Oxidative dechlorination of methoxychlor by ligninolytic enzymes from white-rot fungi

Article abstract of DOI:10.1016/j.chemosphere.2003.11.035

Ligninolytic enzymes, manganese peroxidase (MnP), laccase, and lignin peroxidase (LiP), from white-rot fungi were used in an attempt to treat methoxychlor (MC), a chemical widely used as a pesticide. MnP and laccase in the presence of Tween 80 and 1-hydroxybenzotriazole (HBT), respectively, and LiP were found to degrade MC, and MnP-Tween 80 decreased MC levels by about 65% after a 24-h treatment. MC was converted into methoxychlor olefin (MCO) and 4,4′-dimethoxybenzophenone by MnP-Tween 80 or laccase-HBT treatment. These results indicate that ligninolytic enzymes from white-rot fungi can catalyze the oxidative dechlorination of MC. Moreover, a metabolite MCO was also degraded by MnP-Tween 80 or laccase-HBT treatment.

Full text of DOI:10.1016/j.chemosphere.2003.11.035

Chemosphere 55 (2004) 641–645
www.elsevier.com/locate/chemosphere
Short Communication
Oxidative dechlorination of methoxychlor
by ligninolytic enzymes from white-rot fungi
Hirofumi Hirai, Sawako Nakanishi, Tomoaki Nishida *
Department of Forest Resources Science, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
Received 27 January 2003; received in revised form 29 September 2003; accepted 12 November 2003
Abstract
Ligninolytic enzymes, manganese peroxidase (MnP), laccase, and lignin peroxidase (LiP), from white-rot fungi were
used in an attempt to treat methoxychlor (MC), a chemical widely used as a pesticide. MnP and laccase in the presence
of Tween 80 and 1-hydroxybenzotriazole (HBT), respectively, and LiP were found to degrade MC, and MnP-Tween 80
0
decreased MC levels by about 65% after a 24-h treatment. MC was converted into methoxychlor olefin (MCO) and 4,4 -
dimethoxybenzophenone by MnP-Tween 80 or laccase-HBT treatment. These results indicate that ligninolytic enzymes
from white-rot fungi can catalyze the oxidative dechlorination of MC. Moreover, a metabolite MCO was also degraded
by MnP-Tween 80 or laccase-HBT treatment.
Ó 2003 Elsevier Ltd. All rights reserved.
Keywords: Methoxychlor; Manganese peroxidase; Laccase; Lignin peroxidase; Dechlorination
1
. Introduction
neonatal or the adult organism to high doses of MC may
place the male reproductive system at risk, and MC has
been reported to affect the reproductive success of male
rats via decreasing sperm counts (Chapin et al., 1997).
There is a great interest in the lignin-degrading white-
rot fungi and their ligninolytic enzymes, because their
industrial potential for degrading recalcitrant environ-
mental pollutants, such as polychlorinated dibenzodi-
oxin, DDT (Bumpus et al., 1985), chlorophenols (Joshi
and Gold, 1993), and polycyclic aromatic carbons
(Bezalel et al., 1996; Collins et al., 1996), have become
increasingly recognized. Recently, we demonstrated that
ligninolytic enzymes such as manganese peroxidase
(MnP) and laccase in the presence of Tween 80 and 1-
hydroxybenzotriazole (HBT), respectively, are effective
in removing the estrogenic activities of bisphenol A,
nonylphenol (Tsutsumi et al., 2001) and steroidal hor-
mones (Suzuki et al., 2003). Herein we present the
application of MnP-Tween 80, laccase-HBT, and lignin
peroxidase (LiP) to the treatment of MC and describe
the dechlorination of MC without the production of
hydroxy metabolites.
Human exposure to environmental contaminants
that negatively affect the male reproductive system is
increasing (Sharpe, 1993), and many of these contami-
nants, such as organochlorine pesticides, accumulate in
the fatty tissues of the body. Methoxychlor (MC), 1,1,1-
trichloro-2,2-bis(p-methoxyphenyl)ethane, is a newly de-
veloped pesticide that is intended as a replacement for
1
,1,1-trichloro-2,2-bis(chlorophenyl)ethane (DDT), the
use of which has been regulated internationally since the
970s. MC displays both estrogenic and antiandrogenic
1
activities in vivo and in vitro (Maness et al., 1998) and
is considered to be a proestrogen that, when conver-
ted into mono- and bis-hydroxy metabolites, exhibits
greater estrogenic properties than does the MC parental
compound (Bulger et al., 1978). Because of the estro-
genicity of MC metabolites, exposure of either the
*
Corresponding author. Tel./fax: +81-54-238-4852.
E-mail address: aftnisi@agr.shizuoka.ac.jp (T. Nishida).
0
045-6535/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2003.11.035

