Elucidation of metabolic pathways of xenobiotics (pesticides, pharmaceuticals and industrial pollutants) in human, animals and plants and chemical identification of corresponding metabolites are required for comprehensive (eco-) toxicological evaluation of the compounds prior to their usage. The most important metabolic products are oxidized metabolites, and most of these are formed by catalytic activity of P450s (cytochrome P450 mono-oxygenases). In human, 11 P450 isoenzymes exhibiting broad and overlapping substrate specificities are responsible for approx. 90% of drug metabolism. As support for inevitable metabolism studies with intact organisms under relevant conditions, tobacco cell cultures were transformed separately with cDNA sequences of human P450 isoenzymes CYP1A1, CYP1A2 and CYP3A4. The resulting P450-transgenic cell suspensions were used for metabolism studies with pesticides, industrial pollutants, a secondary plant metabolite and human sex hormones. A summary of basic results is provided; these are discussed regarding application of the method for screening of the oxidative metabolism of xenobiotics and the large-scale production of metabolites.

Role of cytochromes P450 in xenobiotic metabolism

Commonly, xenobiotics are defined as organic compounds that are not synthesized by action of enzymes present in organisms and that are thus foreign to the biosphere. Xenobiotics comprise pesticides, pharmaceuticals and industrial pollutants; they arrive at organisms accidentally or due to intended usage. Since xenobiotics may exert adverse effects on living species, examination of their toxicity and metabolism prior to usage are today demanded by regulatory authorities of all countries. In case of pesticides for example, metabolism data are needed at early stages of development of candidates, because metabolites arising in organisms exposed to the compounds may exhibit an unacceptable (residual) toxicity. A nocuous metabolite of a pesticide candidate has significant influence on decision making in the further development of the chemical. The same holds true for pharmaceuticals and roughly for industrial pollutants. On the other hand, metabolism plays a crucial role in tolerance, resistance and susceptibility, e.g. with herbicides and insecticides, as well as in various phenomena observed among individuals in case of drugs and carcinogens. Regarding all aspects of xenobiotic metabolism, thorough chemical identification of metabolites is required. Several in vitro systems, including plant cell cultures, have been developed in order to generate rapidly a broad spectrum of metabolites for their identification [15]. These screening procedures considerably support inevitable studies conducted subsequently or in parallel with organisms under relevant conditions. In addition, databases were established, which enable the prediction of metabolites of pharmaceuticals and pesticides [6].

Xenobiotic metabolism in human, animals and higher plants is usually subdivided into three phases: transformation (phase I), conjugation (phase II) and excretion in human/animals or compartmentation in plants (phase III) [79]. Typical phase I reactions are oxidation, hydrolysis and reduction. The resulting primary metabolites are those that are of utmost importance e.g. in the evaluation of pesticide candidates due to their possible toxicological properties. In contrast, conjugates (phase II) are commonly considered as innocuous. The most relevant phase I processes are oxidative reactions leading to compounds that are less lipophilic than the parent chemicals. Often, these phase I products are subjected to conjugation, such as glycosylation. In human, animals and plants, P450s (cytochrome P450 mono-oxygenases) are mainly responsible for oxidative transformation of xenobiotics. Typical reactions are N-, O- and S-dealkylation, aromatic and aliphatic hydroxylation, epoxidation, oxidative desulfuration and sulfoxidation [1019]. Reactions catalysed by P450s are initial crucial steps leading to detoxification, inactivation and excretion; these reactions, however, can also result in intended or accidental activation. As extension of the in vitro systems mentioned, straightforward generation by similar systems of oxidized metabolites for chemical identification is thus desirable, not least because of difficulties occasionally arising during chemical synthesis of the compounds as references.

