The rapid alveolar absorption of diesel soot-adsorbed benzo[a]pyrene: bioavailability, metabolism and dosimetry of an inhaled particle-borne carcinogen

P. Gerde,3, B.A. Muggenburg1,, M. Lundborg and A.R. Dahl2,

Institute of Environmental Medicine, Division of Inhalation Toxicology, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden and National Institute for Working Life, Solna, Sweden,
1 Lovelace Respiratory Research Institute, PO Box 5890, Albuquerque, NM 87185, USA and
2 Battelle Memorial Institute, 505 King Avenue, Columbus, OH 43201-26, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 
Exposure to diesel exhaust may contribute to lung cancer in humans. It remains unclear whether the carbonaceous core of the soot particle or its coat of adsorbed/condensed organics contributes most to cancer risk. Equally unclear are the extent and rate at which organic procarcinogens desorb from soot particles in the lungs following inhalation exposure and the extent of their metabolic activation in the lungs. To explore the basic relationship between a model polycyclic aromatic hydrocarbon (PAH) and a typical carrier particle, we investigated the rate and extent of release and metabolic fate of benzo[a]pyrene (BaP) adsorbed on the carbonaceous core of diesel soot. The native organic content of the soot had been denuded by toluene extraction. Exogenous BaP was adsorbed onto the denuded soot as a surface coating corresponding to 25% of a monomolecular layer. Dogs were exposed by inhalation to an aerosol bolus of the soot-adsorbed BaP. Following deposition in the alveolar region a fraction of BaP was rapidly desorbed from the soot and quickly absorbed into the circulation. Release rates then decreased drastically. When coatings reached ~16% of a monolayer the remaining BaP was not bioavailable and was retained on the particles after 5.6 months in the lung. However, the bioavailability of particles transported to the lymph nodes was markedly higher; after 5.6 months the surface coating of BaP was reduced to 10%. BaP that remained adsorbed on the soot surface after this period was ~30% parent compound. In contrast, the rapidly released pulse of BaP, which was quickly absorbed through the alveolar epithelium after inhalation, appeared mostly unmetabolized in the circulation, along with low concentrations of phase I and phase II BaP metabolites. However, within ~1 h this rapidly absorbed fraction of BaP was systemically metabolized into mostly conjugated phase II metabolites. The results indicate that absorption through the alveolar epithelium is an important route of entry to the circulation of unmetabolized PAHs.

Abbreviations: BaP, benzo[a]pyrene; LSC, liquid scintillation counting; PAHs, polycyclic aromatic hydrocarbons.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 
Diesel soot particles are important contributors to the pollution of some workplace atmospheres, as well as to urban air, and exposures are suspected of increasing the risk of lung cancer in humans (1,2). With relative risk ratios usually <2, the association is, however, not without dispute, mainly because of possible confounding effects of smoking (3). Nevertheless, because of the large populations at risk of exposure, the association warrants further investigation.

Diesel exhaust particles consist of an aggregated core of ultrafine carbonaceous particles surrounded by an adsorbate/condensate of different hydrocarbons (4). The adsorbate/condensate contains low concentrations of polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs (5), some of which are carcinogenic. Yet, the mechanism by which diesel exhaust may induce lung cancer remains unclear. The adsorbed PAHs were early suspects as causative agents (6) and diesel exhaust was later shown to be a pulmonary carcinogen in rats following chronic exposure by inhalation (5,7). However, the same effect was also seen with carbon black and titanium dioxide particles at similar exposure levels. The lung cancer incidence in rats correlated closely with an increased level of inflammation in the lung and build-up of particle deposits (8). Other rodent species, such as hamsters and mice, were less sensitive (9). It therefore remains unclear whether suspected lung cancers in humans are linked to a genotoxic effect of the adsorbed organics or to an inflammatory process induced by the carbonaceous core particles of the soot (10). Thus, the PAHs in the organic coat of diesel soot may play a role in carcinogenesis, but the extent of its contribution to the carcinogenic process is unclear. The levels of extractable PAHs on diesel soot are quite low in comparison with their levels on other air pollution aerosols, such as coal tar pitch, for which lung cancer risk has been assessed with greater certainty (11). Vostal (12) even questioned whether the carcinogenic PAHs on diesel soot are bioavailable at all in the lungs. Whereas PAHs can be easily extracted from the soot particles with organic solvents, such as toluene or dichloromethane, results are tentative when liquids simulating the lining layer of the lung are used (13,14). The most common model solutions for the liquid lining of the lung have been dispersions of surfactant liposomes in saline. Usually low or undetected amounts of PAHs have been released from the diesel soot into these model solutions. Yet, elevated levels of DNA adducts of PAHs in white blood cells have been observed in humans following exposure to diesel exhaust (15,16). To further complicate the issue, in some cases exposure to carrier particles with much higher levels of organic extractable PAHs, such as tar pitch, has not led to elevated levels of DNA adducts in white blood cells (17,18). In the light of seemingly conflicting results from exposure to particle-associated PAHs, we sought to answer four fundamental questions regarding their toxicity: (i) do PAHs have to be released from their carrier aerosols to become toxic, i.e. metabolized, to surrounding tissues; (ii) if so, how is the potential toxic level of PAHs on such particles quantitated; (iii) what is the extracting capacity of organic solvents in vitro compared with lung lining fluids in vivo; (iv) once released from particles, how fast are PAHs absorbed from the peripheral lung into the circulation and is this fraction of PAHs metabolized in the lungs or elsewhere?

