Formation and Removal of Pentachlorophenol-Derived Protein Adducts in Rodent Liver under Acute, Multiple, and Chronic Dosing Regimens

Chin-Hsiang Tsai*, Po-Hsiung Lin{dagger}, Melissa A. Troester* and Stephen M. Rappaport*,1

* Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27599-7400; and {dagger} Department of Environmental Engineering, National Chung-Hsing University, Taichung, Taiwan

Received December 18, 2002; accepted February 4, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the kinetics of production and elimination of chlorinated quinone adducts of liver cytosolic proteins derived from pentachlorophenol (PCP), following oral administration under acute dosing (0–40 mg/kg body weight [bw] in Sprague-Dawley rats, 0–120 mg/kg bw in F344 rats, and 0–60 mg/kg bw in B6C3F1 mice), multiple dosing (0–60 mg/kg bw/day for 5 days in F344 rats and B6C3F1 mice), and chronic feeding (60 mg/kg bw/day for 6 months in F344 rats). We measured adducts of both tetrachloro-1,2-benzoquinone (Cl4-1,2-BQ) and tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) following reduction of cysteinyl adducts by Raney nickel and gas chromatography-mass spectrometry. Ratios of Cl4-1,2-BQ to Cl4-1,4-BQ adducts were much greater in mice (0.8–2) than in F344 rats (0.04–0.07), indicating that Cl4-1,2-BQ is an important PCP-binding species in mice but not rats. Following acute administration of 20 mg PCP/kg bw to Sprague-Dawley rats and B6C3F1 mice, the time course of adduct elimination over 14 days followed biphasic kinetics, with a rapid phase representing at least 92% of the adduct burden. Using data from acute experiments, we predicted adduct levels in rats and mice after the multiple- and chronic-dosing regimens. The agreement between predicted and observed levels was good (intraclass correlation coefficients of predicted and observed pairs of logged adduct levels were 0.812–0.921). These results provide evidence that the kinetics of liver protein adducts were not influenced by the dosing regimen of PCP, a recognized toxicant of the liver.

Key Words: pentachlorophenol; quinone; liver protein adducts; protein turnover; adduct stability.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After decades of widespread use as a wood preservative, pentachlorophenol (PCP) has become a ubiquitous environmental contaminant (Seiler, 1991Go; WHO, 1987Go). PCP is a recognized toxicant of the liver (reviewed by Lin et al., 1996Go) and has produced liver tumors in mice and mesotheliomas in rats (Chhabra et al., 1999Go; McConnell et al., 1991Go; NTP, 1989Go, 1999Go). It is also suspected that long-term exposures to PCP may cause human cancers (Greene et al., 1978; Hardell and Sandstörm, 1979Go; Hardell et al., 1994Go; Pearce et al., 1986Go; Roberts, 1990Go).

Although the mechanism by which PCP produces tumors is unclear, metabolic activation to chlorinated quinones is thought to play a role (Ehrlich, 1990Go; Lin et al., 2001Go; Witte et al., 1985Go). As shown in Figure 1AGo, PCP is metabolized to tetrachlorohydroquinone (Cl4HQ) and tetrachlorocatechol (Cl4CAT), which can be oxidized to tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) and tetrachloro-1,2-benzoquinone (Cl4-1,2-BQ) via the respective semiquinone intermediates (Renner and Hopfer, 1990Go; Van Ommen et al., 1986Go, 1988Go). Alternatively, PCP can be converted into quinones directly, via peroxidases, hydroperoxides (Samokyszyn et al., 1995Go; Tsai et al., 2001Go), or P450-catalyzed oxidation (Rietjens et al., 1997Go), without the involvement of hydroquinones.



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FIG. 1. (A) Proposed pathways for PCP metabolism. (B) The formation of quinone adduct in proteins followed by reaction with Raney nickel and HFBI derivatization. Note that YSH could be a protein and/or nonprotein thiol such as GSH and that quinone adducts can continue to react with YSH to form multiple-S-substituted adducts.

