Myeloperoxidase-catalyzed formation of PCDD/F from chlorophenols

Article abstract of DOI:10.1016/S0045-6535(99)00340-9

Chlorophenols (CP) are transformed in vitro to polychlorinated dibenzo- p-dioxins and dibenzofurans (PCDD/F) by a biochemical-catalyzed oxidation. This is shown for 2,4,5-tri-, 2,3,4,6-tetra-and pentachlorophenol with myeloperoxidase recovered from human leucocytes in the presence of hydrogen peroxide. The yield, the reaction, and the PCDD/F-pattern found depend on the CP. The formation rates are in the μmol-per-mol range for all substrates. The experiments confirm the suspicion that a biochemical formation of PCDD/F from precursors such as CPs can take place in the human body and that this metabolic pathway may lead to a higher inner exposure with PCDD/F than is now assumed. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0045-6535(99)00340-9

Chemosphere 40 (2000) 963±968
Myeloperoxidase-catalyzed formation of PCDD/F from
chlorophenols
*

Jurgen Wittsiepe , Yvonne Kullmann, Petra Schrey, Fidelis Selenka,
Michael Wilhelm
Ruhr-Universitat Bochum, Abteilung fur Hygiene, Sozial- und Umweltmedizin, Universitatsstraûe 150, D-44801 Bochum, Germany
Abstract
Chlorophenols (CP) are transformed in vitro to polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) by
a biochemical-catalyzed oxidation. This is shown for 2,4,5-tri-, 2,3,4,6-tetra-and pentachlorophenol with myeloper-
oxidase recovered from human leucocytes in the presence of hydrogen peroxide. The yield, the reaction, and the PCDD/
F-pattern found depend on the CP. The formation rates are in the lmol-per-mol range for all substrates.
The experiments con®rm the suspicion that a biochemical formation of PCDD/F from precursors such as CPs can
take place in the human body and that this metabolic pathway may lead to a higher inner exposure with PCDD/F than
is now assumed. Ó 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction
growing granulocyte cells in the bone marrow can be
higher.
Several studies on humans and cows indicate a higher
fecal excretion of PCDD/F, especially higher chlorinated
PCDD, in relation to the dietary intake (Moser et al.,
1996; Fries et al., 1996, 1997; Schrey et al., 1998). A
biochemical formation of PCDD/F in the organisms
from precursors such as chlorophenols (CP) would be
one explanation of these ®ndings. Biochemical forma-
tion of PCDD/F from chlorophenols have been ob-
In the present study we investigated the in vitro
formation of PCDD/F from 2,4,5-trichlorophenol
(2,4,5-TrCP), 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP)
and pentachlorophenol (PCP) catalyzed by myeloper-
oxidase recovered from human leucocytes in the pres-
ence of hydrogen peroxide.

served in sewage sludge (Oberg et al., 1992), compost
2. Material and methods

(Oberg et al., 1992, 1993) and in in vitro studies cata-
lyzed by horseradish peroxidase (Svenson et al., 1989a,
Myeloperoxidase recovered from human leucocytes
and lyophylized from 0.02 M sodium acetate bu€er at
pH 6.0 (MPO; EC 1.11.1.7, M 6908/Lot 126H9402) was
purchased from Sigma. 2,4,5-trichlorophenol (99.9%
certi®ed, C 177745/Lot 40615) and 2,3,4,6-tetrachloro-
phenol (97.2% certi®ed, C 173746/Lot 59426) were ob-
tained from Promochem (Wesel, Germany) and
pentachlorophenol (certi®ed reference material, P 16-23/
Lot 127/Sn 1439) was from International Physical Lab-
oratory. Methanolic solutions of the chlorophenols were
made in concentrations of 2, 5 or 15 mg/ml.

