- Email: [email protected]
520 Relationship between blood lead concentration and free protoporphirin in erythrocytes in workers exposed to low lead levels
Poster Session P26. Heavy metals 24 hours under sub-toxic conditions. The intracellular GSH-content was monitored by sensitive HPLC with fluorescence detection. Supported by Philip Morris External Research 2002 Program MLU: 321003 517
EXPRESSION OF MRP1–5 IN HUMAN LUNG CELLS CULTURE WITH AN APICAL AIR SURFACE AND ITS CORRELATION TO CADMIUM AND ZINC IN RESPECT TO THE GLUTATHIONE POOL.
E. Stehfest, A.W. Torky, H. Foth. Institute of Environmental Toxicology, Martin Luther University Halle, Halle (Saale), Germany In treatment of lung cancer very often a failure of chemotherapy is observed. This phenomenon is caused by proteins such as multi drug resistance related proteins, MRP, a sub group of the ABC-binding cassette transport proteins. The expression, localization and function of the different MRP isoforms in the lung are widely unknown. At least MRP1–4 are supposed to be involved in the regulation of the glutathione pool and transport of conjugates. MRP1–7 are expressed on their mRNA level in peripheral lung cell (PLC) cultures in different quantities (real-time PCR). MRP1–5 could be determined in the cell culture qualitative by immuno-histochemistry. Trough modulation of the glutathione pool by Buthionine sulfoximine and N-acetylcysteine, the transport activity of MRP1 was modulated. Cadmium (2,5 and 5 µM) and zinc (10 – 20 µM) affected the MRP-expression in long term experiments (4 – 6 weeks). Under these conditions a clear raising (between doubling up to tripling) of the glutathione pool was found. To investigate the peripheral lung cells under more physiological conditions the cells were cultured in a alternative way: insert dishes with a semi permeable membrane provide a culture system with a wet (medium) and a dry (air) surface. These cultures were characterized by immuno-histochemistry as human lung cells and show a very different morphology than cells which were grown in plastic dishes covered with medium. Under these culture condition the lung cells develop their natural polarization. That makes it possible to localize the subcellular distribution of different mrp-isomers on their natural position and allows so to draw more profound conclusions about their specific function. Up to now we found MRP1, 3 and 5 localized in the cell membrane, especially in higher densities on cell-cell-contact sites. Research described in this abstract was supported by Philip Morris Incorporated, MLU: 328001 518
LOCALIZATION AND FUNCTION OF MRP1–5 IN HUMAN LUNG CELL CULTURES IN VITRO
A.W. Torky, E. Stehfest, H. Foth. Institute of Environmental Toxicology, Martin Luther University Halle, Halle (Saale), Germany, Tumor cell resistance to cytotoxic drugs is considered one of the major obstacles to successful chemotherapy. This phenomenon can be produced by ABC–(ATP-Binding-Cassette) transpotproteins. Prominent members of this family are transport-proteins summerized in the group of the multidrug-resistance-associated proteins. Inhibition of this transport is from major interest to provide a successfull therapy of cancer patients. The different functions of the isomers are closely related to their localiztion in the cell. We detected the MRP isomers 1–5 in peripheral human lung cell cultures by immuno-histochemistry. They are expressed in different manners and mainly found on the cell membrane. MRP1 was also detected intra-cellularly. We used a recently established model of an air/liquid interface culture of human lung cells to demonstrate where the single MRPisomers are situated (apical, baso-lateral or intracellular). Preliminary studies showed baso-lateral localization of MRP1 and 5. MRP is supposed to be involved in many toxicologically relevant processes by transport of metals as glutathion complexes. We used the heavy metal nickel because of its occupational relevance and iron because of its physiological importance to investigate their effect on MRP. Nickel and Iron were tested by means of MTT-test
s139
(vitality test) in order to work in concentrations below the acute cell toxicity. Lung cell cultures did not show any signs of toxicity up to nickel concentrations of 500µM and iron concentrations of 20µM in 24 hours test. We will verify how nickel (10µM) and iron (10µM) influence the expression of MRP1–5 mRNA in human lung cell cultures in short term experiments (24 hours) and long-term experiments (4 – 6 weeks) as other metals did show adaptation reaction. Supported by Philip Morris External Research 2002 Program MLU: 321003 and DFG Graduate College 519
EFFECTS OF THE LEAD ACETATE TREATMENT IN THE REPRODUCTIVE TRACT OF THE MALE RATS.
