Irgasan DP 300 (5-chloro-2-(2,4-dichlorophenoxy)-phenol) induces cytochrome P450s and inhibits haem biosynthesis in rat hepatocytes cultured on Matrigel.

Article abstract of DOI:10.1080/004982597240271

1. The effect of Irgasan DP 300 (5-chloro-2-(2,4-dichlorophenoxy)phenol) on cytochrome P450 (P450) induction and haem biosynthesis was studied in rat hepatocytes cultured on Matrigel. 2. Irgasan DP 300 significantly induced 7-benzyloxyresorufin O-debenzylase activity, followed by 7-pentoxyresorufin O-depentylase and 7-ethoxyresorufin O-deethylase activities. 4-Nitrophenol hydroxylase, testosterone 6 beta-hydroxylase and methoxyresorufin O-demethylase activities were also slightly increased. The maximum induction of these enzyme activities was obtained at the same concentration of 125 microM in the culture medium. 3. Immunochemical blots using anti-rat cytochrome P450 antibodies revealed that Irgasan DP 300 preferably induced CYP2B1/2 along with a slight increase in 3A. These results indicate that Irgasan DP 300 is a phenobarbital-type inducer. 4. In the absence of exogenous 5-aminolevulinic acid (ALA), slight increases in protoporphyrin IX (2.6-fold) and coproporphyrin III (1.3-fold) were observed in the Irgasan DP 300-treated cultures. In contrast, when 75 microM ALA was present, Irgasan DP 300 (250 microM) caused an extensive accumulation of uroporphyrin I (13-fold). 5. Irgasan DP 300 inhibited rat hepatic uroporphyrinogen III synthase in vitro. 6. These results indicate that Irgasan DP 300 produced accumulation of hydroxymethylbilane in rat hepatocytes by inhibiting uroporphyrinogen III synthase, and consequently an accumulation of uroporphyrin I.

Full text of DOI:10.1080/004982597240271

xenobiotica , 1997, vol. 27, no. 7, 681±692
Irgasan‡ DP 300 (5-chloro-2-(2,4-dichlorophenoxy)-
phenol) induces cytochrom e P450s and inhibits
haem biosynthesis in rat hepatocytes cultured on
M atrigel
H. JINNO‹*, N. HANIOKA‹, S. ONODERAŒ, T. NISHIMURA‹
and M. ANDO‹
‹
Division of Environmental Chemistry, National Institute of Health Sciences, 1-18-1
Kamiyoga, Setagaya-ku, Tokyo 158, Japan
Œ Faculty of Pharmaceutical Sciences, Science University of Tokyo, 1±3 Kagurazaka,
Shinjyuku-ku, Tokyo, 163, Japan
Received 31 January 1997
1
. The eåect of Irgasan DP 300 (5-chloro-2-(2,4-dichlorophenoxy)phenol) on cyto-
chrome P450 (P450) induction and haem biosynthesis was studied in rat hepatocytes
cultured on Matrigel.
2
. Irgasan DP 300 signi®cantly induced 7-benzyloxyresoru®n O-debenzylase activity,
followed by 7-pentoxyresoru®n O-depentylase and 7-ethoxyresoru®n O-deethylase ac-
tivities. 4-Nitrophenol hydroxylase, testosterone 6b-hydroxylase and methoxyresoru®n O-
demethylase activities were also slightly increased. The maximum induction of these
enzyme activities was obtained at the same concentration of 125 lm in the culture medium.
3
. Immunochemical blots using anti-rat cytochrome P450 antibodies revealed that
Irgasan DP 300 preferably induced CYP2B1}2 along with a slight increase in 3A. These
results indicate that Irgasan DP 300 is a phenobarbital-type inducer.
4
. In the absence of exogenous 5-aminolevulinic acid (ALA), slight increases in
protoporphyrin IX (2±6-fold) and coproporphyrin III (1±3-fold) were observed in the
Irgasan DP 300-treated cultures. In contrast, when 75 lm ALA was present, Irgasan DP
3
00 (250 lm) caused an extensive accumulation of uroporphyrin I (13-fold).
5
. Irgasan DP 300 inhibited rat hepatic uroporphyrinogen III synthase in vitro.
6
. These results indicate that Irgasan DP 300 produced accumulation of hydroxy-
methylbilane in rat hepatocytes by inhibiting uroporphyrinogen III synthase, and
consequently an accumulation of uroporphyrin I.
Introduction
Irgasan DP 300 (5-chloro-2-(2,4-dichlorophenoxy)phenol) (®gure 1) is widely
used as a bacteriostatic ingredient in household products, such as soap and
toothpaste. In addition to Irgasan DP 300 being an environmental pollutant (Levin
and Nilsson 1977, Miyazaki et al. 1984), it has been of interest because of the
chemical structure `predioxin ’, or polychloro-2-phenoxyphenol, which is readily
cyclized to polychlorodibenzo-p-dioxins by pyrolysis and photolysis (Rappe and
Nilsson 1972, Langer et al. 1973, Nilsson et al. 1974, Kanetoshi et al. 1988).
The toxic eåect of Irgasan DP 300 is rather low, with an acute oral LD&! of
approximately 4000 mg}kg in rat and mouse (Lyman and Furia 1969) and an acute
i.p. LD ¯ 1090 mg}kg in mouse (Kanetoshi et al. 1992). Another biological eåect
&
!
of concern is the induction of cytochrome P450-dependent monooxygenase
*
Author for correspondence.
049±8254}97 $12±00 ’ 1997 Taylor & Francis Ltd
0