We chose to address these problems using a single component surrogate for PAHs adsorbed onto organic-denuded core particles of diesel soot. Our aim was to understand the exposure and dosimetry of PAHs adsorbed onto particles in order to comprehend how early events, such as exposure, deposition, bioavailability and metabolism, may influence later events, such as adduction to DNA and mutation patterns. We have not, however, directly addressed the issue of whether an inhaled PAH on diesel soot is a human lung carcinogen but have not excluded the possibility that particles may contribute to lung cancer risk by means other than acting as carriers of genotoxic PAHs. Benzo[a]pyrene (BaP) is a natural first choice for a PAH surrogate. BaP is a proven carcinogen in several animal models (19) and mutations in lung tumors of smokers have a pattern of `hot spots' at the same codons as those of tumors induced in mice with BaP (20). The dog was chosen for the present study mainly because its lungs are large enough to allow control of its breathing pattern and the study of regional deposition of inhaled materials. Dogs also develop airway epithelial changes from exposure to tobacco smoke similar to those seen in human smokers (21).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 
Experimental design
Three dogs were exposed to a single breath bolus of aerosolized diesel soot that deposited on the airways and in the alveolar or peripheral region of the lung. Clearance of BaP from the lung following this deposition pattern was monitored by repeatedly sampling blood from both sides of the systemic circulation for 1 h. In a study to be reported elsewhere, at respectively 154, 168 and 190 days after the alveolar exposures, the same dogs were exposed only in the airspace of the temporarily occluded trachea to a bolus of the same soot-adsorbed BaP. After these experiments the dogs were killed and soot from the experiments presented here was recovered from lung parenchyma and tracheo-bronchial lymph nodes.

Collection of diesel soot
Diesel soot was collected from a three cylinder, 3.8 l tractor engine (Model 1113 TR; Bolinder-Munktell) working at 80% of its rated 41.2 kW output at the Swedish Engine Test Center, Uppsala. The engine was run at 1600 r.p.m. on diesel fuel (Swedish environment class MK 3) and the exhaust was diluted 11-fold with air before precipitation on a Tepcon electrostatic filter (Model 2200; ActAir, Cardiff, UK) at 44°C. The entire exhaust flow of 1600 kg/h was passed through the filter. About 40 g of soot was scraped from the Teflon-coated electrodes and stored in the dark at –20°C.

Preparation of the BaP-coated diesel soot
The collected diesel soot was pre-extracted twice with toluene in a Soxhlet apparatus for 6 h, then dried at 130°C for 70 h. The specific surface area of the organic-denuded soot (Brunauer–Emmett–Teller method; ref. 22, pp. 531–539) was 137 ± 1 m2/g (n = 3). Two identical batches of BaP-coated soot were prepared, one with unlabeled BaP for size determination of the aerosol and one with tritium-labeled BaP (463.3 d.p.m./pg uniformly labeled, TRK 662 batches 0.8xSP2 + 0.2xB99A, 98% purity; Amersham) for the exposures.

BaP from stock solution was evaporated to dryness under a stream of nitrogen and redissolved in methanol. Denuded diesel soot (10.2 mg) and the BaP (150 ng) in 4 ml of methanol were added to a melt-seal vial. The soot/methanol suspension was gently agitated for 2 h and evaporated to dryness under a stream of nitrogen. The vial was melt-sealed under a reduced nitrogen atmosphere and heated to 240°C for 2 h to allow the BaP to adsorb evenly over the soot surface. After cooling the cake of soot was finely ground with a spatula before further use. No crystals of BaP were observed under a fluorescence microscope. A triplicate sample of the soot was extracted in a Soxhlet apparatus with toluene for 24 h to determine the re-extractable fraction of BaP on the soot. The extracted samples of soot were combusted to determine the remaining 3H activity on the particles. The resulting batch of [3H]BaP-coated soot contained 14.5 ± 0.1 ng BaP/µg soot, corresponding to a 25% coating of BaP on the soot surface based on 100 Å2/molecule. The level of surface coating was chosen to give an about equal distribution between firmly adsorbed and easily extractable BaP that would allow both fractions to be accurately quantitated in the model systems used.

