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Archives of Toxicology (v.80, #3)
Toxicological comments to the discussion about REACH by Helmut Greim; Michael Arand; Herman Autrup; Hermann M. Bolt; James Bridges; Erik Dybing; Rémi Glomot; Vito Foa; Rolf Schulte-Hermann (pp. 121-124).
The intention of REACH is to identify hazardous properties in order that a reliable risk assessment can be made and measures taken to deal with chemicals posing a significant risk.The recent debate has centered on ways in which the well established in vivo methods for risk assessment can be bypassed.The evidence that the available alternatives would support such replacement is weak. Progress to improve their value for risk assessment purposes is bound to be slow because the issues are very complex. As a group of European Toxicologists we strongly support the need for more research support in these areas, but we believe that over claims for progress is damaging their development.to reduce very substantially the estimation of hazard and risk with inevitable adverse consequences for human health and environmental protection, orto continue the existing methods until properly validated new methods are available.
Keywords: REACH; European Union; European Commission; Chemical substances legislation
Toxicological comments to the discussion about REACH by Helmut Greim; Michael Arand; Herman Autrup; Hermann M. Bolt; James Bridges; Erik Dybing; Rémi Glomot; Vito Foa; Rolf Schulte-Hermann (pp. 121-124).
The intention of REACH is to identify hazardous properties in order that a reliable risk assessment can be made and measures taken to deal with chemicals posing a significant risk.The recent debate has centered on ways in which the well established in vivo methods for risk assessment can be bypassed.The evidence that the available alternatives would support such replacement is weak. Progress to improve their value for risk assessment purposes is bound to be slow because the issues are very complex. As a group of European Toxicologists we strongly support the need for more research support in these areas, but we believe that over claims for progress is damaging their development.to reduce very substantially the estimation of hazard and risk with inevitable adverse consequences for human health and environmental protection, orto continue the existing methods until properly validated new methods are available.
Keywords: REACH; European Union; European Commission; Chemical substances legislation
Non-destructive micromethod for MRP1 functional assay in human lung tumor cells by Ekkehard Stehfest; Abdelrahman Torky; Felix Glahn; Heidi Foth (pp. 125-133).
Defense against toxic endo- and xenobiotics is a major concern of all living species and ABC transporters play a vital role in this defense system. Multidrug resistance associated proteins 1 (MRP1) is a cellular detoxifying factor supposed to transport a wide range of compounds across cell membranes either as GSH conjugates or as co-transport accompanying glutathione transposition. The cellular localization of MRP1 is a determining factor whether the transport function can take place. In this study we have undertaken experiments on the transport activity of MRP1 in cultured human lung tumor cells in order to check whether MRP1 is expressed as a functionally active protein. For this purpose we have adapted a quantitative fluorescence imaging assay to conditions where a small number of attached cells should be repeatedly measured by a non-destructive method. In cultured A549, H358 and H322 cells MRP1 is located in the cell membrane as observed by immunocytochemistry. Efflux of 5,6-carboxy-2′-7′-dichloro-fluorescein (CDF) from lung cells was sensitive toward the MRP1 inhibitor MK571 while verapamil had no effect. On the other hand, efflux of Rhodamin 123, a Pgp-glycoprotein substrate, from lung cells reacted to inhibition by verapamil, while MK571 had no effect. Modulation of glutathion content of lung cells by N-acetyl cystein and buthionine sulfoximine shifted CDF efflux toward higher or lower rates, respectively. These experiments confirm that MRP1 function can be followed in the attached cells in vitro under non-toxic concentrations of the substrates without the need to harvest and destroy the cells.
Keywords: Tumor lung cells; MRP1; Immunocytochemistry; Transport function; Glutathione; N-acetyl cystein (NAC); Buthionin sulfoximine (BSO)
Non-destructive micromethod for MRP1 functional assay in human lung tumor cells by Ekkehard Stehfest; Abdelrahman Torky; Felix Glahn; Heidi Foth (pp. 125-133).
