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Oxidative stress and neurotoxicity markers in Paracentrotus lividus from Bizerte lagoon (North Tunisia)
Abstracts / Toxicology Letters 280S (2017) S202–S213
P-06-01-22 Acute and chronic effect of Diclofenac in D. magna and D. longispina João Joaquim Pharmacy, Instituto Politécnico de Coimbra/ESTeSC – Coimbra Health School, Coimbra, Portugal The continued increase in drug consumption leads to a greater risk of environmental contamination, and this is a concern as it may interfere with specific biological systems. The objective was to identify and evaluate the effects produced by Sodium Diclofenac, an active substance, in non-target aquatic organisms. Were performed acute and chronic tests with cladocerans, to compare the survival, reproduction, and growth. Diclofenac, are effective in the survivorship and reproduction of the two cladoceran species used in this study. D. magna seems to be more tolerant (EC50 = 134.087 mg/L), and we found more sensibility to Diclofenac in D. longispina (EC50 = 35.353 mg/L). Diclofenac significantly affects the fecundity (LOEC = 38.745 mg/L) of D. magna and the somatic growth rate (LOEC = 57.713 mg/L), at the last concentration tested. For D. longispina the sublethal endpoints significantly affected was fecundity (LOEC = 5.0 mg/L), maturation (LOEC = 2.5 mg/L). In the chronic exposure, the number and size of neonates of first brood are impaired, in both species. Diclofenac impairs in the survivorship, reproduction, and growth of the cladoceran species. However, the concentration levels used to produce these effects in acute and chronic tests are much higher, if we compare with the concentration levels detected in the aquatic environment. Diclofenac affects more D. longispina at individual level endpoints (fecundity and maturation), in opposite to D. magna, fecundity is impaired, and the somatic growth rate is slightly, but significantly affected by the extreme concentration tested. http://dx.doi.org/10.1016/j.toxlet.2017.07.573 P-06-01-23 Role of nitric oxide and methyl jasmonate in minimizing heat stress toxicity in wheat (Triticum aestivum L.) Mehar Fatma, Baishnab C. Tripathy School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Heat stress toxicity in plants has become a major concern worldwide because different global circulation models have predicted that greenhouse gases will gradually increase the world’s average ambient temperature and will lead to global warming substantially influencing the growth of plants. Plant exposed to high temperature, has impaired chlorophyll biosynthesis due to down regulation of gene involved in its biosynthesis pathway. This influences the growth, development and yield of crops and therefore has become a major concern in the world. In order to understand the course of action that give rise to tolerance of high temperature, the role of nitric oxide (NO) in methyl jasmonate (MJ) signaling pathway on stomatal response and photosynthetic performance was studied in wheat (Triticum aestivum L.) in the presence or absence of heat stress. The combined application of 100 M NO (as sodium nitroprusside) and 50 M MJ more prominently influenced stomatal behavior, photosynthetic and growth performance both in the optimal and heat stress. Heat-stressed plants had disorganized chloroplast thylakoids, but combined application of NO and MJ resulted in well-developed chloroplast thylakoids and properly stacked grana. These plants also showed increased production of
S209
cysteine (Cys), methionine (Met), reduced glutathione (GSH) and antioxidant system that help in reducing oxidative stress. These results suggested that NO and MJ influenced photosynthesis under heat stress by regulating oxidative stress by its effects on an antioxidant system and NO generation. http://dx.doi.org/10.1016/j.toxlet.2017.07.574 P-06-01-24 Introducing a microcystin congener toxicity equivalent Dorothy Yu Huang, Vichaya Charoensuk, David Donnelly, Xu Zhang, Dalia El-Mougi, David Kinniburgh Alberta Centre for Toxicology, University of Calgary, Calgary, Canada Microcystins (MCs) are hepatotoxins produced by several species of cyanobacteria. Depending on the amino acids present at the second and fourth positions of the ring structure, there have been over 200 members identified in this family sharing similar mechanism of action. MC-LR is the most studied congener with significant toxicological data. Therefore, many countries set guidelines based on MC-LR concentration. In recent years, with the availability of more