Extraction of diesel soot in 1-octanol in vitro
To simulate the extent and rate at which soot-adsorbed hydrocarbons are released in the lining layer fluid of the lung we determined the desorption of BaP from the labeled soot in 1-octanol. The experiment was conducted in a vertical, cylindrical glass reactor 27 mm in diameter with four low vertical glass baffles. The solution was stirred at 400 r.p.m. with a two-bladed impeller (15 mm diameter and 12 mm blade heights). Seventeen milliliters of analytical grade 1-noctanol was added to the reactor and adjusted to 37°C. About 120 µg BaP-coated soot was added to the octanol and the mixture was stirred immediately. After 20 s the stirring was stopped briefly and 0.5 ml of the suspension was quickly removed and centrifuged at 13 000 g (Microcentaur; MSE, UK) for 8 min. From the clear supernatant, three 50 µl samples were transferred to scintillation vials for liquid scintillation counting (LSC) using Ultima Gold scintillation fluid (Packard Instruments, Meridian, CT). The sampling procedure was repeated at 12, 24, 35, 46, 58, 70 and 90 min and 2, 3, 4, 5, 6, 24 and 48 h after addition of the soot. The actual time point of separation of particles from the supernatant was counted from 1 min after the sample was removed from the reactor.

Generation of re-aerosolized diesel soot
Single breath boluses of aerosolized diesel soot were generated by means of a novel patented method (23; Figure 1Go). The aerosol generator consisted of a pressure chamber, a spherical powder chamber and a fast release valve. The pressure chamber was connected to the powder chamber via the valve and the powder chamber had a narrow conduit leading to ambient pressure in the aerosol generator syringe. Nitrogen (220 ml) compressed to 80 atm was loaded into the pressure chamber and soot was loaded into the powder chamber. When the valve was opened the soot was explosively suspended and pressurized by nitrogen equilibrating the pressure between the pressure and powder chambers. While in suspension the soot agglomerates ejected through the narrow conduit into the aerosol generator syringe. Passage through the small orifice in the ejecting conduit allowed an explosive, yet controlled, decompression of a fraction of the suspension at a time, generating micron-sized particles. At the same time, the volume of the decompressed carrier nitrogen pushed up the plunger of the syringe. After decompression the aerosol was ready for use. (Additional details of this method will be published elsewhere.) The average mass mean aerodynamic diameter of the administered diesel soot, as determined repeatedly with unlabeled soot, was 1.3 ± 0.2 µm (n = 5). The aerosol for the alveolar exposures was generated and passed through tubing identical to that used during the exposures. The aerosol was sized in a quartz crystal cascade impactor (QCM Cascade Impactor System, Model PC-2; California Instruments, Sierra Madre, CA). The re-aerosolized soot consisted of densely packed aggregates of soot spherules as observed by transmission electron microscopy. Diameters of individual spherules typically ranged from 0.01 to 0.04 µm (4). Assuming a spherule density of 2 g/ml indicated that most of the measured specific surface area was external surface with little intra-spherular porosity. The dimensions of the intra-aggregate porosity of the soot were likely to be too large to contribute significantly to the overall mass transfer resistance of adsorbed BaP from micron-sized particles to surrounding lung medium (24). Delayed release of adsorbed BaP was, thus, likely to be caused by the kinetics of surface desorption.



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Fig. 1. Apparatus for exposing dogs, with the alveolar region as the target, to a bolus of radiolabeled BaP adsorbed onto organic-denuded diesel soot. Approximately 220 ml of aerosol from the generator was followed by 90 ml of clean air from the large syringe to help push the aerosol into the alveolar region. Following the single breath exposure blood-borne clearance was monitored by repeatedly sampling blood from both sides of the systemic circulation for 1 h.

 
The dose of diesel soot deposited during the exposures was determined using a total filter (Millipore AA 1.2 µm, 25 mm) at the end of the endotracheal tube in the exposure system. An aerosol bolus of the radiolabeled BaP/diesel soot was forced through the filter at the same flow rate used for the exposures. The amount of BaP on the filter was determined by total combustion and measurement of radioactivity by LSC. Converted to the amount of soot, the deposition was 36 ± 20 µg (n = 6). The test series for particle deposition were done in parallel with the exposures. To reduce the risk of tritium cross-contamination, the aerosol generator was cleaned between experiments not by rinsing with solvent, but by repeatedly ejecting gas without loading particles, then catching loosened particles on a total filter.

