The partial pressure of oxygen affects biomarkers of oxidative stress in cultured rainbow trout (Oncorhynchus mykiss) hepatocytes.

Oxidative stress, the imbalance between production of reactive oxygen species and the cellular detoxification of these reactive compounds, is believed to be involved in the pathology of various diseases. Several biomarkers for oxidative stress have been proposed to serve as tools in toxicological and ecotoxicological research. Not only may exposure to various pro-oxidants create conditions of cellular oxidative stress, but hyperoxic conditions may also increase the production of reactive oxygen species. The objective of the current study was to determine the extent to which differences in oxygen partial pressure would affect biomarkers of oxidative stress in a primary culture of hepatocytes from rainbow trout (Oncorhynchus mykiss). Membrane integrity, metabolic activity, levels of total and oxidized glutathione (tGSH/GSSG) was determined, as well as mRNA expression levels of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), gamma-glutamyl-cystein synthetase (GCS) and thioredoxin (TRX). The results show that different biomarkers of oxidative stress are affected when the cell culture is exposed to atmospheric oxygen, and that changes such as increased GSSG content and induction of GSSG-R and GSH-Px can be reduced by culturing the cells under lower oxygen tension. Oxygen tension may thus influence results of in vitro based cell research and is particularly important when assessing parameters in the antioxidant defence system. Further research is needed to establish the magnitude of this effect in different cellular systems.

Toxicogenomic responses in rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals and a synthetic mixture.

As more salmon gene expression data has become available, the cDNA microarray platform has emerged as an appealing alternative in ecotoxicological screening of single chemicals and environmental samples relevant to the aquatic environment. This study was performed to validate biomarker gene responses of in vitro cultured rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals, and to investigate effects of mixture toxicity in a synthetic mixture. Chemicals used for 24h single chemical- and mixture exposures were 10 nM 17alpha-ethinylestradiol (EE2), 0.75 nM 2,3,7,8-tetrachloro-di-benzodioxin (TCDD), 100 microM paraquat (PQ) and 0.75 microM 4-nitroquinoline-1-oxide (NQO). RNA was isolated from exposed cells, DNAse treated and quality controlled before cDNA synthesis, fluorescent labelling and hybridisation to a 16k salmonid microarray. The salmonid 16k cDNA array identified differential gene expression predictive of exposure, which could be verified by quantitative real time PCR. More precisely, the responses of biomarker genes such as cytochrome p4501A and UDP-glucuronosyl transferase to TCDD exposure, glutathione reductase and gammaglutamyl cysteine synthetase to paraquat exposure, as well as vitellogenin and vitelline envelope protein to EE2 exposure validated the use of microarray applied to RNA extracted from in vitro exposed hepatocytes. The mutagenic compound NQO did not result in any change in gene expression. Results from exposure to a synthetic mixture of the same four chemicals, using identical concentrations as for single chemical exposures, revealed combined effects that were not predicted by results for individual chemicals alone. In general, the response of exposure to this mixture led to an average loss of approximately 60% of the transcriptomic signature found for single chemical exposure. The present findings show that microarray analyses may contribute to our mechanistic understanding of single contaminant mode of action as well as mixture effects, but that its use in screening of complex environmental samples will need to be further evaluated.

Expression of human protein tyrosine phosphatase epsilon in leucocytes: a potential ERK pathway-regulating phosphatase.

The expression of protein tyrosine phosphatase epsilon (PTPepsilon) was studied in human tissues and blood cells. High mRNA expression was observed in peripheral blood leucocytes, particularly in monocytes and granulocytes which revealed at least four distinct transcripts. In lymphocytes, PTPepsilon expression was induced after 12-O-tetradecanoylphorbol-13-acetate (TPA) or antigen-receptor stimulation, indicating that PTPepsilon plays a role in the events taking place after antigen engagement. Previously, PTPepsilon has been shown to be involved in regulating voltage-gated potassium channel activity, insulin-receptor signalling and Janus kinase-signal transducers and activators of transcription (STAT) signalling. Transfection of cells with different PTPepsilon constructs and activator protein-1 reporter gene indicates that the catalytic activity of PTPepsilon is involved in the regulation of the mitogen-activated protein kinase cascade. In particular, the extracellular signal-regulated kinases (ERK1/2) were shown to be inhibited in both phosphorylation status and enzymatic activity after overexpression of PTPepsilon. Thus, PTPepsilon emerges as a phosphatase with a potential to regulate the ERK1/2 pathway either directly or indirectly through its catalytic activity.