Top predators - Glaucous Gull - Project title
	Genotoxic effects in Glaucous Gulls.
Co-ordinating institution
	NTNU
Final report
	Krøkje, Åse, Bingham, Chris and Gabrielsen, Geir Wing: Genotoxic effects in Glaucus Gulls
Summary and results
	Summary Glaucous gull (Larus hyperboreus), which is one of the top-predators in the arctic marine ecosystem, has at Svalbard very high concentrations of polychlorinated biphenyls (PCBs). PCBs have caused hepatocellular carcinoma when fed to birds. DNA-adduct formation has been shown to be higher in quail hepatocytes than in rat hepatocytes, after exposure of PCBs in primary cultures. These studies suggest that PCBs have a genotoxic effect in wild birds. The aim of the current study is to show if the complex mixture of pollutants at Svalbard has genotoxic effects in Glaucous gulls. During this project effective methods of peripherial blood lymphocyte (PBL) culture and chromosome preparation for light microscopy for Glaucous gull have been established. Alternative methods for preparing chromosome spreads from bone marrow have also been established. The results from the PBL cultures indicate a higher frequency of chromosome aberrations in the exposed group compared to the control. The same trend was shown both for the female and the male gulls. The 32P-postlabeling DNA-adduct analysis of liver tissue indicates that the adduct level was higher in the exposed group than in the control group. The current study may indicate that the complex mixture of pollutants at Svalbard has genotoxic effects in Glaucous gulls   Scientific resultats The aim of the project was to study the genotoxic effect of the complex mixture of pollutants at Svalbard in Glaucous gull with use of chromosomal analysis of blood and bone marrow samples and with DNA adduct analysis of liver tissue. The methodology had been developed with chicken as a pilot study in preparation for the field-experiment at Svalbard on Glaucous gull. Such methods need to be adapted and re-optimised at each step for another species. Therefore the projects sub-aim had to be such an adaptation of methods to Glaucous gull.   The project has produced some important results: We have been successful in establishing effective methods of peripherial blood lymphocyte (PBL) culture and chromosome preparation for light microscopy for Glaucous gull. The results from the PBL cultures indicate a higher frequency of chromosome aberrations in exposed group compared to the control group. The same trend was shown both for female and male gulls. We have established alternative methods for preparing chromosome spreads from bone marrow of Glaucous gull. We have established 32P-postlabeling method for analysis of DNA adducts from the liver tissue of Glaucous gull. The results of the DNA adduct analysis indicate a higher level of adducts in the exposed group compared to the control group. The biomarker DNA adducts, representing the "biological effective dose" of DNA-reactive compounds (index of exposure), indicate that Glaucous gulls have been exposed to some compounds which have been able to bind to DNA. The results indicate that the level of adducts is higher in the exposed group than in the control group. The other biomarker chromosome aberrations, involving gross alteration of the genetic material possibly induced by clastogenic and spindle impairing chemicals (index of early effect), indicate that the pollutants the exposed gulls were fed have caused some genotoxic effects. The co-operation between the different institutions involved in the Glaucous gull projects to solve different aspects related to biological effects of persistant organic pollutants on individuals and populations of glaucous gulls has worked out quite well. Since several parameters were measured on the same individuals, we have the possibility to correlate all this parameters. It is important to co-ordinate at one time as many relevant studies as possible during each such field experiment so as to make efficient use of the available material, for both economic and ethical reasons, i.e. wherever possible all relevant parameters should be studied using the same biological material. The funding we got from The Effects programme has mainly been used to cover the expenses (travelling, accommodations, etc.) for the fieldwork and chemicals for the analysis. The salary for Bingham and Krøkje has been covered by NTNU and for Østby by the Norwegian Research Council. Especially the chromosome analysis is very time consuming and consumes most of the salary expenses in the project. Relevance for monitoring We have developed methods for obtaining cytogenetic data from lymphocytes of bone marrow and peripherial blood. With the latter, samples can be obtained on several occasions non-destructively, so long as culturing facilities are available. If such facilities ever fail in the field during a course of studies, then the bone marrow method can be suitable as this does not require such culturing facilities. Since lymphocytes from peripherial blood can be sampled non-destructively it is well suited for monitoring studies. The DNA adducts has so far only been measured in liver tissue. It might be possible to use the method on peripherial blood lymphocytes as well, thereby allowing non-destructive sampling for DNA adduct analysis. Extrapolation of results from one species to another is not necessarily correct because of differences in physiology and sensitivity. However, measuring genotoxic effects in Glaucous gulls with high and low levels of POPs will increase the understanding of what importance POPs exposure in polar regions has on organisms.