Defense against toxic endo- and xenobiotics is a major concern of all living species and ABC transporters play a vital role in this defense system. Multidrug resistance associated proteins 1 (MRP1) is a cellular detoxifying factor supposed to transport a wide range of compounds across cell membranes either as GSH conjugates or as co-transport accompanying glutathione transposition. The cellular localization of MRP1 is a determining factor whether the transport function can take place. In this study we have undertaken experiments on the transport activity of MRP1 in cultured human lung tumor cells in order to check whether MRP1 is expressed as a functionally active protein. For this purpose we have adapted a quantitative fluorescence imaging assay to conditions where a small number of attached cells should be repeatedly measured by a non-destructive method. In cultured A549, H358 and H322 cells MRP1 is located in the cell membrane as observed by immunocytochemistry. Efflux of 5,6-carboxy-2′-7′-dichloro-fluorescein (CDF) from lung cells was sensitive toward the MRP1 inhibitor MK571 while verapamil had no effect. On the other hand, efflux of Rhodamin 123, a Pgp-glycoprotein substrate, from lung cells reacted to inhibition by verapamil, while MK571 had no effect. Modulation of glutathion content of lung cells by N-acetyl cystein and buthionine sulfoximine shifted CDF efflux toward higher or lower rates, respectively. These experiments confirm that MRP1 function can be followed in the attached cells in vitro under non-toxic concentrations of the substrates without the need to harvest and destroy the cells.
Keywords: Tumor lung cells; MRP1; Immunocytochemistry; Transport function; Glutathione; N-acetyl cystein (NAC); Buthionin sulfoximine (BSO)
Superantigen-primed T cells exposed to 2,3,7,8–tetrachlorodibenzo-p-dioxin (TCDD) replicate poorly following recall encounter by Laura Faulconer; Iris Camacho; Mitzi Nagarkatti; Prakash S. Nagarkatti (pp. 134-145).
The current study investigated the effect of tetrachlorodibenzo-p-dioxin (TCDD) on the ability of staphylococcal enterotoxin A (SEA)-primed T cells to divide by dual-labeling the cells with 5,6–carboxyfluorescein diacetate succinimidyl ester (CFSE) and antibodies against the specific T cell receptors. C57BL/6 wild-type mice were injected ip with TCDD (10 μg/kg body weight) followed by hind footpad injections of SEA (10 μg/footpad). The draining popliteal lymph nodes (PLN) were harvested 1–4 days posttreatment, labeled with CFSE and cultured for 1–4 days without further stimulation or in the presence of the recall antigen. TCDD-exposed SEA-reactive Vβ3+ and Vβ11+ T cells showed decreased cell divisions upon in vitro culture in the absence of any stimulation, which correlated with increased levels of apoptosis. The recall cell-division response was also defective in SEA-reactive T cells isolated from TCDD-exposed mice. However, during the recall response, cells from TCDD-exposed mice did not exhibit a defect in apoptosis, suggesting the defective recall response may result from a state of anergy rather than increased apoptosis. Using AhR knockout (KO) mice, we found AhR involvement in the regulation of defective cell division and apoptosis induced by TCDD. Together, these data demonstrate, while TCDD-induced apoptosis may account for the decreased primary T cell proliferative response, that the reduced cell division seen during subsequent exposure to recall antigen may result from a state of anergy. The study also demonstrates that a combined use of superantigen and CFSE may offer a simple and useful tool to monitor the ability of immunotoxicants to alter the proliferative responsiveness of antigen-specific T cells.
Keywords: TCDD; Immunosuppression; CFSE
Superantigen-primed T cells exposed to 2,3,7,8–tetrachlorodibenzo-p-dioxin (TCDD) replicate poorly following recall encounter by Laura Faulconer; Iris Camacho; Mitzi Nagarkatti; Prakash S. Nagarkatti (pp. 134-145).
