Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Distribution, retention, and elimination of pyrene in rats after inhalation C. E. Mitchell & K. W. Tu To cite this article: C. E. Mitchell & K. W. Tu (1979) Distribution, retention, and elimination of pyrene in rats after inhalation, Journal of Toxicology and Environmental Health, 5:6, 1171-1179, DOI: 10.1080/15287397909529822 To link to this article: http://dx.doi.org/10.1080/15287397909529822
Published online: 20 Oct 2009.
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Date: 07 November 2015, At: 19:43
DISTRIBUTION, RETENTION, AND ELIMINATION OF PYRENE IN RATS AFTER INHALATION C. E. Mitchell, K. W. Tu
Downloaded by [Universite Laval] at 19:43 07 November 2015
Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, Albuquerque, New Mexico
Pyrene was measured in tissues of Fischer 344 rats at various times after inhalation of pyrene aerosols (500 µg/l; mass median diameter, 0.3-0.8 µm) for 7 h. Significant quantities of pyrene were found in nasal turbinates, trachea, lungs, kidney, and liver immediately after exposure. Clearance from the respiratory tract was rapid; concentrations in the trachea and lungs 48 h after exposure were 20 and 5% of the concentrations present ½ h after exposure. Pyrene also cleared from liver and kidney at a relatively rapid rate; concentrations in these tissues 48 h after exposure were approximately 10% of those ½ h after exposure. Concentrations in the gastrointestinal tract 24 h after exposure were 4 times those found ½ h after exposure. Pyrene cleared from the gastrointestinal tract approximately 4 d after exposure. Thus, Inhaled pyrene is rapidly cleared from the respiratory tract by mucociliary action from the trachea and bronchi and by translocation from the respiratory tract to the liver and kidney; it is eliminated primarily through the gastrointestinal tract.
INTRODUCTION Although there have been relatively few studies of the chemical and physical characteristics of polycyclic aromatic hydrocarbon (PAH) aerosols, epidemiologic data suggest that inhalation, with deposition of PAH aerosols in the respiratory tract, is a major route of exposure [Committee on Biological Effects of Atmospheric Pollutants (CBEAP), 1972]. Pyrene is a major PAH in air, cigarette smoke, gasoline and diesel exhaust, and tars and ash from coal conversion technologies (Environmental Protection Agency, 1975; Mariich and Lenkevich, 1973; Diehl et al., 1967). Higher concentrations of PAH have been found in urban atmospheres and near industrial operations using fossil fuel (CBEAP, 1972). The higher incidences of urban and industry-related lung cancer suggest that inhalation of PAH may be associated with an increased frequency of cancer (CBEAP, 1972). We acknowledge the excellent technical assistance of Mr. Rafael Harpaz. Research was supported in part by the Environmental Protection Agency through Interagency Agreement EPA-IAG-D5-E61 under Department of Energy contract EY-76-C-04-1013 and in part by the Department of Energy under contract EY-76-C-04-1013. It was conducted in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. Requests for reprints should be sent to C. E. Mitchell, Inhalation Toxicology Research Institute, P.O. Box 5890, Albuquerque, New Mexico 87115. 1171 Journal of Toxicology and Environmental Health, 5:1171-1179, 1979 Copyright © 1979 by Hemisphere Publishing Corporation 0098-4108/79/051171-09$Z25
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Although pyrene is not considered a potent carcinogen, it may be a cofactor in carcinogenesis (Weinstein and Troll, 1977). Studies with laboratory animals indicate that initiation and development of lung cancer can be related to PAH in the lung (Stenback, 1974). Absorption, distribution, and clearance of inhaled PAH may depend on the form of the material in the exposure environment (Chang, 1943). In the present study, we measured clearance of pyrene during the first 4 d after inhalation of pyrene in particulate form, the form in which it primarily exists in the atmosphere (CBEAP, 1972).
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MATERIAL AND METHODS An aerosol generator was designed to produce pyrene aerosols at high, constant mass concentrations for several hours. This system (Fig. 1) consisted of a heating column, a chamber, and a Pyrex sample boat. N2 flowed through the tube of the boat as a driving gas. The boat was 10 cm long, 2 cm in diameter, and had a circular opening 1.5 cm in diameter. Approximately 6 g reagent grade crystalline pyrene was placed in the sample boat and heated to 250°C. The flow rate of the driving gas (N 2 ) through the tube of the boat was 0.4 l/min and that of the vapor carrier gas (N 2 ) was 3 l/min. Warm air (17 l/min) flowed through the chamber. The input air mixed with the outcoming vapor from the heating column in region R, where the vapor condensed to form aerosols, which were then diluted to the desired concentrations. The dilution air also provided a sheath to minimize wall losses. Thermal diffusion of vapor in the condensation section was minimized by reducing the temperature gradient in this area. The aerosol entered an animal exposure chamber, where it was uniformly mixed with dilution air. Uniform mixing was confirmed by visibly viewing the aerosol with a fluorescent lamp (366 nm wavelength) and by gravimetric analysis of filter samples obtained at different positions within the chamber. Infrared absorption spectra, fluorescence spectra, and gas chromatographic analysis of the aerosol filter samples showed that pyrene was thermostable. The particle size distribution measured by a laser active scattering aerosol spectrometer (Particle Measuring System, Inc., Boulder, Colo.) is shown in Fig. 2. The count median diameter (CMD) was 0.2-0.5 /urn, and the geometric standard deviation was 1.4-1.6. The
To
p Chamber
FIGURE 1. Diagram of aerosol generation system: G, aerosol generator; M, mixing and diluting chamber; T, thermometer; B, sample boat; and R, region of aerosol formation. A hollow tube at one end of the sample boat was used to introduce N2 as a driving gas for the vapor.
