- Email: [email protected]
Biomarkers to detect environmental pollution
Poster Session 2M. Ecotaxicology CHANGES OF CYTOTOXICITY AND IP2M-2851 UVB-INDUCED MITOGEN RESPONSE OF FISH (ROACH, RUTILUS
RUTILUS) LEUKOCYTES Ilmari E. Jokinen *, Tuula M. Aahonen, Eveliina Markkula, Harri M. Salo. Department of Biological and Environmental Science,
University of Jyvaskyla, Jyvaskyla',Finland Exposure to ultraviolet B (UVB) radiation induces local and systemic changes of immunological functions in mammals. Very little is known of the effects of UV radiation on the immune system of lower vertebrates. In this study adult cyprinid fish, roach, were exposed to a single dose of UVB. Fish were sampled 1, 3, 7 and 14 days after exposure and leukocytes from spleen and anterior kidney were separated by density gradient. Natural cytotoxic cell (NCC) activity by cells from the kidney were assayed and mitogen stimulated cultures of splenic lymphocytes were performed. Cells were cultured for 3 days and proliferative responses monitored by incorporation of radioactive thymidine. After exposure to UVB NCC activity decreased and remained lower compared to controls for 14 days followed. Concanavalin A-driven proliferative responses of exposed fish decreased during the first day but increased later and reached the level of controls. When stimulated with lipopolysaccharide (LPS) an increase on day 1 was noticed followed with a steep decreased on days 3 to 14. These results show altered immune functions after exposure to UVB suggesting potential of increased UVB to compromize integrity of fish immune system.
Keywords: ultraviolet-light; UVB; fish; natural cytotoxicity; proliferative response; mitogens
P2M-286 ] EVALUATION OF THE ECOTOXICITY OF INDUSTRIAL WASTES BY MICROTOX BIOASSAY
B. Coya *, E. Marafi6n, H. Sastre. Department of Chemical and
Environmental Engineering, Universityof Oviedo, 33204 Gij6n, Spain The purpose of this study is to assess the potential toxic effects of leachate generated by industrial solid wastes in Asturias, Spain. The wastes studied were residues from the coal industry, such as coal rejects tailings from slag heaps and from washeries, ash and slag from a power station (LADA) and residues from the metallurgical industry. Leachate was obtained from solid waste samples using the Extract Procedure protocol, in an attempt to simulate naturally occurring leachate, and the toxicity of the leachates was subsequently determined using the Microtox test. This is a bioassay based on the reduction of light emitted by a non-pathogenic strain of luminescent marine bacterium, Photobacterium phosphoreum, upon exposure to a toxic substance. Results are reported as the Effective Concentration values, ECso, the concentration at which 50% loss of light emission is observed. The values of the ECs0 found in the samples were correlated with analytical metal data in order to ascertain the different metal concentrations in the samples. Although these concentrations were noticeable, all the ECs0 values found were above 25576 mg/1. According to European legislation, a residue is classified as ecotoxic if the ECs0 of its leachate is < 3000 mg/l. The leachates were hence classified as "non-ecotoxic".
Keywords: ecotoxicity; bioassay; heavy metals; Microtox; industrial waste
79
P2M-287 ] BIOMARKERS TO DETECT ENVIRONMENTAL POLLUTION
Juan L6pez-Barea. Department of Biochemistry and Molecular
Biology, Faculty of Veterinary, Universityof C6rdoba, Spain Biomarkers are parameters assessed in fluids, cells, tissues, organisms or ecosystems to indicate the presence of pollutants or the responses of bioindicators. Molecular biomarkers, such as induction of biotransforming enzymes or some particular isoenzymes, are early-warning signals of ecological distress. Xenobiotics can be biotransformed into more reactive metabolites, responsible for their toxic or genotoxic effects, which are assessed with bacterial mutagenesis tests. In animals from selected areas of the Spanish littoral we have studied new molecular biomarkers of exposure and effect. In bivalve molluscs, direct-acting oxidative polar genotoxins are detected and an inverse relationship with detoxifying and antioxidative enzymes is found. Fish from polluted areas show oxidized glutathione status and higher activity of detoxifying and antioxidative enzymes, such as GSH-transferase and cytochrome P450 1A1. These increased activities correlated with highly induced promutagen activation capability. Specific induction of particular isoenzymes was also studied. Fish from polluted areas show three new Cu,Zn-SOD forms after electrophoretic separation and staining for enzymatic activity. These new SOD isoenzymes are formed by oxidative attack and are good biomarkers of oxidative damage. A new HPLC procedure allows the separation and "on line" assay of 11 glutathione transferase isoenzymes and significant differences are observed between the profiles of fish from control and polluted areas. The new biomarkers have been validated by comparison with those previously well-established and by exposure to model xenobiotics under controlled conditions. We are now developing new molecular biomarkers based in Molecular Biology approaches. Thus, oxidative DNA damages are being assessed by analyzing 8-oxo desoxiguanosine in DNA hydrolyzates by HPLC with electrochemical detection. In addition, cytochrome P450, GST and metallothionein gene expression is being analyzed by Northern blotting using as specific probes the corresponding genes isolated by PCR.
