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

Response of Rodents to Inhaled Diluted Diesel Exhaust: Biochemicaland Cytological Changes in Bronchoalveolar Lavage Fluid andin Lung Tissue. HENDERSON, R. F., PICKRELL, J. A., JONES, R.K., SUN, J. D., BENSON, J. M., MAUDERLY, J. L., AND MCCLELLAN,R. O. (1988). Fundam. Appl. Toxicol. 11, 546-567. The effectof long-term (24 months) inhalation of diesel exhaust on thebronchoalveolar region of the respiratory tract of rodents wasassessed by serial (every 6 months) analysis of bronchoalveolarlavage fluid (BALF) and of lung tissue from F344/Crl rats andCD-I mice (both sexes) exposed to diesel exhaust diluted tocontain 0, 0.35, 3.5, or 7.0 mg soot/m³. The purpose of thestudy was twofold. One was to assess the potential health effectsof inhaling diluted exhaust from light-duty diesel engines.The second was to determine the usefulness of BALF analysisin detecting the early stages in the development ofnonon-cogeniclung disease and differentiating them from the normal repairprocesses. No biochemical or cytological changes in BALF orin 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 inflammatoryresponse was measured in both species by dose-dependent increasesin inflammatory cells, cytoplasmic and lysosomal enzymes, andprotein in BALF. Histologically, after 1 year of exposure, therats had developed focal areas of fibrosis associated with thedeposits of soot, while the mice, despite a higher lung burdenof soot than the rats, had only a fine fibrillar thickeningof an occasional alveolar septa in the high-level exposure group.Higher increases in BALF ß-glucuronidase activityand in hydroxyproline content accompanied the greater degreeof fibrosis in the rat BALF levels of glutathione (GSH) andglutathione reductase activity increased in a dose-dependentfashion and were higher in mice than in rats. Lung tissue GSHwas depleted in a dose-dependent fashion in rats but was slightlyincreased in mice. This depletion may have played a role inthe greater fibrogenic response observed in rats. Other tissuechanges in enzymatic activity were small compared to changesobserved in BALF. The exposure did not increase the cytochromeP-450 content of the lung in either species. The results suggestthat, for the noncarcinogenic health effects reported in thispaper, there is a threshold of exposure below which adverseeffects were not observed. This threshold was well above environmentallyrelevant levels of diesel exhaust but may be in the range ofsome occupational exposures. The analysis of BALF proved a usefuladjunct to the chronic toxicity study to quantitate the inflammatorychanges accompanying the developmentof pulmonary disease.     

Lung Tissue Responses and Sites of Particle Retention Differ between Rats and Cynomolgus Monkeys Exposed Chronically to Diesel Exhaust and Coal Dust

Several chronic inhalation bioassays of poorly soluble, nonfibrousparticles have resulted in an increased incidence of lung tumorsin rats, no increase in lung tumors in Syrian hamsters, andinconsistent results in mice. These results have raised concernsthat rats may be more prone than other species to develop persistentpulmonary epithelial hyperplasia, metaplasia, and tumors inresponse to the accumulation of inhaled particles. In addition,particle deposition and the rate of particle clearance fromthe lung differ between rats and primates, as does the anatomyof the centriacinar region. For these reasons, the usefulnessof pulmonary carcinogenicity data from rats exposed to highconcentrations of particles for quantitatively predicting lungcancer risk in humans exposed to much lower environmental oroccupational concentrations has been questioned. The purposeof this investigation was to directly compare the anatomicalpatterns of particle retention and the lung tissue responsesof rats and monkeys exposed chronically to high occupationalconcentrations of poorly soluble particles. Lung sections frommale cynomolgus monkeys and F344 rats exposed 7 hr/day, 5 days/weekfor 24 months to filtered ambient air, diesel exhaust (2 mgsoot/m³), coal dust (2 mg respirable particulate material/m³),or diesel exhaust and coal dust combined (1 mg soot and 1 mgrespirable coal dust/m³) were examined histopathologically.The relative volume density of particulate material and thevolume percentage of the total particulate material in definedpulmonary compartments were determined morphometrically to assessthe relative amount and the anatomic distribution of retainedparticulate material. In all groups, relatively more particulatematerial was retained in monkey than in rat lungs. After adjustmentfor differences between rat and monkey controls, the coal dust-and the combined diesel exhaust and coal dustexposed monkeysretained more particulate material than the coal dust- and thecombined diesel exhaust and coal dust-exposed rats, respectively.There was no significant difference in the relative amount ofretained particulate material between diesel exhaustexposedmonkeys and rats. Within each species, the sites of particleretention and lung tissue responses were the same for dieselsoot, coal dust, and the combined material. Rats retained agreater portion of the particulate material in lumens of alveolarducts and alveoli than monkeys. Conversely, monkeys retaineda greater portion of the particulate material in the interstitiumthan rats. Rats, but not monkeys, had significant alveolar epithelialhyperplastic, inflammatory, and septal fibrotic responses tothe retained particles. These results suggest that intrapulmonaryparticle retention patterns and tissue reactions in rats maynot be predictive of retention patterns and tissue responsesin primates exposed to poorly soluble particles at concentrationsrepresenting high occupational exposures.     

