Peroxidase-catalyzed in vitro formation of polychlorinated dibenzo-p-dioxins and dibenzofurans from chlorophenols

Article abstract of DOI:10.1016/S0378-4274(99)00066-1

Chlorophenols (CP) are transformed in vitro to polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) by a peroxidase-catalyzed oxidation. This is shown for 2,4,5-tri-, 2,3,4,6-tetra- and pentachlorophenol with plant horseradish peroxidase and with myeloperoxidase recovered from human leukocytes, each in the presence of hydrogen peroxide. The yield, the reaction and the PCDD/F-pattern found are dependent on the CP. The amounts of PCDD/F formed within 4 or 24 h are in the μmol/mol-range for all substrates and both peroxidases. The experiments suggest that 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 to PCDD/F than up to now assumed based on intake data for PCDD/F. Copyright (C) 1999 Elsevier Science Ireland Ltd.

Full text of DOI:10.1016/S0378-4274(99)00066-1

Toxicology Letters 106 (1999) 191–200
Peroxidase-catalyzed in vitro formation of polychlorinated
dibenzo-p-dioxins and dibenzofurans from chlorophenols
Ju¨rgen Wittsiepe *, Yvonne Kullmann, Petra Schrey, Fidelis Selenka,
Michael Wilhelm
Ruhr-Uni6ersita¨t Bochum, Abteilung fu¨r Hygiene, Sozial- und Umweltmedizin, Uni6ersita¨tsstraße 150, D-44801 Bochum, Germany
Received 5 January 1999; received in revised form 10 March 1999; accepted 17 March 1999
Abstract
Chlorophenols (CP) are transformed in vitro to polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F)
by a peroxidase-catalyzed oxidation. This is shown for 2,4,5-tri-, 2,3,4,6-tetra- and pentachlorophenol with plant
horseradish peroxidase and with myeloperoxidase recovered from human leukocytes, each in the presence of hydrogen
peroxide. The yield, the reaction and the PCDD/F-pattern found are dependent on the CP. The amounts of PCDD/F
formed within 4 or 24 h are in the mmol/mol-range for all substrates and both peroxidases. The experiments suggest
that 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 to PCDD/F than up to now assumed based on intake data
for PCDD/F. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Peroxidase; Horseradish peroxidase; Myeloperoxidase; Hydrogen peroxide; Enzymatic formation; Poly-
chlorinated dibenzo-p-dioxins; Polychlorinated dibenzofurans; Octachlorodibenzo-p-dioxin; 2,3,7,8-Tetra-
chlorodibenzo-p-dioxin; Chlorophenols; Pentachlorophenol; 2,3,4,6-Tetrachlorophenol; 2,4,5-Trichlorophenol
tachlorophenol (PCP) treated wood, (Fries et al.,
1996; Fries et al. 1997) indicate a higher fecal
1. Introduction
excretion of polychlorinated dibenzo-p-dioxins
and dibenzofurans (PCDD/F), especially higher
Several mass balance studies on humans (Moser
et al., 1996; Schrey et al., 1998) and studies on
chlorinated PCDD, in relation to the amount
cattle, which were in contact or fed with pen-
ingested. The results point to the possible bio-
chemical formation of PCDD/F in the organisms
* Corresponding author. Fax: +49-234-7094-199.
from precursors such as chlorophenols (CP) or
E-mail address: wittsiepe@hygiene.ruhr-uni-bochum.de (J.
the known contaminants of technical-grade CP,
Wittsiepe)
0378-4274/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0378-4274(99)00066-1

192
J. Wittsiepe et al. / Toxicology Letters 106 (1999) 191–200
i.e. chlorinated phenoxyphenols. Biochemical for-
mations of PCDD/F from chlorophenols and
other precursors have been observed in sewage
2,4,5-TrCP (99.9% chemically pure, certified, Dr
Ehrenstorfer) and 2,3,4,6-TeCP (97.2% chemically
pure, certified, Dr Ehrenstorfer) were obtained
from Promochem (Wesel, Germany) and PCP
(certified reference material) was from Interna-
tional Physical Laboratory (UK). Methanolic so-
lutions of the chlorophenols were made with
concentrations of 2, 5 or 15 mg/ml.