 
Chlorinated quinones from PCP metabolism react with macromolecules to form adducts (Bodell and Pathak, 1998Go; Lin et al., 1996Go; Tsai et al., 2001Go; Van Ommen et al., 1988Go; Waidyanatha et al., 1996Go). Since the residence times of these reactive quinones are very short in vivo, longer-lived protein adducts offer the means to estimate doses of PCP-quinones to the liver (a target tissue). Dose-dependent production of cysteinyl adducts has been reported in liver cytosolic proteins (hereafter simply "cytosolic proteins") of rats and mice following a single administration of PCP (0–40 mg/kg bw; Lin et al., 1999Go). Moreover, following oral administration of [14C]PCP, five times more PCP was covalently bound to liver proteins of mice than to those of rats (Tsai et al., 2002Go).

In order to use protein adducts for dosimetry, it is necessary to employ kinetic relationships that rely upon rates of adduct formation and elimination in vivo (Ehrenberg et al., 1983Go; Fennell et al., 1992Go; Granath et al., 1992Go; Troester et al., 2001Go). We previously estimated rates of formation and elimination of selected PCP-derived adducts (i.e., those from Cl4-1,4-BQ) of cytosolic proteins from rats and mice following a single dose of 0–40 mg PCP/kg bw (Lin et al., 1997Go, 1999Go). Those studies did not include adducts of Cl4-1,2-BQ, due to the lack of appropriate reference standards.

Since PCP is hepatotoxic, it is reasonable to postulate that the rates of adduct formation and/or elimination could be altered under multiple or chronic dosing regimens. For example, Walker et al.(1992)Go presented evidence that hemoglobin adducts of ethylene oxide were eliminated more rapidly following multiple administration of ethylene oxide than single administration, and attributed this to enhanced turnover of older erythrocytes. Since adducts of cytosolic proteins should be partially eliminated with the turnover of hepatocytes, we examined whether early hepatotoxic effects of PCP, arising from multiple or chronic administration, might increase the rates of elimination of quinone adducts. Although the validity of extrapolating adduct dosimetry from acute to multiple- and/or chronic-dosing scenarios is an important issue, it has rarely been addressed experimentally.

In the current investigation, we measured PCP-derived quinone adducts in rats and mice following oral administration under acute (0–120 mg/kg bw), multiple (30, 60 mg/kg bw/day for 5 days), and chronic (60 mg/kg bw/day for 6 months) dosing with PCP. This allowed us to compare adduct levels arising from both Cl4-1,4-BQ and Cl4-1,2-BQ in rats and mice and also to compare adduct levels following multiple or chronic dosing with those predicted from acute administration.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
PCP (99% pure), bis-tris-propane, and Raney nickel (pore size 50 mm, 50% slurry in water) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Protease XIV (Pronase E), 4,6-dichlororesorcinol, and diethylenetriaminepentaacetic acid (DTPA) were obtained from the Sigma Chemical Co. (St. Louis, MO). Methyl-tert-butyl ether (MTBE) and ascorbic acid were obtained from Fisher Scientific (Pittsburg, PA). N-Heptafluorobutyrylimidazole (HFBI) was purchased from Pierce (Rockford, IL). Isotopically labeled protein-bound internal standards were synthesized, as described by Lin et al.(1996)Go, for [13C6]Cl4-1,4-BQ, and by Tsai et al.(2001)Go, for [13C]Cl4-1,2-BQ. All other chemicals were the same as those reported by Tsai et al.(2001)Go.

Animal Studies
Adducts were measured in cytosolic proteins obtained from new experiments and in stored-liver proteins from previous studies described by Lin et al. (1996Go, 1997Go, 1999Go). The specimens from prior studies were assayed to include measurements of adducts of Cl4-1,2-BQ, which had not been analyzed previously.