Clearance of Diesel Soot Particles from Rat Lung after a Subchronic Diesel Exhaust Exposure

Clearance of Diesel Soot Particles from Rat Lung after a ChronicDiesel Exhaust Exposure. Griffis, L.C., Wolff, R.K., Henderson,R.F., Griffith, W.C., Mokler, B.V. and McClellan, R.O. (1983).Fundam. Appl. Toxicol. 3:99-103. The participate exhaust ofdiesel engines consists of 0.1–0.2 µm mass mediandiameter particles composed of a carbonaceous core and adsorbedorganic compounds. A technique was needed to determine accumulatedlung burdens of particles in animals exposed to diesel exhaustas a determinant of dose. A method was developed for determininglung burdens of diesel soot particles in rats at 1 day, andat 1, 5, 15, 33 and 52 weeks after cessation of a subchronicexposure to diluted diesel exhaust. Lung tissue was dissolvedin tetramethylammonium hydroxide and the diesel soot separatedby centrifugation. The soot was suspended in water by sonicationand the light absorption of samples was compared to standardsuspensions of diesel soot. Recovery from lungs spiked with50–1000 µg of soot was 89±5%. Rats exposedover a period of 18 weeks to diluted diesel exhaust at averagenet diesel particle concentrations of 150, 940 and 4100 µg/m³had lung burdens of 35,220 and 1890 µg/g lung, respectively,one day after the last exposure. The long-term clearance ratesof soot had estimated half-times of 87±28, 99±4days, for the low and medium exposure groups, respectively.The clearance half-time for the high level exposure group of165 ± 8 days was significantly longer (P < 0.0001)than those of the other two groups.     

Carcinogenicity Studies of Diesel Engine Exhausts in Laboratory Animals: A Review of Past Studies and a Discussion of Future Research Needs

Diesel engines play a vital role in world economy, especially in transportation. Exhaust from traditional diesel engines using high-sulfur fuel contains high concentrations of respirable carbonaceous particles with absorbed organic compounds. Recognition that some of these compounds are mutagenic has raised concern for the cancer-causing potential of diesel exhaust exposure. Extensive research addressing this issue has been conducted during the last three decades. This critical review is offered to facilitate an updated assessment of the carcinogenicity of diesel exhaust and to provide a rationale for future animal research of new diesel technology. Life-span bioassays in rats, mice, and Syrian hamsters demonstrated that chronic inhalation of high concentrations of diesel exhaust caused lung tumors in rats but not in mice or Syrian hamsters. In 1989, the International Agency for Research on Cancer (IARC) characterized the rat findings as “sufficient evidence of animal carcinogenicity,” and, with “limited” evidence from epidemiological studies, classified diesel exhaust Category 2A, a “probable human carcinogen.” Subsequent research has shown that similar chronic high concentration exposure to particulate matter generally considered innocuous (such as carbon black and titanium dioxide) also caused lung tumors in rats. Thus, in 2002, the U.S. Environmental Protection Agency (EPA) concluded that the findings in the rats should not be used to characterize the cancer hazard or quantify the cancer risk of diesel exhaust. Concurrent with the conduct of the health effects studies, progressively more stringent standards have been promulgated for diesel exhaust particles and NO_x. Engine manufacturers have responded with new technology diesel (improved engines, fuel injection, fuels, lubricants, and exhaust treatments) to meet the standards. This review concludes with an outline of research to evaluate the health effects of the new technology, research that is consistent with recommendations included in the U.S. EPA 2002 health assessment document. When this research has been completed, it will be appropriate for IARC to evaluate the potential cancer hazard of the new technology diesel.     

Effects of diesel exhaust on lung inflammation related to bacterial endotoxin in mice

We have previously shown that intratracheal instillation of diesel exhaust particles enhances lung inflammation and lung expression of proinflammatory cytokines and chemokines related to bacterial endotoxin (lipopolysaccharide) in mice. The present study was designed to elucidate the effects of inhalation of diesel exhaust on lung inflammation related to lipopolysaccharide. ICR mice were exposed for 12 hr to clean air or diesel exhaust at a soot concentration of 0.3, 1.0, or 3.0 mg/m(3) after intratracheal challenge with 125 microg/kg of lipopolysaccharide. Lung inflammation and lung expression of proinflammatory chemokines such as macrophage chemoattractant protein-1 and keratinocyte chemoattractant were evaluated 24 hr after intratracheal administration. Diesel exhaust inhalation decreased lipopolysaccharide-elicited inflammatory cell recruitment into the bronchoalveolar lavage fluid as compared with clean air inhalation. Histological study demonstrated that exposure to diesel exhaust did not affect lipopolysaccharide-enhanced neutrophil recruitment into the lung parenchyma. Lipopolysaccharide instillation elevated lung expression of macrophage chemoattractant protein-1 and keratinocyte chemoattractant under clean air or diesel exhaust inhalation. However, diesel exhaust exposure did not influence but rather did suppress these levels in the presence of lipopolysaccharide. These results suggest that short-term exposure to diesel exhaust did not exacerbate lung inflammation related to bacterial endotoxin.     