Animals
Three 1-year-old beagle dogs, two female and one male, purchased from Marshall Farms (North Rose, NY), were used in this study. The dogs weighed 8.0 ± 0.9 kg. Food was withheld for 18 h prior to the procedures. Each dog was given 0.2 ml of acepromazine s.c. Anesthesia was induced 15 min later with isoflurane using a face mask.

Exposures
When light surgical anesthesia was achieved an endotracheal tube was put in place and a surgical level of anesthesia was maintained using isoflurane inhaled via the tube. Dogs were prepared for aseptic procedures. A surgical cut-down procedure over the femoral artery and vein was done and catheters filled with heparinized saline were passed into and positioned in the posterior vena cava close to the right chamber of the heart (blood entering the lungs) and in the thoracic aorta (blood leaving the lungs). The anesthetized dog was connected to the exposure apparatus shown in Figure 1Go. Immediately before exposure the dog was hyperventilated for 3 min to obtain an equally long period of apnea, then the line to the anesthesia machine was closed. With its exit tube closed, the aerosol generator was triggered and ~220 ml of aerosol was generated. As soon as the generator syringe was filled the aerosol bolus was gently pushed into the apneic dog over a 4–5 s period. The aerosol bolus was immediately followed by 90 ml of clean air from the large syringe to help push the aerosol into the alveolar region. The aerosol was allowed to precipitate in the inflated lungs for ~2 min while the dog was still apneic. Next, the dog was ventilated several times through a total filter for a short period to remove the remaining suspended aerosol, then reconnected to the anesthesia machine.

Blood sampling began at the moment the clean air was injected after the aerosol bolus. During a 1 h period blood (~1 ml/sample) was sampled with increasing intervals from every 12 s to every 5 min from both sides of the systemic circulation. Immediately following sampling ~0.2 ml of each blood sample was transferred to 2 ml paper thimbles (Packard Instruments) for determination of total radioactivity. The samples were immediately frozen at –80°C, as were the remaining fractions of the blood intended for metabolite analysis.

After the blood sampling period the catheters were removed, the blood vessels repaired and the surgical incisions closed. The dogs were carefully observed during recovery. Then 5.6 months after the alveolar exposures, the dogs were killed following exposure of the occluded trachea to the same preparation of diesel soot with adsorbed BaP. At this point the four tracheo-bronchial lymph nodes and some peripheral lung tissue were sampled for analysis of soot from the presented peripheral lung exposures. These tissues were stored at –80°C until processing.

Radiochemical analysis and metabolite fractionation
Before further analysis the samples were dried by vacuum distillation with collection of water, including tritiated water, for LSC. When dry, the BaP-derived tritium in blood and tissue samples was measured after combustion by LSC of the tritiated water generated according to a previously published method (25).

The metabolites in blood and tissues were determined as previously described (26). In brief, the dried blood samples were resuspended in saline and extracted five times with ethyl acetate by centrifugation between extractions and the organic fraction was decanted. The organic fraction contained parent compound and its lipophilic phase I metabolites. The aqueous fraction was centrifuged and the supernatant and separated pellet were combusted separately. Water-soluble radioactivity came from conjugated phase II metabolites and the radioactivity of the separated pellet gave the covalently bound fraction of BaP. To improve uptake of BaP and its lipophilic metabolites into the HPLC elution fluid from the pooled organic fraction the lipids were saponified with calcium oxide and re-extracted with ethyl acetate. After centrifugation and washing of the precipitate by resuspending with ethyl acetate and precipitating, the organic extract was dried and redissolved in HPLC elution fluid. BaP and metabolites were separated using a Beckman Ultrasphere C18 column (5 µmx4.6 mmx25 mm) eluted with a methanol/water gradient from 55 to 100% (v/v). The chromatograms showed separation of BaP from its phase I metabolites and peaks for individual metabolites.

Recovery of diesel soot from tissues
Soot was recovered by a procedure modified from that of Sun et al. (27) to analyze the content and nature of the BaP still adsorbed on the diesel soot retained in tissues. The tracheo-bronchial lymph nodes or ~1 g peripheral lung tissue from each dog were homogenized in separate experiments with a Tissuemizer in 10 ml of distilled water. Five milliliters of the homogenate was layered on top of 15 ml of 25% sucrose solution in a 38 ml centrifuge tube (polycarbonate, 1x3.5 inches). The tubes were centrifuged for 2 h at 100 000 g in an ultracentrifuge (Beckman L8-60M).