M.J. Ochoa 1 , J.L. Morán 2 , C. Morán 2 , A. Handal 2 . 1 Escuela de Biología Universidad Autónoma de Puebla, Puebla, México. Laboratorio de Investigaciones Biológicas2 del Instituto de Ciencias de la Benemérita Universidad Autónoma de Puebla, Puebla, México The aim of this investigation was to analyze the effects of the lead treatment in the morphology and physiology of the male reproductive tract. Male rats of 1-month of age the stock CII-ZV. They were separated in four experimental groups of eight animal each one including the control group. To each group respectively was administered daily 0.0, 0.003, 0.03 and 0.6 g/l of lead acetate in the drinking water, during four months. The different groups were weighed and sacrificed by decapitation. To each one, were weighed and dissected the testes. Of each animal was collected the blood to analyze the lead concentration. The results showed in Sertoli cells, nuclei appeared fragmentated. Diminution in the number of the Leyding cells and differences in the arrangement of the spermatozoids into seminiferous tubules. These results suggest that increase in the lead concentration in the blood increase testicular atrophy, cellular degeneration, and reductions in the spermatic count. 520
RELATIONSHIP BETWEEN BLOOD LEAD CONCENTRATION AND FREE PROTOPORPHIRIN IN ERYTHROCYTES IN WORKERS EXPOSED TO LOW LEAD LEVELS
Z. Paskalev 1 , D. Aposolova 2 , S. Pavlova 2 , D. Adjaraov 2 . 1 National Center of Radiobiology and Radiation Protection, Center of Occupational Diseases, Clinic of Toxicology, Sofia, Bulgaria In recent years the medical literature has focused its attention on biological effects resulting from low to moderate levels of Pb exposure. It is well known that elevations in PbB are associated to an increase in free protoporphyrin in erythrocytes levels (EPP). A significant correlation between these two indices can be observed the biological response (EPP). In the present study, the relationships between PbB and EPP were evaluated in 192 workers of a battery factory consisting of males with varying degrees of occupational lead poisoning. The number of workers with PbB lower than 2,1 µmol/L (Group I) was 115 workers /mean age 42, range 24 – 56 years), and PbB higher than 2,1 (umol/L) to 4,6 (umol/L) – 67 workers (Group II) (mean age 55, range 31 – 60 years). For each of the workers examined in this study we determined the PbB (umol/L) and EPP (nmol) gHb, using the method of Pionrellis. The length of exposure was from 2 to 15 years. Simultaneous measurement of free protoporphyrin in erythrocytes showed hight diagnostic sensitivity in detecting lead poisoning in occupationally exposed subjects. The results shown high interindividual variability of the EPP. In our study, we used the mean of PbB and EPP values and standard deviation for group I and group II. For each group statistically significant correlation was found between PbB concentration and EPP, expressed in the following equation: For group I: EPP (nmol/gHB) = (1,6 ± 0,8) ± (28,3 ± 8,2) PbB (µmol/L) correlation coefficient r = (0,54 ± 0,11). For group II EPP (nmol/gHG) = (2,8±0,9) ± (34,1±10,8) PbB (nmol/L) correlation coefficient = (0,54 ± 0,11). In fact, free protoporphyrin in erythrocytes levels increase exponentially when there is a sharp increase in PbB. In this study, we observed that an increase in EPP level is
s140
Poster Session P26. Heavy metals
associated with lead intoxication since Pb interference in Fe incorporation in heme molecules determines a gradualaccumulataion of portoporphyrin in erythrocytes. 521
EFFECT OF CHRONIC LEAD EXPOSURE ON PROAPOPTOTIC BAX AND ANTIAPOPTOTIC BCL-2 PROTEIN EXPRESSION IN RAT HIPPOCAMPUS IN VIVO
A.M. Sharifi, S. Baniasadi, M. Jorjani, F. Rahimi, M. Bakhshayesh. Department of Pharmacology & Cellular and Molecular Research Center, Iran University of Medical Sciences,Tehran, Iran Despite reduction in environmental lead, chronic lead exposure still posess a public health hazard, particularly in children, with devastating effects on developing CNS. To investigate the mechanism of this neurotoxicity, young and adult rats were used to study whether exposure to low concentrations of lead could induce apoptosis in hippocampus. 2–4 and 12–14- week-old rats received lead acetate in concentration of 500 ppm for 40 days. Control animals received deionized distilled water. In lead treated groups, the Blood lead levels was increased by 3–4 folds. Light and electron microscopical study of hippocampus revealed increased apoptotic cells. Western blot analysis of Bax and Bcl-2 (pro- and antiapoptotic gene products respectively) indicated higher expression of Bax protein and no significant change in bcl-2 expression and accordingly increased the Bax/Bcl-2 ratio compared to control group, confirming the histological study. In conclusion these data suggest that neurotoxicity of chronic lead exposure in hippocampus in vivo may partly be due to facilitation of apoptosis. 522