Chronic Inhalation Exposure of Wistar Rats and two Different Strains of Mice to Diesel Engine Exhaust,Carbon Black,and Titanium Dioxide

Abstract

Wistar rats were exposed for 2 yr to diesel engine exhaust, carbon black (Printex 90, Degussa, FR. G), and ultraline TiO₂ (P25, Degussa, FRG) and were subsequently kept in clean air for 6 mo. Particle exposure concentration was increased during the course of the experiment for carbon black and TiO₂ to reach particle lung loads similar to those found in the diesel soot-exposed rats. The average particle exposure concentrations for diesel soot, carbon black, and TiO₂ were 7, 11.6, and 10 mg/m³, respectively. Lung tumor rates in these rats increased with increasing cumulative particle exposure (mg/m³ x h) independent of the type of particle employed. The exposure to 2.5 mg/m¹ diesel soot also induced a significantly increased lung tumor rate, but 0.8 mg/m³ diesel soot did not. With this study, it could be demonstrated that the carbon core of diesel soot is mainly responsible for the occurrence of diesel engine exhaust-related lung tumors; the role of diesel soot-attached polycyclic aromatic hydrocarbons (PAH) and NO₂-PAH is probably of minor importance in the rat lung. Agglomerates of ultrafine carbon and TiO₂ particles seem particularly suited to exert toxic effects primarily on alveolar macrophages and alveolar lung particle clearance. Although such lung toxic effects were also seen with the lowest diesel soot exposure concentration (0.8 mg/m³) used, no increased lung tumor rate was detected in this group of rats. Whether this result implies a threshold for the particle-related lung tumor induction mechanism as already discussed by Vostal (1986) or whether the tumor effect was simply not observed because of statistical reasons needs further research on the possible mode of action of ultra-fine insoluble particles in the lung. NMR. I mice that were kept in the same exposure atmospheres (high diesel soot, carbon black, TiO₂) as the rats did not show an increased lung tumor rate. Furthermore, there was no treatment-related tumor response in NMRI nor in C57BL/6N mice exposed to diesel exhaust containing 4.5 mg/m³ diesel soot or to the same exhaust dilution but devoid of soot particles. C57BU6N mice were exposed for 24 mo and were subsequently kept in clean air for another 6 mo. Not only the average survival time but also the particle load per gram lung wet weight of the C57BU6N mice was very similar to rats exposed to 7 mg/m³ diesel soot.     

Carcinogenicity studies of diesel engine exhausts in laboratory animals: a review of past studies and a discussion of future research needs

Diesel engines play a vital role in world economy, especially in transportation. Exhaust from traditional diesel engines using high-sulfur fuel contains high concentrations of respirable carbonaceous particles with absorbed organic compounds. Recognition that some of these compounds are mutagenic has raised concern for the cancer-causing potential of diesel exhaust exposure. Extensive research addressing this issue has been conducted during the last three decades. This critical review is offered to facilitate an updated assessment of the carcinogenicity of diesel exhaust and to provide a rationale for future animal research of new diesel technology. Life-span bioassays in rats, mice, and Syrian hamsters demonstrated that chronic inhalation of high concentrations of diesel exhaust caused lung tumors in rats but not in mice or Syrian hamsters. In 1989, the International Agency for Research on Cancer (IARC) characterized the rat findings as "sufficient evidence of animal carcinogenicity," and, with "limited" evidence from epidemiological studies, classified diesel exhaust Category 2A, a "probable human carcinogen." Subsequent research has shown that similar chronic high concentration exposure to particulate matter generally considered innocuous (such as carbon black and titanium dioxide) also caused lung tumors in rats. Thus, in 2002, the U.S. Environmental Protection Agency (EPA) concluded that the findings in the rats should not be used to characterize the cancer hazard or quantify the cancer risk of diesel exhaust. Concurrent with the conduct of the health effects studies, progressively more stringent standards have been promulgated for diesel exhaust particles and NOx. Engine manufacturers have responded with new technology diesel (improved engines, fuel injection, fuels, lubricants, and exhaust treatments) to meet the standards. This review concludes with an outline of research to evaluate the health effects of the new technology, research that is consistent with recommendations included in the U.S. EPA 2002 health assessment document. When this research has been completed, it will be appropriate for IARC to evaluate the potential cancer hazard of the new technology diesel.     

