Metabolism Of Vinyl Acetate Research Articles

Vinyl acetate decreases intracellular pH in rat nasal epithelial cells.

Vinyl acetate is a synthetic organic ester that has been shown to produce nasal tumors in rats following exposure to 600 ppm in air. The proposed mechanism of action involves the metabolism of vinyl acetate by carboxylesterases and the production of protons leading to cellular acidification. While vinyl acetate-induced decreases in intracellular pH (pHi) have been demonstrated in rat hepatocytes, comparable data from nasal epithelial cells do not exist. Using an in vitro assay system, we have determined the effects of vinyl acetate exposure on pHi in respiratory and olfactory nasal epithelial cells from rats. The respiratory and olfactory epithelial cells were isolated from dissected maxillo- and ethmoturbinates by enzyme digestion. The cells were plated; loaded with the pH-sensitive dye, carboxyseminaphthorhodafluor-1 (SNARF-1); and observed using confocal microscopy. Individual cellular analysis demonstrated that both respiratory and olfactory epithelial cells responded to vinyl acetate exposures with a dose-dependent decrease in pHi. Changes occurred at 100 microM but reached a plateau above 250 microM. Maximal decreases in pHi were 0.3 pH unit in respiratory epithelial cells. While pHi values were normally distributed for the respiratory epithelial cells, the olfactory epithelial cells demonstrated a bimodal distribution, indicating at least two populations of cells, with only one population of cells responding to vinyl acetate. Acidification in these cells did not plateau but continued to increase at 1000 microM. Bis(p-nitrophenyl)phosphate (BNPP), a carboxylesterase inhibitor, was able to attenuate the vinyl acetate-induced decrease in pHi. Data obtained from the isolated cells were validated using tissue explants. These results are consistent with the proposed mode of action for vinyl acetate and supply further data for developing appropriate risk assessments for vinyl acetate exposure.

Physiologically Based Pharmacokinetic (PBPK) Models for Nasal Tissue Dosimetry of Organic Esters: Assessing the State-of-Knowledge and Risk Assessment Applications with Methyl Methacrylate and Vinyl Acetate

Mathematical models have been developed to describe nasal epithelial tissue dosimetry with two compounds, vinyl acetate (VA) and methyl methacrylate (MMA), that cause toxicity in these tissues These models couple computational fluid dynamics (CFD) calculations that map airflow patterns within the nose with physiologically based pharmacokinetic (PBPK) models that integrate diffusion, metabolism, and tissue interactions of these compounds. Dose metrics estimated in these models for MMA and VA, respectively, were rates of MMA metabolism per volume of tissue and alterations in pH in target tissues associated with VA hydrolysis and metabolism. In this article, four scientists who have contributed significantly to development of these models describe the many similarities and relatively few differences between the MMA and VA models. Some differences arise naturally because of differences in target tissues, in the calculated measures of tissue dose, and in the modes of action for highly extracted vapors (VA) compared with poorly extracted vapors (MMA). A difference in the approach used to estimate metabolic parameters from human tissues provides insights into interindividual extrapolation and identifies opportunities for studies with human nasal tissues to enhance current risk assessments. In general, the differences in model structure for these two esters were essential for describing the biology of the observed responses and in accounting for the different measures of target tissue dose. This article is intended to serve as a guide for understanding issues of optimum model structure and optimal data sources for these nasal tissue dosimetry models. We also hope that it leads to greater international acceptance of these hybrid CFD/PBPK modeling approaches for improving risk assessment for many nasal toxicants. In general, these models predict either equivalent (VA) or lower (MMA) nasal tissue doses in humans compared with tissue doses at equivalent exposure concentrations in rats.

Time- and concentration-dependent increases in cell proliferation in rats and mice administered vinyl acetate in drinking water.

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 approximately 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.

Vinyl acetate-induced intracellular acidification: implications for risk assessment.

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 (pH(i)) at noncytotoxic levels, but that prolonged exposure to reduced pH(i) 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 pH(i). Quantitative fluorescence imaging ratio microscopy showed that exposure of hepatocytes to vinyl acetate concentrations ranging from 10 to 1000 microM caused rapid and sustained reductions of approximately 0.03 to 0.65 pH units. Cellular acidification was rapidly reversed to control pH(i) upon removal of vinyl acetate. There was minimal accumulation of protons during the exposure period, as suggested by minor differences in pH(i) of cells with or without prior exposure to vinyl acetate. The effect of vinyl acetate on pH(i) 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 pH(i) is an appropriate metric dose for use in quantitative risk assessments of cancer and noncancer human health risk assessment.

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

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

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

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

Physiologically Based Modeling of Vinyl Acetate Uptake, Metabolism, and Intracellular pH Changes in the Rat Nasal Cavity

