (programmed cell death) in the dynamics of cell death/renewal, its role is likely minor in comparison to the observed CP response, since apoptotic bodies were not observed microscopically.

The observation that cytotoxicity is not prevalent following VA exposure suggests that an alternative mechanism(s) is operative to explain the increase in oral cavity CP. Numerous studies suggest that mechanisms other than cytotoxicity can moderate CP. Some of these mechanisms involve Na+/H+ exchange, Ca+2 ion channels, intracellular pH, and protein kinases. Conceptually, reductions in intracellular pH can moderate the regulation of cell replication and transformation. Syrian hamster embryo cells for example, cultured at a reduced pH (6.7 vs. 7.4), show a marked increase in lifespan as measured by the number of population doublings and days in culture before cellular senescence (Kerckaert et al., 1996Go). High intracellular hydrogen ion concentrations can displace Ca+2 from intracellular binding sites (Battle et al., 1993Go) and the displaced Ca+2 from the growth-and-differentiation factor protein can block the intracellular signaling mechanism leading to differentiation (Isfort et al., 1993Go) and increased CP in Syrian hamster ovary cells (Isfort et al., 1996Go). Decreasing intracellular pH by approximately 0.25 units through addition of potassium ferricyanide (an impermeable electron acceptor) or addition of sodium propionate to reduce external pH elicited a mitogenic response in cultured PC12 cells (Thomas et al., 1996Go). These authors suggested that CP occurs upon activation of a mitogen-activation protein kinase through intracellular pH reduction. Thus, blocking differentiation or stimulating mitogen activation pathways could promote a sustained cellular proliferation without cytotoxicity. Although it is possible that the observed CP response was a result of tissue exposure to low pH drinking water solutions, we are not aware that such a response has been demonstrated for the upper digestive tract.

Comparable dose-, species-, or time-dependent studies on the effects of VA hydrolysis products on the upper digestive tract mucosa were not found in the literature and the limited data available only hint at possible interactions. Although the oral cavity was not examined, Mori (1952) reported that rats fed a rice diet containing acetic acid (50 cc/kg rice) exhibited progressively severe pathological changes in the forestomach. Acanthosis or keratosis, progressing into hyperplastic squamous epithelium, was seen after 30-days of feeding. Inflammation and mitotic figures were commonly noted in the lamina propria and submucosa along with basal layer thickening (Mori, 1952Go). In rats consuming 120 mM (324 mg/kg/day) acetaldehyde in drinking water for 8 months, a 1.5-fold increase in CP in the tongue, epiglottis and forestomach was produced; other than basal cell hyperplasia in these tissues, no histopathological changes were observed (Homann et al., 1997Go). Together, these limited studies suggest that both acetic acid and acetaldehyde can increase CP that may or may not be accompanied by cytotoxic changes in digestive tract mucosa. It remains to be determined if these changes reflect either their acidity, osmolality or irritancy, or altered nutritional status or cell signaling pathways (potentially involving changes in intracellular pH) due to high local concentrations of these substances for prolonged periods of time.

In conclusion, VA produced a toxicologically significant increase in CP in the oral cavity mandibular mucosa of mice, following 92 days of exposure at concentrations of 10,000 ppm or greater. The minimal CP response in the rat oral cavity may reflect the weak carcinogenic response (i.e., 0–6% incidence) previously reported in rats chronically administered 10,000 ppm VA in drinking water. The absence of cytotoxicity in the upper digestive tract mucosa suggests that the increased CP may be due to a mitogenic response, ostensibly from the loss of cell growth controls in oral cavity mucosa. These findings may conceivably be due to either the intracellular metabolism of VA with the concomitant decrease in intracellular pH or a direct effect of low pH dosing solutions on oral mucosal cell turnover. Further studies are needed to elucidate the interactions of VA with upper digestive tract tissues for cell injury and modulating cell proliferation.