After centrifugation the upper water/tissue homogenate layer plus some of the sucrose layer was transferred to combustion thimbles and air dried for analysis of radioactivity by total combustion. Two 1.0 ml samples were taken from the sucrose layer and counted directly after adding 20 ml of Ultima Gold. The rest of the sucrose layer was carefully decanted from the precipitated particles, which were wiped up with small balls of cotton and placed on filter paper to air dry. When dry the cotton balls were folded into filter paper, placed in a Soxhlet apparatus and refluxed with toluene for 24 h. Samples of the extract were evaporated to dryness, then dissolved again in the elution fluid for HPLC. Drying removed the tritiated water. The remaining amount of radioactivity was considered equivalent to the molar amount of parent compound and metabolites introduced by BaP. Using HPLC, the BaP-related radioactivity recovered from the particles was separated into parent compound, organic-extractable metabolites and water-soluble metabolites (void volume) by comparison of elution times with standards.

The filter paper extracted with the Soxhlet apparatus was air dried for 24 h, then combusted. For each sample the total amount of radioactivity in the toluene extract (MTE) was compared with that in the combusted filter paper containing the extracted particles. We assumed that the fraction of total radioactivity not extracted with toluene in a Soxhlet apparatus was the same whether the particles had resided in the lungs or not. This allowed the amount of soot retained in the tissues to be calculated, as well as the total amount of radioactivity expected to have been associated with this soot before exposure (MTOT), assuming a homogeneous distribution. By comparing MTOT with MTE, the amount of BaP recovered in the toluene extract, and with MEP, the amount remaining on the extracted particles as measured by combustion, the fractional bioavailability (BA) of soot-adsorbed BaP in the tissues was calculated according to:


We assumed that the fraction of firmly adsorbed BaP not removed by Soxhlet extraction in toluene was the same whether the particles had resided in tissues or not. Therefore, the non-extractable fraction of BaP could be used as an indicator of the amount of soot particles in a sample, which in turn allowed us to calculate an approximate long-term BA of BaP on the soot. One possible error would be if over time in vivo the radiolabeled BaP migrated from the tightly bound sites to loosely bound sites and then desorbed. The low standard deviation between samples from two types of tissues, lymph nodes and peripheral lung, in three different dogs indicates a clear limit to release affected by this potential pathway.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 
After the bolus with denuded diesel soot and adsorbed BaP was deposited in the peripheral lung, desorbing BaP-equivalents appeared rapidly in the systemic circulation (Figure 2Go). The concentration in the blood peaked at 2.1 ± 0.6 min (n = 3) in all three dogs. However, dog C had an ~4-fold higher systemic level of BaP in the blood than the other two, most likely because of a greater amount of diesel soot in the aerosol bolus (Figure 2CGo). For each dog clearance of BaP-equivalents from the lungs was estimated by trapezoid integration of the net increase in concentration of BaP in blood after passage through the lungs multiplied by the cardiac output (1.42 l/min) over the period when a net influx of BaP-equivalents entered the circulation. Whereas cumulative clearance was higher in dog C than in dogs A and B, the fractional retention of BaP-equivalents, as determined by integration, was similar in all three dogs. The first half-time of absorption was 4.3 ± 0.8 min (n = 3) (Figure 3Go). The net influx into the circulation, as indicated by the difference between the concentration of BaP in blood leaving the lungs and blood entering the lungs, peaked at 1.8 ± 0.8 min (n = 3). For dogs A and B ~30% of the estimated deposited amount of BaP-equivalents was cleared to the blood during the early period when a net influx from the lungs to the blood could be measured. This is similar to the fraction of BaP released from the soot particles during extraction in 1-octanol in vitro (Figure 4Go).



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Fig. 2. The concentration of BaP-equivalent activity in blood as a function of time in dogs (A)–(C) following inhalation exposure of the alveolar region to an aerosol bolus of BaP adsorbed onto organic-denuded diesel soot. Samples from blood exiting and entering the lungs were drawn at the ascending aorta and the posterior vena cava, respectively. Note that the concentration scale in (C) is different from those in (A) and (B).

 


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Fig. 3. Fractional retention of the readily bioavailable BaP-equivalent adsorbed onto denuded diesel soot in the dogs' lungs as a function of time. The dashed curve shows the corresponding fractional retention of microcrystalline BaP (data from ref. 43). Error bars show SD (n = 3).

 


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Fig. 4. The release of BaP from diesel soot in a well-stirred solution of 1-octanol at 37°C, in toluene during Soxhlet extraction and in tissues following 5.6 months retention in vivo. Surface concentration as a function of time is given as a fraction of a monomolecular layer of BaP adsorbed onto the soot surface. The initial surface concentration on the soot was 25% of a monomolecular layer. Error bars show SD (n = 4 in vitro, n = 3 in vivo).