1173
PYRENE, P0LYCYCL1C AROMATIC HYDROCARBONS
MMD = 0.45 ^m (calculated) = 1.4
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i
-
o
FRACTION (Percent)
60
_
i
/t\
i
i
i
i i
11
1
or LU CD
|
20
n
'1
10 "
10"
PARTICLE DIAMETER,/xm
FIGURE 2. Particle size distribution of pyrene aerosols. Pyrene aerosols were produced at 250°C with a flow rate of 3.4 l/min through the heating column. The particle size distribution was measured by a laser active scattering aerosol spectrometer.
calculated mass median diameter (MMD) was 0.3-0.8 jum. Electron microscopy of pyrene aerosols showed that the particles were irregular in shape, as shown previously (Tu and Kanapilly, 1979). Groups of 8 male Fischer 344 rats (COB CD F/Crl BR), 8-10 wk old, were placed in wire restrainers in a doubly contained inhalation exposure chamber. The restrainers allowed some movement but there was no significant grooming during the exposure. The mass concentration of pyrene inside the restrainer was 350 or 500 ytg per liter of air as measured by filter sampling. Particle number concentration was 1 X 10 7 to 3 X 10 7 cm" 3 inside the restrainer. Animals were exposed for 60 min. Immediately after exposure they were removed from the exposure chamber to an adjacent housing facility. Free access to food (Lab-Blox, Allied Mills, Chicago, III.) and water was provided throughout the experiments except during exposure in the inhalation chamber. Animals were sacrificed by CO 2 asphyxiation at various times after exposure. Table 1 shows the numbers of animals and the times when animals exposed to pyrene were sacrificed. After sacrifice,
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C. E.MITCHELL AND K.W.TU
TABLE 1. Experimental Studies"
Design
for
Pyrene
Inhalation
Exposure group
No. of animals
Sacrifice time after inhalation
A B
8 4 4 4 4
30 min 30 min 1d 2d 4d
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J Male Fischer 344 rats, 8-10 wk old, were exposed to 350-500 Mg pyrene per liter of air.
the animal pelts were lightly swabbed with ethyl alcohol to remove external contamination. This procedure was efficient for removing external pyrene aerosols in the concentrations used in this study. Turbinates, trachea, lungs, liver, kidney, skeletal muscle (distal biceps femoris), pelt, and gastointestinal (Gl) tract were then carefully removed and placed in preweighed vials. Dissecting instruments were cleaned between tissue samples to avoid transfer of pyrene. Weights of the tissue samples were obtained and each sample was homogenized with hexane and shaken for \ h. The hexane extract was washed with 25% dimethyl sulfoxide (DMSO) in H 2 O (v/v), which was very effective for removing interfering substances. Approximately 92% of the pyrene was recovered in the hexane layer after this procedure. The sensitivity was 0.01 jug per milliliter of solvent extract. Pyrene in the hexane extract was quantitated spectrofluorometrically at excitation and emission wavelengths of 323 and 387 nm, respectively. The pyrene-equivalent fluorescence (observed at 323 and 387 nm) was expressed as micrograms of pyrene per gram of wet tissue. To measure Gl absorption of pyrene, two male rats were anesthetized by inhalation of halothane and nitrous oxide in O 2 . A fine suspension of pyrene in a gelatin-saline solution was obtained by adding gelatin-saline to an acetone solution of pyrene, followed by evaporation of the acetone with N 2 . A gavage tube was inserted in the stomach and 50 /xg pyrene suspended in gelatin-saline was administered. Animals were sacrificed 24 h after the administration. The Gl tract, lungs, live, kidneys, and trachea were removed and analyzed for pyrene. To assess absorption of pyrene deposited on the pelt, 2 male rats 8-10 wk old were used. Pyrene (20 mg) in acetone was added to the dorsal pelt of the rats in a 2 X 3 in area. The acetone (microliter quantities) dried as quickly as it was applied to the pelt. This was facilitated with a warm air stream as the pyrene was applied to the pelt to minimize transfer of pyrene through the skin with acetone. The objective was to deposit crystalline pyrene on the pelt. The animals were sacrificed 24 h later and the same tissues used in the gavage study were analyzed for pyreneequivalent fluorescence.
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TABLE 2. Pyrene-Equivalent Fluorescence in Various Organs and Tissues of Rats \ h Following Inhalation of Pyrene Aerosols 0
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Total amount in tissue Organ or tissue
(Mg)
Nasal turbinate Trachea Lung Kidney Liver Stomach Intestine Muscle
2.5 ± 0.64 6 3.1 ± 1.3 16.0± 5.5 16.3 ± 4 . 9 42.2+15.0 73.4 + 25.9 55.6 + 26.9 123.0±43.0 c
Amount (jug/g wet weight) 15.5 27.9 15.0 10.6 6.1 28.9 5.2 1.7
No. of animals 8 8 8 8 7 8 8 8
±4.3* ± 8.1 + 5.0 + 3.2 ± 2.3 ±11.7 ± 2.5 + 0.6
°Male Fischer 344 rats were exposed to a pyrene aerosol concentration of 500 Mg/1 for 1 h. 6 Mean + SD. c Calculated by assuming that 40% of the body weight is muscle mass.