Original project description
	Summary Glaucous gulls at Svalbard are exposed to a complex mixture of pollutants, including polychlorinated biphenyls (PCBs). PCBs have shown carcinogenic effect when fed to birds, and a genotoxic effect when cells are exposed in vitro. With use of chromosome aberration analysis and DNA-adduct analysis the current study will show if the complex mixture of pollutants at Svalbard has genotoxic effects in glaucous gulls. Introduction Chemical analyses have shown that glaucous gulls (Larus hyperboreus) at Svalbard have very high concentrations of polychlorinated biphenyls (PCBs) (Gabrielsen et al., 1995). PCBs cause hepatocellular carcinoma when fed to birds (Brunn et al., 1987). Dubois et al. (1995) showed that DNA-adduct formation was higher in quail hepatocytes than in rat hepatocytes or Hep G2 cells, after exposure of PCBs in primary cultures. These studies suggest that PCBs have a genotoxic effect in wild birds. Genotoxic compounds represent a specific challenge since the effects not only strike exposed individuals in the form of tumors and reduced reproductivity, but damage can also be transmitted to descendants. Basically the release of genotoxins into the environment should be avoided because massive exposure may affect the reproductive capacity of many species, and modest exposure may lead to an enhanced instability of ecosystems (Würgler & Kramers, 1992). Development of methods which can provide early warning about effects on the genetic material before serious effects on the ecosystem occur are therefore very important. In genetic toxicology, genotoxic observations are principally divided into the following categories; structural and numerical chromosome aberrations and gene mutations.   Methods Chromosome aberrations Chromosome aberrations involve gross alteration of the genetic material and are generally detected by using light microscopy to examine metaphase chromosomes in appropriately prepared cells. The methodology for chromosome aberration studies has been established in our group, and has been used on lymphocytes from wild reindeer for studying genotoxic effects of exposure of radioactivity (Espelien, 1991) and heavy metals (Espelien et al., 1995). In our studies in the heavily polluted Lapland Biospheric Reserve on the Kola Peninsula, chromosome aberration studies were used on root-tips from spruce (Krøkje & Gullvåg, 1994; Krøkje et al., in prep.) and on lymphocytes from small mammals (Krøkje et al., 1997; Krøkje & Bingham, 1998; Østby et al., in prep.). Last winter we produced good slides of lymphocyte chromosomes prepared from blood from grey seals from Froan and Estonia. Even though the methods for preparing of lymphocytes are established in our group, the methods of preparation of lymphocytes for genetic investigation have to be optimized for each species. For field studies it is important that the methods work under field or primitive laboratory conditions. In both these aspects our methods have been successful in our previous field studies. DNA-adducts The mechanisms for most genotoxic compounds involve covalent binding to the genetic material (DNA), producing DNA-adducts. DNA-adducts are markers for the biological effective dose or for the actual amount of mutagens which have interacted with DNA, and can act as "early warning-systems" for identifying genotoxic damage. As dosimeters, DNA-adducts are very sensitive (Randerrath and Randerrath, 1990). The glaucous gulls at Svalbard are exposed to a complex mixture of chemicals. These compounds may interact with each other in ways which are difficult to predict. DNA-adducts represent a "fingerprint" of a genotoxic exposure, and perhaps give an answer to which kind of exposure has caused DNA-damage in one single individual. With analysis of DNA-adducts we may get information about which component in the mixture has been bound to DNA and is the most probable cause of genetic damage. Even if the genotoxic compounds