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Interactions of excitatory neurotransmitters and xenobiotics in excitotoxicity and oxidative stress
18
Symposium 15. Functional Consequences ofAltered Gene Expression for Exposure
reduction in sex hormone biosynthesis and bioavailability, inhibition of protein tyrosine kinases and inhibition of angiogenesis. None of these actions have been demonstrated in vivo. and the concentrations required in vitro are generally much higher than those reported in human or animal serum, and therefore their biologicalsignificanceis still questionable. Furthermore, it has not been unequivocally established that the effects of phytoestrogen-rich diets are explicitly due to phytoestrogens, rather than other coinciding dietary components, and there is no direct evidence for the protective action of phytoestrogens per se, against hormonally dependent diseases in humans or experimental animals. In addition, it cannot yet be explained why phytoestrogens would exert only beneficial effects in humans, but would be devoid of the well-knownadverse effects of estrogens.
SIS. Functional Consequences of Altered Gene Expression for Exposure
IS15/L1 I THE ROLE OF APOPTOSIS IN TOXIC CELL KILLING IN THE IMMUNE SYSTEM
S. Orrenius. Institute ofEnvironmental Medicine, Karolinska lnstitutet. Stocklwlm, Sweden
Apoptosis is a special form of cell death, which is now widely recognized as being a distinct process of importancein normal physiology and pathology. In addition, many drugs and toxic chemicals kill cells by apoptosis. Thymocytes and lymphocytesare particularly sensitiveto apoptosiscaused by various immunetoxicants, including dithiocarbarnates and organotin compounds. In the current paradigm for apoptotic cell death, the activity of a family of cysteine proteases, caspases, orchestrates the multiple downstream events (such as cell shrinkage, membrane blebbing, glutathione depletion and chromatin fragmentation) that comprise apoptosis. Caspase activation may result from direct interaction of procaspases with activated cell surface receptor complexes (TNF-RI or CD95) or imported proteases (granzyme B). In other forms of apoptosis, caspase activation is believed to be mediated by factors released from the mitochondria, notably cytochrome c. The possible significanceof these mechanismsfor caspase activation in apoptosis triggered by immune toxicants will be discussed.
IS 15/L21 LEAD-INDUCED ALTERATIONS IN RETINALcGMP PDE GENEEXPRESSION AND ACTIVITY RESULTIN CELL-SPECIFIC APOPTOSIS DURING DEVELOPMENT
D.A. Fox *, D. Srivastava, A.T.Poblenz, L. He. University of Houston, Houston, TX, USA Developmental lead exposure results in a selective loss of rod photoreceptorsand bipolar cells by apoptosis. One candidatemechanism is a lead-induced decrease in retinal cGMP (phosphodiesterase) PDE activityand/or expression resulting in calcium overload and then apoptosis. In developmentally lead-exposed rats, northern blot hybridization shows that the onset of Jl-PDEexpression was delayed 1-2 days and altered throughout development and adulthood while the onset of PDE activity was not chan~ed but its activity was lower from day 14 onwards. Retinal [Ca "l was increased at all ages examined. To model these results, in vitro studies used retinas incubated in buffers with Ca2+ and/or Pb2+. With increasing [Ca2+] or [Pb2+], high molecular weight DNA fragmentation (50--600 kbp), DNA condensation (acridine orange staining) only in rods, and CPP32-like activity were enhanced. With the lowest [Ca2+] and [Pb2+], an additive effect on all apoptotic measures was observed. These results demonstrate that selective rod apoptosis can be produced both in vivo and in vitro by lead exposure and, importantly
from a toxicological and public health viewpoint,suggest that Ca2+ can amplify this effect These in vivo and in vitro findings show that lead-inducedalterations in cGMP PDE gene expression and enzyme activity result in Ca2+ overload that underlies the apoptotic rod cell death seen during developmental lead exposure. Supported by NllI Grant ES03183.
I
S 15/L31INTERACTIONS OF EXCITATORY NEUROTRANSMITTERS AND XENOBIOTICS IN EXCITOTOXICITY AND OXIDATIVE STRESS
K.M. Savolainen*, J. Naarala, S. Eerikainen, J. Loikkanen. Department of Pharmacology and Toxicology, University ofKuopio, and Department ofEnvironmental Medicine , National Public Health Institute, Kuopio, Finland
Both glutamate (Glu) and acetylcholine (ACh) are ubiquitous cerebral excitatory neurotransmitters. Stimulation of Glu receptor subtypes is associated with several brain conditions, e.g. convulsions, trauma and stroke, as well as Parkinson's and Alzheimer's disease. Also alterations in the activity of cerebral cholinergic muscarinic receptors are associated with Alzheimer's and Parkinson's disease, and with epileptic convulsions. Increased stimulation of both glutamatergic and cholinergicmuscarinic receptors also leads to neuronal activation, production of reactive oxygen species (ROS) and subsequent oxidative stress. Oxidativestress is associated with decreased levelsof neuronalglutathione(GSH) and neuronaldamage in several cell types in vitro. Glu alone and together with lead (PbAc) increases the production of ROS, decreases neuronal GSH, and neuronal viability. Also stimulation of cholinergic receptors by carbachol (CCh) causes neuronal stimulation and subsequent oxidative stress. Glu, Glu + PbAc, CCh, and CCh + PbAc increase also binding of transcription factors NFkappaB and/or AP-l to their target sequences in the DNA. H202 and tertbutylhydroperoxide also induced oxidative stress. Compounds that induce oxidative stress seem to induce DNA fragmentation, and thus apoptosis may be involved. These events are most likely associated with alterations in gene expression that have marked effects on neuronal functions and on the regulation of apoptosis. Supported by the Academyof Finland.