Cytochrome P450 isoenzymes and models for human and mammalian metabolism of xenobiotics

A number of P450 isoenzymes are responsible for the metabolism of xenobiotics in human and (laboratory) animals. In human, 11 were regarded as the most important species catalysing more than 90% of all P450 reactions observed with xenobiotics; these P450s are prevailingly expressed in the liver, but also in other organs [13,20,21]. The crucial human isoenzymes are CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C10, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. They exhibit broad and overlapping substrate specificities. Thus certain xenobiotic metabolites are produced by action of more than one of these isoenzymes; different kinetic parameters, however, are observed [14,15]. Though great similarities exist between human and mammals used as models (e.g. mouse, rat, rabbit, dog, monkey and pig), differences exist that are caused by expression levels and substrate specificities [21,22]. Subtle differences are additionally observed among human individuals e.g. due to genetic polymorphism [23]. On the whole, these findings render difficult detailed studies on the metabolism of pharmaceuticals and xenobiotics.

Besides animal models, data on catalytic activities of P450 isoenzymes towards xenobiotics are derived from studies using bacteria or yeasts heterologously expressing P450 genes [20,24]. Both expression systems show no or low endogenous P450 activity. In addition, microsomes containing selected human P450s are commercially available, which are produced by means of bacterial (Escherichia coli) or baculovirus expression systems [25]. As supplier of necessary electrons, all systems mentioned require co-expression of NADPH-P450 reductase to obtain catalytically active P450s.

These remarks may show that in vitro systems covering all details of the biotransformation of xenobiotics by P450 isoenzymes in human and mammals are not feasible. All the more, this holds for the P450-related metabolism in human, animals and plants as a whole. So, detection of involvement of genetic polymorphism or elucidation of insecticide resistance mechanisms requires sophisticated approaches other than in vitro systems for generation of principal oxidized metabolites.

Plant cell suspension cultures for metabolic profiling of xenobiotics

Heterotrophic plant cell suspension cultures were often utilized in order to obtain qualitative data on the metabolism of xenobiotics, especially of pesticides in plants [13,5]. Advantages are the lack of interfering photochemical and microbial transformation of the compounds. The absence of chlorophyll and other pigments facilitates extraction and identification of xenobiotic metabolites. Heterotrophically grown plant cells are not injured by moderate concentrations of most herbicides. Additionally, plant cell suspensions can be grown in scaled-up assays (up to 50 g fresh weight) or air-lift fermenters (∼500 g fresh weight) [5,26]. Plant cell cultures, most favourably of different species, are thus convenient in vitro systems for screening of metabolic profiles of xenobiotics in plants and for production of corresponding metabolites.

Nevertheless, though xenobiotic turnover in plant cell cultures is usually higher than in plants, large-scale production of pesticide metabolites by plant cells for thorough identification including (conventional) 1H-NMR is not possible with many compounds. This is mainly due to the fact that often phase I reactions, especially those catalysed by P450s, are delicate processes proceeding slowly. In contrast, glycosylation of xenobiotic substrates, such as suitable phase I metabolites, occurs rapidly. Corresponding P450s activities in plant cell cultures range mainly between low and moderate. As yet, their induction is poorly understood; information on substrate specificities is limited concerning xenobiotics [2733].

Expression of human and mammalian P450s in plant tissues

Regarding human and mammals, a considerable amount of data is available on the transformation of pharmaceuticals, xenobiotics and pesticides by P450 isoenzymes. In human, these belong to families CYP1–CYP4 [1317,20,21]; as already mentioned, most of these reactions are catalysed by 11 isoenzymes [13,20]. P450 species CYP1A1, CYP1A2 and CYP3A4 were estimated to metabolize approx. 1, 4 and 52% respectively of all drugs in human [13]. In the last 15 years, studies were published on the overexpression of human and mammalian P450 isoenzymes of families CYP1–CYP3 in higher plants, such as tobacco, potato or rice [20,29,3443]. The plants were transformed with a single (e.g. CYP1A1 or CYP2E1) or several P450s genes (e.g. CYP1A1, CYP2B6 and CYP2C19). Partially, plants were additionally transformed with NADPH-cytochrome P450 reductase, or P450 and reductase were expressed as fusion enzyme. Objectives of these investigations were the production of either herbicide-resistant plants (e.g. chlortoluron) or plants suitable for (phyto-) remediation of contaminated areas (e.g. halogenated hydrocarbons and herbicides). Due to broad substrate specificities of human and mammalian P450s, the transgenic plants exhibited considerable enhancement of metabolic activity towards several herbicides (multiple resistance) or a range of contaminants (trichloro- and dibromo-ethylene). Negative effects of the transgenes on plant growth were observed only in one study [34]. All experiments showed that co-expression of NADPH-cytochrome P450 reductase was not required, since the human or mammalian P450s co-operated sufficiently well with endogenous plant reductases.

