Journal of Toxicology and Environmental Health
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Pharmacokinetics and bioavailability of pyrene in the rat J. R. Withey , F. C. P. Law & L. Endrenyi To cite this article: J. R. Withey , F. C. P. Law & L. Endrenyi (1991) Pharmacokinetics and bioavailability of pyrene in the rat, Journal of Toxicology and Environmental Health, 32:4, 429-447, DOI: 10.1080/15287399109531494 To link to this article: http://dx.doi.org/10.1080/15287399109531494
Published online: 20 Oct 2009.
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PHARMACOKINETICS AND BIOAVAILABILITY OF PYRENE IN THE RAT J. R. Withey Environmental Health Directorate, Ottawa, Ontario, Canada
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F. C. P. Law Simon Fraser University, Burnaby, British Columbia, Canada L. Endrenyi University of Toronto, Toronto, Ontario, Canada Groups of 6 male Wistar rats, of about 400 g body weight, were dosed with 14C-labeled pyrene, dissolved in an Emulphor/water solvent vehicle, at 5 different dose levels by the intravenous or oral routes. Appropriate mathematical models were fitted to blood concentration-time data for [14C]pyrene and pyrene per se and dose-trend analyses were carried out. Areas under these curves were used to assess the bioavailability of the orally administered doses. Tissue concentrations, measured at the termination of the blood sampling period, gave a quantitative measure of the distribution of the administered dose. Attempts to repeat these studies with similar doses of tritiumlabeled benzo[a]pyrene were frustrated by the lack of meaningful blood-level data. Dose trends for the derived pharmacokinetic parameters for pyrene revealed that the kinetics were nonlinear and strongly suggestive of enterohepatic recycling. Biliary excretion, measured in a separate experiment, gave support to this hypothesis. The bioavailability of the orally administered doses was between 50 and 60%. Over a 6-d period postdosing, some 45 and 40% of the administered dose was excreted via the urine and feces, respectively, irrespective of the route of administration. Distribution to the tissues of the 14C-label was highest in the perirenal fat, intermediate in the liver, kidneys, and lungs, and lowest in the heart, testes, spleen, and brain.
INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are organic compounds of two or more fused benzene rings in which adjacent rings share two or We thank Dr. R. Burnett for his helpful discussion of the statistical analysis, Ms. Leung and Mr. J. Lam for their technical assistance, and Mr. D. Jol for the animal maintenance. The authors are also indebted to the financial support from the Canadian Federal Panel on Energy Research and Development. Requests for reprints should be sent to Dr. J. R. Withey, Environmental and Occupational Toxicology Division, Bureau of Chemical Hazards, Environmental Health Centre, Tunney's Pasture, Ottawa, Ontario, Canada K1A 0L2.
429 Journal of Toxicology and Environmental Health, 32:429-447, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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). R. WITHEY ET AL.
more carbon atoms. Nonaromatic rings may also be present. They are ubiquitously distributed throughout the environment and have been found in ambient air, environmental water, foods, and indoor air (Santadonato et al., 1981). Identified sources that release PAHs to the air include fossil fuel-fired steam generators, home heating stacks, municipal incinerators, cement plants, and coke oven plants (Federal Register, 1971). Mobile sources include emissions from the exhausts of aircraft and automobile engines (Begeman and Colucci, 1962; Grimmer et al., 1972; Spindt, 1977). Fugitive emissions from roadway dust of bituminous road surfaces have also been identified (Waibel, 1976). Forest fires are considered the largest natural source of atmospheric PAH pollution (Lee et al., 1977; Zedeck, 1980). Because PAHs have high boiling points and, consequently, a low vapor pressure at ambient temperatures, their presence in air arises largely as a consequence of adsorption to particulate matter, as in wood smoke for example, or from their formation as aerosol condensates. Contaminated lake water has been estimated to contain concentrations of PAHs that were from 5 to 20 times larger than those in groundwater, and those in sewage waters were 10,000 times higher (Borneff, 1977). Their limited water solubility precludes high levels of contamination, and the highest concentration found in groundwater was only 0.04 /xg/l, although one sample of drinking water was found to contain 138.5 jug/I (U.S. EPA, 1980). PAHs were also found in raw or uncooked foods, such as vegetables, fruits, or fish, and their content was proportional to the level of PAH pollution in their local environment (Kolar et al., 1975). They were also found in cooking oils that had been used for the deep frying of foods (Lijinski and Ross, 1967). Smoked fish and meats had appreciable levels (10-100 ppb) of PAHs (Howard et al., 1966; Masuda and Kuratsune, 1971). Significant levels of PAHs have been recorded in certain soils and marine sediments (Youngblood and Blumer, 1975). Marine plankton, some sampled from depths of more than 40 m, have been shown to contain PAHs, some almost certainly originating from oil spills (Mallet et al., 1963). Canadian tar sands have been shown to contain a variety of PAHs. Some 97, including pyrene, were identified in a recent report (Poirier and Das, 1984). High levels of PAHs have also been determined in the fat (3.5 ppb) and livers (0.8 ppb) of human cadavers (Obana et al., 1981). In view of the wide distribution of PAHs in the environment and their potential for exposure and uptake via multiple routes, it was decided that an investigation of the bioavailability and uptake of model PAH compounds via all possible routes would permit a better evaluation of exposure and risk assessment for such compounds. Benzo[a]pyrene (a known human carcinogen) and pyrene (a noncarcinogen) were
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therefore chosen as model PAH compounds, and their pharmacokinetics and bioavailability were assessed after administration by iv, oral (intragastric), inhalation, and percutaneous routes. The work reported herein concerns only the data generated after the administration of pyrene by the oral and iv routes.
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METHODS Male Wistar rats (Crl: (WI)BR), specific-pathogen free (Charles River Canada Inc., St. Constant, Quebec), of approximately 400 g body weight, were surgically prepared with a polyethylene (PE-10) indwelling jugular cannula and dosed with 2, 4, 6, 9, or 15 mg/kg of 14C-labeled pyrene. The specific activity of the [4,5,9,10-14C]-pyrene was 43 mCi/mmol. Six animals per dose group were used and the pyrene was administered as 1.0 ml of a solution prepared in 20% Emulphor:80% physiological saline. An activity of 4 /xCi of [14C]pyrene/mg "cold" pyrene was used. A maximum of 15 blood samples (0.5 ml) was collected at 15, 30, 45, and 60 min, then every hour up to 6 h and then at suitably spaced time intervals postdosing until levels were below the detectable limits. The same number of animals per dose group was administered the same doses and a similar number of blood samples was taken from animals dosed by gastric intubation. Animals were placed in metabolism cages immediately after dosing. At the termination of the sampling period the animals in each dose group were divided into two equal groups. Three rats were then killed and their hearts, livers, lungs, spleens, kidneys, brains, testes, and perirenal fat were removed and frozen (-10°C) for subsequent analysis of their [14C]pyrene equivalent content at a later date. The other 3 in the dose group were maintained in metabolism cages and their urine and feces were collected at 0.5, 1.0, 2.0, 4.0, and 6.0 d postdosing and analyzed for their [14C]pyrene equivalent content. [14C]Pyrene in blood was analyzed using the method of Rubin (1973). Blood samples (0.1 ml) were digested with 1.0 ml of a mixture of protosol and ethanol (1:2) at 60°C for 30 min. The mixture was then cooled in an ice bath and decolorized with 30% hydrogen peroxide (0.5 ml). After the addition of Biofluor (New England Nuclear, Dupont Canada, Lachine, Quebec) (14 ml) and 0.5 N HCI (0.5 ml), the radioactivity in the mixture was determined with a Beckman LS-8000 liquid scintillation counter (Beckman Instruments Inc., Fullerton, Calif.). The radiolabel counts were transformed to pyrene equivalents from calibration curve data and will be referred to as "total 14C." Unchanged pyrene was extracted by a method that was modified from that described by Mitchell and Tu (1979). Exactly 0.35 ml of blood was placed into a centrifuge tube containing 2.0 ml of 0.5 N sulfuric acid. The mixture was extracted with 2.0 ml of hexane that contained a
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known amount of 1,2-benzanthracene, which served as an internal standard for the HPLC analysis. A 1.0 ml portion of the hexane extract was removed from the supernatant and 20 /il was injected directly into a Hewlett Packard 1090 liquid chromatograph interfaced with a 3392A Hewlett Packard integrator and a Hewlett Packard 85 personal computer [Hewlett Packard (Canada) Ltd., Mississauga, Ontario]. The column was a 100 m x 4.6 mm internal diameter ODS-Hypersil (5 /*m) column equipped with a diode-array detector that had a setpoint at 230 nm. Column temperature was 40°C. The eluent (methanol: water, 73:27) had a flow rate of 1.5 ml/min. The analytical data obtained in this way will be referred to as "free pyrene." Radioactivity in the urine was determined directly with a Beckman LS-8000 LSC after the addition of Biofluor. The radiolabel in the air dried feces or wet tissues was determined in a model 306 Packard Tricarb sample oxidizer (Packard Instrument Co. Ltd., Downers Grove, III.). The 14 CO2 produced from each sample was trapped in a solution of Carbosorb (7 ml) and Permafluor V (13 ml) and counted in the liquid scintillation counter. The blood concentration data for 14C, free pyrene, and free pyrene subtracted from 14C at each time point were plotted against time on semilogarithmic paper. It was evident from a visual inspection of these plots that these curves could be described by, at least, an equation containing two exponential components. Those following iv administration were therefore fitted to C, = Ae'at + Be~m
(1)
where C, is the concentration in blood at any time t, a and /3 are the hybrid rate coefficients for the initial and terminal elimination phases and A and B are the preexponential coefficients. Similarly, after oral administration, the blood data were fitted to Q = Ae~al' + Be"*3'' - (A +
fi)e"M'
(2)
where Q, A, B, a, and /? have a similar meaning as those defined above, /ca is the absorption rate coefficient, and t' = t - flag with f,ag being a lag time for the apparent initiation of absorption. This equation was somewhat similar to that used by Modica et al. (1983), except for the additional term introduced by us to account for the absorption phase and lag time. The rate coefficients for the assumed two-compartment model, kn, k2V and ke (where ke, kn, and /c21 are the coefficients for elimination from compartment 1 and the transfer coefficients from compartments 1 to 2 and 2 to 1, respectively), were calculated from the relationships presented by Mayersohn and Gibaldi (1971).
