Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by human cytochrome P450-dependent monooxygenase systems

Article abstract of DOI:10.1021/jf020415z

Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by monooxygenase systems dependent on 12 forms of human cytochrome P450 (CYP) was examined with the recombinant yeast microsomes containing each of the human CYP. The metabolites of PCDDs were analyzed by HPLC and GC-MS. Remarkable metabolism by the multiple CYP forms was observed toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. The metabolism contained multiple reactions such as hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, and hydroxylation with elimination of a chloride substituent. Although major CYPs in human liver such as CYP2C8, CYP2C9, and CYP3A4 showed no significant metabolism toward the PCDDs, CYP1A1 and CYP1A2 showed high catalytic activity toward DD and mono-, di-, and trichloroDDs. The kinetic parameters K_m^app and V_max of the CYP1A1-dependent 8-hydroxylation activity of 2,3,7-trichloro-DD (2,3,7-triCDD) were estimated to be 0.30 μM and 51 (mol/min/mol of P450), respectively, suggesting that 2,3,7-triCDD was a good substrate for CYP1A1. However, none of the CYPs showed any detectable activity [<0.01 mol/min/mol of P450)]) toward 2,3,7,8-tetraCDD. Substrate-induced absorption spectrum and inhibition studies indicated that CYP1A1 could bind 2,3,7,8-tetraCDD with considerably high affinity. It was strongly suggested that the long half-life (7.1 years) of 2,3,7,8-tetraCDD in humans was due to an extremely low activity of CYPs toward 2,3,7,8-tetraCDD in addition to its chemical stability.

Full text of DOI:10.1021/jf020415z

5496 J. Agric. Food Chem. 2002, 50, 5496−5502
Metabolism of Polychlorinated Dibenzo-p-dioxins (PCDDs) by
Human Cytochrome P450-Dependent Monooxygenase Systems
KUNIYO INOUYE,*,‡ RAKU SHINKYO,‡ TEISUKE TAKITA,‡ MIHO OHTA,§ AND
‡
TOSHIYUKI SAKAKI
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan, and Laboratory of Nutrition,
Koshien College, 4-25 Kawarabashi-cho, Nishinomiya 663-8107, Japan
Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by monooxygenase systems dependent
on 12 forms of human cytochrome P450 (CYP) was examined with the recombinant yeast microsomes
containing each of the human CYP. The metabolites of PCDDs were analyzed by HPLC and GC-
MS. Remarkable metabolism by the multiple CYP forms was observed toward dibenzo-p-dioxin (DD)
and mono-, di-, and trichloroDDs. The metabolism contained multiple reactions such as hydroxylation
at an unsubstituted position, hydroxylation with migration of a chloride substituent, and hydroxylation
with elimination of a chloride substituent. Although major CYPs in human liver such as CYP2C8,
CYP2C9, and CYP3A4 showed no significant metabolism toward the PCDDs, CYP1A1 and CYP1A2
showed high catalytic activity toward DD and mono-, di-, and trichloroDDs. The kinetic parameters
Kmapp and Vmax of the CYP1A1-dependent 8-hydroxylation activity of 2,3,7-trichloro-DD (2,3,7-triCDD)
were estimated to be 0.30 µM and 51 (mol/min/mol of P450), respectively, suggesting that 2,3,7-
triCDD was a good substrate for CYP1A1. However, none of the CYPs showed any detectable activity
[<0.01 mol/min/mol of P450)] toward 2,3,7,8-tetraCDD. Substrate-induced absorption spectrum and
inhibition studies indicated that CYP1A1 could bind 2,3,7,8-tetraCDD with considerably high affinity.
It was strongly suggested that the long half-life (7.1 years) of 2,3,7,8-tetraCDD in humans was due
to an extremely low activity of CYPs toward 2,3,7,8-tetraCDD in addition to its chemical stability.
KEYWORDS: Dioxin; PCDD; cytochrome P450; CYP; recombinant yeast
INTRODUCTION
prepared from control and 3-methylcholanthrene-treated rats.
They suggested that CYP1A1 and CYP1A2 play an important
role in the metabolism of PCDDs. Thus, cytochrome P450-
dependent monooxygenases appear to be key enzymes for the
metabolism of PCDDs. It is known that the body distribution
of PCDDs is strongly species dependent, with the liver and
adipose tissue being the major storage sites (7, 11). In addition,
the tissue distribution of PCDDs is dose dependent. The liver-
to-adipose tissue distribution ratio increases with increasing dose
(12, 13). Thus, a remarkable species difference between humans
and rats on the half-life of 2,3,7,8-tetraCDD (3, 14, 15) appears
to be based on the difference of tissue distribution of PCDDs,
enzymatic properties of cytochromes P450, and their expression
levels. To our knowledge, no papers identifying the metabolites
of PCDDs by each human CYPs have been published, although
studies on the metabolism of PCDDs by human CYPs are
essential for human risk assessment of PCDDs.