New studies.
Male F344 rats (approximately 200 g) and B6C3F1 mice (approximately 25 g) were obtained from Charles River Breeding Laboratories (Raleigh, NC). Animals were kept in polycarbonate cages on a 12-h light/dark cycle and food and water were provided ad libitum. For the acute study, 6 animals per group received a single dose of PCP in corn oil by gavage (rats: 0, 30, 60, or 120 mg PCP/kg bw; mice: 0, 30, or 60 mg PCP/kg bw). Three animals from each group were sacrificed 4 h (30 and 60 mg/kg only) and 24 h after administration by exsanguination via cardiac puncture while anesthetized with methoxyflurane. For the multiple-dosing study, three animals per group of rats and mice received one gavage dose of PCP in corn oil per day (0, 30, or 60 mg PCP/kg bw) for 5 consecutive days. Animals were sacrificed 4 h after the last administration by exsanguination via cardiac puncture while anesthetized with methoxyflurane. Immediately after the collection of blood, livers were perfused with 0.25 M sucrose solution (1 ml/10 g bw), excised, washed in 0.25 M sucrose solution, and stored at -80°C until further processing.

Previous studies.
Tissues were reanalyzed from two previous acute studies of Sprague-Dawley rats and B6C3F1 mice and one previous chronic feeding study of F344 rats (Lin et al., 1996Go, 1997Go, 1999Go). In the first acute study, groups of 3 rats and 3 mice received a single dose of PCP in corn oil by gavage (0, 5, 10, 20, or 40 mg/kg bw), and were sacrificed 24 h after administration. In the second acute study, groups of 3 rats and mice received a single dose of PCP in corn oil by gavage (20 mg/kg bw) and were sacrificed at 0.02, 0.04, 0.08, 0.17, 0.33, 1, 2, 7, and 14 days after administration. In the chronic feeding study, conducted by the National Toxicology Program (1999)Go, livers were collected from 10 male F344 rats that had been fed a diet containing 1000 ppm of PCP (equivalent to 60 PCP mg/kg bw/day) for 6 months. Cytosolic proteins from these animals had been stored at -80°C for 36 months prior to analysis in the current investigation.

Isolation of liver cytosolic proteins.
All fresh livers from new and old experiments were perfused with 0.25 M sucrose and stored at -80°C until further processing. Livers were thawed and homogenized in 4 volumes of phosphate buffer with 8–10 strokes using a Potter-Elhjem tissue grinder in an ice bath, and centrifuged at 2000 x g to remove the nuclei. The supernatant was centrifuged at 15,000 x g for 20 min to remove mitochondria and the resulting supernatant was centrifuged at 105,000 x g for 60 min to remove microsomes. The 105,000 x g supernatant was dialyzed and dried as described in Tsai et al.(2001)Go.

Measurement of quinone adducts.
In what follows, we designate the products of mono-S- and multi-S-substituted quinone adducts as Clx-1,4-BQ-Y(4-x) and Clx-1,2-BQ-Y(4-x), where Y denotes a sulfhydryl group and 1 <= x <= 3 represents the number of chlorine atoms remaining on the aromatic ring.

PCP-derived adducts of Cl4-1,4-BQ and Cl4-1,2-BQ with cytosolic proteins were measured as described in Lin et al.(1999)Go. As shown in Figure 1BGo, the method relies upon catalytic reduction of each sulfur-bound adduct by Raney nickel to release the adducted moiety as a hydroquinone, which is then reacted with HFBI to produce a volatile derivative, suitable for GC-MS analysis. Briefly, 1 ml of 0.9 M ascorbic acid, 1 ml of 1 M bis-tris-propane buffer (pH 7.0), and the isotopically labeled protein-bound internal standards (5–10 mg containing 152 to 2050 pmol adduct/g protein) were added to purified proteins. The mixture was digested with pronase E at 37°C for 4 h. After adding 1 ml of 0.9 M ascorbic acid containing 1 mM DTPA and acidification of the sample to pH 1–2 with hydrochloric acid, the samples were extracted with MTBE to remove interfering compounds. Following the addition of 100 pg (100 µl of a 1 ng/ml solution) of 4,6-dichlororesorcinol, an unbound internal standard, the samples were reacted with Raney nickel at room temperature for 10 min. After acidifying the medium to pH 1–2, samples were extracted with MTBE, derivatized with HFBI, and analyzed by GC-MS with negative-ion-chemical ionization (NICI).

