nitrophenols and vinyl-acetate

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.

Topics: Animals; Benzopyrans; Carboxylesterase; Cells, Cultured; Dose-Response Relationship, Drug; Drug Antagonism; Enzyme Inhibitors; Hydrogen-Ion Concentration; Male; Microscopy, Confocal; Naphthols; Nitrophenols; Olfactory Mucosa; Rats; Rats, Inbred Strains; Rhodamines; Vinyl Compounds

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.

Topics: Animals; Carboxylesterase; Carboxylic Ester Hydrolases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hepatocytes; Hydrogen-Ion Concentration; In Vitro Techniques; Male; Models, Biological; Nitrophenols; Rats; Rats, Inbred Strains; Risk Assessment; Time Factors; Vinyl Compounds

Vinyl acetate induces nasal tumors in rats, but not mice. Species differences in airflow patterns, physiology, and biochemistry complicate extrapolation of nasal dosimetry from rats to humans. Physiologically based pharmacokinetic modeling of vinyl acetate dosimetry in rats suggested the presence of a saturable metabolic removal pathway in rat nasal mucus. We explored the possibility that this pathway is either a cytochrome P-450 2E1 (CYP2E1) or high-affinity carboxylesterase. Nasal extraction of vinyl acetate vapor (150 ppm) was measured in the surgically isolated nasal cavity of anesthetized rats. Vinyl acetate (150 ppm) was extracted with 73% efficiency in controls. Pretreatment of rats with the CYP2E1 inhibitor diallyl sulfide (DAS) had no effect on extraction, despite significantly reducing CYP2E1 activity. Pretreatment with bis(p-nitrophenyl) phosphate (BNPP), a carboxylesterase inhibitor, reduced extraction to approximately 41%. Acetaldehyde production was similarly unaffected by DAS but was reduced to 55% of control by BNPP. Rat nasal mucus carboxylesterase activity had a K(m) value (32 microM) similar, within a factor of 2, to the value predicted by the physiologically based model, although V(max) was significantly lower than the model prediction. Histochemical observations support the inference that the high-affinity carboxylesterase is bound to the luminal plasma membrane of nasal tissue and is not readily released by nasal lavage, providing an explanation for the low V(max) of the lavage enzyme. This high-affinity isoenzyme could be important in the removal of odorants from the sensory cell-rich nasal olfactory epithelium.

Topics: Algorithms; Animals; Carboxylic Ester Hydrolases; Cytochrome P-450 CYP2E1; Enzyme Inhibitors; In Vitro Techniques; Inhalation Exposure; Models, Biological; Nasal Lavage Fluid; Nasal Mucosa; Nitrophenols; Rats; Rats, Inbred Strains; Vinyl Compounds

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 pHi of 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.

Topics: Acetaldehyde; Acetic Acid; Animals; Computer Simulation; Enzyme Inhibitors; Hydrogen-Ion Concentration; Male; Models, Biological; Mucous Membrane; Nasal Cavity; Nitrophenols; Rats; Solubility; Vinyl Compounds

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 degrees 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 cross-linking agent, acetaldehyde, and the cytotoxic metabolite, acetic acid.

Topics: Acetaldehyde; Animals; Carboxylic Ester Hydrolases; Cell Survival; DNA; Dose-Response Relationship, Drug; Male; Nasal Mucosa; Nitrophenols; Nose; Proteins; Rats; Semicarbazides; Turbinates; Vinyl Compounds