INFLUENCE OF HEAVY METALS FROM MASS GRAVES BONES ON IDENTIFICATION BY GENOMIC DNA
D. Sutlovic 1 , S. Andjelinovi´c 1 , M. Definis Gojanovi´c 1 , J. Pavlov 2 . 1 Department of Pathology and Forensic Medicine, Split University Hospital and School of Medicine, Split, Croatia, 2 Public Health Institute of Split-Dalmatia County, Split, Croatia The identification process of dead bodies or human remains is being conducted under different circumstances. Exhumation and war victim identification have a special connotation. Different identification methods are used depending on the case circumstance and the state of postmortem body changes. One of the methods is the identification by DNA typing from different biological samples (genotyping). Considering to the fact that every person inherits half of the genetic material from the mother and half from the father, DNA typing can verify the relationship between the examined persons. A particular problem is the isolation and DNA typing from human remains found in mass graves, that had undergone the degradation process, as well as postmortem DNA contamination with bacteria, fungi, humic acids, metals etc. This study analyzed the possible influence of metal ions on the successfulness of DNA typing of bone samples from mass graves. The study included 30 bone samples from mass graves and the 5 fresh bone samples. Successful and unsuccessful DNA typing has been determined the concentration of iron, cooper, lead and cadmium ions and their possible correlation. The influence of single metal ions the and influence of different combinations and concentrations of iron, cooper, cadmium and lead ions on DNA typing has been analyzed through in vitro experiments with fresh bone suspensions and metal ions. The results revealed that iron, cooper, lead and cadmium ions, if present in bone samples from mass graves, do not inhibit DNA amplification, while they inhibit the DNA amplification only if they are present in the amplification reaction mix.
523
GENETIC VARIABILITY OF δ-AMINOLEVULINIC ACID DEHYDRATASE (δ-ALA) AND THE WHOLE BLOOD LEAD CONCENTRATION IN NORTHEAST SPAIN.
M. Torra 1,2 , C. Barrot 2 , M. Ortega 2 , A. Xifró 2 , E. Huguet 2 , J. Corbella 1,2 , M. Gené 2,3 . 1 Servei de Toxicologia, Hospital Clínic de Barcelona; 2 Escola Professional de Medicina del Treball de la Universitat de Barcelona; 3 Unitat Bàsica de Prevenció, Hospital Universitari de Bellvitge, C/ Villarroel 170, 08036 Barcelona. Spain The second enzyme of the heme biosynthetic pathway δ-aminolevulinic acid dehydratase, is a cytosolic enzyme that catalyses the condensation of two molecules of 5-aminolevulinat (ALA) to form porphobilinogen. The inhibition of ALA-D activity by lead is a sensitive indicator of exposure and has been used as a diagnostic tool. The gene that encodes ALAD exists in two polymorphic forms that may modify lead toxicokinetics, bioaccumulation and ultimately influences individual susceptibility to lead poisoning. This gene is located in chromosome 9q34, which has two codominant alleles: ALAD1 and ALAD2. The ALAD2 allele contains a G → C transversion at position 177 of the coding region, resulting in the substitution of a positive charged lysine by a neutral aspargine at amnioacid 59 concluding in a higher affinity for lead by the ALAD-2. This substitution is responsible of three different isoenzyme phenotypes: ALAD1–1, ALAD1–2 and ALAD2–2. Another mutation consisting in a T → C transversion, without translation changes, increases the gene polymorphic degree. Venous blood samples were obtained from 100 healthy subjects residing in Barcelona, Spain. Blood lead concentration was determined by means of a graphite oven atomic absorption spectrometry. The ALAD1 and ALAD2 alleles were detected by amplification of a 916bp region of genomic DNA and digested with the restriction endonucleases MspI and RsaI. The cleavage products are then analyzed on agarose gel using ethidium bromide and ultraviolet detection. To investigate the possible relation between the blood lead concentration and the ALA-D polymorphism, a model of multiple regression was applied. The preliminary results, obtained until now, seem to indicate that the presence of the ALAD2 allele only contributes to modify the toxicokinetics of lead at high exposure levels. 524
