Chem. Res. Toxicol. 1995,8, 349-355
349
Peroxidase-CatalyzedOxidation of Pentachlorophenol Victor M. Samokyszyn,*>+James P. Freeman,+ Krishna Rao Maddipati,g and Roger V. Lloyd" Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 72079, Cayman Chemical Corporation, Ann Arbor, Michigan, 48108, and Department of Chemistry, University of Memphis, Memphis, Tennessee 38152 Received July 22, 1994@ Pentachlorophenol (PCP) was shown to function as a reducing substrate for horseradish peroxidase (HRP) and to stimulate the HRP-catalyzed reduction of 5-phenyld-penten-1-yl hydroperoxide (PPHP) to 5-phenyl-4-penten-1-01. HRP catalyzed the hydroperoxide-dependent oxidation of PCP, using H202, PPHP, or ethyl hydroperoxide as substrates, as evidenced by UV spectroscopic and reverse phase HPLC analysis of reaction mixtures. The major oxidation product was tetrachloro-1,4-benzoquinonewhich was identified on the basis of electronic absorption spectroscopy, mass spectrometry, and cochromatography with authentic standard. HRP-catalyzed oxidation of PCP yielded relatively stable, ESR-detectable pentachlorophenoxyl radical intermediates whose ESR spectra consisted of a symmetrical single line without hyperfine structure. Substitution of natural abundance isotopically-labeled PCP with 13Clabeled PCP resulted in broadening of the ESR signal line width from 6.1 G to 13.5 G. ESR spin trapping studies, with a-(l-oxy-4-pyridyl)-Ntert-butylnitrone (4-POBN) as the spin t r a p demonstrated identical spectra using natural abundance isotopically-labeled PCP versus 13Clabeled PCP, suggesting oxy1 addition, rather t h a n carbon-centered radical addition to 4-POBN. The computer simulation of the observed spectra is consistent with two distinct 4-POBN adducts, with relative abundances of -3:1, and hyperfine coupling constants of uN = (14.61 G)/uH = 1.83 G and aN = (14.76 G)hH = 5.21 G, respectively. Mechanisms for the hydroperoxide-dependent, HRP-catalyzed oxidation of PCP are presented that are consistent with these results.
Introduction Pentachlorophenol (PCP;I CAS Registry No. 87-86-512 is a major industrial and agricultural biocide that has been used primarily as a wood preservative (1-3). In addition, PCP was registered for use as a n insecticide, fungicide, acaricide, herbicide, molluscicide, slimicide, and disinfectant ( I ) . Its worldwide usage and relative stability make PCP a ubiquitous environmental pollutant that has contaminated air, soil, and water and resulted in persistent, widespread exposure through drinking water and food. In fact, worldwide production is estimated a t between 25 000 and 90 000 tons (41, and PCP has been detected in the general North American population in urine, blood, milk, and adipose tissue (1, 5-7). Significant in vivo generation of PCP may also occur through metabolism of hexachlorobenzene (8)or hexachlorocyclohexane (9), which are ubiquitous environmental contaminants. *Correspondence should be addressed to this author at Department of Pharmacology and Toxicology, Mail Slot 638, University of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205. Tel: (501)686-5810; FAX: (501) 686-8970. ' University of Arkansas for Medical Sciences. National Center for Toxicological Research. 5 Cayman Chemical Corp. 'I University of Memphis. Abstract published in Advance ACS Abstracts, February 1, 1995. Abbreviations: PCP, pentachlorophenol; HRP, horseradish peroxidase; ESR, electron spin resonance spectroscopy; 4-POBN, a-(1-oxy4-pyridyl)-N-tert-butylnitrone; PPHP, 5-phenyl-4-penten-1-yl hydroperoxide; PPA, 5-phenyl-4-penten-1-01; IS, internal standard; HPLC, high performance liquid chromatography; tR, retention time. CAS Registry Nos. supplied by the author.