6
82
H. Jinno et al.
Figure 1. Chemical structure of Irgasan DP 300.
activities, such as aminopyrine N-demethylase activity in mouse (Miller et al. 1981),
and 7-ethoxycoumarin O-deethylase and p-nitrophenetole O-deethylase activities
in rat (Kanetoshi et al. 1992). To date, however, no information has been available
on the P450 isozymes induced by Irgasan DP 300.
The present study examined the induction of P450 by Irgasan DP 300 by
assessing both enzyme activity and the apoprotein, using rat hepatocytes cultured on
Matrigel, a laminin-rich reconstituted basement membrane. This extracellular
matrix was adopted on the basis of the maintenance and induction of P450 during
primary culture (Schuetz et al. 1988, Kocarek et al. 1993). We also demonstrate the
impact of Irgasan DP 300 on hepatocellular haem biosynthesis, which has been
demonstrated to be disturbed by
tetrachlorodibenzo-p-dioxins (TCDD) (Poland and Glover 1973, Goldstein et al.
973), polychlorinated biphenyl (PCB) congeners (Vos and Koeman 1970,
a variety of chemicals such as 2,3,7,8-
1
Goldstein et al. 1974) and diphenyl ether herbicides (Matringe et al. 1989).
M aterials and m ethods
Materials
Irgasan DP 300 (99±5 %) was obtained from Ciba-Geigy (Basle, Switzerland). Matrigel was purchased
from Nippon Becton Dickinson (Tokyo, Japan). Resoru®n, 7-ethoxyresoru®n, 7-pentoxyresoru®n and
6
b-hydroxytestosterone were purchased from Sigma (St Louis, MO, USA). 7-Methoxyresoru®n and 7-
benzyloxyresoru®n were from Molecular Probes (Eugene, OR, USA). Testosterone, 4-nitrophenol and
-nitrocatechol were from Wako Pure Chemicals (Osaka, Japan). Goat anti-rat CYP1A1}2, 2B1}2, 2E1
and 3A2}1 antibodies were purchased from Daiichi Pure Chemicals (Tokyo, Japan). Porphobilinogen
PBG), uroporphyrin I (URO I), uroporphyrin III (URO III) and coproporphyrin III (COPRO III)
4
(
were purchased from Porphyrin Products (Logan, UT, USA), protoporphyrin IX (PROTO IX) from
Sigma, and 5-aminolevulinic acid (ALA) from Wako. All other chemicals were of the highest purity
commercially available.
Isolation and culture of rat hepatocytes
Culture dishes (60 mm diameter; Iwaki Glass, Chiba, Japan) were coated with 100 ll undiluted
Matrigel as described by Li et al. (1991). Hepatocytes were prepared from male Wistar rats (Charles
River Japan, Kanagawa, Japan), weighing 180±210 g, by the two-step collagenase perfusion method
(
Seglen 1976). The cells were plated at a density of 2¬10’ cell}dish in 4 ml Williams’ E medium
supplemented with 10 % newborn calf serum, 10 nm insulin, 10 nm dexamethsone and 30 mg}l
kanamycin. Hepatocytes were incubated in a humidi®ed atmosphere of 95 % air and 5 % CO# at 37 °C.
After 4 h in culture, media were renewed with Williams’ E medium with the same supplement. After a
total preincubation period of 24 h, media were refreshed and the chemicals dissolved in dimethyl
sulphoxide (DMSO) were added to the cultures (®nal DMSO concentration 0±5 % v}v). Thereafter,
media and the chemicals were refreshed every 24 h.
Enzyme assays
After the exposure to chemicals for 48 h, hepatocyte cultures were washed twice with ice-cold PBS.
The cells and Matrigel were then scraped with a rubber policeman in 4 ml 0±2 m sucrose±0±05 m Tris
HCl, pH 7±4 (buåered sucrose) and placed on ice. After the cells were freed from Matrigel by repeated