8
sludge (Oberg et al., 1992; Schramm et al., 1996),
8
compost (Oberg et al., 1992, 1993; Scha¨fer et al.,
1993), in in vitro studies catalyzed by horseradish
peroxidase (HRP) (Svenson et al., 1989a; Svenson
8
et al. 1989b; Oberg et al., 1990; Wagner et al.,
8
1990), bovine lactoperoxidase (LP) (Oberg et al.,
1990) or more complex enzymatic systems such as
whey or culture filtrates from fungi (Wagner et
al., 1990). The formation of octachlorodibenzo-p-
dioxin from reagent or technical grade pen-
tachlorophenol and from nonachloro-2-phenoxy-
phenol, a contaminant of technical PCP, has been
observed in rats (Feil and Tiernan, 1997; Huwe
et al., 1998).
Peroxidases are omnipresent bi-substrate en-
zymes in nature. Within the catalyzed reactions
the presence of hydrogen peroxide is necessary
because it is used as the electron accepting sub-
strate. In the present study we investigated the in
vitro formation of PCDD/F from the commercial
significant CPs 2,4,5-trichlorophenol (2,4,5-
TrCP), 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP)
and pentachlorophenol catalyzed by plant HRP
and by myeloperoxidase (MYP) from human
leukocytes, each in the presence of hydrogen
peroxide.
2.2. Composition and incubation conditions of the
experiments
The reactions with HRP were incubated over 24
h in a total volume of 10 ml of a dimethyl succinic
acid (DSA) buffer at pH 4.0 at room temperature
as described by Wagner et al. (1990). All experi-
ments with myeloperoxidase were carried out in a
potassium dihydrogen phosphate buffer (pH 5.4)
with a total volume of 2.0 ml in 20 ml glass test
tubes at 37°C. The incubation time was 4 h in
these experiments. The incubation was started
with the addition of hydrogen peroxide. All series
were performed with two blank controls contain-
ing either the chlorophenol substrate or the perox-
idase while all other additions were left
unchanged. The detailed lists of composition are
shown in Tables 1 and 2.
In the present study the MYP catalyzed forma-
tion of PCDD/F from different chlorophenols,
from chlorophenol substrate mixtures and a com-
parison with the catalyzation by HRP are de-
scribed for the first time. The experiments with
HRP were repeated here for better comparison
because formations of PCDD/F might be influ-
enced by impurities of the substrates.
2.3. Analysis
After incubation 100 ml of a standard solution
containing 17 C12-labelled PCDD/F congeners
13
(2.5 or 5.0 pg/ml) in toluene, 3 ml saturated am-
monium sulphate solution and 3 ml ethanol were
added to an 2-ml aliquot of the HRP incubations
or to the total amount of the MYP incubations.
The samples were extracted three times with 4 ml
hexane. The hexane extracts were dried over
sodium sulphate and purified using three column
clean up steps: a combination of neutral, acidic
and basic silica gels, alumina and activated char-
coal (Nygren et al. 1988; Ho¨ckel and Hagenmaier,
1995). After addition of 2 ml dodecane as keeper
the cleaned extracts were evaporated to dryness
using a nitrogen stream and 10 ml toluene contain-
ing 2.5 pg/ml 1,2,3,4-[13C12] tetraCDD were added
as recovery standard.
2. Materials and methods
2.1. Enzymes and substrates
HRP Type II (P 8250/Lot 16H9522) was ob-
tained from Sigma. MYP recovered from human
leukocytes and lyophylized from 0.02 M sodium
acetate buffer at pH 6.0 (MPO; EC 1.11.1.7, M
6908/Lot 126H9402) was purchased from Sigma.