1989b; Oberg et al., 1990; Wagner et al., 1990) or bovine

lactoperoxidase (Oberg and Rappe, 1992).
Myeloperoxidase is a component of neutrophile
granulocytes, a subgroup of the leucocytes in the human
organism. The content of myeloperoxidase is up to
5%dry matter in the peripheral cells, whereas the content in
* Corresponding author. Fax: +49-234-7094-199.
E-mail address: wittsiepe@hygiene.ruhr-uni-bochum.de (J.
Wittsiepe).
All reactions were carried out in a potassium dihy-
drogen phosphate bu€er (pH 5.4) with a total volume
0045-6535/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 4 5 - 6 5 3 5 ( 9 9 ) 0 0 3 4 0 - 9

964
J. Wittsiepe et al. / Chemosphere 40 (2000) 963±968
of 2.0 ml in 20 ml glass test tubes at 37°C. The incu-
bation was started with the addition of hydrogen per-
oxide. The incubation time was 4 h in all experiments.
The series were performed with two blank controls
containing either the chlorophenol substrate or the
myeloperoxidase while all other additions were left
unchanged. The detailed list of composition is shown in
Table 1.
8000±10000 at 10%; electron impact ionization at 40 eV;
per¯uorokerosene lock mass check; observation of 2
ions each for native and labelled isomers; setting of 5
time windows; GC-parameters: column: J&W Scienti®c,
DB-5, 60 m, 0.1 lm ®lm thickness; temperature pro-
gram: 180°C (3 min), 5°C/min, 220°C (16 min), 5°C/min,
235°C (7 min), 5°C/min, 280°C (15 min); injector
program: 60°C (60 s), 12°C/s, 330°C (10 min), split o€
(1 min); split on (2 min); injection volume: 4 ll.
After incubation 100 ll of a standard solution con-
13
taining 17
C
labelled PCDD/F congeners (2.5 or 5.0
12
pg/ll) in toluene, 3 ml saturated ammonium sulphate
solution and 3 ml ethanol were added. The samples were
extracted three times with 4 ml of hexane. The dried
hexane extracts were puri®ed using standard methods
including modi®ed silicagels, alumina and activated
charcoal. After addition of 2 ll of dodecane the cleaned
3. Results and discussion
The results of the PCDD/F homologue groups of
major interest and the sum of the PCDD/F are given in
Tables 2±5 for the experiments with 2,4,5-TrCP, 2,3,4,6-
TeCP, PCP and the multisubstrate experiments.
The PCDD/F concentrations in the chlorophenol free
blank (No. 0) was near the detection limit for all cong-
eners and very small amounts were detected in the my-
eloperoxidase free blank samples (No. 1, 4 and 6). In all
other samples a signi®cant formation of PCDD/F was
observed.
extracts were evaporated to dryness using a nitrogen
13
stream and 10 ll of toluene containing 2.5 pg/ll
1.2,3,4-TetraCDD were added as external standard.
C
12
The analytical instrument system consisted of a VG
AutoSpec high-resolution mass spectrometer and a
Hewlett Packard 5890 series II gas chromatograph
equipped with a Gerstel KAS 2 vaporization system:
MS-parameters: single ion recording mode; resolution
Table 1
List of experiments
Experiment number
0 (CP ± blank)
KH2PO4 bu€er [mM]
0.375
Myeloperoxidase [UÃ]
1.0
Chlorophenol [mM]
±
H2O2 [mM]
0.5
2,4,5-Trichlorophenol
1 (blank)
0.375
0.375
0.375
±
1.0 (2,4,5-TrCP)
1.0 (2,4,5-TrCP)
0.1 (2,4,5-TrCP)
1.0
1.0
0.1
2
3
1.0
1.0
2,3,4,6-Tetrachlorophenol
4 (blank)
5
0.375
0.375
±
1.0
1.0 (2,3,4,6-TeCP)
1.0 (2,3,4,6-TeCP)
1.0
1.0
Pentachlorophenol
6 (blank)
0.375
0.375
0.375
0.375
0.375
0.375
±
1.0 (PCP)
0.1 (PCP)
1.0 (PCP)
0.1 (PCP)
1.0 (PCP)
1.0 (PCP)
1.0
0.1
0.5
1.0
1.0
2.0
7
1.0
1.0
1.0
1.0
1.0
8
9
10
11
Equimolar multisubstrate experiments
12
0.375
1.0
1.0 (2,4,5-TrCP)
1.0 (PCP)
1.0
13
14
0.375
0.375
1.0
1.0
0.1 (2,4,5-TrCP)
1.0 (2,3,4,6-TeCP)
1.0 (PCP)
0.1
1.0
15
16
0.375
0.375
1.0
1.0
0.1 (2,3,4,6-TeCP)
0.1 (PCP)
0.1
1.0
1.0 (2,4,5-TrCP)
1.0 (2,3,4,6-TeCP)
1.0 (PCP)
* Units as speci®ed by the manufacturer.