Polychlorinated dibenzo-p-dioxins (PCDDs) are the family
consisting of 75 structurally similar chlorinated compounds.
They are known as environmental contaminants due to the
extreme toxicity of some members of the family. Of these
compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin CDD (tetra-
CDD) is known to be the most toxic one. Recently, the World
Health Organization (WHO) established a tolerable daily intake
of 1-4 pg of TEQs/kg of body weight (1). Human exposure to
dioxins appears to be predominantly from meat, fish, and dairy
products (2). In the past two decades, the metabolism of PCDDs
has been studied in vivo using experimental animals (3-9) and
in vitro using liver slices and liver microsomal fractions (10).
Major metabolites were hydroxylated products, glucuronide
conjugates, and sulfate conjugates (4, 6). They involve the initial
insertion of a single oxygen atom into the PCDD molecule to
form an epoxide, probably by cytochrome P450 (CYP)-
dependent monooxygenases. Recently, Hu and Bunce (10)
studied the metabolism of PCDDs in the microsomal fractions
We have established a yeast expression system for human
CYPs and shown its usefulness for the prediction of drug
metabolism in humans (16-19). Addition of a drug to the yeast
cell culture or microsomal fraction prepared from the recom-
binant yeast cells could supply sufficient amounts of metabolites
to determine their chemical structures. In this study, the yeast
* Author to whom correspondence should be addressed (telephone +81-
75-753-6266; fax +81-75-753-6265; e-mail inouye@kais.kyoto-u.ac.jp).
‡ Kyoto University.
§ Koshien College.
10.1021/jf020415z CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/08/2002

Metabolism of PCDDs
J. Agric. Food Chem., Vol. 50, No. 19, 2002 5497
expression system was applied to the prediction of the metabo-
lism of PCDDs in humans. The detailed metabolism of various
types of PCDDs by human CYPs is demonstrated.
Table 1. Metabolism of PCDDs by 12 Forms of Human Cytochrome
P450
activity (mol/min/mol of P450)
CYP DD 1-MC 2-MC 2,3-DC 2,7-DC 2,3,7-TC 2,3,7,8-TC
MATERIALS AND METHODS
b
1A1
1A2
1B1
2A6
2B6
2C8
2C9
2C18
1.0
5.6
−
0.9
−
−
0.5
−
3.4
0.2
−
−
−
1.7
−
−
2.9
1.7
0.7
−
10.3
5.0
0.6
−
−
−
0.7
−
3.6
2.2
0.7
−
18.4
1.6
0.5
−
0.8
−
3.4
2.7
6.7
2.5
0.2
−
20.0
2.2
1.1
−
−
−
−
−
−
−
44.7
5.5
3.5
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
Materials. Polychlorinated-p-dioxins (PCDDs), 2,3,7,8-tetraCDD,
2,3,7-triCDD, and 2,7-DCDD were purchased from Cambridge Isotope
Laboratories Inc. (Andover, MA). 2,3-DichloroDD (DCDD), 1-mono-
chloroDD (MCDD), and 2-MCDD were purchased from AccuStandard
Inc. (New Haven, CT). Dibenzo-p-dioxin (DD) was purchased from
Wako Pure Chemical Industries, Ltd. Co. (Osaka, Japan). The
recombinant yeast microsomes containing each human CYP1A1,
CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19,
CYP2D6, CYP2E1, and CYP3A4 were purchased from Sumika
Chemical Analysis Service (Osaka, Japan) (17, 18). Specific content
of P450 (picomoles per milligram of protein) and standard activity
(moles per minute per mole of P450) of each microsomal fraction
measured by Sumika Chemical Analysis Service were as followss:
CYP1A1, 16.2 pmol/mg of protein and 9.3 mol/min/mol of P450 for
7-ethoxyresorufin O-deethylation; CYP1A2, 40.0 pmol/mg of protein
and 2.4 mol/min/mol of P450 for 7-ethoxyresorufin O-deethylation;
CYP2A6, 23.1 pmol/mg of protein and 6.8 mol/min/mol P450 for
coumarin 7-hydroxylation; CYP2B6, 11.7 pmol/mg of protein and 2.1
mol/min/mol of P450 for 7-ethoxycoumarin O-deethylation; CYP2C8,
145.0 pmol/mg of protein and 3.2 mol/min/mol of P450 for tolbutamide
hydroxylation; CYP2C9, 102.0 pmol/mg of protein and 12.4 mol/min/
mol of P450 for tolbutamide hydroxylation; CYP2C18, 71.3 pmol/mg
of protein and 5.5 mol/min/mol of P450 for tolbutamide hydroxylation;
CYP2C19, 29.8 pmol/mg of protein and 16.6 mol/min/mol of P450
for S-mephenytoin 4′-hydroxylation; CYP2D6, 12.8 pmol/mg of protein
and 4.8 mol/min/mol of P450 for debrisoquin 4-hydroxylation;
CYP2E1, 67.1 pmol/mg of protein and 5.5 mol/min/mol of P450 for
chlorzoxazone 6-hydroxylation; and CYP3A4, 50.6 pmol/mg of protein
and 26.2 mol/min/mol of P450 for testosterone 6â-hydroxylation.