The current study investigated the effect of tetrachlorodibenzo-p-dioxin (TCDD) on the ability of staphylococcal enterotoxin A (SEA)-primed T cells to divide by dual-labeling the cells with 5,6–carboxyfluorescein diacetate succinimidyl ester (CFSE) and antibodies against the specific T cell receptors. C57BL/6 wild-type mice were injected ip with TCDD (10 μg/kg body weight) followed by hind footpad injections of SEA (10 μg/footpad). The draining popliteal lymph nodes (PLN) were harvested 1–4 days posttreatment, labeled with CFSE and cultured for 1–4 days without further stimulation or in the presence of the recall antigen. TCDD-exposed SEA-reactive Vβ3+ and Vβ11+ T cells showed decreased cell divisions upon in vitro culture in the absence of any stimulation, which correlated with increased levels of apoptosis. The recall cell-division response was also defective in SEA-reactive T cells isolated from TCDD-exposed mice. However, during the recall response, cells from TCDD-exposed mice did not exhibit a defect in apoptosis, suggesting the defective recall response may result from a state of anergy rather than increased apoptosis. Using AhR knockout (KO) mice, we found AhR involvement in the regulation of defective cell division and apoptosis induced by TCDD. Together, these data demonstrate, while TCDD-induced apoptosis may account for the decreased primary T cell proliferative response, that the reduced cell division seen during subsequent exposure to recall antigen may result from a state of anergy. The study also demonstrates that a combined use of superantigen and CFSE may offer a simple and useful tool to monitor the ability of immunotoxicants to alter the proliferative responsiveness of antigen-specific T cells.
Keywords: TCDD; Immunosuppression; CFSE
Involvement of the extracellular signal-regulated protein kinase pathway in phosphorylation of p53 protein and exerting cytotoxicity in human neuroblastoma cells (SH-SY5Y) exposed to acrylamide by Takeo Okuno; Masato Matsuoka; Tomoyuki Sumizawa; Hideki Igisu (pp. 146-153).
Using human neuroblastoma SH-SY5Y cells, effects of acrylamide on p53 protein and intracellular signal transducting pathways were examined. Acrylamide increased p53, phosphorylated p53, and p53-associated protein murine double minute 2 (MDM2). The phosphorylation of p53 was specific for the Ser15 site. Among mitogen-activated protein kinases (MAPKs), acrylamide caused phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 but not c-Jun NH2-terminal kinase. Nevertheless, blocking p38 pathway by LL-Z1640-2 did not suppress the phosphorylation of p53 at Ser15. In contrast, a specific inhibitor of ERK kinase (U0126 or PD98059) could abolish the accumulation as well as the phosphorylation of p53 at Ser15. Elevation of MDM2 was also abolished by U0126. An inhibitor of phosphatidylinositol 3-kinase-related kinase (PIKK) pathway (wortmannin) suppressed the increase of p53 and its phosphorylation at Ser15. Hence, acrylamide increases p53 protein and its phosphorylation at Ser15 through ERK and/or PIKK pathways. On the other hand, U0126 and PD98059 suppressed to some extent the cytotoxicity of acrylamide evaluated by trypan blue exclusion and lactate dehydrogenase (LDH) leakage, whereas neither LL-Z1640-2 nor wortmannin was effective in suppressing the toxicity. Thus, ERK pathway seems to play a role both in causing the phosphorylation of p53 at Ser15 and in the cytotoxicity of acrylamide in SH-SY5Y cells.
Keywords: Acrylamide; SH-SY5Y cells; p53; Phosphorylation; ERK
Involvement of the extracellular signal-regulated protein kinase pathway in phosphorylation of p53 protein and exerting cytotoxicity in human neuroblastoma cells (SH-SY5Y) exposed to acrylamide by Takeo Okuno; Masato Matsuoka; Tomoyuki Sumizawa; Hideki Igisu (pp. 146-153).
