Comparative gavage subchronic toxicity studies of o-chloroaniline and m-chloroaniline in F344 rats and B6C3F1 mice.

ortho-Chloroaniline (o-CA) andmeta-chloroaniline (m-CA) are chemical intermediates for pigment production in the textile industry. Comparative subchronic gavage studies were conducted to determine the effect of structure on toxicity.o-CA orm-CA was administered to 10 animals/sex/species in deionized water at dosages of 0, 10, 20, 40, 80, and 160 mg/kg for 13 weeks. Blood samples for clinical pathology were collected after 3 and 23 days in rats and at study termination (Day 93) in rats and mice. No mortalities occurred that could be directly attributed to treatment. Transient clinical signs of toxicity observed after dosing included cyanosis in rats and ataxia and tremors in mice. Methemoglobin formation was directly related to dosage (rats and mice) and duration of treatment (rats). At study termination, Heinz body formation in erythrocytes in association with decreased hemoglobin, hematocrit, and red blood cell count was a prominent treatment-related effect. Enlarged spleens (gross necropsy observation) and increased spleen weight were treatment effects of each chemical in both species. Microscopic lesions typical of increased red blood cell production were found in hematopoietic tissues (bone marrow, spleen, and liver), while lesions due to increased red cell destruction were found in these tissues and also the kidneys (rats). Microscopic changes were more frequently seen and severe, and involved more body organs, in rats than mice, and in m-CA-treated animals thano-CA-treated animals. Sex differences in lesion incidence/severity were not evident.

DNA-protein crosslink formation in rat nasal epithelial cells by hexamethylphosphoramide and its correlation with formaldehyde production.

Hexamethylphosphoramide (HMPA) is an aprotic polar solvent and nasal carcinogen in rats. The metabolism of HMPA to formaldehyde, another nasal carcinogen in rats, was found to be approximately 6 times greater in microsomes from olfactory tissues than from respiratory tissues (isolated from both male and female rats). HMPA was shown to induce formation of DNA-protein crosslinks (DPXLS) in isolated rat nasal epithelial cells. Using a filter binding assay, we demonstrated that microsomal activation is necessary for HMPA-induced crosslink formation between plasmid DNA and calf thymus histones, presumably through metabolic N-demethylation of HMPA and the formation of formaldehyde. Both formaldehyde production and DPXL formation were inhibited by pre-incubation of nasal mucosal extracts with metyrapone, an inhibitor of cytochrome P-450. Significant dose-dependent increases in DPXL formation were observed in respiratory and olfactory epithelial cells exposed to > or = 0.5 and 1 mM HMPA, respectively, for 3 h at 37 degrees C. This resulted in DPXL accumulation at 18-20% higher levels than untreated cells. Increases in DPXL formation in rat nasal epithelial cells cultured with 1 mM HMPA were inhibited by over 70% by co-administration of metyrapone. These data suggest that metabolic liberation of formaldehyde from HMPA is involved in the mechanism of HMPA-induced nasal carcinogenesis. Comparative studies showed formaldehyde to be more potent than HMPA in the induction of DPXL in nasal epithelium. However, induction of tumor formation after two years at 50 ppb HMPA and 6 ppm formaldehyde show the former to be active at several-fold lower concentrations. Therefore, other mechanisms are likely to be involved in HMPA nasal carcinogenesis.

A microscopic and ultrastructural evaluation of dibasic esters (DBE) toxicity in rat nasal explants.

Dibasic esters (DBE) solvent has been demonstrated to induce a mild degeneration of the olfactory, but not the respiratory epithelium of the rat nasal cavity following a 90-day inhalation exposure. Previous work has demonstrated that acid phosphatase release is a reliable index of DBE-induced cytotoxicity in an in vitro system of rat nasal explants. In the present study, rat nasal explants were examined microscopically and ultrastructurally following incubation in varying concentrations of a representative DBE, dimethyl adipate (DMA). DMA-induced microscopic and ultrastructural changes in rat nasal explants correlated well with biochemical perturbations associated with DBE exposure in a previous study. In both studies, olfactory epithelium was more susceptible to DMA-induced toxicity than respiratory epithelium and DMA-induced nasal toxicity was attenuated by pretreatment with a carboxylesterase inhibitor. The results of this study support the hypothesis that DBE and potentially other inhaled organic esters induce nasal toxicity via a common mechanism, carboxylesterase-mediated production of toxic acid metabolites. It was established that carboxylesterase-rich sustentacular cells are the primary target cells for DBE toxicity in rat nasal explants. It was proposed that degeneration of nasal olfactory sensory cells observed in rats following 90-day inhalation exposure to DBE may be secondary to necrosis and loss of sustentacular cells.