J. Wittsiepe et al. / Chemosphere 40 (2000) 963±968
965
Table 2
Myeloperoxidase-catalyzed formation of PCDD/F from 2,4,5-Trichlorophenol
Experiment number
1 (blank) [pg]
PentaCDD
HexaCDD
HeptaCDD
PentaCDF
HexaCDF
PCDD/F
<0.5
150
9.2
140
17
74
<0.5
89
1.9
310
83.2
782
2
[pg]
[lmol/molTrCP
[pg]
Ã
Ã
]
]
0.21
24
0.34
0.17
30
0.27
0.07
7.4
0.00
0.13
18
0.26
0.41
53
0.68
0.99
138
1.55
3
[lmol/molTrCP
* Blank (experiment 1) corrected values.
Table 3
Myeloperoxidase-catalyzed formation of PCDD/F from 2,3,4,6-Tetrachlorophenol
Experiment number
HexaCDD
HeptaCDD
OctaCDD
PCDD/F
4 (blank) [pg]
5
17
260
0.31
220
2200
2.33
65
1100
1,320
4610
[pg]
[lmol/molTeCP
Ã
]
1.13
3.77
* Blank (experiment 4) corrected values.
Table 4
Myeloperoxidase-catalyzed formation of PCDD/F from Pentachlorophenol
Ã
Ã
PCDD/F [lmol/molPCP]
Experiment number
OctaCDD [pg]
PCDD/F [pg]
OCDD [lmol/molPCP
]
6 (blank)
140
1600
155
1660
7
8
15.9
3.65
15.9
10.7
6.48
16.4
3.88
16.0
11.1
6.80
3500
1600
3670
1600
9
10
11
10,000
6100
10,400
6350
* Blank (experiment 6) corrected values.
Table 5
Myeloperoxidase-catalyzed formation of PCDD/F from chlorophenol mixtures
Experiment number
12 [pg]
PentaCDD
HexaCDD
HeptaCDD
OctaCDD
HexaCDF
880
0.59
200
1.33
450
0.30
29
0.19
890
0.40
PCDD/F
280
0.20
120
0.84
12
680
0.43
100
13000
7.64
1300
7.64
3800
2.23
84
1400
0.76
140
16000
9.70
1830
11.3
14800
8.34
492
[lmol/molRCP
13 [pg]
[lmol/molRCP
14 [pg]
[lmol/molRCP
15 [pg]
[lmol/molRCP
16 [pg]
[lmol/molRCP
]
]
]
]
]
0.64
150
0.76
9700
5.27
310
0.01
0.29
0.00
36
0.10
7.0
0.04
1100
0.47
0.49
3000
1.18
1.69
910
0.33
2.78
6760
2.70
0.02
The monosubstrate experiments with PCP showed a
predominant formation of OctaCDD. The amount of
OctaCDD increased with the amount of the substrate.
But the highest formation rates were observed with the
lower PCP concentrations (exp. 7 and 9), whereas the
H2O2 concentration showed only in¯uence on the
PCDD/F formation with the higher dose (1 mM) (exp. 8,
10, 11) but not the lower dose (0.1 mM) (exp. 7 and 9)
experiments.
homologue groups were the HexaCDF, followed by
HexaCDD, PentaCDD and PentaCDF (2,4,5-TrCP) or
the HeptaCDD, OctaCDD and HexaCDD (2,3,4,6-
TeCP). A comparison of the PCDD/F formation rates
on a molar basis for the monosubstrate experiments
with 1 mM concentrations (exp. 2, 5 and 10) is given in
Fig. 1.
The combination of equimolar mixtures of 2,4,5-
TrCP and PCP resulted in a formation of HeptaCDD as
major component, whereas OctaCDD, followed by
HeptaCDD were the main components in the experi-
ments with equimolar mixtures of 2,3,4,6-TeCP and
The formation of PCDD/F was lower in the mono-
substrate experiments with 2,4,5-TrCP and 2,3,4,6-TeCP
and showed di€erent homologue patterns. The main