Tobacco cell suspension cultures as expression systems for human P450 isoenzymes

The present approach combines the importance of P450s in xenobiotic metabolism, the broad substrate specificities of corresponding human P450s, the convenient plant cell culture in vitro system and the ease of expressing catalytically active P450s in plant tissues. It is intended as a method to screen rapidly and qualitatively the principal patterns of oxidized xenobiotic metabolites and to produce metabolites of concern on a larger scale for thorough chemical identification. As an illustration of this approach, the human P450s responsible for xenobiotic metabolism may be visualized as the Duke Ellington Orchestra. Such big bands play tunes that consist merely of melody line, chordal background and rhythm; these plain principal traits are embellished by arrangement to give a sound rich in details. If the same tunes are played by Chick Corea's Akoustic Band, a trio, its competent players focus on the principal traits of songs. The underlying questions of the present investigation were whether it is feasible to reduce the human P450 orchestra to a small ensemble in order to cover qualitatively the principal patterns of the oxidative metabolism of xenobiotics and which players are needed for this ‘oxidative metabolic profiling’ concept. To make the first move, tobacco cell suspension cultures were transformed with human CYP1A1, CYP1A2 and CYP3A4 and examined in xenobiotic metabolism studies.

Transformation of tobacco cell cultures with cDNA of human CYP1A1, CYP1A2 and CYP3A4

The cDNA sequences for human CYP1A1, CYP1A2 and CYP3A4 were obtained as bacterial vectors pCW1A1, pCW1A2 and pCW3′A4 respectively from Professor F.P. Guengerich (Vanderbilt University School of Medicine, Nashville, TN, U.S.A.). Construction of the plasmids was described including modification at the 5′-end (N-terminus) for improved expression in E. coli [24,44,45]. The P450 sequences were separately cloned into binary plant expression vector pSPAM (pPAM derivative; GenBank® accession no. AY027531) and put under constitutive control of the transcriptionally enhanced CaMV (cauliflower mosaic virus) promoter 35 SS; the resulting plasmids were spsA1, spsA2 and spRNY3A4 respectively [4648]. Each spsA1, spsA2 and spRNY3A4 were introduced into Agrobacterium tumefaciens. The transformed bacteria were subsequently used to transform directly tobacco cell suspension cultures (Nicotiana tabacum L.; PC-120; DSMZ, Braunschweig, Germany) by co-cultivation [4749]. After selection, the P450-transgenic cell suspension cultures Nt-CYP1A1, Nt-CYP1A2 and Nt-CYP3A4 (tobacco cell cultures expressing human CYP1A1, CYP1A2 and CYP3A4 respectively) were obtained (where Nt is Nicotiana tabacum). Successful transformation was confirmed by PCR, while expression of the P450s was verified using commercially available antibodies. The suspensions were subcultivated until constant growth parameters were achieved [3]. In case of Nt-CYP1A1 and Nt-CYP1A2, activity of the human P450s was determined by means of the ECOD (7-ethoxycoumarin O-de-ethylase) assay [47,48]. After plating cells on solidified medium, the same assay was used to select clones with high activity [47,48]. As yet, a similar examination of Nt-CYP3A4 was impossible, since all marker substrates tested proved to be inexpedient with the tobacco cells.

Oxidative metabolic profiling using CYP1A1-, CYP1A2- and CYP3A4-transgenic tobacco cell suspension cultures

After establishment, the P450-transgenic cell cultures Nt-CYP1A1, Nt-CYP1A2 and Nt-CYP3A4 were used to examine the metabolism of a number of xenobiotics; as reference, similar studies were performed with the Nt-NT (non-transformed tobacco culture). All studies were executed according to established procedures [3,5]. In basic experiments, application was 20–50 μg per assay (6–7 g fresh weight of cells and 25 ml of medium), and incubation at 27°C was for 24 or 48 h; corresponding results are shown in Table 1. Additional experiments were performed for extended periods (e.g. 96 h) and with up to 500 μg per assay.