NADPH-cyt.c reductase activity indicating the contents of yeast
NADPH-P450 reductase of each microsome was in the range of 1000-
2000 nmol of cyt.c reduced/min/mg of protein. Only CYP3A4
microsomes contain human cyt.b5 at the level of 220 pmol/mg of protein
(18). The microsomes prepared from the recombinant yeast harboring
the plasmid which contains yeast NADPH-P450 reductase gene (20)
were purchased from Sumika Chemical Analysis Service and used as
control microsomes. The microsomes containing human CYP1B1
prepared from recombinant Sf9 cells were purchased from Daiichi
Chemical Co. (Tokyo, Japan). The specific content of P450 and
7-ethoxyresorufin O-deethylation activity were 65 pmol/mg of protein
and 4.1 mol/min/mol of P450, respectively. NADPH was from Oriental
Yeast (Tokyo, Japan). All other chemicals were of the best com-
mercially available grade.
Measurement of Reduced CO Difference Spectra and Substrate-
Induced Difference Spectra. The reduced CO difference spectra were
measured with a Shimadzu UV-2200 spectrophotometer (Kyoto, Japan)
according to the following procedure as described previously (21-
25). The substrate-induced difference spectra of the microsomal fraction
containing 0.33 µM P450 were measured in the presence of 3.1 µM
2,3,7-triCDD or 2,3,7,8-tetraCDD. The solution contains dimethyl
sulfoxide (DMSO) at a final concentration of 2.0%.
Measurement of Catalytic Activity toward PCDDs. The substrates
DD, 1-MCDD, and 2-MCDD were dissolved in ethanol, and 2,3-DCDD,
2,7-DCDD, and 2,3,7-TriCDD were dissolved in acetone, whereas
2,3,7,8-tetraCDD was dissolved in DMSO. The reaction mixture
contains each PCDD at 0-10 µM with 1% of the organic solvent and
the microsomes containing 10 or 33 nM of each human CYP in 50
mM potassium phosphate buffer, pH 7.4. On the metabolism by
CYP2E1, DD, 1-MCDD, and 2-MCDD were dissolved in methanol,
and 2,3-DCDD, 2,7-DCDD, and 2,3,7-triCDD were dissolved in
DMSO. The final concentration of the organic solvents in the reaction
mixtures containing CYP2E1 was 0.5%. For the metabolism of 2,3,7,8-
tetraCDD, P450 concentration and 2,3,7,8-tetraCDD were adjusted to
0.10 and 0.78 µM, respectively. In the experiments to examine the
inhibitory effect of 2,3,7,8-tetraCDD, both substrate and 2,3,7,8-
2C19 6.3
2D6
2E1
3A4
1.2
1.9
−
−
−
−
−
a Each value represents the total activity including multiple metabolites at a
substrate concentration of 10 µM except for 2,3,7,8-tetraCDD (0.78 µM) as
described under Materials and Methods. b −, not detected (<0.01).
tetraCDD were dissolved in DMSO, and the final concentration of
DMSO was adjusted to 1%. The reaction was initiated by the addition
of NADPH at a final concentration of 0.5 mM. Aliquots of the reaction
mixture were collected after various time intervals and extracted with
4 volumes of chloroform/methanol (3:1). The organic phase was
recovered and dried. The resulting residue was solubilized with
acetonitrile and applied to HPLC performed on a YMC-Pack ODS-
AM (4.6 mm i.d. × 300 mm) (YMC Co., Tokyo, Japan). The mobile
phase was acetonitrile/0.01% TFA aqueous solution (50:50, v/v) for 5
min followed by a linear gradient of 50-100% acetonitrile for 15 min
and 100% acetonitrile for 10 min. The flow rate was 1.0 mL/min, and
the elution was detected by measuring absorption at 227 nm. The
column temperature was maintained at 40 °C.