and their metabolites are not otherwise detectable or have been eliminated from tissues, DNA-adducts may provide long term stable evidence of genotoxic damage causation. It can be a long time before genetic damage that is initiated is phenotypically manifested. Various methods are available to measure DNA-adducts, including immunoassays, spectrometry and postlabeling. The most common and most sensitive method for detection of DNA-adducts is the 32P-postlabeling technique (Gupta, 1993; Qu et al., 1997). This method can detect several types of adducts and is the only available method to detect unknown adducts. 32P-postlabeling is therefore the best method suitable for the analysis of adducts from complex mixtures. The 32P-postlabeling technique involve 32P-labeling of the modified nucleotide (the adduct) after the adduct has been produced. Assuming that the 32P-postlabeling method is performed optimally, it is able to detect aromatic adducts down to 1 adduct per 109-1010 nucleotide. This corresponds to about 1 adduct per cell. With exposure for a mixture of more or less unknown mutagens it is not sufficient that the method is only sensitive, but it must be able to detect most of the adducts, independent of their chemical structure (Gupta & Early, 1988). Since several organic pollutants need metabolic activation to show genotoxic effects, compounds which influence the activation system also have an effect on adduct production. Cytochrome P-450 is the most important enzyme system for activation of secondary genotoxic compounds. The highest concentration of P-450 enzymes involved in biotransformation of xenobiotic compounds are found in liver, but P-450 enzymes are present in virtually all tissues. It is well known that PCBs and organochlorines can induce the P-450 system. Very high concentrations of PCB have been found in hepatic tissue of glaucous gulls (Gabrielsen et al., 1995), and the liver may probably also have a high concentration of DNA-adducts. The adduct level may reflect the net effect of bioactivation of premutagens and detoxification of reactive metabolic intermediates. Measuring of adducts may identify species/individuals with high sensitivity caused either by high levels of bioactivation or reduced levels of detoxification. DNA-adduct techniques have been used on human material, on experimental animals and in in vitro -tests. The method can principally be used on all cells, assuming that it is always possible to isolate DNA. The 32P-postlabeling technique is now under establishment in our group. One dr. scient.-student (L. Østby) and one cand. scient.-student (C. Aalmo) are going to use the technique in their work.   Experiment This project will be coordinated with Gabrielsens project; Effects of persistant organic pollutants (POPs) on the immune response and retenoid- and thyroid hormone status of glaucous gulls (Larus hyperboreus). The two projects will use material from the same individuals. A total of 40 individuals is needed for this experiment, further divided in two groups with 20 birds in each. One group will be a control group, the other a persistant organic pollutant (POP) exposed group. The coordination of the projects will provide us with the opportunity to correlate the results from our analysis with the results from the chemical analysis which are included in the other project. For the analysis of chromosome aberrations, blood samples and bone marrow from the femurs will be used. From these samples lymphocytes will be isolated for cell culture. After the preparation of metaphase enriched cultures, slides for light microscopy will be prepared after standard hypotonic and fixation treatment of cells. 