THE CHANGES IN THE PARAMETERS OF ENERGETIC STATUS OF THE STELLATE STURGEON Acipenser stellatus Palls SPERMATOZOA AFTER SHORT-TIME EXPOSURE TO LEAD IONS IN VITRO
I. Baranowska-Bosiacka 1 , A. Rzemieniecki 2 , M. Rutkowska 1 , G.J. Dietrich 3 , A. Ciereszko 3 , J. Głogowski 3 , J. Domagala 2 , A.J. Hlynczak 1 . 1 Department of Biochemistry, University of Szczecin, Poland, 2 Department of Zoology, University of Szczecin, Felczaka 3a St, 70–412 Szczecin, Poland, 3 Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10–747 Olsztyn, Poland Objectives: The aim of this work was to evaluate the influence of short-time exposure to lead ions on the parameters of energetic status of stellate sturgeon (Acipenser stellatus, Palls) spermatozoa. Material: The experiment was carried out on milt samples obtained from three 5-year-old stellate surgeon males. Methods: Sperm production was stimulated by intraperitoneal injection of 18–20 µg [D-Ala6 ,Pro9 Net] m-GnRH+ 8–10 mg of metoclopramide (formulated in Ovopel). Milt was collected 48 h after injection. Spermatozoa were separated from semen plasma by centrifugation and resuspended in sperm immobilizing solution (20mM Tris-HCl buffer, pH 8,0, containing 400 mM saccharose). Then solutions of lead acetate dissolved in the immobilizing solution were added to the final lead ion concentrations: of 1, 10, 100 µg Pb/dl. Samples were incubated in standard conditions for 0 min; 1h; 4 h; 24 h. The concentrations of ATP, ADP, AMP, adenosine (Ado), GTP, GDP, GMP, guanosine (Guo), IMP, inosine (Ino), NAD, NADP, hypoxantine (Hyp) in spermatozoa were measured using HPLC. Adenylate (guanylate) energy charge- AEC(GEC) and total adenine (guanine) nucleotide-TAN(TGN) were calculated.
EXPRESSION OF MRP1–5 IN HUMAN LUNG CELLS CULTURE WITH AN APICAL AIR SURFACE AND ITS CORRELATION TO CADMIUM AND ZINC IN RESPECT TO THE GLUTATHIONE POOL.
E. Stehfest, A.W. Torky, H. Foth. Institute of Environmental Toxicology, Martin Luther University Halle, Halle (Saale), Germany In treatment of lung cancer very often a failure of chemotherapy is observed. This phenomenon is caused by proteins such as multi drug resistance related proteins, MRP, a sub group of the ABC-binding cassette transport proteins. The expression, localization and function of the different MRP isoforms in the lung are widely unknown. At least MRP1–4 are supposed to be involved in the regulation of the glutathione pool and transport of conjugates. MRP1–7 are expressed on their mRNA level in peripheral lung cell (PLC) cultures in different quantities (real-time PCR). MRP1–5 could be determined in the cell culture qualitative by immuno-histochemistry. Trough modulation of the glutathione pool by Buthionine sulfoximine and N-acetylcysteine, the transport activity of MRP1 was modulated. Cadmium (2,5 and 5 µM) and zinc (10 – 20 µM) affected the MRP-expression in long term experiments (4 – 6 weeks). Under these conditions a clear raising (between doubling up to tripling) of the glutathione pool was found. To investigate the peripheral lung cells under more physiological conditions the cells were cultured in a alternative way: insert dishes with a semi permeable membrane provide a culture system with a wet (medium) and a dry (air) surface. These cultures were characterized by immuno-histochemistry as human lung cells and show a very different morphology than cells which were grown in plastic dishes covered with medium. Under these culture condition the lung cells develop their natural polarization. That makes it possible to localize the subcellular distribution of different mrp-isomers on their natural position and allows so to draw more profound conclusions about their specific function. Up to now we found MRP1, 3 and 5 localized in the cell membrane, especially in higher densities on cell-cell-contact sites. Research described in this abstract was supported by Philip Morris Incorporated, MLU: 328001 518
LOCALIZATION AND FUNCTION OF MRP1–5 IN HUMAN LUNG CELL CULTURES IN VITRO