*
@
The major target organs for PCP toxicity include the liver, kidneys, hematopoietic system, pulmonary system, and central nervous system. PCP is a general cytotoxic agent because of its ability to uncouple mitochondrial oxidative phosphorylation (10). In addition, PCP undergoes cytochrome P-450-dependent metabolic activation in vitro and in vivo to genotoxic tetrachlorobenzenediols and to electrophilic tetrachlorobenzoquinones, which have been shown to react with protein- and DNA-derived nucleophiles (11-18). Alternatively, PCP may undergo oxidative metabolic activation by mammalian peroxidases including prostaglandin H synthase, myeloperoxidase, salivary peroxidase, lactoperoxidase, and/or uterine peroxidase. These heme peroxidases reduce hydroperoxides to the corresponding alcohols resulting in the generation of peroxidase compound I which is two oxidizing equivalents above the resting ferric state (reviewed in ref 19). Compound I consists of a ferryl intermediate (Fe(IV)=O) and, depending on the peroxidase, a porphyrin n cation radical or protein radical. Peroxidase compound I undergoes one-electron reduction by peroxidase reducing substrates, yielding the ferryl intermediate compound I1 which undergoes reduction to the resting ferric state. Both compounds I and I1 exhibit redox potentials of -1 V and oxidize numerous compounds by electron transfer mechanisms including phenols, catechols, hydroquinones, aromatic amines, 1,3-dicarbonyls, and others (20,Zl). Aust and co-workers (22) as well as Hammel and Tardone (23)have previously demonstrated PCP oxidation to tetrachloro-1,4-benzoquinone(2,3,5,6-tetrachloro-
0893-228x/95/2708-0349$09.00/00 1995 American Chemical Society
350 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
2,5-cyclohexadiene-l,4-dione j by lignin peroxidases derived from the fungus Phanerochaete chrysosporium. PCP oxidation by lactoperoxidase has also been reported (24j, but the oxidation products or mechanism of oxidation were not investigated. We have undertaken the mechanistic investigation of hydroperoxide-dependent oxidation of PCP by horseradish peroxidase as a model peroxidase system. Although various phenols function as peroxidase reducing substrates (20,211, PCP is novel because of the bulkiness of the chlorine substituents as well as their polar and/or resonance contributions. We report that PCP functions as a reducing substrate for HRP and undergoes hydroperoxide-dependent, HRP-catalyzed oxidative dechlorination to the electrophilic tetrachloro-1,4benzoquinone. We also report that the mechanisms of oxidation involves the intermediacy of relatively stable ESR-detectable pentachlorophenoxyl radicals which are trappable by the nitrone spin trap a-(l-oxy-4-pyridyl)N-tert-butylnitrone (4-POBN). Mechanisms consistent with these results are proposed for the generation of the tetrachlorobenzoquinone.
Materials and Methods Materials. Horseradish peroxidase (HRP, EC 1.11.1.7, type VI, A402/&80 = 3.1) was purchased from Sigma (St. Louis, MO) and was quantitated by measurement of the Soret absorbance at 402 n m (E402 = 9.5 x lo4 M-' cm-' (25)). The HRP was chromatographed over Sephadex G-25 (Sigma) to remove any contaminating reducing substrates which may be present in the Sigma HRP. Pentachlorophenol (PCP), tetrachloro-1,4-benzoquinone (2,3,5,6-tetrachloro-2,5-cyclohexadiene-1,4-dione), a-(loxy-4-pyridyl)-N-tert-butylnitrone (4-POBN), cinnamyl alcohol (4-phenyl-3-buten-l-ol), and hydrogen peroxide (30%) were purchased from Aldrich (Milwakee, WI), and the latter was quantitated by iodometric titration using KIO3-standardized &03*-. Caution: It should be noted that PCP is highly toxic, is a n irritant, cancer suspect agent, mutagen, a n d possible teratogen, a n d is readily absorbed through skin. Tetrachloro2,4-benzoquinone is a n irritant, a n d the target organ is liver. Thus, these substances should be handled with gloves a n d protective clothing. Ethyl hydroperoxide was obtained from Accurate Chemical & Scientific Corp. (Westbury, NY). 