congener reference materials and analytical methods, studies have shown that the relative amount of congener varies temporally and geographically. MC-LR is not necessarily the most prevalent congener in different parts of the world. Therefore, it is important to collect more toxicity information in order to revisit the guidelines. In this study, we applied two effect-based assays to evaluate nine microcystin congeners including LR, YR, RR, LY, LF, LW, LA, HtyR, dMeLR, dMeRR, as well as nodularin, tautomycin and okadaic acid. The protein phosphatase inhibition assay detects the toxicity potential at molecular level. The cytotoxicity assay, using the real time cell analyzer, assesses the toxicity at the cellular level. Both assays revealed that different microcystin congeners have different toxicity potentials. By introducing a conversion factor based on the IC50 relative to MC-LR IC50, congener concentration can be reported as MC-LR toxicity equivalent. The conversion factor was applied to several known mixtures. The results agreed well between effect-based assays and the accurate quantification by LC-HRMS. Our study demonstrated the potential application of toxicity equivalent for regulatory decisions. http://dx.doi.org/10.1016/j.toxlet.2017.07.575 P-06-01-25 Oxidative stress and neurotoxicity markers in Paracentrotus lividus from Bizerte lagoon (North Tunisia) Dhouha Boussoufa, Hela Chalouati, Safa Bejaoui, Feriel Ghribi, Mhamed El Cafsi Department of Biology, Unit of Physiology and Aquatic Environment, Tunis Faculty of Sciences, University of Tunis El Manar, Tunis/El Manar II, Tunisia This study was carried out in the Bizerte Lagoon, located on the northern coast of Tunisia. This lagoon supports industry and agriculture activities and several aquaculture farms along its shores. A spatial variation of physiological responses of the sea urchin Paracentrotus lividus collected from three sites was conducted. Lipid peroxidation (LPO), the activity of the antioxidant enzymes (catalase (CAT), glutathione peroxidase (GPx) and glutathione
S210
Abstracts / Toxicology Letters 280S (2017) S202–S213
S-transferase (GST)) and levels of the neurotoxic biomarker acetylcholinesterase (AChE) were evaluated. Samples were collected during autumn 2014 in three contrasting sites regarding their levels of pollution: a reference site (in the Bay of Bizerte), Chaara (in the northern sector of the lagoon, influenced by several industrial activities) and Menzel Bourguiba (in the southern sector, influenced by continental waters). After collection, sea urchins were immediately transferred to the laboratory and the gonads were separated, weighed and prepared for analysis. Relative to the control site, high levels of LPO was found in the gonad tissues of P. lividus collected from Chaara and Menzel Bourguiba indicating the induction of oxidative stress. The activities of antioxidant enzymes like CAT, GPx and GST increased significantly in P. lividus from Chaara and Menzel Bourguiba, whereas AChE activity was significantly higher in Menzel Bourguiba specimens compared to reference site. Measurements of oxidative stress biomarkers and AChE activity in P. lividus revealed differences between the sites in relation to their pollution state confirming previous reports. It was determined that Chaara was the most contaminated site due to their proximity to different pollution sources. http://dx.doi.org/10.1016/j.toxlet.2017.07.576 P-06-01-26 Frequency of sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-MET-STC)-producing airborne Aspergilli from flooded and unflooded area in Croatia Daniela Jakˇsic´ Despot 1 , Miranda Sertic´ 2 , Ana Mornar Turk 2 , ˇ Domagoj Kifer 3 , Biljana Nigovic´ 2 , Maja Segvi c´ Klaric´ 1 1
Department of Microbiology, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia 2 Department of Pharmaceutical Analysis, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia 3 Department of Biophysics, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