Using human neuroblastoma SH-SY5Y cells, effects of acrylamide on p53 protein and intracellular signal transducting pathways were examined. Acrylamide increased p53, phosphorylated p53, and p53-associated protein murine double minute 2 (MDM2). The phosphorylation of p53 was specific for the Ser15 site. Among mitogen-activated protein kinases (MAPKs), acrylamide caused phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 but not c-Jun NH2-terminal kinase. Nevertheless, blocking p38 pathway by LL-Z1640-2 did not suppress the phosphorylation of p53 at Ser15. In contrast, a specific inhibitor of ERK kinase (U0126 or PD98059) could abolish the accumulation as well as the phosphorylation of p53 at Ser15. Elevation of MDM2 was also abolished by U0126. An inhibitor of phosphatidylinositol 3-kinase-related kinase (PIKK) pathway (wortmannin) suppressed the increase of p53 and its phosphorylation at Ser15. Hence, acrylamide increases p53 protein and its phosphorylation at Ser15 through ERK and/or PIKK pathways. On the other hand, U0126 and PD98059 suppressed to some extent the cytotoxicity of acrylamide evaluated by trypan blue exclusion and lactate dehydrogenase (LDH) leakage, whereas neither LL-Z1640-2 nor wortmannin was effective in suppressing the toxicity. Thus, ERK pathway seems to play a role both in causing the phosphorylation of p53 at Ser15 and in the cytotoxicity of acrylamide in SH-SY5Y cells.
Keywords: Acrylamide; SH-SY5Y cells; p53; Phosphorylation; ERK
Induction of oxidative stress and inhibition of plasminogen activator inhibitor-1 production in endothelial cells following exposure to organic extracts of diesel exhaust particles and urban fine particles by Akiko Furuyama; Seishiro Hirano; Eiko Koike; Takahiro Kobayashi (pp. 154-162).
Endothelial cells play important roles in anticoagulant and fibrinolytic systems. Recent studies suggest that increases in ambient particulate matter (PM) levels have been associated with an increase in mortality rate from cardiovascular diseases. We examined the production of heme oxygenase-1 (HO-1) and factors related to the fibrinolytic function by rat heart microvessel endothelial cells exposed to organic extracts of diesel exhaust particles (OE-DEP) and urban fine particles (OE-UFP) to investigate the direct effects of these soluble organic fractions in these PM on the fibrinolytic function of endothelial cells. The cell monolayer exposed to 10 μg/ml OE-DEP produced a larger amount of HO-1 than cells exposed to 10 μg/ml OE-UFP. OE-DEP and OE-UFP exposure reduced plasminogen activator inhibitor-1 (PAI-1) production by the cells but did not affect the production of thrombomodulin, tissue-type plasminogen activator, or urokinase-type plasminogen activator. Increased PAI-1 synthesis in response to treatment with 1.0 ng/ml tumor necrosis factor-α or 0.5 ng/ml transforming growth factor-β1 was reduced by OE-DEP exposure. Suppression of PAI-1 production by OE-DEP exposure was mediated through oxidative stress and was independent of HO-1 activity. These results suggest that exposure to the soluble organic fraction of PM and DEP induced oxidative stress and reduced the PAI-1 production of endothelial cells.
Keywords: Heme oxygenase-1; PAI-1; Fibrinolysis; TGF-β1; TNF-α
Induction of oxidative stress and inhibition of plasminogen activator inhibitor-1 production in endothelial cells following exposure to organic extracts of diesel exhaust particles and urban fine particles by Akiko Furuyama; Seishiro Hirano; Eiko Koike; Takahiro Kobayashi (pp. 154-162).
Endothelial cells play important roles in anticoagulant and fibrinolytic systems. Recent studies suggest that increases in ambient particulate matter (PM) levels have been associated with an increase in mortality rate from cardiovascular diseases. We examined the production of heme oxygenase-1 (HO-1) and factors related to the fibrinolytic function by rat heart microvessel endothelial cells exposed to organic extracts of diesel exhaust particles (OE-DEP) and urban fine particles (OE-UFP) to investigate the direct effects of these soluble organic fractions in these PM on the fibrinolytic function of endothelial cells. The cell monolayer exposed to 10 μg/ml OE-DEP produced a larger amount of HO-1 than cells exposed to 10 μg/ml OE-UFP. OE-DEP and OE-UFP exposure reduced plasminogen activator inhibitor-1 (PAI-1) production by the cells but did not affect the production of thrombomodulin, tissue-type plasminogen activator, or urokinase-type plasminogen activator. Increased PAI-1 synthesis in response to treatment with 1.0 ng/ml tumor necrosis factor-α or 0.5 ng/ml transforming growth factor-β1 was reduced by OE-DEP exposure. Suppression of PAI-1 production by OE-DEP exposure was mediated through oxidative stress and was independent of HO-1 activity. These results suggest that exposure to the soluble organic fraction of PM and DEP induced oxidative stress and reduced the PAI-1 production of endothelial cells.