Table 1
Metabolic turnover of xenobiotics by P450-transgenic tobacco cell suspensions

F, fungicide; H, herbicide; I, insecticide; PAH, polycyclic aromatic hydrocarbon; SH, sex hormone; SPM, secondary plant metabolite; XE, xenoestrogen; n.d., not determined. Values are turnover in percentage of applied amount.

Compound Application per assay (μg) Incubation interval (h) Nt-CYP1A1 (%) Nt-CYP1A2 (%) Nt-CYP3A4 (%) Nt-NT (%) 
Atrazine, H [4720 48 36.3 100.0 n.d. 20.2 
Carbaryl, I 20 24 48.8 99.8 27.5 21.8 
Caffeine, SPM 20 96 0.0 26.6 0.0 1.7 
Cyprodinil, F 20 24 99.8 93.5 n.d. 13.8 
DDT, I 20 48 <1.0 <1.0 <1.0 <1.0 
Diflubenzuron, I 20 48 25.8 89.8 24.8 15.6 
Dimethoate, I 20 48 12.3 30.5 n.d. 17.5 
Oestradiol, SH 20 48 n.d. n.d. 94.2 97.8 
Ethinyloestradiol, SH 20 48 n.d. n.d. 97.2 98.9 
Fluometuron, H 45 48 86.7 95.2 42.6 62.1 
Imidacloprid, I 20 24 n.d. 1.0 0.7 5.5 
Metamitron, H [4820 48 55.3 93.1 31.9 39.4 
Methoxychlor, I 50 24 62.3 95.7 89.9 64.7 
Nitrofen, H 20 48 22.5 20.9 n.d. 18.3 
 20 120 45.3 57.9 n.d. 37.4 
n-Nonylphenol, XE [5020 24 50.1 66.4 n.d. 43.5 
Pyrene, PAH 20 48 100 n.d. n.d. 82.3 
Compound Application per assay (μg) Incubation interval (h) Nt-CYP1A1 (%) Nt-CYP1A2 (%) Nt-CYP3A4 (%) Nt-NT (%) 
Atrazine, H [4720 48 36.3 100.0 n.d. 20.2 
Carbaryl, I 20 24 48.8 99.8 27.5 21.8 
Caffeine, SPM 20 96 0.0 26.6 0.0 1.7 
Cyprodinil, F 20 24 99.8 93.5 n.d. 13.8 
DDT, I 20 48 <1.0 <1.0 <1.0 <1.0 
Diflubenzuron, I 20 48 25.8 89.8 24.8 15.6 
Dimethoate, I 20 48 12.3 30.5 n.d. 17.5 
Oestradiol, SH 20 48 n.d. n.d. 94.2 97.8 
Ethinyloestradiol, SH 20 48 n.d. n.d. 97.2 98.9 
Fluometuron, H 45 48 86.7 95.2 42.6 62.1 
Imidacloprid, I 20 24 n.d. 1.0 0.7 5.5 
Metamitron, H [4820 48 55.3 93.1 31.9 39.4 
Methoxychlor, I 50 24 62.3 95.7 89.9 64.7 
Nitrofen, H 20 48 22.5 20.9 n.d. 18.3 
 20 120 45.3 57.9 n.d. 37.4 
n-Nonylphenol, XE [5020 24 50.1 66.4 n.d. 43.5 
Pyrene, PAH 20 48 100 n.d. n.d. 82.3 

