- Email: [email protected]
Subcellular distribution and some biochemical properties of human liver paraoxonase
Poster Session 1A. Neurotoxicity
26
proteins and lipids amount or the total nucleic acids in pups at birthday, suggesting that brain is able to protect itself from nocive effects of cadmium, but the intoxication was evident in the significant change of indolamines neurotransmitters. We found that the content of 5-HT increased in telencephalon (206%), diencephalon (142%), mesencephalon (312%) and metencephalon (347%), while 5-HIAA overcame 4 times the control value in telencephalon and 8, 14 and 11 times respectively in the rest of brain areas. The ratio 5-HIAA/5-HT also increased in all areas although in telencephalon was not significant with respect to the control. Data suggest that the intoxication of Cd can affect the sinthesis and the degradative process of serotoninergic neurotransmission, that appears in midbrain on 13th gestation day. We also observed the brain areas that showed higher variations were the mesencephalon and metencephalon, where are located the raphe nucleus and their projections.
Keywords: rat; cadmium; brain; 5-HT; development; neurotoxicity ~
CHOLINERGIC RECEPTOR-INDUCED OXIDATIVE BURST IN HUMAN NEUROBLASTOMACELLS
in liver and plasma, using the rat as model; and (2) to determine whether the response of both esterase activities to inducers is similar each one or, on the contrary, they are different enzymes. If the former is true, the hypothesis of a unique enzyme instead two different enzymes would become more consistent. Groups of 5 rats were pre-treated with the inducers according to standard protocols. Control rats were either untreated or administered appropriate volumes of vehicle. After killing, liver microsomes were prepared by differential centrifugation and POX and ARE activities as well as protein content were assayed. PB and 3-MC each increased the POX activity in microsomal fraction (58 and 36%, respectively) and ARE activity (61 and 13%, respectively). In addition, only 3-MC was able to increase the esterases activities in plasma (23% POX and 16% ARE). By contrast, RFP decreased both enzyme activities in liver and plasma. Microsomal fraction proteins did not show significative differences between controls and different inducers. Our results indicate that classical enzyme-inducing agents exert divergent effects on POX and ARE in rat plasma and liver microsomes, and support the previous evidence on the identity between the two esterase activities.
Keywords: enzyme induction; A-esterase; paraoxonase; arylesterase;
Jonne Naarala :, Pirkko Tervo i, Jarkko Loikkanen * l, Kai Savolainen 1.2.1 National Public Health Institute, Laboratory of
microsomal fraction
Toxicology, P.O.B. 95, FIN-70701 Kuopio, Finland; 2 Department of Pharmacology and Toxicology, University of Kuopio, Finland
•
The effects of a muscarinic receptor agonist, carbachol (CCh), on the production of reactive oxygen species (ROS) and the levels of the intracellular glutathione (GSH) were studied in human SH-SY5Y neuroblastoma cell line. ROS and GSH were measured by using fluorescent probes, dichlorofluorescin (DCF) and monochlorobimane (MBCL), respectively. The translocation of protein kinase C (PKC) to the cell membrane was measured by phorbol dibutyrate (PDBu) binding and muscarinic receptor number was assayed by using quinuclidinylbenzilate (QNB) binding in intact SH-SY5Y cells. One millimolar CCh increased ROS production 1.87 fold at 60 min, 2.37 fold at 120 min, and 3.0 fold at 180 min as compared to the control values. However, CCh, at a concentration of 500/zM, caused only a slight increase in ROS production, and values obtained with I00/zM CCh remained at control levels. Intracellular GSH levels decreased by about 20% when the cells were incubated for 120 min with 1 mM CCh. However, GSH levels retumed back to the control level after 180 min incubation. PDBu binding (BMAx) increased by 73% after 20 min incubation with 1 mM CCh as compared to the control cells. Also 500 /~M CCh increased PDBu binding by 33-54% as compared to corresponding controls, i.e. PKC translocation to the membrane was enhanced. QNB binding (BM,xX) increased by 166% after 60 min incubation with 1 mM CCh, and returned back to the control level after 180 min incubation. We conclude that muscarinic receptor stimulation with CCh may cause oxidative burst in human neuroblastoma cells which may be partially routed through PKC, because increase in PDBu binding is a prerequisite for the production of ROM. However, there may be other effector mechanisms that have to be activated prior to the production of ROS. Supported by The Academy of Finland.
