Rémi Thibaut

Effects of fibrates, anti-inflammatory drugs and antidepressants in the fish hepatoma cell line PLHC-1: cytotoxicity and interactions with cytochrome P450 1A.

Effects of 11 pharmaceuticals belonging to three therapeutic classes (lipid regulators from the fibrate group, non-steroidal anti-inflammatory drugs and anti-depressives from the selective serotonin reuptake inhibitors group) were assessed in the fish hepatoma cell line (PLHC-1) by looking at cytotoxicity and interactions with cytochrome P450 1A (CYP1A) function. Among the tested pharmaceuticals, fluoxetine and paroxetine exerted cytotoxic effects, cell viability decreased to 52% and 6% after 24 h of exposure to 20 microM fluoxetine and paroxetine, respectively. The cytotoxicity of both compounds was modulated by cytochrome P450 inhibitors and was dramatically reduced when culture medium was supplemented with reduced glutathione and vitamin E succinate. Additionally, exposure of PLHC-1 cells to some pharmaceuticals led to an early and transient induction of ethoxyresorufin O-deethylase (EROD) activity: bezafibrate and antidepressants induced EROD activity at a concentration of 1 microM whereas clofibrate, ibuprofen and naproxen acted as inducers at a higher concentration (10 microM). These effects might be of toxicological concern since alterations of CYP1A may affect xenobiotic metabolism and toxicity.

Use of effect-directed analysis for the identification of organic toxicants in surface flow constructed wetland sediments.

Wetlands constitute one of the most efficient ecosystems with a great capacity to recycle the organic matter and able to attenuate or mitigate the chemical pollution. However, limited information exists on the ecotoxicological effects that may be caused due to the presence of these pollutants in wetland sediments. In this work, a bioassay-directed approach was used to identify toxicologically active compounds retained in sediments from a surface flow constructed wetland located in the North-Eastern of Spain. Sediment fractionation was accomplished by pressurized-liquid extraction (PLE) followed by semipreparative normal phase high performance liquid chromatography (NP-HPLC). During the extraction procedure, different solvents were sequentially applied in order to selectively extract the compounds as a function of their polarity. The cytotoxicity of the resulting fractions was assessed on the fish hepatoma cell line PLHC-1 by using the thiazolyl blue tetrazolium bromide (MTT) assay, while the presence of CYP1A inducing agents was determined by measuring the activity 7-ethoxyresorufin-O-deethylase (EROD) in exposed cells. Identification of the compounds was performed by gas chromatography coupled to mass spectrometry (GC/MS). Compounds such as polycyclic aromatic hydrocarbons (PAHs), non-steroidal anti-inflammatory drugs (NSAIDs), polycyclic musk fragrances and pesticides were identified in the most toxic fractions.

Assessment of metabolic capabilities of PLHC-1 and RTL-W1 fish liver cell lines

Metabolic capabilities of PLHC-1 and RTL-W1 cell lines were investigated since to date, cytochrome P450 (CYP) 1A and glutathione-S-transferase have been almost the unique biotransformation enzymes reported in these cells. Functionality of CYP3A-, CYP2M- and CYP2K-like enzymes was assessed by studying the hydroxylation of testosterone (T) and lauric acid (LA), and glucuronidation and sulfation capacity was assessed by looking at 1-naphthol (1-N) and T conjugation. Only PLHC-1 cells showed the ability to hydroxylate T at 6β-position (a CYP3A-like catalysed pathway) and LA at (ω-1)-position (a CYP2K-like catalysed pathway). Hydroxysteroid dehydrogenase and steroid reductase enzymes showed comparatively higher activities than CYPs: 5α-dihydrotestosterone, androstenedione and 3β-androstanediol were the major metabolites of T detected in both cell lines. Regarding phase II activities, both cell lines metabolised 1-N to glucuronide and sulfate conjugates. In contrast, when using T as substrate, RTL-W1 formed the glucuronide, whilst PLHC-1 formed the corresponding sulfate. Overall, the observed enzymatic activities are much lower (up to 17.5 × 103 times) than those reported in primary cultures of fish hepatocytes. The present study highlights the need of developing new fish cell lines that could be used as alternative in vitro tools for studying xenobiotic metabolism and toxicity in fish.

UDP-glucuronosyltransferase 1A6 overexpression in breast cancer cells resistant to methotrexate

Methotrexate is a chemotherapeutic agent used in breast cancer treatment, but the occurrence of resistance limits its therapeutic use. A microarrays analysis between sensitive and methotrexate resistant MCF7 and MDA-MB-468 breast cancer cells pointed out the UDP-glucuronosyltransferase 1A (UGT1A) family as a common deregulated node in both cell lines. This family of genes is involved in Phase II metabolism. UGT1A6 was the main isoform responsible for UGT1A family overexpression in these cells. Its overexpression was not due to gene amplification. Transfection of a vector encoding for UGT1A6 in sensitive cells counteracted the cytotoxicity caused by methotrexate. Methotrexate increased the transcriptional activity from a luciferase reporter driven by the UGT1A6 promoter and induced UGT1A6 mRNA and enzymatic activity. Promoter analysis suggested that UGT1A6 induction by methotrexate could be driven by the transcription factors ARNT (HIF-1) and AhR/ARNT. Cells incubated with anticancer drugs susceptible to glucuronidation, such as tamoxifen or irinotecan, together with methotrexate, showed a lesser degree of cytotoxicity, due to UGT1A6 induction. The pharmacological effect of this induction should be taken into account when combining methotrexate with other drugs that are glucuronidated.