Metabolismus von 14 C-Methoxychlor in humanen P450- transgenen und nicht-transgenen Pflanzenzellkulturen zur Produktion endokrin-wirksamer Metaboliten sowie deren Untersuchung auf östrogene Wirkung

Using the estrogenic organochlor insecticide 14C-methoxychlor, the utility of (P450) transgenic plant cell suspension cultures for the determination of the biotransformation of environmental pollutants was investigated in the present study. Research focus was on turnover rates, metabolic profiles and endocrine activity of MXC, including its main metabolites. Analysis was performed using radio-TLC, radio-HPLC and a bioassay (YES). Soluble metabolites were identified and quantified via co-chromatographie. Since MXC is a prochiral and proestrogenic compound, R- and S-enantiomers of corresponding metabolites were separated and examined individually. A carrot cell suspension culture (Dc) was established and transformed separately with cDNA of human Cyp3a4 (3DcA4) and Cyp2c19 (2DcC19) via Agrobacterium tumefaciens. Expression of the P450 genes was succesfully proven by PCR and detection of the corresponding mRNA. The growth of all new cell cultures was monitored by fresh weight, dry weight, conductivity and pH of the media. In addition to the carrot cultures, metabolism studies were performed with soybean (Gm), wheat (Ta) and tobacco cultures (Nt). The latter were also transformed with human P450 isoforms (1NtA1, 1NtA2, 3NtA4, 2NtC19, 2NtB6). The triple demethoxylated derivative of MXC was synthesized as reference substance and its chemical structure was proven by GC-(MS) and 1H-NMR. Metabolism studies showed that all cell cultures had the endogenous enzymatic activity to metabolize MXC. Turnover rates were 87,3% for carrot, 71,3% for tobacco, 26,1% for soybean and 41,7% for wheat (TLC after enzymatic hydrolysis of glycoside conjugates). Regarding metabolic patterns, the non-transformed cultures differed among each other. Whereas mono-OH-, bis-OH- and tris-OH-MXC were found in carrot cultures (18,8%, 9,2% and 4,6%, respectively), only bis-OH-and tris-OH-MXC were detected in tobacco cultures (10,3% and 13,8%, respectively). Amounts of mono-OH-and tris-OH-MXC were nearly the same in soybean and wheat cultures (4,8%, 5,4% mono-OH-MXC and 5,9%, 5,5% tris-OH-MXC, respectively). Whereas the results for Cyp3a4-transgenic cultures remained inconsistent, turnover for the CYP2C19 culture was 10% lower than in the non-transformed culture. Within 24 hours, only 7,7% MXC remained non-metabolized in 2NtB6 cultures. 40,1% of the metabolites formed remained structurally not identified, ca. 20% were identified as mono-OH-MXC, ca. 8% as bis-OH-MXC and ca. 3,5% as tris-OH-MXC. Metabolism studies with Nt, 1NtA1, 1NtA2 and 3NtA4 cultures showed that between 24 h and 72 h of incubation, the amounts of MXC decreased to one ninth to one sixteenth. Furthermore, a decrease in the amount of mono-OH-MXC to one third to one fivth was observed. Excepting the 3NtA4 cultures, amounts of tris-OH-MXC increased in all cultures. Whereas within 24 hours of incubation, tris-OH-MXC emerged in none of the cultures, it was found in all cultures after 72 hours ranging between 10,2% (1NtA1) and 19,9% (1NtA2). Comparison of the turnover of MXC in CYP2C19 cultures of carrot and tobacco revealed a higher metabolic activity of the transgenic carrot culture (+ 16%). Main metabolites were similiar to the non-transformed carrot culture. The higher portion of bis-OH-MXC (+ 6,5%) formed was probably due to the activity of the human enzym. In summary, all cell cultures tested were applicable tools to demonstrate metabolic pathways of MXC and in particular, the P450 isoform specifity. The separation of the R- and S-enantiomers was accomplished by chirale HPLC (CHIRALPAK®AD). The differences in the retention time were 6.8 min for mono-OH-MXC and 13.9 min for tris-OH-MXC. The optical rotation of S-mono-OH-MXC was [alpha] 26589 = +24,1 (c = 1,0, methanol); that of R-mono-OH-MXC [alpha] 26589 = -24,1 (c = 1,0, methanol). The enantioselectivity of the P450 isoforms was demonstrated by the samples of CYP3A4 and CYP2C19 cultures. Via HPLC (NUCLEODEX-gamma-PM), a distinctive separation of the enantiomers of mono-OH-MXC, derived from the 3DcA4 culture, was shown. This suggested the presence of both enantiomers. However, for the Dc and 2DcC19 culture only one signal was detected. According to the results of the 3DcA4 culture, the signal from the 2DcC19 culture was identified as S-mono-OH-MXC. Due to elution with higher retention time, the signal of the Dc culture was identified as R-mono-OH-MXC. With the YES assay, mono-OH-XC as well as bis-OH-MXC and tris-OH-MXC were found to be estrogenic in a concentration-responsive manner. The potency measured (relative to 17ß-estradiol, 10^-6-10^-8 g/L) was 10^-5-10^-4 g/L for mono-OH-MXC, 10^-4-10^-3 g/L for bis-OH-MXC and 10^-3-10^-2 g/L for tris-OH-MXC. These results were supported by the calculated EC50 values of 6,64 x 10^-5 g/L for mono-OH-MXC and 1,2 x 10^-5 g/L for bis-OH-MXC. In contrast to the racemate, the potency of mono-OH-MXC´s enantiomers was about tenfold higher. R-mono-OH-MXC showed a tenfold higher estrogenic activity than S-mono-OH-MXC.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2009

Author dc:creator

• Breuer, Maren Anne

Contributors dc:contributor

• Schuphan, Ingolf

• info:eu-repo/classification/ddc/570
• Insektizid
• Endokrin wirksamer Stoff
• Cytochrom P-450
• Enantiomere
• Zellkultur
• Biowissenschaften, Biologie
• Methoxychlor
• CYP3A4
• CYP2C19
• Pflanzenzellsuspensionskultur
• plant cell suspension culture

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113785%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A51497