GC-MS analysis.
Samples were analyzed by GC-NICI-MS using a HP 5890 gas chromatograph coupled to a HP 5989A mass spectrometer. A DB-5 fused silica capillary column (30 m x 0.25-mm i.d., 1-µm phase thickness, J&W Scientific, Folsom, CA) was used at a carrier gas (He) flow rate of 1 ml/min. Injections (3 µl) were made in the splitless mode. Methane was used as the chemical ionization reagent gas. The injection port and the ion source temperatures were 250°C and 150°C, respectively. The GC oven temperature was held at 75°C for 2 min, increased at 10°C per min to 135°C, held for 8 min, and then increased at 12°C per min to 200°C. Late-eluting compounds were removed by increasing the temperature at 50°C per min to 250°C where it was held for 10 min. HFBI derivatives of all adducts were quantified in the selected ion-monitoring mode using the following ions: Cl-HQ, m/z 339; Cl2-HQs, m/z 373; Cl3-HQ, m/z 409; [13C6]Cl-HQ, m/z 345; [13C6]Cl2-HQs, m/z 379; [13C6]Cl3-HQ, m/z 415; Cl-CATs, m/z 339; Cl2-CATs, m/z 373; Cl3-CATs, m/z 407; [13C]Cl-CATs, m/z 340; [13C]Cl2-CATs, m/z 374; and [13C]Cl3-CATs, m/z 408. Figure 2Go shows NICI mass spectra of 3,4,5-Cl3-CAT-HFB, obtained in scan mode, from a reference standard (Fig. 2AGo) and from a sample of cytosolic protein obtained from a mouse to which 60 mg of PCP/kg bw has been administered (Fig. 2BGo).



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FIG. 2. NICI mass spectra of 3,4,5-Cl3-CAT-HFB from (A) an analytical standard and (B) a sample of cytosolic protein obtained from a mouse to which 60 mg of PCP/kg bw has been administered.

 
Mathematical Models
Adduct production and elimination.
Let A(t) (nmol/g protein), represent the amount of each quinone adduct present at time t, following a single administration of PCP to a rat or mouse. We estimated A(t) for each adduct from a time course study between 0.02 and 14 days in Sprague-Dawley rats and B6C3F1 mice following a single dose of 20 mg PCP/kg bw (samples from Lin et al., 1996Go, 1997Go). For all adducts, we observed biphasic elimination with an initial rapid-elimination phase between 0.2 and 2 days and a slower phase thereafter. Thus, A(t) was modeled as:


(1)

where A1(t) and A2(t) represent adduct levels in the short-lived and long-lived populations at time t, respectively. Assuming instantaneous adduct production and first-order elimination, Equation 1Go can be rewritten in terms of the theoretical initial adduct levels, namely, A1(0) and A2(0), as:


(2)

where k1 and k2 represent the first-order elimination rates (d-1) of A1 and A2, respectively. Estimates of A1(0), A2(0), k1 and k2 were obtained from Equation 2Go by nonlinear regression of the observed adduct levels on t, using software from SlideWrite Plus (Advanced Graphic Software, Carlsbad, CA). The coefficients of determination were 0.930 <= r2 <= 0.996. Also, the proportions of the short- and long-lived adduct populations were estimated at time t from Equation 2Go as {alpha}(t) and [1–{alpha}(t)], respectively, for


(3)

where t = 4 h, and all parameters refer to estimated values.

Predicting adduct levels following multiple and chronic dosing.
Let A(n) represent the level of each adduct after n days of dosing with PCP. Our goal was to predict A(n), based upon data obtained from acute dosing, and then to compare these predictions with observed values after multiple or chronic dosing. Since groups of three animals were sacrificed 4 h after the 5th daily administration of PCP at 30 or 60 mg/kg bw, predictions were based upon the average adduct level, observed in three animals, 4 h after a single administration of 30 or 60 mg/kg bw [denoted A(1), nmol/g]. With repeated daily administrations of PCP, the level of each adduct 4 h after the 5th dosing was predicted as:


(4)

where n = 5, {Delta}t = 1 day and all parameters refer to estimated values. Following chronic administration of PCP by feeding for 6 months, steady-state adduct levels [A(s.s.)] were predicted with the following approximate expression:


(5)

where all parameters refer to estimated values. Observed values were estimated as the average levels among animals following multiple dosing [A(5)] or chronic dosing [A(s.s.)].