RESULTS The pyrene-equivalent fluorescence in tissues of rats sacrificed \ h after 1 h exposure to the pyrene aerosols (500 /ug per liter of air) is shown in Table 2. The highest pyrene concentrations were present in the upper respiratory tract tissues. The trachea contained the highest concentration, 27 Mg/g- Lungs and nasal turbinates contained approximately 15 jug/g. Smaller amounts were present in liver, kidney, and carcass. Table 3 shows the distribution of pyrene in various tissues at different times after inahlation of pyrene aerosols for 1 h (350 Mg/I). Pyrene was cleared very rapidly from the majority of tissues analyzed. At 1 d after exposure the lungs contained 69% of the amount present \ h after TABLE 3. Distribution and Rate of Elimination of Pyrene-Equivalent Inhalation0
Fluorescence in Rats after
Amount (/Jg/g wet weight) Organ or tissue Nasal turbinate Trachea Lung Kidney Liver Stomach Intestine Muscle
O6 8.82 18.01 10.18 8.15 5.40 17.98 3.48 2.32
± 3.25C ± 4.35 ± 3.02 ± 3.05 ± 1.60 ±6.58 ± 2.01 ± 1.15
1d 7.26 + 3.99 15.17 ±5.60 6.62 ± 2.53 3.06 ± 1.06 0.84 ± 0.20 5.76 + 2.10 15.80+ 1.27 5.23 ± 2.26
4d
2d 0.73 2.28 0.61 1.14 0.35 2.69 11.70 0.10
± ± ± ± ± ± ± ±
0.30 1.16 0.17 0.65 0.10 0.90 5.20 0.04
0.65 0.23 0.09 0.81 0.10 1.38 1.28 0.12
°Male Fischer 344 rats were exposed to a pyrene aerosol concentration of 350 Mg/I for 1 h. Zero time equals \ h after exposure. c Mean ± SD for four animals at each sacrifice time.
+ ± + + + + + +
0.25 0.10 0.03 0.12 0.05 0.68 0.13 0.03
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C. E.MITCHELL AND K.W.TU
exposure. This decreased to 5 and 2% of the initial burdens at 2 and 4 d, respectively. The pyrene concentration in the trachea 1 d after exposure was 80% of the initial burden but cleared very rapidly afterward. Pyrene was rapidly cleared from liver and kidney, reaching approximately 17% of the initial burdens 2 d after exposure. The Gl tract contained the highest concentration of pyrene in the tissues assayed 1 d after exposure; however, clearance from this organ was very rapid. Because of the possibility that pyrene was transferred from the gut and pelt to the other tissues by way of the systemic circulation, Gl absorption was also studied (Table 4). No significant increase in pyreneequivalent fluorescence was found in any of the tissues examined 24 h after pyrene administration by gavage. The Gl tract contained approximately half the material that was administered; the remainder was apparently absorbed by the blood and was below detection levels in the various tissues and/or was excreted. When pyrene was administered on the pelt, a significant increase in pyrene-equivalent fluorescence was found only in the Gl tract 24 h later. DISCUSSION This study showed that inhaled pyrene clears rapidly from the respiratory tract of rats and is distributed to other tissues during its removal from the body. The relatively high levels found in the upper respiratory tract indicated that pyrene aerosols in the size range 0.3-0.8
TABLE 4. Pyrene-Equivalent Fluorescence in Tissues of Rats Following Pyrene Administration by Gavage or Application to the Pelt" Amount in tissue Tissue
Pelt application
Gavage
Lung Kidney Liver Trachea Gl tract
ND 6
ND b ND ND ND
0.56 ± 0.20 1.50 + 0.68 0.17 ±0.05 287.00 + 120.00
24 ± 10
a Pyrene (50 pg) was administered by gavage or (20 mg) applied to the pelt. Animals were sacrificed 24 h later and tissues were analyzed for pyrene-equivalent fluorescence. Values are the means ± SD (where appropriate) for three animals and are corrected for nonspecific fluorescence in tissues of control animals. ''Pyrene-equivalent fluorescence above control values not detectable (ND).
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1177
(MMD) are deposited in this region. The presence of pyrene fluorescence in the liver and kidney \ h after a 1-h exposure to pyrene by inhalation indicated rapid absorption and distribution of this material from its initial site of deposition. The rate of clearance from the lung was almost linear for the first 2 d, at which time only 5% of the initial quantity remained. Although the stomach initially contained a significant amount of pyrene, caused by swallowing pyrene during grooming of the pelt and mucociliary clearance from the upper respiratory tract into the esophagus, the rate of clearance from the stomach was rapid; the concentration was 15% of the initial value 2 d after exposure. Turnover of pyrene in the liver and kidney was also rapid. A clearance half-time of approximately 15-18 h was found for the first day following exposure. Because of the possibility of translocation of pyrene from the Gl tract and pelt, clearance of pyrene from these tissues was studied after administration by gavage and application to the pelt. The absence of pyrene in tissues other than the Gl tract in these studies indicated that pyrene levels measured in other tissues after inhalation were not likely to be significantly derived from the Gl tract or the pelt. The large increase in the concentration of pyrene in the Gl tract after its application to the dorsal pelt probably resulted from ingestion of pyrene during grooming. Additional pyrene may have reached the Gl tract by absorption through the skin and excretion via the bile duct. Elimination of internally deposited PAH via the Gl tract has been reported (Chang, 1943; Kotin et al., 1959). Other studies suggested that use of lipid solutions and oils as vehicles may affect Gl absorption, localization, and elimination of PAH (Kotin et al., 1959; Daniel et al., 1967). Our studies indicate that pyrene in an aqueous suspension is poorly absorbed from the Gl tract and are consistent with studies by Chang (1943). Although there have been reports of the clearance of PAH from the respiratory tract after intratracheal instillation, relatively few studies of PAH clearance after inhalation have been reported. Intratracheal instillation results in an uneven distribution of material in the lung, and this material may not be cleared in the same way as inhaled material, which is initially more evenly distributed (Brain et al., 1976). The vehicle for PAH instillation is also likely to have a pronounced effect on PAH absorption and distribution (Kotin et al., 1959; Ho, 1975). Studies of PAH clearance after intratracheal instillation indicated that PAH was cleared from the lung and eliminated from the host primarily through the feces within a few days (Kotin et al., 1959; Creasia et al., 1976; Pylev et al., 1969; Saffiotti et al., 1967). The data in the present study are consistent with these later studies. However, our data showed somewhat longer residence times, particularly in the lung. This may have been related to the specific PAH investigated, the dose, and/or variations in metabolism among animal species. We measured pyrene by a spectrofluorometric procedure and expressed