Mass Spectrometric Analysis of the Metabolites. Isolated me-
tabolites from HPLC effluents were subjected to GC-MS analysis using
a Finnigan Mat Thermo Quest GC with EI mode. A Chrompack Cp-
Sil 24CB-MS (0.32 mm × 30 m) was used as a GC column.
Ah Receptor Assay. The SV40 promoter region of the vector pGV-P
(Toyo Ink Manufacturing Co. Ltd., Tokyo, Japan) was replaced with
five tandem repeats of XRE (26) followed by the nucleotides -164 to
+53 of rat glutathione S-transferase Ya subunit gene (27). The resultant
plasmid was introduced into mouse hepatoma Hepa-1clc7 cells. The
stable transformant was used for the Ah-receptor assay. After the
addition of each PCDD and their metabolites to the cell culture,
luciferase activity was measured by using the luciferase assay system
(Promega Co., Madison, WI).
Other Methods. The concentrations of PCDDs and their metabolites
were estimated by using a molar extinction coefficient of 4.12 × 104
M-1 cm-1 at 227 nm on the basis of spectral analysis of PCDDs. Protein
concentration was determined according to the method of Lowry et al.
(28), using bovine serum albumin as a standard.
RESULTS
Human CYP-Dependent Metabolism of PCDDs. The
recombinant yeast microsomal fraction containing each human
CYP was examined for the metabolism of PCDDs. The control
microsomes with overexpression of yeast NADPH-P450 re-
ductase showed no activity against any of the PCDDs examined
in this study. Remarkable metabolism by multiple P450 forms
was observed toward DD, 1-MCDD, 2-MCDD, 2,3-DCDD, 2,7-
DCDD, and 2,3,7-triCDD as shown in Table 1. The metabolism
contained multiple reactions such as hydroxylation at an
unsubstituted position, hydroxylation with migration of a
chloride substituent, and hydroxylation with elimination of a
chloride substituent. Major CYPs in human liver such as
CYP2C8, CYP2C9, and CYP3A4 showed no significant me-
tabolism toward the PCDDs. On the other hand, CYP2C19 and

5498 J. Agric. Food Chem., Vol. 50, No. 19, 2002
Inouye et al.
Figure 1. HPLC profiles of PCDDs and their metabolites: DD and its metabolite (M1) by human CYP1A2 (A); 2-MCDD and its metabolites (M4 and M5)
by human CYP1A1 (B); 2,7-DCDD and its metabolites by human CYP1A2 (C); 2,3,7-triCDD and its metabolites by human CYP1A2 (D). After reaction,
the reaction mixture was extracted and analyzed by HPLC as described under Materials and Methods.
CYP2D6, which are known to present genetic polymorphism,
showed high catalytic activities toward DD, 1-MCDD, 2-M-
CDD, and 2,3-DCDD. The CYPs in the CYP1 family, CYP1A1,
CYP1A2, and CYP1B1, showed high catalytic activity toward
DD and mono-, di-, and trichloroDDs. However, none of the
CYPs showed detectable activity toward 2,3,7,8-tetraCDD.
Analysis of Metabolites of DD Produced by Human CYPs.
Figure 1A shows HPLC analysis of the metabolite of DD by
CYP1A2. One major metabolite (M1) was observed at a
retention time of 12.3 min. The mass spectrum of the metabolite
showed molecular ions at m/z 200 (M)+, 171 (M - COH)+,
and 144 (M - 2CO)+, suggesting that the metabolite was the
hydroxylated product of DD. Table 2 summarizes the relative
intensities of the major ion fragments The same metabolite was
observed in the reaction mixtures containing CYP1A1, CYP2A6,
CYP2C9, CYP2C19, CYP2D6, or CYP2E1.
Table 2. Relative Intensities of Major Ion Fragments of Metabolites
of PCDDs
no.
compound
m/z
M
M− COH M − Cl M − 2CO M − COCl
a
M1 DD-OH
200 100
234 100
234 100
234 100
234 100
234 100
268 100
268 100
268 100
268 100
268 100
10
6
16
6
12
12
4
3
12
2
11
−
−
12
6
19
4
28
−
18
20
−
3
21
1
22
−
−
56
22
39
14
20
62
35
24
52
21
44
M2 1-MCDD-OH
M3 1-MCDD-OH
M4 2-MCDD-OH
M5 2-MCDD-OH
M10 2-MCDD-OH
M6 2,3-DCDD-OH
M7 2,3-DCDD-OH
M8 2,3-DCDD-OH
M9 2,7-DCDD-OH
31
6
4
62
35
11
29
13
27
b
M11 3,7-DCDD-OH
M12 2,3,7-triCDD-OH 302 100
a −, not detected. b Putative structure of M11 based on the facts that M11 is
different from any M6, M7, M8, and M9 by HPLC analysis.
