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Cyp1A and ABC transporter expression and function in PLHC-1cell line after microcystin-LR and Benzo (a)pyrene exposure

SND-ID: 2024-450. Version: 1. DOI: https://doi.org/10.5878/bfa6-gd21

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Citation

Creator/Principal investigator(s)

Flavia Bieczynski - University of Gothenburg, Department of Biological and Environmental Sciences orcid

Malin Celander - University of Gothenburg, Department of Biological and Environmental Sciences orcid

Maja Edenius - University of Gothenburg, IVL Swedish Environmental Institute

Annika Lindkvist - University of Gothenburg, Department of Biological and Environmental Sciences

Julio C. Painefilú - National University of Comahue, Biological and Geoenvironmental Technologies Patagonian Institute orcid

... Show more..

Flavia Bieczynski - University of Gothenburg, Department of Biological and Environmental Sciences orcid

Malin Celander - University of Gothenburg, Department of Biological and Environmental Sciences orcid

Maja Edenius - University of Gothenburg, IVL Swedish Environmental Institute

Annika Lindkvist - University of Gothenburg, Department of Biological and Environmental Sciences

Julio C. Painefilú - National University of Comahue, Biological and Geoenvironmental Technologies Patagonian Institute orcid

Andrés Venturino - National University of Comahue, Environmental Toxicology and Agrobiotechnology Research Centre of Comahue orcid

Carlos M. Luquet - National University of Comahue, Laboratory of Aquatic Ecotoxicology orcid

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Research principal

University of Gothenburg - Department of Biological and Environmental Sciences rorId

Description

The dataset presented corresponds to a study investigating mixture toxicity between two common aquatic contaminants, microcystin-LR (MCLR) and benzo[a]pyrene (BaP), on the detoxification system of fish. We used the Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cell line as a model for fish liver cells.

Cells were exposed to MCLR (0.01, 1 µM), BaP (0.01, 0.1, 1 µM), or combinations of both chemicals for periods ranging from 1 to 48 hours. We measured the following endpoints:
- Cytochrome P450 1A (CYP1A) function and regulation, assessing ethoxy resorufin-O-deethylase (EROD) activity and CYP1A mRNA expression. EROD activity was normalized to the protein.
- P-glycoprotein (Pgp) function and expression, using the rhodamine 123 (Rh123) accumulation assay and Pgp mRNA expression. Rh123 accumulation was normalized to the protein.
- Cell cytotoxicity, using two fluorescent indicators, 5-Carboxyfluorescein Diacetate-Acetoxymethyl Ester (CFDA-AM) and Alamar Blue (AB).

The dataset includes the following information:

1) CYP1A Activity: This section contains raw data used to calculate CYP1A acti

... Show more..
The dataset presented corresponds to a study investigating mixture toxicity between two common aquatic contaminants, microcystin-LR (MCLR) and benzo[a]pyrene (BaP), on the detoxification system of fish. We used the Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cell line as a model for fish liver cells.

Cells were exposed to MCLR (0.01, 1 µM), BaP (0.01, 0.1, 1 µM), or combinations of both chemicals for periods ranging from 1 to 48 hours. We measured the following endpoints:
- Cytochrome P450 1A (CYP1A) function and regulation, assessing ethoxy resorufin-O-deethylase (EROD) activity and CYP1A mRNA expression. EROD activity was normalized to the protein.
- P-glycoprotein (Pgp) function and expression, using the rhodamine 123 (Rh123) accumulation assay and Pgp mRNA expression. Rh123 accumulation was normalized to the protein.
- Cell cytotoxicity, using two fluorescent indicators, 5-Carboxyfluorescein Diacetate-Acetoxymethyl Ester (CFDA-AM) and Alamar Blue (AB).

The dataset includes the following information:

1) CYP1A Activity: This section contains raw data used to calculate CYP1A activity, expressed as specific EROD activity. The EROD assay is based on the ability of the CYP1A enzyme to catalyze the O-deethylation of ethoxyresorufin to resorufin, a fluorescent product. Resorufin production was monitored over time and quantified using a resorufin standard curve. Each sample value was normalized to total protein content, which was measured using fluorescamine, and bovine serum albumin (BSA) as the protein standard. The files include:
- EROD slopes for each sample
- Protein concentration per sample
- Specific EROD activity
- Standard curves (for BSA and resorufin).
Additionally, the dataset includes results from an experiment involving pre-induction of EROD activity using β-naphthoflavone (BNF). Detailed descriptions of measurement conditions and calculation methods are provided in the accompanying README file (EROD_readme).