Irgasan DP 300 induction of cytochrome P450
683
pipetting, the cells were precipitated by centrifugation, resuspended in 1 ml buåered sucrose, and lysed
by sonication using a Shimadzu USP-300 ultrasonic processor (Kyoto, Japan). Enzyme activities in the
cell lysate were assayed, except for testosterone 6b-hydroxylase (TS6bH) activity, which was assayed in
intact hepatocytes.
7
-Methoxyresoru®n O-demethylase (MROD), 7-ethoxyresoru®n O-deethylase (EROD), 7-pentoxy-
resoru®n O-depentylase (PROD) and 7-benzylozyresoru®n O-debenzylase (BROD) activities were
determined ¯uorometrically from the amount of resoru®n produced (Hanioka et al. 1995). The substrate
concentrations employed were 2 lm for MROD, 4 lm for EROD and PROD, and 1 lm for BROD.
4
-Nitrophenol hydroxylase (PNPH) activity was assayed in 0±6 ml incubation medium consisting of
phosphate buåer (0±1 m, pH 6±8), ascorbic acid (1 mm), NADPH-generating system (1 mm NADP+,
0 mm glucose 6-phosphate, 2 U glucose 6-phosphate dehydrogenase and 5 mm MgCl ), cell lysate
1
#
(
0±1 ml) and 4-nitrophenol (100 lm). The reaction was initiated by the addition of a NADPH-generating
system and carried out at 37 °C for 30 min. The resulting 4-nitrocatechol was determined by hplc
according to Tassaneeyakul et al. (1993).
TS6bH activity was assayed in intact hepatocytes (Wortelboer et al. 1990). The cells were washed
twice with prewarmed Hanks’ balanced salt solution (HBSS) and incubated in 4 ml HBSS containing
2
50 lm testosterone at 37 °C. After 15 min, the reaction was stopped by cooling the medium on ice. An
aliquot (1 ml) of the medium was extracted with 6 ml dichloromethane, and the solvent was evaporated
to dryness under a stream of N . The residue was dissolved in 50 % (v}v) methanol}water and analysed
#
by LC Module 1 (Waters, Tokyo). The samples were injected into a 150¬4±6 mm i.d. Inertsil ODS-80A
(
5 lm) column (GL-Science, Tokyo). The metabolites were eluted with aqueous 25 % methanol for
1
1
0 min, followed by a 25-min linear gradient to 40 % methanol and 3±5 % acetonitrile at a ¯ow rate of
±0 ml}min. 6b-Testosterone was detected by the absorbance at 254 nm. The peak areas were calculated
with a Shimadzu CR-4A Chromatopac.
Immunoblot analysis of P450s
Hepatocytes microsomes were prepared by centrifugation of the cell lysates for 30 min at 105000 g
(
Schuetz et al. 1988). Separation of the microsomal proteins was carried out with the SDS-polyacrylamide
gel electrophoresis (Laemmli 1970), using a 4% stacking gel and a 7±5 % separating gel. The resolved
proteins were electrophoretically transferred to nitrocellulose membranes according to the method of
Towbin et al. (1979) and immunochemically stained using goat anti-rat P450 antibodies. The
nitrocellulose membranes were blocked overnight at 4 °C in Tris-buåered saline containing 0±5 % Tween
2
0 and 3% bovine serum albumin. Thereafter the membranes were incubated with a primary antibody
for 1 h at room temperature, followed by 1-h incubation with anti-goat IgG conjugated with horseradish
peroxidase. Colour was developed with 4-chloro-1-naphthol and hydrogen peroxide.
Hplc analysis of porphyrins in hepatocyte cultures
After the exposure to chemicals for 24 h, hepatocytes were scraped oå the dishes in the culture
medium and brie¯y sonicated. To a portion (1 ml) of the cell lysate was added an equal volume of freshly
prepared acetone}concentrated HCl (97±5}2±5, v}v). After centrifugation, the resulting supernatant was
assayed for porphyrins by the hplc method of Bonkovsky et al. (1986). The hplc consisted of two
Shimadzu LC-10AD pumps, a SCL-10A system controller, a SIL-10A autoinjector and a RF-550
¯
uorescence monitor. The samples were diluted twice with 56 % 0±1 m ammonium
dihydrogenphosphate-44 % methanol, pH 3±4 (solvent A), and an aliquot (200 ll) was injected into an
Inertsil ODS-80A column. A 15-min linear gradient from 70 % solvent A±30 % methanol to 100%
methanol was employed at a ¯ow rate of 1±0 ml}min. Porphyrins were detected by ¯uorescence with
excitation at 398 nm and emission at 620 nm.
URO I and URO III were co-eluted under these hplc conditions. For the separation of the isomers,
2% acetonitrile in 1 m ammonium acetate, pH 5±16 was employed as the mobile phase (Tsai et al. 1987).
1
Assay of uroporphyrinogen III synthase (UROgen III-S) activity in vitro
The inhibition of UROgen III-S by Irgasan DP 300 was demonstrated in vitro according to the
methods of Tsai et al. (1987) and Clement et al. (1982). The cytosolic fraction was prepared from the male
Wistar rat by diåerential centrifugation (20 min, 9000 g ; 60 min, 105000 g) as described previously
(
Hanioka et al. 1995). The intact and the heated (60 °C, 1 h) cytosolic fractions were applied as enzyme
sources of UROgen III-S and hydroxymethylbilane (HMB) synthase respectively. UROgen III-S
activity was measured in 0±6 ml incubation medium consisting of Tris-HCl buåer (0±05 m, pH 7±8), KCl
(
0±06 m), the intact cytosol (5 ll), the heated cytosol (115 ll) and PBG (2±5±12±5 lm). Irgasan DP 300 was
dissolved in methanol and added to the incubation medium (®nal methanol concentration 1±7 %). The
reaction was initiated by the addition of PBG and carried out at 37 °C for 15 min. The reaction was
terminated by adding 0±4 ml 10 % (w}v) tricloroacetic acid±0±5 % (w}v) iodine. After centrifugation,
URO I and URO III in the supernatant were analysed by hplc as described above.