J. Wittsiepe et al. / Toxicology Letters 106 (1999) 191–200
193
ization system (MS-parameters: single ion
recording mode; resolution 8000–10 000 at 10%;
electron impact ionization at 40 eV; perfluoro-
kerosene lock mass check; observation of two ions
The analytical instrument system consisted of a
VG AutoSpec high-resolution mass spectrometer
and a Hewlett-Packard 5890 series II gas chro-
matograph equipped with a Gerstel KAS 2 vapor-
Table 1
List of experiments with HRP (total volume: 10 ml)
Experiment No.
H0 (CP-blank)
DSA buffer (mM)
10
HRP (U)a
2.0
CP (mM)
–
Hydrogen peroxide (mM)
2.0
2,4,5-TrCP
H1 (blank)
H2
10
10
–
2.0
0.1 (2,4,5-TrCP)
0.1 (2,4,5-TrCP)
2.0
2.0
2,3,4,6-TeCP
H3 (blank)
H4
10
10
–
2.0
0.1 (2,3,4,6-TeCP)
0.1 (2,3,4,6-TeCP)
2.0
2.0
PCP
H5 (blank)
H6
10
10
–
2.0
0.1 (PCP)
0.1 (PCP)
2.0
2.0
a Units as specified by the manufacturer.
Table 2
List of experiments with MYP (total volume: 2 ml)
Experiment No. KH2PO4 buffer
(mM)
MYP (U)a
CP (mM)
–
Hydrogen peroxide
(mM)
M0 (CP-blank)
0.375
1.0
0.5
2,4,5-TrCP
M1 (blank)
M2
0.375
0.375
0.375
–
1.0
1.0
1.0 (2,4,5-TrCP)
1.0 (2,4,5-TrCP)
0.1 (2,4,5-TrCP)
1.0
1.0
0.1
M3
2,3,4,6-TeCP
M4 (blank)
M5
0.375
0.375
–
1.0
1.0 (2,3,4,6-TeCP)
1.0 (2,3,4,6-TeCP)
1.0
1.0
PCP
M6 (blank)
M7
M8
M9
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
1.0
1.0
1.0
1.0
1.0
M10
M11
Equimolar multisubstrate experiments
M12
M13
M14
M15
M16
0.375
0.375
0.375
0.375
0.375
1.0
1.0
1.0
1.0
1.0
1.0 (2,4,5-TrCP), 1.0 (PCP)
0.1 (2,4,5-TrCP), 0.1 (PCP)
1.0 (2,3,4,6-TeCP), 1.0 (PCP)
0.1 (2,3,4,6-TeCP), 0.1 (PCP)
1.0 (2,4,5-TrCP), 1.0 (2,3,4,6-TeCP), 1.0 (PCP)
1.0
0.1
1.0
0.1
1.0
a Units as specified by the manufacturer.

194
J. Wittsiepe et al. / Toxicology Letters 106 (1999) 191–200
Table 3
HRP- or MYP-catalyzed formation of PCDD/F from PCP (detailed information on the experiments are given in Tables 1 and 2)
a
a
Experiment No.
OctaCDD (pg)
ꢀPCDD/F (pg)
OCDD (mmol/mol
)
ꢀPCDD/F (mmol/mol
)
PCP
PCP
H5 (bl)
H6
M6 (bl)
M7
M8
M9
64
6800
140
1600
3500
1600
10 000
6100
150
6910
155
1660
3670
1600
10 400
6350
–
14.7
–
15.9
3.65
15.9
10.7
6.48
–
14.9
–
16.4
3.88
16.0
11.1
6.80
M10
M11
a Blank (experiment H5 or M6) corrected values.
Table 4
HRP- or MYP-catalyzed formation of PCDD/F from 2,4,5-TrCP (detailed information on the experiments are given in Tables 1 and
2)
Experiment No.
