Matthew S. Bogdanffy

Efficiency of DNA-histone crosslinking induced by saturated and unsaturated aldehydes in vitro

Using a filter-binding assay based on precipitation of pUC13 plasmid DNA bound to calf-thymus histones, we have determined the efficiency of formation of DNA-protein crosslink formation induced by several aldehyde compounds in vitro. Formaldehyde, glutaraldehyde and acrolein were the most potent, causing 1 crosslink per 2.7 kbp of DNA at 1.5, 8 and 150 microM, respectively. All other compounds tested gave 1 crosslink per plasmid molecule in the mM concentration range as follows: acetaldehyde, 115 mM; propionaldehyde, 295 mM; butyraldehyde, 360 mM; crotonaldehyde, 8.5 mM; trans-2-pentenal, 6.3 mM. Significant decreases in the efficiency of DPXL formation were observed with monofunctional aldehydes of higher carbon chain length. For example, the concentration of formaldehyde needed to give 1 crosslink per molecule was almost 10(5) times less than that of acetaldehyde. Acetaldehyde differs from formaldehyde only by one saturated carbon. The presence of an unsaturated bond between the 2-3 carbons improved the potential for crosslink formation. For example, acrolein was over 500-fold more potent than propionaldehyde. Glutaraldehyde was almost as potent as formaldehyde, indicating that the bifunctional nature of this 5-carbon saturated aldehyde may be crucial to its high efficiency of DNA-protein crosslinking.

Biochemical quantitation and histochemical localization of carboxylesterase in the nasal passages of the fischer-344 rat and B6C3F1 mouse

Inhalation exposure of rats and mice to glycol ether acetates and acrylate esters causes degeneration of the olfactory epithelium but not of the respiratory epithelium. Since these compounds are metabolized via carboxylesterase to acids that are toxic to the olfactory epithelium, the activity and cellular distribution of carboxylesterase in the nasal passages of rats and mice were studied. Olfactory mucosal carboxylesterase in both rats and mice was found to have a Vmax value for the hydrolysis of p-nitrophenyl butyrate approximately 3 to 6 times larger than that for respiratory mucosa. Similarly, the second-order rate constant for binding and catalysis, V/K, was approximately four times greater in olfactory mucosa than in respiratory mucosa of both rats and mice. These data demonstrate that the olfactory mucosa of rats and mice hydrolyze carboxylesters more efficiently than the respiratory mucosae. Enzyme histochemistry was employed to identify the individual cells within the respiratory and olfactory mucosae which contain carboxylesterase activity. All cell types of the respiratory epithelium had some carboxylesterase activity, with varying intensities between individual cell populations. Ciliated and cuboidal epithelial cells were most active in this region. In the olfactory mucosa, however, Bowmans glands stained most intensely, sustentacular cells demonstrated moderate activity, and no activity was detectable in olfactory sensory cells. Together, these data quantitate carboxylesterase activity in nasal mucosal homogenates and localize the enzyme in individual cell types. The data suggest that olfactory mucosa may metabolize carboxylesters to acids more readily than respiratory mucosa. However, such metabolism does not occur in the target cell population, the olfactory sensory neurons, raising the possibility of intercellular migration of toxic acid metabolites.

Histochemical localization of aldehyde dehydrogenase in the respiratory tract of the Fischer-344 rat

Acetaldehyde, a nasal carcinogen and respiratory tract irritant, is metabolized by aldehyde dehydrogenase. The localization of aldehyde dehydrogenase in individual cells of the nasal passages, trachea, and lungs of the Fischer-344 rat was determined histochemically using a cold glycol methacrylate embedding procedure. Aldehyde dehydrogenase activity was detected principally in the nasal respiratory epithelium, especially in the supranuclear cytoplasm of ciliated epithelial cells while olfactory epithelium was almost devoid of enzyme activity. Epithelial cells of the trachea demonstrated little detectable aldehyde dehydrogenase activity, however, the Clara cells of the lower bronchioles showed remarkably high activity. These results corroborate previous biochemical findings and extend them by histochemically identifying particular aldehyde dehydrogenase-positive cell types within the nasal respiratory epithelium. The distribution of nasal lesions induced by acetaldehyde correlated with regional aldehyde dehydrogenase deficiencies suggesting that regional susceptibility to the toxic effects of acetaldehyde may be due, at least in part, to a lack of aldehyde dehydrogenase in the susceptible regions. Furthermore, aldehyde dehydrogenase activity in the Clara cells indicates a possible site for metabolism of aldehydes which penetrate to the lower airways.