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Bioavailability (f) was calculated as the ratio of the areas under the blood level-time curves measured, by means of the trapezoidal rule, after oral and iv administration:
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F = AUC(po)/AUC(iv)
(3)
Data analysis was performed by using the SYSTAT program package (Wilkinson, 1988). Model parameters for each individual animal were estimated by weighted nonlinear regression. The analysis of several blood level-time data sets yielded residuals that increased in proportion to calculated predicted concentrations at high values of concentration but remained almost constant near the detection limit (about 0.05 /ng/ml). Therefore weights of 1/(Cj;red + K) were applied, with K being set to 0.01 for unchanged pyrene and 0.005 for total 14C. In some cases an analysis of variance with respect to dose was carried out on the estimated coefficients followed by Newman-Keuls multiple comparison in some cases and with linear regression in others. The estimates of A, B, a, and (3 were examined for divergence from zero by assuming that the ratio of the estimate to its standard error followed a standard normal distribution. If this ratio did not exceed 1.645, then the estimate was said to be not significant at the 5% level (see Tables 1, 2, 4, and 5). For the oral administration data, estimates of some of the coefficients were not obtained due to a lack of convergence. In these cases, number of significant parameters indicates the number of parameters in which convergence was achieved and the above criterion for statistical significance was met (see Tables 3 and 6). RESULTS Mean blood level-time data for pyrene, after iv or oral administration, were plotted for each dose on semilogarithmic paper and are shown in Figures 1 and 2. Two of these curves obtained after iv administration of the two highest dose levels were of particular interest since they reflected an uneven decline of the average blood levels, which was also evident in some individual curves at all dose levels. For the 2 mg/kg (iv) dose group, the average free pyrene levels in 3 of the 6 rats decreased in a smooth fashion for the first 4 h postdosing, and then increased. For the 15 mg/kg dose group, the average 14C levels increased some 6-10 h postdosing in 4 of the 6 animals. These levels resumed their decline following this intermediate increase. The "rebound" phenomenon was consistent with biliary excretion of part of the absorbed dose and its subsequent partial reabsorption, a process that has been described as enterohepatic recycling. Facilitated uptake of the PAH excreted via the bile or its metabolites by bile-salt micellar solubilization has been observed in a number of studies
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100.0
10.0
-
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o
160
320 4B0 TIME (mini
640
800
960
160
320 480 TIME (mln)
640
800
960
160
320
640
800
960
100.00
480 TIME (min)
FIGURE 1. Time course of mean blood concentration of 14C (upper panel), free pyrene (middle panel), and metabolites (lower panel) after iv administration of pyrene: 2 mg/kg T ; 4 mg/kg A; 6 mg/kg • ; 9 mg/kg • ; 15 mg/kg • .
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100.0
160
320
480
640
600
960
640
800
960
640
800
960
TIME (min) 100.00
160
320
480
TIME (mln) 10.0
160
320
480 TIME (mln)
FIGURE 2. Time course of mean blood concentration of 14C (upper panel), free pyrene (middle panel), and metabolites (lower panel) after oral administration of pyrene: 2 mg/kg T ; 4 mg/kg • ; 6 mg/kg • ; 9 mg/kg • ; 15 mg/kg • .
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(Rahman et al., 1986; Kotin et al., 1959). It was evident, from an inspection of the mean blood level curves for the iv administered dose of pyrene (Fig. 1), that the data obtained at 370 and 480 min were indicative of an increased elimination rate prior to a "rebound" of the blood levels. Blood concentrations predicted from the model and those actually observed were compared for the six animals dosed. The observed values were below the predicted values in all six cases. The average ratios and standard errors of predicted to observed values were 2.21 ± 0.47 and 1.87 ± 0.30, respectively. These values were significantly different from unity (p < .05). As a consequence of the rebound phenomenon, which was not always clearly defined and occurred when blood levels were at or near the limits of detection in many cases, it was considered that a more meaningful analysis of the overall kinetic profile would be obtained if the rebound segment of the curves was ignored. Thus, computation of the preexponential and rate coefficients, for the two-exponential model, was carried out by excluding the data obtained between 6 and 8 h postdosing. Rate and preexponential coefficients for free 14C and pyrene, following iv administration, together with the apparent volume of distribution Vd, are given in Tables 1 and 2. A number of the blood concentration-time data sets obtained after oral dosing, particularly at the lower dose levels, did not support a two-exponential model. Median parameters, together with the number of significant data sets, are therefore given in Table 3. The data obtained from the curve-fitting of the 14C and free pyrene data following iv administration were also used to obtain the individual compartmental transfer and elimination coefficients (Mayersohn and Gibaldi, 1971). Individual values for iv data are given in Tables 4 and 5. Many of the parameter estimates for the coefficients of the twocompartment model following oral dosing were either unobtainable or less than meaningful owing to the sparse nature of some of the data both as a consequence of the analytical limitations for the terminalphase data and the impact of the reabsorption phenomenon. Median values of those data sets that were conducive to further analysis are given in Table 6. The average terminal half-life in the /3 phase was 244 and 478 min for free pyrene and total 14C, respectively, independently of the dose. In contrast the macroparameter a and the compartmental parameters of ke and kn all declined, especially for free pyrene, with increasing dose. This result strongly indicated the nonlinearity of the pyrene kinetics. The areas under the blood level-time curves (AUC), required to calculate the bioavailability, were evaluated by using the trapezoidal rule. In principle, the AUC should have been calculated from zero to infinite time, but since there were only limited data for the terminal phase in many cases, the areas obtained for the time interval 0 to 480 min were
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TABLE 1. Parameter Estimates for 14C (Total) Observations, Following iv Administration, Fitted to a Two-Exponential Model A (/ig/ml)
B
a
(^g/ml)
(min"1)
C D E F Mean
13.8 11.2 4.7 5.9 8.4 8.4 8.7
1.13 1.67 0.22b 0.75h 1.01 1.06 0.98
4
A B C D E F Mean
26.3 26.3 22.9 29.4 19.3 24.9 24.9
6
A B C D E F Mean
9
A B
Dose (mg/kg)
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15a
vd
(ml)
0.0353 0.0336 0.0201 0.0262 0.0322 0.0306 0.0297
0.00146 0.00256 0.000126 0.00213b 0.00166 0.00122 0.00153
133 154 403 296 211 211 235
2.50 1.25 2.25 2.22 1.60 2.10 1.99
0.0304 0.0332 0.0485 0.0411 0.0322 0.0336 0.0365
0.00168 0.00084 0.00215 0.00194 0.00189 0.00172 0.00170
138 145 158 126 190 147 151
52.5 57.1 30.4 56.2 21.1 36.0 42.3
2.44 2.35 1.78 3.13 2.34 3.11 2.53
0.0330 0.0397 0.0372 0.0393 0.0284 0.0277 0.0343
0.00113 0.00169 0.00141 0.00193 0.00235 0.00168 0.00170
110 101 186 101 257 153 151
C D E F Mean
53.5 47.9 61.2 53.4 50.9 50.1 52.9
3.40 2.54 2.58 4.02 3.68 3.83 3.35
0.0271 0.0285 0.0285 0.0273 0.0258 0.0306 0.0280
0.00131 0.00113 0.00072 0.00152 0.00108 0.00167 0.00124
158 178 141 157 164 167 161
A B C D E F Mean
100.2 68.3 55.5 102.1 153.4 76.8 92.7
5.61 2.00 3.81 7.21 15.47 5.89 6.46
0.0194 0.0187 0.0269 0.0273 0.0226 0.0250 0.0233
0.00085 0.000026 0.00106 0.00110 0.00194 0.00147 0.00107
142 213 253 137 88 181 169
A B
2
0 (min- 1 )
Rat
^Observations at 370 and 460 min excluded. fc Not significant at the 5% level.
compared. It was considered that the areas calculated for this time period would approximate a constant proportion of the areas to infinite time and would also be defined by an acceptable number of measured data points in most cases. Mean areas for 14C and free pyrene, together with the ratios of po and iv values (i.e., bioavailability), are given in
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J. R. WITHEY ET AL.
TABLE 2. Parameter Estimates for Pyrene Observations, Following iv Administration, Fitted to a Two-Exponential Model
Dose (mg/kg)
A B C D E F Mean A B C D E F Mean A B C D E F Mean A B C D E F Mean A B C D E F Mean
2
4
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Rat
6
9
15
vd
A
B
(jig/m!)
(ftg/ml)
(min"1)
(min-1)
(ml)
29.4 16.2 11.8a 8.8 6.5 7.5 13.4
0.90 0.59 1.233 0.10a 0.06a 0.01a 0.49
0.0751 0.0547 0.0782 0.0426 0.0388 0.0313 0.0535
0.00734 0.00567 0.01070 -0.00267a 0.00015a -0.00886a 0.00206
66 119 153a 223 302 268 188
23.8 36.7 46.7 35.3 28.9 31.8 33.9
0.97 0.78 1.11 0.99 0.76 0.86 0.91
0.0378 0.0486 0.0797 0.0503 0.0496 0.0461 0.0521
0.00253 0.00315 0.00273 0.00354 0.00300 0.00251 0.00291
160 107 84 110 134 122 119
53.2 60.2 59.9 65.9 30.4 28.1 49.7
1.28 0.97 0.68 0.76 1.35 3.63 1.45
0.0442 0.0461 0.0489 0.0516 0.0330 0.0295 0.0423
0.00313 0.00263 0.00214 0.00330 0.00506 0.00336 0.00327
110 98 99 90 189 190 129
32.3 31.8 54.5 28.4 41.2 45.6 39.0
0.58a 4.57 2.82 5.86 3.17 0.67a 2.95
0.0107 0.0277 0.0317 0.0219 0.0265 0.0278 0.0244
0.00282a 0.00653 0.00351 0.00516 0.00444 0.00173a 0.0403
273 247 157 262 202 193 222
82.0 80.0 26.8 70.7 112.0 56.0 74.3
2.75 3.18 1.17 1.50 5.81 1.28a 2.62
0.0142 0.0250 0.0245 0.0196 0.0164 0.0168 0.0195
0.00201 0.00304 0.00291 0.00110 0.00269 -0.000033 0.00195
176 180 535 207 127 262 248
or
a
Not significant at the 5% level.
