Abstract

During the 1986/87 summer season the level of detoxication enzyme glutathione-s-transferase was tested in antarctic fish species Pagothenia borchgrevinki and Dissostichus mawsoni. The enzymes were tested using a variety of substrates including the lipid peroxide cumene hydroperoxide. These measurements will be used to calculate base levels of enzyme activity. The enzymes of antarctic fish have appreciable activity with 1-chloro-2, 4-dinitrobenzene and cumene hydroperoxide but very low activity with 2,4-dichloro-1-nitrobenzene and 0-nitrobenzly chloride. Frozen samples of antarctic fish liver were taken for further purification and study at Victoria University. This will be compared and combined with research from the 1983/84 season and with other temperate comparative species to help ellucidate the role of these enzymes.

Programme

The research in the Antarctic this season represents the third phase in a three phase programme to study the detoxication systems of antarctic fish. The first two phases involve the thermodynamic characterization of a reaction with the general substrate 1-chloro-2, 4-dinitrobenzene.

The first phase involves the thermodynamic characterization of the spontaneous reaction, so reaction velocity con be calculated at any given pH or temperature. This allows extrapolation to the physiological conditions of antarctic fish. Results show that the spontaneous reaction will proceed 30 times slower under antarctic conditions.

Relevant thermodynamic parameters calculated for the reaction are activation enthalpy 49 KJ mol⁻¹ and enthalpy of glutathione sulphydryl proton 31.6 KJ mol⁻¹.

The second phase involves the thermodynamic characterization of enzymes from Dissostichus mawsoni being compared with those of other organisms with different temperature regimes. Results gained to date give activation energies for the catalysed reaction between 1-chloro-2, 4-dinitrobenzene of 56.8±7.8 KJ mol⁻¹ for D.mawsoni, 47.0±3.8 KJ mol⁻¹ for the moth Galleria mellonella and 34.8±2.6 KJ mol⁻¹ for rat glutathione-s-transferase 1,2.

The third phase involves the characterization of enzyme levels with different substrates. In particular endogenous xenobiotic substrates will be examined. These include the lipid peroxide cumene hydroperoxide, a substrate used in the Antarctic this season.

To summarize, the first phase examines the need for a detoxication system. The second examines any structural evolutionary change unique to the Antarctic and the third examines specialization of enzyme type that may have occurred to facilitate life in the antarctic waters.

Personnel

K. C. Falkner, Biochemistry Department, Victoria University of Wellington.

Scientific Endeavours

This project attempted to test the glutathione-s-transferases of antarctic fish species with a variety of xenobiotic substrates. The substrates chosen were 1-chloro-2, 4-dinitrobenzene, 2,4-dichloro-1-nitrobenzene, p-nitrobenzly chloride and page 18 cumene hydroperoxide. To test for these enzymes fish had to be caught, their livers excised and homogenised, then lightly centrifuged and assayed spectrophotometrically. All work was based at Scott Base and fish were caught in the Cape Armitage area.

Attempts were made to separate the two enzyme systems that break down cumen hydroperoxide; glutathione-s-transferase and selenium dependent glutathione peroxidase. These were unsuccessful as the yield of enzyme from initial purification procedures was too low.

It was possible to repeat the work attempted in the 1983/84 programme where results gained were affected by instrument errors. This gave significantly higher enzyme activity in accordance with that found in frozen samples in New Zealand. The effect of freezing on the glutathione-s-transferases and cumen peroxidase activity were examined as well as the thermal stability of the enzyme in crude homogenates.

Frozen liver samples of antarctic fish species were brought back to New Zealand for further research.

Publication

This research will be incorporated in a PhD thesis titled "Detoxication systems of antarctic fish". The anticipated completion date for the thesis is 25 February 1988. From this thesis it is expected that one paper specifically on the purification and properties of the glutathione-s-transferases will be published. Other papers on the effects of temperature and pH on glutathione-s-transferases will also feature material from antarctic fish.

Future Research

As this project is based on laboratory work in New Zealand the future research for this project is outlined below:

In this project frozen samples brought back this season will be used to repeat the purification procedures developed after the 1983/84 season. It is hoped that the experiments giving the enthalpy of activation for the glutathione-s-transferase will be repeated and attempts will be made to characterize this and the other glutathione-s-transferases present in D.mawsoni.

The glutathione-s-transferases of the fish Geniagnus monopterygius will be purified and characterized as a comparative species. It is hoped sufficient enzyme will be obtained to gain an enthalpy of activation with as least one of the isoenzymes.

Rat glutathione-s-transferases will be used to look at variation within one animal as opposed to variation between species. The glutathione-s-transferases of rat are highly characterized making it a suitable candidate for study.

Management of Science

At present the scope of studies that can be undertaken at Scott Base is limited unless commitment to an antarctic programme spans several years in which equipment can be tested and specialist wannagins built. However there seems quite a lot of scope for studies such as these in which the antarctic is seen primarily as a sample collection urea and only limited scientific work is attempted. Scott Base is well sited to study antarctic flora and fauna so it is unfortunate that the Base has no real biological science laboratory. It would be a great asset to the programme to have a biological laboratory complete with running water and refrigerated areas.