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Critical subcellular targets of dibucaine in mammalian Vero cells
Abstracts / Toxicology Letters 172S (2007) S1–S240
Conclusions: (1) Sub-chronic introducing of phensuccinalum and diacamph activated FRPO and MOGS, and of glyklaside, caused hypoglycemia and depression of antioxidant protection system. (2) ADDs stimulated phagocytic component of immunity but changed cytokine regulation of immune reactions thus increasing risk of allergic pathology advancement. doi:10.1016/j.toxlet.2007.05.162 M38 Elevated urinary and serum d-lactate of diabetics Sirpa Pennanen 1 , Petteri Talasniemi 2 , Heikki Savolainen 3 , Jyrki Liesivuori 4 1 Finnish Institute of Occupational Health, Kuopio, Finland; 2 Univerisity of Kuopio, Kuopio, Finland; 3 Ministry of Social Affairs and Health, Tampere, Finland; 4 Finnish Institute of Occupational Health, Turku, Finland
Methylglyoxal is a reactive alpha-dicarbonyl that is thought to contribute to diabetic complications either as a direct toxin or as a precursor for advanced glycation end products. It is produced primarily from triose phosphates and is detoxified to d-lactate by the glyoxalase pathway. We hypothesise that urine and serum d-lactate may be increased in vivo in diabetics as a result of increased glucose flux. Levels of urinary and serum d-lactate were measured by enzymatic assay from 38 subjects with diabetics. We also measured d-lactate in urine from 42 non-diabetic subjects. Urine d-lactate was significantly elevated (p < 0.0001) in subjects with diabetes as compared with nondiabetic subjects (35.9 ± 24.2 M/mM creatinine versus 18.2 ± 12.0 M/mM creatinine). Plasma d-lactate was also significantly elevated in diabetic subjects as compared with literature concentrations of non-diabetic subjects (39.6 + 23.7 M versus range 5–20 M). These data suggest that diabetic patients have elevated urinary and serum d-lactate concentrations compared to normal subjects. High concentrations of d-lactate can induce severe metabolic acidosis and d-lactic acidosis. d-Lactic acidosis is associated with neurotoxic effects. Mechanism of d-lactic acidosis neurotoxicity is not yet known.
S53
ˇ zi´c 1 , Maja Peraica 1 , Danica Ljubanovi´c 2 , Davor Zeljeˇ 1 Ana-Marija Domijan 1 Institute
for Medical Research and Occupational Health, Zagreb, Croatia; 2 Clinical Hospital Dubrava, Zagreb, Croatia Mycotoxin fumonisin B1 (FB1 ) is nephrotoxic, hepatotoxic and carcinogen in laboratory animals. It is already known that long exposure to this mycotoxin causes apoptosis and increases lipid peroxidation in kidney of experimental animals. The aim of this study was to check whether single dose and short exposure to this mycotoxin cause the same effects. In this study the groups (N = 6) of adult male Wistar rats were given by gavage a single FB1 dose (5, 50, 500 g/kg b.w.) and sacrificed four, 24 and 48 h afterwards. The parameters of oxidative stress (glutathione-GSH and malondialdehyde-MDA), concentration of sphingolipids (sphinganine/sphingosine-Sa/So), DNA lesions (measured with comet assay) and histopathological changes were checked in kidney of treated and control animals. The lowest FB1 dose (5 g/kg b.w.) increased the Sa/So ratio after 4, 24 and 48 h (2.53 + 0.52, 2.93 + 0.42 and 1.44 + 0.13, respectively) as compared to controls (0.62 + 0.10) (P < 0.05). In contrast, GSH and MDA concentrations were not changed even by the highest FB1 dose (500 g/kg b.w.). DNA lesions measured as comet tail length and tail intensity were FB1 dose- and timedependent. The increase of tail intensity occurred at lower dose (5 g/kg) than that of tail length (50 g/kg). The application of higher FB1 doses caused the earlier appearance of DNA damage. Apoptotic cells and mitotic figures were found in cortical and outer stripe of medullar part of the kidney indicating the high potential of FB1 to produce degenerative and regenerative processes, probably due to changes in sphingolipid metabolism. doi:10.1016/j.toxlet.2007.05.164 M40 Critical subcellular targets of dibucaine in mammalian Vero cells ´ Ana Peropadre, Paloma Fern´andez Freire, Oscar Herrero, Jose Manuel P´erez Mart´ın, Maria Jos´e Hazen
doi:10.1016/j.toxlet.2007.05.163
Universidad Aut´onoma de Madrid, Madrid, Spain
M39 The effect of single dose of fumonisin B1 on oxidative stress, sphingolipid metabolism and DNA damage in rat kidney
Dibucaine (2-butoxy-N-[2-(diethylamino)ethyl]-4quinoline carboxamine, CAS No. 85-79-0), the most common amine local anesthetic drug, is known to interact with cellular membranes of the nervous system. This fact is the central point of its therapeutic
