Metabolism of 4-n-Nonylphenol by Non-modified and CYP1A1- and CYP1A2-Transgenic Cell Cultures of Tobacco

Abstract

The metabolism of 14 C-4-n-nonylphenol ( 14 C-4-n-NP), as a model for the xenoestrogen nonylphenol, was investigated in three types of tobacco cell suspension cultures: one genetically non-modified culture (NT) and two cultures constitutively expressing human cytochrome P450 CYP1A1 or CYP1A2. With 1 mg l Ð1 of 14 C-4-n-NP and 24 h of incubation, the xenobiotic was transformed almost completely to glycosides. After glycosidic cleavage, 14 C-4-n-NP and several primary metabolites of 14 C-4-n-NP were liberated. Portions of the primary metabolites were 29.3% (NT culture), 34.3% (CYP1A1 culture), and 50.7% of applied 14 C (CYP1A2 culture). Thus, the endogenous capacity of the tobacco cells to form primary metabolites of 4-n-NP was noticeably higher than that of CYP1A1 or CYP1A2. The results however clearly suggest that 4-n-NP is Ð even though a poor Ð substrate of CYP1A1 and CYP1A2. In order to examine metabolic profiles of 4-n-NP in the NT, CYP1A1 and CYP1A2 cultures, the suspensions were exposed to 10 mg l Ð1 of 14 C-4-n-NP using a two-liquid-phase system with carrier n-hexadecane and 192 h of incubation. Results obtained resembled those of the low concentration study. The oxidative metabolic profiles determined after hydrolytic cleavage using GC-EIMS were similar in the NT, CYP1A1 and CYP1A2 cultures. Main metabolites were side-chain mono-hydroxylated derivatives of 4-n-NP with 6Ј-, 7Ј-and 8Ј-OH-4-n-NP as prominent metabolites. In addition, olefinic side-chain hydroxy, ring methoxylated, keto and ring hydroxylated derivatives were observed. The lack of differences in metabolic profiles among the CYP1A1, CYP1A2 and NT cultures was referred to the low enzymatic activity of CYP1A1 and CYP1A2 as compared to the higher endogenous oxidative capacity of tobacco, as well as to similar metabolic profiles of 4-n-NP produced by CYP1A1 and CYP1A2 and tobacco itself.

4-n-nonylphenol degradation by the genus Metarhizium with cytochrome P450 involvement

“…They have different structures, have versatile catalytic properties, and have a wide range of substrates (Durairaj et al, 2016). Moreover, monooxygenases engagement in the breakdown of nonylphenol has been also confirmed in the microsomes of human liver (Deng et al, 2010), Atlantic salmon (Thibaut et al, 2002), as well as in tobacco cells (Berger et al, 2005). Our study has showed that in Metarhizium sp.…”

“…Moreover, in the elimination of NP by human liver cells, CYP450 was involved in the process of hydroxylation at the 1′ carbon position, identifying the metabolite 4-(1-Hydroxynonyl)phenol, di-TMS (Deng et al, 2010). In transgenic tobacco cell cultures, CYP450 enzymes were involved in hydroxylation of 4-n-NP at the 4′, 5′, 6′, 7′, and 8′ carbon positions of the aliphatic chain as well as in the hydroxylation of the aromatic ring (Berger et al, 2005).…”

Metabolism of imidacloprid and DDT by P450 CYP6G1 expressed in cell cultures of Nicotiana tabacum suggests detoxification of these insecticides in Cyp6g1‐overexpressing strains of Drosophila melanogaster, leading to resistance

“…The authors assayed the ability of CYP6G1 to metabolise imidacloprid and DDT by functional expression in heterotrophic tobacco cell suspension cultures. This system had previously been shown to be successful in supporting activity of constitutively expressed human P450s CYP1A1, CYP1A2 and CYP3A4 and production of high amounts of oxidised xenobiotic metabolites, without requiring coexpression of human NADPH‐P450 reductase and cytochrome b 5 14–17. Two binary plant expression vectors, sp Dm 6g1 and sp Dm 6g1‐his, were constructed by cloning D. melanogaster Cyp6g1 without or with a 3′ His‐tag next to a constitutive promoter into plasmid spsma 15.…”

Comparison of the Biotransformation of the14C-Labelled Insecticide Carbaryl by Non-Transformed and Human CYP1A1-, CYP1A2-, and CYP3A4-Transgenic Cell Cultures ofNicotiana tabacum

“…Experimentally, we started by production of transgenic tobacco-cell-suspension cultures overexpressing human P450s CYP1A1 or CYP1A2 (under control of a constitutive promotor) [9]. These P450-transgenic in vitro systems were then used to examine the metabolism of the herbicides atrazine and metamitron [9], and the xenoestrogen nonylphenol [10]. Objectives were to elucidate metabolic pathways in the transgenic tobacco cultures, to produce higher amounts of the metabolites for identification, and to determine whether the xenobiotics studied are substrates of the respective human P450 isozyme.…”