Keywords: molluscs; fish; oxidative stress; promutagen activation; superoxide dismutase; glutathione transferase
P2M-288 ] TRANSLOCATION OF ALDICARB SULPHONE ACROSS THE DUCK EGG SHELL
L. Murphy McMahon *, C.M. Chambers, P.L. Chambers.
Department of Pharmacology and Therapeutics, Universityof Dublin, Trinity College, Dublin, Ireland Highly water soluble carbamate pesticides have been used to model the hazard presented by water soluble xenobiotics to the developing duck embryo. The shell of fertile 3 day old duck eggs were painted with 15/zmol aldicarb (CAS 116-06-3), or its metabolites, aldicarb sulphone (CAS 1646-88-4) or aldicarb sulphoxide (CAS 1646-87-3). After 24 days incubation, they all produced a statistically significant shortening of the tarsometatarsus and middle web toe [1,2]. Both the metabolites are considered to be responsible for the long term systemic pesticidal action of aldicarb. Aldicarb sulphone (ASN) is the most stable oxidation metabolite in both plants and animals [3]. Detection of N-methyl carbamates in the egg is difficult. We have developed a method for ASN [4]. The samples were dried in Na2SO4 and soxhlet extracted with dichloromethane. A silica SPE column provided clean-up. Quantification was accomplished using HPLC with post column derivatisation coupled with fluorometric detection. Our current limit of detection for ASN is 1.11 x 10-7 g per g fertile duck egg with recovery levels greater than 90% (n = 13). Three day old fertile duck eggs were killed by chilling at 4 ° C for 3 days. The eggs were then painted with 5/zmol ASN and kept at
RUTILUS) LEUKOCYTES Ilmari E. Jokinen *, Tuula M. Aahonen, Eveliina Markkula, Harri M. Salo. Department of Biological and Environmental Science,
University of Jyvaskyla, Jyvaskyla',Finland Exposure to ultraviolet B (UVB) radiation induces local and systemic changes of immunological functions in mammals. Very little is known of the effects of UV radiation on the immune system of lower vertebrates. In this study adult cyprinid fish, roach, were exposed to a single dose of UVB. Fish were sampled 1, 3, 7 and 14 days after exposure and leukocytes from spleen and anterior kidney were separated by density gradient. Natural cytotoxic cell (NCC) activity by cells from the kidney were assayed and mitogen stimulated cultures of splenic lymphocytes were performed. Cells were cultured for 3 days and proliferative responses monitored by incorporation of radioactive thymidine. After exposure to UVB NCC activity decreased and remained lower compared to controls for 14 days followed. Concanavalin A-driven proliferative responses of exposed fish decreased during the first day but increased later and reached the level of controls. When stimulated with lipopolysaccharide (LPS) an increase on day 1 was noticed followed with a steep decreased on days 3 to 14. These results show altered immune functions after exposure to UVB suggesting potential of increased UVB to compromize integrity of fish immune system.
Keywords: ultraviolet-light; UVB; fish; natural cytotoxicity; proliferative response; mitogens
P2M-286 ] EVALUATION OF THE ECOTOXICITY OF INDUSTRIAL WASTES BY MICROTOX BIOASSAY
B. Coya *, E. Marafi6n, H. Sastre. Department of Chemical and
Environmental Engineering, Universityof Oviedo, 33204 Gij6n, Spain The purpose of this study is to assess the potential toxic effects of leachate generated by industrial solid wastes in Asturias, Spain. The wastes studied were residues from the coal industry, such as coal rejects tailings from slag heaps and from washeries, ash and slag from a power station (LADA) and residues from the metallurgical industry. Leachate was obtained from solid waste samples using the Extract Procedure protocol, in an attempt to simulate naturally occurring leachate, and the toxicity of the leachates was subsequently determined using the Microtox test. This is a bioassay based on the reduction of light emitted by a non-pathogenic strain of luminescent marine bacterium, Photobacterium phosphoreum, upon exposure to a toxic substance. Results are reported as the Effective Concentration values, ECso, the concentration at which 50% loss of light emission is observed. The values of the ECs0 found in the samples were correlated with analytical metal data in order to ascertain the different metal concentrations in the samples. Although these concentrations were noticeable, all the ECs0 values found were above 25576 mg/1. According to European legislation, a residue is classified as ecotoxic if the ECs0 of its leachate is < 3000 mg/l. The leachates were hence classified as "non-ecotoxic".
