Patrick J. Sabourin

Effect of dose on the absorption and excretion of [14C]benzene administered orally or by inhalation in rats and mice

The effect of dose on the absorption and excretion of [14C]benzene was studied using 13-week old male F344/N rats, Sprague-Dawley rats, and B6C3F1 mice. Gastrointestinal absorption of benzene administered by gavage was greater than 97% in these species for doses between 0.5 and 150 mg benzene/kg body wt. At oral doses below 15 mg/kg, greater than 90% of the 14C excreted was in the urine as nonethylacetate extractable material. Above 15 mg/kg, in both rats and mice, an increasing percentage of the administered benzene was exhaled unmetabolized, suggesting saturation of metabolic pathways. Above 50 mg/kg, total metabolites (as determined by 14C in the urine, feces, and carcass after 2 days) were not linearly related to administered dose. Total metabolites per unit body weight was equal in F344/N rats and B6C3F1 mice at gavage doses up to 50 mg/kg; however, total metabolites in mice did not increase at higher doses. For inhalation exposures, the percentage of inhaled benzene that was absorbed and retained during a 6-hr exposure decreased from 33 +/- 6% (mean +/- standard deviation) to 15 +/- 9% in rats, and from 50 +/- 15 to 10 +/- 2% in mice as the exposure concentration was increased from approximately 26 to 2600 micrograms/liter (10 to 1000 ppm at 615 Torr, 23 degrees C). Total metabolite formation was exponentially related to the benzene exposure concentration with one-half the maximal amount of metabolite formation occurring at 220 micrograms/liter (84 ppm) for B6C3F1 mice and 650 micrograms/liter (250 ppm) for F344/N rats. Total metabolites were higher in mice than in rats at any of the vapor concentrations used due mainly to the higher amount inhaled by mice. Saturation of overall metabolism in mice but not in rats at high doses by both routes of administration indicates species differences in metabolism of benzene.

A physiological model for simulation of benzene metabolism by rats and mice

Studies conducted by the National Toxicology Program on the chronic toxicity of benzene indicated that B6C3F1 mice are more sensitive to the toxic effects of benzene than are F344 rats. A physiological model was developed to describe the uptake and metabolism of benzene in rats and mice and to determine if the observed differences in toxic effects could be explained by differences in the pathways for metabolism of benzene or by differences in uptake of benzene. Major pathways for elimination of benzene included metabolism to hydroquinone glucuronide or hydroquinone sulfate, phenyl glucuronide or phenyl sulfate, muconic acid, and prephenyl mercapturic acid or phenyl mercapturic acid. Model simulations for total benzene metabolized and for profiles of benzene metabolites were conducted for oral or inhalation exposure and compared to data for urinary excretion of benzene metabolites after exposure of rats and mice to [14C]- or [3H]-benzene by inhalation or gavage. Results for total amount of benzene metabolized, expressed per kilogram body weight, indicated that for inhalation exposure concentrations up to 1000 ppm, mice metabolized at least two to three times as much benzene as did rats. Simulations of oral exposure to benzene resulted in more benzene metabolized per kilogram body weight by rats at oral exposures of greater than 50 mg/kg. Patterns of metabolites formed after either route of exposure were very different for F344/N rats and B6C3F1 mice. Rats primarily formed the detoxification metabolite, phenyl sulfate. Mice formed hydroquinone glucuronide and muconic acid in addition to phenyl sulfate. Hydroquinone and muconic acid are associated with pathways leading to the formation of the putative toxic metabolites of benzene. Metabolic rate parameters, Vmax and Km, were very different for hydroquinone conjugate and muconic acid formation compared to formation of phenyl conjugates and phenyl mercapturic acids. Putative toxication pathways could be characterized as high affinity, low capacity whereas detoxification pathways were low affinity, high capacity. Model simulations suggested that for both rats and mice at lower exposure concentrations hydroquinone and muconic acid represented a larger fraction of the total benzene metabolized than at higher exposure concentrations where detoxification metabolites were predominant. Preferential production of a putative toxic metabolite at low exposure concentrations may have important implications in risk assessment for benzene.