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the results as pyrene-equivalent fluorescence. Some previous reports indicated that pyrene is metabolized to polar compounds (Harper, 1957, 1958), although there have been relatively few studies of the enzyme mechanisms for these transformations. The major pyrene metabolites isolated and characterized to date are 3-hydroxypyrene, 3:10- and 3:8-quinones, and 3:10- and 3:8-dihydroxypyrenes (Harper, 1957). Considerable data indicate that PAH metabolites have different fluorescence spectra and solubilities. The ratio of unaltered to altered PAH varied among organs and body fluids, being very high in the lungs and very low in the urine (Ho, 1975). Because of the differences in solubility and fluorescence between metabolites, it is not known what percentages of the metabolites have been measured. Inspection of the fluorescence spectra of the major pyrene metabolites indicated that they would be overlapped by the excitation and emission settings for pyrene (Harper, 1975). Thus, significant quantities of altered and unaltered materials were probably measured. In addition, the elimination patterns we observed are consistent with the results of studies using both radiotracer and fluorescence analysis to follow the fate of PAH (Kotin et al., 1959; Rigdon and Neal, 1963; Creasia et al., 1976; Pylev et al., 1969; Saffiotti et al., 1967). In conclusion, inhaled pyrene is deposited throughout the respiratory system and rapidly translocated to other tissues. Pyrene elimination occurs primarily via the liver and bile duct to the feces. Low concentrations of pyrene are found in tissues after gavage and after application of pyrene to the dorsal pelt of animals. This suggests that pyrene, which may be ingested during grooming or absorbed through the skin, is rapidly eliminated through the Gl tract. REFERENCES Brain, J. D., Knudson, D. E., Sorokin, S. P., and Davis, M. A. 1976. Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation. Environ. Res. 11:13-33. Chang, L. H. 1943. The fecal excretion of polycyclic hydrocarbons following their administration to the rat. J. Biol. Chem. 151:93-99. Committee on Biological Effects of Atmospheric Pollutants, National Research Council. 1972. Paniculate Polycyclic Organic Matter, pp. 1-361. Washington, D.C.: National Academy of Sciences. Creasia, D. A., Poggenburg, J. K., Jr., and Nettesheim, P. 1976. Elution of benzo(a)pyrene from carbon particles in the respiratory tract of mice. J. Toxicol. Environ. Health 1:967-975. Daniel, P. M., Pratt, O. E., and Prichard, M. M. L. 1967. Metabolism of labelled carcinogenic hydrocarbons in rats. Nature (Lond.) 215:1142-1146. Diehl, E. K., du Breuil, F., and Glenn, R. A. 1967. Polynuclear hydrocarbon emission from coal-fired installations. J. Eng. Power 89:276-282. Environmental Protection Agency. 1975. Scientific and technical assessment report on particulate polycyclic organic matter (PPOM). EPA Publ. EPA-600/6-75-001. Washington, D.C.: Environmental Protection Agency. Harper, K. H. 1957. The metabolism of pyrene. Br. J. Cancer 11:499-507. Harper, K. H. 1958. The intermediary metabolism of pyrene. B. J. Cancer 12:116-120. Ho, W. 1975. Distribution and turnover of benzo(a)pyrene instilled into mouse lungs. Proc. West.
Pharmacol. Soc. 18:302-305.
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Kotin, P., Falk, H. L., and Busser, R. 1959. Distribution, retention and elimination of 14 C-3,4benzpyrene after administration to mice and rats. J. Natl. Cancer Inst. 23:541-555. Mariich, L. I. and Lenkevich, Zh. K. 1973. Capillary chromatography as a method for the rapid determination of the main components in coal tar and coal tar fractions. Zh. Anal. Khim. 28(6):1193-1198. Pylev, L. N., Roe, F. J. C , and Warwick, G. P. 1969. Elimination of radioactivity after intratracheal instillation of tritiated 3,4-benzopyrene in hamsters. Br. J. Cancer 23:103-115. Rigdon, R. H. and Neal, J. 1963. Absorption and excretion of benzpyrene; observations in the duck, chicken, mouse and dog. Tex. Rep. Biol. Med. 2 1 : 2 4 7 - 2 6 1 . Saffiotti, U., Montesano, R., and Tompkins, N. 1967. Benzo(a)pyrene retention in hamster lungs: Studies o n particle size and on total dust load. Proc. Am. Assoc. Cancer Res. 8:57. Stenback, F. 1974. Morphogenesis of experimental lung tumors in hamsters: The effects of carrier dust. In Experimental Lung Cancer, Carcinogenesis and Bioassays, eds. E. Karbe and J. F. Park, pp. 161-172. New York: Springer-Verlag. Tu, K. W. and Kanapilly, G. K. 1979. Generation and characterization of condensation aerosols of vanadium pentoxide and pyrene. Am. Ind. Hyg. Assoc. J. 40:763-769. Weinstein, I. B. and Troll, W. 1977. National Cancer Institute Workshop on tumor promotion and cofactors in carcinogenesis. Cancer Res. 37:3461-3463. Received April 27, 1979 Accepted July 29, 1979
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Distribution, retention, and elimination of pyrene in rats after inhalation C. E. Mitchell & K. W. Tu To cite this article: C. E. Mitchell & K. W. Tu (1979) Distribution, retention, and elimination of pyrene in rats after inhalation, Journal of Toxicology and Environmental Health, 5:6, 1171-1179, DOI: 10.1080/15287397909529822 To link to this article: http://dx.doi.org/10.1080/15287397909529822
Published online: 20 Oct 2009.