Statistical tests.
For the adduct-elimination study, data obtained prior to peak adduct levels were excluded from nonlinear regression analyses. After peak adduct levels are achieved, it can be assumed that adduct formation no longer contributes to the adduct burden and that the rate of change of the adduct concentration is a function of adduct removal only. Agreement between predicted and observed adduct levels, following multiple or chronic dosing, was evaluated as suggested by Lee et al.(1989)Go. Good agreement is indicated when the estimated intraclass correlation coefficient (ICC) for data pairs (in this context, predicted and observed levels of each adduct) approaches unity, when no significant difference can be detected between mean data pairs, and when no bias is detected in a scatter plot of data pairs. In our study, predicted and observed adduct levels from multiple and chronic dosing were obtained for all measured quinone adducts in rats or mice following administration of either 30 or 60 mg PCP/kg bw. Statistical analyses were performed upon the natural logarithms of these values to satisfy assumptions regarding uniform variance and normality. Estimates of the ICCs (from pairs of logged adduct levels), their 95% confidence intervals, and the corresponding paired t-tests were performed with SAS software (v8.03, SAS Institute, Cary, NC).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Characterization of Cl4-1,2-BQ-Derived Adducts in Rodents
Because of the lack of a chemically equivalent internal standard, Cl4-1,2-BQ adducts had not previously been quantified in cytosolic proteins from rats and mice dosed with PCP (Lin et al., 1996Go, 1997Go, 1999Go). In the current study, we applied a refined procedure (Tsai et al., 2001Go), which included isotopically-labeled internal standards of [13C]Cl4-1,2-BQ in addition to [13C6]Cl4-1,4-BQ. One mono-S- (i.e. 3,4,5-Cl3-1,2-BQ-Y) and four multi-S-substituted (i.e., 4,5-Cl2-1,2-BQ-Y2, 3,4-Cl2-1,2-BQ-Y2, 4-Cl-1,2-BQ-Y3, and 3-Cl-1,2-BQ-Y3) adducts arising from Cl4-1,2-BQ were detected in cytosolic proteins of F344 rats and B6C3F1 mice following acute, multiple, and chronic dosing. No Cl4-1,2-BQ-derived adducts were detected in cytosolic proteins from Sprague-Dawley rats.

Production of PCP-Derived Liver Protein Adducts
Acute dosing.
Levels of mono- and multi-S-substituted adducts arising from Cl4-1,4-BQ and Cl4-1,2-BQ were aggregated to obtain total adduct levels as summarized in Figures 3Go (rats) and 4 (mice) for animals sacrificed 24-h after administration (note the separate Y-axes). Saturation of production was observed for adducts of Cl4-1,4-BQ but not of Cl4-1,2-BQ in both species. There was no apparent difference in the production of aggregated Cl4-1,4-BQ adducts between Sprague-Dawley and F344 rats at doses of 0–40 mg PCP/kg bw (Fig. 3Go). Saturated production of Cl4-1,4-BQ adducts was only observed at the highest dose of 120 mg PCP/kg bw in F344 rats (Sprague-Dawley rats were not dosed above 40 mg PCP/kg bw.).



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FIG. 3. Production of chlorinated quinone adducts from liver cytosolic proteins in Sprague-Dawley rats (samples from Lin et al., 1996Go), following a single administration of 0, 5, 10, 20, or 40 mg/kg bw, and in F344 rats, following a single administration of 0, 30, 60, or 120 mg PCP/kg bw. Mean values and standard errors are shown for 3 animals per group for adducts from chlorinated 1,4-benzoquinones in Sprague-Dawley rats (triangle) and F344 rats (diamond), and chlorinated 1,2-benzoquinones in F344 rats (circle).

 
Table 1Go shows adduct levels measured in animals dosed with PCP at 30 or 60 mg/kg body wt and sacrificed 4 h after administration. The results indicate that some adducts were produced at greater levels than others, with Cl3-1,4-BQ-Y being the major product in F344 rats and 3,4,5-Cl3-1,2-BQ-Y predominating in B6C3F1 mice.