Differences in the metabolism and disposition of inhaled [3H]benzene by F344N rats and B6C3F1 mice

Benzene is a potent hematotoxin and has been shown to cause leukemia in man. Chronic toxicity studies indicate that B6C3F1 mice are more susceptible than F334/N rats to benzene toxicity. The purpose of the studies presented in this paper was to determine if there were metabolic differences between F344/N rats and B6C3F1 mice which might be responsible for this increased susceptibility. Metabolites of benzene in blood, liver, lung, and bone marrow were measured during and following a 6-hr 50 ppm exposure to benzene vapor. Hydroquinone glucuronide, hydroquinone, and muconic acid, which reflect pathways leading to potential toxic metabolites of benzene, were present in much greater concentrations in the mouse than in rat tissues. Phenylsulfate, a detoxified metabolite, and an unknown water-soluble metabolite were present in approximately equal concentrations in these two species. These results indicate that the proportion of benzene metabolized via pathways leading to the formation of potentially toxic metabolites as opposed to detoxification pathways was much higher in B6C3F1 mice than in F344 rats, which may explain the higher susceptibility of mice to benzene-induced hematotoxicity and carcinogenicity.

Disposition of three glycol ethers administered in drinking water to male F344N rats

The glycol ethers 2-methoxyethanol (ME), 2-ethoxyethanol (EE), and 2-butoxyethanol (BE) are widely used solvents in industrial and consumer applications. The reproductive, teratogenic, and hematotoxic effects of the glycol ethers are due to the alkoxyacetic acid metabolites of these compounds. The effect of alkyl group length on disposition of these three glycol ethers was studied in male F344/N rats allowed access for 24 hr to 2-butoxy[U-14C]ethanol, 2-ethoxy[U-14C]ethanol, or 2-methoxy[U-14C]ethanol in drinking water at three doses (180 to 2590 ppm), resulting in absorbed doses ranging from 100 to 1450 mumols/kg body wt. Elimination of radioactivity was monitored for 72 hr. The majority of the 14C was excreted in urine or exhaled as CO2. Less than 5% of the dose was exhaled as unmetabolized glycol ether. Distinct differences in the metabolism of the glycol ethers as a function of alkyl chain length were noted. For BE 50-60% of the dose was eliminated in the urine as butoxyacetic acid and 8-10% as CO2; for EE 25-40% was eliminated as ethoxyacetic acid and 20% as CO2; for ME 34% was eliminated as methoxyacetic acid and 10-30% as CO2. Ethylene glycol, a previously unreported metabolite of these glycol ethers, was excreted in urine, representing approximately 10, 18, and 21% of the dose for BE, EE, and ME, respectively. Thus, for longer alkyl chain lengths, a smaller fraction of the administered glycol ether was metabolized to ethylene glycol and CO2. Formation of ethylene glycol suggests that dealkylation of the glycol ethers occurs prior to oxidation to alkoxyacetic acid and, as such, represents an alternate pathway in the metabolism of these compounds that does not involve formation of the toxic acid metabolite.

A high pressure liquid chromatographic method for the separation and quantitation of water-soluble radiolabeled benzene metabolites

The glucuronide and sulfate conjugates of benzene metabolites as well as muconic acid and pre-phenyl- and phenylmercapturic acids were separated by ion-pairing HPLC. The HPLC method developed was suitable for automated analysis of a large number of tissue or excreta samples. p-Nitrophenyl [14C]glucuronide was used as an internal standard for quantitation of these water-soluble metabolites. Quantitation was verified by spiking liver tissue with various amounts of phenylsulfate or glucuronides of phenol, catechol, or hydroquinone and analyzing by HPLC. Values determined by HPLC analysis were within 10% of the actual amount with which the liver was spiked. The amount of metabolite present in urine following exposure to [3H]benzene was determined using p-nitrophenyl [14C]glucuronide as an internal standard. Phenylsulfate was the major water-soluble metabolite in the urine of F344 rats exposed to 50 ppm [3H]benzene for 6 h. Muconic acid and an unknown metabolite which decomposed in acidic media to phenylmercapturic acid were also present. Liver, however, contained a different metabolic profile. Phenylsulfate, muconic acid, and pre-phenylmercapturic acids as well as an unknown with a HPLC retention time of 7 min were the major metabolites in the liver. This indicates that urinary metabolite profiles may not be a true reflection of what is seen in individual tissues.