2) Rhodamine 123 Accumulation: This section includes raw data for calculating rhodamine 123 (Rh123) accumulation in cells, expressed as fluorescent units (FU) per mg of protein. The Rh123 accumulation assay measures the ability of the P-glycoprotein (Pgp) transporter to efflux this fluorescent compound from cells. When chemicals interfere with Pgp transport function, Rh123 accumulates within the cells. The files include:
- Rh123 fluorescence values (FU) per sample
- Protein concentration per sample
- BSA standard curves for protein quantification
Detailed descriptions of the measurement conditions and calculations are provided in the accompanying README file (Rh123_readme).

3) Cytotoxicity: Cytotoxicity was assessed by measuring mitochondrial activity and cell membrane integrity using two fluorescent indicators, Alamar Blue (AB) for mitochondrial activity and CFDA-AM for membrane integrity. This section includes raw fluorescence data (in fluorescent units, FU), which were used to express cytotoxicity as a percentage of FU relative to the control treatment. Detailed descriptions of measurement conditions and calculations are provided in the README file (cytotox_readme).

4) qPCR Data: This section contains raw cycle threshold (Ct) values from real-time polymerase chain reaction (qPCR) used to estimate the mRNA levels of target genes (CYP1A and Pgp). The data is normalized to a reference housekeeping gene, 18S, and presented as fold increase relative to the control treatment. Detailed descriptions of measurement conditions and normalization calculations are provided in the README file (qPCR_readme). Show less..

Data contains personal data

No

Language

Method and outcome

Time period(s) investigated

2022-05-01 – 2024-08-08

Data format / data structure

Species and taxons

Poeciliopsis lucida (fish) hepatocellular carcinoma cell line (plhc-1)

Data collection
  • Mode of collection: Laboratory experiment
  • Description of the mode of collection:
    The data were collected through laboratory experiments conducted at the Department of Biological and Environmental Sciences, University of Gothenburg. All experiments took place in 2022, except for the half-maximal inhibition concentration (IC50) assays for benzo[a]pyrene (BaP) on CYP1A-mediated EROD activity in PLHC-1 cells pre-exposed to BNF (performed in July 2024).

    The PLHC-1 cell line (ATCC® CRL-2406TM) was obtained from LGC Standards (Borås, Sweden). Cells were cultured in tightly capped 75 cm² cell culture flasks with 25 mM HEPES-buffered MEM, pH 7.1, supplemented with 5% (v/v) FBS at 30 ºC. For each assay, cells were seeded as follows:
    - EROD and Rh123 accumulation assay: 48-well plates, 6 × 10⁵ cells/mL in 500 µL per well.
    - Cytotoxicity Analyses: 96-well plates, 5 × 10⁵ cells/mL in 200 µL per well.
    - mRNA Expression Analyses: 6-well plates, 2 × 10⁶ cells/mL in 2 mL per well.

    After a 24-hour incubation post-seeding, media were removed from each well, and cells were rinsed with 10 mM Na-phosphate buffer, pH 7.4, containing 0.9% (w/v) NaCl (PBS). Cells were then exposed to DMSO (control), MCLR, BaP, or their mixture. EROD activity, Rh123 accumulation, cytotoxicity, and mRNA expression were measured on separate plates, with each type of measurement conducted on different days.

    Data were recorded using a VICTORTM 1420 Multilabel Counter (Wallac Sverige AB) for EROD, cytotoxicity, and Rh123 assays, and an iCYCLER instrument (Bio-Rad) for qPCR.
  • Time period(s) for data collection: 2022-05-01 – 2024-08-08
  • Data collector: University of Gothenburg rorId
  • Instrument: VICTORTM 1420 Multilabel Counter - fluorometer VICTORTM 1420 Multilabel Counter from Wallac Sverige AB
  • Instrument: iCYCLER instrument (Bio-Rad) - iCYCLER instrument (Bio-Rad)
  • Source of the data: Biological samples
Geographic coverage
Administrative information