STC-producing Aspergilli, frequently airborne in damp dwellings, could contribute to STC intake by inhalation. In addition, 5-METSTC could be produced simultaneously resulting with alternating toxic effects. The purpose of this study was to compare STC/5-MET-STC producing abilities of airborne Aspergilli (sect. Versicolores) collected in September 2016 from flooded village (5 repaired/5 unrepaired houses and a school) and unflooded control village (5 houses and a school) in Croatia. STC and 5-MET-STC detection were performed by HPLC-DAD-ESI/MS/MS gradient method. STC-producing Aspergilli were more frequent in flooded village compared to control village (40–100%; 702 ± 975 CFU/m3 ). They were isolated from 2/5 unrepaired houses, 5/5 repaired houses and in school from the flooded area, while in the control village they were present in 1/5 houses. STC/5-MET-STC producing abilities were similar for all the isolates (N = 14) despite the location. 10/14 isolates produced both STC and 5-MET-STC (1.97 ± 1.05 g/mg and 5.06 ± 2.90 g/mg, respectively) while 2/14 produced only STC (4.48 ± 2.56 g/mg) and 1/14 only 5-MET-STC (6.05 g/mg). Significantly higher concentrations of STC/5-MET-STCproducing airborne Aspergilli in flooded houses suggest increased health risk due to expected STC/5-MET-STC content in inhalable airborne particles. Health effects due to the co-occurrence of STC and 5-MET-STC should be further explored.
http://dx.doi.org/10.1016/j.toxlet.2017.07.577 P-06-01-27 Assessment of nanoscale zero-valent iron toxicity towards several bacterial species by specific marker of oxidative stress monitoring Jaroslav Semerád 1,2 , Tomáˇs Cajthaml 1,2 1 Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic 2 Institute for Environmental Studies, Faculty of Science, Charles University, Prague, Czech Republic
Nanoscale zero-valent iron (nZVI) is a newly developed nanomaterial used in remediation technologies. As a result of its very high efficiency in degradation of various inorganic and organic pollutants, the worldwide application of nZVI is rapidly increasing. These iron nanoparticles are being introduced in high amounts (up to 20 g/L) to the environment even though their toxicity has not yet been properly investigated. The presented study includes a novel approach for toxicity determination of nZVI and for monitoring of its impact on 6 bacterial species (typical representatives of exposed organisms). The assay is based on oxidative stress marker formation in bacterial cultures after short term exposition to nanoparticles. The monitored marker is one of the most common toxic and mutagenic products of lipid peroxidation/degradation – malondialdehyde. During the toxicity testing of iron nanomaterials the bacterial cultures were extracted after exposition to nZVI and then malondialdehyde in the extract was derivatized. Determination and quantification of the derivatised marker were performed by HPLC with fluorescence detection. The results show high variability in specific toxicity towards the bacteria species even in the same genus. There was not a significant difference in toxicity between Gram positive and negative bacteria, but the production of malondialdehyde shows great dependence on bacterial size (i.e. surface). The results of this study show that the specific oxidative stress marker analysis enables determination and comparison of toxicity of nZVI and its derived nanomaterials on bacteria species before their direct use in the environment. http://dx.doi.org/10.1016/j.toxlet.2017.07.578 P-06-01-28 TiO2 -carbon nanotubes nanohybrid toxicity in Danio rerio embryo Gabriela Silva 1,2,3 , Zaira Clemente 3,2 , Francine Coa 2 , Lais Neto 2 , Vera Castro 3 , Diego Martinez 2,1 , Regina Monteiro 1 1
Center of Nuclear Energy in Agriculture, Piracicaba, Brazil Brazilian Nanotechnology National Laboratory, Brazilian Center for Research in Energy and Materials, Campinas, Brazil 3 Brazilian Agricultural Research Corporation (EMBRAPA), Jaguariuna, Brazil 2
Recent findings indicate that the combination of titanium dioxide (TiO2 ) with carbon nanotubes (CNT) increase the photocatalytic efficiency. Due to the importance of these new technologies, the production of such particles has been encouraged. However, concerns about their toxicity and safety when released into the environment are considerable. Therefore, to understand the role of TiO2 -MWCNT in the environment, our gold was to synthesize TiO2 -MWCNT nanomaterial, by mechanical mixing method,