Cytotoxicity of dibasic esters (DBE) metabolites in rat nasal explants.

Dibasic esters (DBE) is a solvent mixture of dimethyl adipate, dimethyl glutarate, and dimethyl succinate which causes a selective degeneration of the nasal olfactory epithelium in rats following a 90-day inhalation exposure. In short-term cultures of rat nasal explants, it has been demonstrated that DBE cytotoxicity is due to a carboxylesterase-mediated activation. In the present study, the putative toxic metabolites of DBE, the monomethyl esters and the dicarboxylic acids, were evaluated in the nasal explant system at concentrations ranging from 10 to 50 mM. Monomethyl adipate (MMA), monomethyl glutarate (MMG), and monomethyl succinate (MMS) induced increases in nasal explant acid phosphatase release, a biochemical index of cytotoxicity. The nasal explant-mediated metabolism of MMA and MMG to their corresponding diacids paralleled the increases in acid phosphatase release. A carboxylesterase inhibitor, bis(p-nitrophenyl)phosphate (BNPP), inhibited both the cytotoxicity and the hydrolysis of MMA and MMG in the nasal explant system. The metabolism and cytotoxicity of MMS was not attenuated as effectively by BNPP pretreatment. Adipate, glutarate, and succinate induced concentration-related increases in cytotoxicity in the nasal explant system. These dicarboxylic acids were neither metabolized nor utilized significantly by the nasal explants. Diacid-induced cytotoxicity was not attenuated by BNPP pretreatment. This study further established the utility of the nasal explant system for evaluating cytotoxicity of organic esters in vitro. It was established that both the monomethyl ester and diacid metabolites are cytotoxic in rat nasal explants. Finally, it was concluded that although both the monomethyl esters and the diacids contribute to the cytotoxic potential of DBE in vitro, it is critical to establish if one or both of these are formed in vivo in order to identify the ultimate toxic metabolite of DBE.

Deposition of dibasic esters in the upper respiratory tract of the male and female Sprague-Dawley rat.

Inhalation exposure of the male and female rat to high concentrations of a mixture of the dibasic esters dimethyl succinate (DMS), dimethyl glutarate (DMG), and dimethyl adipate (DMA) results in mild olfactory toxicity. This response is thought to be due to the in situ formation of acidic metabolites via nasal carboxylesterases. The current study was designed to provide inhalation dosimetric information for these vapors. Deposition of DMS, DMG, and DMA was measured in the surgically isolated upper respiratory tracts (URT) of ketamine-xylazine-anesthetized male and female rats under constant velocity flow conditions at a flow rate of 100 ml/min. Deposition of acetone was measured in both genders for comparative purposes. URT deposition efficiencies in excess of 98.3% were observed for DMS, DMG, and DMA in animals exposed to each vapor individually. No gender differences in deposition efficiency were observed for these vapors or for acetone. Deposition of DMS, DMG, and DMA was also measured in animals exposed to all three vapors simultaneously. Deposition efficiency under simultaneous exposure conditions ranged between 97.3 and 98.5%. These values were slightly lower (about 1%) than those obtained under individual exposure conditions (p less than 0.0001). The reduced deposition efficiency may have resulted from competitive inhibition of nasal metabolism due to the simultaneous presence of all three carboxylesterase substrate vapors in nasal tissues. If so, inhalation of dibasic ester vapors would be expected to inhibit the uptake of other carboxylesterase substrate vapors without influencing uptake of vapors which are not substrates for this enzyme. Such was observed in studies using DMS, ethyl acetate (the substrate vapor), and isoamyl alcohol (the nonsubstrate vapor). Specifically, simultaneous exposure to DMS markedly inhibited uptake of ethyl acetate without altering uptake of isoamyl alcohol. Gender differences were not observed in URT deposition of any of the six vapors used in the current study, DMS, DMG, DMA, ethyl acetate, isoamyl alcohol, or acetone, suggesting that gender differences in URT deposition may not be widespread among vapors. The high URT deposition efficiencies of the dibasic esters are consistent with the olfactory toxicity resulting from inhalation exposure to these vapors.

Carboxylesterase-dependent cytotoxicity of dibasic esters (DBE) in rat nasal explants.