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TABLE 1 Levels of Liver Cytosolic-Protein Adducts (A(1), nmol/g Protein) Observed in F344 Rats and B6C3F1 Mice 4 h following a Single Administration of Either 30 or 60 mg PCP/kg bw
 
Multiple and chronic dosing.
Tables 2Go and 3Go present adduct levels observed 4 h after the 5th daily administration in F344 rats and B6C3F1 mice at doses of 30 or 60 mg PCP/kg bw and after chronic feeding of F344 rats at 60 mg PCP/kg bw/day for 6 months. The corresponding aggregated adduct levels are illustrated in Figure 5AGo (rats) and 5B (mice). In rats, levels of quinone adducts increased with administered dose in a roughly linear fashion after 5 days of dosing (Fig. 5AGo), consistent with the behavior observed following acute administration over 30–60 mg PCP/kg bw (Fig. 3Go). Likewise, linear production of Cl4-1,4-BQ adducts was observed following multiple administration of PCP to mice (Fig. 5BGo). However, multiple administration led to pronounced saturation of levels of Cl4-1,2-BQ adducts in mice (Fig. 5BGo), mainly due to the most prominent species, i.e., 3,4,5-Cl3-1,2-BQ (Table 3Go).


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TABLE 2 Predicted and Observed Levels of Liver Cytosolic-Protein Adducts (nmol/g Protein) in F344 Rats following Multiple or Chronic Dosing
 

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TABLE 3 Predicted and Observed Levels of Liver Cytosolic-Protein Adducts (nmol/g Protein) in B6C3F1 Mice following Multiple Dosing
 


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FIG. 5. Production of chlorinated quinone adducts from liver cytosolic proteins in F344 rats (A) and B6C3F1 mice (B), following multiple administration of PCP (0, 30, or 60 mg/kg bw/day) for 5 days. Mean values and standard errors are shown for three animals per group for adducts from chlorinated 1,4-benzoquinones (diamond) and chlorinated 1,2-benzoquinones (circle).

 
Steady-state adduct levels are also summarized in Table 2Go for F344 rats, which had been chronically fed 60 mg PCP/kg bw/day for 6 months. Aggregated adduct levels derived from Cl4-1,4-BQ and Cl2-1,2-BQ were 15.0 and 1.07 nmol/g protein, respectively.

Adduct elimination.
The elimination of cytosolic-protein adducts in vivo was investigated from a time course study following a single dose of 20 mg PCP/kg bw. Figure 6Go shows semi-logarithmic plots of these data in rats (Fig. 6AGo) and mice (Figs. 6BGo and 6CGo) up to 14 days after dosing. In both species, the biphasic model adequately fit the data (Equation 2Go), suggesting two adduct populations. Estimates of adduct-elimination-rate constants and the theoretical initial levels of these adducts, obtained by fitting Equation 2Go to the acute time course study, are shown in Table 4Go. Adduct elimination rates were 0.973–1.26 d-1 (rat) and 0.642–1.32 d-1 (mice) for rapid-phase elimination, and 0.227–0.306 d-1 (rats) and 0.015–0.107 d-1 (mice) for slow-phase elimination, depending upon the particular adduct. Rates of elimination could not be estimated for Cl4-1,2-BQ adducts in Sprague-Dawley rats because these adducts were below the detection limits.



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FIG. 6. Time course of adducts of liver cytosolic proteins following a single administration of 20 mg PCP/kg bw. (A) Adducts from chlorinated 1,4-benzoquinones in Sprague-Dawley rats; (B) adducts from chlorinated 1,4-benzoquinones in B6C3F1 mice; (C) adducts from chlorinated 1,2-benzoquinones in B6C3F1 mice. Mean values and standard errors are shown for three animals per group. The curves represent the fits of Equation 2Go to data after peak adduct levels were reached.