Responsible department/unit

Department of Biological and Environmental Sciences

Funding 1

  • Funding agency: CONICET rorId
  • Funding agency's reference number: RD - EX-2021-93553723-APN-CB#CONICET
  • Project name on the application: Effects of the combination of cyanotoxins and polycyclic aromatic hydrocarbons on the multiple xenobiotic resistance system in fish
  • Funding information: Partial Funding Program for Assistant Researchers, CONICET, 2021, to F. Bieczynski

Funding 2

  • Funding agency: National Agency for the Promotion of Research, Technological Development and Innovation rorId
  • Funding agency's reference number: PICT 2019-649
  • Project name on the application: Evaluation of combined effects of contaminants and environmental risk on fish present in the waters of Northern Patagonia
  • Funding information: PICT 2019-649 to F. Bieczynski

Funding 3

  • Funding agency: National Agency for the Promotion of Research, Technological Development and Innovation. rorId
  • Funding agency's reference number: PIP 2753
  • Funding information: PIP 2753 to C. Luquet

Funding 4

  • Funding agency: Wenner-Gren Center rorId
  • Funding agency's reference number: GFOv2024-004
  • Project name on the application: Chemical Interactions on the Detoxification Pathway in Fish Liver Cells Exposed to Mixtures
  • Funding information: GFOv2024-004 to M. C. Celander

Funding 5

  • Funding agency: Carl Trygger Foundation rorId
  • Funding agency's reference number: 942-215-605
  • Funding information: 942-215-605 to M. Edenius
... Show more..

Funding 1

  • Funding agency: CONICET rorId
  • Funding agency's reference number: RD - EX-2021-93553723-APN-CB#CONICET
  • Project name on the application: Effects of the combination of cyanotoxins and polycyclic aromatic hydrocarbons on the multiple xenobiotic resistance system in fish
  • Funding information: Partial Funding Program for Assistant Researchers, CONICET, 2021, to F. Bieczynski

Funding 2

  • Funding agency: National Agency for the Promotion of Research, Technological Development and Innovation rorId
  • Funding agency's reference number: PICT 2019-649
  • Project name on the application: Evaluation of combined effects of contaminants and environmental risk on fish present in the waters of Northern Patagonia
  • Funding information: PICT 2019-649 to F. Bieczynski

Funding 3

  • Funding agency: National Agency for the Promotion of Research, Technological Development and Innovation. rorId
  • Funding agency's reference number: PIP 2753
  • Funding information: PIP 2753 to C. Luquet

Funding 4

  • Funding agency: Wenner-Gren Center rorId
  • Funding agency's reference number: GFOv2024-004
  • Project name on the application: Chemical Interactions on the Detoxification Pathway in Fish Liver Cells Exposed to Mixtures
  • Funding information: GFOv2024-004 to M. C. Celander

Funding 5

  • Funding agency: Carl Trygger Foundation rorId
  • Funding agency's reference number: 942-215-605
  • Funding information: 942-215-605 to M. Edenius

Funding 6

  • Funding agency: Swedish Fund for Research Without Animal Experiments rorId
  • Funding agency's reference number: S2021-0046
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Topic and keywords

Research area

Biochemistry and molecular biology (Standard för svensk indelning av forskningsämnen 2011)

Keywords

Cytochrome p450 enzyme system, Cytotoxicity, Abc transporter, Microcystin-lr, Ethoxy resorufin-o-deethylase activity, Qpcr, Polycyclic aromatic hydrocarbon, Poeciliopsis lucida hepatocellular carcinoma cell line

Publications

Title: Effects of Combined Exposures of Microcystin-Lr and Benzo[A]Pyrene on Detoxification Mechanisms in the Fish Liver Cell Line (Plhc-1).

Authors: Celander, Malin Charlotta and Bieczynski, Flavia and Edenius, Maja and Lindkvist, Annika and Painefilú, Julio C. and Venturino, Andres and Luquet, Carlos M.

This is a preprint article, it offers immediate access but has not been peer reviewed.

Available at SSRN: https://ssrn.com/abstract=4997829 or http://dx.doi.org/10.2139/ssrn.4997829

Journal: Aquatic Toxicology

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Versions

Version 1. 2025-01-31

Version 1: 2025-01-31

DOI: https://doi.org/10.5878/bfa6-gd21

Contact for questions about the data

Published: 2025-01-31