Toxicokinetics of inhaled 1,3-butadiene in monkeys: Comparison to toxicokinetics in rats and mice

1,3-Butadiene is a potent carcinogen in mice and a weaker carcinogen in rats. People are exposed to butadiene through its industrial use--largely in rubber production (over 3 billion pounds of butadiene were produced in 1989)--and because it is common in the environment, occurring in cigarette smoke, gasoline vapor and in the effluents from fossil fuel incineration. Epidemiological studies have provided some evidence for butadiene carcinogenicity in people. Differences in the uptake and metabolism of inhaled butadiene between rodents and primates, including people, might be reflected in differences in its toxicity. In order to compare uptake and metabolism in primates to that in rodents--for which data were already available--we exposed cynomolgus monkeys (Macaca fascicularis) to 14C-labeled butadiene at concentrations of 10.1, 310 or 7760 ppm for 2 hr. Exhaled air and excreta were collected during exposure and for 96 hr after exposure. The uptake of butadiene as a result of metabolism was much lower in monkeys than in rodents. For equivalent inhalation exposures, the concentrations of total butadiene metabolites in the blood were 5-50 times lower in monkey than in the mouse, the more sensitive rodent species, and 4-14 times lower than in the rat. If the toxicokinetics of butadiene in people is more like that of the monkey than that of rodents, then our data suggest that people will receive lower doses of butadiene and its metabolites than rodents following equivalent inhalation exposures to butadiene. This has important implications for assessing the risk to humans of butadiene exposure based on animal studies.

Metabolism of [14C]benzene by cynomolgus monkeys and chimpanzees

Rodent bioassays indicate that B6C3F1 mice are more sensitive to the carcinogenicity of benzene than are rats. The urinary profile of benzene metabolites is different in rats vs mice. Mice produce higher proportions of hydroquinone conjugates and muconic acid, indicators of metabolism via pathways leading to putative toxic metabolites, than do rats. In both species, metabolism to hydroquinone and muconic acid is favored at low concentrations of benzene, indicating that these pathways are easily saturated. These species differences in the metabolism of benzene make it difficult to predict the health risk to humans and how this risk varies with dose. For this reason, the metabolism of [14C]benzene by cynomolgus monkeys and chimpanzees, animals phylogenetically closer to humans than rodents, was studied. Monkeys were dosed ip with 5, 50, or 500 mg [14C]benzene/kg body wt. Urine was collected for up to 24 hr following exposure and was analyzed for benzene metabolites. The proportion of the administered 14C excreted in the urine of monkeys decreased from approximately 50 to 15% as the dose increased. Phenyl sulfate was the major urinary metabolite. The proportion of hydroquinone conjugates and muconic acid in the monkeys urine decreased as the dose increased. The proportion of catechol conjugates was not affected by dose. The proportion of these metabolites in the urine was quite variable from animal to animal, but the proportion of muconic acid was consistently much lower in the monkey than in the mouse or rat. Three chimpanzees were administered 1 mg [14C]benzene/kg body wt, iv; essentially all of the injected 14C was recovered in the urine. Of the total urinary metabolites, 79% were accounted for by phenyl conjugates and less than 15% by hydroquinone conjugates or muconic acid. Catechol conjugates were not detected. The metabolism of benzene appeared to be qualitatively similar but quantitatively different in the species studied. The mouse, the sensitive rodent species, forms the highest levels of hydroquinone conjugates and muconic acid and the chimpanzee, the lowest. In all animal species studied for the effect of dose on benzene metabolism, as the dose decreased, a larger proportion of the benzene metabolites was represented by hydroquinone conjugates and muconic acid.

Effect of repeated benzene inhalation exposures on benzene metabolism, binding to hemoglobin, and induction of micronuclei.