S54
Abstracts / Toxicology Letters 172S (2007) S1–S240
target. Nevertheless, different studies show important secondary effects that might be related to its interaction with the mitochondrial respiratory chain. Noteworthy the precise mechanism of action is not completely elucidated. The present study investigates the toxic potential of dibucaine in mammalian cells, using biochemical and morphological parameters which reveal interference with structures essential for cell survival, proliferation and/or function. Cell growth and viability were assessed by using colorimetric assays, whereas cellular alterations after dibucaine exposure were evaluated using conventional light, fluorescence and transmission electron microscopy. Our cellular model shows that dibucaine is a very potent cytotoxic drug that displays an unusual and interesting mode of action in Vero cells. Microscopic observations correlated well with the biochemical results, indicating that two main cellular targets, lysosomes and cytoskeleton, were severely affected even by low drug exposure. In these same conditions, mitochondria did not show important morphologic or functional alterations. In conclusion, our experimental procedures demonstrate that there are multiple subcellular targets of dibucaine in mammalian cells. Among them, lysosomes seem to play a critical role for its toxic mechanism, but further studies are needed to elucidate the pathways leading to cell death after dibucaine treatments. doi:10.1016/j.toxlet.2007.05.165 M41 Biological pathways and plasma biomarkers for muscle toxicity induced by a PPAR alpha agonist, fenofibrate Plasterer 1 ,
Hamano 2 ,
Damian 1 ,
Thomas Takako Doris Miyoko Okada 2 , Peter Jahasz 1 , Toshinobu Shimizu 2 , Chenhui Zeng 1 , Naoya Masutomi 2 , Stephan Martin 1 , Naohisa Tsutsui 2 , Robert McBurney 1 1 BG
Medicine Inc., Waltham, MA, United States; Laboratory, Mitsubishi Pharma Corportion, Kisarazu, Chiba, Japan
2 Toxicology
Peroxisome proliferators-activated receptor (PPAR) alpha agonists, such as fenofibrate, bezafibrate and gemfibrozil, are known to cause toxicity of skeletal muscle, including muscle pain, muscle weakness and rhabdomyolysis. The effects of PPAR alpha agonists on skeletal muscle can be aggravated by concomitant administration of a variety of drugs, including HMA-COA reduc-
tase inhibitors. The mechanism of PPAR alpha agonists induced muscle toxicity is poorly understood and no reliable plasma biomarker has existed. To discover plasma biomarkers of muscle toxicity by PPAR alpha agonists based on elucidation of its molecular mechanism, this study employed a molecular systems pharmacology/toxicology approach to describe the broad spectrum of molecular changes in femoral muscle and in plasma caused by fenofibrate treatment to rats. Male Sprague–Dawley rats were orally administered 300 mg/kg fenofibrate daily for 28 days. On the next day of the final dosing, the animals were euthanized, plasma samples were collected for clinical chemistry, proteomic (targeted immunoassay and LC-MALDI-MS) and metabolomic (LC/MS and GC/MS) analyses, and muscles were collected for histopathology, transcriptomic (Gene-chip), proteomic (LC-MALDI-MS) and mertabolomic (LC/MS) analyses. After normalization of all omics platform data for plasma and muscle, univariate analysis was performed between fenofibrate and control groups or fenofibrate lesion plus/minus animals defined by histopathological findings in femoral muscle. Finally, Correlation NetworksTM were constructed to determine biomarkers of treatment effects and plasma biomarkers associated with muscle lesion. Fifteen muscle analytes were extracted as lesion marker. These markers were correlated with 712 plasma analytes. Fifty-four muscle–plasma correlations, 33 muscle–muscle correlation and 445 plasma–plasma correlation were also found. In addition, 11 analytes are both muscle-correlated markers of lesion and plasma lesion markers. Among them, tryptophan was identified as a univariate marker in two platforms (LC/MS and GC/MS) as well as correlated to multiple muscle biomarkers. doi:10.1016/j.toxlet.2007.05.166 M42 Do they act similar or independent? In vitro testings of the binary mixture nickel and chlorpyrifos Susanne Rudzok, Olf Herbarth, Sebastian Krejci, Eike Schm¨ucking, Mario Bauer Helmholtz Centre for Environmental Research, UFZ, Leipzig, Germany We are exposed to a variety of chemical cocktails in our environment and the question is: which present more risk to our health than the single compounds? Therefore, mixtures toxicity assessment is a central role in toxicological studies.