A.W. Torky, E. Stehfest, H. Foth. Institute of Environmental Toxicology, Martin Luther University Halle, Halle (Saale), Germany, Tumor cell resistance to cytotoxic drugs is considered one of the major obstacles to successful chemotherapy. This phenomenon can be produced by ABC–(ATP-Binding-Cassette) transpotproteins. Prominent members of this family are transport-proteins summerized in the group of the multidrug-resistance-associated proteins. Inhibition of this transport is from major interest to provide a successfull therapy of cancer patients. The different functions of the isomers are closely related to their localiztion in the cell. We detected the MRP isomers 1–5 in peripheral human lung cell cultures by immuno-histochemistry. They are expressed in different manners and mainly found on the cell membrane. MRP1 was also detected intra-cellularly. We used a recently established model of an air/liquid interface culture of human lung cells to demonstrate where the single MRPisomers are situated (apical, baso-lateral or intracellular). Preliminary studies showed baso-lateral localization of MRP1 and 5. MRP is supposed to be involved in many toxicologically relevant processes by transport of metals as glutathion complexes. We used the heavy metal nickel because of its occupational relevance and iron because of its physiological importance to investigate their effect on MRP. Nickel and Iron were tested by means of MTT-test
s139
(vitality test) in order to work in concentrations below the acute cell toxicity. Lung cell cultures did not show any signs of toxicity up to nickel concentrations of 500µM and iron concentrations of 20µM in 24 hours test. We will verify how nickel (10µM) and iron (10µM) influence the expression of MRP1–5 mRNA in human lung cell cultures in short term experiments (24 hours) and long-term experiments (4 – 6 weeks) as other metals did show adaptation reaction. Supported by Philip Morris External Research 2002 Program MLU: 321003 and DFG Graduate College 519
EFFECTS OF THE LEAD ACETATE TREATMENT IN THE REPRODUCTIVE TRACT OF THE MALE RATS.
M.J. Ochoa 1 , J.L. Morán 2 , C. Morán 2 , A. Handal 2 . 1 Escuela de Biología Universidad Autónoma de Puebla, Puebla, México. Laboratorio de Investigaciones Biológicas2 del Instituto de Ciencias de la Benemérita Universidad Autónoma de Puebla, Puebla, México The aim of this investigation was to analyze the effects of the lead treatment in the morphology and physiology of the male reproductive tract. Male rats of 1-month of age the stock CII-ZV. They were separated in four experimental groups of eight animal each one including the control group. To each group respectively was administered daily 0.0, 0.003, 0.03 and 0.6 g/l of lead acetate in the drinking water, during four months. The different groups were weighed and sacrificed by decapitation. To each one, were weighed and dissected the testes. Of each animal was collected the blood to analyze the lead concentration. The results showed in Sertoli cells, nuclei appeared fragmentated. Diminution in the number of the Leyding cells and differences in the arrangement of the spermatozoids into seminiferous tubules. These results suggest that increase in the lead concentration in the blood increase testicular atrophy, cellular degeneration, and reductions in the spermatic count. 520
RELATIONSHIP BETWEEN BLOOD LEAD CONCENTRATION AND FREE PROTOPORPHIRIN IN ERYTHROCYTES IN WORKERS EXPOSED TO LOW LEAD LEVELS
Z. Paskalev 1 , D. Aposolova 2 , S. Pavlova 2 , D. Adjaraov 2 . 1 National Center of Radiobiology and Radiation Protection, Center of Occupational Diseases, Clinic of Toxicology, Sofia, Bulgaria In recent years the medical literature has focused its attention on biological effects resulting from low to moderate levels of Pb exposure. It is well known that elevations in PbB are associated to an increase in free protoporphyrin in erythrocytes levels (EPP). A significant correlation between these two indices can be observed the biological response (EPP). In the present study, the relationships between PbB and EPP were evaluated in 192 workers of a battery factory consisting of males with varying degrees of occupational lead poisoning. The number of workers with PbB lower than 2,1 µmol/L (Group I) was 115 workers /mean age 42, range 24 – 56 years), and PbB higher than 2,1 (umol/L) to 4,6 (umol/L) – 67 workers (Group II) (mean age 55, range 31 – 60 years). For each of the workers examined in this study we determined the PbB (umol/L) and EPP (nmol) gHb, using the method of Pionrellis. The length of exposure was from 2 to 