2
22
F. W. Wiese et al.
tertiary structures of these proteins do not allow substrate
access to the ferryl intermediate of compound I (Ator and Ortiz
de Montellano 1987; DePillis et al. 1990). If the latter occurred,
this would result in the incorporation of hydroperoxide-derived
oxygen. In fact the latter investigators have reported evidence
suggesting that the peroxidase-catalyzed oxidations by both
HRP and ligninase involve sequential one-electron transfer
mechanisms through the methine carbon at the ␦-edge of the
heme prosthetic group.
Other than 2,6-dichloro-1,4-benzoquinone, our analysis of
peroxidase-dependent, TCP-derived oxidation products failed
to demonstrate any significant generation of other stable
oxidation products including carbon–carbon or carbon–oxygen
radical coupling products (e.g., dimers). These represent major
phenoxyl radical oxidation products involving radical–radical
coupling reactions at unsubstituted carbon centers (McDonald
and Hamilton 1973). Thus, chlorine substitution at the ortho
and para positions in TCP, the carbons characterized by
unpaired electron density in the phenoxyl radical intermediate
DeMarini DM, Brooks HG, Parkes Jr. DG (1990) Induction of
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Inactivation of lignin peroxidase by phenylhydrazine and sodium
azide. Arch Biochem Biophys 280:217–223.
Dunford HB (1991) Horseradish peroxidase: structure and kinetic
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Everse J, Everse KE, Grisham MB (eds) (1991) Peroxidases in
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Fitzloff JF, Portig J, Stein K (1982) Lindane metabolism by human and
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Hammel KE, Tardone PJ (1988) The oxidative 4-dechlorination of
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Biol Interactions 69:333–344
(Figure 7), imposes steric restrictions that hinder intermolecular
coupling reactions. This has previously been observed during
peroxidase-catalyzed oxidation of PCP (Samokyszyn et al.
Li S, Paleologou M, Purdy WC (1991) Determination of the acidity
constants of chlorinated phenolic compounds by liquid chromatog-
raphy. J Chromat Sci 29:66–69
Lin PH, Waidyanatha S, Rappaport SM (1996) Investigation of liver
binding of pentachlorophenol based upon measurements of protein
adducts. Biomarkers 1:109–113
Lin PH, Waidyanatha S, Pollack GM, Rappaport SM (1997) Dosimetry
of chlorinated quinone metabolites of pentachlorophenol in the
livers of rats and mice based upon measurement of protein adducts.
Toxicol Appl Pharmacol 145:399–408
Markey CM, Alward A, Weller PE, Marnett LJ (1987) Quantitative
studies of hydroperoxide reduction by prostaglandin H synthase.
Reducing substrate specificity and the relationship of peroxidase to
cyclooxygenase activities. J Biol Chem 262:6266–6279
Mauk MR, Girotti AW (1974) The protoporphyrin-apoperoxidase
complex. Photooxidation studies. Biochem 13:1757–1763
McDonald PD, Hamilton GA(1973) Mechanisms of phenolic oxidative
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chemistry, part B. Academic Press, New York, NY pp 97–134
Monks TJ, Hanzlik RP, Cohen GM, Ross D, Graham DG
1
995). Mechanistically, this also accounts for the absence of
detectable formation of TCP-derived chlorinated dibenzodiox-
ins and chlorinated dibenzofurans, which have been detected as
major HRP- and lactoperoxidase-catalyzed oxidation products
of 3,4,5- and 2,4,5-trichlorophenol (Oberg et al. 1990). An
additional consequence of steric effects imposed by chlorine
substitution is the relative stability of the TCP-derived phenoxyl
radical intermediates, as evidenced by direct detection by EPR
spectroscopy in reaction mixtures (Figure 6). This is consistent
with our previous report demonstrating the generation of
relatively stable phenoxyl radicals during peroxidase-catalyzed
oxidation of PCP (Samokyszyn et al. 1995).
We predict that 2,6-dichloro-1,4-benzoquinone, the major
peroxidase oxidation product of TCP, may function as an
effective electrophile that reacts with nucleophiles by Michael
addition reactions resulting in the formation of protein and
DNA adducts as well as adducts derived from other cellular
nucleophiles. This is consistent with the general electrophilic
nature of quinones (Monks et al. 1992) as well as the demonstr-
ation by Rappaport and colleagues that the analogous PCP-
derived tetrachlorobenzoquinones react with proteins in vitro
and in vivo by reaction with cysteine sulfhydryls (Waidyanatha
et al. 1994, 1996; Lin et al. 1996, 1997). In addition, we have
recently demonstrated that synthetic 2,6-dichloro-1,4-benzoqui-
none reacts rapidly with low molecular weight primary amine-
and thiol-containing molecular probes (data not shown) and we
are in the process of elucidating the structures of these adducts.
Thus, peroxidases may play a relevant role in the bioactivation
of TCP (and perhaps other chlorophenols) in vivo, which may
contribute to the observed toxicity and carcinogenicity of TCP.
(1992) Quinone chemistry and toxicity. Toxicol Appl Pharmacol
112:2–16
National Toxicology Program (NTP) (1985) CAS No. 85-002, US
Dept. of Health and Human Services
National Toxicology Program (NTP) (1987) CAS No. 88-06-2, US
Dept. of Health and Human Services
National Cancer Institute (NCI) (1979) Bioassay of 2,4,6-trichlorophe-
nol for possible carcinogenicity: NCI-TR-155. NIH Publication
No. 79-1711, National Cancer Institute, Bethesda, MD
Oberg LG, Glas B, Swanson SE, Rappe C, Paul KG (1990) Peroxidase-
catalyzed oxidation of chlorophenols to polychlorinated dibenzo-p-
dioxins and dibenzofurans. Arch Environ Contam Toxicol 19:930–
9
38
Samokyszyn VM, Freeman JP, Maddipati KR, Lloyd RV (1995)
Peroxidase-catalyzed oxidation of pentachlorophenol. Chem Res
Toxicol 8:349–355
Waidyanatha S, McDonald TA, Lin PH, Rappaport SM (1994)
Measurement of hemoglobin and albumin adducts of tetrachloro-
Acknowledgment. This work was supported by NIH Grant
R29ES06765-03.
1
,4-benzoquinone. Chem Res Toxicol 7:463–468
Waidyanatha S, Lin PH, Rappaport SM (1996) Characterization of chlori-
nated adducts of hemoglobin and albumin following administration of
pentachlorophenol to rats. Chem Res Toxicol 9:647–653
Weller P, Markey CM, Marnett LJ (1985) Enzymatic reduction of
5-phenyl-4-pentenyl-hydroperoxide: detection of peroxidases and
identification of peroxidase reducing substrates. Arch Biochem
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