966
J. Wittsiepe et al. / Chemosphere 40 (2000) 963±968
Fig. 1. PCDD/F formation rates of the 1 mM monosubstrate experiments 2, 5 and 10.
PCP. Hepta- and HexaCDD were the main components
in the experiment containing all three chlorophenols.
The results of the experiments 12, 14 and 16 are shown
in Fig. 2.
with MYP showing 15.9 lmol/molPCP. The PCDD/F
pattern found in the experiments with 2,4,5-TrCP is
di€erent between all authors. The observed di€erences
could be an e€ect of the experimental conditions, espe-
cially the di€erent peroxidase and hydrogen peroxide
concentrations used. In addition the discrepancies may
be attributed to di€erent analytical procedures and to
di€erent quality grades of the CPs used.
A comparison of the experiments with studies of
other authors is given in Table 6. All experiments with
PCP gave OctaCDD as the main component. The for-
mation rates in the studies with LP and HRP published


by Oberg et al. (1990) and Oberg and Rappe (1992) were
two to four times higher than in our own experiments
First in vivo studies with rats fed with 0.1 mg/d of
puri®ed PCP over 14 days showed no higher PCDD/F
Fig. 2. PCDD/F formation rates of the multisubstrate experiments 12, 14 and 16.

J. Wittsiepe et al. / Chemosphere 40 (2000) 963±968
967
Table 6
Comparison with data from literaturea


Parameter
Oberg et al. (1990); Oberg and Rappe
(1992)
Svenson et al.
(1989b)
MYP
This study
HRP
LP
HRP
Experimental conditions
2,4,5-TrCP [mM]
0.1
0.1
0.1
0.1
0.1
±
0.1
0.1
0.5
0.1
PCP
Peroxidase [U/ml]
H2O2 [mM]
[mM]
1.3±1.7
0.2
1.3±1.7
0.2
1.3±1.7
9.0
Formation of PCDD/F from 2,4,5-TriCPÃ
PentaCDD [lmol/molTrCP
HexaCDD [lmol/molTrCP
PentaCDF [lmol/molTrCP
]
]
]
]
]
3.60
1.01
0.37
0.04
5.73
1.27
0.86
0.06
0.03
2.43
3.37
5.88
0.37
0.75
10.5
0.34
0.27
0.26
0.68
1.55
HexaCDF
[lmol/molTrCP
R PCDD/F [lmol/molTrCP
Formation of PCDD/F from PCP
OctaCDD [lmol/molPCP
]
34.8
57.9
±
15.9
a LP ˆ lactoperoxidase, HRP ˆ horseradish peroxidase, MYP ˆ myeloperoxidase
* Wagner et al. (1990) found a formation of 5,7 lgRPCDD=F/g2;4;5-TrCP. The formation rate on a molar basis cannot be calculated due to
missing rates for the several homologue groups.
levels in the liver in comparison to a control group.
Administration of reagent grade PCP and technical
grade PCP showed a formation of higher chlorinated
PCDD (Feil and Tiernan, 1997). The main component
was OctaCDD with levels 2.6 or 1042 times higher in
comparison to the control group. In other experiments a
formation of OctaCDD from nonachloro-2-phenoxy-
phenol, which is a known contaminant of technical PCP,
was observed in rats (Huwe et al., 1998). Both studies
con®rm a formation of PCDD/F in vivo from PCP im-
purities.
impurities are in particular responsible for the observed
formations of PCDD/F.
4. Conclusion
The in vitro studies con®rm the suspicion that a
biochemical formation of PCDD/F from precursors
such as chlorophenols can take place in the human
body and that this metabolic pathway may lead to a
higher inner exposure with PCDD/F as up to now
estimated by food analyses or duplicate studies. Thus,
part of the observed higher excretion rates of higher
chlorinated PCDD/F in relation to the dietary intake
found in the mass balances studies (Schrey et al.,
1998, Moser et al., 1996) might be explained by
peroxidase-catalyzed metabolic transformations of
chlorophenols.
All observed formation rates are in the lmol-per-
mol range and so ± at ®rst glance ± they seem to be of
minor importance. But the estimated intake of
pentachlorophenol of unexposed persons in Germany
is in the range of 1±2 lg/d. Chlorophenols can also
be signi®cant metabolites of other chlorinated
substances such as chlorobenzenes and chlorocyclohex-
anes. Typical serum concentrations of pentachlorophe-
nol of unexposed persons from Germany are in the
range of up to 20 lg/l and occupationally exposed in-
dividuals show CP concentrations 10 to 1000 times
higher (Butte and Heinzow, 1995; Kommission Human-
Biomonitoring des Umweltbundesamtes, 1997). On this
background and under consideration of the observed
formation rates in the in vitro experiments additional
PCDD/F formations in the pg/d range would be
expected.
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