PentaCDD
HexaCDD
HeptaCDD
PentaCDF
HexaCDF
ꢀPCDD/F
H1 (bl)
H2
pg
pg
B0.5
560
1.57
B0.5
510
1.30
1.2
9.6
0.02
4.9
62
0.17
0.76
140
0.37
50.3
1340
3.45
a
mmol/molTrCP
M1 (bl)
M2
pg
pg
mmol/mol
pg
B0.5
150
0.21
24
9.2
140
0.17
30
17
74
0.07
7.4
0.00
B 0.5
89
0.13
18
1.9
310
0.41
53
83.2
782
0.99
138
a
a
TrCP
TrCP
M3
mmol/mol
0.34
0.27
0.26
0.68
1.55
a Blank (experiment H1 or M1) corrected values.
each for native and labelled isomers; setting of
five time windows; GC-parameters: column: J&W
Scientific, DB-5, 60 m, 0.1 mm film thickness;
temperature program: 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 off (1 min); split on
(2 min); injection volume: 4 ml).
The PCDD/F concentrations in the chlorophe-
nol free blanks (exp. H0 and M0) were near to the
detection limit for all congeners and very small
amounts were detected in the HRP or MYP free
blank samples (exp. H1, H3, H5, M1, M4 and
M6). In all other samples a significant formation
of PCDD/F—2,3,7,8-chlorosubstituted as well as
non-2,3,7,8-chlorosubstituted
congeners—was
The PCDD/F concentrations were calculated
considering the recovery rates of the internal stan-
dard compounds and response factors.
observed. The formation of 2,3,7,8-tetra-
chlorodibenzo-p-dioxin was only observed in the
experiments using 2,4,5-TrCP.
3. Results and discussion
3.1. Monosubstrate experiments with PCP
The results of the PCDD/F homologue groups
of major interest and the sum of the PCDD/F are
given in the Tables 3–6 for the experiments with
PCP, 2,4,5-TrCP, 2,3,4,6-TeCP and the multisub-
strate experiments.
The monosubstrate experiments with PCP and
both HRP or MYP showed a predominant forma-
tion of OctaCDD (Table 3). The amounts formed
were in the same order of magnitude for both
peroxidases. The amount of OctaCDD built by

J. Wittsiepe et al. / Toxicology Letters 106 (1999) 191–200
195
MYP showed a tendency to increase with the
amount of the substrate. But highest formations
were observed with the lower PCP concentrations
(exp. M7 and M9), whereas the hydrogen perox-
ide concentration only showed an influence on the
PCDD/F formation in the higher dosed (1 mM)
(exp. M8, M10, M11) but not in the lower dosed
(0.1 mM) (exp. M7 and M9) experiments.
2,4,5-TrCP (Table 4) and 2,3,4,6-TeCP (Table 5)
and showed different homologue patterns. The
main homologue groups were the PentaCDD, fol-
lowed by HexaCDD and HexaCDF (2,4,5-TrCP,
HRP), the HexaCDF, PentaCDD, HexaCDD,
and PentaCDF (2,4,5-TrCP, MYP), the Oc-
taCDD, HeptaCDD and HexaCDD (2,3,4,6-
TeCP, HRP) or the HeptaCDD, OctaCDD and
HexaCDD (2,3,4,6-TeCP, MYP). A comparison
of the amounts of PCDD/F formed on a molar
basis for the HRP catalyzed monosubstrate exper-
iments with 0.1 mM concentrations (exp. H2, H4
and H6) is given in Fig. 1 and for the MYP
catalyzed monosubstrate experiments with 1 mM
concentrations (exp. M2, M5 and M10) in
Fig. 2.
