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

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).