Chem. Res. Toxicol. 1994,7, 463-468
463
Measurement of Hemoglobin and Albumin Adducts of Tetrachlorobenzoquinone Suramya Waidyanatha, Thomas A. McDonald, Po-Hsiung Lin, and
Stephen M. Rappaport'
Department of Environmental Science and Engineering, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599- 7400 Received January 7, 1994"
Tetrachloro-l,4-benzoquinone (Cl4BQ),a metabolite of pentachlorophenol (PCP),is believed to play a role in the genotoxicity of PCP. We have developed a method to measure the adducts of CLBQ with cysteine residues of hemoglobin (Hb) and albumin (Alb). This method employs the use of Raney nickel to selectively cleave the sulfur-bound adducts. Adducts of Hb and Alb with CLBQ were measured following modification of rat blood with CLBQ (0-90 pM)in vitro. The formation of both Hb and Alb adducts was linear over the entire range with second-order rate constants of 6.89 and 167 L mol-l h-l, respectively. The proportions of the concentrations of these H b and Alb adducts to those of all covalently-bound products were estimated to be 0.053 and 0.178, respectively, a t initial C4BQ concentrations between 3 and 90 pM. The overall rate of reaction of CLBQ in rat blood (in vitro) was pseudo-first-order with an estimated halftime of 4.35 h. Hb and Alb adducts of CLBQ were also measured in vivo following oral administration of PCP to rats (0-20 mg/kg body wt). Linear production of H b and Alb adducts was observed over the entire range of dosages, with slopes of 0.09 and 8.22 pmol of adduct (g of protein)-l [(mg of PCP)/(kg body wt)]-l, respectively. On the basis of production of Hb adducts in vitro and in vivo, it is estimated that 2.7 X lo-' mol of CLBQ was released to the blood of rats per mole of PCP administered. In reconciling the enhanced production of Alb adducts relative to those of H b in these experiments, it is postulated that the transport of CLBQ is restricted either by cell membranes or by noncovalent binding to proteins. Introduction The worldwide use of pentachlorophenol (PCP)l as a general biocide has been estimated to be about 30000 tons annually (1). Use of such large quantities of an environmentally-stable compound has made PCP a ubiquitous contaminant (I). PCP has been detected in body fluids and tissues of people with and without known exposures ( 2 ) . Metabolism of PCP has been studied extensively both invivo and in vitro (3-8). PCP is oxidativelydechlorinated to produce tetrachlorocatechol and tetrachlorohydroquinone (CLHQ) by liver enzymes from rats and humans in vitro ( 4 , 6 )and by rodents in vivo ( 3 , 5 , 8 ) . CLHQ has been detected, either free or as conjugates, in the urine of PCP-treated rats ( 4 , 8 , 9 )and of workers exposed to PCP (3,7). Reigner et al. (9)reported that 1 % of the total dose of PCP administered to rats iv was recovered in the urine as free CLHQ, and 30% as CLHQ conjugates. The acute toxicity of PCP relates primarily to the uncoupling of oxidative phosphorylationin mitochondria (10). PCP is also known to inhibit cytochrome P-450 (11) and sulfotransferases (12)and is fetotoxic in the rat (13).
* To whom correspondence should beaddressedat the School of Public Health, C.B. No. 7400,University ofNorth Carolinaat Chapel Hill, Chapel Fax: 919/966-4711. Hill, NC 27599-7400.Tel: 919/966-5017; *Abstract published in Advance ACS Abstracts, April 1, 1994. 1 Abbreviations: Alb, albumin;DTPA, diethylenetriaminepentaacetic acid; ECD, electron capture detector; EI, electron ionization; HFBI, N-(heptafluorobutyry1)imidazole;MTBE, methyl tert-butyl ether;NICI, negative ion chemical ionization; PCP, pentachlorophenol;SIM, selected ion monitoring;CWQ, tetrachloro-l,4-benzoquinone;CLHQ, tetrachlorohydroquinone;ChHQ, trichlorohydroquinone; CWQ-Hb, trichlorobenzoquinone adduct of hemoglobin; ClgQ-Alb, trichlorobenzoquinone adduct of albumin. Q893-228J94127Q7-Q463$Q4.5Q/Q
However, current interest in the toxicology of PCP relates largely to the genotoxic potential of this compound because it has been shown to be carcinogenic in mice (14).Elevated levels of chromosomal aberrations have also been reported in the peripheral lymphocytes of workers in a factory where PCP was produced (15). CLHQ has been shown to be cytotoxic in human fibroblasts (16) and to cause single-strand breaks in bacteriophage PM2 DNA (16),in Chinese hamster ovary cells and in human fibroblasts (18). More recently, CLHQ has been shown to induce mutations (19) and micronuclei (20) in V79 Chinese hamster cells. Covalent binding of C4HQ to rat microsomal proteins and DNA (21) and to calf thymus DNA (16) has been reported in vitro. It has been suggested that either the semiquinone or benzoquinone form of CLHQ is responsible for this covalent binding (16,211. Conversion of CLHQ to the quinone species is thought to be mediated by superoxide anion formed by cytochrome P-450 (22)or by autoxidation (23). [These reactions would also produce reactive oxygen species,which are thought to be responsible for induction of DNA-strand breaks (16, 18, 19)J den Besten et al. (24)recently proposed a cytochrome P-450mediated mechanism for direct formation of tetrachloro1,Cbenzoquinone(CLBQ) from PCP via elimination of a halide ion. CLBQ has been detected at trace levels in the urine of rats treated with PCP (25). These studies suggest that the metabolites of PCP, Le., CLHQ, CLBQ, andlor a semiquinone,can be responsible for the genotoxicity of PCP. However, despite the fact that binding is known to occur, specific macromolecular adducts of these species have not been identified thus far. We report here the measurement of cysteinyl adducts of
(In,
0 1994 American Chemical Society
464
Chem. Res. Toxicol., Vol. 7,No. 3, 1994
CldBQ with the blood proteins hemoglobin (Hb) and albumin (Alb). These adducts are detected both in vitro, following incubation of blood with CbBQ, and in vivo, following administration of PCP t o rats. Because formation proceeds through covalent binding to the aromatic ring after an additional dechlorination step, these adducts are designated C13BQ-Hb and C13BQ-Alb, respectively.