Analysis of Metabolites of 1-MCDD Produced by Human
CYPs. In HPLC analysis, one major peak of a metabolite of
1-MCCD was observed in the metabolism by CYP1A1,
CYP1A2, CYP2C8, CYP2C19, CYP2D6, and CYP2E1. How-
ever, the retention times of the metabolites were 14.4 min for
CYP1A1 and CYP2C8 and 14.9 min for CYP1A2, CYP2C19,
CYP2D6, and CYP2E1. The mass spectrum of the metabolite
(M2) by CYP1A1 showed major molecular ions at m/z 234 (M
with 35Cl)+, 199 (M - Cl)+, and 171 (M - COCl)+, suggesting
that the metabolite was a hydroxylated product of 1-MCDD
(Table 1). On the other hand, the mass spectrum of the
metabolite (M3) by CYP2C19 showed major molecular ions at
m/z 234 (M with 35Cl)+, 205 (M - COH)+, and 178 (M -
2CO)+. Although the mass spectra of two metabolites were
clearly different, the relationship between the fragmentation
pattern and the chemical structure has not been elucidated.
Methylated compounds of both metabolites showed molecular
ions at m/z 248 (M with 35Cl)+, 233 (M - CH3)+, and 205
(M - COCH3)+ (Table 3). The presence of the (M - CH3)+
fragment indicates that the methoxy group is in a lateral (C-2,
C-3, C-7, and C-8) position as described by Tulp and Hutzinger
(29).

Metabolism of PCDDs
J. Agric. Food Chem., Vol. 50, No. 19, 2002 5499
Figure 2. GC profile (m/z 233.5−234.5) of the metabolites of 2-MCDD by human CYP1A1 collected from HPLC effluent (A) and mass spectra of
metabolites designated M4 (B) and M5 (C).
Table 3. Relative Intensities of Major Ion Fragments of the Methylated
Metabolites and Putative Positions of OCH Group
3
M −
CH
3
M −
COCH
no.
M2
M3
M4
M5
compound
m/z
M
positiona
3
1-MCDD-OCH
248 100
248 100
248 100
248 100
282 100
282 100
282 100
316 100
51
48
70
54
95
48
48
69
42
42
69
60
51
45
48
60
2,3,7,8
2,3,7,8
3,7,8
3,7,8
7
2
3
8
3
1-MCDD-OCH
3
2-MCDD-OCH
3
2-MCDD-OCH
3
M(6,7,8) 2,3-DCDD-OCH b
3
M(6,7,8) 1,3-DCDD-OCH b
3
M9
M12
2,7-DCDD-OH
2,3,7-triCDD-OCH
3
a The presence of molecular ion (M − CH )+ means that the OCH group is in
3
3
a lateral (C-2, C-3, C-7, and C-8) position. b The relationship between MS data
and putative chemical structures might be reversed.
Figure 3. Mass spectra of the metabolite (M12) of 2,3,7-triCDD by human
CYP1A2 (A) and of the methylated compound of M12 (B). Putative
structures of M12 and the methylated M12 are presented.
Analysis of Metabolites of 2-MCDD Produced by Human
CYPs. Figure 1B shows the HPLC profile of the metabolites
of 2-MCDD by CYP1A1. A broad peak at ∼16 min including
small peaks was observed. The metabolites at the broad peak
were collected from HPLC effluents and analyzed by GC-MS.
Figure 2A shows the GC profile of the metabolites. The mass
spectrum of the metabolite (M4) by CYP1A1 showed molecular
ions at m/z 234 (M with 35Cl)+, 199 (M - Cl)+, and 171 (M -
COCl)+ (Figure 2). On the other hand, the mass spectrum of
metabolite M5 showed molecular ions at m/z 234 (M with
35Cl)+, 205 (M - COH)+, and 178 (M - 2CO)+ (Figure 2).
Methylated compounds of both metabolites showed molecular
ions at m/z 248 (M)+, 233 (M - CH3)+, and 205 (M -
COCH3)+ (Table 3). These results suggest that M4 and M5
contain a hydroxyl group at a distinct site in a lateral position.
Analysis of Metabolites of 2,3-DCDD Produced by Human
CYPs. The HPLC profile of the metabolites of 2,3-DCDD by
CYP1A1 showed a broad peak at ∼17.5 min (data not shown).