P-06-01-22 Acute and chronic effect of Diclofenac in D. magna and D. longispina João Joaquim Pharmacy, Instituto Politécnico de Coimbra/ESTeSC – Coimbra Health School, Coimbra, Portugal The continued increase in drug consumption leads to a greater risk of environmental contamination, and this is a concern as it may interfere with specific biological systems. The objective was to identify and evaluate the effects produced by Sodium Diclofenac, an active substance, in non-target aquatic organisms. Were performed acute and chronic tests with cladocerans, to compare the survival, reproduction, and growth. Diclofenac, are effective in the survivorship and reproduction of the two cladoceran species used in this study. D. magna seems to be more tolerant (EC50 = 134.087 mg/L), and we found more sensibility to Diclofenac in D. longispina (EC50 = 35.353 mg/L). Diclofenac significantly affects the fecundity (LOEC = 38.745 mg/L) of D. magna and the somatic growth rate (LOEC = 57.713 mg/L), at the last concentration tested. For D. longispina the sublethal endpoints significantly affected was fecundity (LOEC = 5.0 mg/L), maturation (LOEC = 2.5 mg/L). In the chronic exposure, the number and size of neonates of first brood are impaired, in both species. Diclofenac impairs in the survivorship, reproduction, and growth of the cladoceran species. However, the concentration levels used to produce these effects in acute and chronic tests are much higher, if we compare with the concentration levels detected in the aquatic environment. Diclofenac affects more D. longispina at individual level endpoints (fecundity and maturation), in opposite to D. magna, fecundity is impaired, and the somatic growth rate is slightly, but significantly affected by the extreme concentration tested. http://dx.doi.org/10.1016/j.toxlet.2017.07.573 P-06-01-23 Role of nitric oxide and methyl jasmonate in minimizing heat stress toxicity in wheat (Triticum aestivum L.) Mehar Fatma, Baishnab C. Tripathy School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Heat stress toxicity in plants has become a major concern worldwide because different global circulation models have predicted that greenhouse gases will gradually increase the world’s average ambient temperature and will lead to global warming substantially influencing the growth of plants. Plant exposed to high temperature, has impaired chlorophyll biosynthesis due to down regulation of gene involved in its biosynthesis pathway. This influences the growth, development and yield of crops and therefore has become a major concern in the world. In order to understand the course of action that give rise to tolerance of high temperature, the role of nitric oxide (NO) in methyl jasmonate (MJ) signaling pathway on stomatal response and photosynthetic performance was studied in wheat (Triticum aestivum L.) in the presence or absence of heat stress. The combined application of 100 M NO (as sodium nitroprusside) and 50 M MJ more prominently influenced stomatal behavior, photosynthetic and growth performance both in the optimal and heat stress. Heat-stressed plants had disorganized chloroplast thylakoids, but combined application of NO and MJ resulted in well-developed chloroplast thylakoids and properly stacked grana. These plants also showed increased production of
S209
cysteine (Cys), methionine (Met), reduced glutathione (GSH) and antioxidant system that help in reducing oxidative stress. These results suggested that NO and MJ influenced photosynthesis under heat stress by regulating oxidative stress by its effects on an antioxidant system and NO generation. http://dx.doi.org/10.1016/j.toxlet.2017.07.574 P-06-01-24 Introducing a microcystin congener toxicity equivalent Dorothy Yu Huang, Vichaya Charoensuk, David Donnelly, Xu Zhang, Dalia El-Mougi, David Kinniburgh Alberta Centre for Toxicology, University of Calgary, Calgary, Canada Microcystins (MCs) are hepatotoxins produced by several species of cyanobacteria. Depending on the amino acids present at the second and fourth positions of the ring structure, there have been over 200 members identified in this family sharing similar mechanism of action. MC-LR is the most studied congener with significant toxicological data. Therefore, many countries set guidelines based on MC-LR concentration. In recent years, with the availability of more congener reference materials and analytical methods, studies have shown that the relative amount of congener varies temporally and geographically. MC-LR is not necessarily the most prevalent congener in different parts of the world. Therefore, it is important to collect more toxicity information in order to revisit the guidelines. In this study, we applied two effect-based assays to evaluate nine microcystin congeners including LR, YR, RR, LY, LF, LW, LA, HtyR, dMeLR, dMeRR, as well as nodularin, tautomycin and okadaic acid. The protein phosphatase inhibition assay detects the toxicity potential at molecular level. The cytotoxicity assay, using the real time cell analyzer, assesses the toxicity at the cellular level. Both assays revealed that different microcystin congeners have different toxicity potentials. By introducing a conversion factor based