Tumours of the respiratory tract in rats and hamsters following chronic inhalation of engine exhaust emissions

The potential carcinogenic effect of inhaled automobile exhaust emissions was examined in rodents. Both rats and hamsters were exposed to the emissions from (1) a gasoline engine, (2) a gasoline engine fitted with a three-way catalytic converter, (3) a diesel engine and (4) a diesel engine with particle filtration. Exposures were for 16 hours per day, 5 days per week, for 2 years. All hamsters were sacrificed at the end of the 2-year exposure period, whereas the rats surviving after 2 years of exposure were maintained for a further 6-month observation period without additional exposure to emissions. Some of the hamsters in each treatment group were pretreated with diethylnitrosamine to induce respiratory tract tumours. No statistically significant changes were seen in the incidence of respiratory tract tumours in emission-exposed hamsters compared to controls. This lack of a treatment-related effect was seen in both the nitrosamine pretreated and the non-pretreated hamsters. There was no increase in the incidence of lung tumours in rats exposed to filtered diesel exhaust or to the exhaust from the gasoline or gasoline-catalyst engines. There was a statistically significant increase in the incidence of lung tumours in rats exposed to diesel engine emissions compared to controls. A clear dose response was evident in both males and females, although the incidence of lung tumours was markedly higher in females (96% in rats surviving beyond 2 years) than in males (44% in rats surviving beyond 2 years). An increased incidence of lung tumours was observed only in rats exposed to mean concentrations of diesel soot particles of either 2200 or 6600 micrograms/m3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diesel Exhaust Is a Pulmonary Carcinogen in Rats Exposed Chronically by Inhalation

Diesel Exhaust Is a Pulmonary Carcinogen in Rats Exposed Chronicallyby Inhalation. MAUDERLY, J. L., JONES, R. K., GRIFFITH, W. C.,HENDERSON, R. F., AND MCCLELLAN, R. O. (1987). Fundam. Appl.Toxicol 9, 208–221. Male and female F344 rats were exposed7 hr/day, 5 day/week for up to 30 months to automotive dieselengine exhaust at soot concentrations of 0.35, 3.5, or 7.0 mg/m³or were sham-exposed to clean air. Rats were terminated at 6-monthintervals to measure lung burdens of diesel soot and for histopathology.Other rats either died or were terminated after 30 months ofexposure. Lungs were fixed, sectioned into 3-mm slices, andexamined by a dissecting microscope to detect tumors. Lesionswere stained and examined by light microscopy. Survival andbody weight were unaffected by exposure. Focal fibrotic andproliferative lung disease accompanied a progressive accumulationof soot in the lung. The prevalence of lung tumors was significantlyincreased at the high (13%) and medium (4%) dose levels abovethe control prevalence (1%). Four tumor types, all of epithelialorigin, were observed: adenoma. adenocarcinoma, squamous cyst,and squamous cell carcinoma. Logistic regression modeling demonstrateda significant relationship between tumor prevalence and bothexposure concentration and soot lung burden. These results demonstratethat diesel exhaust, inhaled chronically at a high concentration,is a pulmonary carcinogen in the rat.     

Chronic effects on the respiratory tract of hamsters, mice and rats after long-term inhalation of high concentrations of filtered and unfiltered diesel engine emissions

A long-term exposure study with hamsters, mice and rats inhaling filtered and unfiltered diesel engine exhaust was carried out to investigate effects of chronic toxicity and, predominantly, carcinogenicity in the respiratory tract. The level of diesel exhaust in the exposure chambers corresponded to a concentration close to 4 mg m-3 in the unfiltered diesel exhaust. Satellite groups of animals were additionally treated with BaP, DBahA or nitrosamines in order to check for syncarcinogenic effects. In hamsters and rats, alveolar lung clearance and mechanical lung function tests as well as biochemical and cytological measurements in lung lavage fluids showed significant changes only after exposure to unfiltered diesel exhaust and, predominantly, in rats. No lung tumors were found in hamsters. Spontaneous tumor rates occurred in mice and both types of diesel exhaust increased the incidence of adenocarcinomas in the lungs. In rats, only the unfiltered diesel exhaust caused a lung tumor incidence. It amounted to 16% with no tumors in the controls. The heavy load of particulate matter in the lungs of rats was caused by an exposure-related impairment of the alveolar lung clearance and may have been instrumental in the induction of squamous cell tumors. However, an effect of particle-associated PAH cannot be excluded. Syncarcinogenic effects of diesel exhaust after initial carcinogen treatment were found only in the respiratory tract of rats.     