Conclusions: (1) Sub-chronic introducing of phensuccinalum and diacamph activated FRPO and MOGS, and of glyklaside, caused hypoglycemia and depression of antioxidant protection system. (2) ADDs stimulated phagocytic component of immunity but changed cytokine regulation of immune reactions thus increasing risk of allergic pathology advancement. doi:10.1016/j.toxlet.2007.05.162 M38 Elevated urinary and serum d-lactate of diabetics Sirpa Pennanen 1 , Petteri Talasniemi 2 , Heikki Savolainen 3 , Jyrki Liesivuori 4 1 Finnish Institute of Occupational Health, Kuopio, Finland; 2 Univerisity of Kuopio, Kuopio, Finland; 3 Ministry of Social Affairs and Health, Tampere, Finland; 4 Finnish Institute of Occupational Health, Turku, Finland
Methylglyoxal is a reactive alpha-dicarbonyl that is thought to contribute to diabetic complications either as a direct toxin or as a precursor for advanced glycation end products. It is produced primarily from triose phosphates and is detoxified to d-lactate by the glyoxalase pathway. We hypothesise that urine and serum d-lactate may be increased in vivo in diabetics as a result of increased glucose flux. Levels of urinary and serum d-lactate were measured by enzymatic assay from 38 subjects with diabetics. We also measured d-lactate in urine from 42 non-diabetic subjects. Urine d-lactate was significantly elevated (p < 0.0001) in subjects with diabetes as compared with nondiabetic subjects (35.9 ± 24.2 M/mM creatinine versus 18.2 ± 12.0 M/mM creatinine). Plasma d-lactate was also significantly elevated in diabetic subjects as compared with literature concentrations of non-diabetic subjects (39.6 + 23.7 M versus range 5–20 M). These data suggest that diabetic patients have elevated urinary and serum d-lactate concentrations compared to normal subjects. High concentrations of d-lactate can induce severe metabolic acidosis and d-lactic acidosis. d-Lactic acidosis is associated with neurotoxic effects. Mechanism of d-lactic acidosis neurotoxicity is not yet known.
S53
ˇ zi´c 1 , Maja Peraica 1 , Danica Ljubanovi´c 2 , Davor Zeljeˇ 1 Ana-Marija Domijan 1 Institute
for Medical Research and Occupational Health, Zagreb, Croatia; 2 Clinical Hospital Dubrava, Zagreb, Croatia Mycotoxin fumonisin B1 (FB1 ) is nephrotoxic, hepatotoxic and carcinogen in laboratory animals. It is already known that long exposure to this mycotoxin causes apoptosis and increases lipid peroxidation in kidney of experimental animals. The aim of this study was to check whether single dose and short exposure to this mycotoxin cause the same effects. In this study the groups (N = 6) of adult male Wistar rats were given by gavage a single FB1 dose (5, 50, 500 g/kg b.w.) and sacrificed four, 24 and 48 h afterwards. The parameters of oxidative stress (glutathione-GSH and malondialdehyde-MDA), concentration of sphingolipids (sphinganine/sphingosine-Sa/So), DNA lesions (measured with comet assay) and histopathological changes were checked in kidney of treated and control animals. The lowest FB1 dose (5 g/kg b.w.) increased the Sa/So ratio after 4, 24 and 48 h (2.53 + 0.52, 2.93 + 0.42 and 1.44 + 0.13, respectively) as compared to controls (0.62 + 0.10) (P < 0.05). In contrast, GSH and MDA concentrations were not changed even by the highest FB1 dose (500 g/kg b.w.). DNA lesions measured as comet tail length and tail intensity were FB1 dose- and timedependent. The increase of tail intensity occurred at lower dose (5 g/kg) than that of tail length (50 g/kg). The application of higher FB1 doses caused the earlier appearance of DNA damage. Apoptotic cells and mitotic figures were found in cortical and outer stripe of medullar part of the kidney indicating the high potential of FB1 to produce degenerative and regenerative processes, probably due to changes in sphingolipid metabolism. doi:10.1016/j.toxlet.2007.05.164 M40 Critical subcellular targets of dibucaine in mammalian Vero cells ´ Ana Peropadre, Paloma Fern´andez Freire, Oscar Herrero, Jose Manuel P´erez Mart´ın, Maria Jos´e Hazen
doi:10.1016/j.toxlet.2007.05.163
Universidad Aut´onoma de Madrid, Madrid, Spain
M39 The effect of single dose of fumonisin B1 on oxidative stress, sphingolipid metabolism and DNA damage in rat kidney
Dibucaine (2-butoxy-N-[2-(diethylamino)ethyl]-4quinoline carboxamine, CAS No. 85-79-0), the most common amine local anesthetic drug, is known to interact with cellular membranes of the nervous system. This fact is the central point of its therapeutic
S54
Abstracts / Toxicology Letters 172S (2007) S1–S240