Table 7. Data for the mean cumulative urinary and fecal excretion, together with their combined values, are presented in Table 8. Concentrations in the principal organs and tissues following iv or po administration were measured in ng equivalents of [14C]pyrene per gram of wet tissue. In order to facilitate comparisons of the distribution to the various tissues, following the administration of the various doses,
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439
it was assumed that the uptake was proportional to the administered dose. Thus, levels of the dose-adjusted uptake in tissue (concentration divided by the dose), given in Table 9, revealed that these were independent of the iv administered dose for all tissues. The highest uptake was to the fat and was about 1.44 ng of [14C]pyrene equivalents/g wet tissue/(mg/kg body weight). Liver and kidney had intermediate values of 0.4 and 0.5 followed by the lung at 0.2. Much lower uptake was found in the heart, testes, spleen, and brain at 0.04, 0.04, 0.04, and 0.013 units, respectively. In contrast, the adjusted uptake to most tissues following oral administration clearly indicated a dose dependence. At the lowest dose (2 mg/kg), uptake to all tissues, except the fat, was two- to threefold that observed following iv administration. However, at doses of 4 mg/kg and higher the uptake was about the same. Uptake by the fat exhibited a different pattern since it was independent of the dose following iv administration. When the dose was administered orally, the dose-adjusted concentrations for the lower doses were only about 56% of those observed after the iv administration route and rose to 100% at the highest dose. In a separate study, in which three rats per route and dose group were surgically prepared with an indwelling biliary cannula, rats dosed with 2 mg/kg of pyrene by the iv and oral route excreted some 37 and 12% respectively via the bile over a six hour period. When the dose was increased to 9 mg/kg, some 27% was excreted via the bile after iv administration and 8% after dosing by the oral route.
TABLE 3. Median Values of the Statistically Significant Parameter Estimates, Following Oral Administration, Fitted to a Two-Exponential Model Dose (mg/kg)
A
B fag/ml)
a
0 (min~1) 14
2 4 6 9 15
a
9(2) 14(3) 12(3) 24(6) 39(4)
0.7(1) 1.1(3) 0.9(3) 2.2(4) 4.0(5)
0.030(1) 0.015(3) 0.013(4) 0.012(6) 0.014(6)
k
abs
T
lag
vd
(min)
(ml)
0.077(1) 0.078(4) 0.087(3) 0.062(6) 0.046(6)
3.9(1) 3.8(4) 4.1(4) 3.8(6) 3.8(6)
380(2) 480(3) 390(5) 390(6)
0.3138(1) 0.084(1) 0.056(1) 0.073(1)
4.5(1) 4.2(3) 4.2(3) 4.4(3)
870(1) 510(2) 300(3) 650(3)
C data 0.0005(2) 0.0009(1) 0.0011(4) 0.0010(5)
Pyrene data 2 4 6 9 15 a
4.7(1) 13(1) 41(1) 27(2)
0.19(1) 2.7(1)
0.019(1) 0.021(2) 0.018(3) 0.010(3)
0.0025(1) 0.0038(1) 0.0055(1) 0.0024(1)
Number of statistically significant parameters are in parentheses.
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J. R. WITHEY ET AL.
TABLE 4. Parameter Estimates for 14C Data, Fitted to a Two-Compartment Model, Following iv Dosing
kn (min~ 1 )
(min"1)
(min~ 1 )
2
A B C D E F
0.0200 0.0165 0.01696 0.0121 0.0182 0.0191
0.0040 0.0066 0.0011b 0.00496 0.0049 0.0045
0.0128 0.01301 0.0024b 0.0117 0.0109 0.00827
4
A
0.0157 0.0198 0.0278 0.0214 0.0154 0.0174
0.0042 0.0023 0.0063 0.0047 0.0042 0.0042
0.0123 0.0120 0.0166 0.0170 0.0145 0.0138
0.0169 0.0172 0.0198 0.0179 0.0122 0.0133
0.0025 0.0032 0.0034 0.0039 0.0049 0.0038
0.0145 0.0210 0.0154 0.0194 0.0134 0.01241
0.0132 0.0143 0.0162 0.0131 0.0140 0.0148
0.0029 0.0025 0.0019 0.0033 0.0028 0.0037
0.0124 0.0129 0.0111 0.0125 0.0102 0.0137
A B C D E F
0.0095 0.01756 0.0148 0.0150 0.0093 0.0117
0.0018 0.00066 0.0027 0.0028 0.0038 0.0032
0.00890 0.00076 0.0105 0.0107 0.0115 0.0117
B
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ke
Rat
u_
Dose (mg/kg)
C D E F A
6
B C D E F 9
A
B C D E 15a
a
Observations at 370 and 460 min excluded. Not significant at the 5% level.
h
DISCUSSION There have been relatively few detailed studies of the pharmacokinetics of the PAHs, and limited conclusions were drawn with respect to the mechanisms involved in their uptake, distribution, and elimination. In some of the earliest studies (Peacock, 1936; Kotin et al., 1959), the intravenous administration of benzo[a]pyrene (BaP) to rats revealed that the principal route of excretion was via the feces and that this was preceded by elimination from the systemic circulation via the biliary
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route by a saturable mechanism. It was also noted (Kotin et al., 1959) that intravenous doses of BaP were cleared from the blood compartment very rapidly, with less than 1% of a 55 /xg/kg dose remaining 10 min after administration. In the studies reported herein, an iv dose of 15 mg/kg of pyrene gave blood levels of unchanged pyrene of about 90 /*g/ml after 10 min and measurable levels (>0.05 /xg/ml) that persisted for up to 26 h postdosing. A similar dose of BaP, administered by the same route, was barely detectable in the blood at 10 min postdosing. These observations confirmed previous low dose studies by Kotin et al. (1959), Mitchell and TABLE 5. Parameter Estimates for Pyrene Data, Fitted to a Two-Compartment Model, Following iv Administration
Dose (mg/kg) 2
Rat A B
C D E F 4
A B C
D E F
A B C
6
D E F
A B C D
9
E
F A B C D
15
E
F a
kn (min"1)
(min" 1 )
0.0142 0.0111 0.0228 -0.0109a 0.0277a -0.0004a
0.0094 0.0074 0.0171a -0.00223 0.0006a -0.0088a
0.0589 0.0418 0.0491 0.05303 0.0107a 0.0317
0.0120 0.0104 0.0298 0.0121 0.0129 0.0134
0.0039 0.0041 0.0045 0.0048 0.0042 0.0037
0.0246 0.0373 0.0482 0.0370 0.0355 0.0316
0.0095 0.0089 0.0091 0.0069 0.0051 0.0109a
0.0041 0.0033 0.0027 0.0039 0.0063 0.0063
0.03371 0.0366 0.0393 0.0443 0.0267 0.0156
0.0004a 0.0054 0.0076 0.0050 0.0054 0.0046
0.0030a 0.0092 0.0049 0.0080 0.0060 0.0021a
0.0103 0.0197 0.0228 0.0141 0.0196 0.0229
0.0020 0.0046 0.0049 0.0047 0.0027 0.0182a
0.0024 0.0039 0.0038 0.0015 0.0034 0.0004a
0.0119 0.0196 0.0187 0.0146 0.0131 - 0.0017a
Not significant at the 5% level.
*21
ke
(min"1)
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J. R. WITHEY ET AL.
TABLE 6. Median Values for the Statistically Significant Parameter Estimates, Following Oral Administration, Fitted to a Two-Compartment Model Dose (mg/kg)
ke (min~1)
k2t (min~1)
*12
(min~1) 14
C-data
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2 4 6 9 15
0.011(2) 0.012(3) 0.0053(5) 0.0063(6)
0.0024(2) 0.0012(3) 0.0021(4) 0.0023(5)
0.0040(2) 0.0078(1) 0.0064(5) 0.0056(4)
Pyrene data 2 4 6 9 15 a
0.0032(1)
0.0035(1) 0.0035(2) 0.0022(1)
0.015(1) 0.017(2) 0.014(3) 0.008(3)
0.0008(1) 0.0031(2)
Number of statistically significant parameters are in parentheses.