6
84
H. Jinno et al.

Irgasan DP 300 induction of cytochrome P450
685
Figure 2. Immunoblot analysis of P450 induction in rat hepatocytes. Proteins from pooled microsomes
were subjected to electrophoresis, electrophoretically transferred to nitrocellulose membranes and
immunochemically detected with anti-rat P450 antibodies. Each lane contained 5 lg (1A1}2 and
2
B1}2) or 10 lg (2E1 and 3A2}1) of microsomal protein. Lanes 1±5 contained microsomal protein
from Irgasan DP 300-treated hepatocytes: 1, 7±8 lm ; 2, 15±6 lm ; 3, 31±3 lm ; 4, 62±5 lm ; 5, 125 lm.
Lanes 6±8 contained proteins from control, MC (5 lm)- and PB (750 lm)-treated hepatocytes
respectively.
Other methods
Protein concentrations were measured by the method of Lowry et al. (1951) using bovine serum
albumin as the standard.
Results
Induction of P450s in Irgasan DP 300-treated hepatocyte cultures
As summarized in table 1, Irgasan DP 300 induced P450-dependent mono-
oxygenase activities in rat hepatocytes. Of the enzyme activities assayed, the BROD
activity was the greatest, followed by PROD and EROD activities. The PNPH,
!
TS6bH and MROD activities were also slightly increased ( 2-fold) in the Irgasan
DP 300-treated hepatocytes. The maximum induction of these enzyme activities
was obtained at the same concentration of 125 lm. At 250 lm, all of the activities
were reduced to the almost undetectable level, re¯ecting the cytotoxity of Irgasan
DP 300 (LC&! ¯ 150 lm ; data not shown).
The induction of P450 was further demonstrated at the apoprotein level by
immunoblot analysis using anti-rat P450 antibodies (®gure 2). In accordance with

6
86
H. Jinno et al.

Irgasan DP 300 induction of cytochrome P450
687
Figure 3. Formation of UROgen I and UROgen III from hydroxymethylbilane.
the increase in BROD activity, Irgasan DP 300 preferably induced CYP2B1}2 along
with a slight increase in 3A in rat hepatocytes. In contrast, 2E1 was not aåected by
Irgasan DP 300, despite the induction of PNPH activity. Of interest was that 1A2
was detected at low levels in the Irgasan DP 300-treated hepatocytes. For this P450
isozyme, the maximum induction was observed at 62±5 lm.
The results of both the enzyme assay and the immunoblot analysis clearly
indicate that Irgasan DP 300 was similar to phenobarbital (PB) as an inducer.
Inhibition of hepatocellular haem biosynthesis by Irgasan DP 300
Porphyrins accumulated in the hepatocyte culture were assayed after 24-h
exposure to Irgasan DP 300. In the absence of exogenous ALA, slight increases of
PROTO IX (2±6-fold) and COPRO III (1±3-fold) were observed in the hepatocyte
cultures treated with Irgasan DP 300 at 125 lm (table 2). In contrast, when 75 lm
ALA was present, Irgasan DP 300 caused an extensive accumulation of URO (13-
fold) at the concentration of 250 lm. As shown in ®gure 3, two isomers of URO,
URO I and URO III, could be formed in the haem biosynthetic pathway. However,
URO I and URO III not separated from each other because both of the isomers co-
eluted under the hplc conditions used for the porphyrin pro®le analysis (ammonium
phosphate±methanol as mobile phase). Complete separation of the isomers was
achieved using ammonium acetate±acetonitrile as the mobile phase and URO I was
the isomer which accumulated in hepatocyte cultures treated with Irgasan DP 300

