James D. Sun

A proposed mechanism of benzene toxicity: Formation of reactive intermediates from polyphenol metabolites☆

Male Fischer-344 rats were given 100 μCi (14 mg/kg) [14C]catechol or [14C]hydroquinone by injection into the lateral tail vein. For a period of at least 24 hr, soluble radioactivity associated with either compound was retained in the bone marrow, but not in the liver or thymus. The amount of covalently bound radioactivity increased with time in all tissues examined and was significantly depressed in liver, white blood cells, and bone marrow in rats pretreated with Aroclor 1254, a regimen which protects against benzene toxicity. Potential enzymatic and nonenzymatic activation pathways for catechol, hydroquinone, and other known benzene metabolites were examined. In air-saturated 50 mm phosphate buffer (pH 7.4) at 37°C, only hydroquinone and 1,2,4-benzenetriol autoxidized. The oxidation product of hydroquinone had an uv absorption maximum (248 nm) identical to that of benzoquinone. With 250 units superoxide dismutase, hydroquinone autoxidation increased fivefold, whereas the oxidation of 1,2,4-benzenetriol was inhibited (4% of control). Epinephrine autoxidation, an indirect measure of superoxide anion generation, was stimulated by 1,2,4-benzenetriol and hydroquinone, but was barely detectable in the presence of catechol. Of the compounds studied, only benzoquinone augmented the oxidation of NADPH by a 3000g rat bone marrow supernatant. These data support a mechanism for benzene toxicity in which the formation of potentially cytotoxic metabolites, semiquinone, and quinone oxidation products and superoxide radicals, result from autoxidation of at least two polyphenol metabolites of benzene, hydroquinone, and 1,2,4-benzenetriol.

Response of Rodents to Inhaled Diluted Diesel Exhaust: Biochemical and Cytological Changes in Bronchoalveolar Lavage Fluid and in Lung Tissue

The effect of long-term (24 months) inhalation of diesel exhaust on the bronchoalveolar region of the respiratory tract of rodents was assessed by serial (every 6 months) analysis of bronchoalveolar lavage fluid (BALF) and of lung tissue from F344/Crl rats and CD-1 mice (both sexes) exposed to diesel exhaust diluted to contain 0, 0.35, 3.5, or 7.0 mg soot/m3. The purpose of the study was twofold. One was to assess the potential health effects of inhaling diluted exhaust from light-duty diesel engines. The second was to determine the usefulness of BALF analysis in detecting the early stages in the development of nononcogenic lung disease and differentiating them from the normal repair processes. No biochemical or cytological changes in BALF or in lung tissue were noted in either species exposed to the lowest, and most environmentally relevant, concentration of diesel exhaust. In the two higher levels of exposure, a chronic inflammatory response was measured in both species by dose-dependent increases in inflammatory cells, cytoplasmic and lysosomal enzymes, and protein in BALF. Histologically, after 1 year of exposure, the rats had developed focal areas of fibrosis associated with the deposits of soot, while the mice, despite a higher lung burden of soot than the rats, had only a fine fibrillar thickening of an occasional alveolar septa in the high-level exposure group. Higher increases in BALF beta-glucuronidase activity and in hydroxyproline content accompanied the greater degree of fibrosis in the rat. BALF levels of glutathione (GSH) and glutathione reductase activity increased in a dose-dependent fashion and were higher in mice than in rats. Lung tissue GSH was depleted in a dose-dependent fashion in rats but was slightly increased in mice. This depletion may have played a role in the greater fibrogenic response observed in rats. Other tissue changes in enzymatic activity were small compared to changes observed in BALF. The exposure did not increase the cytochrome P-450 content of the lung in either species. The results suggest that, for the noncarcinogenic health effects reported in this paper, there is a threshold of exposure below which adverse effects were not observed. This threshold was well above environmentally relevant levels of diesel exhaust but may be in the range of some occupational exposures. The analysis of BALF proved a useful adjunct to the chronic toxicity study to quantitate the inflammatory changes accompanying the development of pulmonary disease.