Dibasic esters (DBE) are a solvent mixture of dimethyl adipate (DMA), dimethyl glutarate (DMG), and dimethyl succinate (DMS) used in the paint and coating industry. Subchronic inhalation toxicity studies have demonstrated that DBE induce a mild degeneration of the olfactory, but not the respiratory, epithelium of the rat nasal cavity. Carboxylesterase-mediated hydrolysis of the individual dibasic esters is more efficient in olfactory than in respiratory mucosal homogenates. In the present study, an in vitro system of cultured rat nasal explants was utilized to determine if DBE toxicity is dependent on a metabolic activation by nonspecific carboxylesterase. Explants from both the olfactory and the respiratory regions of the female rat nasal cavity were incubated for 2 hr in Williams' medium E containing 10-100 mM DMA, DMG, or DMS. DBE caused a dose-related increase in nasal explant acid phosphatase release, a biochemical index of cytotoxicity. HPLC analysis demonstrated parallel increases in the carboxylesterase-mediated formation of monomethyl ester metabolites. Diacid metabolite production in the nasal explant system was not entirely concentration-dependent. Metabolite concentrations and acid phosphatase release were generally greater in olfactory than respiratory tissues. DBE-induced cytotoxicity and acid metabolite production were markedly attenuated in nasal tissue excised from rats which were pretreated with bis(p-nitrophenyl)phosphate, a carboxylesterase inhibitor. This study presents a viable in vitro method for assessing organic ester cytotoxicity in the rat nasal cavity. It was shown that DBE are weak nasal toxicants under the conditions of this system. It was further demonstrated that DBE toxicity is dependent on a carboxylesterase-mediated activation. A similar mechanism was proposed for the nasal toxicity induced by other organic esters following inhalation exposure.

Metabolism of dibasic esters by rat nasal mucosal carboxylesterase.

Inhalation exposure of rats to dibasic esters revealed lesions of the nasal olfactory epithelium similar to those observed with other ester solvents. Female rats are more sensitive to these effects than are male rats. It has been proposed that carboxylesterase conversion of inhaled esters within nasal tissues to organic acids may be a critical biochemical step in converting these chemicals to toxic substances. These experiments measured the kinetic parameters Vmax, KM, Ksi, and V/K for the hydrolysis of the dibasic esters in the target nasal tissue, olfactory mucosa, and nontarget tissue, respiratory mucosa. It was determined that under the conditions of these experiments, diacid metabolites are not formed. Esterase activity was inhibited by pretreatment with bis p-nitrophenyl phosphate. Vmax values for the three dibasic esters were 5- to 13-fold greater in olfactory mucosa than respiratory mucosa for male or female rats. V/K values were 4- to 11-fold greater in olfactory mucosa than respiratory mucosa for male or female rats. V/K was similar between male and female olfactory mucosa when dimethyl glutarate was used as the substrate. With dimethyl succinate or dimethyl adipate as the substrate, V/K for female olfactory tissue was 0.5- or 2-fold that of males, respectively. Differences in V/K were mainly due to decreases in KM associated with increasing carbon chain length. Substrate inhibition was observed at dibasic ester concentrations greater than approximately 25 mM, which are unlikely to be achieved in vivo. These results lend further support to the hypothesis that organic acid accumulation in the target tissue, olfactory mucosa, plays a significant role in the pathogenesis of dibasic ester-induced nasal lesions. This mechanism may be applicable to a wide range of inhaled esters.

Effect of carbon monoxide on the cytochrome P-450-mediated activation of 4-ipomeanol by the isolated perfused rabbit lung.

4-Ipomeanol is a naturally occurring toxin that induces lesions in the lung following its activation to an alkylating metabolite by the pulmonary cytochrome P-450 system. The aim of this study was to determine if an environmentally relevant concentration of carbon monoxide could inhibit the activation of 4-ipomeanol and prevent the associated toxic sequelae in the isolated perfused rabbit lung. The lungs of male New Zealand rabbits were removed and perfused with [14C]-4-ipomeanol for 2 h starting with an initial concentration of 0.1 mM. Lungs were ventilated with either air (control) or 7.5% CO/20% O2. 4-Ipomeanol-derived covalent binding was identical in the control and carbon monoxide treatment groups. Lungs perfused with 4-ipomeanol and ventilated with air or 7.5% CO/20% O2 both displayed alveolar type II cell hyperplasia and alveolar macrophage infiltration. Surprisingly, there was no histological evidence of Clara cell damage in any of the 4-ipomeanol-perfused lungs. These results suggest that the isozymes of pulmonary cytochrome P-450 that act in concert to metabolize 4-ipomeanol are relatively insensitive to inhibition by carbon monoxide.