Chronic inhalation exposure to vinyl acetate (VA) causes lesions in the nasal cavity of the rat. This effect appears to be related to tissue exposure to either acetaldehyde (AAld) or acetic acid (AA) metabolites of VA or both. A physiologically based pharmacokinetic model was constructed to describe the deposition of VA in the nasal cavity of the rat and provide estimates of regional tissue exposure to VA, AAld, and AA. Since formation of AA in the nasal tissue should cause intracellular acidification, a submodel which describes free intracellular hydrogen ion concentration and intracellular pH (pHi) changes was linked to the VA model. The dosimetry model was applied to data from a series of experiments designed to measure the uptake and metabolism of VA in the isolated upper respiratory tract of the rat at exposure concentrations ranging from 73 to 2190 ppm. Extraction of VA from the nasal cavity was nonlinear with respect to exposure concentration and ranged from 36 to 94%, with the greatest deposition occurring at the lowest VA concentrations. Pretreatment with bis(p-nitrophenyl)phosphate, an inhibitor of carboxylesterases, significantly reduced fractional deposition of VA compared to naive rats exposed to similar VA concentrations. The best model fits for VA extraction and AAld appearance were achieved when a second carboxylesterase isozyme, with high-affinity characteristics, was included. Simulations of 6-h inhalation exposures to VA predicted that the order of nasal tissue exposures will be to AA > AAld > VA. In addition, based on measured tissue hydrolysis rates, sufficient acid should be formed by the metabolism of VA to cause significant changes in pHi. VA exposures of 200 and 600 ppm were predicted to result in a pHiof less than 7.2 and 6.7, respectively. This model provides nasal dosimetry estimates needed to develop mechanistically based risk assessment approaches for human exposures to VA vapor.

Cytotoxicity and DNA-Protein Crosslink Formation in Rat Nasal Tissues Exposed to Vinyl Acetate Are Carboxylesterase-Mediated

Vinyl acetate is used in the paint, adhesive, and paper board industries. Vinyl acetate is a nasal carcinogen in rats exposed by inhalation for 2 years to 200 and 600 ppm, but not 50 ppm. Previous studies from our laboratory suggest that rat liver microsome-activated vinyl acetate induces plasmid DNA-histone crosslinks, in vitro, through esterase-mediated metabolism. Since nasal tissues contain high levels of carboxylesterase, tumorigenesis may be related to in situ production of the hydrolysis products acetaldehyde and acetic acid. Vinyl acetate was cytotoxic to both respiratory and olfactory tissues in vitro at 50-200 mM, but not 25 mM, after 2 hr exposure. Pretreatment of rats with the carboxylesterase inhibitor, bis-(p-nitrophenyl) phosphate (BNPP), attenuated the cytotoxic effects and metabolism of vinyl acetate in both tissue types. Semicarbazide, an aldehyde scavenger, was unable to protect the tissues from vinyl acetate-induced cytotoxicity. When the metabolites were tested, acetic acid, but not acetaldehyde, was cytotoxic to both tissues. The induction of DNA-protein crosslink (DPXL) formation by acetaldehyde and vinyl acetate in rat nasal epithelial tissues was detected using a sodium dodecyl sulfate/KCl precipitation technique. Endogenous crosslink levels ranged from 0.5 to 2.0% of total DNA and were considered background. Epithelial cells isolated from both olfactory and respiratory turbinates exhibited dose- and time-dependent increases in DPXL formation when exposed to 10-150 mM acetaldehyde for 1-2 hr at 37°C. Similarly, respiratory and olfactory epithelial cells exposed to 5-75 mM vinyl acetate for 1-2 hr accumulated up to 12- and 15-fold higher crosslink levels than untreated cells, respectively. However, vinyl acetate appears to induce much higher levels of DPXLs at equimolar doses than acetaldehyde. This is thought to be related to stimulation of acetaldehyde-induced DPXL formation by the pH lowering effect of acetic acid production (via vinyl acetate hydrolysis). Pretreatment of the nasal turbinates with 1 mM BNPP reduced 25 mM vinyl acetate-induced DPXL formation by over 75% in both tissues. These data support a hypothesis that carboxylesterase-mediated hydrolysis of vinyl acetate is necessary to generate the active intracellular crosslinking agent, acetaldehyde, and the cytotoxic metabolite, acetic acid.

Degradation of vinyl acetate by soil, sewage, sludge, and the newly isolated aerobic bacterium V2

Vinyl acetate is subject to microbial degradation in the environment and by pure cultures. It was hydrolyzed by samples of soil, sludge, and sewage at rates of up to 6.38 and 1 mmol/h per g (dry weight) under aerobic and anaerobic conditions, respectively. Four yeasts and thirteen bacteria that feed aerobically on vinyl acetate were isolated. The pathway of vinyl acetate degradation was studied in bacterium V2. Vinyl acetate was degraded to acetate as follows: vinyl acetate + NAD(P)+----2 acetate + NAD(P)H + H+. The acetate was then converted to acetyl coenzyme A and oxidized through the tricarboxylic acid cycle and the glyoxylate bypass. The key enzyme of the pathway is vinyl acetate esterase, which hydrolyzed the ester to acetate and vinyl alcohol. The latter isomerized spontaneously to acetaldehyde and was then converted to acetate. The acetaldehyde was disproportionated into ethanol and acetate. The enzymes involved in the metabolism of vinyl acetate were studied in extracts. Vinyl acetate esterase (Km = 6.13 mM) was also active with indoxyl acetate (Km = 0.98 mM), providing the basis for a convenient spectrophotometric test. Substrates of aldehyde dehydrogenase were formaldehyde, acetaldehyde, propionaldehyde, and butyraldehyde. The enzyme was equally active with NAD+ or NADP+. Alcohol dehydrogenase was active with ethanol (Km = 0.24 mM), 1-propanol (Km = 0.34 mM), and 1-butanol (Km = 0.16 mM) and was linked to NAD+. The molecular sizes of aldehyde dehydrogenase and alcohol dehydrogenase were 145 and 215 kilodaltons, respectively.