Keywords: Heme oxygenase-1; PAI-1; Fibrinolysis; TGF-β1; TNF-α
Partial purification and characterization of a novel neurotoxin and three cytolysins from box jellyfish (Carybdea marsupialis) nematocyst venom by J. Sánchez-Rodríguez; E. Torrens; L. Segura-Puertas (pp. 163-168).
This paper describes one neurotoxin and three cytolysins isolated from the venom of the Caribbean box jellyfish Carybdea marsupialis. To assess the cytolytic and neurotoxic activity of the nematocyst venom, several bioassays were carried out, and to evaluate the effect of the toxin, the dose causing 50% lethality (LD50) was determined in vivo using sea crabs (Ocypode quadrata). The proteins with neurotoxic and cytolytic effects were isolated using low-pressure liquid chromatography. The fraction containing the neurotoxic activity was analyzed by SDS-PAGE and showed a single protein band with an apparent molecular weight of 120 kDa (CmNt). To demonstrate the neurotoxic activity of this protein, a small fraction of the purified protein was injected into a crab, and the typical convulsions, paralysis, and death provoked by neurotoxins were observed. Three fractions containing cytolysins had protein bands in SDS PAGE with apparent molecular weights of 220, 139, and 36 kDa, and their cytolytic activity was confirmed with the haemolysis assay.
Keywords: Cnidaria; Carybdea marsupialis ; Nematocysts; Neurotoxins; Cytolysins
Partial purification and characterization of a novel neurotoxin and three cytolysins from box jellyfish (Carybdea marsupialis) nematocyst venom by J. Sánchez-Rodríguez; E. Torrens; L. Segura-Puertas (pp. 163-168).
This paper describes one neurotoxin and three cytolysins isolated from the venom of the Caribbean box jellyfish Carybdea marsupialis. To assess the cytolytic and neurotoxic activity of the nematocyst venom, several bioassays were carried out, and to evaluate the effect of the toxin, the dose causing 50% lethality (LD50) was determined in vivo using sea crabs (Ocypode quadrata). The proteins with neurotoxic and cytolytic effects were isolated using low-pressure liquid chromatography. The fraction containing the neurotoxic activity was analyzed by SDS-PAGE and showed a single protein band with an apparent molecular weight of 120 kDa (CmNt). To demonstrate the neurotoxic activity of this protein, a small fraction of the purified protein was injected into a crab, and the typical convulsions, paralysis, and death provoked by neurotoxins were observed. Three fractions containing cytolysins had protein bands in SDS PAGE with apparent molecular weights of 220, 139, and 36 kDa, and their cytolytic activity was confirmed with the haemolysis assay.
Keywords: Cnidaria; Carybdea marsupialis ; Nematocysts; Neurotoxins; Cytolysins
Induction of thyroid lesions in 14-week toxicity studies of 2 and 4-methylimidazole in Fischer 344/N rats and B6C3F1 mice by Po Chan; Joel Mahler; Greg Travlos; Abraham Nyska; M. Wenk (pp. 169-180).