 

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TABLE 4 Estimated Adduct Elimination Rates and Initial Adduct Levels of Two Populations of Liver Cytosolic-Protein Adducts in Sprague-Dawley Rats and B6C3F1 Mice Following Acute Dosing
 
Predicted vs. observed adduct levels following multiple and chronic dosing.
The adduct levels predicted under Equation 4Go for multiple dosing and Equation 5Go for chronic dosing are also included in Tables 2Go (rats) and 3Go (mice). Scatter plots showing all predicted and observed data pairs are presented in Figures 7AGo (rats) and Go7B (mice). Although the scatter plot for mice shows somewhat more variability from the line of strict equality than that for rats, there is little evidence of bias in either case. Overall, the agreement between predicted and observed values was good as summarized in Table 5Go (ICC: 0.812–0.969 for rats and 0.921–0.939 for mice). For rats in the multiple exposure experiments, observed adduct levels were marginally greater than those predicted (about 34%; p = 0.070 at 30 mg/kg bw and p = 0.012 at 60 mg/kg bw). On the other hand, no statistical evidence of differences was observed between predicted and observed levels for rats chronically dosed with PCP (p = 0.468), nor in mice from the multiple-dosing experiments (p >= 0.156).



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FIG. 7. Scatter plot of predicted and observed data pairs in (A) rats and (B) mice following multiple (30 [circle], 60 [triangle] mg/kg bw/day for 5 days) or chronic (60 [X] mg/kg bw/day for 6 months) administration of PCP. Note that the 45-degree line represents strict equality of predicted and observed adduct levels.

 

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TABLE 5 Agreement between Predicted and Observed Adduct Levels Based on Estimates of the Intraclass Correlation Coefficients (ICC) and Difference between Predicted and Observed Values
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Due to improvements in our assay, we were able to measure Cl4-1,2-BQ in cytosolic proteins from both F344 rats and B6C3F1 mice. Although we had measured Cl4-1,2-BQ adducts following incubation of PCP with microsomes and cumene hydroperoxides in vitro (Tsai et al., 2001Go), the current study confirms that Cl4-1,2-BQ adducts were also produced in vivo. The ratios of Cl4-1,2-BQ adducts to Cl4-1,4-BQ adducts were much greater in mice (0.8–2) than in F344 rats (0.04–0.07), confirming our previous conjecture that Cl4-1,2-BQ was an important PCP-binding species in mice but not rats (Lin et al., 1997Go). This result may explain, in part, the species difference in PCP-derived liver tumorigenesis. Adducts of Cl4-1,2-BQ were not detected in Sprague-Dawley rats at doses up to 40 mg PCP/kg bw.

We measured chlorinated quinone adducts in the livers of Sprague-Dawley or F344 rats and of B6C3F1 mice sacrificed 24 h after a single administration of PCP (Figs. 3Go and 4Go). Comparable amounts of Cl4-1,4-BQ adducts were produced in Sprague-Dawley and F344 rats. Saturation was observed in the formation of Cl4-1,4-BQ adducts in both F344 rats and B6C3F1 mice at administered doses above 40 mg PCP/kg bw. However, no saturation was observed in production of Cl4-1,2-BQ adducts in either F344 rats or B6C3F1 mice.



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FIG. 4. Production of chlorinated quinone adducts from liver cytosolic proteins in B6C3F1 mice, following a single administration of 0–60 mg PCP/kg bw. Mean values and standard errors are shown for three animals per group for adducts from chlorinated 1,4-benzoquinones (diamond) and chlorinated 1,2-benzoquinones (circle).

 
The elimination of cytosolic-protein adducts in vivo was investigated in a time course study following a single dose of PCP to Sprague-Dawley rats and B6C3F1 mice. Semi-logarithmic plots of these data (Fig. 6Go) displayed biphasic elimination kinetics up to 14 days after dosing. From the data in Table 4Go, we estimate that between 91.8 and 99.8 percent of each measured adduct was eliminated during the rapid phase. Estimates of the rapid-phase elimination rate constant (k1= 0.642–1.21 d-1, Table 4Go) are much larger than published rates of liver cytosolic protein turnover, i.e., kCp = 0.11–0.14 d-1 (t1/2 = 5.1–6.2 days) in rats (Arias et al., 1969Go) and kCp = 0.12–0.23 d-1 (t1/2 = 3.0–5.7 days) in mice (Miller et al., 1976Go). This may be due to instability of quinone adducts arising from additional substitution reactions following nucleophilic attack by sulfhydryl moieties to displace chlorines (Lin et al., 1996Go; Van Ommen et al., 1986Go; Waidyanatha et al., 1996Go). Similar behavior has recently been reported for albumin adducts of 1,4-benzoquinone and 1,2- and 1,4-naphthoquinone in F344 rats (Troester et al., 2000Go, 2002Go). Judging by the parallel curves of adduct loss for the various chlorinated quinones (Fig. 6Go), these substitution reactions appear to proceed at similar rates for all adducts.