In the basic studies, Nt-CYP1A1 demonstrated a noticeable turnover above that of Nt-NT with cyprodinil and fluometuron, while Nt-CYP1A2 was distinctly metabolically active towards atrazine, carbaryl, cyprodinil, diflubenzuron, fluometuron, metamitron, methoxychlor and n-nonylphenol. In contrast, the Nt-CYP3A4 cultures transformed only methoxychlor at a significantly higher rate than the Nt-NT cells. Large-scale production of metabolites was already achieved with atrazine and metamitron [4748], but is thought to be possible using Nt-CYP1A2 for several of the compounds examined. Some of the xenobiotics, including DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane], dimethoate and imidacloprid, proved to be stable with Nt-CYP1A1, Nt-CYP1A2 and Nt-CYP3A4. Data obtained for caffeine and nitrofen show that extended incubation periods may be favourable in order to obtain a distinct biotransformation. With most of the xenobiotics studied, identification of metabolites produced in the transgenic cells was possible [TLC, HPLC, GLC–MS and LC–MS (liquid chromatography–MS)]. Partially, primary oxidized metabolized were found as glycosides, which were cleaved enzymatically or chemically prior to analysis.

Concluding remarks

The present approach utilizing P450-transgenic tobacco cell suspension cultures is considered valuable for ‘oxidative metabolic profiling’ of xenobiotics. As compared with commercially available microsomes, advantages are extended incubation periods and scale-up of assays. It remains to be investigated which P450 isoenzymes are useful to cover principal oxidation reactions in human, but also in animals and plants. Thus the present data derived from the CYP3A4-transgenic tobacco culture can be explained only with difficulty, in the light of reports that this isoenzyme catalyses approx. 50% of the oxidative metabolism of drugs. We speculated that either unfavourable substrate specificity, inhibitors of plant origin, or homotropic co-operativity was responsible for rather unexpected results. Increasing concentrations generally resulted in increased transformation rates of xenobiotics, e.g. carbaryl, but did not point definitely to homotropic co-operativity.

8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology: Independent Meeting held at Swansea Medical School, Swansea, Wales, U.K., 23–27 July 2006. Organized and Edited by D. Kelly, D. Lamb and S. Kelly (Swansea, U.K.).