Long-Term Exposure to Diesel Exhaust Enhances Antigen-Induced Eosinophilic Inflammation and Epithelial Damage in the Murine Airway

The histopathologic changes in the murine airway induced bylong-term exposure to diesel exhaust (DE), ovalbumin (OA), orboth were investigated. The relationship between the histopathologicappearances in the airway and immunoglobulin production or localcytokine levels in the lungs was also studied. ICR mice wereexposed to clean air or DE at a soot concentrations of 0.3,1.0, or 3.0 mg/m³ for 34 weeks. Fifteen weeks after exposureto DE, mice were sensitized intraperitoneally with 10 µgof OA and challenged by an aerosol of 1% OA six times at 3-weekintervals during the last 18 weeks of the exposure. DE exposurecaused a dose-dependent increase of nonciliated cell proliferationand epithelial cell hypertrophy in the airway, but showed noeffect on goblet cell proliferation in the bronchial epitheliumand eosinophil recruitment in the submucosa of the airway. OAtreatment induced very slight changes in goblet cell proliferationand eosinophil recruitment. The combination of OA and DE exposureproduced dose-dependent increases of goblet cells and eosinophils,in addition to further increases of the typical changes inducedby DE. OA treatment induced OA-specific IgG₁ and IgE productionin plasma, whereas the adjuvant effects of DE exposure on immunoglobulinproduction were not observed. Inhalation of DE led to increasedlevels of IL-5 protein in the lung at a soot concentration of1.0 and 3.0 mg/m³ with OA, although these increases did notreach statistical significance. We conclude that the combinationof antigen and chronic exposure to DE produces increased eosinophilicinflammation, and cell damage to the epithelium may depend onthe degree of eosinophilic inflammation in the airway.     

Comparative Pulmonary Toxicities and Carcinogenicities of Chronically Inhaled Diesel Exhaust and Carban Black in F344 Rats

Diesel exhaust (DE) is a known pulmonary carcinogen in rats,and the carcinogenic response is known to require the presenceof soot. Many estimates of human lung cancer risk from inhaledDE have been developed from rat bioassay data or from the comparativemutagenic potencies of DE soot extract and known human chemicalcarcinogens. To explore the importance of the DE soot-associatedorganic compounds in the lung tumor response of rats, male andfemale F344 rats were exposed chroni cally to diluted wholeDE or aerosolized carbon black (CB) 16 hr/day, 5 days/week attarget particle concentrations of 2.5 mg/m³ (LDE, LCB) or 6.5mg/m³ (HDE, HCB) or to filtered air. The CB served as a surrogatefor the elemental carbon matrix of DE soot. Considering boththe mass fraction of solvent-extractable matter and its mutagenicityin the Ames Salmonella assay, the mutagenicity in revertantsper unit particle mass of the CB was three orders of magnitudeless than that of the DE soot. Both DE soot and CB particlesaccumulated progressively in the lungs of exposed rats, butthe rate of accumulation was higher for DE soot. In general,DE and CB caused similar, dose-related, nonneoplastic lesions.CB and DE caused significant, exposure concentration-relatedincreases, of similar magnitudes, in the incidences and prevalencesof the same types of malignant and benign lung neoplasms infemale rats. The incidences of neoplasms were much lower inmales than females, and the mci dences were slightly higheramong DE- than CB-exposed males. Survival was shortened in theCB-exposed males, and the short ened survival may have suppressedthe expression of carcinoge nicity as measured by crude incidence.Logistic regression mod eling did not demonstrate significantdifferences between the carcinogenic potencies of CB and DEin either gender. The re sults suggest that the organic fractionof DE may not play an important role in the carcinogenicityof DE in rats.     

Pulmonary Retention of Inhaled Diesel Particles after Prolonged Exposures to Diesel Exhaust

Pulmonary Retention of Inhaled Diesel Particles after ProlongedExposures to Diesel Exhaust CHAN, T. L., LEE, P. S., AND HERING,W. E. (1984). Fundam. Appl. Toxicol. 4, 624–631. The effectof continuous exposure to diluted diesel exhaust on the pulmonaryretention of inhaled diesel particles was studied in male Fischer344 rats. Test animals were first exposed to clean air or diluteddiesel exhaust in exposure chambers at nominal particulate concentrationsof 250 µg/m³ or 6 mg/m³ for 20 hr/day, 7 days/week, forperiods lasting from 7 to 112 days, followed by a nose-onlyexposure to ^l4C-tagged diesel particles for 45 min. At preselectedtime intervals after the radioactive exposure, the ¹⁴C-activitiesin the lungs of groups of four animals were measured to determinethe clearance of the ^l4C-diesel particles up to 1 year. Thepulmonary retention of the radioactive diesel particles wasgreater in animals which had been preexposed to diesel exhaustThe slower alveolar clearance of particle-laden macrophagesand leukocytes can be described by a normal Diphasic clearancemodel. Since some of the macrophages were found sequesteredas aggregates in the pulmonary region, a slow-clearing residualcomponent was included in a modified lung retention model. Whenthese residual fractions were determined and excluded from theactive particulate transport within the lungs, normal alveolarclearance rates were calculated for the animals with a preexposurediesel particulate dose less than 0.8 mg. Slower clearance wasobserved at a dose of 6.5 mg and no clearance was evident ata dose of 11.8 mg in their lungs. In effect, the greater retentionof inhaled particles can be interpreted as-a sign of impairedlung clearance attributable to the prolonged exposures to highconcentrations of diesel exhaust gases and/or the presence ofaccumulated carbonaceous particles residing in sequestered macrophageaggregates in the lungs.     