Ma Carmen Gonzalvo, Femando GII, Antonio F. Herndndez, Enrique Villanueva, Antonio Pla. Department of Legal Medicine
and Toxicology Service, Avda. Madrid, 11 Faculty of Medicine, University of Granada, 18071-Granada, Spain Humans are exposed to a range of xenobiotic esters used as pesticides. Hydrolysis by esterases present in liver microsomes, cytosol and blood is a major route for the detoxification of xenobiotic esters in humans. We have studied the subcellular localization and some biochemical properties of human liver paraoxonase in order to establish a correlation with serum enzyme and the relative importance of both activities in the toxicity of OP compounds. The subcellular localization of paraoxonase (POX) was determined by differential centrifugation. POX activity was determined spectrophotometrically using methods described elsewhere. Kinetic parameters (Km, Vmax), optimum pH and temperature and the effect of EDTA, calcium and some inhibitors were determined. POX activity was found mainly in the microsomal fraction of human liver, that was further used for the biochemical characterization. Human liver POX showed maximum activity at pH 10-10.5 and 37--40°C and was estimulated by NaC1 0.3 M. Km was 0.29 mM and Vmax 99.64 mU/ml. Activity was calcium dependent (optimum [Ca ++] = 1 mM) and was abolished by EDTA and restored by adding calcium. POX activity was inhibited by mercurial compounds, barium, cobalt, lanthanum and zinc.
Keywords: organophosphates; paraoxonase; A-esterases; human liver ~
- ~
INDUCTIONOF A-ESTERASES IN RAT PLASMA AND LIVER MICROSOMES
SUBCELLULAR DISTRIBUTION AND SOME BIOCHEMICAL PROPERTIES OF HUMAN LIVER PARAOXONASE
EFFECTS OF AMIKACIN ON THE RAT'S TECTORIAL MEMBRANE
J. Rueda *, M. Esteban, R. Cantos, M.L. Sala, J.A. Merchttn. Dpto.
Antonio F. Hern~indez, Ma Carmen Gonzalvo, Fernando Gil, Enrique Villanueva, Antonio Pla. Department of Legal Medicine
Histologfa, Instituto de Neurociencias, Facultad de Medicina, Universidad de Alicante, Alicante, Spain
and Toxicology Service, Avda. Madrid, 11 Faculty of Medicine, University of Granada, 18071-Granada, Spain
Amikacin is an aminoglycoside antibiotic with clinical use, mainly at the hospital level. Ototoxicity is the most important secondary effect, and the cellular site of its puttative action remains controversial, although a close relationship with cellular glycoconjugates has been suggested. The auditory receptor is a neuroepithelium, with sensory
The present study was designed: (1) to investigate the effects of the inducing agents phenobarbital (PB), 3-methyl cholanthrene (3-MC), and rifampicin (RFP) on paraoxonase (POX) and arylesterase (ARE)
26
proteins and lipids amount or the total nucleic acids in pups at birthday, suggesting that brain is able to protect itself from nocive effects of cadmium, but the intoxication was evident in the significant change of indolamines neurotransmitters. We found that the content of 5-HT increased in telencephalon (206%), diencephalon (142%), mesencephalon (312%) and metencephalon (347%), while 5-HIAA overcame 4 times the control value in telencephalon and 8, 14 and 11 times respectively in the rest of brain areas. The ratio 5-HIAA/5-HT also increased in all areas although in telencephalon was not significant with respect to the control. Data suggest that the intoxication of Cd can affect the sinthesis and the degradative process of serotoninergic neurotransmission, that appears in midbrain on 13th gestation day. We also observed the brain areas that showed higher variations were the mesencephalon and metencephalon, where are located the raphe nucleus and their projections.
Keywords: rat; cadmium; brain; 5-HT; development; neurotoxicity ~
CHOLINERGIC RECEPTOR-INDUCED OXIDATIVE BURST IN HUMAN NEUROBLASTOMACELLS
in liver and plasma, using the rat as model; and (2) to determine whether the response of both esterase activities to inducers is similar each one or, on the contrary, they are different enzymes. If the former is true, the hypothesis of a unique enzyme instead two different enzymes would become more consistent. Groups of 5 rats were pre-treated with the inducers according to standard protocols. Control rats were either untreated or administered appropriate volumes of vehicle. After killing, liver microsomes were prepared by differential centrifugation and POX and ARE activities as well as protein content were assayed. PB and 3-MC each increased the POX activity in microsomal fraction (58 and 36%, respectively) and ARE activity (61 and 13%, respectively). In addition, only 3-MC was able to increase the esterases activities in plasma (23% POX and 16% ARE). By contrast, RFP decreased both enzyme activities in liver and plasma. Microsomal fraction proteins did not show significative differences between controls and different inducers. Our results indicate that classical enzyme-inducing agents exert divergent effects on POX and ARE in rat plasma and liver microsomes, and support the previous evidence on the identity between the two esterase activities.
