Biomarker protein expression in primary cultures of salmon (Salmo salar L.) hepatocytes exposed to environmental pollutants.

Abstract Primary cultures of salmon (Salmo salar L.) hepatocytes were analysed using (35)s-methionine/cysteine incorporation and SDS-PAGE gel electrophoresis (1 and 2-D) and Western blotting after treatment with representative environmental pollutants (benzo(a)pyrene (BaP), 2,3,3', 4,4'-pentachlorobiphenyl (PCB-105)1 arsenite (AsO2-) and cadmium (Cd)). The results demonstrated striking similarities in changes in protein expression after treatment with the different pollutants. Hsp70 (Hsp72/73) proteins were induced after treatment with all the compounds as shown by (35)S-methionine/cysteine labelling. However, high background levels of these proteins were shown with Western blotting and an anti-Hsp70 antibody, indicating a slow turnover of these proteins. The Hsp70s in salmon hepatocytes were extremely susceptible to degradation in urea used in 2-D electrophoresis, resulting in peptide fragments of 45-46 kDa. In addition to these Hsp70 fragments, arsenite induced several proteins of 42,38, and in the 30-32 kDa range. CYPlA (58 kDa) and an unidentified protein of 16 kDa were furthermore induced after treatment with the organic xenobiotics (BaP, PCB and the model compound β-naphthoflavone, BNF). CYPlA was expressed in a dose-dependent manner, and was resolved into several protein spots in 2-D Western blotting. Elevated levels of metallothionein and haem oxygenase (HO) were indicated in Western blots after treatment with cadmium or arsenite (only HO). The hepatocytes showed cytoplasmic protrusions after treatment with 35 μM arsenite and 100 μM Cd, indicative of cells entering apoptosis.

[1]  H. K. Mitchell,et al.  Self-degradation of heat shock proteins. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[2]  P. O’Farrell.  High resolution two-dimensional electrophoresis of proteins. , 1975, The Journal of biological chemistry.

[3]  E1a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex. , 1989, Molecular and cellular biology.

[4]  N G Anderson,et al.  Effects of toxic agents at the protein level: quantitative measurement of 213 mouse liver proteins following xenobiotic treatment. , 1987, Fundamental and applied toxicology : official journal of the Society of Toxicology.

[5]  A. Goksøyr,et al.  Use of cytochrome P450 1A (CYP1A) in fish as a biomarker of aquatic pollution. , 1995, Archives of toxicology. Supplement. = Archiv fur Toxikologie. Supplement.

[6]  D. Friend,et al.  HIGH-YIELD PREPARATION OF ISOLATED RAT LIVER PARENCHYMAL CELLS , 1969, The Journal of cell biology.

[7]  W. Welch,et al.  How cells respond to stress. , 1993, Scientific American.

[8]  P. Sorger,et al.  Trimerization of a yeast transcriptional activator via a coiled-coil motif , 1989, Cell.

[9]  O. Pellegrini,et al.  Cadmium induced apoptosis in a human T cell line , 1994 .

[10]  A. Goksøyr,et al.  Use of cytochrome P450 1A (CYP1A) in fish as a biomarker of aquatic pollution , 1994 .

[11]  R. Voellmy,et al.  Transduction of the stress signal and mechanisms of transcriptional regulation of heat shock/stress protein gene expression in higher eukaryotes. , 1994, Critical reviews in eukaryotic gene expression.

[12]  C. Hogstrand,et al.  Binding and detoxification of heavy metals in lower vertebrates with reference to metallothionein. , 1991, Comparative biochemistry and physiology. C, Comparative pharmacology and toxicology.

[13]  G. Poste,et al.  Induction of 32- and 34-kDa stress proteins by sodium arsenite, heavy metals, and thiol-reactive agents. , 1986, The Journal of biological chemistry.

[14]  M. Pesonen,et al.  Toxic effects of bleached and unbleached paper mill effluents in primary cultures of rainbow trout hepatocytes. , 1992, Ecotoxicology and environmental safety.

[15]  R. Morimoto,et al.  Antiproliferative prostaglandins activate heat shock transcription factor. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[16]  R. Kothary,et al.  Induction of a novel set of polypeptides by heat shock or sodium arsenite in cultured cells of rainbow trout, Salmo gairdnerii. , 1982, Canadian journal of biochemistry.

[17]  B. Sanders,et al.  Stress proteins in aquatic organisms: an environmental perspective. , 1993, Critical reviews in toxicology.

[18]  S. Sakiyama,et al.  Increase in the synthesis of a Mr 32,000 protein in BALB/c 3T3 cells after treatment with tumor promoters, chemical carcinogens, metal salts, and heat shock. , 1986, Cancer research.