Interlaboratory validation of a new assay for DNA-protein crosslinks

In 1992, a simple and sensitive assay for detecting DNA-protein crosslinks was developed [1]. In an effort to facilitate the greater use of the assay, a number of studies were conducted to evaluate its reliability and reproducibility. During this work, the assay was used to assess whether various metals and other compounds could induce crosslinks in cultured human lymphocytes (Epstein-Barr virus-transformed Burkitts Lymphoma cell line). Potassium permanganate, mercury chloride, lead nitrate, magnesium perchlorate, aluminum chloride, and cadmium chloride did not induce DNA-protein crosslinks at either cytotoxic or non-cytotoxic levels. Copper sulfate, arsenic trioxide, and potassium chromate induced DNA-protein crosslinks only at cytotoxic concentrations. Acute lethality of the cells was assessed immediately after exposure to metals by trypan blue exclusion while long-term lethality was assessed by cell proliferation and trypan blue exclusion following an incubation period of 5 days after exposure to the metal compound. All metals exhibited more toxicity in the long-term lethality assay compared to the short-term assay. The cultured human lymphocytes treated with various doses of lead acetate, cadmium chloride, arsenic trioxide and copper sulfate, as well as cis-platinum and chromate, were sent to four different laboratories to compare the reliability and reproducibility of the DNA-protein crosslink assay. Depending on the chemical studied, there were quantitative differences in the results observed among the various laboratories using the assay. However, all laboratories generally showed that cis-platinum, chromate, arsenic trioxide and copper sulfate induced DNA-protein crosslinks at levels that produced acute cytotoxicity, whereas cadmium chloride and lead acetate did not.

Degeneration and recovery of rat olfactory epithelium following inhalation of dibasic esters

Dibasic esters (DBE) are solvent mixtures used in the paint and coating industry. To evaluate the potential subchronic toxicity of DBE, groups of male and female rats were exposed for periods of up to 13 weeks to DBE concentrations of 0, 20, 76, or 390 mg/m3. After approximately 7 and 13 weeks of exposure, 10 rats per sex per group were subjected to clinical chemical, hematological, and urine analyses. Following 7 or 13 weeks of exposure, 10 or 20 rats per sex per group, respectively, were euthanized. An additional 10 rats were euthanized following a 6-week recovery period. A standard profile of tissues, including four levels of nasal cavity, was evaluated histopathologically. After 7 weeks of exposure, slight degeneration of the olfactory epithelium was observed in both male and female rats at 76 and 390 mg/m3. After 13 weeks, degeneration of the olfactory epithelium was present at all DBE concentrations in female rats, but only at the mid and high concentrations in male rats. The severity and incidence of the lesions were concentration related for both sexes with female rats being more sensitive than males. Following the recovery period, histological changes compatible with repair in the olfactory mucosa included an absence of degeneration, focal disorganization of the olfactory epithelium, and respiratory metaplasia. All other tissues were macroscopically normal. No other signs of toxicity were indicated by the other parameters evaluated. Inhalation studies of other esters demonstrate similar pathology in the olfactory epithelium. Since olfactory mucosa is rich in carboxylesterase activity, acids may be the toxic metabolites of these compounds. This hypothetical mechanism may explain the sensitivity of olfactory tissue to the effects of DBE.

Chronic toxicity and oncogenicity inhalation study with vinyl acetate in the rat and mouse.

Vinyl acetate was evaluated for chronic toxicity and oncogenicity in male and female rats and mice in a 104-week study. Target concentrations were 0, 50, 200, and 600 ppm. The study included interim terminations at approximately 53 and 83 weeks and a group whose exposure was terminated at 70 weeks and allowed a 15-week recovery period. Over the course of the exposures, body weight gain was consistently depressed in all 600 ppm groups and in the 200 ppm mice. Except for female rats of the 600 ppm exposure group, recovery animals showed significant improvements in weight gain relative to controls. There were no changes in hematological parameters of either species that could be unequivocally related to treatment. The only effect noted on clinical chemical parameters during the study were decreases in blood glucose in the 600 ppm females. There were no adverse effects on survival in either species. Increases in lung weight were noted in rats and mice primarily in the 600 ppm groups. These changes were associated with bronchial exfoliation, macrophage accumulation, and fibrous plaques and buds extending into the airway lumen, and bronchial/bronchiolar epithelial disorganization. The most significant histopathological changes were noted in the nasal cavity. In the olfactory epithelium of both rats and mice, the main nonneoplastic changes included epithelial atrophy, regenerative effects (squamous metaplasia and respiratory metaplasia of olfactory epithelium), basal cell hyperplasia, and epithelial nest-like infolds. No nonneoplastic changes were observed in the respiratory epithelium of rats, while squamous metaplasia at the naso/maxilloturbinate region was prevalent in mice. Nonneoplastic changes were similar in the recovery groups. Oncogenic responses to vinyl acetate exposure were mainly confined to the nasal cavity in rats and included endo- and exophytic papillomas, squamous cell carcinoma, carcinoma in situ in olfactory regions, and endophytic papilloma in respiratory regions. Squamous cell carcinomas were also found either in areas normally covered by cuboidal epithelium or areas of unknown origin. One squamous cell carcinoma was found in the larynx of a rat of the 600 ppm groups. One squamous cell carcinoma was found in the lung of a mouse of the 600 ppm group.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of Vinyl Acetate Metabolism in Rat and Human Nasal Tissues by an in Vitro Gas Uptake Technique