Tu (1979), and Mitchell (1982,1983). Similar results were obtained following administration by the oral route. A comparison of the pharmacokinetics and bioavailability of the two selected model PAH compounds was therefore precluded. The mean blood concentration-time curves following iv and oral TABLE 7. Mean Areas under the Blood Concentration-Time Curves (min • jig/ml), over the Time Period 0-480 min Following iv and po Administration
iv UOSG
(mg/kg) 2 4 6 9 15
14
C
604 (184) 1350 (215) 2073 (501) 3129 (283) 6240 (2636)
Pyrene 371 (147) 919 (76) 1591 (125) 2198 (424) 4824 (2446)
Note. Six animals per dose group. - , Data did not extend to 480 min. b Standard deviations are in parentheses.
a
Bioavailability (%)
po 14
Pyrene
14
Pyrene
1136 (312) 1349 (234) 2272 (361) 4629 (1484)
501 (145) 713 (154) 1412 (363) 3464 (1226)
84
54
65
49
73
64
74
72
C
C
PHARMACOKINETICS OF PYRENE
443
TABLE 8. Mean Cumulative Percent Dose Excreted via the Urine, Feces, and Combined Urine at
Feces at d
Combined at d
0.5
1
2
4
6
2
4
6
2
4
6
iv
2 4 6 9 15
24 21 23 19 24
40 29 34 29 40
45 35 40 36 48
47 36 41 37 51
47 37" 41 37 51
32 36 35 49 32
33 39 39 52 38
34 41 40 52 38
77 71 75 86 81
80 75 81 89 88
81 67" 81 90 89
po
2 4 6
29 14 14 18 18
40 27 22
44 34 31 34 38
45 36 34 34 40
45 35" 34 34" 40
38 47 19 20 25
39 50 25 21 27
39 51" 26 21 28
82 82 50 54 63
84 85 58 56 67
84 92" 60 56" 68
Route
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d
Dose (mg/kg)
9 15
31 31
Note. Three animals per dose group except where noted. "Urine or fecal data available from only one animal and only one pair of combined data was used.
administration (Figs. 1 and 2) and the elimination data (Tables 1-4) revealed that the terminal (/3) phases were parallel, independently of the dose. The terminal rate was highest for pyrene, very low for the apparent metabolites and bound material and, consequently, intermediate for the 14C material. In contrast, the initial (a) phase was not parallel across doses, the slopes at higher doses being significantly shallower than those at lower doses. This behavior clearly indicated a nonlinearity in the kinetics with dose, presumably due to saturable metabolic or physiological processes. TABLE 9. Mean Tissue Levels of 14C and Pyrene, Expressed as a Dose-Adjusted Parameter in ftg/g Wet Tissue/(mg/kg body weight) after iv and po Administration Dose
Brain
Lung
Heart
Liver
2 4 6 9 15
0.0135 0.0168 0.0104 0.0111 0.0127
0.130 0.174 0.269 0.224 0.218
0.0405 0.0552 0.0409 0.0383 0.0343
0.259 0.415 0.437 0.287 0.359
2 4 6 9 15
0.0347 0.0149 0.0102 0.0411 0.0171
0.380 0.151 0.227 0.083 0.125
0.0878 0.0588 0.0366 0.0384 0.0425
0.640 0.523 0.342 0.300 0.326
Spleen
Kidney
Testis
Fat
0.0397 0.0470 0.0533 0.0437 0.0494
0.418 0.661 0.624 0.421 0.394
0.0378 0.0530 0.0536 0.0276 0.0423
1.25 1.64 1.37 1.32 1.62
0.0722 0.0434 0.0321 0.0464 0.0436
1.027 0.724 0.576 0.460 0.476
0.0970 0.0634 0.0301 0.0419 0.0404
0.702 1.071 0.764 1.067 1.713
iv Route
po Route
Note. Three animals per dose group.
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For reasons that have already been pointed out, the terminal phase of some of the data obtained after oral dosing was not clearly defined. This was particularly the case for 2 animals in the 15 mg/kg dose group where the rebound phenomenon was clearly evident between 6 and 8 h postdosing. Nevertheless, reasonable data fits were obtained in most cases for the biexponential model, similar to that used in the recent study by Modica et al. (1983). The apparent increase in the rate of excretion, prior to the rebound phenomenon, may be due to an increase in biliarly flow as a consequence of the action of the highly lipid dose of pyrene and its significant biliary excretion (Greenberger and Isselbacher, 1987). Other parameters that were consistent across doses were the absorption rate (with a half-life of about 10 min) and a lag time of about 4 min. In addition, the average apparent volume of distribution both for pyrene and 14C following administration by the iv route was about 180 ml and were also remarkably similar at all dose levels (Table 1). The apparent volume of distribution following oral administration (Table 3) was about 390 ml, which gave a value of about 50% for the bioavailability. This value was similar to that deduced from the areas under the blood concentration-time curves. The areas under the curve for the iv and oral data (Tables 1 and 3) showed, not unexpectedly, that values for the iv data were greater than those following oral administration. When these values were adjusted, by dividing by the respective administered doses, a significant (p < .05) increase with dose was noted for pyrene following iv or oral administration. Significance was nearly reached for 14C following iv but not after oral dosing. Increases in the dose-adjusted areas with increasing dose, particularly in the case of the pyrene data, again suggested that the kinetics were nonlinear. The mean values for the bioavailability for pyrene (Table 7) were about 60% while that for the 14C data were around 74%. Relatively stable values calculated from the volume of distribution for pyrene also supported these values. These observations were contrary to the observations of Mitchell and Tu (1979), who claimed that an orally administered aqueous suspension of pyrene was poorly absorbed from the gastrointestinal tract of the rat. At the lowest administered dose (2 mg/kg) both urinary and fecal excretion of pyrene were qualitatively identical after iv and oral administration. For either route, about 45 and 40% of the administered dose was excreted via the urine and feces over the 6-d collection period. This was contrary to the observation, in several communications, that the feces was the principal route of excretion for PAHs (Kotin et al., 1959; Sun et al., 1982; Sanders et al., 1984), although the doses used in the present study (2-15 mg/kg) were very much larger than those administered in previous studies (between 5 and 515 pg/kg). Distribution of 14C to the tissues was very much as anticipated from
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445
previous studies (Kotin et ah, 1959; Mitchell and Tu, 1979). High levels were found in the fat, probably as a consequence of the high lipid solubility of pyrene, followed by the liver and kidney, probably due to the high metabolic activity of these tissues (Daniel et al., 1967; Smith and Doody, 1981; Tyrer et al., 1981; Modica et al., 1983). Uptake to the fat and to the other tissues probably involved some redistribution so that part of the absorbed dose would be transferred from tissues having a high blood flow but low capacity to those having a low blood flow and high capacity. Precise evaluation of model kinetic parameters was complicated by their nonlinear dose dependence and enterohepatic recycling. These factors would also make the application of physiologically based pharmacokinetics somewhat complicated. Clearly, the pharmacokinetics of pyrene was remarkably different from that of benzo[a]pyrene. In particular, the risk assessment methodology that has been recommended for mixtures of compounds with a similar chemical structure, like the PAHs, in which the monitored PAH levels were converted (using potency factors) to benzo[a]pyrene equivalents, must be questionable (U.S. EPA, 1984; Thorslund and Charnley, 1988).
REFERENCES Begeman, C. R., and Colucci, J. M. 1962. Apparatus for determining the contribution of the automobile to the benzene-soluble organic matter in air. Natl. Cancer Inst. Monogr. 9:17-57. Borneff, J. 1977. Fate of carcinogens in aquatic environments. In Fate of Pollutants in the Air and Water Environments, Part 2, ed. I. H. Suffet, pp. 393-408. New York: Wiley. Daniel, P. M., Pratt, O. E., and Prichard, M. M. L. 1967. Metabolism of labeled carcinogenic hydrocarbons in rats. Nature 215:1142-1146. Federal Register. 1971. Determination of particulate emissions from stationary sources. 36(247). Greenberger, N. J., and Isselbacher, K. J. 1987. Disorders of absorption. In Harrison's Principles of Internal Medicine, 11th ed., eds. E. Braunwald, K. J. Isselbacher, R. G. Petersdorf, j . D. Wilson, J. B. Martin, and A. S. Fauchi, pp. 1260-1276. New York: McGraw-Hill. Grimmer, V. C., Hiddebrandt, A., and Bohnke, H. 1972. Sampling and analysis of polycyclic aromatic hydrocarbons in automobile exhaust gas. Erdöl und Kohle, Erdgas Petrochem. Brennstoff-Chem. 25:442-447, 531-536. Howard, J. W., White, R. H., Fry, B. E., and Turicchi, E. W. 1966. Extraction and estimation of polycyclic aromatic hydrocarbons in smoked foods. II. Benzo(a)pyrene. J. Assoc. Off. Anal. Chem. 49:611-617. Kolar, L R., Ticha, J., and Hanns, F. 1975. Pollution of soil, agricultural plants and vegetables by 3,4benzopyrene in the Ceske Budejovice. Cask. Hyg. 20:135-139. Kotin, P., Falk, H. L., and Busser, R. 1959. Distribution, retention, and elimination of 14C-3,4Benzpyrene after administration to rats and mice. J. Natl. Cancer Inst. 24:541-555. Lee, M. L., Prado, G. P., Hasard, J. B., and Hites, R. A. 1977. Source identification of urban airborne polycyclic aromatic hydrocarbons by gas chromatographic mass spectrometry and high resolution mass spectrometry. Biomed. Mass Spectrom. 4(3):182-186. Lijinski, W., and Ross, A. E. 1967. Production of carcinogenic polynuclear aromatic hydrocarbons in cooking of food. Food Cosmet. Toxicol. 5:343-347. Mallet, L., Pendian, A., and Perdian, J. 1963. Pollution by BaP type PAH of the western region of the Arctic Ocean. C. R. Hebd. Seances Acad. Sci (Paris) 256:3487-3489.