Fifteen-day and 14-week studies of 2-methylimidazole (2MI) and 4-methylimidazole (4MI) were conducted because of widespread human exposure via ingestion of food products containing the compounds and lack of toxicity data. Groups of five male and five female Fischer rats and B6C3F1 mice were administered 2MI by dosed feed at 0, 1,200, 3,300, or 10,000 ppm or 4MI at 0, 300, 800, or 2,500 ppm for 15 days, and groups of 10 male and 10 female Fischer rats and B6C3F1 mice were administered 2MI or 4MI at 0, 625, 1,250, 2,500, 5,000 or 10,000 ppm for 14 weeks. In the 15-day studies, 2MI induced thyroid follicular-cell hyperplasia and pituitary pars-distalis hypertrophy in rats and thyroid follicular-cell hypertrophy and spleen hematopoietic-cell proliferation in mice; 4MI induced no histopathological changes in rats and mice. In the 14-week studies, 2MI increased concentrations of thyroid-stimulating hormone (TSH) and decreased those of thyroxine (T4) and triiodothyroxine (T3) in male and female rats according to the dosage. Incidences of diffuse follicular-cell hyperplasia of the thyroid gland increased significantly in male rats exposed to 1,250 ppm or greater and female rats exposed to 2,500 ppm or greater. Thyroid follicular-cell adenoma was diagnosed in two males in the 10,000-ppm group. A dose-related anemia occurred in female rats. In mice, follicular-cell hypertrophy of the thyroid gland, anemia, splenic hematopoietic-cell proliferation, and hemosiderin in kidney tubules appeared. In rats, 4MI induced tremors and ataxia in the high-dose groups. Serum T3, T4, and TSH levels were not altered, and no thyroid lesions occurred. Anemia, hepatocytic vacuolation, testicular degeneration, and prostatic atrophy were observed. In mice, anemia, liver cytoplasmic vacuolization, and renal degeneration and dilation occurred. Our studies demonstrated that, in rats and mice, 2MI induces thyroid hyperplasia and hypertrophy, and both 2MI and 4MI induce anemia; 2MI induces thyroid follicular-cell adenoma in male rats.
Keywords: 2 And 4-methylimidazole; Thyroid lesions; Rats; Mice
Induction of thyroid lesions in 14-week toxicity studies of 2 and 4-methylimidazole in Fischer 344/N rats and B6C3F1 mice by Po Chan; Joel Mahler; Greg Travlos; Abraham Nyska; M. Wenk (pp. 169-180).
Fifteen-day and 14-week studies of 2-methylimidazole (2MI) and 4-methylimidazole (4MI) were conducted because of widespread human exposure via ingestion of food products containing the compounds and lack of toxicity data. Groups of five male and five female Fischer rats and B6C3F1 mice were administered 2MI by dosed feed at 0, 1,200, 3,300, or 10,000 ppm or 4MI at 0, 300, 800, or 2,500 ppm for 15 days, and groups of 10 male and 10 female Fischer rats and B6C3F1 mice were administered 2MI or 4MI at 0, 625, 1,250, 2,500, 5,000 or 10,000 ppm for 14 weeks. In the 15-day studies, 2MI induced thyroid follicular-cell hyperplasia and pituitary pars-distalis hypertrophy in rats and thyroid follicular-cell hypertrophy and spleen hematopoietic-cell proliferation in mice; 4MI induced no histopathological changes in rats and mice. In the 14-week studies, 2MI increased concentrations of thyroid-stimulating hormone (TSH) and decreased those of thyroxine (T4) and triiodothyroxine (T3) in male and female rats according to the dosage. Incidences of diffuse follicular-cell hyperplasia of the thyroid gland increased significantly in male rats exposed to 1,250 ppm or greater and female rats exposed to 2,500 ppm or greater. Thyroid follicular-cell adenoma was diagnosed in two males in the 10,000-ppm group. A dose-related anemia occurred in female rats. In mice, follicular-cell hypertrophy of the thyroid gland, anemia, splenic hematopoietic-cell proliferation, and hemosiderin in kidney tubules appeared. In rats, 4MI induced tremors and ataxia in the high-dose groups. Serum T3, T4, and TSH levels were not altered, and no thyroid lesions occurred. Anemia, hepatocytic vacuolation, testicular degeneration, and prostatic atrophy were observed. In mice, anemia, liver cytoplasmic vacuolization, and renal degeneration and dilation occurred. Our studies demonstrated that, in rats and mice, 2MI induces thyroid hyperplasia and hypertrophy, and both 2MI and 4MI induce anemia; 2MI induces thyroid follicular-cell adenoma in male rats.
Keywords: 2 And 4-methylimidazole; Thyroid lesions; Rats; Mice