15 years. Simultaneous measurement of free protoporphyrin in erythrocytes showed hight diagnostic sensitivity in detecting lead poisoning in occupationally exposed subjects. The results shown high interindividual variability of the EPP. In our study, we used the mean of PbB and EPP values and standard deviation for group I and group II. For each group statistically significant correlation was found between PbB concentration and EPP, expressed in the following equation: For group I: EPP (nmol/gHB) = (1,6 ± 0,8) ± (28,3 ± 8,2) PbB (µmol/L) correlation coefficient r = (0,54 ± 0,11). For group II EPP (nmol/gHG) = (2,8±0,9) ± (34,1±10,8) PbB (nmol/L) correlation coefficient = (0,54 ± 0,11). In fact, free protoporphyrin in erythrocytes levels increase exponentially when there is a sharp increase in PbB. In this study, we observed that an increase in EPP level is
s140
Poster Session P26. Heavy metals
associated with lead intoxication since Pb interference in Fe incorporation in heme molecules determines a gradualaccumulataion of portoporphyrin in erythrocytes. 521
EFFECT OF CHRONIC LEAD EXPOSURE ON PROAPOPTOTIC BAX AND ANTIAPOPTOTIC BCL-2 PROTEIN EXPRESSION IN RAT HIPPOCAMPUS IN VIVO
A.M. Sharifi, S. Baniasadi, M. Jorjani, F. Rahimi, M. Bakhshayesh. Department of Pharmacology & Cellular and Molecular Research Center, Iran University of Medical Sciences,Tehran, Iran Despite reduction in environmental lead, chronic lead exposure still posess a public health hazard, particularly in children, with devastating effects on developing CNS. To investigate the mechanism of this neurotoxicity, young and adult rats were used to study whether exposure to low concentrations of lead could induce apoptosis in hippocampus. 2–4 and 12–14- week-old rats received lead acetate in concentration of 500 ppm for 40 days. Control animals received deionized distilled water. In lead treated groups, the Blood lead levels was increased by 3–4 folds. Light and electron microscopical study of hippocampus revealed increased apoptotic cells. Western blot analysis of Bax and Bcl-2 (pro- and antiapoptotic gene products respectively) indicated higher expression of Bax protein and no significant change in bcl-2 expression and accordingly increased the Bax/Bcl-2 ratio compared to control group, confirming the histological study. In conclusion these data suggest that neurotoxicity of chronic lead exposure in hippocampus in vivo may partly be due to facilitation of apoptosis. 522
INFLUENCE OF HEAVY METALS FROM MASS GRAVES BONES ON IDENTIFICATION BY GENOMIC DNA
D. Sutlovic 1 , S. Andjelinovi´c 1 , M. Definis Gojanovi´c 1 , J. Pavlov 2 . 1 Department of Pathology and Forensic Medicine, Split University Hospital and School of Medicine, Split, Croatia, 2 Public Health Institute of Split-Dalmatia County, Split, Croatia The identification process of dead bodies or human remains is being conducted under different circumstances. Exhumation and war victim identification have a special connotation. Different identification methods are used depending on the case circumstance and the state of postmortem body changes. One of the methods is the identification by DNA typing from different biological samples (genotyping). Considering to the fact that every person inherits half of the genetic material from the mother and half from the father, DNA typing can verify the relationship between the examined persons. A particular problem is the isolation and DNA typing from human remains found in mass graves, that had undergone the degradation process, as well as postmortem DNA contamination with bacteria, fungi, humic acids, metals etc. This study analyzed the possible influence of metal ions on the successfulness of DNA typing of bone samples from mass graves. The study included 30 bone samples from mass graves and the 5 fresh bone samples. Successful and unsuccessful DNA typing has been determined the concentration of iron, cooper, lead and cadmium ions and their possible correlation. The influence of single metal ions the and influence of different combinations and concentrations of iron, cooper, cadmium and lead ions on DNA typing has been analyzed through in vitro experiments with fresh bone suspensions and metal ions. The results revealed that iron, cooper, lead and cadmium ions, if present in bone samples from mass graves, do not inhibit DNA amplification, while they inhibit the DNA amplification only if they are present in the amplification reaction mix.
