John S. Dutcher

Species differences in the disposition of inhaled butadiene.

Recent chronic inhalation carcinogenicity studies of butadiene indicated that B6C3F1 mice are more sensitive to the tumorigenic effects of inhaled butadiene than are Sprague-Dawley rats. Tumors in mice included lymphomas, hemangiosarcomas, alveolar/bronchiolar adenomas and carcinomas, and hepatocellular adenomas and carcinomas whereas in rats tumors included mammary tumors, thyroid follicular cell adenomas, uterine tumors, and exocrine pancreatic adenomas. The purpose of this investigation was to determine if there were differences in the uptake and disposition of inhaled butadiene between rats and mice and if these differences were consistent with the differences in the species susceptibility to inhaled butadiene. Male Sprague-Dawley rats and B6C3F1 mice were exposed nose only to concentrations in the range of 0.14 to 13,000 micrograms [14C]butadiene/liter air (0.08 to 7100 ppm; 25 degrees C, 620 torr) for 6 hr. Blood samples were taken during exposure and urine, feces, and expired air were collected for up to 65 hr after exposure. In both rats and mice there was a significant (p less than 0.001) concentration-related decrease in the percentage of butadiene retained at the cessation of a 6-hr exposure with increasing butadiene exposure concentration, suggesting saturable metabolism of this chemical. At all concentrations of butadiene tested, mice retained about 4 to 7 times the amount (mumol/kg body wt) of butadiene and metabolites than did rats. In both species and at all butadiene concentrations tested, urine and exhaled air were the major routes of excretion of 14C, together accounting for 75 to 85% of the total 14C eliminated. In mice, for all concentrations tested, elimination of 14C in urine, feces, and exhaled air increased with increasing butadiene exposure concentration, although the increase was not proportional to exposure concentration. However, exposure of rats to 13,000 micrograms butadiene/liter air resulted in a leveling off in the amount of 14C that was eliminated in urine and a concomitant increase in exhalation of 14CO2. Analysis of blood samples taken during exposure indicated that the blood of mice contained 2 to 5 times the concentration of 1,2-epoxy-3-butene than did the blood of rats. The data from this study indicate that species differences exist in the amount retained and metabolism of inhaled butadiene.

Species and strain differences in target organ alkylation and toxicity by 4-ipomeanol. Predictive value of covalent binding in studies of target organ toxicities by reactive metabolites.

Abstract The organ specificities of the in vivo covalent binding of 4-ipomeanol were closely correlated with the patterns of organ-specific damage by 4-ipomeanol in several different animal species and strains. In all species tested, the lung was a major target for 4-ipomeanol covalent binding and toxicity. In the hamster and the mouse, 4-ipomeanol caused liver necrosis and kidney necrosis, respectively, in addition to pulmonary damage. Correspondingly, high levels of covalent binding of 4-ipomeanol occurred in these target organs in these species. These in vivo results, in addition to studies of the in vitro covalent binding of 4-ipomeanol in microsome preparations from the various target tissues, were consistent with the view that the organ-specific toxicities of 4-ipomeanol were caused by a highly reactive 4-ipomeanol metabolite(s) primarily produced in situ in the respective target tissues. The present results suggest that studies of both the in vivo and the in vitro covalent binding of 4-ipomeanol may have some utility in predicting the target organ specificity of 4-ipomeanol toxicity in other species. The present investigations also have identified some relevant new in vivo toxicity models for future studies of the relationships between the metabolism and the toxicity of 4-ipomeanol.

Excretion and metabolism of 1-nitropyrene in rats after oral or intraperitoneal administration☆

Many nitro-substituted polycyclic aromatic hydrocarbons (NPAHs) have been identified as environmental pollutants and have been found to be mutagens and carcinogens in bacteria and mammalian systems. They require metabolism to express their biological activity. The metabolism and excretion of 1-nitropyrene (NP), a prevalent NPAH, by Fischer-344 rats after intraperitoneal (ip) or oral administration was studied. Radiolabeled NP was administered to rats (10 mg NP/kg body wt), and urine and feces were collected for 7 days. After ip administration of [14C]NP, 60% of the radioactivity was found in the urine and 20% in the feces. Likewise, 55 and 35% of the orally administered 14C was found in urine and feces, respectively. Both urine and feces were analyzed by high-pressure liquid chromatography for metabolites. The majority of the radioactivity in both urine and feces was associated with very polar metabolites, none accounting for more than 10% of the dose. Small amounts (less than 1% of the dose) of aminopyrene (AP), acetylaminopyrene, and NP were detected. A urinary metabolite (3-8% of the dose) was found that converted to acetylaminopyrene phenol (two isomers) when urine was heated overnight at 37 degrees C at pH 4.5. More of this metabolite (2.2 times) as well as AP (1.8 times), was excreted after oral than after ip administration of NP. The NP metabolites found in this study demonstrate that reduction of the nitro group is a significant route of NP metabolism in rats. Since nitroreduction appears to be necessary in the activation of NPAHs to bacterial mutagens, this indicates that similar metabolic pathways are present in rats (catalyzed by mammalian and/or gut bacterial enzymes) and that activation of NPAHs to carcinogens or toxins by nitroreduction is possible.