Submit your article to this journal
Article views: 4
View related articles
Citing articles: 11 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [Universite Laval]
Date: 07 November 2015, At: 19:43
DISTRIBUTION, RETENTION, AND ELIMINATION OF PYRENE IN RATS AFTER INHALATION C. E. Mitchell, K. W. Tu
Downloaded by [Universite Laval] at 19:43 07 November 2015
Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, Albuquerque, New Mexico
Pyrene was measured in tissues of Fischer 344 rats at various times after inhalation of pyrene aerosols (500 µg/l; mass median diameter, 0.3-0.8 µm) for 7 h. Significant quantities of pyrene were found in nasal turbinates, trachea, lungs, kidney, and liver immediately after exposure. Clearance from the respiratory tract was rapid; concentrations in the trachea and lungs 48 h after exposure were 20 and 5% of the concentrations present ½ h after exposure. Pyrene also cleared from liver and kidney at a relatively rapid rate; concentrations in these tissues 48 h after exposure were approximately 10% of those ½ h after exposure. Concentrations in the gastrointestinal tract 24 h after exposure were 4 times those found ½ h after exposure. Pyrene cleared from the gastrointestinal tract approximately 4 d after exposure. Thus, Inhaled pyrene is rapidly cleared from the respiratory tract by mucociliary action from the trachea and bronchi and by translocation from the respiratory tract to the liver and kidney; it is eliminated primarily through the gastrointestinal tract.
INTRODUCTION Although there have been relatively few studies of the chemical and physical characteristics of polycyclic aromatic hydrocarbon (PAH) aerosols, epidemiologic data suggest that inhalation, with deposition of PAH aerosols in the respiratory tract, is a major route of exposure [Committee on Biological Effects of Atmospheric Pollutants (CBEAP), 1972]. Pyrene is a major PAH in air, cigarette smoke, gasoline and diesel exhaust, and tars and ash from coal conversion technologies (Environmental Protection Agency, 1975; Mariich and Lenkevich, 1973; Diehl et al., 1967). Higher concentrations of PAH have been found in urban atmospheres and near industrial operations using fossil fuel (CBEAP, 1972). The higher incidences of urban and industry-related lung cancer suggest that inhalation of PAH may be associated with an increased frequency of cancer (CBEAP, 1972). We acknowledge the excellent technical assistance of Mr. Rafael Harpaz. Research was supported in part by the Environmental Protection Agency through Interagency Agreement EPA-IAG-D5-E61 under Department of Energy contract EY-76-C-04-1013 and in part by the Department of Energy under contract EY-76-C-04-1013. It was conducted in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. Requests for reprints should be sent to C. E. Mitchell, Inhalation Toxicology Research Institute, P.O. Box 5890, Albuquerque, New Mexico 87115. 1171 Journal of Toxicology and Environmental Health, 5:1171-1179, 1979 Copyright © 1979 by Hemisphere Publishing Corporation 0098-4108/79/051171-09$Z25
1172
C. E. MITCHELL AND K. W. TU
Although pyrene is not considered a potent carcinogen, it may be a cofactor in carcinogenesis (Weinstein and Troll, 1977). Studies with laboratory animals indicate that initiation and development of lung cancer can be related to PAH in the lung (Stenback, 1974). Absorption, distribution, and clearance of inhaled PAH may depend on the form of the material in the exposure environment (Chang, 1943). In the present study, we measured clearance of pyrene during the first 4 d after inhalation of pyrene in particulate form, the form in which it primarily exists in the atmosphere (CBEAP, 1972).
Downloaded by [Universite Laval] at 19:43 07 November 2015
MATERIAL AND METHODS An aerosol generator was designed to produce pyrene aerosols at high, constant mass concentrations for several hours. This system (Fig. 1) consisted of a heating column, a chamber, and a Pyrex sample boat. N2 flowed through the tube of the boat as a driving gas. The boat was 10 cm long, 2 cm in diameter, and had a circular opening 1.5 cm in diameter. Approximately 6 g reagent grade crystalline pyrene was placed in the sample boat and heated to 250°C. The flow rate of the driving gas (N 2 ) through the tube of the boat was 0.4 l/min and that of the vapor carrier gas (N 2 ) was 3 l/min. Warm air (17 l/min) flowed through the chamber. The input air mixed with the outcoming vapor from the heating column in region R, where the vapor condensed to form aerosols, which were then diluted to the desired concentrations. The dilution air also provided a sheath to minimize wall losses. Thermal diffusion of vapor in the condensation section was minimized by reducing the temperature gradient in this area. The aerosol entered an animal exposure chamber, where it was uniformly mixed with dilution air. Uniform mixing was confirmed by visibly viewing the aerosol with a fluorescent lamp (366 nm wavelength) and by gravimetric analysis of filter samples obtained at different positions within the chamber. Infrared absorption spectra, fluorescence spectra, and gas chromatographic analysis of the aerosol filter samples showed that pyrene was thermostable. The particle size distribution measured by a laser active scattering aerosol spectrometer (Particle Measuring System, Inc., Boulder, Colo.) is shown in Fig. 2. The count median diameter (CMD) was 0.2-0.5 /urn, and the geometric standard deviation was 1.4-1.6. The
To
p Chamber
FIGURE 1. Diagram of aerosol generation system: G, aerosol generator; M, mixing and diluting chamber; T, thermometer; B, sample boat; and R, region of aerosol formation. A hollow tube at one end of the sample boat was used to introduce N2 as a driving gas for the vapor.