Deposition, retention, and biological fate of inhaled benzo(a)pyrene adsorbed onto ultrafine particles and as a pure aerosol

Abstract The effect of ultrafine, airborne, carrier particles on the deposition, retention, and biological fate of inhaled polycyclic aromatic hydrocarbons (PAHs) was studied. Using a radiolabeled model PAH, [3H]benzo(a)pyrene ([3H]BaP), Fischer-344 rats were exposed by nose-only inhalation (30 min) to this compound, as a coating (15% by mass) on insoluble 67Ga2O3 particles or as a pure aerosol. These aerosols were produced by vapor condensation methods in a dynamic aerosol generation system. The concentrations of [3H]BaP in the coated and homogeneous aerosols were 0.6 and 1.0 μg/liter of air, respectively, while the mass median diameter of both these aerosols was approximately 0.1 μm. Pulmonary retention of 3H radioactivity was longer in animals exposed to the [3H]BaP coated on the 67Ga2O3 particles. The time required to clear 90% of the initial lung burden of 67Ga2O3-associated 3H radioactivity detected 30 min postexposure was approximately 1 day as compared to 4 hr for animals exposed to the pure [3H]BaP aerosol. Tracheal clearance of 90% of the 67Ga2O3-associated 3H radioactivity (as a fraction of the amount detected 30 min postexposure) required 1 day, while only 1.5 hr were required to clear the same percentage of 3H radioactivity from the tracheas of rats exposed to the pure [3H]BaP aerosol. The rates of clearance of this 3H material to other tissues suggested that a substantial amount of the [3H]BaP coated on 67Ga2O3 was cleared from lungs by mucociliary clearance and subsequent ingestion, whereas the majority of the pure [3H]BaP aerosol was cleared by direct absorption into blood. In both cases, the ultimate fate of the majority of the [3H]BaP and its metabolites was excretion in feces. However, clearance of the 67Ga2O3-associated [3H]BaP by ingestion may have been the cause for the higher levels and longer retention times of 3H radioactivity in stomach, liver, and kidneys when compared to the levels found in these same tissues from animals exposed to the pure [3H]BaP aerosol. Thus, particle association of BaP not only increased the respiratory tract retention of this PAH, but also increased the dose of this compound and its metabolites to stomach, liver, and kidneys.

Lung retention and metabolic fate of inhaled benzo(a)pyrene associated with diesel exhaust particles

Polycyclic aromatic hydrocarbons (PAHs) are a class of compounds considered to have human carcinogenic potential and have been found associated with many respirable, environmental particle pollutants. The effect of these ultrafine, insoluble, carrier particles on the lung retention and metabolic fate of inhaled PAHs was investigated with a radiolabeled model PAH, [3H]benzo(a)pyrene (3H-BaP). Fischer-344 rats were exposed (30 min) by nose-only inhalation to 3H-BaP adsorbed (approximately 0.1% by mass) onto diesel engine exhaust particles. These aerosols were generated in a dynamic aerosol generation system by vapor condensation methods. The total mass concentration of these aerosols was 4-6 micrograms/liter of air with a mass median diameter of 0.14 micron. Lung clearance of the inhaled particle-associated 3H radioactivity occurred in two phases. The initially rapid clearance of this inhaled radiolabel had a half-time of less than 1 hr. The second, long-term component of lung clearance had a half-time of 18 +/- 2 days and represented 50 +/- 2% of the 3H radioactivity that had initially deposited in lungs. In contrast, previous inhalation studies with a pure 3H-BaP aerosol showed that greater than 99% of the 3H radioactivity deposited in lungs was cleared within 2 hr after exposure (Sun et al., Toxicol. Appl. Pharmacol. 65, 231-244, 1982). By HPLC analysis, the majority of diesel soot-associated 3H radioactivity retained in lungs was BaP (65-76%) with smaller amounts of BaP-phenol (13-17%) and BaP-quinone (5-18%) metabolites also being detected. No other metabolites of BaP were detected in lungs of exposed rats. Tissue distribution and excretion patterns of 3H radioactivity were qualitatively similar to previous inhalation studies with 3H-BaP coated Ga2O3 aerosols (Sun et al., 1982). These findings suggest that inhaled PAHs may be retained in lungs for a greater period of time when these compounds are associated with diesel engine exhaust particles. In addition, these compounds retained in lungs can be metabolized in lungs. These results may have significant implications for the health risks that may be involved with human exposure to particle-associated organic pollutants.