Route of exposure alters inflammation and lung function responses to diesel exhaust

Abstract

Context: Mice are commonly used in studies investigating the effects of diesel exhaust exposure on respiratory health. A plethora of studies in this field has resulted in a range of exposure protocols, from inhalation of diesel exhaust, to the administration (via various routes) of diesel exhaust particles in solution.

Objective: In this study, we compared the physiological consequences of short-term exposure to diesel exhaust via inhalation to those due to exposure to the same diesel exhaust particles suspended in solution and delivered intranasally.

Materials and methods: Adult BALB/c mice were exposed to diesel exhaust via inhalation for 2 hours per day for 8 days. A representative, simultaneous sample of particles was collected and a second group of mice then exposed to them suspended in saline. A low and a high-dose were studied, with these matched based on respiratory parameters. Six and twenty-four hours after the last exposure we measured bronchoalveolar inflammation, lung volume, lung function and the amount of elemental carbon in alveolar macrophages.

Results: Exposure via either route elicited pulmonary inflammation and changes in lung function. We identified significant differences in response between the two routes of exposure, with mice exposed via inhalation generally displaying more realistic dose-response relationships. Mice exposed via intranasal instillation responded more variably, with little influence of dose.

Conclusions: Our results suggest that selection of the route of exposure is of critical importance in studies such as this. Further, inhalation exposure, while more methodologically difficult, resulted in responses more akin to those seen in humans.     

Pulmonary Inflammation and DNA Adducts in Rats Inhaling Diesel Exhaust or Carbon Black

Abstract

Inhaled diesel exhaust has been shown to be carcinogenic to rats. Two mechanisms have been suggested: a genotoxic effect of organic compounds associated with the diesel soot, and an epigenetic effect resulting from large accumulations of particles that produce chronic inflammation and epithelial proliferation in the lung. To gain insights into the possible roles of these two mechanisms, we compared inflammatory responses and DNA adducts in lungs of rats exposed to diesel exhaust (33% extractable organic compounds by mass) and carbon black (0.04% extractable), which are similar carbonaceous particles with virtually no organic compounds. Male rats were exposed 7 h/day, 5 days/wk for 12 wk to diesel exhaust or carbon black, both at a concentration of 70.0 mg/m³, or to filtered air: Both diesel exhaust and carbon black exposures caused mild inflammation in the lung, as evidenced by increases in numbers of neutrophils and in the protein content of bronchoalveolar lavage fluids. The levels of DNA adducts, as measured by ³²P postlabeling, were increased more by exposure to diesel exhaust than to carbon black. Although it remains unclear whether the carcinogenicity of diesel exhaust is related primarily to DNA injury or primarily to the loading of the lung with particles, our findings are consistent with an initiation-promotion mechanism that involves both DNA damage and lung inflammation.     

THE RELEVANCE OF THE RAT LUNG RESPONSE TO PARTICLE OVERLOAD FOR HUMAN RISK ASSESSMENT: A Workshop Consensus Report

On 23-24 March 1998, the International Life Sciences Institute (ILSI) Risk Science Institute convened a workshop entitled ''Relevance of the Rat Lung Response to Particle Overload for Human Risk Assessment. The workshop addressed the numerous study reports of lung tumors in rats resulting from chronic inhalation exposures to poorly soluble, nonfibrous particles of low acute toxicity and not directly genotoxic. These poorly soluble particles, indicated by the acronym PSPs (e.g., carbon black, coal dust, diesel soot, nonasbestiform talc, and titanium dioxide), elicit tumors in rats when deposition overwhelms the clearance mechanisms of the lung resulting in a condition referred to as ''overload. These PSPs have been shown not to induce tumors in mice and hamsters, and the available data in humans are consistently negative. The objectives were twofold: (1) to provide guidance for risk assessment on the interpretation of neoplastic and nonneoplastic responses of the rat lung to PSPs; and (2) to identify important data gaps in our understanding of the lung responses of rats and other species to PSPs. Utilizing the five critical reviews of relevant literature that follow herein and the combined expertise and experience of the 30 workshop participants, a number of questions were addressed. The consensus views of the workshop participants are presented in this report. Because it is still not known with certainty whether high lung burdens of PSPs     