6
42
H. Hirai et al. / Chemosphere 55 (2004) 641–645
2.3. Enzyme treatment
2
. Materials and methods
2
.1. Chemicals
For reactions with MnP, 10 ml reaction mixture
consisted of 10 nkat MnP, 0.1 ml of 10 mM substrate in
dimethylsulfoxide (DMSO), 1 mM MnSO , 0.1% Tween
MC, methoxychlor olefin [1,1-dichloro-2,2-bis(p-
4
0
methoxyphenyl)ethylene; MCO], and 4,4 -dimethoxy-
benzophenone (DMB) used in this study were extra-pure
grade and were purchased from Wako Pure Chemicals,
Sigma Chemical Company, and Tokyo Chemical Ind-
ustry, respectively. All other chemicals were also extra-
pure grade, obtained from commercial sources, and used
without further purification.
80, 4 nkat glucose oxidase, and 2.5 mM glucose in 50
mM malonate buffer (pH 4.5). The laccase reaction
mixture (10 ml) contained 10 nkat laccase, 0.1 ml of 10
mM substrate in DMSO, and 0.2 mM HBT in 50 mM
malonate buffer (pH 4.5). The LiP reaction mixture (10
ml) contained 10 nkat LiP, 0.1 ml of 10 mM substrate in
DMSO, 4 nkat glucose oxidase, and 2.5 mM glucose in
2
0 mM succinate buffer (pH 3.0). Reactions were carried
out at 30 °C for 24 h. The amounts of MC, MCO, and
DMB were determined by high-performance liquid
chromatography (HPLC) analysis, and the identifica-
tions of the metabolites were carried out by gas chro-
matography–mass spectrometry (GC–MS) analysis.
2
.2. Enzyme assay and preparation
Laccase activity was determined by monitoring the
oxidation of 2,6-dimethoxyphenol (DMP) at 470 nm.
The reaction mixture contained 1 mM DMP and 50 mM
malonate buffer (pH 4.5). MnP activity was determined
in the same manner except that the reaction mixture
2.4. Instrumentation
contained 1 mM MnSO
4
and 0.2 mM H
2
O
2
. LiP activity
HPLC analyses were conducted with a Wakosil-II
5C18HG (Wako) column, isocratic elution with 80%
methanol aqueous at a flow rate of 1 ml/min, and
detection at 280 nm. GC–MS was carried out at 70 eV on
a Shimadzu QP-5050A equipped with a 30-m fused
capillary column (TC-1, Shimadzu). The oven tempera-
ture was programmed to increase from 100 to 270 °C at a
rate of 10 °C/min. Products were identified by comparing
their retention times on GC and comparing mass frag-
mentation patterns with those of authentic compounds.
was determined by monitoring the oxidation of veratryl
alcohol (VA) at 310 nm. The reaction mixture contained
1
0
mM VA, 20 mM succinate buffer (pH 3.0), and
.2 mM H . One katal (kat) of enzyme activity is
2
O
2
the amount of enzyme producing 1 mol of the qui-
ꢀ1
ꢀ
1
none dimer (49.3 mM cm ) or veratraldehyde (9.3
ꢀ
1
ꢀ1
mM cm ) from DMP or VA, respectively, per second.
Partially purified MnP was isolated from cultures of
Phanerochaete chrysosporium (ME-446) by DEAE Sep-
harose CL-6B (Pharmacia) column chromatography
(Wariishi et al., 1992). LiP was partially purified from
cultures of P. chrysosporium by Mono Q HR5/5 (Phar-
3. Results and discussion
macia) column chromatography (Wariishi and Gold,
1
990). Partially purified laccase was prepared from cul-
We applied the ligninolytic enzymes, 10 nkat of MnP,
laccase, and LiP, to the treatment of MC. As Table 1
indicates, MnP-Tween 80 system decreased levels of MC
by about 65% after a 24-h treatment and was much more
effective in degrading MC than were either LiP or lac-
tures of Trametes versicolor (IFO-6482) as described in
our previous report (Nishida et al., 1999). Each purified
ligninolytic enzyme preparation contained no other lig-
ninolytic enzyme activities.
Table 1
Degradation of methoxychlor by ligninolytic enzymes
Enzymes
MnP
Reaction
time (h)
Decrease of
MC (%)
Production of metabolites (%)
MCO
DMB
12
2
42.6 ± 2.3
64.9 ± 4.1
4.2 ± 0.5
6.2 ± 0.4
n.d.
trace
4
LiP
12
4
15.2 ± 1.6
28.4 ± 2.1
n.d.
n.d.
n.d.
n.d.
2
Laccase
12
2
12.8 ± 2.4
23.3 ± 2.6
0.4 ± 0.1
0.7 ± 0.1
n.d.
trace
4
n.d.; not detected.
Trace; <0.1%.
Values are averages of three experiments ± deviations.