 
Initially, soot-adsorbed BaP was quickly released into 1-octanol in our in vitro dissolution system. Within minutes the concentration on the soot decreased from the initial 25% to ~18% surface coating, but in the next 48 h the concentration only dropped to ~16% surface coating. At this time 36% of the BaP was desorbed. The in vitro extraction thus showed that desorption from the particles is most intense at the moment of deposition and that the rate of release then decreases with the inverse of time after deposition. Twenty-four hours after deposition into 1-octanol the release rate of BaP dropped to ~1/10 000th of the initial release rate (Figure 5Go).



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Fig. 5. The rate of release of soot-adsorbed BaP into 1-octanol as a function of time, presented as a fraction of the adsorption at t = 0. The fitted decay function is f(x) = 4.239x10–4/x1.191 (r2 = 0.911).

 
Tritiated water, indicating metabolism, appeared rapidly in the blood, with a net influx from the lungs in all three dogs during the first minute after exposure. The level of tritiated water in the systemic blood increased rapidly up to some 40 min after exposure, then leveled off (Figure 6Go). In all three dogs there was an early net influx of tritiated water from the lungs, indicating a rapid onset of metabolism in airway mucosa. In dog A this net influx persisted throughout the 1 h blood sampling period. However, the dominant fraction of BaP-equivalent activity entering the blood during the rapid initial pulse was parent compound, with smaller amounts of phase I and phase II metabolites (Figure 7Go). Note that the release in Figure 6Go is related only to the readily available fraction of BaP, whereas the release in Figure 7Go is related to the total amount of BaP on the soot. The rapid initial pulse of BaP decreased quickly towards a much lower and steadier level that slowly tapered off. Within 40 min of exposure the slowly declining fraction of radioactivity in the blood became dominated by phase II metabolites and bound radioactivity (Figure 7Go).



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Fig. 6. The concentration of tritiated water in blood, expressed as Bq tritium/ml, as a function of time in dogs (A)–(C) following exposure to an aerosol bolus of denuded diesel soot with adsorbed BaP. Note that the concentration scale in (C) is different from those in (A) and (B).

 


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Fig. 7. The metabolite pattern of BaP in the systemic blood at increasing times after deposition of an aerosol bolus of denuded diesel soot with adsorbed BaP. Each pair of bars shows, respectively, the average concentration of different metabolite categories in blood (pmol BaP-equivalent activity) entering (IN) and exiting (OUT) the lungs at that particular time. Error bars show SD (n = 3) of the metabolite categories given as a fraction of total radioactivity, not of absolute concentration.

 
Diesel soot was recovered by ultracentrifugation from tissues of the peripheral lung and lymph nodes after 5.6 ± 0.6 months (n = 3) retention time. Of the originally adsorbed BaP 37 ± 3 (n = 3) and 59 ± 2% (n = 3) had desorbed from the soot in peripheral lung and lymph nodes, respectively (Figure 4Go). During centrifugation of peripheral lung tissue and lymph nodes 99 ± 0.5 and 91 ± 12% (n = 3), respectively, of the radioactivity was recovered from below the sucrose layer, i.e. from the particles. The rest was in the tissue homogenate-containing layer on top. This suggests that most of the tissue-retained radioactivity came from particle-associated BaP rather than tissue-bound BaP. For dogs A and B, but not dog C, the amount of soot recovered from the lung tissues corresponded to a long-term retention of soot in the lungs of ~77%, which is close to the measured long-term retention of inert particles in the dog lung (28). Analysis of BaP-equivalents extracted from soot retained in the lungs for 5.6 months showed that about one-third remained as parent compound on the particles (Table IGo), with nearly half being lipophilic metabolites/decay products of BaP (Figure 8Go).


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Table I. Distribution between major metabolite groups of BaP-associated radioactivity adsorbed on soot recovered from respiratory tract tissues after 5.6 months retention in vivo following exposure of the alveolar region(% ± SD, n = 3)
 


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Fig. 8. Composition of BaP-equivalent activity extracted from soot surfaces after long-term retention in lung tissues. The quantitative distribution is shown in Table IGo. (A) HPLC chromatogram of extracted BaP and metabolite standards. (B) In the toluene extract of diesel soot recovered from the lung parenchyma 5.6 months after bolus inhalation in the alveolar region of BaP adsorbed on denuded diesel soot. (C) In the toluene extract of diesel soot recovered from the tracheo-bronchial lymph nodes 5.6 months after bolus inhalation in the alveolar region of BaP adsorbed on denuded diesel soot.