1173
PYRENE, P0LYCYCL1C AROMATIC HYDROCARBONS
MMD = 0.45 ^m (calculated) = 1.4
Downloaded by [Universite Laval] at 19:43 07 November 2015
i
-
o
FRACTION (Percent)
60
_
i
/t\
i
i
i
i i
11
1
or LU CD
|
20
n
'1
10 "
10"
PARTICLE DIAMETER,/xm
FIGURE 2. Particle size distribution of pyrene aerosols. Pyrene aerosols were produced at 250°C with a flow rate of 3.4 l/min through the heating column. The particle size distribution was measured by a laser active scattering aerosol spectrometer.
calculated mass median diameter (MMD) was 0.3-0.8 jum. Electron microscopy of pyrene aerosols showed that the particles were irregular in shape, as shown previously (Tu and Kanapilly, 1979). Groups of 8 male Fischer 344 rats (COB CD F/Crl BR), 8-10 wk old, were placed in wire restrainers in a doubly contained inhalation exposure chamber. The restrainers allowed some movement but there was no significant grooming during the exposure. The mass concentration of pyrene inside the restrainer was 350 or 500 ytg per liter of air as measured by filter sampling. Particle number concentration was 1 X 10 7 to 3 X 10 7 cm" 3 inside the restrainer. Animals were exposed for 60 min. Immediately after exposure they were removed from the exposure chamber to an adjacent housing facility. Free access to food (Lab-Blox, Allied Mills, Chicago, III.) and water was provided throughout the experiments except during exposure in the inhalation chamber. Animals were sacrificed by CO 2 asphyxiation at various times after exposure. Table 1 shows the numbers of animals and the times when animals exposed to pyrene were sacrificed. After sacrifice,
1174
C. E.MITCHELL AND K.W.TU
TABLE 1. Experimental Studies"
Design
for
Pyrene
Inhalation
Exposure group
No. of animals
Sacrifice time after inhalation
A B
8 4 4 4 4
30 min 30 min 1d 2d 4d
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J Male Fischer 344 rats, 8-10 wk old, were exposed to 350-500 Mg pyrene per liter of air.
the animal pelts were lightly swabbed with ethyl alcohol to remove external contamination. This procedure was efficient for removing external pyrene aerosols in the concentrations used in this study. Turbinates, trachea, lungs, liver, kidney, skeletal muscle (distal biceps femoris), pelt, and gastointestinal (Gl) tract were then carefully removed and placed in preweighed vials. Dissecting instruments were cleaned between tissue samples to avoid transfer of pyrene. Weights of the tissue samples were obtained and each sample was homogenized with hexane and shaken for \ h. The hexane extract was washed with 25% dimethyl sulfoxide (DMSO) in H 2 O (v/v), which was very effective for removing interfering substances. Approximately 92% of the pyrene was recovered in the hexane layer after this procedure. The sensitivity was 0.01 jug per milliliter of solvent extract. Pyrene in the hexane extract was quantitated spectrofluorometrically at excitation and emission wavelengths of 323 and 387 nm, respectively. The pyrene-equivalent fluorescence (observed at 323 and 387 nm) was expressed as micrograms of pyrene per gram of wet tissue. To measure Gl absorption of pyrene, two male rats were anesthetized by inhalation of halothane and nitrous oxide in O 2 . A fine suspension of pyrene in a gelatin-saline solution was obtained by adding gelatin-saline to an acetone solution of pyrene, followed by evaporation of the acetone with N 2 . A gavage tube was inserted in the stomach and 50 /xg pyrene suspended in gelatin-saline was administered. Animals were sacrificed 24 h after the administration. The Gl tract, lungs, live, kidneys, and trachea were removed and analyzed for pyrene. To assess absorption of pyrene deposited on the pelt, 2 male rats 8-10 wk old were used. Pyrene (20 mg) in acetone was added to the dorsal pelt of the rats in a 2 X 3 in area. The acetone (microliter quantities) dried as quickly as it was applied to the pelt. This was facilitated with a warm air stream as the pyrene was applied to the pelt to minimize transfer of pyrene through the skin with acetone. The objective was to deposit crystalline pyrene on the pelt. The animals were sacrificed 24 h later and the same tissues used in the gavage study were analyzed for pyreneequivalent fluorescence.
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TABLE 2. Pyrene-Equivalent Fluorescence in Various Organs and Tissues of Rats \ h Following Inhalation of Pyrene Aerosols 0
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Total amount in tissue Organ or tissue
(Mg)
Nasal turbinate Trachea Lung Kidney Liver Stomach Intestine Muscle
2.5 ± 0.64 6 3.1 ± 1.3 16.0± 5.5 16.3 ± 4 . 9 42.2+15.0 73.4 + 25.9 55.6 + 26.9 123.0±43.0 c
Amount (jug/g wet weight) 15.5 27.9 15.0 10.6 6.1 28.9 5.2 1.7
No. of animals 8 8 8 8 7 8 8 8
±4.3* ± 8.1 + 5.0 + 3.2 ± 2.3 ±11.7 ± 2.5 + 0.6
°Male Fischer 344 rats were exposed to a pyrene aerosol concentration of 500 Mg/1 for 1 h. 6 Mean + SD. c Calculated by assuming that 40% of the body weight is muscle mass.