3.2. Monosubstrate experiments with 2,4,5-TrCP
and 2,3,4,6-TeCP
In comparison to the experiments with PCP the
formation of PCDD/F by HRP or MYP was
lower in the monosubstrate experiments with
Table 5
HPR- or MYP-catalyzed formation of PCDD/F from 2,3,4,6-TeCP (detailed information on the experiments are given in Tables 1
and 2)
Experiment No.
HexaCDD
HeptaCDD
OctaCDD
ꢀPCDD/F
H3 (bl)
H4
pg
pg
mmol/mol
2.7
43
0.10
14
180
0.39
23
2000
4.30
189
2530
5.12
a
a
TeCP
TeCP
M4 (bl)
M5
pg
pg
mmol/mol
17
260
0.31
220
2200
2.33
65
1100
1.13
1320
4610
3.77
a Blank (experiment H3 or M4) corrected values.
Table 6
MYP-catalyzed formation of PCDD/F from CP mixtures (detailed information on the experiments are given in Table 2)
Experiment No.
M12
PentaCDD
HexaCDD
HeptaCDD
OctaCDD
HexaCDF
ꢀPCDD/F
pg
280
0.20
680
0.43
13 000
7.64
1400
0.76
880
0.59
16 000
9.70
mmol/molS CP
M13
M14
M15
M16
pg
120
0.84
100
0.64
1300
7.64
140
0.76
200
1.33
1830
11.3
mmol/molS CP
pg
12
0.01
150
0.10
3800
2.23
9700
5.27
450
0.30
14 800
8.34
mmol/molS CP
pg
0.29
0.00
7.0
0.04
84
0.49
310
1.69
29
0.19
492
2.78
mmol/molS CP
pg
36
0.02
1100
0.47
3000
1.18
910
0.33
890
0.40
6760
2.70
mmol/molS CP

196
J. Wittsiepe et al. / Toxicology Letters 106 (1999) 191–200
Fig. 1. PCDD/F formation of the 0.1 mM monosubstrate experiments H2, H4 and H6 with HRP (detailed information on the
experiments are given in Table 1)
3.3. MYP-catalyzed multisubstrate experiments
with mixtures of 2,4,5-TrCP, 2,3,4,6-TeCP and
PCP
taCDD as the main component. The formations
observed in the studies with LP and HRP pub-
8
8
lished by Oberg et al. (1990) and Oberg and
Rappe (1992) were two to four times higher as in
our own experiments with HRP and MYP show-
ing both about 15 mmol/molPCP. The PCDD/F
pattern found in the experiments with 2,4,5-TrCP
is different between authors. The differences ob-
served could be an effect of the experimental
conditions, especially the different peroxidase and
hydrogen peroxide concentrations used. In addi-
tion the discrepancies may be attributed to differ-
ent analytical procedures and to different quality
grades of the CPs used.
Within the MYP catalyzed experiments series
(Table 6) the combination of equimolar mixtures
of 2,4,5-TrCP and PCP resulted in the formation
of HeptaCDD as the major component, whereas
OctaCDD, followed by HeptaCDD, were the
main components in the experiments with
equimolar mixtures of 2,3,4,6-TeCP and PCP.
Hepta- and HexaCDD were the main components
in the experiment containing all three chlorophe-
nols. The results of the experiments M12, M14
and M16 are shown in Fig. 3.
3.5. Comparison with in 6i6o studies
3.4. Comparison with other in 6itro studies
First in vivo studies with rats fed with 0.1
mg/day of purified PCP over 14 days showed no
higher PCDD/F levels in the liver in comparison
to a control group. Administration of reagent
A comparison of the experiments with HRP
and MYP with studies of other authors is given in
Table 7. All experiments with PCP gave Oc-

J. Wittsiepe et al. / Toxicology Letters 106 (1999) 191–200
197
grade PCP and technical grade PCP showed the
formation of higher chlorinated PCDD (Feil and
Tiernan, 1997). The main component was Oc-
taCDD with levels 2.6 or 1042 times higher in
comparison to the control group. In other experi-
ments the formation of OctaCDD from
nonachloro-2-phenoxyphenol, which is a known
contaminant of technical PCP, was observed in
rats (Huwe et al., 1998). Both studies confirm the
formation of PCDD/F in vivo from PCP
impurities.
