Transcriptional profiling of Chinese hamster ovary (CHO) cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Sadowska, Agnieszka; Nynca, Anna; Ruszkowska, Monika; Paukszto, Łukasz; Myszczyński, Kamil; Świgońska, Sylwia; Orlowska, Karina; Molcan, Tomasz; Jastrzebski, J; Ciereszko, Renata E.

doi:10.1016/j.reprotox.2021.07.012

Cited by 7 publications

(3 citation statements)

References 75 publications

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“…However, a connection to further CYP-independent hormonal metabolic pathways is less understood. Further evidence for our hypothesis is provided by transcriptome analyses of CHO cells, in which the expression of 22 CYP enzymes was detected [55]. CYP51 and CYP20A1 were the most abundant.…”

Section: Discussionmentioning

confidence: 60%

Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies

Schulz,

Herzog,

Kubick

et al. 2023

Cells

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In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising “humanised” in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine.

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Section: Discussionmentioning

confidence: 60%

Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies

Schulz,

Herzog,

Kubick

et al. 2023

Cells

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“…With 10% (CYP1A2), 5% (CYP2B6) and 20% (CYP3A4) participation in CYP metabolism, these CYPs are among the most important representatives of their respective gene families in research and industry 5 . Transcriptome data suggests, that no homologs of these three human CYPs are expressed in CHO cells 57 . Accordingly, as for CYP3A4, no significant activity of the other CYPs was measured in lysates of parental CHO or CHO-CPR before the CYP synthesis.…”

Section: Discussionmentioning

confidence: 99%

Synthesis of mono Cytochrome P450 in a modified CHO-CPR cell-free protein production platform

Knauer,

Schulz,

Zemella

et al. 2024

Sci Rep

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Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup.

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“…The effect of in vivo exposure to TCDD on miRNA expression in the reproductive organs and its impact on reproductive functions has not been previously studied. However, there are a few reports on the in vitro effects of TCDD on miRNA expression in cells such as Chinese Hamster Ovary (CHO) cells ( Sadowska, et al, 2021 ) and human Sertoli cells ( Ribeiro, et al, 2018 ), the former study showing no significant changes in miRNA expression while the latter showing significant alterations. The present study investigated the miRNA expression in the ovaries and testes of adult mice exposed to TCDD and the genes targeted by those miRNAs.…”

Section: Introductionmentioning

confidence: 99%

Effect of TCDD exposure in adult female and male mice on the expression of miRNA in the ovaries and testes and associated reproductive functions

Hall,

Mattison,

Singh

et al. 2023

Front. Toxicol.

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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant found widely across the world. While animal and human studies have shown that exposure to TCDD may cause significant alterations in the reproductive tract, the effect of TCDD on the expression of miRNA in the reproductive organs has not been previously tested. In the current study, we exposed adult female or male mice to TCDD or vehicle and bred them to study the impact on reproduction. The data showed that while TCDD treatment of females caused no significant change in litter size, it did alter the survival of the pups. Also, TCDD exposure of either the male or female mice led to an increase in the gestational period. While TCDD did not alter the gross morphology of the ovaries and testes, it induced significant alterations in the miRNA expression. The ovaries showed the differential expression of 426 miRNAs, of which 315 miRNAs were upregulated and 111 miRNA that were downregulated after TCDD exposure when compared to the vehicle controls. In the testes, TCDD caused the differential expression of 433 miRNAs, with 247 miRNAs upregulated and 186 miRNAs downregulated. Pathway analysis showed that several of these dysregulated miRNAs targeted reproductive functions. The current study suggests that the reproductive toxicity of TCDD may result from alterations in the miRNA expression in the reproductive organs. Because miRNAs also represent one of the epigenetic pathways of gene expression, our studies suggest that the transgenerational toxicity of TCDD may also result from dysregulation in the miRNAs.

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Transcriptional profiling of Chinese hamster ovary (CHO) cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Cited by 7 publications

References 75 publications

Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies

Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies

Synthesis of mono Cytochrome P450 in a modified CHO-CPR cell-free protein production platform

Effect of TCDD exposure in adult female and male mice on the expression of miRNA in the ovaries and testes and associated reproductive functions

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