Metabolism of the Nonylphenol Isomer [Ring-U-¹⁴c]-4-(3′,5′-Dimethyl-3′-Heptyl)-Phenol by Cell Suspension Cultures of Agrostemma githago and Soybean

Abstract

The biotransformation of the nonylphenol isomer [ring-U-¹⁴C]-4-(3′,5′-dimethyl-3′-heptyl)-phenol (4-353-NP, consisting of two diastereomers) was studied in soybean and Agrostemma githago cell suspension cultures. With the A. githago cells, a batch two-liquid-phase system (medium/n-hexadecane 200:1, v/v) was used, in order to produce higher concentrations and amounts of 4-353-NP metabolites for their identification; 4-353-NP was applied via the n-hexadecane phase. Initial concentrations of [¹⁴C]-4-353-NP were 1 mg L⁻¹ (soybean), and 5 and 10 mg L⁻¹ (A. githago). After 2 (soybean) and 7 days (A. githago) of incubation, the applied 4-353-NP was transformed almost completely by both plant species to four types of products: glycosides of parent 4-353-NP, glycosides of primary 4-353-NP metabolites, nonextractable residues and unknown, possibly polymeric materials detected in the media. The latter two products emerged especially in soybean cultures. Portions of primary metabolites amounted to 19–22% (soybean) and 21–42% of applied ¹⁴C (A. githago). After liberation from their glycosides, the primary 4-353-NP metabolites formed by A. githago were isolated by HPLC and examined by GC-EIMS as trimethylsilyl derivatives. In the chromatograms, eight peaks were detected which due to their mass spectra, could be traced back to 4-353-NP. Seven of the compounds were side-chain monohydroxylated 4-353-NP metabolites, while the remaining was a (side-chain) carboxylic acid derivative. Unequivocal identification of the sites of hydroxylation/oxidation of all transformation products was not possible. The main primary metabolites produced by A. githago were supposed to be four diastereomers of 6′-hydroxy-4-353-NP (about 80% of all products identified). It was concluded that plants contribute to the environmental degradation of the xenoestrogen nonylphenol; the toxicological properties of side-chain hydroxylated nonylphenols remain to be examined.

Keywords:

• Plant cell culture
• Agrostemma githago
• Soybean
• Metabolism
• Nonylphenol isomer; 4-(3′,5′-Dimethyl-3′-heptyl)-phenol
• Two-liquid-phase system
• Metabolic profiling
• Hydroxylated nonylphenol metabolites

Acknowledgments

For the synthesis of [ring-U-¹⁴C]-4(3′,5′-dimethyl-3′-heptyl)-phenol, the authors wish to thank R. Vinken and the “Aachen Graduate College: Elimination of Endocrine-Disrupting Substances from Waste-Water” (AGEESA) funded by the Deutsche Forschungsgemeinschaft.