Comparison of acute ozone-induced nasal and pulmonary inflammatory responses in rats☆

The centriacinar pulmonary lesion induced by ozone has been extensively characterized, but little is known about the effects of this oxidant gas in the upper airways. The present study was designed to compare the effects of acute ozone exposure in the nose and lungs of rats. We examined the cellular inflammatory responses in the nasal cavity and lower respiratory tract by means of nasal and bronchoalveolar lavage and morphometric quantitation of neutrophils within the nasal mucosa and pulmonary terminal bronchioloalveolar duct regions (i.e., centriacinar). Rats were exposed to 0.0, 0.12, 0.8, or 1.5 ppm ozone for 6 hr and were sacrificed immediately or 3, 18, 42, or 66 hr following exposure. Eighteen hours after exposure, increased numbers of neutrophils, as compared to controls, were recovered from nasal lavage fluid (NLF) of rats exposed to 0.12 ppm ozone. There was no change in the number of neutrophils recovered from bronchoalveolar lavage fluid (BALF) at any time after exposure. Rats exposed to 0.8 ppm ozone had more neutrophils in NLF than controls immediately after exposure, but no concomitant increase in BALF neutrophils at that time. However, as the number of neutrophils in BALF increased (maximum at 42 hr), the number of neutrophils recovered from NLF decreased (minimum at 42 hr). Rats exposed to 1.5 ppm ozone had no significant increases in nasal neutrophils in NLF at any time after exposure but had greatly increased numbers of neutrophils in BALF 3, 18, and 42 hr after exposure. The number of neutrophils recovered by nasal and bronchoalveolar lavage accurately reflected the tissue neutrophil response at sites within the nasal cavity and lung that were injured by acute ozone exposure. Our results suggest that at high ozone concentrations (0.8 and 1.5 ppm), the acute nasal inflammatory response is attenuated by a simultaneous, competing, inflammatory response within the centriacinar region of the lung. Analysis of nasal lavage fluid for changes in cellular composition may be a useful indicator of acute exposure to ambient levels of ozone, but at higher ozone levels, the nasal cellular inflammatory response may underestimate the effects of ozone on nasal and pulmonary epithelia.

Effects of the Long-Term Depletion of Reduced Glutathione in Mice Administered L-Buthionine-S,R-sulfoximine

Abstract Previous methods to deplete in vivo concentrations of reduced glutathione (GSH) have not been able to lower tissue GSH levels for extended periods, have been toxic, and can alter the metabolism of xenobiotics. A possible alternative to lower in vivo concentrations of GSH may be the use of buthionine-S,R-sulfoximine (BSO) in the drinking water of laboratory animals to inhibit the biosynthesis of GSH. It has been previously reported that 20 m m BSO in the drinking water given to mice was able to lower GSH levels in a variety of tissues after 15 days. In order to more fully characterize the in vivo depletion of GSH in tissues by ingestion of BSO and determine if this method would be suitable in studies requiring depressed levels of GSH for extended periods, we added different amounts of this agent to the drinking water given to mice for various times up to 28 days. We found that ingested BSO at the highest concentrtion used in drinking water (30 m m ) was able to maximally lower GSH concentrations in mouse lungs, lung lavage fluid, liver, kidneys, and blood to 59.0 ± 3.6%, 35.0 ± 5.1%, 44.3 ± 1.5%, 69.5 ± 3.9%, and 70.0 ± 6.0% of control mice, respectively, for up to 28 days. These lowered concentrations of tissue GSH returned to control levels after mice were returned to untreated drinking water for 7 days. The potential toxicity of such treatments was also evaluated. Levels of alkaline phosphatase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, and glutathione reductase in lungs and lung lavage fluid, and total and differential cell counts from lung lavage fluid were not different between control and BSO-treated mice. This showed that BSO treatment did not produce indications of lung injury as measured by these biochemical parameters. Serum aspartyl transferase and γ-glutamyl transpeptidase activities were unaffected by the BSO treatments, indicating normal liver functions. Lung and liver cytochrome P-450 concentrations were also not different between controls and BSO-treated animals. Thus, BSO in the drinking water of mice was able to effectively lower in vivo levels of GSH without eliciting acute toxic responses.

S-Phenylcysteine formation in hemoglobin as a biological exposure index to benzene