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Masuda, Y., and Kuratsune, M. 1971. Polycyclic aromatic hydrocarbons in smoked fish, "Katsuobushi." GANN 62:27-30. Mayersohn, M., and Gibaldi, M. 1971. Mathematical models in pharmacokinetics. Am. J. Pharmacol. Educ. 35:19-27. Mitchell, C. E. 1982. Distribution and retention of benzo(a)pyrene in rats after inhalation. Tox. Lett. 11:35-42. Mitchell, C. E. 1983. The metabolic fate of benzo(a)pyrene in rats after inhalation. Toxicol. 28:65-73. Mitchell, C. E., and Tu, K. W. 1979. Distribution, retention, and elimination of pyrene in rats after inhalation. J. Toxicol. Environ. Health 5:1171-1179. Modica, R., Fiume, M., Cuaitani, A., and Bartosek, I. 1983. Comparative kinetics of benz(a)anthracene, chrysene and triphenylene in rats after oral administration. Toxicol. Lett. 18:103-109. Obana, H., Hori, S., Kashimoto, T., and Kunita, N. 1981. Polycyclic aromatic hydrocarbons in human fat and liver. Bull. Environ. Contam. Toxicol. 27:23-27. Peacock, P. R. 1936. Evidence regarding the mechanism of elimination of 1:2-benzpyrene, 1:2:5:6 dibenzanthracene and anthracene from the blood-stream of injected animals. Bat. J. Exp. Pathol. 17:164-172. Poirier, M. A., and Das, B. S. 1984. Characterization of polynuclear aromatic hydrocarbons in bitumen, heavy oil fractions boiling above 350°C by G.C.-M.S. Fuel 63:361-367. Rahman, A., Barroman, J. A., and Rahimtula, A. 1986. The influence of bile on the bioavailability of polynuclear aromatic hydrocarbons from the rat intestine. Can. J. Physiol. Pharmacol. 64:12141218. Rubin, I. B. 1973. A simplified method for the determination of labeled alkane hydrocarbons in mammalian tissue after experience to radiolabeled cigarette smoke. Anal. Lett. 6:387-396. Sanders, C. L., Skinner, C., and Gelman, R. A. 1984. Percutaneous absorption of [7, 1014 C]benzo(a)pyrene and [7, 12-14C]dimethylbenz(a)anthracene in mice. Environ. Res. 33:353360. Santodonato, J., Howard, P., and Basu, D. 1981. Health and ecological assessment of polynuclear aromatic hydrocarbons. J. Environ. Pathol Toxicol. 5(1):1-376. Smith, I. C., and Doody, M. C. 1981. Kinetics of benzo(a)pyrene transfer between human plasma lipoproteins. In Chemical Analysis and Fate: Polynuclear Aromatic Hydrocarbons, 5th Int. Symp., eds. M. Cooke and A. J. Dennis, pp. 615-624. Columbus, OH.: Battelle Press. Spindt, R. A. 1977. Polynuclear aromatic content of heavy duty diesel engine exhaust gases. U.S. Nat. Tech. Inform. Serv. PB-267774. Sun, J. D., Wolff, R. K., and Kanapilly. 1982. Deposition, retention and biological fate of inhaled benzo(a)pyrene adsorbed onto ultrafine particles and as a pure aerosol. Toxicol. Appl. Pharmacol. 65:231-244. Thorslund, T., and Charnley, G. 1988. Comparative potency approach for estimating the cancer risk associated with exposure to mixtures of polycyclic aromatic hydrocarbons. ICF-Clement Associates, Washington, D.C. Tyrer, H. W., Cantrell, E. T., Horres, R., Lee, I. P., Peirano, W. B., and Danner, R. M. 1981. Benzo(a)pyrene metabolism in mice exposed to diesel exhaust: I. Uptake and distribution. Environ. Int. 5:307-311. U.S. Environmental Protection Agency. 1980. Ambient Water Quality Criteria for Polynuclear Aromatic Hydrocarbons. EPA 440/5-80-069. Office of Water Regulation and Standards, Washington, D.C. U.S. Environmental Protection Agency. 1984. Health Effects Assessment for Polycyclic Aromatic Hydrocarbons (PAH's). EPA 540/1-86-013. Environmental Criteria and Assessment Office, Cincinnati, Ohio. Waibel, M. 1976. Air and water contamination by road abrasion. Zentralbl. Bakteriol. Hyg. 1:B 163:458-469. Wilkinson, L. 1988. SYSTAT:The System for Statistics. [Computer program]. Evanston, Ill.: SYSTAT, Inc.
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Youngblood, W. W., and Blumer, M. 1975. Polycyclic aromatic hydrocarbons in the environment: Homologous series in soils and recent marine sediments. Geochim. Cosmochim. Acta 39:1303-1314. Zedeck, M. S. 1980. Polycyclic aromatic hydrocarbons, a review. J. Environ. Pathol. Toxicol. 3:537567.
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Received August 1, 1990 Accepted November 2, 1990
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Pharmacokinetics and bioavailability of pyrene in the rat J. R. Withey , F. C. P. Law & L. Endrenyi To cite this article: J. R. Withey , F. C. P. Law & L. Endrenyi (1991) Pharmacokinetics and bioavailability of pyrene in the rat, Journal of Toxicology and Environmental Health, 32:4, 429-447, DOI: 10.1080/15287399109531494 To link to this article: http://dx.doi.org/10.1080/15287399109531494
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PHARMACOKINETICS AND BIOAVAILABILITY OF PYRENE IN THE RAT J. R. Withey Environmental Health Directorate, Ottawa, Ontario, Canada
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F. C. P. Law Simon Fraser University, Burnaby, British Columbia, Canada L. Endrenyi University of Toronto, Toronto, Ontario, Canada Groups of 6 male Wistar rats, of about 400 g body weight, were dosed with 14C-labeled pyrene, dissolved in an Emulphor/water solvent vehicle, at 5 different dose levels by the intravenous or oral routes. Appropriate mathematical models were fitted to blood concentration-time data for [14C]pyrene and pyrene per se and dose-trend analyses were carried out. Areas under these curves were used to assess the bioavailability of the orally administered doses. Tissue concentrations, measured at the termination of the blood sampling period, gave a quantitative measure of the distribution of the administered dose. Attempts to repeat these studies with similar doses of tritiumlabeled benzo[a]pyrene were frustrated by the lack of meaningful blood-level data. Dose trends for the derived pharmacokinetic parameters for pyrene revealed that the kinetics were nonlinear and strongly suggestive of enterohepatic recycling. Biliary excretion, measured in a separate experiment, gave support to this hypothesis. The bioavailability of the orally administered doses was between 50 and 60%. Over a 6-d period postdosing, some 45 and 40% of the administered dose was excreted via the urine and feces, respectively, irrespective of the route of administration. Distribution to the tissues of the 14C-label was highest in the perirenal fat, intermediate in the liver, kidneys, and lungs, and lowest in the heart, testes, spleen, and brain.
INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are organic compounds of two or more fused benzene rings in which adjacent rings share two or We thank Dr. R. Burnett for his helpful discussion of the statistical analysis, Ms. Leung and Mr. J. Lam for their technical assistance, and Mr. D. Jol for the animal maintenance. The authors are also indebted to the financial support from the Canadian Federal Panel on Energy Research and Development. Requests for reprints should be sent to Dr. J. R. Withey, Environmental and Occupational Toxicology Division, Bureau of Chemical Hazards, Environmental Health Centre, Tunney's Pasture, Ottawa, Ontario, Canada K1A 0L2.
429 Journal of Toxicology and Environmental Health, 32:429-447, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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). R. WITHEY ET AL.
more carbon atoms. Nonaromatic rings may also be present. They are ubiquitously distributed throughout the environment and have been found in ambient air, environmental water, foods, and indoor air (Santadonato et al., 1981). Identified sources that release PAHs to the air include fossil fuel-fired steam generators, home heating stacks, municipal incinerators, cement plants, and coke oven plants (Federal Register, 1971). Mobile sources include emissions from the exhausts of aircraft and automobile engines (Begeman and Colucci, 1962; Grimmer et al., 1972; Spindt, 1977). Fugitive emissions from roadway dust of bituminous road surfaces have also been identified (Waibel, 1976). Forest fires are considered the largest natural source of atmospheric PAH pollution (Lee et al., 1977; Zedeck, 1980). Because PAHs have high boiling points and, consequently, a low vapor pressure at ambient temperatures, their presence in air arises largely as a consequence of adsorption to particulate matter, as in wood smoke for example, or from their formation as aerosol condensates. Contaminated lake water has been estimated to contain concentrations of PAHs that were from 5 to 20 times larger than those in groundwater, and those in sewage waters were 10,000 times higher (Borneff, 1977). Their limited water solubility precludes high levels of contamination, and the highest concentration found in groundwater was only 0.04 /xg/l, although one sample of drinking water was found to contain 138.5 jug/I (U.S. EPA, 1980). PAHs were also found in raw or uncooked foods, such as vegetables, fruits, or fish, and their content was proportional to the level of PAH pollution in their local environment (Kolar et al., 1975). They were also found in cooking oils that had been used for the deep frying of foods (Lijinski and Ross, 1967). Smoked fish and meats had appreciable levels (10-100 ppb) of PAHs (Howard et al., 1966; Masuda and Kuratsune, 1971). Significant levels of PAHs have been recorded in certain soils and marine sediments (Youngblood and Blumer, 1975). Marine plankton, some sampled from depths of more than 40 m, have been shown to contain PAHs, some almost certainly originating from oil spills (Mallet et al., 1963). Canadian tar sands have been shown to contain a variety of PAHs. Some 97, including pyrene, were identified in a recent report (Poirier and Das, 1984). High levels of PAHs have also been determined in the fat (3.5 ppb) and livers (0.8 ppb) of human cadavers (Obana et al., 1981). In view of the wide distribution of PAHs in the environment and their potential for exposure and uptake via multiple routes, it was decided that an investigation of the bioavailability and uptake of model PAH compounds via all possible routes would permit a better evaluation of exposure and risk assessment for such compounds. Benzo[a]pyrene (a known human carcinogen) and pyrene (a noncarcinogen) were
PHARMACOKINETICS OF PYRENE
431
therefore chosen as model PAH compounds, and their pharmacokinetics and bioavailability were assessed after administration by iv, oral (intragastric), inhalation, and percutaneous routes. The work reported herein concerns only the data generated after the administration of pyrene by the oral and iv routes.