A similar HPLC pattern was observed in the metabolism of
CYP1A2, CYP1B1, CYP2B6, CYP2C9, CYP2C18, CYP2C19,
CYP2D6, and CYP2E1. The metabolites by CYP1A1 were
collected from HPLC effluents and analyzed by GC-MS. The
GC profile of the metabolites indicated the presence of three
distinct metabolites designated M6, M7, and M8 with the same
molecular mass (Table 2). However, only two metabolites were
detected after methylation. The methoxy group of both me-
tabolites was in a lateral position, suggesting that one of the
metabolite would arise via an NIH shift after enzymatic
formation of an epoxide as described by Petroske et al. (30)
(Table 3). It is noted that only two distinct metabolites are
possible even if the NIH shift is considered. Further studies are
needed to reveal the chemical structure of the third metabolite.
Analysis of Metabolites of 2,7-DCDD Produced by Human
CYPs. HPLC analysis showed a metabolite of 2,7-DCDD
designated M9 by CYP1A1 (data not shown). The mass spectra
of M9 and its methylated compound suggested that M9 was
the hydroxylated product of 2,7-DCDD at the C-3 position
(Tables 2 and 3), although the possibility of the hydroxylated
product with migration of a chloride substituent could not be
discarded. On the other hand, CYP1A2 showed another
metabolite (M10 in Figure 1C). The mass spectrum of M10
was quite similar to that of M5. These results suggest that the
metabolite contains a hydroxyl group with elimination of a
chloride substituent of 2,7-DCDD, namely, 7-hydroxy-2-MCDD.
Analysis of Metabolites of 2,3,7-TriCDD Produced by
Human CYPs. Figure 1D shows the HPLC profile of the
metabolites of 2,3,7-triCDD by CYP1A2, indicating the presence
of two metabolites designated M11 and M12. Although the mass
spectrum of M11 was quite similar to that of M8, the
metabolites M11 and M8 were clearly separated by HPLC. The
mass spectra of M12 and its methylated compound suggested
that M12 was the hydroxylated product of 2,3,7-triCDD at the
C-8 position (Figure 3). On the metabolism of 2,3,7-triCDD
by CYP1A1 and CYP1B1, the 8-hydroxylated product of 2,3,7-
triCDD (M12) was observed as a metabolite, but M11 was not
detected (data not shown).
Substrate-Induced Difference Spectra of CYP1A1. Addi-
tion of 2,3,7-triCDD or 2,3,7,8-tetraCDD to the microsomal
fraction containing CYP1A1 induced a typical type I spectrum

5500 J. Agric. Food Chem., Vol. 50, No. 19, 2002
Inouye et al.
Figure 4. 2,3,7-TriCDD- (s) and 2,3,7,8-tetraCDD-induced (- - -) difference
spectra of CYP1A1. The difference spectra of the microsomal fraction
containing 0.33 µM P450 were measured in the presence of 3.1 µM
2,3,7-triCDD or 2,3,7,8-tetraCDD as described under Materials and
Methods.
Figure 5. Hanes−Woolf plots (S/v vs S) of CYP1A1-dependent 8-hy-
droxylation of 2,3,7-triCDD in the presence (O) and absence (b) of 2,3,7,8-
tetraCDD (0.78 µM). The units of S and v are µM and (mol/min/mol of
P450), respectively.
min/mol of P450, respectively. These results indicate that both
2,7-DCDD and 2,3,7-triCDD are good substrates for CYP1A1
and CYP1A2.
Table 4. Kinetic Parameters for 2,7-DCDD 3-Hydroxylation Activity and
2,3,7-TriCDD 8-Hydroxylation Activity by Human CYP1A1 and CYP1A2
a
Inhibitory Effect of 2,3,7,8-TetraCDD to 8-Hydroxylation
of 2,3,7-TriCDD. Hanes-Woolf plots (S/V vs S) of the
CYP1A1-dependent 8-hydroxylation of 2,3,7-triCDD in the
presence and absence of 2,3,7,8-tetraCDD (0.78 µM) indicated
the competitive inhibition by 2,3,7,8-tetraCDD (Figure 5). The
apparent Ki value of 2,3,7,8-tetraCDD for the CYP1A1-
dependent 8-hydroxylation of 2,3,7-triCDD was estimated to
be 0.61 µM. These results strongly suggest that 2,3,7,8-tetraCDD
binds to the substrate-binding pocket of CYP1A1 with consider-
ably high affinity.
Vmax
CYP
substrate
Kmapp a (µM)
(mol/min/mol of P450)
1A1
1A1
1A2
1A2
2,7-DCDD
2,3,7-TriCDD
2,7-DCDD
0.24 ± 0.11
0.30 ± 0.08
0.06 ± 0.01
0.23 ± 0.04
20.5 ± 6.5
50.8 ± 2.7
2.2 ± 0.2
3.9 ± 0.7
2,3,7-triCDD
app
a Km and Vmax values represent the mean ± SD from three separate
experiments.
as shown in Figure 4, indicating a change of the heme iron of
CYP1A1 from a low-spin state to a high-spin state upon binding
of 2,3,7-triCDD and 2,3,7,8-tetraCDD. These results are con-
sistent with the fact that human CYP1A1 purified from the
recombinant yeast cells used in this study is a low-spin state
based on the absolute spectrum (Dr. S. Imaoka, personal
communication). The magnitude of absorbance difference
between 390 and 420 nm for 2,3,7,8-tetraCDD was ∼45% of
that for 2,3,7-triCDD. The dissociation constant Kd for 2,3,7-
triCDD was estimated to be 0.65 µM. It should be noted that
2,3,7,8-tetraCDD bound to the substrate-binding pocket of
CYP1A1, whereas no detectable activity of CYP1A1 was
observed toward 2,3,7,8-tetraCDD. On the other hand, no
spectral change of CYP1A2 was observed with the addition of
2,3,7-triCDD and 2,3,7,8-tetraCDD, probably due to a high-
spin form of CYP1A2 even in the absence of a substrate.
Kinetic Analysis of 3-Hydroxylation of 2,7-DCDD and
8-Hydroxylation of 2,3,7-TriCDD by CYP1A1 and CYP1A2.
The apparent Michaelis constant designated Kmapp and Vmax for
the CYP1A1-dependent 3-hydroxylation activity toward 2,7-
DCDD were estimated to be 0.24 µM and 20.5 mol/min/mol
of P450, respectively. These values for the CYP1A1-dependent
8-hydroxylation activity toward 2,3,7-triCDD were estimated
to be 0.30 µM and 50.8 mol/min/mol of P450, respectively
(Table 4). On the other hand, the Kmapp and Vmax values for the
CYP1A2-dependent 3-hydroxylation activity toward 2,7-DCDD
were estimated to be 0.06 µM and 2.2 mol/min/mol of P450,
respectively, and those for the CYP1A2-dependent 8-hydrox-
ylation activity toward 2,3,7-triCDD were 0.23 µM and 3.9 mol/
Ah Receptor Assay. Addition of 8-hydroxy-2,3,7-triCDD to
the cell culture at a final concentration of 50 nM showed no
effect on the luciferase activity, whereas 50 nM 2,3,7-triCDD
clearly demonstrated a positive effect. It was indicated that the
magnitude of the effect of 8-hydroxy-2,3,7-triCDD was <10%
of that of 2,3,7-triCDD and 0.001% of that of 2,3,7,8-tetraCDD.
These results indicated that 8-hydroxylation of 2,3,7-triCDD
was a detoxication reaction.
DISCUSSION
To our knowledge, this is the first identification of metabolites
of PCDDs by human CYPs. Remarkable metabolism by multiple
P450 forms was observed toward dibenzo-p-dioxin (DD) and
mono-, di-, and trichloroDDs. The metabolism contained
multiple reactions such as hydroxylation at an unsubstituted
position, hydroxylation with migration of a chloride substituent
(NIH shift), and hydroxylation with elimination of a chloride
substituent. From the results summarized in Table 3, the
hydroxyl group is predominantly added to a lateral position of
PCDDs in CYP-dependent metabolism. These results are quite
similar to those obtained in rats as reported by Tulp and
Hutzinger (4). Major CYPs in human liver such as CYP2C8,
CYP2C9, and CYP3A4 did not show high catalytic activity
toward PCDDs. The fact that CYP2C19 and CYP2D6 showed
high activities toward DD, 1-MCDD, 2-MCDD, and 2,3-DCDD
is pharmacologically interesting, because they are polymorphic
with significant percentages of populations deficient. It was
noted that CYP1A1 showed multiple metabolites of 2,3-DCDD,

Metabolism of PCDDs
J. Agric. Food Chem., Vol. 50, No. 19, 2002 5501
whereas Tulp and Hutzinger (4) observed only one metabolite
of 2,3-DCDD in rats. One of the metabolites was considered to
be 2-hydroxy-1,3-DCDD arising via an NIH shift after enzy-
matic formation of an epoxide (Table 3). Although 2,3-DCDD
was a good substrate for multiple P450 species, 2,7-DCDD was
metabolized by only the CYP1 family. These results indicated
that the presence of a chloride substituent in both rings of
PCDDs greatly diminished the activity of CYPs except for the
CYP1 family. Both CYP1A1 and CYP1A2 showed high
catalytic activity toward DD and mono-, di-, and trichloroDDs.
From the contents of CYP1A1, CYP1A2, and CYP1B1 in
human liver, a major CYP for the metabolism of 2,7-triCDD
and 2,3,7-tetraCDD in human liver appears to be CYP1A2.