on the IC50 relative to MC-LR IC50, congener concentration can be reported as MC-LR toxicity equivalent. The conversion factor was applied to several known mixtures. The results agreed well between effect-based assays and the accurate quantification by LC-HRMS. Our study demonstrated the potential application of toxicity equivalent for regulatory decisions. http://dx.doi.org/10.1016/j.toxlet.2017.07.575 P-06-01-25 Oxidative stress and neurotoxicity markers in Paracentrotus lividus from Bizerte lagoon (North Tunisia) Dhouha Boussoufa, Hela Chalouati, Safa Bejaoui, Feriel Ghribi, Mhamed El Cafsi Department of Biology, Unit of Physiology and Aquatic Environment, Tunis Faculty of Sciences, University of Tunis El Manar, Tunis/El Manar II, Tunisia This study was carried out in the Bizerte Lagoon, located on the northern coast of Tunisia. This lagoon supports industry and agriculture activities and several aquaculture farms along its shores. A spatial variation of physiological responses of the sea urchin Paracentrotus lividus collected from three sites was conducted. Lipid peroxidation (LPO), the activity of the antioxidant enzymes (catalase (CAT), glutathione peroxidase (GPx) and glutathione
S210
Abstracts / Toxicology Letters 280S (2017) S202–S213
S-transferase (GST)) and levels of the neurotoxic biomarker acetylcholinesterase (AChE) were evaluated. Samples were collected during autumn 2014 in three contrasting sites regarding their levels of pollution: a reference site (in the Bay of Bizerte), Chaara (in the northern sector of the lagoon, influenced by several industrial activities) and Menzel Bourguiba (in the southern sector, influenced by continental waters). After collection, sea urchins were immediately transferred to the laboratory and the gonads were separated, weighed and prepared for analysis. Relative to the control site, high levels of LPO was found in the gonad tissues of P. lividus collected from Chaara and Menzel Bourguiba indicating the induction of oxidative stress. The activities of antioxidant enzymes like CAT, GPx and GST increased significantly in P. lividus from Chaara and Menzel Bourguiba, whereas AChE activity was significantly higher in Menzel Bourguiba specimens compared to reference site. Measurements of oxidative stress biomarkers and AChE activity in P. lividus revealed differences between the sites in relation to their pollution state confirming previous reports. It was determined that Chaara was the most contaminated site due to their proximity to different pollution sources. http://dx.doi.org/10.1016/j.toxlet.2017.07.576 P-06-01-26 Frequency of sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-MET-STC)-producing airborne Aspergilli from flooded and unflooded area in Croatia Daniela Jakˇsic´ Despot 1 , Miranda Sertic´ 2 , Ana Mornar Turk 2 , ˇ Domagoj Kifer 3 , Biljana Nigovic´ 2 , Maja Segvi c´ Klaric´ 1 1
Department of Microbiology, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia 2 Department of Pharmaceutical Analysis, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia 3 Department of Biophysics, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
STC-producing Aspergilli, frequently airborne in damp dwellings, could contribute to STC intake by inhalation. In addition, 5-METSTC could be produced simultaneously resulting with alternating toxic effects. The purpose of this study was to compare STC/5-MET-STC producing abilities of airborne Aspergilli (sect. Versicolores) collected in September 2016 from flooded village (5 repaired/5 unrepaired houses and a school) and unflooded control village (5 houses and a school) in Croatia. STC and 5-MET-STC detection were performed by HPLC-DAD-ESI/MS/MS gradient method. STC-producing Aspergilli were more frequent in flooded village compared to control village (40–100%; 702 ± 975 CFU/m3 ). They were isolated from 2/5 unrepaired houses, 5/5 repaired houses and in school from the flooded area, while in the control village they were present in 1/5 houses. STC/5-MET-STC producing abilities were similar for all the isolates (N = 14) despite the location. 10/14 isolates produced both STC and 5-MET-STC (1.97 ± 1.05 g/mg and 5.06 ± 2.90 g/mg, respectively) while 2/14 produced only STC (4.48 ± 2.56 g/mg) and 1/14 only 5-MET-STC (6.05 g/mg). Significantly higher concentrations of STC/5-MET-STCproducing airborne Aspergilli in flooded houses suggest increased health risk due to expected STC/5-MET-STC content in inhalable airborne particles. Health effects due to the co-occurrence of STC and 5-MET-STC should be further explored.