Materials and Methods Chemicals. [1w6]Phenol (99+%)was purchased from Cambridge Isotope Laboratories (Woburn,MA). SbCb, hydroquinone (99+%),C&BQ (99%) , PCP (99%),Raney nickel (pore size 50 pm, 50% slurry in water), 1,3-bis[[tris(hydroxymethyl)methyllaminolpropane (bis-tris-propanebuffer) (99+% ), and magnesium chloride hexahydrate were from Aldrich Chemical Co. (Milwaukee, WI). (Heptafluorobutyry1)imidazole(HFBI) was purchased from Pierce (Rockford,IL). Protease XJY (Pronase E), Sephadex G-25 (20-80 pm), and diethylenetriaminepentaacetic acid (DTPA) were obtained from Sigma Chemical Co. (St. Louis, MO). Ascorbic acid, hydrogen peroxide (30%), acetic acid, hydrochloric acid (concentrated), acetone (nanograde),hexane, and methylene chloride (pesticidegrade) were from Fisher Scientific(Pittsburgh, PA). Methyl tert-butyl ether (MTBE) was from Mallinckrodt, Inc. (Paris, KY), anhydrous NazSO, was from J. T. Baker Co. (Marrietta, GA), and dichlororesorcinol was from TCI America (Portland, OR). All reagents were used without further purification. Water was purified with a Milli-Qsystem (Waters, Millipore Division, Bedford, MA). Synthesis of Trichlorohydroquinone. Trichlorohydroquinone (ClaHQ) was synthesized according to the method of Rettig and Latscha (26)with some modifications. Toa suspension of hydroquinone (0.550g, 5 mmol) in 50 mL of methylene chloride was added a 3.3-fold molar excess of SbCls (4.93 g, 16.5 mmol), and the mixture was stirred under nitrogen at room temperature for 40 min. The reaction mixture was extracted several times with 25% aqueous acetic acid to remove the antimony salts. The organic phase was taken to dryness and recrystallized from acetone/water. A portion of the product, dissolvedin ethyl acetate and analyzed by gas chromatography-electron ionization mass spectrometry (GC-ELMS), indicated the presence of both C13HQ and CLBQ. ClsHQ was purified by reversed-phase HPLC on a CIScolumn (Partisil M9 10/50 ODs-2, Whatman, Hillsboro, OR) using 67 % methanol in 1% aqueous acetic acid. To overcome the problems associated with oxidation of the product, HPLC fractions were collected in a solution of ascorbic acid (100 mM). Methanol was removed from the HPLC fractions under a stream of nitrogen, and the aqueous layer was extracted with ethyl acetate. The organic extract was immediately dried under nitrogen to a constant weight. The structure and the purity of the product were confirmed by GC-ELMS [m/z 212 (M)+, 100; 176 (M HCl)+, 13; 148 (M - CHC10)+,31; 113 (M - CHClZO)+,331 and proton NMR [6 7.10 ppm (lH, C&Cls), 8.55 and 8.95 ppm (2H, OH)]. Stock solutions of ClSHQ were prepared in 100 mM ascorbic acid/l mM DTPA and stored at -20 "C prior to use. Synthesis of [1F~]Tetrachlorobenzoquinone.[laC6]Tetrachlorobenzoquinone ( [13C6]CWQ)was synthesized according to the method of Lubbecke and Boldt (27). The product, confirmed by TLC (by comparing with the R, value of unlabeled CLBQ) and GC-EI-MS [m/z 250 (M)+,55; 215 (M - C1)+, 30; 186 (M COCl)+, 13; 157 (M - CZOzCl)+,29; 122 (M - C2OzC12)+,32; 90 (CSClO)+,1001,was used without further purification. The purity was estimated to be 48% by GC-EI-MS. Synthesis of Isotopically-Labeled Internal Standards. [W8]ClsBQ-Hb and ["CS] ClaQ-Alb were synthesized according to the following method. To aqueous solutions of rat globin or human Alb (25 mg/mL) at 37 "C was added in two portions a 10 in acetone,to give a final [13C61C&mg/mL solutionof [13c6]c~& BQ concentration of 2 mM. The mixture was incubated at 37 "C for 3 h. Reaction of [13C6]C&B&with cysteine residues of Hb