Abbreviations

     
  • DDT

    1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane

  •  
  • Nt-NT

    non-transformed tobacco culture

  •  
  • P450

    cytochrome P450 mono-oxygenase

References

References
1
Swisher
B.A.
LeBaron
H.M.
Mumma
R.O.
Honeycutt
R.C.
Duesing
J.J.
Biotechnology and Agricultural Chemistry
1987
Washington, DC
American Chemical Society
(pg. 
18
-
40
)
2
Komossa
D.
Langebartels
C.
Sandermann
H.
Trapp
S.
McFarlane
J.C.
Plant Contamination: Modelling and Simulation of Organic Chemical Processes
1995
Boca Raton, FL
CRC Press
(pg. 
69
-
103
)
3
Schmidt
B.
Hall
J.C.
Hoagland
R.E.
Zablotowicz
R.E.
Pesticide Biotransformation in Plants and Microorganisms: Similarities and Divergences
2001
Washington, DC
American Chemical Society
(pg. 
40
-
56
)
4
Schocken
M.J.
Hall
J.C.
Hoagland
R.E.
Zablotowicz
R.E.
Pesticide Biotransformation in Plants and Microorganisms: Similarities and Divergences
1991
Washington, DC
American Chemical Society
(pg. 
30
-
39
)
5
Schmidt
B.
AGCHEMFORUM: Metabolism of Agrochemicals in Plants and Animals – Recent Developments and Experimental Approaches
2002
London
IBC Life Sciences
6
Lee
P.
AGCHEMFORUM: Metabolism of Agrochemicals in Plants and Animals – Recent Developments and Experimental Approaches
2002
London
IBC Life Sciences
7
Bounds
S.V.J.
Hudson
D.H.
Roberts
T.
Metabolism of Agrochemicals in Plants
2000
Chichester, U.K.
Wiley
(pg. 
179
-
209
)
8
Hatzios
K.K.
Hatzios
K.K.
Regulation of Enzyme Systems Detoxifying Xenobiotics in Plants
1996
Dordrecht
Kluwer
(pg. 
1
-
5
)
9
Ioannides
C.
Ioannides
C.
Enzyme Systems that Metabolize Drugs and Other Xenobiotics
2002
Chichester, U.K.
Wiley
(pg. 
1
-
32
)
10
Schuler
M.A.
Crit. Rev. Plant Sci.
1996
, vol. 
15
 (pg. 
235
-
284
)
11
Frear
D.S.
Drug Metab Drug Interact.
1995
, vol. 
12
 (pg. 
329
-
357
)
12
Barrett
M.
Drug Metab Drug Interact.
1995
, vol. 
12
 (pg. 
299
-
315
)
13
Anzenbacher
P.
Anzenbacherová
E.
Cell Mol. Life Sci.
2001
, vol. 
58
 (pg. 
737
-
747
)
14
Lewis
D.F.V.
Cytochromes P450: Structure
1996
London
Function and Mechanism, Taylor & Francis
15
Rendic
S.
Drug Metab Rev.
2002
, vol. 
34
 (pg. 
83
-
448
)
16
Hodgson
E.
J. Biochem. Mol. Toxicol.
2001
, vol. 
15
 (pg. 
296
-
299
)
17
Hodgson
E.
J. Biochem. Mol. Toxicol.
2003
, vol. 
17
 (pg. 
201
-
206
)
18
Bollwell
G.P.
Bozak
K.
Zimmerlin
A.
Phytochemistry
1994
, vol. 
37
 (pg. 
1491
-
1506
)
19
Mansuy
D.
Comp. Biochem. Physiol. C
1998
, vol. 
121
 (pg. 
5
-
14
)
20
Inui
H.
Shiota
N.
Motoi
Y.
Ido
Y.
Inoue
T.
Kodama
T.
Ohkawa
Y.
Ohkawa
H.
J. Pestic. Sci.
2001
, vol. 
26
 (pg. 
28
-
40
)
21
Zuber
R.
Anzenbacherová
E.
Anzenbacher
P.
J. Cell. Mol. Med.
2002
, vol. 
6
 (pg. 
189
-
198
)
22
Guengerich
F.P.
Chem.-Biol Interact.
1997
, vol. 
106
 (pg. 
161
-
182
)
23
Daly
A.K.
J. Mol. Med.
1995
, vol. 
73
 (pg. 
539
-
553
)
24
Parikh
A.
Gillam
E.M.J.
Guengerich
F.P.
Nat. Biotechnol.
1997
, vol. 
15
 (pg. 
784
-
788
)
25
Gonzales
F.J.
Korzekwa
K.R.