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METHODS Male Wistar rats (Crl: (WI)BR), specific-pathogen free (Charles River Canada Inc., St. Constant, Quebec), of approximately 400 g body weight, were surgically prepared with a polyethylene (PE-10) indwelling jugular cannula and dosed with 2, 4, 6, 9, or 15 mg/kg of 14C-labeled pyrene. The specific activity of the [4,5,9,10-14C]-pyrene was 43 mCi/mmol. Six animals per dose group were used and the pyrene was administered as 1.0 ml of a solution prepared in 20% Emulphor:80% physiological saline. An activity of 4 /xCi of [14C]pyrene/mg "cold" pyrene was used. A maximum of 15 blood samples (0.5 ml) was collected at 15, 30, 45, and 60 min, then every hour up to 6 h and then at suitably spaced time intervals postdosing until levels were below the detectable limits. The same number of animals per dose group was administered the same doses and a similar number of blood samples was taken from animals dosed by gastric intubation. Animals were placed in metabolism cages immediately after dosing. At the termination of the sampling period the animals in each dose group were divided into two equal groups. Three rats were then killed and their hearts, livers, lungs, spleens, kidneys, brains, testes, and perirenal fat were removed and frozen (-10°C) for subsequent analysis of their [14C]pyrene equivalent content at a later date. The other 3 in the dose group were maintained in metabolism cages and their urine and feces were collected at 0.5, 1.0, 2.0, 4.0, and 6.0 d postdosing and analyzed for their [14C]pyrene equivalent content. [14C]Pyrene in blood was analyzed using the method of Rubin (1973). Blood samples (0.1 ml) were digested with 1.0 ml of a mixture of protosol and ethanol (1:2) at 60°C for 30 min. The mixture was then cooled in an ice bath and decolorized with 30% hydrogen peroxide (0.5 ml). After the addition of Biofluor (New England Nuclear, Dupont Canada, Lachine, Quebec) (14 ml) and 0.5 N HCI (0.5 ml), the radioactivity in the mixture was determined with a Beckman LS-8000 liquid scintillation counter (Beckman Instruments Inc., Fullerton, Calif.). The radiolabel counts were transformed to pyrene equivalents from calibration curve data and will be referred to as "total 14C." Unchanged pyrene was extracted by a method that was modified from that described by Mitchell and Tu (1979). Exactly 0.35 ml of blood was placed into a centrifuge tube containing 2.0 ml of 0.5 N sulfuric acid. The mixture was extracted with 2.0 ml of hexane that contained a
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J. R. WITHEY ET AL.
known amount of 1,2-benzanthracene, which served as an internal standard for the HPLC analysis. A 1.0 ml portion of the hexane extract was removed from the supernatant and 20 /il was injected directly into a Hewlett Packard 1090 liquid chromatograph interfaced with a 3392A Hewlett Packard integrator and a Hewlett Packard 85 personal computer [Hewlett Packard (Canada) Ltd., Mississauga, Ontario]. The column was a 100 m x 4.6 mm internal diameter ODS-Hypersil (5 /*m) column equipped with a diode-array detector that had a setpoint at 230 nm. Column temperature was 40°C. The eluent (methanol: water, 73:27) had a flow rate of 1.5 ml/min. The analytical data obtained in this way will be referred to as "free pyrene." Radioactivity in the urine was determined directly with a Beckman LS-8000 LSC after the addition of Biofluor. The radiolabel in the air dried feces or wet tissues was determined in a model 306 Packard Tricarb sample oxidizer (Packard Instrument Co. Ltd., Downers Grove, III.). The 14 CO2 produced from each sample was trapped in a solution of Carbosorb (7 ml) and Permafluor V (13 ml) and counted in the liquid scintillation counter. The blood concentration data for 14C, free pyrene, and free pyrene subtracted from 14C at each time point were plotted against time on semilogarithmic paper. It was evident from a visual inspection of these plots that these curves could be described by, at least, an equation containing two exponential components. Those following iv administration were therefore fitted to C, = Ae'at + Be~m
(1)
where C, is the concentration in blood at any time t, a and /3 are the hybrid rate coefficients for the initial and terminal elimination phases and A and B are the preexponential coefficients. Similarly, after oral administration, the blood data were fitted to Q = Ae~al' + Be"*3'' - (A +
fi)e"M'
(2)
where Q, A, B, a, and /? have a similar meaning as those defined above, /ca is the absorption rate coefficient, and t' = t - flag with f,ag being a lag time for the apparent initiation of absorption. This equation was somewhat similar to that used by Modica et al. (1983), except for the additional term introduced by us to account for the absorption phase and lag time. The rate coefficients for the assumed two-compartment model, kn, k2V and ke (where ke, kn, and /c21 are the coefficients for elimination from compartment 1 and the transfer coefficients from compartments 1 to 2 and 2 to 1, respectively), were calculated from the relationships presented by Mayersohn and Gibaldi (1971).
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Bioavailability (f) was calculated as the ratio of the areas under the blood level-time curves measured, by means of the trapezoidal rule, after oral and iv administration:
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F = AUC(po)/AUC(iv)
(3)
Data analysis was performed by using the SYSTAT program package (Wilkinson, 1988). Model parameters for each individual animal were estimated by weighted nonlinear regression. The analysis of several blood level-time data sets yielded residuals that increased in proportion to calculated predicted concentrations at high values of concentration but remained almost constant near the detection limit (about 0.05 /ng/ml). Therefore weights of 1/(Cj;red + K) were applied, with K being set to 0.01 for unchanged pyrene and 0.005 for total 14C. In some cases an analysis of variance with respect to dose was carried out on the estimated coefficients followed by Newman-Keuls multiple comparison in some cases and with linear regression in others. The estimates of A, B, a, and (3 were examined for divergence from zero by assuming that the ratio of the estimate to its standard error followed a standard normal distribution. If this ratio did not exceed 1.645, then the estimate was said to be not significant at the 5% level (see Tables 1, 2, 4, and 5). For the oral administration data, estimates of some of the coefficients were not obtained due to a lack of convergence. In these cases, number of significant parameters indicates the number of parameters in which convergence was achieved and the above criterion for statistical significance was met (see Tables 3 and 6). RESULTS Mean blood level-time data for pyrene, after iv or oral administration, were plotted for each dose on semilogarithmic paper and are shown in Figures 1 and 2. Two of these curves obtained after iv administration of the two highest dose levels were of particular interest since they reflected an uneven decline of the average blood levels, which was also evident in some individual curves at all dose levels. For the 2 mg/kg (iv) dose group, the average free pyrene levels in 3 of the 6 rats decreased in a smooth fashion for the first 4 h postdosing, and then increased. For the 15 mg/kg dose group, the average 14C levels increased some 6-10 h postdosing in 4 of the 6 animals. These levels resumed their decline following this intermediate increase. The "rebound" phenomenon was consistent with biliary excretion of part of the absorbed dose and its subsequent partial reabsorption, a process that has been described as enterohepatic recycling. Facilitated uptake of the PAH excreted via the bile or its metabolites by bile-salt micellar solubilization has been observed in a number of studies
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100.0
10.0
-
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o
160
320 4B0 TIME (mini
640
800
960
160
320 480 TIME (mln)
640
800
960
160
320
640
800
960
100.00
480 TIME (min)
FIGURE 1. Time course of mean blood concentration of 14C (upper panel), free pyrene (middle panel), and metabolites (lower panel) after iv administration of pyrene: 2 mg/kg T ; 4 mg/kg A; 6 mg/kg • ; 9 mg/kg • ; 15 mg/kg • .
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100.0
160
320
480
640
600
960
640
800
960
640
800
960
TIME (min) 100.00
160
320
480
TIME (mln) 10.0
160
320
480 TIME (mln)
FIGURE 2. Time course of mean blood concentration of 14C (upper panel), free pyrene (middle panel), and metabolites (lower panel) after oral administration of pyrene: 2 mg/kg T ; 4 mg/kg • ; 6 mg/kg • ; 9 mg/kg • ; 15 mg/kg • .
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(Rahman et al., 1986; Kotin et al., 1959). It was evident, from an inspection of the mean blood level curves for the iv administered dose of pyrene (Fig. 1), that the data obtained at 370 and 480 min were indicative of an increased elimination rate prior to a "rebound" of the blood levels. Blood concentrations predicted from the model and those actually observed were compared for the six animals dosed. The observed values were below the predicted values in all six cases. The average ratios and standard errors of predicted to observed values were 2.21 ± 0.47 and 1.87 ± 0.30, respectively. These values were significantly different from unity (p < .05). As a consequence of the rebound phenomenon, which was not always clearly defined and occurred when blood levels were at or near the limits of detection in many cases, it was considered that a more meaningful analysis of the overall kinetic profile would be obtained if the rebound segment of the curves was ignored. Thus, computation of the preexponential and rate coefficients, for the two-exponential model, was carried out by excluding the data obtained between 6 and 8 h postdosing. Rate and preexponential coefficients for free 14C and pyrene, following iv administration, together with the apparent volume of distribution Vd, are given in Tables 1 and 2. A number of the blood concentration-time data sets obtained after oral dosing, particularly at the lower dose levels, did not support a two-exponential model. Median parameters, together with the number of significant data sets, are therefore given in Table 3. The data obtained from the curve-fitting of the 14C and free pyrene data following iv administration were also used to obtain the individual compartmental transfer and elimination coefficients (Mayersohn and Gibaldi, 1971). Individual values for iv data are given in Tables 4 and 5. Many of the parameter estimates for the coefficients of the twocompartment model following oral dosing were either unobtainable or less than meaningful owing to the sparse nature of some of the data both as a consequence of the analytical limitations for the terminalphase data and the impact of the reabsorption phenomenon. Median values of those data sets that were conducive to further analysis are given in Table 6. The average terminal half-life in the /3 phase was 244 and 478 min for free pyrene and total 14C, respectively, independently of the dose. In contrast the macroparameter a and the compartmental parameters of ke and kn all declined, especially for free pyrene, with increasing dose. This result strongly indicated the nonlinearity of the pyrene kinetics. The areas under the blood level-time curves (AUC), required to calculate the bioavailability, were evaluated by using the trapezoidal rule. In principle, the AUC should have been calculated from zero to infinite time, but since there were only limited data for the terminal phase in many cases, the areas obtained for the time interval 0 to 480 min were
PHARMACOKINETICS OF PYRENE
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TABLE 1. Parameter Estimates for 14C (Total) Observations, Following iv Administration, Fitted to a Two-Exponential Model A (/ig/ml)
B
a
(^g/ml)
(min"1)
C D E F Mean
13.8 11.2 4.7 5.9 8.4 8.4 8.7
1.13 1.67 0.22b 0.75h 1.01 1.06 0.98
4
A B C D E F Mean
26.3 26.3 22.9 29.4 19.3 24.9 24.9
6
A B C D E F Mean
9
A B
Dose (mg/kg)
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15a
vd
(ml)
0.0353 0.0336 0.0201 0.0262 0.0322 0.0306 0.0297
0.00146 0.00256 0.000126 0.00213b 0.00166 0.00122 0.00153
133 154 403 296 211 211 235
2.50 1.25 2.25 2.22 1.60 2.10 1.99
0.0304 0.0332 0.0485 0.0411 0.0322 0.0336 0.0365
0.00168 0.00084 0.00215 0.00194 0.00189 0.00172 0.00170
138 145 158 126 190 147 151
52.5 57.1 30.4 56.2 21.1 36.0 42.3
2.44 2.35 1.78 3.13 2.34 3.11 2.53
0.0330 0.0397 0.0372 0.0393 0.0284 0.0277 0.0343
0.00113 0.00169 0.00141 0.00193 0.00235 0.00168 0.00170
110 101 186 101 257 153 151
C D E F Mean
53.5 47.9 61.2 53.4 50.9 50.1 52.9
3.40 2.54 2.58 4.02 3.68 3.83 3.35
0.0271 0.0285 0.0285 0.0273 0.0258 0.0306 0.0280
0.00131 0.00113 0.00072 0.00152 0.00108 0.00167 0.00124
158 178 141 157 164 167 161
A B C D E F Mean
100.2 68.3 55.5 102.1 153.4 76.8 92.7
5.61 2.00 3.81 7.21 15.47 5.89 6.46
0.0194 0.0187 0.0269 0.0273 0.0226 0.0250 0.0233
0.00085 0.000026 0.00106 0.00110 0.00194 0.00147 0.00107
142 213 253 137 88 181 169
A B
2
0 (min- 1 )
Rat
^Observations at 370 and 460 min excluded. fc Not significant at the 5% level.