2nd division controls will be established by our differential staining methods adapted from Wolf and Perry (1974), Chen and Lin (1995) and Edelman and Lin (1986) by Østby et al. (in prep.). Both blood and bone marrow sources of nucleated cells are to be used in the current study in order to cover for unknown technical contingencies in the field, as this will be the first cytogenetic study of this species in this particular field site. For the analysis of DNA-adducts liver tissue will be used. The plan is that Akvaplan-niva´s project "Biological effects of POPs on Svalbard glaucous gulls (Larus hyperboreus)" will take liver tissue from the same individuals for their analysis of metabolic enzymes. The tissue samples will be frozen down immediately after the birds have been killed, and will be kept frozen for further analysis at the laboratory at NTNU. A coordination with Akvaplan-niva´s project will provide us with the opportunity to correlate the results from the DNA-adduct analysis with their results from the enyme analysis. The funds allocated to us for 1999 are sufficient for the single field experiment described above but not also for a separate pilot study, so this current study must be considered such a pilot study, in which the experimental methodology, which has worked well on other species in field will be optimized for the first time for glaucous gulls. Successful pilot studies on chicken have already been undertaken in this laboratory in preparation for this work.   Total budget NOK 200 000,-. NOK 100 000,- is applied for from the Effects Programme, while NOK 100 000,- will be covered by NTNU.   References Brunn, H., E. Schmidt, M. Reinacler, D. Manz & E. Eigenbrodt (1987). Histology and histochemistry of the liver of chickens after DENA induced hepatocarcinogenesis and ingestion of low chlorinated biphenyls. Arch. Toxicol., 60, 337-342. Chen, J.F., & Y.J. Lin (1985). Improved light source for induction of sister chromatid differentiation. Cytobios, 44, 73-87. Dubois, M., A. Pfohl-Leszkowicz, Y. Grosse & P. Kremers (1995). DNA adducts and P450 induction in human , rat and avian liver cells after exposure to polychlorobiphenyls. Mutat. Res., 345, 181-190. Edelman, J.R., & Y.J. Lin (1986). Simplified differential staining of mouse sister chromatids. Cytobios, 46, 147-153. Espelien, I.S. (1991) Kromosomaberasjoner hos norsk villrein etter Tsjernobyl-ulykken. Cand. scient.-oppgave ved Universitetet i Trondheim. Espelien, I.S., O. Strand, T. Skogland, J. Kolacz & Å. Krøkje (1995). Chromosome aberrations in Norwegian wild reindeer exposed to low-dose radiation following the Chernobyl accident. Nordic Environ. Mutagen Society, Beito, 16.-19.3 1995. Gabrielsen, G.W., J.U. Skaare, A. Polder & V. Bakken (1995). Chlorinated hydrocarbons in glaucous gulls (Larus Hyperboreus) in the southern part of Svalbard. Sci. Total. Environ, 160/161, 337-346. Gupta, R.C. (1993). Recent advances with the 32Ppost-labelling DNA adduct assay. Presentert på New DNA and Cytogenetic technology, Barossa Valley, 1.- 4.3.1993. Gupta, R.C. & Earley, K. (1988). 32P-adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures. Carcinogenesis, 9 (9), 1687-1693. Krøkje, Å. & C. Bingham (1998). Genotoxic pollution registered as increased levels of chromosome aberrations in plants ans small mammals. 8th Annual Meeting of SETAC-Europe, Bordeaux, p. 255. Krøkje, Å., C. Bingham & C. Østby (1997). Genetic effects of high and low exposure to heavy metals. Mutat. Res. 379, Suppl.1, 112. Krøkje, Å. & B. Gullvåg (1994). Genotoksisk belastning i jord. Effektstudier, med mål å finne frem til akseptable grenser for genotoksisk belastning fra langtransportert luftforurensning. Utredning for Direktoratet for naturforvaltning. Fagrapport nr. 47. (In Norwegian) Krøkje, Å., G. Hofsli, R. Røsbak & B. Gullvåg. Genotoxic effects of pollution on root tips from wild grown plants. (In prep.). Randerath, K. & Randerath, E. (1990). Detection of human DNA adducts by 32P-postlabeling. -I: Sutherland, B. M., Woodhead, A. D. (red.), DNA Damage and Repair in Human Tissues. Plenum Press, New York, s. 13-33. Wolf, S., & P. Perry (1974). New giemsa method for differential staining of sister chromatids. 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