Annu. Rev. Pharmacol. Toxicol.
1995
, vol. 
35
 (pg. 
369
-
390
)
26
Knops
M.
Schuphan
I.
Schmidt
B.
Plant Sci.
1995
, vol. 
109
 (pg. 
215
-
224
)
27
Durst
F.
Benveniste
I.
Lesot
A.
Salaün
J.P.
Werck-Reichhart
D.
Hatzios
K.K.
Regulation of Enzymatic Systems Detoxifying Xenobiotics in Plants
1997
Dordrecht
Kluwer
(pg. 
19
-
34
)
28
Werck-Reichhart
D.
Hahn
A.
Didierjean
L.
Trends Plant Sci.
2000
, vol. 
5
 (pg. 
116
-
123
)
29
Ohkawa
H.
Tsujii
H.
Ohkawa
Y.
Pestic Sci.
1999
, vol. 
55
 (pg. 
857
-
854
)
30
Robineau
T.
Batard
Y.
Nedelkina
S.
Cabello-Hurtado
F.
LeRet
M.
Sorokine
O.
Didierjean
L.
Werck-Reichhart
D.
Plant Physiol.
1998
, vol. 
118
 (pg. 
1049
-
1056
)
31
Benveniste
I.
Bronner
R.
Wang
Y.
Compagnon
V.
Michler
P.
Schreiber
L.
Salaün
J.P.
Durst
F.
Pinot
F.
Planta
2005
, vol. 
221
 (pg. 
881
-
890
)
32
Didierjean
L.
Gondet
L.
Perkins
R.
Lau
S.-M.C.
Schaller
H.
O'Keefe
D.P.
Werck-Reichhart
D.
Plant Physiol.
2002
, vol. 
130
 (pg. 
179
-
189
)
33
Siminszky
B.
Corbin
F.T.
Ward
E.R.
Fleischmann
T.J.
Dewey
R.E.
Proc. Natl. Acad. Sci. U.S.A.
1999
, vol. 
95
 (pg. 
1750
-
1755
)
34
Saito
K.
Noji
M.
Ohmori
S.
Imai
Y.
Murakoshi
I.
Proc. Natl. Acad. Sci. U.S.A.
1991
, vol. 
88
 (pg. 
7041
-
7045
)
35
Shiota
N.
Nagasawa
A.
Sakaki
T.
Yabusaki
Y.
Ohkawa
H.
Plant Physiol.
1994
, vol. 
106
 (pg. 
17
-
23
)
36
Inui
H.
Shiota
N.
Ishige
T.
Ohkawa
Y.
Ohkawa
H.
Breed Sci.
1998
, vol. 
48
 (pg. 
135
-
143
)
37
Inui
H.
Ueyama
Y.
Shiota
N.
Ohkawa
Y.
Ohkawa
H.
Pestic Biochem. Physiol.
1999
, vol. 
64
 (pg. 
33
-
46
)
38
Inui
H.
Kodama
T.
Ohkawa
Y.
Ohkawa
H.
Pestic Biochem. Physiol.
2000
, vol. 
66
 (pg. 
116
-
129
)
39
Shiota
N.
Kodama
T.
Ohkawa
Y.
Ohkawa
H.
Biosci. Biotechnol. Biochem.
2000
, vol. 
64
 (pg. 
2025
-
2033
)
40
Doty
S.L.
Shang
T.Q.
Wilson
A.M.
Tangen
J.
Westergreen
A.D.
Newman
L.A.
Strand
S.E.
Gordon
M.P.
Proc. Natl. Acad. Sci. U.S.A.
2000
, vol. 
97
 (pg. 
6287
-
6291
)
41
Inui
H.
Shiota
N.
Ido
Y.
Inoue
T.
Hirose
S.
Kawahigashi
H.
Ohkawa
Y.
Ohkawa
H.
Pestic Biochem. Physiol.
2001
, vol. 
71
 (pg. 
156
-
169
)
42
Yamada
T.
Ishige
T.
Shiota
N.
Inui
H.
Ohkawa
H.
Ohkawa
Y.
Theor. Appl. Genet.
2002
, vol. 
105
 (pg. 
515
-
520
)
43
Hirose
S.
Kawahigashi
H.
Ozawa
K.
Shiota
N.
Inui
H.
Ohkawa
H.
Ohkawa
Y.J.
Agric. Food Chem.
2005
, vol. 
53
 (pg. 
3461
-
3467
)
44
Guengerich
F.P.
Martin
M.V.
Guo
Z.
Chun
Y.J.
Methods Enzymol.
1996
, vol. 
272
 (pg. 
35
-
41
)
45
Barnes
H.J.
Methods Enzymol.
1996
, vol. 
272
 (pg. 
3
-
14
)
46
Rademacher
T.
Häusler
R.
Hirsch
H.J.
Zhang
L.
Lipka
V.
Weier
D.
Kreuzaler
F.
Peterhänsel
C.
Plant J.
2002
, vol. 
32
 (pg. 
25
-
39
)
47
Bode
M.
Stöbe
P.
Thiede
B.
Schuphan
I.
Schmidt
B.
Pest Manag Sci.
2004
, vol. 
60
 (pg. 
49
-
58
)
48
Bode
M.
Haas
M.
Faymonville
M.
Thiede
B.
Schuphan
I.
Schmidt
B.
J. Environ. Sci. Health B
2006
, vol. 
41
 (pg. 
201
-
222
)
49
An
G.
Plant Physiol.
1985
, vol. 
79
 (pg. 
568
-
570
)
50
Berger
A.
Russ
A.S.
Schuphan
I.
Schmidt
B.
Z. Naturforsch. [C]
2006
, vol. 
60
 (pg. 
883
-
892
)