13CLabeled PCP (13C6,99%) was purchased from Cambridge Isotope Laboratories (Woburn, MA), and 5-phenyl-4-penten-1-ylhydroperoxide (PPHP) and 5-phenyl-4-penten-1-01(PPA) were synthesized as described by Weller et al. (21). Enzymatic reactions were carried out a t 25 "C (except ESR experiments which were conducted a t ambient temperature) in 0.1 M phosphate buffer (pH 7.0) which had been treated with iminodiacetic acid chelating resin (Sigma) to minimize any contaminating redoxactive transition metals. All other reagents were obtained through commerical sources. Analytical Methods. Electronic absorption spectra were obtained using a Hewlett-Packard 8452A diode array spectrophotometer equipped with Pelltier temperature control. Analytical reverse phase HPLC analysis was carried out using a Waters ,uBondapak C18 125 A 10 ,um 3.9 x 300 mm HPLC column and a Waters 600E HPLC with a 994 programmable photodiode array detector. The latter allowed the acquisition of on-line electronic absorption spectra of eluents as well a s peak integration. Electron ionization mass spectra were obtained using a Finnigan MAT 4500 quadrupole mass spectrometer, employing 70 V electron energy and 150 "C ion source temperature. The samples were applied i n solution to a rhenium wire direct exposure probe (Finnigan MAT) and data collected a s the probe current was ramped from 0 to 650 mA a t 5 d s . The acquired spectra were compared with the NIH/EPA/NBS mass spectral library (INCOS Library Search, version 8; 49 000 spectra). ESR spectra were obtained using a Varian E-104 spectrometer custom-interfaced with a n IBM-compatible computer for
Samokyszyn et al.
4
I.S. PPA
PPHP
0.6
1
T 0.5
-z 6
9+
-
0.4
0.3
P
P
Y I
gd
0.2
0.I
t 0
5
10
0.0
A
B
C
D
Time (min)
Figure 1. PCP-dependent stimulation of HRP-catalyzed reduction of PPHP. Left, reverse phase HPLC elution profile of PPHP, PPA, and the internal standard (IS) cinnamyl alcohol (4-phenyl3-buten-1-01), Chromatographic conditions are described in the Materials and Methods section. Right, PPHP (100 ,uM) was incubated in 0.1 M phosphate buffer (pH 7.0) a t 25 "C (A) with HRP (100 nM) alone; (B) with P C P (200 yM) alone; CC) with HRP plus PCP; (D) with HRP plus ascorbic acid (200 ,uM). Reaction mixtures were worked-up and analyzed by reverse phase HPLC a s described in the Materials and Methods section. data acquisition and analysis. All spectra were stored on the computer for later analysis, and signal intensities were measured from the stored spectra using software written by Duling (NIEHS) ( 2 6 ) . All reactions were carried out a t room temperature. Immediately after preparation, solutions were aspirated into a 10.5-mm Wilmad flat cell centered in the TMllo microwave cavity. To avoid the possibility of transition metal contamination, polyethylene tubing rather t h a n stainless steel was used for the aspiration. Because sample aspiration permits the flat cell to be cleaned and refilled without being disturbed, reproducibility between scans was greatly improved. Nitroxide spectra, in systems employing 4-POBN a s