Alterations in Particle Accumulation and Clearance in Lungs of Rats Chronically Exposed to Diesel Exhaust

Alterations in Particle Accumulation and Clearance in Lungsof Rats Chronically Exposed to Diesel Exhaust. WOLFF, R. K.,HENDERSON, R. F., SNIPES, M. B., GRIFFITH, W. C., MAUDERLY,J. L., CUDDIHY, R. G., AND MCCLELLAN, R. 0. Fundam Appl. Toxicol9, 154–166. F344 rats were chronically exposed to dieselexhaust at target soot concentrations of 0 (control, C), 0.35(low, L), 3.5 (medium, M), and 7.0 (high, H) mg/m³ Accumulatedlung burdens of diesel soot were measured after 6, 12, 18, and24 months of exposure. Parallel measurements of particle depositionand clearance were made to provide insight into the mechanismsof particle accumulation in lungs. The fractional depositionof inhaled ⁶⁷Ga₂O₃ particles after 6, 12, 18, and 24 monthsof exposure and of inhaled ¹³⁴Cs-fused aluminosilicate particlesafter 24 months were similar for all groups. Progressive increasesin lung burdens of soot particles were observed in M and H exposedrats, reaching levels of 11.5 ± 0.5 and 20.5 ±0.8 mg/lung (SE), respectively, after 24 months. Rats in theL group had smaller relative increases in lung burden, reachinglevels of 0.60 ± 0.02 mg/lung after 24 months. Trachealmucociliary clearance measurements, using ^99mTc-macroaggitatedalbumin deposited in the trachea, showed no changes at anytime.There were statistically significant increases inclearance half-timesof inhaled radiolabeled particles of ⁶⁷Ga₂O₃ as early as 6 monthsat the H level and 18 months at the M level; no significantchanges were seen at the L level. Rats inhaled fused aluminosilicateparticles labeled with ¹³⁴Cs after 24 months of diesel exhaustexposure to measure long-term components of pulmonary clearance.The long-term clearance half-times were 79 ± 5, 81 ±5, 264 ± 50, and 240 ± 50 days (± SE) forthe C, L, M, and H groups, respectively. Differences were significantbetween the C and both the M and H exposure groups (p <0.01).Lung burdens of diesel soot were more than expected at the Hand M levels and were also associated with impaired particleclearance while smaller responses were observed in both burdensand clearance at the L level.     

Particle Clearance and Histopathology in Lungs of F344/N Rats and B6C3F1 Mice Inhaling Nickel Oxide or Nickel Sulfate

The goals of this study were to (1)determine the effects ofrepeated inhalation of relatively insoluble nickel oxide (NiO)and highly soluble nickel sulfate hexahydrate (NiSO₄ 6H₂O) onlung particle clearance, (2)investigate the effects of repeatedinhalation of NiO or NiSO₄ on the pulmonary clearance of subsequentlyinhaled ⁸⁵Sr-labeled microspheres, (3)correlate the observedeffects on clearance with accumulated Ni lung burden and associatedpathological changes in the lung, and (4)compare responses inF344 rats and B6C3F₁, mice. Male F344/N rats and B6C3F₁ micewere exposed whole-body to either NiO or NiSO₄ 6H₂O 6 hr/day,5 days/week for up to 6 months. NiO exposure concentrationswere 0, 0.62, and 2.5 mg NiO/m for rats and 0, 1.25, and 5.0mg NiO/ m for mice. NiSO₄ 6H₂O/m³ exposure concentrations were0, 0.12, and 0.5 mg NiSO₄ 6H₂ for rats and 0, 0.25, and 1.0mg NiSO₄ 6H₂O/m³ for mice. After 2 and 6 months of whole-bodyexposure, groups of rats and mice were acutely exposed nose-onlyto 63 (NiO-exposed animals only), ⁶³NiSO₄ 6H₂O (Ni SO₄ 6H₂Oanimals only), or to ⁸⁵Sr-labeled polystyrene latex (PSL) microspheres(both NiO- and NiSO₄ 6H₂O-exposed animals) to evaluate lungclearance. In addition, groups of rats and mice were euthanizedafter 2 and 6 months of exposure and at 2 and 4 months afterthe whole-body exposures were completed to evaluate histopathologicalchanges in the left lung and to quantitate Ni in the right lung.Repeated inhalation of NiO results in accumulation of Ni inlungs of both rats and mice, but to a greater extent in lungsof rats. During the 4 months after the end of the whole-bodyexposures, some clearance of the accumulated Ni burden occurredfrom the lungs of rats and mice exposed to the lower, but notthe higher NiO exposure concentrations. Clearance of acutelyinhaled ⁶³NiO was also impaired in both rats and mice, withthe extent of impairment related to both exposure concentrationand duration. However, the clearance of acutely inhaled ⁸⁵SrPSL microspheres was not impaired. The repeated inhalation ofNiO resulted in alveolar macrophage (AM) hyperplasia with accumulationof NiO particles in both rats and mice, chronic alveolitis inrats, and interstitial pneumonia in mice. These lesions persistedthroughout the 4-month recovery period after the NiO whole-bodyexposures were terminated. In contrast, repeated inhalationof NiSO₄ 6H₂O did not result in accumulation of Ni in lungsof either rats or mice and did not affect the clearance of ⁶³NiSO₄6H₂Oinhaled after either 2 or 6 months of NiSO₄ 6H₂O exposure. Clearanceof the ⁸⁵Sr-labeled microspheres was significantly impairedonly in rats exposed to the microspheres after 2 months of exposureto NiSO₄ 6H₂O Histopathological changes in rats were qualitativelysimilar to those seen in NiO-exposed rats. Only minimal histopathologicalchanges were observed in NiSO₄ 6H₂O mice. These results suggestthat repeated inhalation of NiO at levels resulting in AM hyperplasiaand alveolitis may impair clearance of subsequently inhaledNiO. The potential effects of repeated inhalation of solubleNiSO₄ 6H₂O on the clear ance of subsequently inhaled poorlysoluble particles are less clear.     

