This study presents a broad investigation of the effects of different types of anoxia in the Rainbow Trout Hypodermal Fibroblasts cell line, including a comparison of two widely used anoxia models. Anoxia was obtained either by flushing with N₂ (FN) or by adding NaN₃ to the medium, since chemical anoxia obtained by NaN₃ is a widely used model for simulating anoxia (Ossum et al., 2004).

The effects of anoxia were studied by protein expression analysis using 2-dimensional gel electrophoresis followed by MS/MS. In this way we were able to separate more than 1500 protein spots with an apparent range of molecular masses from 10 to 120 kDa and pI values from 4 to 7. In response to 24 h of anoxia obtained by FN we found that the sexpression of more than 30 proteins changed, including proteins involved in cell metabolism as well as protective proteins.

Chemical anoxia for 30 minutes and subsequent reoxygenation overnight (CO) induced changes in the expression of more than 40 proteins. Two hours of anoxia obtained by FN followed by reoxygenation overnight (AO), resulted in changes in the abundance of more than 30 proteins. Surprisingly the response to AO was very different to the response to CO, actually only the levels of five proteins changed similarly.

In summery we found almost 100 different proteins to be involved in the different aspects of anoxia. We also found a remarkable difference between the effects induced by CO compared to AO, showing the existence of great differences in expression patterns depending on the nature of the stimuli. This calls to circumspection when results obtained by different types of anoxia are compared.

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