Metabolism of nonylphenol by human P450-recombinant yeast and assessment of the xeno-hormone potency of different isomers and their chlorinated derivates

The purpose of this study was to obtain a better understanding of the behaviour, the characteristics, the metabolism and the metabolites or derivatives of 4-nonylphenol isomers (4NP) regarding the risk of exposure for human health to these compounds. The development of efficient molecular tools, making it possible to analyze individual bioconversion steps by well-characterized enzymes, such as cytochrome P450 monooxygenases, appeared critical for valuable toxicological predictions. The first aim of the present work was to establish a recombinant yeast cell culture, able to express the human proteins CYP2C19 and CYP2B6 (cytochromes P450) for model degradation studies. Two different systems were created: System 1: the YR and the cytochrome P450 monooxyigenase were inserted in one plasmid under the control of a strong galactose-inducible promoter. The vector was used to transform the yeast and in this way the two genes would be over-expressed in the yeast cells cultures pT2b6 (expressing Cyp2b6) and pU2c19 (expressing Cyp2c19). System 2: the YR was regulated at a genomic level in the yeast under the control of a strong galactose-inducible promoter (system obtained from Dr. Pompon, France). The cytochrome P450 monooxygenase was inserted in a vector and this construct was used to transform the yeast. In this way only the cytochrome P450 monooxygenase was over-expressed in the yeast cells cultures PY2b6 (expressing Cyp2b6) and PY2c19 (expressing Cyp2c19). The transgenic yeast cell cultures were necessary in order to perform metabolism studies with 4-NP isomer. To verify protein expression and activity, model metabolism experiments were executed with the substrates simazine (herbicide) and methoxychlor (insecticide), for which information regarding transformation by human CYP2C19 and CYP2B6 were published in the literature using yeast system 2. The yeast cells transformed with Cyp2c19 showed the ability of metabolize simazine to a more polar compound, which according to the HPLC retention time was supposed to be hydroxy-simazine. The yeast cell culture transformed with Cyp2c6 showed the ability to metabolize methoxychlor to the more polar compound catechol-methoxychlor according to HPLC data. This system was then used to perform metabolism studies with a branched isomer of nonylphenol, p353-NP (4-(3’,5’-dimethyl-3’heptyl)-phenol). The yeast cells culture PY2c19 was not capable to metabolize this branched isomer, while PY2b6 was. HPLC analysis showed the presence of two peaks, one was identified by GC-EIMS analysis as a side chain hydroxylated metabolite, while the other HPLC peak could not be identified. The system 1 (pU2c19 and pT2b6 cells cultures) was used to perform the same metabolism studies, but in the recombinant cell cultures were not capable to metabolize applied simazine and nonylphenol. In vivo metabolism studies using recombinant yeast transformed with human P450s were proven to be fast, reliable and reproducible, and were therefore suitable for screening the metabolism of different xenobiotics. NPs were detected in drinking water and wastewater, therefore they are subjected to all disinfection processes during the preparation of drinking water and during processes in wastewater treatment plants. The disinfection procedure often includes the use of sodium hypochlorite; during this process many by-products can be produced including chlorinated derivatives of NPs. Thus, the second aim of the present work was, to assess the xeno-hormone potency of different pure isomers of NPs and their mono-chlorinated and di-chlorinated derivatives. I reported the defined mono- and di-chlorination of four discrete NP isomers and the evaluation of their relative estrogenic and androgenic potency using a yeast screen. p353-NP (4-(3’,5’-dimethyl-3’heptyl)-phenol) and p4n-MClNP (2-chlor-4-nonyl-phenol) isomers showed the highest and the lowest estrogenic potency, respectively among all compounds examined. The p363-MClNP (2-chlor-4-(3’,6’-dimethyl-3’-heptyl)-phenol ) estrogenic potency comparable to the parent non-chlorinated isomer; all the dichlorinated compounds (p-DClNP) showed a considerably lower estrogenic potency than the parent compounds. All substances exhibited a positive response in the anti-androgenic screen. The isomer p363-NP (4-4-(3’,6’-dimethyl-3’heptyl)-phenol and its corresponding mono and di-chlorinated derivative were almost inactive; the other compounds showed positive response, but the mono- and di-chlorinated derivatives exhibit lower anti-androgenic potency. My results indicated that for better understanding of the xero-hormone potency of chlorinated NP isomers the use of pure compounds is mandatory. In fact, the environmental concentrations of the different NP isomers can differ from the composition of the technical mixtures themselves (e.g. due to degradation and adsorption processes), leading to an incorrect xero-hormone potency assessment of their corresponding derivatives.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2008

Author dc:creator

• Cormio, Paola Giulia

Contributors dc:contributor

• Schuphan, Ingolf

• info:eu-repo/classification/ddc/540
• Nonylphenol
• Cytochrom P-450
• Endokrin wirksamer Stoff
• Hefeartige Pilze
• Chemie
• cytochrome P450
• yeast
• endocrine disruptor

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-208056%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A50281