Mutagenicity of used crankcase oils from diesel and spark ignition automobiles

The Salmonella mutagenicity assay was used to compare the mutagenic activity of used crankcase oil (UCO) from diesel and spark-ignition (gasoline) engine passenger cars. UCO samples were obtained during periodic oil changes from 9 spark-ignition and 10 diesel-powered vehicles. Five samples of unused motor oil were also tested. Direct tests of UCO did not detect mutagenic activity in Salmonella typhimurium strain TA-98. Therefore, an extraction procedure was used to concentrate the mutagens and remove interfering chemicals. Extracts were tested both with and without Aroclor-1254-induced rat liver homogenate fraction (S-9). Dose-dependent mutagenicity with and without S-9 was observed in both diesel and spark-ignition engine UCO extracts. Mutagenic activity was also found in unused oil extracts, but it was lower than that in UCO extracts and generally required addition of S-9. The mutagenic potency of diesel UCO extracts was similar to that of gasoline UCO extracts, both with and without addition of S-9. This indicated that potential health risks associated with disposal, handling, and recycling of diesel UCO may not be significantly different from those of UCO from gasoline engines.

Chemical and biological properties of diesel exhaust particles collected during selected segments of a simulated driving cycle

Particle emissions, percentage of organic extractable materials, and mutagenicities of extracts from a diesel engine operating on a test stand have been determined for the full Federal Test Procedure driving cycle and several individual segments thereof. Particle samples were collected using a computer controlled high volume sampler. Extracts of the exhaust particles were screened for the potent mutagens nitropyrene/nitrofluoranthenes by mass spectrometry/mass spectrometry (MS/MS). Results indicate that a long acceleration from 0-55 mph produced approximately seven times more particles per second than the full cycle. Also, the 0- to 55-mph acceleration and a subsequent 55-mph cruise produced significantly higher amounts of mutagens than other segments or the full FTP cycle. A direct correlation of both NOx levels and temperature with mutagenicity was noted (r = 0.89 and r = 0.89). The specific activities of the extracts showed decreases or remained unchanged when assayed in TA-98 NR or TA-98 1,8 DNP6, nitroreductase deficient strains of TA-98. Three extracts were found to have high levels of nitropyrenes/nitrofluoranthenes, and two of the three had high specific activities in TA-98.

Fractionation, chemical analysis, and mutagenicity testing of low-Btu coal gasifier tar.

A tar from a low-Btu coal gasifier was subjected to parallel fractionation, chemical analysis, and bacterial mutagenicity (Salmonella) assay. Like other coal-derived tars, it was a complex mixture containing some high-molecular-weight material and several classes of organic compounds as major constituents. The results of bacterial mutagenicity testing of fractions and subfractions of the tar suggest that neutral nitrogen-containing compounds, phenols, organic bases, polycyclic aromatic hydrocarbons, and some uncharacterized high-molecular-weight materials are mutagenic to Salmonella and therefore are potentially mutagenic in higher systems.

Influence of ethanol and methanol gasoline blends on the mutagenicity of particulate exhaust extracts.

TheSalmonella mutagenicity test was used to evaluate the influence of alcohol fuel extenders on the genetic toxicity of particulate exhaust extracts. Four spark-ignition engine equipped vehicles were operated on gasoline alone, 10% blends of ethanol or methanol in gasoline, and a commercially available “gasohol.” The tests were conducted on a chassis dynomometer and the particulate exhaust was collected on high volume filters after dilution in a tunnel. The vehicles used were a 1980 Chevrolet Citation, a 1980 Mercury Monarch, a 1981 Ford Escort and a 1981 Oldsmobile Cutlass. Dichloromethane extracts of the exhaust particles from all tests were mutagenic inSalmonella typhimurium strains TA 100 and TA 98. The extracts were less mutagenic in the nitroreductase deficient strains TA 98NR and TA 98DNPR suggesting that nitro substituted polycyclic aromatic hydrocarbons may be responsible for part of the mutagenicity. In all the alcohol blended fuel tests, the mass of particle associated organics emitted from the exhaust was lower than that observed during the control tests using gasoline alone. Thus, in most cases, estimates of the emission of mutagenic combustion products from the exhaust were lower in the alcohol blend tests.