Metabolism of benzene is thought to be necessary to produce the toxic effects, including carcinogenicity, associated with benzene exposure. To extrapolate from the results of rodent studies to potential health risks in man, one must know how benzene metabolism is affected by species, dose, dose rate, and repeated versus single exposures. The purpose of our studies was to determine the effect of repeated inhalation exposures on the metabolism of [14C]benzene by rodents. Benzene metabolism was assessed by characterizing and quantitating urinary metabolites, and by quantitating 14C bound to hemoglobin and micronuclei induction. F344/N rats and B6C3F1 mice were exposed, nose-only, to 600 ppm benzene or to air (control) for 6 hr/day, 5 days/week for 3 weeks. On the last day, both benzene-pretreated and control animals were exposed to 600 ppm, 14C-labeled benzene for 6 hr. Individual benzene metabolites in urine collected for 24 hr after the exposure were analyzed. There was a significant decrease in the respiratory rate of mice (but not rats) pretreated with benzene which resulted in lower levels of urinary [14C]benzene metabolites. The analyses indicated that the only effects of benzene pretreatment on the metabolite profile in rat or mouse urine were a slight shift from glucuronidation to sulfation in mice and a shift from sulfation to glucuronidation in rats. Benzene pretreatment also had no effect, in either species, on formation of [14C]benzene-derived hemoglobin adducts. Mice and rats had similar levels of hemoglobin adduct binding, despite the higher metabolism of benzene by mice. This indicates that hemoglobin adduct formation occurs with higher efficiency in rats. After 1 week of exposure to 600 ppm benzene, the frequency of micronucleated, polychromatic erythrocytes (PCEs) in mice was significantly increased. Exposure to the same level of benzene for an additional 2 weeks did not further increase the frequency of micronuclei in PCEs. These results indicate that repeated exposures to benzene, such as might be encountered by humans as a result of occupational or environmental exposures, are not likely to change or increase benzene metabolism.

Effects of the respiratory tract on inhaled materials

Inhalation exposure is often compared to intravenous or oral routes of administration with regard to the biological fate of inhaled materials. Such comparisons, however, overlook the contribution of respiratory tract enzymes to the metabolic fate and toxicity of inhaled materials. The effect of respiratory tract metabolism on the toxicity of inhaled materials is thought to be substantial for many compounds for the following reasons. (1) High concentrations of xenobiotic metabolizing enzymes occur in the nose and substantial concentrations occur in the lung. (2) The respiratory tract tissues are the first exposed to inhaled materials and are exposed to the highest concentrations (barring tissue specific uptake). (3) The products of respiratory tract metabolism may have different toxicities from those of hepatic metabolism. (4) Tissues at risk to toxic metabolites formed in the respiratory tract are different from those formed in the liver. These four reasons for concluding that respiratory tract metabolism may influence the toxicity of inhaled materials are backed by a solid body of expanding experimental data. Therefore, a complete assessment of the fate of inhaled materials should include assessment of potential contributions of respiratory tract metabolism.

Effect of exposure concentration on the disposition of inhaled butoxyethanol by F344 rats

The glycol ethers are a class of solvents widely used due to their range of vapor pressures and miscibility in aqueous and organic media. Butoxyethanol (BE) causes anemia and lowered hematocrits in rats due to direct hemolysis of red blood cells. Exposure to BE is most likely to occur by dermal contact or by inhalation. In this paper, we report the uptake, metabolism, and excretion of BE following 6-hr exposure at different inhaled concentrations. The uptake and metabolism of BE were essentially linear up to 438 ppm. The majority of the inhaled butoxy-[14C]ethanol was eliminated in the urine with butoxyacetic acid (BAA) being the major urinary metabolite, accompanied by lesser amounts of ethylene glycol and BE glucuronide. A small proportion (5-8%) of the retained BE was exhaled as 14CO2. Most (greater than 80%) of the [14C]BE-derived material in blood was in the plasma. BAA was the major metabolite of BE in plasma. Ratios of ethylene glycol to BAA in plasma were higher than those in urine. The BE-derived 14C in plasma rapidly became associated with the acid-precipitable (protein) fraction, probably due to binding of metabolites to proteins or incorporation of the BE metabolites into the carbon pool. These results indicate that, in rats, overall metabolism of BE to BAA, the hemolytic metabolite, was linearly related to the exposure concentration up to a concentration that caused severe toxicity (438 ppm). Assuming that the toxicity of inhaled BE is directly proportional to the formation of BAA, the toxicity of inhaled BE can be expected to be linearly related to the exposure concentration up to exposure concentrations that cause mortality.