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Abstract No.: ThP-269
Session: Toxicology
Presentation date: Thu, Aug 31, 2006
Presentation time: 09:50 – 11:20

In Vitro Metabolism and Interaction of Metalaxyl by Human Liver Microsomes and Homogenates

Petri Reponen1,2, Khaled Abass1,3, Jorma Jalonen2, Olavi Pelkonen1

1 Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland
2 Structural Chemistry, Department of Chemistry, University of Oulu, Oulu, Finland
3 Pesticides Department, Menoufia University, Menoufia, Egypt

Correspondence address: Petri Reponen, University of Oulu, Department of Pharmacology and Toxicology, PL 5000, Oulun yliopisto, 90014 Finland.

Keywords: Environmental; Metabolism, Metabolites; Pesticides; Toxicology.

Novel aspect: Metabolism of pesticide with human liver microsomes and homogenates.

 

Metalaxyl [N-(2,6-dimethylphenyl)-N-(methoxyacetyl)alanine methyl ester] is a systemic fungicide in the phenylamide:acylalanine group. Its principal mode of action is to inhibit protein synthesis in fungi by interference with the synthesis of ribosomal RNA. It is presently used on a wide range of crops in several countries for controlling diseases caused by air and soil born agents.

The main objectives of the present study were to 1) elucidate the effects of metalaxyl on different human liver cytochrome P450 (CYP) enzymes, 2) identify and (semi)quantify the potential metalaxyl metabolites in vitro and 3) identify the human CYPs responsible for metalaxyl metabolism.

Studies were performed with pooled human liver microsomes and homogenates, and with cDNA-expressed human P450 enzymes. Inhibition assays were analyzed either by fluorometry or by UV-HPLC with help of common CYP-selective substrates. Metalaxyl and its metabolites were tentatively identified and analyzed by LC-MS (TOF, Micromass LCT). The exact masses of the metabolites were measured.

The IC50 values of metalaxyl for 7-pentoxyresorufin-O-dealkylation (CYP2B) and bupropion hydroxylation (2B6) were 48.9 and 41.7 ÁM, respectively. The IC50 values for all the other model reactions were higher than 100 ÁM.

The principal biotransformation reactions in human liver microsomes and homogenates were hydroxylation, demethylation, second demethylation and lactone formation. In human liver microsomes predominant metabolites were presumably two hydroxylated derivatives. On the basis of previous rat studies these could be N-(2-hydroxymethyl-6-methylphenyl)-N-(methoxyacetyl)alanine methyl ester and/or N-(2,6-dimethyl-5-hydroxyphenyl)-N-(methoxyacetyl)alanine methyl ester. In homogenates, the second demethylation metabolite N-(2,6-dimethylphenyl)-N-(hydroxyacetyl)alanine and the lactone 4-(2,6-dimethylphenyl)-3-methylmorpholine-2,5-dione were predominant. Moderate amounts of the demethylation metabolite were also found. It was either N-(2,6-dimethylphenyl)-N-(methoxyacetyl)alanine or N-(2,6-dimethyphenyl)-N-(hydroxyacetyl)alanine methyl ester. The carcinogenic 2,6-dimethylaniline was not detected as a metabolite in human liver microsomes or homogenates.

Among the 9 major human CYP enzymes, CYP3A4 was the only isoform responsible for metalaxyl hydroxylation, while CYP2B6 was the major isoform responsible for demethylation, second demethylation and lactone formation.