523
GENETIC VARIABILITY OF δ-AMINOLEVULINIC ACID DEHYDRATASE (δ-ALA) AND THE WHOLE BLOOD LEAD CONCENTRATION IN NORTHEAST SPAIN.
M. Torra 1,2 , C. Barrot 2 , M. Ortega 2 , A. Xifró 2 , E. Huguet 2 , J. Corbella 1,2 , M. Gené 2,3 . 1 Servei de Toxicologia, Hospital Clínic de Barcelona; 2 Escola Professional de Medicina del Treball de la Universitat de Barcelona; 3 Unitat Bàsica de Prevenció, Hospital Universitari de Bellvitge, C/ Villarroel 170, 08036 Barcelona. Spain The second enzyme of the heme biosynthetic pathway δ-aminolevulinic acid dehydratase, is a cytosolic enzyme that catalyses the condensation of two molecules of 5-aminolevulinat (ALA) to form porphobilinogen. The inhibition of ALA-D activity by lead is a sensitive indicator of exposure and has been used as a diagnostic tool. The gene that encodes ALAD exists in two polymorphic forms that may modify lead toxicokinetics, bioaccumulation and ultimately influences individual susceptibility to lead poisoning. This gene is located in chromosome 9q34, which has two codominant alleles: ALAD1 and ALAD2. The ALAD2 allele contains a G → C transversion at position 177 of the coding region, resulting in the substitution of a positive charged lysine by a neutral aspargine at amnioacid 59 concluding in a higher affinity for lead by the ALAD-2. This substitution is responsible of three different isoenzyme phenotypes: ALAD1–1, ALAD1–2 and ALAD2–2. Another mutation consisting in a T → C transversion, without translation changes, increases the gene polymorphic degree. Venous blood samples were obtained from 100 healthy subjects residing in Barcelona, Spain. Blood lead concentration was determined by means of a graphite oven atomic absorption spectrometry. The ALAD1 and ALAD2 alleles were detected by amplification of a 916bp region of genomic DNA and digested with the restriction endonucleases MspI and RsaI. The cleavage products are then analyzed on agarose gel using ethidium bromide and ultraviolet detection. To investigate the possible relation between the blood lead concentration and the ALA-D polymorphism, a model of multiple regression was applied. The preliminary results, obtained until now, seem to indicate that the presence of the ALAD2 allele only contributes to modify the toxicokinetics of lead at high exposure levels. 524
THE CHANGES IN THE PARAMETERS OF ENERGETIC STATUS OF THE STELLATE STURGEON Acipenser stellatus Palls SPERMATOZOA AFTER SHORT-TIME EXPOSURE TO LEAD IONS IN VITRO
I. Baranowska-Bosiacka 1 , A. Rzemieniecki 2 , M. Rutkowska 1 , G.J. Dietrich 3 , A. Ciereszko 3 , J. Głogowski 3 , J. Domagala 2 , A.J. Hlynczak 1 . 1 Department of Biochemistry, University of Szczecin, Poland, 2 Department of Zoology, University of Szczecin, Felczaka 3a St, 70–412 Szczecin, Poland, 3 Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10–747 Olsztyn, Poland Objectives: The aim of this work was to evaluate the influence of short-time exposure to lead ions on the parameters of energetic status of stellate sturgeon (Acipenser stellatus, Palls) spermatozoa. Material: The experiment was carried out on milt samples obtained from three 5-year-old stellate surgeon males. Methods: Sperm production was stimulated by intraperitoneal injection of 18–20 µg [D-Ala6 ,Pro9 Net] m-GnRH+ 8–10 mg of metoclopramide (formulated in Ovopel). Milt was collected 48 h after injection. Spermatozoa were separated from semen plasma by centrifugation and resuspended in sperm immobilizing solution (20mM Tris-HCl buffer, pH 8,0, containing 400 mM saccharose). Then solutions of lead acetate dissolved in the immobilizing solution were added to the final lead ion concentrations: of 1, 10, 100 µg Pb/dl. Samples were incubated in standard conditions for 0 min; 1h; 4 h; 24 h. The concentrations of ATP, ADP, AMP, adenosine (Ado), GTP, GDP, GMP, guanosine (Guo), IMP, inosine (Ino), NAD, NADP, hypoxantine (Hyp) in spermatozoa were measured using HPLC. Adenylate (guanylate) energy charge- AEC(GEC) and total adenine (guanine) nucleotide-TAN(TGN) were calculated.