Waidyanatha et al. and Alb forms [13C6]Cl~BQ-Hb and [1SC&13BQ-Alb, following removal of a chlorine atom from the aromatic nucleus. The modified proteins were purified as described below. Stock solutions of ["C6] ClsBQ-Hb (10 pg/mL) and [13C6]CbBQ-Alb (200 pg/mL) were prepared and stored at -20 "C prior to use. The modified proteins were digested and assayed for labeled adduct levelsaccording to the Raney nickel procedure (seebelow), using ClsHQ as the calibration standard, and were found to contain 0.610 pmol of [13C6]Cl&)/g of globin (SE = 0.02, N = 18) and 39.9 nmol of [13C6]C13HQ/gof Alb (SE = 1.39, N = 18). Reaction of [13C61C13BQ-Hb and [13C&lsBQ-Alb with Raney nickel releases [13c&@&, which is reduced to [1SC61C&H&with ascorbic acid. Standard curves were prepared by extracting CISHQ, [13C6]ClsHQ,and dichlororesorcinolfrom solutions equivalent to those of the samples. Standards were prepared over a working range of 0-30 ng of ClsHQ. Elimination of C4BQ from Rat Blood in Vitro. F344 rata were anesthetizedwithmethoxyfluorane. The blood was obtained by cardiac puncture into a heparinized syringe. Blood from two rata was then combined. To 10 mL of the blood at 37 OC was added 100 pL of 2 mM C W Q in ethanol, to give a final concentration of 20 pM. The sample was mixed by gentle inversion of the tube. Aliquota (0.5 mL) of the reaction mixture were removed at different time intervals (0-22 h) and transferred to vials containing 1 mL of ethyl acetate and 100 ng of dichlororesorcinol, the internal standard. The samples were vigorously mixed for 30 sand centrifuged for 2 min. The organic layer was transferred to a clean vial, dried with anhydrous NazSOr, and analyzed by gas chromatography with electron capture detection (GC-ECD). This experiment was conducted three times. Modification of Rat Blood with CldBQ in Vitro. One hundred microliters of solutions of CWQ in ethanol were added to 4.4-mL portions of fresh blood obtained from F344 rata, to give final concentrations of 0,1,3,10,30,60,and 90 pM. Samples were incubated at 37 "C for 3 h and were mixed every 15 min by gentle inversion of the tubes. Administration of PCP to Rats. Twenty male SpragueDawley rata (300-350 g) were divided into 5 groups of four. PCP (720 mg) was dissolved in 8 mL of 0.6 M NaOH and diluted to 20 mL with 10 mM isotonic phosphate-buffered saline. Dilutions were prepared from this stock solution with isotonic phosphate-buffered saline for dosing the animals. A single, oral dosage of PCP in approximately 2 mL of solution was administered via gastric intubation at 0,2.5,5,10, and 20 mg/kg. The animals were anesthetized with methoxfluorane 48 h following administration, and blood was collected by cardiac puncture into a heparinized syringe. Isolation of Globin and Alb. The red blood cells were separated from the plasma by centrifuging at 800g for 15 min and were washed three times with an equal volume of saline (0.9 % NaC1). The red blood cells and plasma were stored at -80 "C prior to analysis. Globin was isolated, purified, and quantified as described by Rappaport et al. (28).Alb was isolated from the plasma according to Rappaport et al. (28) with the exception that the protein was purified by dialysis against 4 X 4 L of 1mM ascorbic acid at 4 "C using Spectra-Por 1dialysis tubing (MWCO 12000 - 14000). The dialyzed protein was lyophilized and weighed. Measurementof Adducts. The procedure was adapted from the method of McDonald et al. (29) for measurement of Hb and Alb adducts of benzoquinone. To purified samples of globin or Alb dissolved in 3 mL of water, 1mL of 0.9 M ascorbic acid and 1mL of 1M bis-tris-propane buffer (pH 7) were added. One pg of [13C6]C13BQ-Hbor 50 pg of [13C6]C13BQ-Albwas added to the mixture, and the pH was adjusted to 7 by the addition of 150 pL of 5 M NaOH. Protease XIV (Pronase E, 8% w/w) was added, and the protein was digested at 37 "C for 4 h with continuous stirring. To the digested protein was added 1mL of 0.9 M ascorbic acid in 1mM DTPA, and the mixture was extracted with 10 mL of MTBE to remove lipophilic impurities and noncovalently bound interferingmolecules. Ten picomoles of dichlororesorcinol,