Keywords: ecotoxicity; bioassay; heavy metals; Microtox; industrial waste
79
P2M-287 ] BIOMARKERS TO DETECT ENVIRONMENTAL POLLUTION
Juan L6pez-Barea. Department of Biochemistry and Molecular
Biology, Faculty of Veterinary, Universityof C6rdoba, Spain Biomarkers are parameters assessed in fluids, cells, tissues, organisms or ecosystems to indicate the presence of pollutants or the responses of bioindicators. Molecular biomarkers, such as induction of biotransforming enzymes or some particular isoenzymes, are early-warning signals of ecological distress. Xenobiotics can be biotransformed into more reactive metabolites, responsible for their toxic or genotoxic effects, which are assessed with bacterial mutagenesis tests. In animals from selected areas of the Spanish littoral we have studied new molecular biomarkers of exposure and effect. In bivalve molluscs, direct-acting oxidative polar genotoxins are detected and an inverse relationship with detoxifying and antioxidative enzymes is found. Fish from polluted areas show oxidized glutathione status and higher activity of detoxifying and antioxidative enzymes, such as GSH-transferase and cytochrome P450 1A1. These increased activities correlated with highly induced promutagen activation capability. Specific induction of particular isoenzymes was also studied. Fish from polluted areas show three new Cu,Zn-SOD forms after electrophoretic separation and staining for enzymatic activity. These new SOD isoenzymes are formed by oxidative attack and are good biomarkers of oxidative damage. A new HPLC procedure allows the separation and "on line" assay of 11 glutathione transferase isoenzymes and significant differences are observed between the profiles of fish from control and polluted areas. The new biomarkers have been validated by comparison with those previously well-established and by exposure to model xenobiotics under controlled conditions. We are now developing new molecular biomarkers based in Molecular Biology approaches. Thus, oxidative DNA damages are being assessed by analyzing 8-oxo desoxiguanosine in DNA hydrolyzates by HPLC with electrochemical detection. In addition, cytochrome P450, GST and metallothionein gene expression is being analyzed by Northern blotting using as specific probes the corresponding genes isolated by PCR.
Keywords: molluscs; fish; oxidative stress; promutagen activation; superoxide dismutase; glutathione transferase
P2M-288 ] TRANSLOCATION OF ALDICARB SULPHONE ACROSS THE DUCK EGG SHELL
L. Murphy McMahon *, C.M. Chambers, P.L. Chambers.
Department of Pharmacology and Therapeutics, Universityof Dublin, Trinity College, Dublin, Ireland Highly water soluble carbamate pesticides have been used to model the hazard presented by water soluble xenobiotics to the developing duck embryo. The shell of fertile 3 day old duck eggs were painted with 15/zmol aldicarb (CAS 116-06-3), or its metabolites, aldicarb sulphone (CAS 1646-88-4) or aldicarb sulphoxide (CAS 1646-87-3). After 24 days incubation, they all produced a statistically significant shortening of the tarsometatarsus and middle web toe [1,2]. Both the metabolites are considered to be responsible for the long term systemic pesticidal action of aldicarb. Aldicarb sulphone (ASN) is the most stable oxidation metabolite in both plants and animals [3]. Detection of N-methyl carbamates in the egg is difficult. We have developed a method for ASN [4]. The samples were dried in Na2SO4 and soxhlet extracted with dichloromethane. A silica SPE column provided clean-up. Quantification was accomplished using HPLC with post column derivatisation coupled with fluorometric detection. Our current limit of detection for ASN is 1.11 x 10-7 g per g fertile duck egg with recovery levels greater than 90% (n = 13). Three day old fertile duck eggs were killed by chilling at 4 ° C for 3 days. The eggs were then painted with 5/zmol ASN and kept at