    ACKNOWLEDGMENTS
 
This work was funded by the Vinyl Acetate Toxicology Group. Sponsoring companies include: Celanese, Ltd., E.I. du Pont de Nemours and Company, Millenium Petrochemicals, Dow Chemical Company, AT Plastics, Borden, Inc., Exxon Biomedical Services, Inc., Rohm and Haas Company, and Air Products and Chemicals, Inc. The authors also wish to extend their thanks to D. Janney and S. Craven for their technical assistance in the cell proliferation assays and M. Wilford for help in preparation of this manuscript.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (302) 366-6420. E-mail: rudolph.valentine{at}usa.dupont.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Battle, D. C., Peces, R., LaPointe, M. S., Ye, M., and Daugirdas, J. T. (1993). Cytosolic free calcium regulation in response to acute changes in intracellular pH in vascular smooth muscle. Am. J. Physiol. 264, C932–C943.[Abstract/Free Full Text]

Bogdanffy, M. S., Gladnick, N. L., Kegelman, T., and Frame, S. R. (1997). Four-week inhalation cell proliferation study of the effects of vinyl acetate on rat nasal epithelium. Inhal. Toxicol. 9, 331–350.[ISI]

Bogdanffy, M. S., Plowchalk, D. R., Sarangapani, R., Starr, T. B., and Andersen, M. E. (2001). Mode-of-action–based dosimeters for interspecies extrapolation of vinyl acetate inhalation risk. Inhal. Toxicol. 13, 377–396.[ISI][Medline]

Bogdanffy, M. S., Sarangapani, R., Plowchalk, D. R., Jarabek, A., and Andersen, M. E. (1999). A biologically based risk assessment for vinyl acetate-induced cancer and noncancer inhalation toxicity. Toxicol. Sci. 51, 19–35.[Abstract]

Bogdanffy, M. S., and Taylor, M. L. (1993). Kinetics of nasal carboxylesterase-mediated metabolism of vinyl acetate. Drug Metab. Dispos. 21, 1107–1111.[Abstract]

Bogdanffy, M. S., Tyler, T. R., Vinegar, M. B., Rickard, R. W., Carpanini, F. M. B., and Cascieri, T. C. (1994). Chronic toxicity and oncogenicity study with vinyl acetate in the rat: In utero exposure in drinking water. Fundam. Appl. Toxicol. 23, 206–214.[ISI][Medline]

Butterworth, B. E. (1990). Consideration of both genotoxic and nongenotoxic mechanisms in predicting carcinogenic potential. Mutat. Res. 239, 117–132.[ISI][Medline]

Cohen, S. M., and Ellwein, L. B. (1990). Cell proliferation in carcinogenesis. Science 249, 1007–1011.[ISI][Medline]

Dellarco, V. L. (1988). A mutagenicity assessment of acetaldehyde. Mutat. Res. 195, 1–20.[ISI][Medline]

Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 1096–1121.[ISI]

Gratzner, H. G. (1982). Monoclonal antibody to 5-bromo- and 5-iododeoxyridine: A new reagent for detection of DNA replication. Science 218, 474–475.[ISI][Medline]

Homann, N., Kärkkäinen, P., Koivisto, T., Nosova, T., Jokelainen, K., and Salaspuro, M. (1997). Effects of acetaldehyde on cell regeneration and differentiation of the upper gastrointestinal tract mucosa. J. Natl. Cancer Inst. 89, 1692–1697.[Abstract/Free Full Text]

Isfort, R. J., Cody, D. B., Asuith, T. N., Ridder, G. M., Stuard, S. B., and LeBoeuf, R. A. (1993). Induction of protein phosphorylation, protein synthesis, immediate-early-gene expresssion, and cellular proliferation by intracellular pH modulation. Implications for the role of hydrogen ions in signal transduction. Eur. J. Biochem. 213, 349–357.[Abstract]