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 465
Measurement of Hb and Alb Adducts of ClaQ an unbound internal standard, was added, and the sample was reacted with Raney nickel at room temperature for 10 min with continuous shaking. The amount of Raney nickel used, 75 mg/ mL of the reaction mixture, was found to be optimal in preliminary experiments. The reaction mixture was extracted with 8 mL of MTBE. The ether was reduced to about 3-4 mL under a stream of nitrogen, treated with anhydrous NaZS04, and transferred to a clean 4-mL vial with a Teflon-lined cap. The extract was reduced to dryness, and the vial was sealed under nitrogen. The samples were derivatized by adding 40 pL of HFBI to the dry residue and heating in a sealed vial for 30 min at 85 "C. Samples were cooled to room temperature, 1mL of hexane was added, and the solutions were stored at -20 "C until ready for analysis. Immediately prior to analysis, the hexane was washed with 0.5 mL of deionized water and concentrated to about 100 pL for the analysisby gas chromatography-negative-ionchemicalionization mass spectrometry (GC-NICI-MS). GC-ECD Analysis. Samples from the ClJiQ-elimination experiment were analyzed with a HP 5890 series I1 gas chromatograph equipped with an electron capture detector. A DB-5 fused-silica capillary column (30 m, 0.242 mm i.d., l-pm phase thickness, J & W Scientific, Inc., Folsom, CA) was used at a carrier gas (He) flow rate of 1mL/min. Injections were made in the splitless mode. The injector and detector temperatures were 250 and 300 "C, respectively. The column temperature was programmed as follows: 100 "C, 1min; 100-200 OC, 10 OC/min; 200 OC, 10 min; 200-250 "C, 10 "C/min; 250 "C, 8 min. GC-MS analysis. The synthesized C13HQ and [13C&&BQ were characterized by GC-ELMS using a HP 5890 series I1 gas chromatograph equipped with a HP 5971A mass-selective detector. The GC column and the carrier gas were the same as those described above. The injections were made in the split mode. The injection-port temperature and the source temperature were 250 and 180 "C, respectively. During the analysis, the column was maintained at 50 "C for 1 min and ramped to 280 "C at 25 "C/min. The mass spectrometer was scanned from 50 to 600 amu. The samples from all other experiments were analyzed by GCNICI-MS in the selected ion monitoring (SIM) mode using a HP 5989A MS engine equipped with a HP 5890 series I1 gas chromatograph. The column was the same type as described above. Injections were made in the splitless mode, and the carrier gas (He) flow rate was 1 mL/min. Injection-port and transferline temperatures were 250 and 280 "C, respectively. The column temperature was programmed as follows: 75 "C, 1 min; 75 "C200 "C, 8 "C/min; 200 "C, 1min. Late-eluting compounds were removed by raising the oven temperature to 250 OC for 10 min. The ion source was maintained at 150 "C, and the chemical ionization reagent gas, methane, was set at a pressure of 2 Torr. The ion monitored in NICI-MS for dichlororesorcinol was m/z 373, which corresponds to loss of C~FTCO (m/z 197) fragment from the molecular ion. Instead of monitoring m/z 407 and 413, which are the correspondingfragments arisingfrom the molecular ions of ClsHQ and [13Ce]C13HQ,respectively, the ions 409 and 415 were monitored to avoid the interference from the isotope cluster of C13HQ on the m/z 413 peak of [13C&&H&. Quantitation was based on peak areas relative to the bound internal standard with a sensitivity of less than 0.1 pmol of adduct/g of protein.