Effect of carbon monoxide on the cytochrome P-450-mediated metabolism of aniline and p-nitroanisole in the isolated perfused rabbit lung.

Carbon monoxide (CO), an environmental pollutant, inhibits the cytochrome P-450-mediated metabolism of xenobiotics in vitro. In recent years, the importance of the lung in the metabolic disposition of certain airborne and systemically administered xenobiotics has been demonstrated. The purpose of this investigation was to establish a threshold for the CO-induced inhibition of cytochrome P-450-mediated activities in the isolated perfused rabbit lung and to determine if these reactions are equally sensitive to this toxicant in this model. Neither the mixed-function oxidase-mediated hydroxylation nor the acetylation of aniline was altered by exposure to 7.5% CO/20% O2 for 2.5 h in the isolated perfused rabbit lung. p-Nitroanisole O-demethylation by isolated rabbit lungs ventilated with 7.5% CO/20% O2 was significantly decreased (approximately 37%) in comparison to controls. That these reactions are not similarly influenced by carbon monoxide may indicate that the constitutive isozymes of cytochrome P-450 in the rabbit lung are differentially sensitive to CO-induced inhibition.

The effect of carbon monoxide on aminopyrine metabolism in the isolated perfused rabbit lung.

Carbon monoxide (CO) is a ubiquitous environmental pollutant widely recognized for its ability to inhibit cytochrome P450-mediated metabolism of xenobiotics in vitro. In recent years, the importance of the lung in the metabolic disposition of certain airborne and systemically administered xenobiotics has been demonstrated. The purpose of this investigation was to establish a threshold for the CO-induced inhibition of cytochrome P450-mediated activity in the isolated perfused rabbit lung and to determine if hemoglobin would alter the carbon monoxide-cytochrome P450 interaction. On the basis of its half-life and the stoichiometry of its metabolism, aminopyrine was shown to be a good substrate for monitoring mixed function oxidase activity in the intact rabbit lung. First-order rate constants for aminopyrine metabolism were significantly lower in isolated rabbit lungs perfused with either artificial medium (39%) or whole blood (67%) and ventilated with a 7.5% CO/20% O2 mixture for 2.5 hr than in the respective control lungs ventilated with breathing air. The threshold level (7.5% CO) for this inhibition is the same in lungs perfused with artificial medium and in whole blood-perfused lungs and is well above environmentally relevant levels of exposure.

Comparison of in vivo and in vitro methods for assessing the effects of repeated dosing with carbon tetrachloride on the hepatic drug-metabolizing enzyme system.

The effect of 7 daily i.p. injections of 0, 2, 20, or 200 microliters/kg carbon tetrachloride on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay, employing lindane (gamma-hexachlorocyclohexane), and by a battery of in vitro enzyme assays. The data in this study indicated that repeated administration of CCl4 for 7 days significantly increased phase I and phase II reactions in vivo and in vitro. Though there were differences between the responses of the in vivo and in vitro assays, this is the first report of increased hepatic drug-metabolizing enzyme activity from repeated treatment with CCl4.

Comparison of in vivo and in vitro methods for assessing the effects of carbon tetrachloride on the hepatic drug-metabolizing enzyme system.

The effect of a single i.p. injection of 0, 20, 200, and 1000 microliter/kg carbon tetrachloride on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay employing lindane (gamma-hexachlorocyclohexane) and by a battery of in vitro enzyme assays. The data in this study indicated that carbon tetrachloride had a biphasic influence on the phase I reactions with the lowest dose inducing a significant increase in enzyme activity while the highest dose produced significant inhibition. Significant CCl4-induced reductions in glucuronyltransferase and sulfotransferase activities were also observed while the effect on glutathione-S-transferase was ambiguous. The in vivo and in vitro assays showed good agreement.

Comparison of in vivo and in vitro methods for assessing the effects of bromobenzene on the hepatic-metabolizing enzyme system.

The effect of daily i.p. injections of 0, 0.05, 0.5 and 5.0 mmol/kg bromobenzene for 1 week on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay employing lindane (gamma-hexachlorocyclohexane) and by a battery of in vitro enzyme assays. The data in this study indicated that repeated pretreatment with bromobenzene for 1 week stimulated a dose-related increase in phase I reactions while inducing phase II reactions at the high dose (5 mmol/kg bromobenzene). The in vivo and in vitro assays showed good agreement.