the spin trap, were also simulated and plotted with programs written by Duling ( 2 6 ) . The composition of reaction mixtures employed in the ESR experiments and the instrumental parameters are described in the figure legends. PPHP Reduction Assays. The PPHP reduction assay is a modification of the method reported by Marnett and co-workers ( 2 0 , 2 1 ) . Incubations were conducted in triplicate. PPHP (100 ;tM) was incubated with HRP (100 nM), PCP (200 pM), or HRP + PCP in a total volume of 2.0 mL. Incubation of PPHP with HRP and ascorbic acid (200pM)served as a positive control. After 6 min, PPHP and PPA were isolated by solid phase extraction using 3 mL octadecylsilyl columns (J.T. Baker & Co.) and eluted with HPLC-grade methanol (2 x 1 mLj. An internal chromatographic standard (IS) of cinnamyl alcohol (4-phenyl4-buten-1-01, 25 /tL of 8 mM stock solution in methanol) was added to each sample, and the samples were filtered and chromatographed by reverse phase HPLC using isocratic 58% methanol a s the mobile phase a t a flow rate of 2.0 m u m i n . Eluents were detected by absorbance at 257 nm. Elution profiles of IS, PPA, and PPHP are shown in Figure 1 (left). The concentrations of PPA and PPHP were calculated relative to IS by peak integration, and PPHP reduction is expressed as [PPAl/ ([PPA] [PPHP]) (Figure 1, right). Reverse Phase HPLC of PCP Metabolites. PCP (175 ,uM) was incubated with HRP (1 ,uM) (or no addition) for 3 min followed by the addition of Hz02 (350 ,uM). Aliquots (1.0 mL) were removed after 0, 1, 5 , 10, and 15 min, mixed with 0.1 mL of 6 N HC1, saturated with NaCI, and extracted with HPLC-
+
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 351
HRP-Catalyzed Oxidation of PCP grade ethyl acetate (3 x 1.0 mL). The solvent was removed under a stream of argon, and the residue was dissolved in HPLC-grade acetonitrile(100 pL) and filtered through 0.45 pm Nylon filters (ScientificResources, Inc., Eatontown, NJ). Reverse phase HPLC was carried out using a mobile phase consisting of 33% solvent B in solvent A at t = 0 and a linear gradient to 72% B over 30 min followed by isocratic conditions for an additional 15 min at a flow rate of 1.0 mumin. Solvent A consisted of 10:90:0.1 CH3CN/H20/CF3COOH,and solvent B consisted of 1OO:O.l CH&N/CF3COOH. Eluents were detected at 290 nm. Reaction mixtures for metabolite isolation for the aquisition of mass spectra were scaled up 100-fold.
*.O T
1.61
p
Ill
Results PCP was shown to function as a peroxidase reducing substrate for HRP as evidenced by its ability to stimulate the HRP-catalyzed reduction of 5-phenyl-4-penten-1-yl hydroperoxide (PPHP) to 5-phenyl-4-penten-1-01 (PPA) (Figure 1, right). The extent of reduction is expressed as [PPAY([PPAl+ [PPHP]). Use of PPHP as a peroxidase probe was originally developed and utilized by Marnett and co-workers to evaluate the peroxidase reducing substrate efficiency of various compounds for HRP (201, prostaglandin H synthase (21,27), and lactoperoxidase (28). The method involves incubation of PPHP with peroxidase in the presence and absence of test compound, isolation of PPHP and PPA by solid phase extraction, and quantitation of PPHP reduction to PPA by reverse phase HPLC analysis (Figure 1, left). The method is essentially as reported by Marnett et al. (20) with several minor modifications as described in the Materials and Methods section. In the absence of exogenous compounds, HRP catalyzed only 6 2~ 2% reduction of PPHP to PPA, underscoring the requirement of reducing equivalents for peroxidase