target. Nevertheless, different studies show important secondary effects that might be related to its interaction with the mitochondrial respiratory chain. Noteworthy the precise mechanism of action is not completely elucidated. The present study investigates the toxic potential of dibucaine in mammalian cells, using biochemical and morphological parameters which reveal interference with structures essential for cell survival, proliferation and/or function. Cell growth and viability were assessed by using colorimetric assays, whereas cellular alterations after dibucaine exposure were evaluated using conventional light, fluorescence and transmission electron microscopy. Our cellular model shows that dibucaine is a very potent cytotoxic drug that displays an unusual and interesting mode of action in Vero cells. Microscopic observations correlated well with the biochemical results, indicating that two main cellular targets, lysosomes and cytoskeleton, were severely affected even by low drug exposure. In these same conditions, mitochondria did not show important morphologic or functional alterations. In conclusion, our experimental procedures demonstrate that there are multiple subcellular targets of dibucaine in mammalian cells. Among them, lysosomes seem to play a critical role for its toxic mechanism, but further studies are needed to elucidate the pathways leading to cell death after dibucaine treatments. doi:10.1016/j.toxlet.2007.05.165 M41 Biological pathways and plasma biomarkers for muscle toxicity induced by a PPAR alpha agonist, fenofibrate Plasterer 1 ,
Hamano 2 ,
Damian 1 ,
Thomas Takako Doris Miyoko Okada 2 , Peter Jahasz 1 , Toshinobu Shimizu 2 , Chenhui Zeng 1 , Naoya Masutomi 2 , Stephan Martin 1 , Naohisa Tsutsui 2 , Robert McBurney 1 1 BG
Medicine Inc., Waltham, MA, United States; Laboratory, Mitsubishi Pharma Corportion, Kisarazu, Chiba, Japan
2 Toxicology
Peroxisome proliferators-activated receptor (PPAR) alpha agonists, such as fenofibrate, bezafibrate and gemfibrozil, are known to cause toxicity of skeletal muscle, including muscle pain, muscle weakness and rhabdomyolysis. The effects of PPAR alpha agonists on skeletal muscle can be aggravated by concomitant administration of a variety of drugs, including HMA-COA reduc-
tase inhibitors. The mechanism of PPAR alpha agonists induced muscle toxicity is poorly understood and no reliable plasma biomarker has existed. To discover plasma biomarkers of muscle toxicity by PPAR alpha agonists based on elucidation of its molecular mechanism, this study employed a molecular systems pharmacology/toxicology approach to describe the broad spectrum of molecular changes in femoral muscle and in plasma caused by fenofibrate treatment to rats. Male Sprague–Dawley rats were orally administered 300 mg/kg fenofibrate daily for 28 days. On the next day of the final dosing, the animals were euthanized, plasma samples were collected for clinical chemistry, proteomic (targeted immunoassay and LC-MALDI-MS) and metabolomic (LC/MS and GC/MS) analyses, and muscles were collected for histopathology, transcriptomic (Gene-chip), proteomic (LC-MALDI-MS) and mertabolomic (LC/MS) analyses. After normalization of all omics platform data for plasma and muscle, univariate analysis was performed between fenofibrate and control groups or fenofibrate lesion plus/minus animals defined by histopathological findings in femoral muscle. Finally, Correlation NetworksTM were constructed to determine biomarkers of treatment effects and plasma biomarkers associated with muscle lesion. Fifteen muscle analytes were extracted as lesion marker. These markers were correlated with 712 plasma analytes. Fifty-four muscle–plasma correlations, 33 muscle–muscle correlation and 445 plasma–plasma correlation were also found. In addition, 11 analytes are both muscle-correlated markers of lesion and plasma lesion markers. Among them, tryptophan was identified as a univariate marker in two platforms (LC/MS and GC/MS) as well as correlated to multiple muscle biomarkers. doi:10.1016/j.toxlet.2007.05.166 M42 Do they act similar or independent? In vitro testings of the binary mixture nickel and chlorpyrifos Susanne Rudzok, Olf Herbarth, Sebastian Krejci, Eike Schm¨ucking, Mario Bauer Helmholtz Centre for Environmental Research, UFZ, Leipzig, Germany We are exposed to a variety of chemical cocktails in our environment and the question is: which present more risk to our health than the single compounds? Therefore, mixtures toxicity assessment is a central role in toxicological studies.