RESULTS The pyrene-equivalent fluorescence in tissues of rats sacrificed \ h after 1 h exposure to the pyrene aerosols (500 /ug per liter of air) is shown in Table 2. The highest pyrene concentrations were present in the upper respiratory tract tissues. The trachea contained the highest concentration, 27 Mg/g- Lungs and nasal turbinates contained approximately 15 jug/g. Smaller amounts were present in liver, kidney, and carcass. Table 3 shows the distribution of pyrene in various tissues at different times after inahlation of pyrene aerosols for 1 h (350 Mg/I). Pyrene was cleared very rapidly from the majority of tissues analyzed. At 1 d after exposure the lungs contained 69% of the amount present \ h after TABLE 3. Distribution and Rate of Elimination of Pyrene-Equivalent Inhalation0
Fluorescence in Rats after
Amount (/Jg/g wet weight) Organ or tissue Nasal turbinate Trachea Lung Kidney Liver Stomach Intestine Muscle
O6 8.82 18.01 10.18 8.15 5.40 17.98 3.48 2.32
± 3.25C ± 4.35 ± 3.02 ± 3.05 ± 1.60 ±6.58 ± 2.01 ± 1.15
1d 7.26 + 3.99 15.17 ±5.60 6.62 ± 2.53 3.06 ± 1.06 0.84 ± 0.20 5.76 + 2.10 15.80+ 1.27 5.23 ± 2.26
4d
2d 0.73 2.28 0.61 1.14 0.35 2.69 11.70 0.10
± ± ± ± ± ± ± ±
0.30 1.16 0.17 0.65 0.10 0.90 5.20 0.04
0.65 0.23 0.09 0.81 0.10 1.38 1.28 0.12
°Male Fischer 344 rats were exposed to a pyrene aerosol concentration of 350 Mg/I for 1 h. Zero time equals \ h after exposure. c Mean ± SD for four animals at each sacrifice time.
+ ± + + + + + +
0.25 0.10 0.03 0.12 0.05 0.68 0.13 0.03
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exposure. This decreased to 5 and 2% of the initial burdens at 2 and 4 d, respectively. The pyrene concentration in the trachea 1 d after exposure was 80% of the initial burden but cleared very rapidly afterward. Pyrene was rapidly cleared from liver and kidney, reaching approximately 17% of the initial burdens 2 d after exposure. The Gl tract contained the highest concentration of pyrene in the tissues assayed 1 d after exposure; however, clearance from this organ was very rapid. Because of the possibility that pyrene was transferred from the gut and pelt to the other tissues by way of the systemic circulation, Gl absorption was also studied (Table 4). No significant increase in pyreneequivalent fluorescence was found in any of the tissues examined 24 h after pyrene administration by gavage. The Gl tract contained approximately half the material that was administered; the remainder was apparently absorbed by the blood and was below detection levels in the various tissues and/or was excreted. When pyrene was administered on the pelt, a significant increase in pyrene-equivalent fluorescence was found only in the Gl tract 24 h later. DISCUSSION This study showed that inhaled pyrene clears rapidly from the respiratory tract of rats and is distributed to other tissues during its removal from the body. The relatively high levels found in the upper respiratory tract indicated that pyrene aerosols in the size range 0.3-0.8
TABLE 4. Pyrene-Equivalent Fluorescence in Tissues of Rats Following Pyrene Administration by Gavage or Application to the Pelt" Amount in tissue Tissue
Pelt application
Gavage
Lung Kidney Liver Trachea Gl tract
ND 6
ND b ND ND ND
0.56 ± 0.20 1.50 + 0.68 0.17 ±0.05 287.00 + 120.00
24 ± 10
a Pyrene (50 pg) was administered by gavage or (20 mg) applied to the pelt. Animals were sacrificed 24 h later and tissues were analyzed for pyrene-equivalent fluorescence. Values are the means ± SD (where appropriate) for three animals and are corrected for nonspecific fluorescence in tissues of control animals. ''Pyrene-equivalent fluorescence above control values not detectable (ND).
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(MMD) are deposited in this region. The presence of pyrene fluorescence in the liver and kidney \ h after a 1-h exposure to pyrene by inhalation indicated rapid absorption and distribution of this material from its initial site of deposition. The rate of clearance from the lung was almost linear for the first 2 d, at which time only 5% of the initial quantity remained. Although the stomach initially contained a significant amount of pyrene, caused by swallowing pyrene during grooming of the pelt and mucociliary clearance from the upper respiratory tract into the esophagus, the rate of clearance from the stomach was rapid; the concentration was 15% of the initial value 2 d after exposure. Turnover of pyrene in the liver and kidney was also rapid. A clearance half-time of approximately 15-18 h was found for the first day following exposure. Because of the possibility of translocation of pyrene from the Gl tract and pelt, clearance of pyrene from these tissues was studied after administration by gavage and application to the pelt. The absence of pyrene in tissues other than the Gl tract in these studies indicated that pyrene levels measured in other tissues after inhalation were not likely to be significantly derived from the Gl tract or the pelt. The large increase in the concentration of pyrene in the Gl tract after its application to the dorsal pelt probably resulted from ingestion of pyrene during grooming. Additional pyrene may have reached the Gl tract by absorption through the skin and excretion via the bile duct. Elimination of internally deposited PAH via the Gl tract has been reported (Chang, 1943; Kotin et al., 1959). Other studies suggested that use of lipid solutions and oils as vehicles may affect Gl absorption, localization, and elimination of PAH (Kotin et al., 1959; Daniel et al., 1967). Our studies indicate that pyrene in an aqueous suspension is poorly absorbed from the Gl tract and are consistent with studies by Chang (1943). Although there have been reports of the clearance of PAH from the respiratory tract after intratracheal instillation, relatively few studies of PAH clearance after