1–2 mg/day (Butte and Heinzow, 1995; HBM-
Kommission, 1997) and in the mass balance stud-
ies on humans (Schrey et al., 1998) the fecal
excretion of OCDD of adults was on average
2300 pg/day higher than the dietary intake.
Chlorophenols can also be significant metabolites
of other chlorinated substances such as
chlorobenzenes and chlorocyclohexanes. Typical
serum concentrations of pentachlorophenol for
unexposed persons from Germany range up to 20
mg/l and occupational exposed individuals show
CP concentrations 10 to 1,000 times higher
(HBM-Kommission, 1997). From this back-
ground and under consideration of the observed
amounts formed in the in vitro experiments addi-
tional PCDD/F formation in the pg/day range
would be expected, although kinetic data are not
available at the moment.
3.6. Assessment and conclusion for human
exposure
All observed formations in the in vitro studies
are in the mmol/mol range and so—at first
glance—they seem to be of minor importance,
but the estimated intake of pentachlorophenol for
unexposed persons in Germany is in the range of
It should be noted, that in all cases the presence
of hydrogen peroxide as second substrate is neces-
Fig. 2. PCDD/F formation of the 1 mM monosubstrate experiments M2, M5 and M10 with MYP (detailed information on the
experiments are given in Table 2)

198
J. Wittsiepe et al. / Toxicology Letters 106 (1999) 191–200
Fig. 3. PCDD/F formation of the multisubstrate experiments M12, M14 and M16 with MYP (detailed information on the
experiments are given in Table 2)
Table 7
Comparison with data from literature
8
Parameter
Oberg et al. (1990) and
Svenson et al. (1989b) HRP
HRP
This study
8
Oberg and Rappe (1992)
LP
HRP
HRP
MYP
Experimental conditions
2,4,5-TrCP (mM)
PCP (mM)
Peroxidase (U/ml)
Hydrogen peroxide (mM)
0.1
0.1
1.3–1.7
0.2
0.1
0.1
1.3–1.7
0.2
0.1
–
1.3–1.7
9.0
0.1
0.1
0.2
2.0
0.1
0.1
0.5
0.1
Formation of PCDD/F from 2,4,5-TriCPa
PentaCDD (mmol/mol
HexaCDD (mmol/mol
PentaCDF (mmol/mol
HexaCDF (mmol/mol
ꢀPCDD/F (mmol/mol
)
)
)
)
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
1.57
1.30
0.17
0.37
3.45
0.34
0.27
0.26
0.68
1.55
TrCP
TrCP
TrCP
TrCP
)
TrCP
Formation of PCDD/F from PCP
OctaCDD (mmol/mol
)
34.8
57.9
–
14.7
15.9
PCP
a Wagner et al. (1990) found a formation of 5,7 mgS PCDD/F/g2,4,5-TrCP. The formation on a molar basis cannot be calculated due
to missing data for the several homologue groups.

J. Wittsiepe et al. / Toxicology Letters 106 (1999) 191–200
199
metabolism in mammalian organs. Physiol. Rev. 59, 527–
605.
sary. Cellular and subcellular hydrogen peroxide
concentrations at steady state are in the range
1–100 nM (Chance et al., 1979). The formations
of PCDD/F in the human body can first takeplace
in the digestive tract, where peroxidases can be
present as part of microorganisms, vegetable or
animal food (Wagner et al., 1990). Anyway, dif-
ferent peroxidase systems can be found in many
organs and cells. The MYP used in this study is a
component of human neutrophile granulocytes.
The content of MYP is up to 5%dry matter in the
peripheral cells, whereas the content in growing
granulocyte cells in bone marrow can be higher
(Marquardt and Scha¨fer, 1994). This shows that
there are many locations where the formation of
PCDD/F by peroxidase systems can take place.