turnover. Slightly greater extents of reduction were obtained (10 f 1%reduction) with HRP alone when the enzyme preparations were not desalted over Sephadex G-10, suggesting the presence of contaminating reductants in the HRP preparation obtained from Sigma. Addition of PCP resulted in 46 f 2%reduction of PPHP, comparable to that of ascorbic acid (52 f 4% reduction), which has previously been shown to function a s a n effective peroxidase reducing substrate for HRP (20). In contrast, PCP alone (in the absence of HRP) failed to stimulate any hydroperoxide reduction. PCP in aqueous solution exhibits electronic absorption maxima a t 252 and 322 nm (Figure 2). Incubation of PCP with HRP and H ~ 0 2resulted in significant timedependent spectral changes of the PCP chromophore characterized by loss of both absorption maxima, increased absorbance at wavelengths below and above the PCP absorbance dt 322 nm, and, after 15 min, the appearance of a shouldei a t -270 nm (Figure 2). In contrast, no absorbance changes were observed when PCP was incubated with H202 alone (not shown). Furthermore, identical HRP-catalyzed absorbance changes were detected using HzOz, ethyl hydroperoxide, or PPHP as peroxidase substrates (data not shown). The products generated in reaction mixtures of PCP, HRP, and HZOz were acidified, extracted with ethyl acetate, and analyzed by reverse phase HPLC as described in the Materials and Methods section. Extractions were carried out a t 0, 1, 5, 10, and 15 min, corresponding approximately to the time course of uv spectral changes in Figure 2. The HPLC product profile, obtained a t 15 min and employing detection a t 290 nm, demonstrated the formation of a single major product ( t ~
250
300
350
450
400
Wavelength (nm)
Figure 2. UV spectroscopic changes associated with HRPcatalyzed oxidation of PCP. PCP (175 pM) was incubated at 25 "C in 0.1 M phosphate (pH 7.0) with HRP (1pM) and H202 (350 pM). Spectra were obtained at t = 0 and 15 s and 1, 10, and 15
min.
1-
0.07
I
0
I
0
10
20 TIME (min)
30
40
10
20 TIME (min)
30
40
Figure 3. Reverse phase HPLC elution profiles of ethyl acetate-extractablematerial generated after a 15 min incubation of PCP (175 pM), HRP (1yM), and H202 (350 pM) (upper) or H202 alone (lower).Incubations were worked up and analyzed as described in the Materials and Methods section. = 20.2 min) and a minor product ( t =~43.3 min) (Figure 3, upper) which were virtually absent in reaction mixtures devoid of HRP (Figure 3, lower). The component eluting a t 27.6 min represents unreacted PCP. Time
course HPLC analysis demonstrated the time-dependent disappearance of PCP and time-dependent increases in formation of the major and minor products (data not shown). The major oxidation product eluting a t 20.2 min
352 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
Samokyszyn et al.
il
220
300
260
340
Complete System
F=i
Wavelength (nm)
Figure 5. ESR spectrum of pentachlorophenoxyl radicals generated during hydroperoxide-dependent, HRP-catalyzed oxidation of PCP. PCP (1mM) was incubated at ambient temperature i n 0.1 M phosphate buffer (pH 7.0) with HRP (1 mg/mL) and H202 (0.5 mM). Controls consisted of incubations devoid of HRP, HzO2, or PCP. Spectra were obtained using a modulation amplitude of 2.5 G, time constant of 1.0 s, reciever gain of lo4, microwave power of 20 mW, and a scan time of 4 min over 100
I_
-
xl o o ]
G. 21 I
I90
Ill
1
x
50
IO0
I50
200
250
m/z Figure 4. Electronic absorption spectrum (upper) and mass spectrum (lower) of major P C P metabolite eluting at 20.2 min.