Effect of flavanone on mixed-function oxidase and conjugation reactions in rats.

Flavanone administered to female Fischer 344 rats (1.00 mmol/kg per day for seven days) did not induce ethylmorphine demethylation, aniline hydroxylation, hexobarbital oxidation or aldrin epoxidation. Flavanone at 0.05 mmol/kg per day for seven days increased glutathione-S-transferase activity with 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy) propane as substrates. Flavanone at 0.05 mmol/kg per day for seven days increased the glucuronidation of 1-naphthol, and at 0.20 mmol/kg increased the glucuronidation of chloramphenicol. Flavanone administration did not alter sulphotransferase activity with 2-naphthol as the substrate and did not enhance glutathione-S-transferase or glucuronyltransferase activity when added in vitro.

A comparison of in vitro and in vivo methods for evaluating alterations in hepatic drug metabolism following mercuric chloride administration.

Mercuric chloride was administered once i.p. to female Fischer-344 rats at doses of 0, 0.2, 0.6 and 1.8 mg/kg. Although there were no alterations in the urinary excretion of lactate dehydrogenase, significant elevations in the activities of urinary (U) alkaline phosphatase, glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) indicated that mercuric chloride was nephrotoxic. There was no evidence of hepatotoxicity as hepatic glucose-6-phosphatase and serum sorbitol dehydrogenase were essentially unaffected by mercuric chloride administration. The activities of ethylmorphine demethylase, hexobarbital oxidase and aldrin epoxidase determined in vitro were not inhibited by mercuric chloride although aniline hydroxylase activity was decreased. Of the four phase-II reactions measured, only the glucuronidation of chloramphenicol was diminished by treatment with mercuric chloride. Results from the in vivo studies on the metabolism of lindane, which indicated no change in the excretion of free or conjugated metabolites, were in close agreement with the in vitro data suggesting that the nephrotoxic effects of mercuric chloride do not alter the urinary excretion of the model substrate lindane.

Comparison of in vivo and in vitro methods for assessing effects of allyl alcohol on the liver.

Allyl alcohol was administered intraperitoneally (i.p.) to female Fischer 344 rats at doses of 0, 3, 10 and 30 mg/kg daily for 7 days. Plasma sorbitol dehydrogenase was minimally elevated. No dose-related changes were observed in hexobarbital oxidation, aniline hydroxylation, or ethylmorphine demethylation. Aldrin epoxidation was slightly elevated. Naphthol glucuronidation and glutathione-S-transferase activity with 1,2-dichloro-4-nitrobenzene were increased. Results from in vivo studies on the metabolism of lindane were in close agreement with the in vitro measurements suggesting that daily treatment for one week with allyl alcohol at doses of 3, 10 and 30 mg/kg has no significant effect on phase I pathways, has a selective effect on phase II pathways and, under the conditions of this experiment, has minimal hepatotoxic effects in these rats.

Comparison of in vitro methods and the in vivo metabolism of lindane for assessing the effects of repeated administration of ethanol on hepatic drug metabolism.

In vitro methods of assessing alterations in drug metabolism and the measurement of lindane metabolites in urine were compared for their ability to determine the influence of ethanol on drug metabolism. Ethanol was administered intraperitoneally (i.p.) to young adult female rats daily for 7 days at doses of 0.12, 0.60 and 3.0 ml/kg. No alterations were observed in ethylmorphine demethylation, hexobarbital oxidation or glucuronyltransferase. Aniline hydroxylation was decreased at the high dose level and aldrin epoxidation was increased at the intermediate dose. In vivo only the high dose of ethanol produced significant changes with significant increases observed for the oxidation of lindane to alcohol metabolites, the glucuronidation of the alcohol but not the chlorophenol metabolites, and glutathione conjugation. The latter increase was also observed in vitro. The in vivo and in vitro data suggest a minimal effect of ethanol on drug metabolism at low levels of administration.

Comparison of in vivo and in vitro methods for assessing the effects of phenobarbital on the hepatic drug-metabolizing enzyme system.

The effect of daily i.p. injections of 0, 1, 10 and 80 mg/kg phenobarbital for 1 week on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay employing lindane (gamma-HCH) and by a battery of in vitro enzyme assays. Comparison of the dose-response curves of the in vivo and in vitro assays indicated that urinary metabolites of lindane provided a good index of phenobarbital-induced change in both phase I and phase II reactions.