Keywords: enzyme induction; A-esterase; paraoxonase; arylesterase;
Jonne Naarala :, Pirkko Tervo i, Jarkko Loikkanen * l, Kai Savolainen 1.2.1 National Public Health Institute, Laboratory of
microsomal fraction
Toxicology, P.O.B. 95, FIN-70701 Kuopio, Finland; 2 Department of Pharmacology and Toxicology, University of Kuopio, Finland
•
The effects of a muscarinic receptor agonist, carbachol (CCh), on the production of reactive oxygen species (ROS) and the levels of the intracellular glutathione (GSH) were studied in human SH-SY5Y neuroblastoma cell line. ROS and GSH were measured by using fluorescent probes, dichlorofluorescin (DCF) and monochlorobimane (MBCL), respectively. The translocation of protein kinase C (PKC) to the cell membrane was measured by phorbol dibutyrate (PDBu) binding and muscarinic receptor number was assayed by using quinuclidinylbenzilate (QNB) binding in intact SH-SY5Y cells. One millimolar CCh increased ROS production 1.87 fold at 60 min, 2.37 fold at 120 min, and 3.0 fold at 180 min as compared to the control values. However, CCh, at a concentration of 500/zM, caused only a slight increase in ROS production, and values obtained with I00/zM CCh remained at control levels. Intracellular GSH levels decreased by about 20% when the cells were incubated for 120 min with 1 mM CCh. However, GSH levels retumed back to the control level after 180 min incubation. PDBu binding (BMAx) increased by 73% after 20 min incubation with 1 mM CCh as compared to the control cells. Also 500 /~M CCh increased PDBu binding by 33-54% as compared to corresponding controls, i.e. PKC translocation to the membrane was enhanced. QNB binding (BM,xX) increased by 166% after 60 min incubation with 1 mM CCh, and returned back to the control level after 180 min incubation. We conclude that muscarinic receptor stimulation with CCh may cause oxidative burst in human neuroblastoma cells which may be partially routed through PKC, because increase in PDBu binding is a prerequisite for the production of ROM. However, there may be other effector mechanisms that have to be activated prior to the production of ROS. Supported by The Academy of Finland.
Ma Carmen Gonzalvo, Femando GII, Antonio F. Herndndez, Enrique Villanueva, Antonio Pla. Department of Legal Medicine
and Toxicology Service, Avda. Madrid, 11 Faculty of Medicine, University of Granada, 18071-Granada, Spain Humans are exposed to a range of xenobiotic esters used as pesticides. Hydrolysis by esterases present in liver microsomes, cytosol and blood is a major route for the detoxification of xenobiotic esters in humans. We have studied the subcellular localization and some biochemical properties of human liver paraoxonase in order to establish a correlation with serum enzyme and the relative importance of both activities in the toxicity of OP compounds. The subcellular localization of paraoxonase (POX) was determined by differential centrifugation. POX activity was determined spectrophotometrically using methods described elsewhere. Kinetic parameters (Km, Vmax), optimum pH and temperature and the effect of EDTA, calcium and some inhibitors were determined. POX activity was found mainly in the microsomal fraction of human liver, that was further used for the biochemical characterization. Human liver POX showed maximum activity at pH 10-10.5 and 37--40°C and was estimulated by NaC1 0.3 M. Km was 0.29 mM and Vmax 99.64 mU/ml. Activity was calcium dependent (optimum [Ca ++] = 1 mM) and was abolished by EDTA and restored by adding calcium. POX activity was inhibited by mercurial compounds, barium, cobalt, lanthanum and zinc.
Keywords: organophosphates; paraoxonase; A-esterases; human liver ~
- ~
INDUCTIONOF A-ESTERASES IN RAT PLASMA AND LIVER MICROSOMES
SUBCELLULAR DISTRIBUTION AND SOME BIOCHEMICAL PROPERTIES OF HUMAN LIVER PARAOXONASE
EFFECTS OF AMIKACIN ON THE RAT'S TECTORIAL MEMBRANE
J. Rueda *, M. Esteban, R. Cantos, M.L. Sala, J.A. Merchttn. Dpto.
Antonio F. Hern~indez, Ma Carmen Gonzalvo, Fernando Gil, Enrique Villanueva, Antonio Pla. Department of Legal Medicine
Histologfa, Instituto de Neurociencias, Facultad de Medicina, Universidad de Alicante, Alicante, Spain
and Toxicology Service, Avda. Madrid, 11 Faculty of Medicine, University of Granada, 18071-Granada, Spain
Amikacin is an aminoglycoside antibiotic with clinical use, mainly at the hospital level. Ototoxicity is the most important secondary effect, and the cellular site of its puttative action remains controversial, although a close relationship with cellular glycoconjugates has been suggested. The auditory receptor is a neuroepithelium, with sensory
The present study was designed: (1) to investigate the effects of the inducing agents phenobarbital (PB), 3-methyl cholanthrene (3-MC), and rifampicin (RFP) on paraoxonase (POX) and arylesterase (ARE)