cochromatographed with authentic tetrachloro-1,4-benzoquinone. In addition, the metabolite and authentic tetrachloro-1,4-benzoquinoneexhibited identical ultraviolet absorption spectra, characterized by a n absorption maximum a t 288 nm (Figure 4, upper), and identical mass spectra characterized by a molecular ion a t m / z 244 and a base peak ion a t m / z 87 (Figure 4, lower). The molecular ion region contained multiple ions with masses and intensities indicative of a compound containing 4 chlorine atoms. The mass spectra matched the INCOS library spectrum of tetrachloro-1,4-benzoquinone (2,3,5,6tetrachloro-2,5-cyclohexadiene-1,4-dione, CAS No. 11875-2) with a 99.7% fit. We have not elucidated the structure of the minor metabolite (tR = 43.3 min) which was characterized by absorption maxima a t 217 nm and 291 nm (A217/A291 = 3.8) and which may represent a dimeric product. We are attempting to obtain sufficient mass of this product for mass spectrometric, I3C-NMR, and infrared spectroscopic analysis. It is interesting that HPLC analysis of reaction mixtures a t 15 min indicated the presence of significant amounts of PCP (Figure 3, upper), whereas UV spectroscopic analysis of reaction mixtures, corresponding to the same time point, demonstrated the absence of the PCP chromophore (Figure 2). Incubation of PCP with HRP and HzOz resulted in ESR-detectable formation of relatively stable PCPderived phenoxyl radical intermediates which, under the conditions in Figure 5 (1 mM PCP, 1 mg/mL HRP ("25 pM HRP), 0.5mM HzOz), exhibited a half-life of 33-45 min. The ESR signal consisted of a relatively sym-
metrical single line characterized by a 6.1 G line width and devoid of any hyperfine structure. This specturm is consistent with the previously reported spectrum for photochemically-generated, PCP-derived phenoxyl radicals, indicating similar line widths and the absence of detectable hyperfine splittings by the 35Cland 37Clnuclei (29). Furthermore, the ESR signal was clearly not that of the tetrachloro-1,4-semiquinoneradical, which exhibits a spectral line width of 0.82 G in the same buffer system (data not shown). In contrast, no signal was observed in the absence of HRP or PCP and only a very weak signal was observed in systems devoid of HzOz (Figure 5). The latter may reflect limited autoxidation of PCP, catalyzed by trace transition metal contaminants, resulting in the generation of low levels of hydrogen peroxide. Substitution of natural abundance isotopically-labeled PCP with I3C-labeledPCP resulted in broadening of the ESR signal line width from 6.1 to 13.5 G (not shown). This represents the first report of the detection of enzymatically-generated pentachlorophenoxyl radicals. The successful application of spin trapping techniques to phenoxyl radical-generating systems is also previously unreported. We have carried out ESR spin trapping studies utilizing 4-POBN as the spin trap. Reaction mixtures containing HRP, 4-POBN, PCP, and H20z resulted in ESR-detectable nitroxide formation corresponding to 4-POBN radical adducts (Figure 6). In contrast, a-phenyl-N-tert-butylnitroneor 2-methyl-2nitrosopropane, when substituted for 4-POBN, failed to yield ESR-detectable nitroxides (data not shown). These differences in reactivity of the various spin traps may be related to differences in solubility because 4-POBN, a-phenyl-N-tert-butylnitrone, and 2-methyl-2-nitrosopropane exhibit octanoUwater partition coefficients (K,) of 0.09, 10.4, and 8.20, respectively (30). As shown in Figure 6, reaction mixtures devoid of HRP, HzOz, or PCP failed to yield any detectable nitroxide spectra. Computer simulation of the 4-POBN adduct ESR spectrum indicated the formation of two distinct adducts generated in a ratio of -3:l characterized by aN = (14.61 G)/uH = 1.83 G (Species I) and aN= (14.76 G)/uH= 5.21 G (Species 111, respectively (Figure 7). Comparison of the Species I
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 353
HRP-Catalyzed Oxidation of PCP
20 Gauss
Figure 6. ESR spectrum of 4-POBN nitroxide adducts generated during hydroperoxide-dependent, HRP-catalyzed oxidation
of PCP. Reaction conditions are as described i n Figure 5 except that 4-POBN (50 mM) was also included. Spectra were obtained using a modulation amplitude of 1.6 G, time constant of 0.5 s, receiver gain of 8 x lo3,microwave power of 20 mW, and a scan time of 4 min over 80 G.
Figure 9. ESR spectrum of 4-POBN-PCP nitroxide adducts generated during hydroperoxide-dependent, HRP-catalyzed oxidation of 13C-labeledPCP. Conditions are as described in Figure 6 except that natural abundant isotopically-labeled PCP was substituted with '3C-labeled P C P (l3C6, 99%). Spectra were obtained using a modulation amplitude of 1.6 G, time constant of 1.0 s, receiver gain of 8 x lo3, microwave power of 20 mW, and a scan time of 4 min over 100 G.