Benzene is metabolized to intermediates that bind to hemoglobin, forming adducts. These hemoglobin adducts may be usable as biomarkers of exposure. In this paper, we describe the development of a gas chromatography/mass spectroscopy assay for quantitating the binding of the benzene metabolite, benzene oxide, to cysteine groups in hemoglobin. We used this assay to study the hemoglobin adduct, S-phenylcysteine (SPC), in the blood of rats and mice exposed to benzene either by inhalation or by gavage. We were able to detect SPC in the hemoglobin of exposed rats and mice, to show the linearity of the exposure dose-response relationship, and to establish the sensitivity limits of this assay. For the same exposure regime, rats showed considerably higher levels of SPC than did mice. As yet, we have not been able to detect SPC in the globin of humans occupationally exposed to benzene. We attempted to determine whether the SPC found in hemoglobin originated from the metabolism of benzene within or outside of the red blood cell. We hypothesized that the greatest red blood cell metabolism would be associated with peripheral reticulocytes, which retain high metabolic capacity. After exposing rats to benzene, we isolated the red blood cells and used discontinuous Percoll gradients to fractionate them into age groups. No differences in SPC levels were found among any of the fractions, suggesting that the SPC found in globin originates from the metabolism of benzene to benzene oxide in a location external to the red blood cell. To our knowledge, this is the first demonstration of the nonenzymatic binding of the benzene metabolite, benzene oxide, to protein. The products of the interaction of benzene oxide with protein are particularly appealing as potential biomarkers. These adducts are stable both in vivo and during isolation, and because high background levels in unexposed individuals are unlikely, the presence of such adducts should be a specific indicator of exposure to benzene. If SPC can be detected in humans, the present assay, with some modification, may be amenable to routine monitoring of worker exposures to benzene.

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.

INFLUENCE OF ADSORPTION TO CARBON BLACK PARTICLES ON THE RETENTION AND METABOLIC ACTIVATION OF BENZO(a)PYRENE IN RAT LUNGS FOLLOWING INHALATION EXPOSURE OR INTRATRACHEAL INSTILLATION

AbstractThere is a need to define the influence of adsorption of organic compounds onto particles on the biological fate of these compounds when inhaled. In this study, rats were exposed by nose-only inhalation (2 h) to 2 mg/m3 or 20 mg/m3 of pure [14C]BaP or [14C]BaP adsorbed onto carbon black particles at 0.2 mg/m3, 2.0 mg/m3, or 20 mg/m3 (total mass concentration ≃ 100 mg/m3). In separate studies, rats were intratracheally instilled with amounts of pure [14C]BaP and [14C]BaP on carbon black that were similar to those deposited in lungs by inhalation. In all cases, clearance of 14C from lungs had a rapid short-term component and a slower long-term component of clearance. As a percentage of the calculated initial lung deposition after inhalation or instillation, the long-term retention of 14C was similar for all amounts of pure [14C]BaP used and also similar for both methods of administration. The long-term lung retention of 14C as a percentage of the initial lung deposition was increased 16–60 times as ...

Inhalation of 1-nitropyrene associated with ultrafine insoluble particles or as a pure aerosol: A comparison of deposition and biological fate

A large number of the environmental particulate pollutants in the atmosphere, including diesel engine exhaust, have a complex mixture of organic compounds associated with them. Organic solvent extracts of many of these particulate pollutants have been shown to contain mutagenic activity which does not require metabolic activation in the Ames bioassay. Much of this direct-acting mutagenic activity has been attributed to nitroaromatic compounds present in these extracts. In the studies reported here, the direct-acting mutagen, [3H]nitropyrene (3H-NP), was used as a model nitroaromatic compound. Rats were exposed to this radiolabeled compound by nose-only inhalation either as a coating (approximately 6% by mass) on relatively inert, ultrafine 67Ga2O3 particles or as a homogeneous ultrafine aerosol. The tissue deposition, retention, and biological fate of each aerosol were investigated and compared. Respiratory tract clearance of 3H radioactivity from each exposure was very rapid with no apparent differences seen in the lung retention of this inhaled compound between each exposure over the course of these studies. Higher 3H-radioactivity levels were seen in stomach and large intestines of rats exposed to the 67Ga2O3-associated 3H-NP than in the same tissues from rats exposed to the pure 3H-NP aerosol. Rats exposed to the 3H-NP-67Ga2O3 aerosol excreted the majority of the deposited 3H radioactivity in the feces (75 +/- 18%), whereas pure 3H-NP exposed animals excreted a major portion of the radiolabeled in the urine (76 +/- 18%). It appeared that the major portion of the pure 3H-NP aerosol was cleared from the respiratory tract by direct absorption into blood, while the 67Ga2O3-associated 3H-NP was cleared by both blood absorption and mucociliary clearance followed by ingestion and fecal excretion. These differences in the deposition and biological fate between the particle-associated NP and the pure NP aerosol may have important implications in terms of the metabolic fate of inhaled nitroaromatic compounds and the health risks associated with human exposures to particulate environmental pollutants that contain this class of compounds.