 

    Discussion and conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 
The results indicate that a particle-adsorbed fraction of BaP in the lungs is essentially non-reactive and must be released in order to participate in toxicity to the surrounding lung tissues. Of the radioactivity recovered from the soot surface after 5.6 months retention in lung tissues, nearly one-third remained as BaP parent compound (Table IGo). When added as a solute to the tracheal mucosa (29) parent BaP was reduced to similar fractions within a mere 3 h; an ~1000-fold difference in reactivity. The fraction of metabolites or decay products of BaP adsorbed on the soot surface after 5.6 months retention in tissues should be taken as an indication of stability rather than reactivity. With the short range of the sorptive forces in liquid media, once desorbed and before binding to the active site of the P450 enzyme the metabolic fate of the BaP molecule should be independent of a previous history of particle adsorption (30). It is likely that the relationship between particle-bound BaP/low reactivity and free BaP/high reactivity in the lungs also holds for a collection of genotoxic PAHs on native soot at much lower concentrations. The bioavailability of genotoxic PAHs on soot particles is thus a critical factor in determining the dose of such agents to the lungs.

Bioavailability of PAHs on soot is inversely related to their strength of adsorption. During combustion in diesel engines ultrafine soot particles are generated and PAHs are formed in the gas phase. Subsequently, when the exhaust is rapidly cooled outside the combustion zone PAHs are adsorbed or condensed onto the soot particles (31). PAHs are strongly adsorbed at a limited number of high energy sites, which cover only a fraction of the soot surface (32). Once these surfaces are occupied, adsorption proceeds at sites of decreasing binding energies.

Because the average residence time of adsorbed PAH molecules on the soot surfaces is likely to increase exponentially with increasing binding energy (22,33), adsorbed PAHs will likely belong to one of two principal fractions; one which will be quickly released upon deposition in the lungs and one which will be retained on the soot for long periods (30).

The major fraction of bioavailable BaP was released from the soot within minutes of deposition, both in the alveolar region and in 1-octanol in vitro (Figures 3 and 4GoGo). For dogs A and B the cumulative amount cleared from the alveolar region to the blood during the first 30 min after exposure was similar to the fraction released into 1-octanol. The short-term bioavailability is likely to be similar in alveoli and bronchi considering that surfactant is the likely prime agent of extraction for particle-associated PAHs in the alveolar region, as well as in the mucous lining layer of the conducting airways (3436).

For long-term bioavailability, diesel soot recovered from the peripheral lung after 5.6 months had a 16% surface coating of BaP, which is about the same as the fraction of BaP remaining on the soot after a 48 h extraction into 1-octanol (Figure 4Go). As is the case in 1-octanol in vitro, this suggests that although the release of BaP from the soot continues over time, it enters a slow phase soon after deposition in the lungs. However, soot recovered from the tracheo-bronchial lymph nodes after 5.6 months had significantly less BaP retained on the surfaces; only 10% of the monolayer remained. This result implies that the ultimate bioavailability of adsorbed carcinogens on particles is dependent on the microenvironment in which the particles have resided in the lungs and a single liquid in vitro cannot be used to simulate release of particle-associated hydrocarbons in all respiratory tract tissues. A higher bioavailability of BaP in tracheo-bronchial lymph nodes could result from a large fraction of soot particles translocated there in pulmonary macrophages (37). The acidic and oxidative environment of macrophage phagolysosomes (38,39) may be a better medium for extraction than the fluids of the lung parenchyma.

In vitro extraction of particle-associated PAHs with organic solvents of either an aromatic or highly non-polar nature is likely to overestimate the tissue dose in vivo. Toluene, the aromatic solvent used as a reference, removed the most BaP, but not even Soxhlet extraction for 24 h decreased BaP below a surface coating of 7% of a monomolecular layer on the soot surface (Figure 4Go). In addition, the denuded soot is likely to contain some native adsorbed organics that were not removed during the pre-extraction procedure. To measure the intense short-term bioavailability, extraction in slightly polar 1-octanol seems to give a better estimate of the readily bioavailable fraction of BaP on soot particles residing in the lungs and airways than does extraction in aromatic or non-polar solvents.

Most of the bioavailable BaP on the soot was released within minutes of deposition in the lung (Figure 4Go). The type of epithelium on which the soot particle was deposited governs subsequent absorption into the circulation. While BaP was adsorbed onto the denuded soot at a relatively high surface concentration to allow for quantitation, as a surrogate for all PAHs, the total inhaled amount was not high. Compared with numerous previous studies on the co-carcinogenicity of PAHs and particles by intra-tracheal instillation (40) or inhalation (27), the present study may be the first time the kinetics of release, absorption and metabolism of a PAH in the lungs have been measured following low level deposition of an aerosol bolus. The average density of deposition of soluble BaP in the alveolar region was estimated to be ~4 ng/m2, assuming a 30% bioavailability of the BaP deposited over 40 m2 lung surface (41). This deposition is within an order of magnitude of estimates for deposition of `total' PAHs in humans after smoking a single cigarette (29).