6
88
H. Jinno et al.
Figure 4. Hplc chromatogram of URO in hepatocyte cultures treated with Irgasan DP 300 (250 lm)
and ALA (75 lm). URO I and URO III were separated using 12 % acetonitrile in 1 m ammonium
acetate, pH 5±16 as the mobile phase.
Figure 5. Lineweaver±Burk plot for the UROgen III-S activity in the presence of Irgasan DP 300.
Each point represents the mean³SD from three determinations.
in the presence of exogenous ALA (®gure 4). These results suggested that Irgasan
DP 300 possibly caused the accumulation of HMB as a result of UROgen III-S
inhibition.
In vitro inhibition of UROgen III-S activity by Irgasan DP 300
To test the possible inhibition of UROgen III-S activity by Irgasan DP 300, a
coupled enzyme assay (Tsai et al. 1987) was carried out using cytosolic fractions
prepared from rat liver. In this assay system, PBG (substrate) was ®rst converted to
HMB by HMB synthase in the heat-treated cytosolic fraction, then cyclized to
UROgen III by UROgen III-S in the intact cytosolic fraction. Figure 5 shows a
Lineweaver±Burk plot for the UROgen III-S activity observed in the presence of

Irgasan DP 300 induction of cytochrome P450
689
Irgasan DP 300. This plot demonstrated that Irgasan DP 300 directly inhibited
UROgen III-S in a non-competitive or a mixed (competitive}non-competitive)
manner.
Discussion
Both the induction of P450 and the inhibition of haem biosynthesis have been
demonstrated in experimental animals intoxicated with polychlorinated aromatic
hydrocarbons. For example, TCDD and certain members of the PCB family
speci®cally induce CYP1A1}2 in the liver (Goldstein and Linko 1984, Dragnev et
al. 1995), mediated by the aryl hydrocarbon (Ah) receptor (reviewed by Landers and
Bunce 1991), and additionally cause hepatic URO III accumulation (Poland and
Glover 1973, Goldstein et al. 1973, 1974, Vos and Koeman 1970). However the
mechanisms involved in the inhibition of UROgen III decarboxylase have not been
fully elucidated (Cantoni et al. 1984). Diphenyl ether herbicides, another class of
polychlorinated aromatic hydrocarbons, have been shown to inhibit proto-
porphyrinogen IX oxidase, resulting in the accumulation of PROTO IX (Matringe
et al. 1989). Some of the diphenyl ethers have also been demonstrated to induce
hepatic P450s (Jacobs et al. 1992, Krijt et al. 1993). In view of the structural
similarity to Irgasan DP 300, we studied the eåect of Irgasan DP 300, a typical 2-
phoenoxyphenol, on hepatic P450 levels and haem biosynthesis.
In previous in vivo studies, Irgasan DP 300 has been reported to increase
aminopyrine N-demethylase activity in mouse (Miller et al. 1981), suggesting that
Irgasan DP 300 is a PB-type inducer. Kanetoshi et al. (1992) reported that Irgasan
DP 300 signi®cantly induced p-nitrophenetole O-deethylase (2±3-fold), biphenyl 2-
hydroxylase (2±3-fold), 7-ethoxycoumarin O-deethylase (2±0-fold), p-nitroanisole
O-demethylase (1±7-fold) and biphenyl 4-hydroxylase (1±3-fold) activities in rat.
From these latter results, however, it is impossible to identify the P450 isozymes
induced by Irgasan DP 300. In the present study, the induction of P450 was ®rst
demonstrated by enzyme assays of MROD, EROD, PROD, BROD, PNPH and
TS6bH activities, which have been established as sensitive and highly selective
markers for the induction of P450 isozymes. Yang et al. (1988) reported that puri®ed
rat CYP1A1 and 1A2 preferentially metabolize ethoxyresoru®n and methoxy-
resoru®n respectively. Dealkylation of pentoxyresoru®n and benzyloxyresoru®n has
been shown to be catalysed by puri®ed rat 2B1 (Wolf et al. 1986, Yang et al. 1988).
The predominant contribution of 2E1 to 4-nitophenol hydroxylation in the rat is
generally accepted (Dicker et al. 1990). Rat 3A has been puri®ed and shown to
metabolize testosterone to the 6b-hydroxy derivative (Halvorson et al. 1990). Of
these enzyme activities, BROD activity was the most increased in the Irgasan DP
3
00-treated hepatocytes (table 1), indicating the possible induction of 2B1}2. The
results obtained from our immunochemical study were mainly consistent with those
from enzyme assays. Irgasan DP 300 preferably induced 2B1}2 along with a slight
increase in 3A (®gure 2), in accordance with the increase in BROD activity. Thus,
Irgasan DP 300 was identi®ed as a PB-type inducer in rat hepatocytes. 2E1 was not
aåected by Irgasan DP 300, despite the induction of PNPH activity. P450 isozymes
other than 2E1, such as 2B1}2 and 3A, might catalyse 4-nitrophenol hydroxylation
to a minor extent, and contribute to this discrepancy between PNPH activity and
2
E1 apoprotein.
Exogenous ALA substantially altered the impact of Irgasan DP 300 on
hepatocellular haem biosynthesis. In the absence of exogenous ALA, Irgasan DP