It is also worth noting that first-order rates of adduct elimination have generally been estimated from relations developed for albumin by Granath et al.(1992)Go:


(6)

where k' (d-1) is the first-order rate constant of adduct elimination. Like albumin, the turnover of cytosolic proteins has been shown to follow first-order kinetics (Arias et al., 1969Go). Therefore, under Equation 6Go, it would be assumed that cytosolic-protein adducts would be eliminated from a single adduct population either at rate k' = kCp, for stable adducts, or at rate k' = kCp + k for unstable adducts, where k represents the rate of adduct instability. However, in contrast to albumin, cytosolic proteins include multiple different intracellular proteins with diverse functions. A first-order model (consistent with Arias et al., 1969Go) assumes that the degradation of the pool of cytosolic proteins can be approximated by a single first-order rate constant kCp (representing the sum of individual protein turnover rate constants). In our study, biphasic kinetics (Equation 2Go) were observed, indicating the presence of two adduct populations with markedly different rate constants for removal (short-lived and long-lived). These distinct adduct populations may reflect different sites of protein adduction, with some sites being more accessible for additional reactions (leading to greater instability) than others. Alternatively, certain adducted intracellular proteins may be targeted for degradation. Jelinsky and Samson (1999)Go presented evidence in S. cerevisiae that treatment with alkylating agents induced transcription of genes involved in eliminating and replacing damaged proteins. Suggestions of biphasic kinetics have been reported for various quinone-protein adducts arising from metabolites of PCP (cytosolic-protein and albumin adducts; Lin et al., 1997Go; Waidyanatha et al., 1996Go), and naphthalene (albumin adducts; Troester et al., 2002Go). In this paper, we propose a model that employs biphasic kinetics to describe adduct stability. We note that such an empirical model cannot offer mechanistic insight as to whether protein degradation or multiple substitution reactions are responsible for adduct turnover. The value of this empirical model is in allowing predictions of adduct levels to be made.

Under multiple and chronic dosing regimens, levels of adducts accumulate daily until a steady state is reached. Using data from acute experiments, we predicted adduct levels in rats and mice after 5 daily administrations of 30 or 60 mg PCP/kg bw and following chronic feeding of rats at 60 mg PCP/kg bw. We then compared these predictions with adduct levels measured in animals dosed with PCP according to the same regimens (Fig. 7Go). The agreement between predicted and observed adduct levels was quite good overall, although observed values were marginally (about 34%) greater than those predicted in rats in the multiple dosing experiments (Table 5Go). These results provide evidence that liver cytosolic-protein adducts were not unduly influenced by the dosing regimen of PCP, a recognized toxicant of the liver.

In conclusion, we confirm that PCP metabolism leads to the production of multiple adducts arising from both Cl4-1,2-BQ and Cl4-1,4-BQ in rats and mice. Production of Cl4-1,4-BQ adducts was more pronounced in rats and displayed evidence of saturable kinetics in both species. Production of Cl4-1,2-BQ adducts was more pronounced in mice and was proportional to dose over the full ranges investigated in both species. Biphasic elimination of cytosolic protein adducts was observed following administration of PCP, suggesting different sites of adduction or degradation of damaged proteins. Finally, adduct levels observed following multiple or chronic administration of PCP were consistent with those predicted from acute administration, indicating that the kinetics of adduct production and elimination were not affected by the dosing regimen.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (919) 966-0521. E-mail: stephen_rappaport{at}unc.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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