http://dx.doi.org/10.1016/j.toxlet.2017.07.577 P-06-01-27 Assessment of nanoscale zero-valent iron toxicity towards several bacterial species by specific marker of oxidative stress monitoring Jaroslav Semerád 1,2 , Tomáˇs Cajthaml 1,2 1 Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic 2 Institute for Environmental Studies, Faculty of Science, Charles University, Prague, Czech Republic
Nanoscale zero-valent iron (nZVI) is a newly developed nanomaterial used in remediation technologies. As a result of its very high efficiency in degradation of various inorganic and organic pollutants, the worldwide application of nZVI is rapidly increasing. These iron nanoparticles are being introduced in high amounts (up to 20 g/L) to the environment even though their toxicity has not yet been properly investigated. The presented study includes a novel approach for toxicity determination of nZVI and for monitoring of its impact on 6 bacterial species (typical representatives of exposed organisms). The assay is based on oxidative stress marker formation in bacterial cultures after short term exposition to nanoparticles. The monitored marker is one of the most common toxic and mutagenic products of lipid peroxidation/degradation – malondialdehyde. During the toxicity testing of iron nanomaterials the bacterial cultures were extracted after exposition to nZVI and then malondialdehyde in the extract was derivatized. Determination and quantification of the derivatised marker were performed by HPLC with fluorescence detection. The results show high variability in specific toxicity towards the bacteria species even in the same genus. There was not a significant difference in toxicity between Gram positive and negative bacteria, but the production of malondialdehyde shows great dependence on bacterial size (i.e. surface). The results of this study show that the specific oxidative stress marker analysis enables determination and comparison of toxicity of nZVI and its derived nanomaterials on bacteria species before their direct use in the environment. http://dx.doi.org/10.1016/j.toxlet.2017.07.578 P-06-01-28 TiO2 -carbon nanotubes nanohybrid toxicity in Danio rerio embryo Gabriela Silva 1,2,3 , Zaira Clemente 3,2 , Francine Coa 2 , Lais Neto 2 , Vera Castro 3 , Diego Martinez 2,1 , Regina Monteiro 1 1
Center of Nuclear Energy in Agriculture, Piracicaba, Brazil Brazilian Nanotechnology National Laboratory, Brazilian Center for Research in Energy and Materials, Campinas, Brazil 3 Brazilian Agricultural Research Corporation (EMBRAPA), Jaguariuna, Brazil 2
Recent findings indicate that the combination of titanium dioxide (TiO2 ) with carbon nanotubes (CNT) increase the photocatalytic efficiency. Due to the importance of these new technologies, the production of such particles has been encouraged. However, concerns about their toxicity and safety when released into the environment are considerable. Therefore, to understand the role of TiO2 -MWCNT in the environment, our gold was to synthesize TiO2 -MWCNT nanomaterial, by mechanical mixing method,