The Fate of Inhaled Azodicarbonamide in Rats

Azodicarbonamide (ADA) is widely used as a blowing agent in the manufacture of expanded foam plastics, as an aging and bleaching agent in flour, and as a bread dough conditioner. Human exposures have been reported during manufacture as well as during use. Groups of male F344/N rats were administered ADA by gavage, by intratracheal instillation, and by inhalation exposure to determine the disposition and modes of excretion of ADA and its metabolites. At 72 hr following gavage, 30% of the administered ADA was absorbed whereas following intratracheal instillation, absorption was 90%. Comparison between groups of rats exposed by inhalation to ADA to achieve body burdens of 24 or 1230 micrograms showed no significant differences in modes or rates of excretion of [14C]ADA equivalents. ADA was readily converted to biurea under physiological conditions and biurea was the only 14C-labeled compound present in excreta. [14C]ADA equivalents were present in all examined tissues immediately after inhalation exposure, and clearance half-times on the order of 1 day were evident for all tissues investigated. Storage depots for [14C]ADA equivalents were not observed. The rate of buildup of [14C]ADA equivalents in blood was linearly related to the lung content as measured from rats withdrawn at selected times during a 6-hr inhalation exposure at an aerosol concentration of 25 micrograms ADA/liter. In a study extending 102 days after exposure, retention of [14C]ADA equivalents in tissues was described by a two-component negative exponential function. The results from this study indicate that upon inhalation, ADA is rapidly converted to biurea and that biurea is then eliminated rapidly from all tissues with the majority of the elimination via the urine.

Effect of Vapor Concentration on the Disposition of Inhaled 2,3-Dichloropropene in Fischer-344 Rats

2,3-Dichloropropene (DCP) is an intermediate used in the manufacture of carbamate herbicides and there is potential for human exposure during the manufacturing process. DCP is a known mutagen in bacteria systems and some structural analogs of DCP are carcinogenic. Since little is known about the disposition of DCP in animals after inhalation, studies were conducted in male Fischer-344 rats to determine the effect of vapor concentration on absorption and excretion. Uptake and elimination of 14C was studied in rats after nose-only inhalation of 17, 240, or 1650 nmol of [14C]DCP vapor/liter of air (0.4, 6, or 40 ppm, respectively, at 760 mm and 25 degrees C) for 6 hr. The percentage of inhaled DCP absorbed averaged 38% and was not statistically different at any vapor concentration, although minute volume was lower during exposure to 1650 nmol/liter. Urine, feces, and expired air were collected from rats for 65 hr after exposure. Rats were sacrificed and tissues, carcass, excreta, and expired air were analyzed for 14C. Routes of 14C excretion were independent of vapor concentration, with 50% of the 14C excreted in urine, 13% in feces, approximately 7% as CO2, and less than 1% as DCP in expired air. Rates of 14C excretion were also independent of vapor concentration, with the half-times averaging 9.9 hr (urine), 13.6 hr (feces), and 0.9 hr (14CO2). Sixty hours after inhalation, 29% of the initial body burden of 14C remained in the carcass. Most was associated with the pelt, but some 14C was found in all tissues. Respiratory tract, GI tract, liver, and kidney were tissues with the highest 14C contents. The results indicate that DCP metabolism and excretion rates are relatively constant throughout the vapor concentration range studied. This suggests that results from more detailed pharmacokinetic studies (and possibly toxicity studies) at one DCP concentration may be extrapolated to other concentrations within this range.

Binding of nitropyrenes and benzo[a]pyrene to mouse lung deoxyribonucleic acid after pretreatment with inducing agents.

In assessing the biological effects of exposure to a complex chemical mixture, it is important to determine how the behavior of one compound may be influenced by the presence of other compounds in the mixture. In this study the effect of pre-exposure to an organic extract of diesel exhaust or to selected compounds in diesel exhaust on the binding of diesel exhaust compounds to DNA was determined. The amount of radiolabel covalently bound to mouse lung DNA following intratracheal administration of radiolabeled benzo[a]pyrene (BaP), 1-nitropyrene, 1,3,6-trinitropyrene, or a mixture of dinitropyrene was determined following pretreatment with benzo[a]pyrene, 1-nitropyrene, and diesel exhaust extract. Male CD-1 mice, 15-18 weeks of age, received 10 mg/kg of putative inducing agents by intratracheal instillation and, after 24 hr, 0.03 to 1.2 mg/kg radiolabeled putative DNA binding agents. Lung DNA was extracted, and covalent binding was quantitated by liquid scintillation spectroscopy. 1-Nitropyrene was a potent lung DNA binding agent in the absence of inducing agents [Covalent Binding Index (CBI) = 970] and was extremely potent after benzo[a]pyrene pretreatment (CBI = 21,540, comparable to the CBI for aflatoxin B1). Similar results were obtained for DNA binding of dinitropyrene and trinitropyrene with and without BaP pretreatment. DNA binding of BaP was lower (CBI = 40) and less inducible (BaP-pretreatment CBI = 230). Pretreatment with diesel extract caused an elevation in the binding of benzo[a]pyrene but little or no elevation in the binding of the nitropyrenes. Pretreatment with 1-nitropyrene did not increase significantly DNA binding of any of the agents tested. These results indicate that nitropyrenes bind readily to lung DNA and this binding may be increased in the presence of respirable mixtures, especially those containing inducing agents such as BaP.