[19]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[20]  R. Tyrrell,et al.  Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R. Morimoto,et al.  Detection of three protein binding sites in the serum-regulated promoter of the human gene encoding the 70-kDa heat shock protein. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Pesonen,et al.  Expression of P4501A1 in a primary culture of rainbow trout hepatocytes exposed to beta-naphthoflavone or 2,3,7,8-tetrachlorodibenzo-p-dioxin. , 1992, Archives of biochemistry and biophysics.

[23]  W. Bonner,et al.  A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. , 1974, European journal of biochemistry.

[24]  T. Andersson,et al.  Biotransformation of 7-ethoxycoumarin in isolated perfused rainbow trout liver. , 1983, Drug metabolism and disposition: the biological fate of chemicals.

[25]  Y. Sasaguri,et al.  Cytopathological changes induced by cadmium-exposure in canine proximal tubular cells: a cytochemical and ultrastructural study. , 1994, Nephron.

[26]  R. Morimoto,et al.  Hemin-induced transcriptional activation of the HSP70 gene during erythroid maturation in K562 cells is due to a heat shock factor-mediated stress response. , 1989, Molecular and cellular biology.

[27]  E. Craig,et al.  Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions , 1980, Cell.

[28]  N. W. Shworak,et al.  Molecular cloning and characterization of a constitutively expressed heat-shock-cognate hsc71 gene from rainbow trout. , 1992, European journal of biochemistry.

[29]  M. Lovett,et al.  Examining the function and regulation of hsp 70 in cells subjected to metabolic stress , 1992, The Journal of cell biology.

[30]  M. Nakao,et al.  PHOPHORUS METABOLISM IN HUMAN ERYTHROCYTE:I. PAPER-CHROMATOGRAPHIC SEPARATION OF ACID-SOLUBLE PHOPHORUS COMPOUNDS INCORPORATING P , 1959 .

[31]  J. Sumpter,et al.  Vitellogenin synthesis in cultured hepatocytes; an in vitro test for the estrogenic potency of chemicals , 1993, The Journal of Steroid Biochemistry and Molecular Biology.

[32]  G. Shyamala,et al.  Estrogen dependent expression of heat shock transcription factor: Implications for uterine synthesis of heat shock proteins , 1995, The Journal of Steroid Biochemistry and Molecular Biology.

[33]  O. Pellegrini,et al.  Cadmium induces apoptosis in a human T cell line. , 1994, Toxicology.

[34]  S. Sassa,et al.  The potent heme oxygenase inducing action of arsenic and parasiticidal arsenicals. , 1981, Pharmacology.

[35]  Angelika Görg,et al.  Two‐dimensional electrophoresis. The current state of two‐dimensional electrophoresis with immobilized pH gradients , 1988 .

[36]  N. Vilaboa,et al.  cAMP increasing agents prevent the stimulation of heat-shock protein 70 (HSP70) gene expression by cadmium chloride in human myeloid cell lines. , 1995, Journal of cell science.

[37]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[38]  J. Frazier,et al.  Isolated fish hepatocytes — model systems for toxicology research☆ , 1990 .

[39]  A. Wyllie,et al.  Apoptosis: A Basic Biological Phenomenon with Wide-ranging Implications in Tissue Kinetics , 1972, British Journal of Cancer.

[40]  N Leighanderson,et al.  Effects of toxic agents at the protein level: Quantitative measurement of 213 mouse liver proteins following xenobiotic treatment*1 , 1987 .

[41]  T. Buchman,et al.  Induction of heat shock response leads to apoptosis in endothelial cells previously exposed to endotoxin. , 1993, The American journal of physiology.

[42]  A. Kappas,et al.  Studies on the mechanism of induction of haem oxygenase by cobalt and other metal ions. , 1976, The Biochemical journal.

[43]  K. Arizono,et al.  Effects of the environmental pollutants on heme oxygenase activity and cytochrome P-450 content in fish , 1990, Bulletin of environmental contamination and toxicology.

[44]  M. Maines.  Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications , 1988, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[45]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[46]  A. Tanimoto,et al.  Cell Death and Regeneration of Renal Proximal Tubular Cells in Rats with Subchronic Cadmium Intoxication , 1993, Toxicologic pathology.

[47]  A. Wyllie,et al.  Cell death: the significance of apoptosis. , 1980, International review of cytology.

[48]  J. Winfield,et al.  Brief Definitive Report AUTOANTIBODIES TO THE CONSTITUTIVE 73-kD MEMBER OF THE hsp70 FAMILY OF HEAT SHOCK PROTEINS IN SYSTEMIC LUPUS ERYTHEMATOSUS , 2022 .

[49]  J. Whitlock,et al.  Mechanistic aspects of dioxin action. , 1993, Chemical research in toxicology.

[50]  G. Igloi,et al.  Structure of a gene encoding heat-shock protein HSP70 from the unicellular alga Chlamydomonas reinhardtii. , 1992, Gene.