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# Monooxygenases responsible for the metabolism of chlorotoluron in wheat microsomes

Christian Mougin, René Scalla, Ferdinand Cabanne

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Christian Mougin, René Scalla, Ferdinand Cabanne. Monooxygenases responsible for the metabolism of chlorotoluron in wheat microsomes. Seventh International Congress of Pesticide Chemistry IUPAC, Aug 1990, Hambourg, Germany. 1990. [hal-01600554](https://hal.science/hal-01600554)

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International Union of Pure
and Applied Chemistry (IUPAC)

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Seventh International Congress of Pesticide Chemistry

August 5 – 10, 1990

Hamburg, Fed. Rep. of Germany

Dr. C. Mougin
I.N.R.A.
Laboratoire des Herbicides
BV 1540

F-21034 Dijon

Scientific Committee

Dr. H. Frehse, Chairman
Bayer AG
Geb. 6610 (Monheim)
D-5090 Leverkusen,
Bayerwerk
Fed. Rep. of Germany
Phone: (49)-2173-383761
Telex 85 103-275 by d

Fax: (49)-2173-383780
May 23, 1990
Dr. Fre/ks
IU - 2199
dks0229

Your Poster "MONOOXYGENASES RESPONSIBLE FOR THE METABOLISM ..."

Serial No. 913, Subtopic/Poster Code 06E-13

Dear Poster Author,

Your Poster-Abstract was received and registered, at GDCh, under the Serial No. given. The Abstract was reviewed and accepted by the Scientific Committee and assigned to the Subtopic denoted above. Your Poster will be identified in the Abstracts Book and at the Congress (Poster boards) under the same alphanumeric Poster Code. You are requested to mount/demount, and be present at your Poster at the time indicated in the attached Timetable.

Further Correspondence and Information

All further correspondence and information concerning this Poster will be sent to you only, as the corresponding author. It is your obligation to inform your co-author(s) accordingly.
Mougin, C.; Scalla, R.; and Cabanne, F.
Laboratoire des herbicides, I.N.R.A., BV 1540, 21034 Dijon, France

MONOOXYGENASES RESPONSIBLES FOR THE METABOLISM OF CHLOROTOLURON IN WHEAT MICROSOMES

Microsomes isolated from achlorophyllous cells of wheat (Triticum aestivum L. var. Koga II) metabolized the herbicide chlorotoluron by two enzymatic reactions, namely a ring-methyl hydroxylation and a *N*-demethylation. These activities, and also lauric acid in-chain hydroxylation and concentrations of cytochromes P-450, were increased following cell treatment with cyometrinil or 2,4-D. The last compound was also an inducer of cinnamic acid 4-hydroxylase.

Cofactor requirements and susceptibility to inhibitors of the reactions lead to the assumption that chlorotoluron ring-methyl hydroxylase and *N*-demethylase probably belong to the family of cytochrome P-450 monooxygenases. However, the properties of the *N*-demethylase appeared to be somewhat different from those of ring-methyl hydroxylase. *N*-demethylation was not sensitive to CO and could be supported by cumene hydroperoxide acting as oxygen donor.

Other extraction conditions and buffer compositions, as well as incubations of microsomes with chlorotoluron in the presence of inhibitors of peroxydases, peroxygenases, flavin-containing monooxygenases and cytochromes P-450 confirmed the involvement of cytochrome P-450(s) in the oxidative metabolism of chlorotoluron. However, no inhibition of ring-methyl hydroxylation and *N*-demethylation could be obtained with polyclonal antibodies prepared against NADPH cytochrome P-450 reductase purified from Jerusalem artichoke.

Substrate specificity of enzymes oxidizing chlorotoluron was investigated. Wheat cell microsomes were not able to metabolize diclofop, which has been shown to be hydroxylated in wheat plants by a P-450 monooxygenase, nor to transform diuron, a structural analog of chlorotoluron.

Until now, cell treatments with classical inducers of P-450(s) systems such as phenobarbital and aminopyrine did not lead to any specific induction of chlorotoluron metabolism. Microsomes from wheat seedlings exhibited only basal rates of ring-methyl hydroxylation and *N*-demethylation without any detectable effects of inducers.

Cellular localisation of activities showed that they belong to the endoplasmic reticulum. Improvement of specific activities is in progress through enrichment of the endoplasmic reticulum in the microsomal fraction.
Mougin, C.; Scalla, R.; and Cabanne, F.

Laboratoire des herbicides, I.N.R.A., BV 1540, 21034 Dijon, France

## MONOOXYGENASES RESPONSIBLES FOR THE METABOLISM OF CHLOROTOLURON IN WHEAT MICROSOMES

Microsomes isolated from achlorophyllous cells of wheat (Triticum aestivum L. var. Koga II) metabolized the herbicide chlorotoluron by two enzymatic reactions, namely a ring-methyl hydroxylation and a *N*-demethylation. These activities, and also lauric acid in-chain hydroxylation and concentrations of cytochromes P-450, were increased following cell treatment with cyometrinil or 2,4-D. The last compound was also an inducer of cinnamic acid 4-hydroxylase.

Cofactor requirements and susceptibility to inhibitors of the reactions lead to the assumption that chlorotoluron ring-methyl hydroxylase and *N*-demethylase probably belong to the family of cytochrome P-450 monooxygenases. However, the properties of the *N*-demethylase appeared to be somewhat different from those of ring-methyl hydroxylase. *N*-demethylation was not sensitive to CO and could be supported by cumene hydroperoxide acting as oxygen donor.

Other extraction conditions and buffer compositions, as well as incubations of microsomes with chlorotoluron in the presence of inhibitors of peroxydases, peroxygenases, flavin-containing monooxygenases and cytochromes P-450 confirmed the involvement of cytochrome P-450(s) in the oxidative metabolism of chlorotoluron. However, no inhibition of ring-methyl hydroxylation and *N*-demethylation could be obtained with polyclonal antibodies prepared against NADPH cytochrome P-450 reductase purified from Jerusalem artichoke.

Substrate specificity of enzymes oxidizing chlorotoluron was investigated. Wheat cell microsomes were not able to metabolize diclofop, which has been shown to be hydroxylated in wheat plants by a P-450 monooxygenase, nor to transform diuron, a structural analog of chlorotoluron.

Until now, cell treatments with classical inducers of P-450(s) systems such as phenobarbital and aminopyrine did not lead to any specific induction of chlorotoluron metabolism. Microsomes from wheat seedlings exhibited only basal rates of ring-methyl hydroxylation and *N*-demethylation without any detectable effects of inducers.

Cellular localisation of activities showed that they belong to the endoplasmic reticulum. Improvement of specific activities is in progress through enrichment of the endoplasmic reticulum in the microsomal fraction.