It must be mentioned that the CPs used in the
present study were of high purity and the influ-
ence of minor impurities in the CPs on the forma-
tion of PCDD/F was not examined. It can not be
excluded that specific impurities are in particular
responsible for the observed formation of PCDD/
F.
Feil, V.J., Tiernan, T., 1997. Pentachlorophenol as a source of
dioxins and furans. Organohalogen Compd. 33, 353–354.
Fries, G.F., Paustenbach, D.J., Wenning, R.J., Mathur, D.B.,
Luksemburg, W.J., 1996. Transport of chlorinated dioxin
and furan contaminants in pentachlorophenol-treated
wood to milk and adipose tissue of dairy cattle.
Organohalogen Compd. 29, 447–452.
Fries, G.F., Dawson, T.E., Paustenbach, D.J., Mathur, D.B.,
Luksemburg, W.J., 1997. Biosynthesis of hepta- and octa-
chlorodioxins in cattle and evidence for lack of involve-
ment by rumen microorganisms. Organohalogen Compd.
33, 296–301.
HBM-Kommission (Kommission Human-Biomonitoring des
Umweltbundesamtes), 1997. Stoffmonographie Pentachlor-
phenol-Referenz-
und
Human-Biomonitoring-Werte
(HBM). Bundesgesundhbl. 6, 212–222.
Ho¨ckel, J., Hagenmaier, H., 1995. Selective separation of
2,3,7,8-substituted tetra- to hexaCDD/CDF from other
PCDD/PCDF by fractionation on Alumina B Super 1 for
dioxin analysis. Organohalogen Compd. 23, 139–140.
Huwe, J.K., Feil, V.J., Tiernan, T.O., 1998. In vivo formation
of octachlorodibenzo-p-dioxin from
a
predioxin.
Organohalogen Compd. 36, 93–95.
Marquardt, H., Scha¨fer, G. (Eds.), 1994. Lehrbuch der
Toxikologie. BI-Wissenschaftsverlag, Mannheim.
Moser, G.A., Schlummer, M., McLachlan, M.S., 1996. Hu-
man absorption of PCDDs, PCDFs, and PCBs from food.
Organohalogen Compd. 29, 385–388.
Nygren, M., Hansson, M., Sjo¨stro¨m, M., et al., 1988. Devel-
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4. Conclusion
The in vitro studies confirm the suspicion that
the biochemical formation of PCDD/F from pre-
cursors such as chlorophenols can take place in
the human body and that this metabolic pathway
may lead to a higher inner exposure to PCDD/F
than 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 dietary intake found in the
mass balances studies (Moser et al., 1996; Schrey
et al., 1998) might be explained by peroxidase-cat-
8
Oberg, L.G., Rappe, C., 1992. Biochemical formation of
PCDD/Fs from chlorophenols. Chemosphere 25, 49–52.
8
Oberg, L.G., Glas, B., Swanson, S.E., Rappe, C., Paul, K.G.,
1990. Peroxidase-catalyzed oxidation of chlorophenols to
polychlorinated dibenzo-p-dioxins and dibenzofurans.
Arch. Environ. Contam. Toxicol. 19, 930–938.
8
Oberg, L.G., Andersson, R., Rappe, C., 1992. De novo forma-
tion of hepta- and octachlorodibenzo-p-dioxins from pen-
tachlorophenol
in
municipal
sewage
sludge.
Organohalogen Compd. 9, 351–354.
alyzed
metabolic
transformations
of
8
Oberg, L.G., Wagman, N., Andersson, R., Rappe, C., 1993.
De novo formation of PCDD/Fs in compost and sewage
sludge—a status report. Organohalogen Compd. 11, 297–
302.
chlorophenols.
Scha¨fer, K., McLachlan, M.S., Reisinger, M., 1993. An inves-
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