HRP-catalyzed reduction of PPHP (Figure 1). In fact, the index of peroxidase reducing substrate efficiency ([PPAY ([PPAI [PPHPI)) of PCP was comparable to that of ascorbic acid. The ESR spectroscopic studies (Figure 5 ) indicate that hydroperoxide-dependent, HRP-catalyzed oxidation of PCP results in the generation of PCP-derived pentachlorophenoxyl radical intermediates. The relative stability of the PCP phenoxyl radicals, as reflected in the relatively long half-life of the ESR signal (tvz -30-45 m i d , probably reflects the steric hindrance imposed by the chlorine substituents, thus inhibiting bimolecular carbon-carbon radical coupling. Collectively, these results suggest that PCP reacts with the higher oxidation states of the peroxidase (compounds I and/or 11) by H-atom or electron abstraction from the hydroxyl group, yielding the PCP-derived phenoxyl radical intermediate (eq 1).
+
il
jl
'Ih
1
C
Figure 7. Computer simulation of 4-POBN-derived nitroxide spectrum. (A) Experimental spectrum; (B) computer simulated spectrum; (C) species I component of computer simulation, U N = (14.61 G)hH = 1.83 G; (D)species I1 component of computer simulation, uN = (14.76 G ) h H = 5.21 G.
OH
0
C$l
- 0' Figure 8. Structure of the hydroxyl-4-POBN adduct.
spectrum with the Spin Trap Data Base (31) indicates that that species I is almost identical to the hydroxyl4-POBN adduct (Figure 8). Interestingly, substitution of naturally abundant isotopically-labeledPCP with 13Clabeled PCP in the complete 4-POBN system yielded identical ESR spectra (Figure 91, demonstrating that the PCP phenoxyl radical does not react with 4-POBN to generate carbon-centered radical adducts.
Discussion The results indicate that PCP functions as a reducing substrate for HRP as evidenced by the PCP-dependent,
tt
c1
Cl cl&;
c1
CI
We have also demonstrated ESR spin trapping of the pentachlorophenoxylradicals using 4-POBN as the spin trap. Interestingly, the major adduct (-70%) exhibited hyperfine coupling constants (aN = (14.62 G)/aH= 1.83 G )which are characteristic of the hydroxyl radical adduct (Figures 6-8). Furthermore, identical spectra were generated using natural abundance-labeled PCP (Figure 6) versus W-labeled PCP (Figure 91, eliminating the possibility of PCP-derived carbon-centered radical adducts because the latter would have resulted in additional hyperfine splitting by the 13C nuclei (S = fl/z). The inability to generate PCP carbon radical adducts again reflects steric hindrance imposed by the chlorine sub-
354 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
Samokyszyn et al.
Scheme 1
Scheme 2 0
CI
"@ C1
CI
Pentachlorophenoxyl Radical
C1
I
Bimolecular Radical
c
1
$kt
+N /
- 0'
I
t
H20
OH
c1y5& CI
stituents. These results suggest that the minor adduct consists of a large (possibly polymeric), stericallyhindered radical derived from PCP, while the hydroxyl adduct is formed by hydrolysis of the initial pentachlorophenoxyl radical adduct. There are a t present no entries in the Spin Trap Data Base (31)consistent with the minor species. This mechanism is consistent with the formation of hydroxyl-4-POBN nitroxide adducts, in a system devoid of hydroxyl radicals, and is shown in Scheme 1 as occuring via a putitive S Nmechanism ~ resulting in stereochemical inversion of one of the enatiomeric PCP adducts. In addition, the hyperfine coupling constants of the minor 4-POBN adduct (aN= (14.76 G)hH= 5.21 G) is consistent with the Heller-McConnell equation for a nitroxide adduct which is more sterically hindered compared with the hydroxyl adduct:
A,, = Bo
+ B, cos'
0
(2)