Results Elimination of C4BQ from Whole Blood in Vitro. Assuming that t h e 20 p M initital concentration of C4BQ is very low relative t o t h a t of t h e pool of available nucleophilic sites in blood and that C4BQ is uniformly distributed in t h e blood, then t h e overall rate of reaction with blood proteins can be considered pseudo-first-order, with rate constant k e which represents t h e s u m of all firstorder rate constants of individual reactions. Since t h e concentration of Cl4BQ at any time t ([CLBQlt) =
P
500 25
d
R
400
: 0
-E
P
300
R
20
:
15
Y
0
E
10 100
5
0
0
0 0
10 20 30 40 50 60 70 80 90 [C $Wo (P M1
Figure 1. Formation of tetrachloro-l,4-benzoquinoneadducts of Hb and Alb in rat blood in vitro. (Note the different scales of the two ordinate axes.) [CLBQIoe-ket, k, can be determined as the slope of the linear regression of ln[C4BQlt on time t. I n rat blood at 37 "C, k, was found to be 0.161 h-' (SE = 0.011, N = 3). The corresponding elimination half-time was 4.35 h (SE = 0.309). Reaction of Cl4BQ with Hb and Alb in Vitro. The adduction of Cl4BQ with Hb and Alb in vitro was investigated by incubating C4BQ with r a t blood at initial concentrations between 0 a n d 90 p M . The upper end of t h e concentration range was limited by the solubility of C4BQ in blood. T h e levels of adducts are presented in Figure 1as nmol of adduct/g of protein versus the initial concentration of CLBQ in blood. For both H b and Alb, linear production of adducts was observed over t h e entire range of dosages with slopes of 0.247 (R2= 0.986) and 5.82 (R2= 0.996) nmol of adduct (g of protein)-l ( p M CLBQ)-l, respectively. T h e relative rates of formation of CLBQ adducts with Hb and Alb in r a t blood can be inferred from the secondorder rate constants for t h e respective reactions. These constants were estimated from the relationship described by Rappaport et al. (28) by using t h e slopes of the doseresponse curves in Figure 1and assuming concentrations of Hb and Alb in rat blood t o be 153 and 16.3 mg/mL (30), respectively. T h e second-order rate constants for t h e reactions of C4BQ with Hb and Alb were 6.89 and 167 L mol-' h-l, respectively, suggesting that t h e reactivity of Alb toward this quinone was much greater than that of Hb in whole r a t blood. Formation of C4BQ Adducts with Hb and Alb in Vivo. T h e formation of adducts of C4BQ with Hb and Alb in vivo was investigated in Sprague-Dawley rats at administered levels of 0,2.5, 5,10, a n d 20 mg of PCP/kg body wt. Figure 2 shows t h e levels of these adducts plotted against t h e dosages of PCP. Each point in the plots represents a n individual animal. Linear production of C4BQ adducts was observed with both H b and Alb over t h e entire range of dosages, with slopes of 0.09 (R2 = 0.901) and 8.22 pmol of adduct (g of protein)-l [(mg of PCP)/(kg body wt)l-l (R2= 0.817), respectively.
Discussion
This application of Raney nickel to cleave t h e cysteinebound adducts of Cl4BQ extends earlier work involving protein adducts of styrene 7,8-oxide (28, 31) and benzoquinone (29). Given t h e well-known affinities of quinones for sulfhydryl groups (321,we applied this assay with the
466
Chem. Res. Toricol., Vol. 7, No. 3, 1994 250 A
2
Waidyanatha et al.
1 A
n
200
I
[C4BQlo
m
:
-: O
;
(PM)
150 n
100
?
U
a
-
m ,
Table 1. Ratios of the Molar Concentrations of Cysteine Adducts of Hb and Alb to the Concentration of Total Products. Formed in Vitro
m
50
3
0
0
0 0
5
10
15
20
1 3 10 30 60 90
[ClsBQ-Hbl/
[ClaQ-Mbl/
[total products] 0.041 0.159 0.180 0.170 0.180 0.200
[total products] 0.021 0.035 0.043 0.065 0.056 0.068
4The concentration of total products (pM)was estimated as [C4BQl&,t,where k, is the pseudo-first-ordereliminationconstant (0.161 h-9 and t is the time of incubation (3 h).
Dosage (mg PCP/kg body wt)
Figure 2. Formation of tetrachloro-l,4-benzoquinoneadducts of Hb and Alb following administration of PCP to SpragueDawley rats. (Notethe different scales of the two ordinate axes.) expectation that it would provide the greatest sensitivity for detecting adducts of C@Q in the blood. The procedure utilizes ascorbic acid to reduce the cleaved quinone species to the corresponding hydroquinone and employs HFBI as the derivatizing agent; both steps resulted from previous experience with the analogous benzoquinone adducts (29). It had previously been shown that CLHQ could be oxidized in vitro to C4BQ (22,23),which was believed to be at least one of the products responsible for macromolecular binding (21,221. This study, in which adducts of C4BQ were unambiguously detected following reaction of Cl4BQ with rat blood in vitro, confirms that C4BQ reacted with free sulfhydryl groups of Hb and Alb. However, second-order rate constants indicate that C4BQ was much less reactive, in whole rat blood in vitro, toward cysteine residues of rat Hb (6.89 L mol-l h-I) than were 1,4-benzoquinone (180 L mol-l h-l) (29) and styrene 7,8-oxide (72 L mol-' h-l) (28). On the other hand, C4BQ was more reactive toward the cysteine residue of rat Alb (167 L mol-' h-1) than were 1,4-benzoquinone (74 L mol-l h-1) and styrene 7,8-oxide (63 L mol-' h-l) (28,29). This observation that Cl4BQ reacts more readily in rat blood with the free cysteine residue of Alb (Cys 34) than with those of Hb (particularly Cys 125 of the 6 chain of rat globin) is clearly at odds with our earlier work involving 1,4-benzoquinone (29) and styrene 7,8-oxide (28) and requires further investigation. The molar concentration of all reaction products produced by incubation of CLBQ with whole blood in vitro can be calculated from the relationship: total products (pM)= [C14BQ]oketwhere [CldBQlorepresents the initial concentration of C4BQ (MM),k, is the pseudo-first-order rate constant for reaction of C4BQ in blood (0.161 h-'1, and t is the time of incubation (3 h). The ratios of the molar concentrations of cysteine adducts of Hb and Alb to the concentration of total products can be used to estimate the fractions of total binding represented by these adducts. These ratios are given in Table 1for values of 1and 90 pM. The ratios are essentially [ C ~ B Q Ibetween O constant for initial C14BQ concentrations between 3 and 90 pM,i.e., at 0.053 for Hb (SE = 0.006, N = 5) and 0.178 for Alb (SE = 0.007, N = 5). (Note that these estimates could be somewhat higher than the true ratios because the values of t used in the above relationship did not take into account the time required for processing the blood samples after the formal period of incubation had ended.) When PCP was administered to rats, the ratio of cisBQ-Alb to C13BQ-Hb was 9.7, as indicated by the ratio of
the corresponding slopes from Figure 2 (after adjusting adduct levels to a molar basis, assuming 153mg of Hb/mL and 16.3 mg of Alb/mL). This ratio is much higher than the value of 2.6 which was found in vitro (from the slopes in Figure 1after adjusting adduct levels to a molar basis). The apparent increase in the rate of production of Alb adducts in vivo is consistent with the notion that CLBQ reacts significantly with Alb inside the hepatocyte following metabolism of PCP. Such behavior has been suggested in the case of the reactive metabolite of aflatoxin B1 (33,341. However, unlike the metabolite of aflatoxin B1, CLBQ is relatively stable both in vitro and in vivo, and no particular preference would be expected for reaction inside the hepatocyte. Indeed, in similar experimentswith styrene and its moderately reactive metabolite, styrene 7,8-oxide, the corresponding ratios of slopes were not significantly different (in vivo: Alb adduct/Hb adduct = 0.09; in vitro: Alb adduct/Hb adduct = 0.13) (28). The anomalous behavior of C4BQ in producing adducts of Hb and Alb in vitro and in vivo suggests that factors other than the intrinsic reactivity of this molecule are playing some role. One consistent explanation is that CLBQ is not easily transported through cell membranes. The resulting restrictions to transport into the erythrocyte would reduce production of C13BQ-Hb in whole blood and out of the hepatocyte would increase the hepatic production of C13BQ-Albin vivo. Another possibility is that C4BQ binds nonspecificallyto Alb and other proteins in much the same manner as does PCP (35). If this were the case, then the residence time of CLBQ would increase in the plasma or the liver cytosol, thereby enhancing the prospects for reaction with Alb. More work is clearly needed to determine the source of these discrepancies. However, as a purely practical matter, the increased production of C13BQ-Alb relative to that of CLBQ-Hb suggests that the Alb adduct might be preferred for biomonitoring of humans exposed to PCP. Since reaction of CllBQ and Hb can only take place in the blood, the integrated dose, D H b , of C4BQ to the blood can be estimated using the following relationship (28,36): [Cl,BQ-Hbl DHb
=
v
-
kC1,BQ-Hb
LH 1
where [ClsBQ-Hbl is the concentration of the Hb adduct in nM, assuming that the concentration of Hb in rat blood was 153 mg/mL (28), kClaQ-Hb is the second-order rate constant (6.89 L mol-' h-I) and [Hbl is the molar concentration of Hb (2.32 X M) in rat blood. Values Of D H b were estimated for each group of rats to which PCP was administered and then regressed upon the corresponding dosage of PCP. The estimated slope, repre-
Measurement of H b and Alb Adducts of ClaQ
senting the integrated dose per unit of administered dosage, was 0.843 nM C4BQ-h [(mg of PCP)/(kg body
wt)l-'. Since DHb represents the integral of the blood concentration of C4BQ over the course of the in vivo experiment, the average blood concentration can be estimated as D m / t = 18 pM C4BQ [(mg of PCP)/(kg body wt)l-', where t is the time, following administration of PCP, that the blood was collected (48 h). After converting the dosage of PCP to a molar basis and assuming that a rat has 0.058 L of blood/kg body w t (30),this concentration represents 2.7 X 10-7 mol of CLBQ/mol of PCP administered. This small proportion of the administered dose of PCP which is bioactivated to Cl&Q casts some doubt upon the notion that C4BQ is solely responsible for the genotoxicity of PCP. We also note that the proportion of PCP which was available to the blood as C4BQ (2.7 X lo-' mol/mol) is about 10-fold greater than that of benzene which found its way to the blood of rats as 1,4-benzoquinone (2.3 X lV mol/mol) in similar experiments (37). We conclude by emphasizing that previous inferences regarding the macromolecular binding of C4BQ in vivo relied entirely upon indirect evidence, i.e., upon results obtained in vitro (16, 21), upon measurements of the precursor molecule, CkHQ, in rodents (3, 5, 8) and in humans (3, 7) following exposure to PCP, and upon measurement of C4BQ in the urine of rats following administration of PCP (25). Our observation that the levels of C4BQ adducts in rats were proportional to the dosage of PCP, therefore, provides the first direct evidence that PCP is metabolized in vivo to C4BQ which subsequently binds with macromolecules. The low levels of adduction observed in vivo may be due to slow conversion of C4HQ to C4BQ and/or to efficient conjugation and excretion of C4HQ prior to oxidation. In fact, it has been suggested that the rate-limiting step in the process of covalent binding is the oxidation of the hydroquinone to its semiquinone or benzoquinone (21).Thus, additional work is needed to define both the metabolic pathway through which PCP gives rise to C4BQ and the transport behavior of this quinone species after it is formed in vivo.