Surprisingly, the activity of CYP1A1 toward PCDDs increased
CYPs in addition to its high affinity for Ah receptor. Tai et al.
(34) reported that human CYP1A1 showed much lower activity
toward 2,3,7,8-tetrachlorodibenzofuran (TCDF) than rat CYP1A1.
Thus, it might be possible to assume that a remarkable species
difference between humans and rats in the half-life of 2,3,7,8-
TCDD is mainly due to the difference of enzymatic properties
of the CYP1 family, although further analysis is needed.
SAFETY
2,3,7,8-TetraCDD is known to be extremely toxic. Thus,
appropriate measures should be used to minimize exposure
during handling of 2,3,7,8-tetraCDD.
app
the number of chloride substituents from 0 to 3. The Km
ABBREVIATIONS USED
() 0.30 µM) and Vmax () 50.8 mol/min/mol P450) values for
the 2,3,7-triCDD 8-hydroxylation indicate that 2,3,7-triCDD is
a good substrate for CYP1A1. It should be noted that 8-hydroxy-
2,3,7-triCDD () 2-hydroxy-3,7,8-triCDD) is one of the major
metabolites of 2,3,7,8-tetraCDD found in bile of 2,3,7,8-
tetraCDD-treated dogs (6). The Ah receptor assay demonstrated
that the magnitude of the effect of 8-hydroxy-2,3,7-triCDD was
<10% of that of 2,3,7-triCDD and 0.001% of that of 2,3,7,8-
tetraCDD. These results indicated that 8-hydroxylation of 2,3,7-
triCDD was a detoxication reaction. Remarkable metabolism
showing hydroxylation with elimination of a chloride substituent
was observed in the CYP1A2-dependent metabolism of 2,7-
DCDD and 2,3,7-triCDD. Recently, Yanagita et al. (31) reported
dehalogenation of chlorinated ethylenes and ethanes by rat
CYP1A2 and its mutants under anaerobic conditions. To
examine the possibility of the elimination of a chloride sub-
stituent by reduced heme iron of CYP1A2 without monooxy-
genation, metabolism of 2,7-DCDD or 2,3,7-triCDD by CYP1A2
was observed under N2 gas. However, no metabolites were
observed, suggesting that the elimination of a chloride substitu-
ent is closely linked to the monooxygenase activity of CYP1A2.
The major metabolites of 2,3,7,8-TCDD in the rats and dogs
were determined to be 2-hydroxy-1,3,7,8-tetraCDD and 2-hy-
droxy-3,7,8-triCDD () 8-hydroxy-2,3,7-triCDD in this paper)
(32). However, we have not detected a metabolite of 2,3,7,8-
tetraCDD by any of the CYPs. From the detection limits, the
catalytic activity of CYPs toward 2,3,7,8-tetraCDD is <0.01
mol/min/mol of P450. On the other hand, the substrate-induced
difference spectra (Figure 4) demonstrated the binding of
2,3,7,8-tetraCDD to the substrate-heme pocket of CYP1A1. In
addition, 2,3,7,8-tetraCDD showed competitive inhibition toward
8-hydroxylation of 2,3,7-triCDD with the apparent Ki value of
0.61 µM. Thus, it is strongly suggested that CYP1A1 can bind
2,3,7,8-tetraCDD, although it shows no detectable catalytic
activity toward 2,3,7,8-tetraCDD. We have not observed spectral
change of CYP1A2 by the addition of 2,3,7,8-tetraCDD,
probably due to the high-spin form of CYP1A2 even in the
absence of 2,3,7,8-tetraCDD. However, CYP1A2-dependent
2,3,7-triCDD 8-hydroxylation was also significantly inhibited
by 2,3,7,8-tetraCDD (data not shown), suggesting that CYP1A2
could bind 2,3,7,8-tetraCDD. These results appear to be
consistent with the finding that CYP1A2 is the sequestering
protein for 2,3,7,8-tetraCDD in mice liver (33).
P450 or CYP, cytochrome P450; PCDD, polychlorinated
dibenzo-p-dioxin; MCDD, monochlorodibenzo-p-dioxin; DCDD,
dichlorodibenzo-p-dioxin; triCDD, trichlorodibenzo-p-dioxin;
tetraCDD, tetrachlorodibenzo-p-dioxin.
ACKNOWLEDGMENT
We express our gratitude to Dr. H. Kaneko and Dr. H.
Matsunaga of Sumitomo Chemical Co., Ltd., for Ah receptor
assay of the metabolites and useful discussions. We thank Dr.
S. Imaoka of Osaka City University Medical School for giving
valuable information on the spectral and enzymatic properties
of human CYPs.
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Received for review April 8, 2002. Revised manuscript received June
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