H. Hirai et al. / Chemosphere 55 (2004) 641–645
643
Table 2
Mass spectra and GC retention times of enzymatic metabolites of methoxychlor
Metabolites
GC retention
time (min)
Mass spectrum m=z (relative intensity, %)
þ
MC (starting compound)
MCO
29.59
348 (0.8), 346 M (2.4), 274 (2.4), 228 (16.4), 227
(100), 212 (4.8), 196 (2.4), 153 (2.0), 152 (2.8)
þ
310 (44.4), 308 M (68.0), 273 (26.4), 238 (100),
27.31
2
23 (43.2), 195 (35.2), 186 (11.2), 163 (11.6), 152
42.4), 119 (14.4)
(
þ
DMB
25.71
243 (5.2), 242 M (30.8), 227 (10.0), 211 (12.8),
35 (100), 107 (14.8), 92 (12.8), 77 (20.0)
1
case-HBT system. However, 1 nkat of each ligninolytic
enzymes hardly degraded MC. The metabolites from
these reaction mixtures were analyzed with the GC–MS.
MnP-Tween 80 or laccase-HBT system converted MC
into two products (Table 2). One of the products was
identified as MCO by GC–MS from a comparison of its
retention time and mass spectrum with those of an
authentic compound. This result suggests that a Cl
radical is eliminated from MC to yield the metabolite
treatments into MCO and DMB. A possible mechanism
for the dechlorination of MC by MnP/Tween 80 or
laccase/HBT is shown in Fig. 1. MnP or laccase have
been reported to oxidize nonphenolic compounds in the
presence of unsaturated fatty acid (Tween 80) or HBT,
respectively, although either enzyme alone cannot oxi-
dize such compounds (Bao et al., 1994; Bourbonnais
et al., 1998). Thus, a lipid peroxyl radical or an HBT
radical is produced in the reaction mixture containing
MnP or laccase, respectively. A phenylalkyl radical,
which would be oxidatively produced by these radicals,
could be an intermediate for the formation of both
MCO and DMB. In one reaction, MCO would be
formed by the cleavage of a C–Cl bond and the elimi-
nation of a Cl radical. In the other, molecular oxygen
would be incorporated into the intermediate, and then
MCO. The other product was identified as DMB and
ꢁ
resulted from the loss of a CCl
3
group from MC. MCO
and DMB, however, were not detected in the reaction
mixture after treatment with the LiP enzyme. Although
the derivatization with N,O-bis(trimethylsilyl)trifluoro-
acetimide or diazomethane of ethyl acetate extracts from
the reaction mixtures was carried out, no derivative that
included mono- and bis-hydroxy metabolites was de-
tected in any reaction mixtures containing the lignino-
lytic enzymes. The amount of MCO produced in the
reaction mixture containing MnP-Tween 80 or Laccase-
HBT system was 6.2% or 0.7%, respectively, and trace
amount of DMB was detected in the reaction mixture
containing MnP-Tween 80 or laccase-HBT (Table 1).
Recent in vitro studies have demonstrated that 2,2-bis(4-
chlorophenyl)-1,1-dichloroethylene (DDE), which is the
most stable metabolite of DDT, can act as an andro-
gen receptor antagonist to inhibit the normal effect of
androgens (Kelce et al., 1995; Chedrese and Feyles,
DMB could be formed by the cleavage of the C–CCl
3
bond. On the other hand, no metabolite was not
detected in the reaction mixture although LiP could
degrade MC. The LiP oxidation of a variety of non-
phenolic aromatics to their corresponding aryl cation
radicals has been well established (Valli et al., 1992), and
LiP oxidation of 3,4-dimethoxytoluene and 1,4-dimeth-
oxybenzene generated to the corresponding dimeric
products (Joshi and Gold, 1996). Probably, MC is oxi-
dized to the corresponding aryl cation radical by LiP,
and the dimeric products would be produced by the
coupling of two cation radicals. Similar reaction prob-
ably occurs in the degradation of MCO by MnP-Tween
80 or Laccase-HBT since the formation of phenylalkyl
radical in MCO is not occurred. Therefore, no meta-
bolite including other monomeric products was detected
in LiP oxidation of MC, and in MnP-Tween 80 or lac-
case-HBT treatment of MCO.
2
001). MCO is a metabolite of MC and bears a struc-
tural resemblance to DDE, and its accumulation in the
environment is, like DDE, considered undesirable. We
therefore attempted to treat MCO with three ligninolytic
enzymes. The levels of MCO decreased by about 15%
and 5% after a 24-h treatment with MnP-Tween 80 and
laccase-HBT, respectively. On the other hand, LiP did
not degrade the metabolite MCO. Subsequent metabo-
lite studies were carried out with GC–MS, but no meta-
bolite was detected in reaction mixtures after a 24-h
treatment with MnP-Tween 80, laccase-HBT, or LiP.
In the present study, we have demonstrated the oxi-
dative dechlorination of MC by the ligninolytic enzymes
MnP-Tween 80 and laccase-HBT without the produc-
tion of hydroxy metabolites. MC was converted by these
Levels of the metabolite MCO, which structurally
resembles antiandrogenic DDE, were also decreased by
MnP-Tween 80 or laccase-HBT treatment. In vivo, MC
is converted to hydroxy metabolites [e.g., 1,1,1-tri-
chloro-2,2-bis(p-hydroxyphenyl)ethane] that have much
higher estrogenic activities than does MC (Welch et al.,
1969; Bulger et al., 1978). In the present study, no
hydroxylated metabolite of MC was detected in reaction
mixtures after MnP-Tween 80 and laccase-HBT