Species differences in release of arachidonate metabolites in response to inhaled diluted diesel exhaust

In life-span studies in CD-1 mice and F344/Crl rats, inhaled diluted diesel exhaust was highly fibrogenic in rats but not in mice. This was the case despite the higher lung burden, in mg soot/g lung, achieved in mice compared to rats. We tested the hypothesis that the greater fibrogenicity of the soot in rats was due in part to greater release of mediators of inflammation from alveolar cells in rats compared to mice. Female F344/rats and B6C3F1 mice were exposed for up to 17 days to diluted diesel exhaust containing 3.5 mg/m3 of soot. The lungs of control and soot-exposed animals were lavaged after 2, 12 or 17 days of exposure. The presence of leukotriene (LT)B4, LTC4, prostaglandin (PG)E2, PGF2 alpha and thromboxane (TX) B2 in the lavage fluids and LTB4 and PGF2 alpha in cultured lavage cell supernatants was determined. The total amount of each lavage fluid constituent was normalized to lung weight for species comparisons. Control rats had higher levels of TXB2 (16-fold), and LTB4 (6-fold) and PGE2 (2-fold) than control mice, but control mice had higher amounts of LTC4 (4-fold). Control rats and mice had approximately the same amounts of PGF2 alpha/g lung in bronchoalveolar lavage fluid (BALF). Rats exposed to diesel exhaust had increases in BALF PGF2 alpha and LTB4 that were highest after 2 days of exposure and decreased thereafter. Mice had lesser increases in both parameters. Rat cells recovered from lavage fluid released larger amounts of LTB4 into culture supernatants than mouse cells. The data were consistent with the hypothesis that soot-laden rat alveolar cells release greater quantities of mediators of inflammation than do the alveolar cells in mice.     

The relevance of the rat lung response to particle overload for human risk assessment: a workshop consensus report. ILSI Risk Science Institute Workshop Participants

On 23-24 March 1998, the International Life Sciences Institute (ILSI) Risk Science Institute convened a workshop entitled "Relevance of the Rat Lung Response to Particle Overload for Human Risk Assessment." The workshop addressed the numerous study reports of lung tumors in rats resulting from chronic inhalation exposures to poorly soluble, nonfibrous particles of low acute toxicity and not directly genotoxic. These poorly soluble particles, indicated by the acronym PSPs (e.g., carbon black, coal dust, diesel soot, nonasbestiform talc, and titanium dioxide), elicit tumors in rats when deposition overwhelms the clearance mechanisms of the lung resulting in a condition referred to as "overload." These PSPs have been shown not to induce tumors in mice and hamsters, and the available data in humans are consistently negative. The objectives were twofold: (1) to provide guidance for risk assessment on the interpretation of neoplastic and nonneoplastic responses of the rat lung to PSPs; and (2) to identify important data gaps in our understanding of the lung responses of rats and other species to PSPs. Utilizing the five critical reviews of relevant literature that follow herein and the combined expertise and experience of the 30 workshop participants, a number of questions were addressed. The consensus views of the workshop participants are presented in this report. Because it is still not known with certainty whether high lung burdens of PSPs can lead to lung cancer in humans via mechanisms similar to those of the rat, in the absence of mechanistic data to the contrary it must be assumed that the rat model can identify potential carcinogenic hazards to humans. Since the apparent responsiveness of the rat model at overload is dependent on coexistent chronic active inflammation and cell proliferation, at lower lung doses where chronic active inflammation and cell proliferation are not present, no lung cancer hazard is anticipated.