Acknowledgment. This work was supported by the National Institute for Environmental Health Sciences through Grant P42ES05948. The authors are most grateful to Avram Gold and the chemistry core of P42ES05948 for assistance with the synthesis of the analytes. The authors would also like to thank Zuliang Jin for his assistance with the animal experiment.
References World Health Organization (WHO) (1987) Enuironmental Health Criteria, 71, Pentachlorophenol, WHO, Geneva. Ahlborg, U. G., and Thunberg, T. M. (1980) Chlorinated phenols: occurrence, toxicity, metabolism, and environmental impact. CRC Crit. Rev. Toxicol. 7, 1-35. Ahlborg, U. G., Lindgren, J. E., and Mercier, M. (1974) Metabolism of pentachlophenol. Arch. Toxicol. 32, 271-281. Ahlborg, U. G., Larsson, K., and Thunberg, T. (1978) Metabolism of pentachlophenol in vivo and in vitro. Arch. Toxicol. 40, 45. Jacobson, I., and Yllner, S. (1971) Metabolism of W-pentachlorophenol in the mouse. Acta Pharmacol. Toxicol. 29, 513-524. Juhl, U., Witte, I., and Butte, W. (1985) Metabolism of pentachlorophenol to tetrachlorohydroquinone by human liver homogenate. Bull. Enuiron. Contam. Toxicol. 35, 596-601. Edgerton, T. R., Moseman, R. F., Linder, R. E., and Wright, L. H. (1979) Multiresidue method for the determination of chlorinated phenol metabolites in urine. J. Chromatogragr. 170,331-342.
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 467 Reigner, B., Rigod, J. F., and Tozer, T. (1990) Simultaneous assay of pentachlorophenol and ita metabolite, tetrachlorohydroquinone, by gas chromatography without derivatization. J. Chromatogragr. 533,111-124. Reigner, B. G., Gungon, R. A., Hoag, M. K., and Tozer, T. (1992) Pentachlorophenol toxicokinetics after intravenous and oral administration to rats. Xenobiotica 21, 1547-1558. Weinbach, E. C. (1954) Theeffect of pentachlorophenolon oxidative phosphorylation. J. Biol. Chem. 210, 545-550. Arrhenius, E., Renberg, L., Johansson, L., and Zetterqvist, M. A. (1977) Disturbance of microsomal detoxication mechanisms in liver by chlorophenol pesticides. Chem.-Biol. Interact. 18, 35-38. Mulder, G. J., and Meerman, J. H. N. (1978) Glucuronidation and sulphation in vivo and in vitro: selective inhibition of sulphation by drugs anddeficiency of inorganicsulphate. In ConjugationReactions in Drug Biotransformation (Aito, A., Ed.) p 389, Elsevier, Amsterdam. Schwetz, B. A,, Keepler, P. A., and Gehring, P. J. (1974) The effect of purified and commercial grade pentachlorophenol on rat embryonal and fetal development. Toxicol. Appl. Pharmacol. 28,151161. McConnel, E. E. (1989) Toxicology and carcinogenesis studies of two pentachlorophenol technical-grade mixtures in B6C3F1 mice. U. S. National Toxicology Program, Department of Health and Human Services, NIH Publication No. 89-2804. Bauchinger, M. J., Presp, J., Schmid, E., and Hauf, R. (1982) Chromosome changes in lymphocytes after occupational exposure to pentachlophenol. Mutat. Res. 102, 83-88. Witte, I., Juhl, U., and Butte, W. (1985) DNA-damaging properties and cytotoxicity in human fibroblasts of tetrachlorohydroquinone, a pentachlorophenol metabolite. Mutat. Res. 145, 71-75. Ehrlich, W. (1990)The effectofpentachlorophenoland itsmetabolite tetrachlorohydroquinone on cell growth and the induction of DNA damage in Chinese hamster ovary cells. Mutat. Res. 244,299-302. (18) Carstens, C.-P., Blum, J. K., and Witte, I. (1990)The role of hydroxyl radicals in tetrachlorohydroquinone induced DNA strand break formation in PM2 DNA and human fibroblasts. Chem.-Biol. Interact. 