where Bo and Bz are constants (Bo 0 and Bz x 26 G for nitroxides), and 8 is the dihedral angle formed by the C-Np-orbital and the N-C PH planes (32). Collectively, these results are consistent with a 4-POBN-PCP phenoxyl radical adduct involving oxygen-centered radical addition. However, it should be pointed out that the trapping of phenoxy1 radicals by nitrone spin traps is unprecedented. Thus, we are currently attempting to isolate and unequivocally identify the structures of the 4-POBN adducts. We have also shown that hydroperoxide-dependent, HRP-catalyzed oxidation of PCP yields tetrachloro-1,4benzoquinone as a major product (Figures 3 and 4). However, we have not investigated the source of oxygen in the tetrachlorobenzoquinone because benzoquinone carbonyls, bonded to two adjacent chlorine-substituted vinylic carbons, exchange rapidly with water (23). However, the source of the tetrachlorobenzoquinone oxygen is probably HzO because we have shown that hydroperoxide (HzOz, ethyl hydroperoxide, PPHPI-dependent, HRP-catalyzed oxidation of PCP is not associated with uptake of molecular oxygen as measured with a Clark electrode (data not shown). In addition, the tertiary structure of HRP does not allow ferryl oxygen transfer to substrates from the peroxidase compound I intermediate (33-35). Essentially, the protein shields substrate access to the ferryl oxygen but does allow access to the 6-meso carbon of the porphyrin ring for electron transfers to compounds I and 11. Furthermore, Hammel and
0
Tardone (23) have shown that 2,4,6-trichlorophenol is oxidized by lignin peroxidase (which exhibits similar active site topoloigy to HRP (36)) to 2,6-dichloro-1,4benzoquinone and that the source of the benzoquinone oxygen is water. Tetrachloro-1,4-benzoquinonein our system may arise by the elimination of HC1 from a gemchlorohydrin intermediate (Scheme 2). The gem-chlorohydrin may arise through subsequent HRP-catalyzed one-electron oxidation of pentachlorophenoxyl radicals to PCP-derived carbocation intermediates which react with water. Kinetically, this is not unreasonable considering the relative persistence of the pentachlorophenoxyl radicals (Figure 53. However, a n alternative mechanism for gem-chlorohydrin formation is represented by hydrolysis of a putative ether arising by C-0 coupling of pentachlorophenoxyl radicals (Scheme 2). This mechanism is consistent with the detection of unreacted PCP (Figure 3, upper) a t a time point corresponding complete disappearance of the PCP W chromophore (Figure 2). The putative ether intermediate has been synthesized by oxidation of PCP by nitric acid in solutions of trifluoroacetic anhydride and trifluoroacetic acid a t -20 "C (37) and by oxidation of PCP by lead dioxide a t room temperature (38). We are attempting to determine if the minor metabolite eluting a t 43.3 min (Figure 3, upper) may represent this PCP-derived dimer. We have demonstrated the peroxidase-catalyzed oxidation of PCP to the electrophilic tetrachloro-1,4-benzoquinone. The tetrachlorobenzoquinone reacts with nucleophiles by Micheal addition reactions (16,17,39) and has been detected in vivo in mammalian systems as a PCP metabolite (18,19). Thus, peroxidases may play a role in the bioactivation of PCP. We are currently investigating the hydroperoxide-dependent cooxidation of PCP by several mammalian peroxidases including myeloperoxidase, lactoperoxidase, and PGH synthase.
References (1)Bronstein, A. C., and Sullivan, J. B. (1992) Fungicides and biocides. In Hazardous Materials Toxicology. Clinical Principles ofEnuironmenta1 Health (Sullivan, J . B., Jr., and Krieger, G. R., Eds.) pp1070-1072, Williams & Wilkins, Baltimore. (2). Crosby, D. G., Benyon, K. I., Greve, P. A., Korte, F., Still, G. G., and Vouk, J.W. (1981) Environmental chemistry of pentachlorophenol. Pure Appl. Chem. 53, 1051-1080. ( 3 ) IPCS (1987) Pentachlrophenol. Environmental Health Criteria, Vol. 71, WHO, Geneva.
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