compared. It was considered that the areas calculated for this time period would approximate a constant proportion of the areas to infinite time and would also be defined by an acceptable number of measured data points in most cases. Mean areas for 14C and free pyrene, together with the ratios of po and iv values (i.e., bioavailability), are given in
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TABLE 2. Parameter Estimates for Pyrene Observations, Following iv Administration, Fitted to a Two-Exponential Model
Dose (mg/kg)
A B C D E F Mean A B C D E F Mean A B C D E F Mean A B C D E F Mean A B C D E F Mean
2
4
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Rat
6
9
15
vd
A
B
(jig/m!)
(ftg/ml)
(min"1)
(min-1)
(ml)
29.4 16.2 11.8a 8.8 6.5 7.5 13.4
0.90 0.59 1.233 0.10a 0.06a 0.01a 0.49
0.0751 0.0547 0.0782 0.0426 0.0388 0.0313 0.0535
0.00734 0.00567 0.01070 -0.00267a 0.00015a -0.00886a 0.00206
66 119 153a 223 302 268 188
23.8 36.7 46.7 35.3 28.9 31.8 33.9
0.97 0.78 1.11 0.99 0.76 0.86 0.91
0.0378 0.0486 0.0797 0.0503 0.0496 0.0461 0.0521
0.00253 0.00315 0.00273 0.00354 0.00300 0.00251 0.00291
160 107 84 110 134 122 119
53.2 60.2 59.9 65.9 30.4 28.1 49.7
1.28 0.97 0.68 0.76 1.35 3.63 1.45
0.0442 0.0461 0.0489 0.0516 0.0330 0.0295 0.0423
0.00313 0.00263 0.00214 0.00330 0.00506 0.00336 0.00327
110 98 99 90 189 190 129
32.3 31.8 54.5 28.4 41.2 45.6 39.0
0.58a 4.57 2.82 5.86 3.17 0.67a 2.95
0.0107 0.0277 0.0317 0.0219 0.0265 0.0278 0.0244
0.00282a 0.00653 0.00351 0.00516 0.00444 0.00173a 0.0403
273 247 157 262 202 193 222
82.0 80.0 26.8 70.7 112.0 56.0 74.3
2.75 3.18 1.17 1.50 5.81 1.28a 2.62
0.0142 0.0250 0.0245 0.0196 0.0164 0.0168 0.0195
0.00201 0.00304 0.00291 0.00110 0.00269 -0.000033 0.00195
176 180 535 207 127 262 248
or
a
Not significant at the 5% level.
Table 7. Data for the mean cumulative urinary and fecal excretion, together with their combined values, are presented in Table 8. Concentrations in the principal organs and tissues following iv or po administration were measured in ng equivalents of [14C]pyrene per gram of wet tissue. In order to facilitate comparisons of the distribution to the various tissues, following the administration of the various doses,
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it was assumed that the uptake was proportional to the administered dose. Thus, levels of the dose-adjusted uptake in tissue (concentration divided by the dose), given in Table 9, revealed that these were independent of the iv administered dose for all tissues. The highest uptake was to the fat and was about 1.44 ng of [14C]pyrene equivalents/g wet tissue/(mg/kg body weight). Liver and kidney had intermediate values of 0.4 and 0.5 followed by the lung at 0.2. Much lower uptake was found in the heart, testes, spleen, and brain at 0.04, 0.04, 0.04, and 0.013 units, respectively. In contrast, the adjusted uptake to most tissues following oral administration clearly indicated a dose dependence. At the lowest dose (2 mg/kg), uptake to all tissues, except the fat, was two- to threefold that observed following iv administration. However, at doses of 4 mg/kg and higher the uptake was about the same. Uptake by the fat exhibited a different pattern since it was independent of the dose following iv administration. When the dose was administered orally, the dose-adjusted concentrations for the lower doses were only about 56% of those observed after the iv administration route and rose to 100% at the highest dose. In a separate study, in which three rats per route and dose group were surgically prepared with an indwelling biliary cannula, rats dosed with 2 mg/kg of pyrene by the iv and oral route excreted some 37 and 12% respectively via the bile over a six hour period. When the dose was increased to 9 mg/kg, some 27% was excreted via the bile after iv administration and 8% after dosing by the oral route.
TABLE 3. Median Values of the Statistically Significant Parameter Estimates, Following Oral Administration, Fitted to a Two-Exponential Model Dose (mg/kg)
A
B fag/ml)
a
0 (min~1) 14
2 4 6 9 15
a
9(2) 14(3) 12(3) 24(6) 39(4)
0.7(1) 1.1(3) 0.9(3) 2.2(4) 4.0(5)
0.030(1) 0.015(3) 0.013(4) 0.012(6) 0.014(6)
k
abs
T
lag
vd
(min)
(ml)
0.077(1) 0.078(4) 0.087(3) 0.062(6) 0.046(6)
3.9(1) 3.8(4) 4.1(4) 3.8(6) 3.8(6)
380(2) 480(3) 390(5) 390(6)
0.3138(1) 0.084(1) 0.056(1) 0.073(1)
4.5(1) 4.2(3) 4.2(3) 4.4(3)
870(1) 510(2) 300(3) 650(3)
C data 0.0005(2) 0.0009(1) 0.0011(4) 0.0010(5)
Pyrene data 2 4 6 9 15 a
4.7(1) 13(1) 41(1) 27(2)
0.19(1) 2.7(1)
0.019(1) 0.021(2) 0.018(3) 0.010(3)
0.0025(1) 0.0038(1) 0.0055(1) 0.0024(1)
Number of statistically significant parameters are in parentheses.
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TABLE 4. Parameter Estimates for 14C Data, Fitted to a Two-Compartment Model, Following iv Dosing
kn (min~ 1 )
(min"1)
(min~ 1 )
2
A B C D E F
0.0200 0.0165 0.01696 0.0121 0.0182 0.0191
0.0040 0.0066 0.0011b 0.00496 0.0049 0.0045
0.0128 0.01301 0.0024b 0.0117 0.0109 0.00827
4
A
0.0157 0.0198 0.0278 0.0214 0.0154 0.0174
0.0042 0.0023 0.0063 0.0047 0.0042 0.0042
0.0123 0.0120 0.0166 0.0170 0.0145 0.0138
0.0169 0.0172 0.0198 0.0179 0.0122 0.0133
0.0025 0.0032 0.0034 0.0039 0.0049 0.0038
0.0145 0.0210 0.0154 0.0194 0.0134 0.01241
0.0132 0.0143 0.0162 0.0131 0.0140 0.0148
0.0029 0.0025 0.0019 0.0033 0.0028 0.0037
0.0124 0.0129 0.0111 0.0125 0.0102 0.0137
A B C D E F
0.0095 0.01756 0.0148 0.0150 0.0093 0.0117
0.0018 0.00066 0.0027 0.0028 0.0038 0.0032
0.00890 0.00076 0.0105 0.0107 0.0115 0.0117
B
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ke
Rat
u_
Dose (mg/kg)
C D E F A
6
B C D E F 9
A
B C D E 15a
a
Observations at 370 and 460 min excluded. Not significant at the 5% level.