Physiologically based pharmacokinetic (PBPK) models require estimates of catalytic rate constants controlling the metabolism of xenobiotics. Usually, these constants are derived from whole tissue homogenates wherein cellular architecture and enzyme compartmentation are destroyed. Since the nasal cavity epithelium is composed of a heterogeneous cell population measurement of xenobiotic metabolizing enzymes using homogenates could yield artifactual results. In this article a method for measuring rates of metabolism of vinyl acetate, a metabolism-dependent carcinogen, is presented that uses whole-tissue samples and PBPK modeling techniques to estimate metabolic kinetic parameters in tissue compartments. The kinetic parameter estimates were compared to those derived from homogenate experiments using two methods of tissue normalization. When the in vitro gas uptake constants were compared to homogenate-derived values, using a normalization procedure that does not account for tissue architecture, there was poor agreement. Homogenate-derived values from rat nasal tissue were 3- to 23-fold higher than those derived using the in vitro gas uptake method. When the normalization procedure for the rat homogenate-derived values took into account tissue architecture, a good agreement was observed. Carboxylesterase activity in homogenates of human nasal tissues was undetectable. Using the in vitro gas uptake technique, however, carboxylesterase activity was detected. Rat respiratory carboxylesterase and aldehyde dehydrogenase activities were about three and two times higher than those of humans, respectively. Activities of the rat olfactory enzymes were about equivalent to those of humans. K(m) values did not differ between species. The results suggest that the in vitro gas uptake technique is useful for deriving enzyme kinetic constants where effects of tissue architecture are preserved. Furthermore, the results suggest that caution should be exercised when scaling homogenate-derived values to whole-organ estimates, especially in organs of cellular heterogeneity.

ras gene mutations in vinyl chloride-induced liver tumours are carcinogen-specific but vary with cell type and species

Previous studies have shown that a high proportion (5/6) of human liver angiosarcomas (ASL) associated with exposure to vinyl chloride (VC) contains a GC→AT mutation at the Ki‐ras codon 13. This mutation, however, has not been found in 5 ASL or 2 hepatocellular carcinomas (HCC) induced in rats by VC. These 2 HCC did contain a mutation at codon 61 of the Ha‐ras gene. In order to extend this study and further explore the mechanisms of tumour induction, an additional 6 ASL and 6 HCC induced in rats by VC were analysed for ras gene point mutations, as well as 10 rat and 10 murine ASL induced by vinyl fluoride (VF), and 5 ASL, 6 Kupffer cell sarcomas, 4 HCC and 2 cholangiocellular carcinomas induced by Thorotrast in rats. Tumour DNA was analysed by PCR‐SSCP and direct sequencing. None of the rodent ASL contained a mutation at codon 13 of the Ki‐ras gene showing that the ras gene mutational pattern is species‐specific. The CAA→CTA mutation, previously found at codon 61 of the Ha‐ras gene in rat HCC, was observed in 5 further VC‐induced HCC but was not detected in the Thorotrast‐induced HCC, suggesting carcinogen‐specificity. This mutation was also absent in VC‐induced ASL, which supports the cell‐specificity of the ras mutational pattern in chemically induced tumours. No predominant mutation was detected in VF‐ and Thorotrast‐induced tumours. Thus, a given mutation in a tumour may be carcinogen‐specific but also depend on the species and the cell type. Int. J. Cancer 85:223–227, 2000. ©2000 Wiley‐Liss, Inc.

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.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.

Formation and stability of acetaldehyde-induced crosslinks between poly-lysine and poly-deoxyguanosine.

The amino acid residue and nucleoside specificity of acetaldehyde-induced DNA-protein crosslinks (DPXLs) were studied using a modified filter binding assay. A 40% inhibition of acetaldehyde-induced pUC13 plasmid DNA-calf thymus histone crosslink formation was achieved by addition of 50 mM lysine (free base), while arginine was unable to affect crosslink formation at concentrations to 150 mM. Polymers (5-mers) of lysine (poly-lys5) were able to substitute for histones in acetaldehyde-induced plasmid crosslink formation, being equally effective at equimolar concentrations. Homopolymers (6-mers) of deoxyguanosine (poly-dG6) (but not deoxyadenosine, deoxycytidine or thymidine) served as an efficient substrate for acetaldehyde-induced DPXL formation, using either calf thymus histones or poly-lys5 as the protein source. Acetaldehyde-induced crosslinks between poly-dG6 and poly-lys5 were formed rapidly, but were unstable at 37 degrees C (a half-life or 1.5-2 h). Stability of these crosslinks was unaffected by pH at a range of 5.5-9.0 at 37 degrees C for 2 h. Results presented here suggest that unstable complexes of deoxyguanosine and lysine constitute a major portion of the DPXLs formed by acetaldehyde in vitro.