6
3
90
H. Jinno et al.
00 slightly increased PROTO IX and COPRO III in the hepatocyte cultures.
When ALA (75 lm) was added alone, the hepatocytes accumulated mostly PROTO
IX and COPRO III, in accordance with previous results in mouse (Sinclare et al.
1
990), rat and chick embryo hepatocytes (De Matteis et al. 1988). In the present
study, Irgasan DP 300 (250 lm), together with exogenous ALA, induced an
extensive accumulation of URO at the expense of COPRO III and PROTO IX. The
URO isomer accumulated in the culture was URO I. In the haem biosynthetic
pathway, HMB is cyclized to UROgen III by UROgen III synthase, associated with
the intermolecular rearrangement of the d-pyrrole group (Battersby et al. 1982, Hart
and Battersby 1985). Another isomer UROgen I, a reduced form of URO I, is
generated from HMB by spontaneous cyclization, or non-enzymatic reaction
(
Battersby et al. 1982). Therefore, the accumulation of URO I we observed suggests
that Irgasan DP 300 may interfere with the UROgen III synthesis from HMB by
UROgen III synthase. As a test of this hypothesis, a coupled-enzyme assay of
UROgen III synthase activity was carried out in vitro. Although the mode of
inhibition was not fully elucidated, it was demonstrated that Irgasan DP 300 directly
inhibited UROgen III synthase. Thus, we showed that Irgasan DP 300 inhibited
hepatocellular UROgen III synthase, resulting in the accumulation of URO I. The
accumulation of URO I has also been reported in the rare human disease of
congenital erythropoietic porphyria (CEP) which is characterized by the de®cient
activity of UROgen III synthase (Deybach et al. 1981). The clinical syndrome of
CEP is dominated by extreme photosensitivity and haemolytic anemia. Irgasan DP
3
00 may induce such manifestations in vivo.
Acknow ledgem ent
This study was supported by grants from the Environment Agency, Japan.
References
Battersby , A. R., Fookes , C. J. R., Gustafson -Potter , K. E., McDonald , E. and Matcham ,
G. W. J., 1982, Biosynthesis of porphyrins and related macrocycles. Part 18. Proof by
spectroscopy and synthesis that unrearranged hydroxymethylbilane is the product from deaminase
and the substrate for cosynthetase in the biosynthesis of uroporphyrinogen-III. Journal of
Chemical Society Perkin Transaction I, 2427±2444.
Bonkovsky , H. L., Wood, S. G., Howell , S. K., Sinclair , P. R., Lincoln , B., Healey , J. F. and
Sinclair , J. F., 1986, High-performance liquid chromatographic separation and quantitation of
tetrapyrroles from biological materials. Analytical Biochemistry, 155, 56±64.
Cantoni , L., dal Fiume , D., Rizzardini , M. and Ruggieri , R., 1984, In vitro inhibitory eåect on
porphyrinogen carboxylyase of liver extracts from TCDD treated mice. Toxicology Letters, 20,
2
11±217.
Clement , R. P., Kohashi , M. and Piper , W. N., 1982, Rat hepatic uroporphyrinogen III cosynthase :
puri®cation, properties, and inhibition by metal ions. Archives of Biochemistry and Biophysics, 214,
6
57±667.
De Matteis , F., Harvey, C., Reed, C. and Hempenius , R., 1988, Increased oxidation of
uroporphyrinogen by an inducible liver microsomal system. Possible relevance to drug-induced
uroporphyria. Biochemical Journal, 250, 161±169.
Deybach , J. C., De Verneuil , H., Phung , N., Nordmann , Y., Puissant , A. and Boffety , B., 1981,
Congenital erythropoietic porphyria (Gunther’s disease) : enzymatic studies on two cases of late
onset. Journal of Laboratory and Clinical Medicine, 97, 551±558.
Dicker , E., McHugh, T. and Cederbaum , A. I., 1990, Increased oxidation of p-nitrophenol and aniline
by intact hepatocytes isolated from pyrazole-treated rats. Biochimca et Biophysica Acta, 1035,
2
49±256.