inhalation have been reported. Intratracheal instillation results in an uneven distribution of material in the lung, and this material may not be cleared in the same way as inhaled material, which is initially more evenly distributed (Brain et al., 1976). The vehicle for PAH instillation is also likely to have a pronounced effect on PAH absorption and distribution (Kotin et al., 1959; Ho, 1975). Studies of PAH clearance after intratracheal instillation indicated that PAH was cleared from the lung and eliminated from the host primarily through the feces within a few days (Kotin et al., 1959; Creasia et al., 1976; Pylev et al., 1969; Saffiotti et al., 1967). The data in the present study are consistent with these later studies. However, our data showed somewhat longer residence times, particularly in the lung. This may have been related to the specific PAH investigated, the dose, and/or variations in metabolism among animal species. We measured pyrene by a spectrofluorometric procedure and expressed
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the results as pyrene-equivalent fluorescence. Some previous reports indicated that pyrene is metabolized to polar compounds (Harper, 1957, 1958), although there have been relatively few studies of the enzyme mechanisms for these transformations. The major pyrene metabolites isolated and characterized to date are 3-hydroxypyrene, 3:10- and 3:8-quinones, and 3:10- and 3:8-dihydroxypyrenes (Harper, 1957). Considerable data indicate that PAH metabolites have different fluorescence spectra and solubilities. The ratio of unaltered to altered PAH varied among organs and body fluids, being very high in the lungs and very low in the urine (Ho, 1975). Because of the differences in solubility and fluorescence between metabolites, it is not known what percentages of the metabolites have been measured. Inspection of the fluorescence spectra of the major pyrene metabolites indicated that they would be overlapped by the excitation and emission settings for pyrene (Harper, 1975). Thus, significant quantities of altered and unaltered materials were probably measured. In addition, the elimination patterns we observed are consistent with the results of studies using both radiotracer and fluorescence analysis to follow the fate of PAH (Kotin et al., 1959; Rigdon and Neal, 1963; Creasia et al., 1976; Pylev et al., 1969; Saffiotti et al., 1967). In conclusion, inhaled pyrene is deposited throughout the respiratory system and rapidly translocated to other tissues. Pyrene elimination occurs primarily via the liver and bile duct to the feces. Low concentrations of pyrene are found in tissues after gavage and after application of pyrene to the dorsal pelt of animals. This suggests that pyrene, which may be ingested during grooming or absorbed through the skin, is rapidly eliminated through the Gl tract. REFERENCES Brain, J. D., Knudson, D. E., Sorokin, S. P., and Davis, M. A. 1976. Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation. Environ. Res. 11:13-33. Chang, L. H. 1943. The fecal excretion of polycyclic hydrocarbons following their administration to the rat. J. Biol. Chem. 151:93-99. Committee on Biological Effects of Atmospheric Pollutants, National Research Council. 1972. Paniculate Polycyclic Organic Matter, pp. 1-361. Washington, D.C.: National Academy of Sciences. Creasia, D. A., Poggenburg, J. K., Jr., and Nettesheim, P. 1976. Elution of benzo(a)pyrene from carbon particles in the respiratory tract of mice. J. Toxicol. Environ. Health 1:967-975. Daniel, P. M., Pratt, O. E., and Prichard, M. M. L. 1967. Metabolism of labelled carcinogenic hydrocarbons in rats. Nature (Lond.) 215:1142-1146. Diehl, E. K., du Breuil, F., and Glenn, R. A. 1967. Polynuclear hydrocarbon emission from coal-fired installations. J. Eng. Power 89:276-282. Environmental Protection Agency. 1975. Scientific and technical assessment report on particulate polycyclic organic matter (PPOM). EPA Publ. EPA-600/6-75-001. Washington, D.C.: Environmental Protection Agency. Harper, K. H. 1957. The metabolism of pyrene. Br. J. Cancer 11:499-507. Harper, K. H. 1958. The intermediary metabolism of pyrene. B. J. Cancer 12:116-120. Ho, W. 1975. Distribution and turnover of benzo(a)pyrene instilled into mouse lungs. Proc. West.
Pharmacol. Soc. 18:302-305.
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Kotin, P., Falk, H. L., and Busser, R. 1959. Distribution, retention and elimination of 14 C-3,4benzpyrene after administration to mice and rats. J. Natl. Cancer Inst. 23:541-555. Mariich, L. I. and Lenkevich, Zh. K. 1973. Capillary chromatography as a method for the rapid determination of the main components in coal tar and coal tar fractions. Zh. Anal. Khim. 28(6):1193-1198. Pylev, L. N., Roe, F. J. C , and Warwick, G. P. 1969. Elimination of radioactivity after intratracheal instillation of tritiated 3,4-benzopyrene in hamsters. Br. J. Cancer 23:103-115. Rigdon, R. H. and Neal, J. 1963. Absorption and excretion of benzpyrene; observations in the duck, chicken, mouse and dog. Tex. Rep. Biol. Med. 2 1 : 2 4 7 - 2 6 1 . Saffiotti, U., Montesano, R., and Tompkins, N. 1967. Benzo(a)pyrene retention in hamster lungs: Studies o n particle size and on total dust load. Proc. Am. Assoc. Cancer Res. 8:57. Stenback, F. 1974. Morphogenesis of experimental lung tumors in hamsters: The effects of carrier dust. In Experimental Lung Cancer, Carcinogenesis and Bioassays, eds. E. Karbe and J. F. Park, pp. 161-172. New York: Springer-Verlag. Tu, K. W. and Kanapilly, G. K. 1979. Generation and characterization of condensation aerosols of vanadium pentoxide and pyrene. Am. Ind. Hyg. Assoc. J. 40:763-769. Weinstein, I. B. and Troll, W. 1977. National Cancer Institute Workshop on tumor promotion and cofactors in carcinogenesis. Cancer Res. 37:3461-3463. Received April 27, 1979 Accepted July 29, 1979