644
H. Hirai et al. / Chemosphere 55 (2004) 641–645
+
H , e-
O
O
C
O
C
H
3
CO
OCH
3
H
3
CO
OCH
3
Cl
C
Cl
DMB
Cl
CCl
3
OH
+
H , e-
O
2
H
C
H
3
CO
OCH
3
H CO
C
OCH3
3
Cl
C
Cl
Cl
C
Cl
Cl
Cl
MC
Cl
H
3
CO
C
C
OCH
3
MCO
Cl
Cl
Fig. 1. Possible mechanism for dechlorination of methoxychlor (MC) by MnP-Tween 80 or laccase-HBT treatment. DMB indicates
0
4,4 -dimethoxybenzophenone; MCO, methoxychlor olefin.
treatments. To our knowledge, very little is actually
known about the microbial or enzymatic degradation of
MC. This is the first report to describe the degradation
of MC and its metabolite, MCO, by the ligninolytic
enzymes MnP and laccase from white-rot fungi.
Moser, V.C., Burka, L.T., Collins, B.J., 1997. The effects of
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Chedrese, J.P., Feyles, F., 2001. The diverse mechanism of
action of dichlorodiphenyldichloroethylene (DDE) and
methoxychlor in ovarian cells in vitro. Reprod. Toxicol
1
5, 693–698.
Collins, P.J., Kotterman, M.J., Field, J.A., Dobson, A.W.,
996. Oxidation of anthracene and benzo[a]pyrene by
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