h
DISCUSSION There have been relatively few detailed studies of the pharmacokinetics of the PAHs, and limited conclusions were drawn with respect to the mechanisms involved in their uptake, distribution, and elimination. In some of the earliest studies (Peacock, 1936; Kotin et al., 1959), the intravenous administration of benzo[a]pyrene (BaP) to rats revealed that the principal route of excretion was via the feces and that this was preceded by elimination from the systemic circulation via the biliary
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route by a saturable mechanism. It was also noted (Kotin et al., 1959) that intravenous doses of BaP were cleared from the blood compartment very rapidly, with less than 1% of a 55 /xg/kg dose remaining 10 min after administration. In the studies reported herein, an iv dose of 15 mg/kg of pyrene gave blood levels of unchanged pyrene of about 90 /*g/ml after 10 min and measurable levels (>0.05 /xg/ml) that persisted for up to 26 h postdosing. A similar dose of BaP, administered by the same route, was barely detectable in the blood at 10 min postdosing. These observations confirmed previous low dose studies by Kotin et al. (1959), Mitchell and TABLE 5. Parameter Estimates for Pyrene Data, Fitted to a Two-Compartment Model, Following iv Administration
Dose (mg/kg) 2
Rat A B
C D E F 4
A B C
D E F
A B C
6
D E F
A B C D
9
E
F A B C D
15
E
F a
kn (min"1)
(min" 1 )
0.0142 0.0111 0.0228 -0.0109a 0.0277a -0.0004a
0.0094 0.0074 0.0171a -0.00223 0.0006a -0.0088a
0.0589 0.0418 0.0491 0.05303 0.0107a 0.0317
0.0120 0.0104 0.0298 0.0121 0.0129 0.0134
0.0039 0.0041 0.0045 0.0048 0.0042 0.0037
0.0246 0.0373 0.0482 0.0370 0.0355 0.0316
0.0095 0.0089 0.0091 0.0069 0.0051 0.0109a
0.0041 0.0033 0.0027 0.0039 0.0063 0.0063
0.03371 0.0366 0.0393 0.0443 0.0267 0.0156
0.0004a 0.0054 0.0076 0.0050 0.0054 0.0046
0.0030a 0.0092 0.0049 0.0080 0.0060 0.0021a
0.0103 0.0197 0.0228 0.0141 0.0196 0.0229
0.0020 0.0046 0.0049 0.0047 0.0027 0.0182a
0.0024 0.0039 0.0038 0.0015 0.0034 0.0004a
0.0119 0.0196 0.0187 0.0146 0.0131 - 0.0017a
Not significant at the 5% level.
*21
ke
(min"1)
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TABLE 6. Median Values for the Statistically Significant Parameter Estimates, Following Oral Administration, Fitted to a Two-Compartment Model Dose (mg/kg)
ke (min~1)
k2t (min~1)
*12
(min~1) 14
C-data
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2 4 6 9 15
0.011(2) 0.012(3) 0.0053(5) 0.0063(6)
0.0024(2) 0.0012(3) 0.0021(4) 0.0023(5)
0.0040(2) 0.0078(1) 0.0064(5) 0.0056(4)
Pyrene data 2 4 6 9 15 a
0.0032(1)
0.0035(1) 0.0035(2) 0.0022(1)
0.015(1) 0.017(2) 0.014(3) 0.008(3)
0.0008(1) 0.0031(2)
Number of statistically significant parameters are in parentheses.
Tu (1979), and Mitchell (1982,1983). Similar results were obtained following administration by the oral route. A comparison of the pharmacokinetics and bioavailability of the two selected model PAH compounds was therefore precluded. The mean blood concentration-time curves following iv and oral TABLE 7. Mean Areas under the Blood Concentration-Time Curves (min • jig/ml), over the Time Period 0-480 min Following iv and po Administration
iv UOSG
(mg/kg) 2 4 6 9 15
14
C
604 (184) 1350 (215) 2073 (501) 3129 (283) 6240 (2636)
Pyrene 371 (147) 919 (76) 1591 (125) 2198 (424) 4824 (2446)
Note. Six animals per dose group. - , Data did not extend to 480 min. b Standard deviations are in parentheses.
a
Bioavailability (%)
po 14
Pyrene
14
Pyrene
1136 (312) 1349 (234) 2272 (361) 4629 (1484)
501 (145) 713 (154) 1412 (363) 3464 (1226)
84
54
65
49
73
64
74
72
C
C
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TABLE 8. Mean Cumulative Percent Dose Excreted via the Urine, Feces, and Combined Urine at
Feces at d
Combined at d
0.5
1
2
4
6
2
4
6
2
4
6
iv
2 4 6 9 15
24 21 23 19 24
40 29 34 29 40
45 35 40 36 48
47 36 41 37 51
47 37" 41 37 51
32 36 35 49 32
33 39 39 52 38
34 41 40 52 38
77 71 75 86 81
80 75 81 89 88
81 67" 81 90 89
po
2 4 6
29 14 14 18 18
40 27 22
44 34 31 34 38
45 36 34 34 40
45 35" 34 34" 40
38 47 19 20 25
39 50 25 21 27
39 51" 26 21 28
82 82 50 54 63
84 85 58 56 67
84 92" 60 56" 68
Route
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d
Dose (mg/kg)
9 15
31 31
Note. Three animals per dose group except where noted. "Urine or fecal data available from only one animal and only one pair of combined data was used.
administration (Figs. 1 and 2) and the elimination data (Tables 1-4) revealed that the terminal (/3) phases were parallel, independently of the dose. The terminal rate was highest for pyrene, very low for the apparent metabolites and bound material and, consequently, intermediate for the 14C material. In contrast, the initial (a) phase was not parallel across doses, the slopes at higher doses being significantly shallower than those at lower doses. This behavior clearly indicated a nonlinearity in the kinetics with dose, presumably due to saturable metabolic or physiological processes. TABLE 9. Mean Tissue Levels of 14C and Pyrene, Expressed as a Dose-Adjusted Parameter in ftg/g Wet Tissue/(mg/kg body weight) after iv and po Administration Dose
Brain
Lung
Heart
Liver
2 4 6 9 15
0.0135 0.0168 0.0104 0.0111 0.0127
0.130 0.174 0.269 0.224 0.218
0.0405 0.0552 0.0409 0.0383 0.0343
0.259 0.415 0.437 0.287 0.359
2 4 6 9 15
0.0347 0.0149 0.0102 0.0411 0.0171
0.380 0.151 0.227 0.083 0.125
0.0878 0.0588 0.0366 0.0384 0.0425
0.640 0.523 0.342 0.300 0.326
Spleen
Kidney
Testis
Fat
0.0397 0.0470 0.0533 0.0437 0.0494
0.418 0.661 0.624 0.421 0.394
0.0378 0.0530 0.0536 0.0276 0.0423
1.25 1.64 1.37 1.32 1.62
0.0722 0.0434 0.0321 0.0464 0.0436
1.027 0.724 0.576 0.460 0.476
0.0970 0.0634 0.0301 0.0419 0.0404
0.702 1.071 0.764 1.067 1.713
iv Route
po Route
Note. Three animals per dose group.
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For reasons that have already been pointed out, the terminal phase of some of the data obtained after oral dosing was not clearly defined. This was particularly the case for 2 animals in the 15 mg/kg dose group where the rebound phenomenon was clearly evident between 6 and 8 h postdosing. Nevertheless, reasonable data fits were obtained in most cases for the biexponential model, similar to that used in the recent study by Modica et al. (1983). The apparent increase in the rate of excretion, prior to the rebound phenomenon, may be due to an increase in biliarly flow as a consequence of the action of the highly lipid dose of pyrene and its significant biliary excretion (Greenberger and Isselbacher, 1987). Other parameters that were consistent across doses were the absorption rate (with a half-life of about 10 min) and a lag time of about 4 min. In addition, the average apparent volume of distribution both for pyrene and 14C following administration by the iv route was about 180 ml and were also remarkably similar at all dose levels (Table 1). The apparent volume of distribution following oral administration (Table 3) was about 390 ml, which gave a value of about 50% for the bioavailability. This value was similar to that deduced from the areas under the blood concentration-time curves. The areas under the curve for the iv and oral data (Tables 1 and 3) showed, not unexpectedly, that values for the iv data were greater than those following oral administration. When these values were adjusted, by dividing by the respective administered doses, a significant (p < .05) increase with dose was noted for pyrene following iv or oral administration. Significance was nearly reached for 14C following iv but not after oral dosing. Increases in the dose-adjusted areas with increasing dose, particularly in the case of the pyrene data, again suggested that the kinetics were nonlinear. The mean values for the bioavailability for pyrene (Table 7) were about 60% while that for the 14C data were around 74%. Relatively stable values calculated from the volume of distribution for pyrene also supported these values. These observations were contrary to the observations of Mitchell and Tu (1979), who claimed that an orally administered aqueous suspension of pyrene was poorly absorbed from the gastrointestinal tract of the rat. At the lowest administered dose (2 mg/kg) both urinary and fecal excretion of pyrene were qualitatively identical after iv and oral administration. For either route, about 45 and 40% of the administered dose was excreted via the urine and feces over the 6-d collection period. This was contrary to the observation, in several communications, that the feces was the principal route of excretion for PAHs (Kotin et al., 1959; Sun et al., 1982; Sanders et al., 1984), although the doses used in the present study (2-15 mg/kg) were very much larger than those administered in previous studies (between 5 and 515 pg/kg). Distribution of 14C to the tissues was very much as anticipated from
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previous studies (Kotin et ah, 1959; Mitchell and Tu, 1979). High levels were found in the fat, probably as a consequence of the high lipid solubility of pyrene, followed by the liver and kidney, probably due to the high metabolic activity of these tissues (Daniel et al., 1967; Smith and Doody, 1981; Tyrer et al., 1981; Modica et al., 1983). Uptake to the fat and to the other tissues probably involved some redistribution so that part of the absorbed dose would be transferred from tissues having a high blood flow but low capacity to those having a low blood flow and high capacity. Precise evaluation of model kinetic parameters was complicated by their nonlinear dose dependence and enterohepatic recycling. These factors would also make the application of physiologically based pharmacokinetics somewhat complicated. Clearly, the pharmacokinetics of pyrene was remarkably different from that of benzo[a]pyrene. In particular, the risk assessment methodology that has been recommended for mixtures of compounds with a similar chemical structure, like the PAHs, in which the monitored PAH levels were converted (using potency factors) to benzo[a]pyrene equivalents, must be questionable (U.S. EPA, 1984; Thorslund and Charnley, 1988).
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Received August 1, 1990 Accepted November 2, 1990