Irgasan DP 300 induction of cytochrome P450
691
Dragnev , K. H., Nims , R. W., Fox, S. D., Lindahl , R. and Lubet , R. A., 1995, Relative potencies of
induction of hepatic drug-metabolizing enzyme genes by individual PCB congeners. Toxicology
and Applied Pharmacology, 132, 334±342.
Goldstein , J. A., Hickman , P. and Jue, D. L., 1974, Experimental hepatic porphyria induced by
polychlorinated biphenyls. Toxicology and Applied Pharmacology, 27, 437±448.
Goldstein , J. A., Hickman , P. and Vos, J. G., 1973, Hepatic porphyria induced by 2,3,7,8-
tetrachlorodibenzo-p-dioxin in the mouse. Research Communication of Chemical and Pathological
Pharmacology, 6, 919±928.
Goldstein , J. A. and Linko , P., 1984, Diåerential induction of two 2,3,7,8-tetrachlorodibenzo-p-
dioxin-inducible forms of cytochrome P-450 in extrahepatic versus hepatic tissues. Molecular
Pharmacology, 25, 185±191.
Halvorson , M., Greenway , D., Eberhart , D., Fitzgerald , K. and Parkinson , A., 1990, Reconstitution
of testosterone oxidation by puri®ed rat cytochrome P450p (IIIA1). Archives of Biochemistry and
Biophysics, 277, 166±180.
Hanioka , N., Jinno , H., Takahashi , A., Nakano , K., Yoda, R., Nishimura , T. and Ando , M., 1995,
Interaction of tetrachloroethylene with rat hepatic microsomal P450-dependent monooxygenases.
Xenobiotica , 25, 151±165.
Hart, G. J. and Battersby , A. R., 1985, Puri®cation and properties of uroporphyrinogen III synthase
(
co-synthetase) from Euglena gracilis. Biochemical Journal, 232, 151±160.
Jacobs, J. M., Sinclair , P. R., Gorman , N., Jacobs, N. J., Sinclair , J. F., Bement , W. J. and Walton ,
H., 1992, Eåects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes.
Journal of Biochemical Toxicology, 7, 87±95.
Kanetoshi , A., Katsura, E., Ogawa, H., Ohyama , T., Kaneshima , H. and Miura, T., 1992, Acute
toxicity, percutaneous absorption and eåects on hepatic mixed function oxidase activities of
2
,4,4«-trichloro-2 «-hydroxydiphenyl ether (Irgasan DP300) and its chlorinated derivatives.
Archives of Environmental Contamination and Toxicology, 23, 91±98.
Kanetoshi , A., Ogawa, H., Katsura, E., Kaneshima , H. and Miura, T., 1988, Formation of
polychlorinated dibenzo-p-dioxins upon combustion of commercial textile products containing
2
2
,4,4«-trichloro-2 «-hydroxydiphenyl ether (Irgasan‡ DP300). Journal of Chromatography, 442,
89±299.
Kocarek, T. A., Schuetz, E. G. and Guzelian , P. S., 1993, Expression of multiple forms of cytochrome
P450 mRNAs in primary cultures of rat hepatocytes maintained on matrigel. Molecular
Pharmacology, 43, 328±334.
Krijt , J., van Holsteijn , I., Hassing , I., Vokurka, M. and Blaauboer , B. J., 1993, Eåect of diphenyl
ether herbicides and oxadiazon on porphyrin biosynthesis in mouse liver, rat primary hepatocyte
culture and HepG2 cells. Archives of Toxicology, 67, 255±261.
Laemmli , U. K., 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage
T4. Nature, 227, 680±685.
Landers , J. P. and Bunce, N. J., 1991, The Ah receptor and the mechanism of dioxin toxicity.
Biochemical Journal, 276, 273±287.
Langer, H. G., Brady, T. P. and Briggs , P. R., 1973, Formation of dibenzodioxins and other
condensation products from chlorinated phenols and derivatives. Environmental Health
Perspectives , 5, 3±7.
Levin , J. O. and Nilsson , C. A., 1977, Chromatographic determination of polychlorinated phenols,
phenoxyphenols, dibenzofurans and dibenzodioxins in wood-dust from worker environments.
Chemosphere, 6, 443±448.
Li, D., Schuetz, E. T. and Guzelian , P. S., 1991, Hepatocyte culture in study of P450 regulation.
Methods in Enzymology, 206, 335±344.
Lowry, O. H., Rosenbrough , N. J., Farr, A. L. and Randall , R. J., 1951, Protein measurement with
Folin phenol reagent. Journal of Biological Chemistry, 193, 265±275.
Lyman , F. L. and Furia, T., 1969, Toxicology of 2,4,4«-trichloro-2 «-hydroxydiphenyl ether. Industrial
Medicine and Surgery, 38, 64±71.
Matringe , M., Camadro , J. M., Labbe, P. and Scalla, R., 1989, Protoporphyrinogen oxidase as a
molecular target for diphenyl ether herbicides. Biochemical Journal, 260, 231±235.
Miller , T. L., Lorusso , D. J. and Deinzer , M. L., 1981, Induction of enzymes of the hepatic mixed-
function oxidase system by hydroxychlorodiphenyl ether contaminants of technical PCP.
Federation Proceedings, 40, 630.
Miyazaki , T., Yamagishi , T. and Matsumoto , M., 1984, Residues of 4-chloro-1-(2,4-
dichlorophenoxy)-2-methoxybenzene (triclosan methyl) in aquatic biota. Bulletin of Environ-
mental Contamination and Toxicology, 32, 227±232.
Nilsson , C. A., Anderson , K., Rappe, C. and Westermark , S. O., 1974, Chromatographic evidence for
the formation of chloro-dioxins from chloro-2-phenoxyphenols. Journal of Chromatography, 96,
1
37±147.
Poland , A. and Glover , E., 1973, 2,3,7,8-Tetrachlorodibenzo-p-dioxin : a potent inducer of d-
aminolevulinic acid synthetase. Science, 179, 476±477.