Soot-adsorbed BaP was rapidly absorbed when deposited and desorbed on the alveolar air–blood barrier in a process not likely different from absorption of instantaneously dissolving microcrystals of BaP (42). The faster rate of absorption of crystalline BaP compared to soot-adsorbed BaP may reflect the difference between instantaneous dissolution of BaP crystals in the lungs and the decreasing desorption rate of BaP from the soot surface (Figure 3Go).

The intense pulse of BaP entering the circulation during early clearance was mostly unmetabolized. This fraction of BaP was then quickly taken up by distal compartments and seemed to be intensely metabolized. The delayed absorption followed by a first order decline in the concentration is a strong indication that: (i) resistance is caused primarily by slow diffusion through the air–blood barrier and not by slow dissolution or desorption from particles; (ii) penetration occurs mostly through the thinnest barrier available, the air–blood barrier of type I cells (42,43). With a square relationship between characteristic time of penetration and barrier thickness (44), absorption from the bronchiolar epithelium should be considerably slower and tracheal absorption of BaP in the dog is known to be much slower (29). Thus, the metabolic pattern of BaP entering the circulation during early clearance following exposure (Figure 7Go) primarily reflects the influence of passage through the alveolar type I epithelium, despite likely deposition of the soot also on the bronchial/bronchiolar epithelium. More mobile tritiated water may well have quickly penetrated the thicker bronchial/bronchiolar sections of the air–blood barrier during the same exposures (Figure 6Go).

Based on the presented low level exposures of dogs to aerosol boluses of BaP adsorbed on denuded diesel soot, a general scheme of the dosimetry of similar sized PAHs can be outlined (Figure 9Go). Driven primarily by physicochemical and biochemical mechanisms, the dosimetry should be similar for PAHs with lipophilicities similar to BaP. After release of the bioavailable PAHs from the particles the lungs showed a marked biphasic dosimetry. During exposure to typical carrier aerosols some 80% of the readily bioavailable fraction of PAHs is likely to be deposited on the alveolar type I epithelium (45) and rapidly become systemic. Subsequently, this fraction of PAHs is absorbed by metabolizing tissues, primarily the liver (46), but likely also other tissues such as white blood cells (47,48). Only ~20% of the desorbed PAHs is likely to be absorbed into the metabolizing epithelium of the tracheo-bronchial/bronchiolar region. However, because of the slow penetration of lipophilic PAHs into the capillary bed below the entrance epithelium of the conducting airways (29), the concentration in directly exposed airway cells will be much higher than in cells exposed via the systemic circulation (Figure 9Go). Assuming an even 20% tracheo-bronchial deposition over a 30 µm thick epithelium covering 1 m2 of the bronchial tree, the initial concentration in these cells will be ~40-fold higher than the 80% absorbed from the alveolar region into the 5 l of circulating blood of a human. After rapid distribution of the systemic fraction into, say, 20 kg of tissues in the vicinity of blood capillaries, this difference has grown to ~200 times. Thus, in the case of inhalation exposure to solid core particles with adsorbed PAHs critical exposures of lung tissues are likely dominated by the fraction of PAHs rapidly desorbed from carrier particles that are deposited on the lining layer of the conducting airways.



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Fig. 9. A schematic of the dosimetric fate of an inhaled bioavailable fraction of PAH in the lungs. A larger fraction, typically 80–90% of the carrier aerosol, is deposited in the alveolar region. Desorbing PAHs are rapidly absorbed into the circulating blood, with little influence of local metabolism, while the site of entry concentration is briefly elevated. A smaller fraction of 10–20% of the inhaled bioavailable PAHs is deposited, slowly absorbed and extensively metabolized in the airway epithelium at prolonged elevation of the local tissue concentration.

 
In conclusion:

  1. the short-term bioavalability of BaP in the lungs is mimicked by extraction in 1-octanol;
  2. highly lipophilic carcinogens such as BaP have a dual dosimetry in the lungs, with 80% deposited in the alveolar region and rapidly passed into the blood without much metabolism while 20% is deposited on the conducting airways and is slowly absorbed under intense metabolism;
  3. PAHs adsorbed on soot is one of two principal fractions, one rapidly bioavailable and one tightly bound.


    Notes
 
3 To whom correspondence should be addressedEmail: Per.Gerde{at}imm.ki.sc Back


    Acknowledgments
 
The authors gratefully acknowledge the contribution of John Anthony Stephens as Senior Technical Associate and the advice of Dr Margaret G.Ménache on statistical analysis. This research was conducted in facilities fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. This research was sponsored by the Health Effects Institute (grant EPI 1351901) with contributing funding from the Swedish Council for Working Life Research (grant 95-0192) under Cooperative Agreement DE-FC04-96AL76406 with the US DOE.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 

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Received November 6, 2000; revised January 15, 2001; accepted January 16, 2001.