74, 305-314. (19) Jansson, K., and Jansson, V. (1991) Induction of mutation in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol. Mutat. Res. 260,83-87. (20) Jansson, K., and Jansson, V. (1992) Induction of micronuclei in V79 Chinese hamster cella by tetrachlorohydroquinone,a metabolite of pentachlorophenol. Mutat. Res. 279,205-208. (21) Van Ommen, B., Adang, A., Muller, F., and Van Bladeren, P. J. (1986) The microsomal metabolism of pentachlorophenol and ita covalent binding to protein and DNA. Chem.-Biol. Interact. 60, 1-11. (22) Van Ommen, B., Voncken, J. W., Muller, F., and Van Bladeren, P. J. (1988) The oxidation of tetrachloro-l,4-hydroquinoneby microsomesand purified cytochrome P-45Ob.Implications for covalent binding to protein and involvement of reactive oxygen species. Chem.-Biol. Interact. 65, 247-259. (23) Pritsos, C. A., Pointon, M., and Pardini, R. S. (1987) Interaction of chlorinated phenolics and quinones with the mitochondrial respiration: a comparison of the 0- and p-chlorinated quinones and hydroquinones. Bull. Enuiron. Contam. Toxicol. 38,847-855. (24) den Besten, C., van Bladeren, P. J., Duizer, E., Vervoort, J., and Rietiens, I.M.C.M. (1993) Cvtochrome P450-mediated oxidation of penkluorophenol tetrduorobenzoquinone as the primary reaction product. Chem. Res. Toxicol. 6, 674-680. Renner, G., and Hopfer, C. (1990) Metabolic studies on pentachlorophenol in rata. Xenobiotica 20,573-582. Rettig, Von G.,and Latacha, H. P. (1980) Reaktionen von Antimon(V)-chlorid mit Hydrochinon. (Reactions of antimony(\') chloride with hydroquinone). Chem.-Ztg. 104, 13-14. Lubbecke, H., and Boldt, P. (1978) Halogenation and oxidative halogenation of phenols with hydrogen halidedhydrogen peroxide: Synthesis of p-chloranil and p-bromanil. Tetrahedron 34, 15771579. Rappaport, S. M., Ting, D., Jin, Z., Yeowell-OConnell, K., Waidyanatha, S., and McDonald, T. (1993) Application of Raney nickel to measure adducts of styrene oxide with hemoglobin and albumin. Chem. Res. Toxicol. 6, 238-244. McDonald, T. A., Waidyanatha, S., and Rappaport, S. M. (1993) Measurement of adducta of benzoquinone with hemoglobin and albumin. Carcinogenesis 14, 1927-1932. Ringler, D. H., and Dabich, L. (1979) Hematology and Clinical Biochemistry. In The Laboratory Rat, Volume 1, Biology and Diseases (Baker, H. J., Lindsey, J. R., and Weisbroth, S. H., Eds.) Academic Press, New York.
468 Chem. Res. Toxicol., Vol. 7, No.3, 1994 (31) Ting, D.,Smith, M. T., Doane-Setzer, P., and Rappaport, S. M. (1990)Analysis of styrene odde-globin adducta baaed upon reaction with h e y nickel. Carcinogenesis 11,755-760. (32) Hinson, J. A., and Roberta, D. W. (1992)Role of covalent and noncovalent interactions in cell toxicity: Effects on proteins. Annu. Reu. Pharmacol. Toricol. 32,471-510. (33) Tannenbaum, S.R., and Skipper, P. L. (1984)Biological aspects to the evaluation of risk doeimetry of cncinogena in man. Fundam. Appl. Toricol. 4,53674373. (34) Wild, C. P., Garner, R. C., Montesano, R., and Tursi, F. (1986) Aflatoxin B1binding to p h m a albumin and liver D N A upon chronic administration to rata. Carcinogenesia 7,853-858.
Waidyanatha et al. (35) Hoben, H.J., Ching, S. A.,Young,R. A.,and Casarett, L.J. (1976) A study of the inhalation of pentachlorophenol by rata. V. Protein binding study ofpentachlorophenol.Bull. Enuiron. Contam. Toricol. 16,225-232. (36) Osterman-Golkar, S.,Ehrenberg, L., Segerback, D.,and Halhtrom, I. (1976) Evaluation of genetic risks of alkylating agents. 11. Haemoglobin aa a dcae monitor. Mutot. Res. 34,l-10. (37) McDonald, T. A., Waidyanatha, S., and Rappaport, S. M. (1993) Production of benzoquinone adducta with hemoglobin and bonemarrow proteins followingadministration of [W~lbenzeneto rata. Carcinogenesis 14, 1921-1925.