Isfort, R. J., Cody, D. B., Stuard, S. B., Ridder, G. M., and LeBoeuf, R. A. (1996). Calcium functions as a transcriptional and mitogenic repressor in Syrian hamster embryo cells: Roles of intracellular pH and calcium ion in controlling embryonic cell differentiation and proliferation. Exp. Cell Res. 226, 363–371.[ISI][Medline]

Jonckheere, A. R. (1954). A distribution-free K-sample test against ordered alternatives. Biometrika 41, 133–145.[ISI]

Kerckaert, G. A., LeBoeuf, R. A., and Isofort, R. J. (1996). pH effects on the lifespan and transformation frequency of Syrian hamster embryo (SHE) cells. Carcinogenesis 17, 1819–1824.[Abstract]

Kuykendall, J. R., and Bogdanffy, M. S. (1992a). Reaction kinetics of DNA-histone crosslinking by vinyl acetate and acetaldehyde. Carcinogenesis 13, 2095–2100.[Abstract]

Kuykendall, J. R., and Bogdanffy, M. S. (1992b). Efficiency of DNA-histone crosslinking induced by saturated and unsaturated aldehydes in vitro. Mutat. Res. 283, 131–136.[ISI][Medline]

Kuykendall, J. R., Taylor, M. L., and Bogdanffy, M. S. (1993). Cytotoxicity and DNA-protein crosslink formation in rat nasal tissues exposed to vinyl acetate are carboxylesterase-mediated. Toxicol. Appl. Pharmacol. 123, 283–292.[ISI][Medline]

Lehman, E. I. (1975). Nonparametrics: Statistical Methods Based on Ranks. Holden-Day, San Francisco.

Levene, H. (1960). Robust test for equality of variances. In Contributions to Probability and Statistics (I. Olkin, Ed.), pp. 278–292. Stanford University Press, Palo Alto.

Lijinsky, W., and Reuber, M. D. (1983). Chronic toxicity studies of vinyl acetate in Fischer rats. Toxicol. Appl. Pharmacol. 68, 43–53.[ISI][Medline]

Maltoni, C., Ciliberti, A., Lefemine, G., and Soffritti, M. (1997). Results of a long-term experimental study on the carcinogenicity of vinyl acetate monomer in mice. Ann. N.Y. Acad. Sci. 837, 209–238.[Free Full Text]

Mori, K. (1952). The production of gastric lesions in the rat by acetic acid feeding. Gann 43, 443–448.

Morris, J. B., Symanowicz, P. T., and Sarangapani, R. (2001). Regional distribution and kinetics of vinyl acetate hydrolysis in the oral cavity of the rat and mouse. Toxicol. Lett. 126, 31–39.[ISI]

Plowchalk, D. R., Andersen, M. E., and Bogdanffy, M. S. (1997). Physiologically based modeling of vinyl acetate uptake, metabolism, and intracellular pH changes in the rat nasal cavity. Toxicol. Appl. Pharmacol. 142, 386–400.[ISI][Medline]

Sano, K., Sekine, J., Pe, M. B., and Inokuchi, T. (1992). Bromodeoxyuridine immunohistochemistry for evaluating age-related changes in the rat mandibular condyle decalcified by intravenous infusion. Biotech. Histochem. 67, 297–302.[ISI][Medline]

Shapiro, S. S., and Wilk, M. B. (1965). An analysis of variance test for normality (complete samples). Biometrika 52, 591–611.[ISI]

Snedecor, G. W. and Cochran, W. G. (1965). Statistical Methods, 6th ed. Iowa State University Press, Ames, IA.

Thomas, D., Ritz, M. F., Malviya, A. N., and Gaillard, S. (1996). Intracellular acidification mediates the proliferative response of PC12 cells induced by potassium ferricyanide and involves MAP kinase activation. Int. J. Cancer 15, 547–542.

U.S. EPA (1997). Carcinogenesis Study of Vinyl Acetate (Drinking Water Study) in Rats and Mice, with cover letter from Japan Bioassay Research Center dated 1/31/97. EPA/OTS, FYI-OTS-0297–1286. U. S. Environmental Protection Agency.