6
92
Irgasan DP 300 induction of cytochrome P450
Rappe, C. and Nilsson , C. A., 1972, An artifact in the gas chromatographic determination of impurities
in pentachlorophenol. Journal of Chromatography, 67, 247±253.
Schuetz, E. G., Li, D., Omiecinski , C. J., Muller -Eberhard , U., Kleinman , H. K., Elswick , B. and
Guzelian , P. S., 1988, Regulation of gene expression in adult rat hepatocytes cultured on a
basement membrane matrix. Journal of Cellular Physiology, 134, 309±323.
Seglen , P. O., 1976, Preparation of isolated rat liver cells. Methods in Cell Biology, 13, 29±83.
Sinclair , P. R., Bement , W. J., Lambrecht , R. W., Gorman , N. and Sinclair , J. F., 1990, Chlorinated
biphenyls induce cytochrome P450IA2 and uroporphyrin accumulation in cultures of mouse
hepatocytes. Archives of Biochemistry and Biophysics, 281, 225±232.
Tassaneeyakul , W., Veronese , M. E., Birkett , D. J. and Miners , J. O., 1993, High-performance
liquid chromatographic assay for 4-nitrophenol hydroxylation, a putative cytochrome P-4502E1
activity, in human liver microsomes. Journal of Chromatography, 616, 73±78.
Towbin , H., Staehelin , T. and Gordon , J., 1979, Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets : procedure and some applications. Proceeding of the
National Academy of Sciences, USA, 76, 4350±4354.
Tsai, S., Bishop , D. F. and Desnick , R. J., 1987, Coupled-enzyme and direct assays for
uroporphyrinogen III synthase activity in human erythrocytes and cultured lymphoblasts.
Analytical Biochemistry, 166, 120±133.
Vos, J. G. and Koeman , J. H., 1970, Comparative toxicologic study with polychlorinated biphenyls in
chicken with special reference to porphyria, edema formation, liver necrosis, and tissue residues.
Toxicology and Applied Pharmacology, 17, 656±668.
Wolf, C. R., Seilman , S., Oesch , F., Mayer, R. T. and Burke, M. D., 1986, Multiple forms of
cytochrome P-450 related to forms induced marginally by phenobarbital. Diåerences in structure
and in the metabolism of alkoxyresoru®ns. Biochemical Journal, 15, 27±33.
Wortelboer , H. M., de Kruif , C. A., van Iersel , A. J., Falke, H. E., Noordhoek , J. and Blaauboer ,
B. J., 1990, The isozyme pattern of cytochrome P450 in rat hepatocytes in primary culture,
comparing diåerent enzyme activities in microsomal incubations and intact monolayers.
Biochemical Pharmacology, 40, 2525±2534.
Yang, H. Y, Namkung , M. J. and Juchau, M. R., 1988, Cytochrome P-450-dependent bio-
transformation of a series of phenoxazone ethers in the rat conceptus during early organogenesis:
evidence for multiple P-450 isoenzymes. Molecular Pharmacology, 34, 67±73.

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