IS15/L41
METHAMPHETAMINE TOXICITY AND ITS MOLECULAR MECHANISMS OF ACTION
*,,
A. Stadlin J.L. Cader'. I Dept ofAnatomy, Chinese University of Hong Kong. Shatin, N.T., Hong Kong, China; 2Molecular Neuropsychiatry Section, NIHIN/DAl/Rp, Baltimore , MD 21224, USA Methamphetamine (METH) is a drug of abuse that has long-lasting neurotoxic effects on the brain monoaminergic systems. It was reported that METH would induce the release of dopamine (DA) and glutamate within the striatum and chronic METH exposure will result in DA depletion, striatal terminal degeneration and astrogliosis. The mechanisms of this neurotoxicityare not well defined althoughevidence suggested that free radicals may play an important role in METH-mediated toxicity. For example, it was shown that METH-induced decrease of DA levels and DA terminal toxicity was attenuated in CuZn-superoxide transgenic mice implicating superoxide radicals in METH-induced neurotoxicity. Further evidence suggested that the degenerative changes observed in the DA striatal terminals were due to free radicals-mediated apoptosis and energy depletion resulting from astrogliosis. bcl-2-expressingimmortalized neural cells were shown to ameliorate METH-induced DNA fragmentation and apoptotic cell death and p53-knockout mice were also shown to protect against long-term METH·induced terminal degeneration. This furthersupports the activation of apoptotic factors like bcl-2 and p53 in the neurotoxiceffects ofMETH . Although there
Symposium 15. Functional Consequences ofAltered Gene Expression for Exposure
reduction in sex hormone biosynthesis and bioavailability, inhibition of protein tyrosine kinases and inhibition of angiogenesis. None of these actions have been demonstrated in vivo. and the concentrations required in vitro are generally much higher than those reported in human or animal serum, and therefore their biologicalsignificanceis still questionable. Furthermore, it has not been unequivocally established that the effects of phytoestrogen-rich diets are explicitly due to phytoestrogens, rather than other coinciding dietary components, and there is no direct evidence for the protective action of phytoestrogens per se, against hormonally dependent diseases in humans or experimental animals. In addition, it cannot yet be explained why phytoestrogens would exert only beneficial effects in humans, but would be devoid of the well-knownadverse effects of estrogens.
SIS. Functional Consequences of Altered Gene Expression for Exposure
IS15/L1 I THE ROLE OF APOPTOSIS IN TOXIC CELL KILLING IN THE IMMUNE SYSTEM
S. Orrenius. Institute ofEnvironmental Medicine, Karolinska lnstitutet. Stocklwlm, Sweden
Apoptosis is a special form of cell death, which is now widely recognized as being a distinct process of importancein normal physiology and pathology. In addition, many drugs and toxic chemicals kill cells by apoptosis. Thymocytes and lymphocytesare particularly sensitiveto apoptosiscaused by various immunetoxicants, including dithiocarbarnates and organotin compounds. In the current paradigm for apoptotic cell death, the activity of a family of cysteine proteases, caspases, orchestrates the multiple downstream events (such as cell shrinkage, membrane blebbing, glutathione depletion and chromatin fragmentation) that comprise apoptosis. Caspase activation may result from direct interaction of procaspases with activated cell surface receptor complexes (TNF-RI or CD95) or imported proteases (granzyme B). In other forms of apoptosis, caspase activation is believed to be mediated by factors released from the mitochondria, notably cytochrome c. The possible significanceof these mechanismsfor caspase activation in apoptosis triggered by immune toxicants will be discussed.
IS 15/L21 LEAD-INDUCED ALTERATIONS IN RETINALcGMP PDE GENEEXPRESSION AND ACTIVITY RESULTIN CELL-SPECIFIC APOPTOSIS DURING DEVELOPMENT
D.A. Fox *, D. Srivastava, A.T.Poblenz, L. He. University of Houston, Houston, TX, USA Developmental lead exposure results in a selective loss of rod photoreceptorsand bipolar cells by apoptosis. One candidatemechanism is a lead-induced decrease in retinal cGMP (phosphodiesterase) PDE activityand/or expression resulting in calcium overload and then apoptosis. In developmentally lead-exposed rats, northern blot hybridization shows that the onset of Jl-PDEexpression was delayed 1-2 days and altered throughout development and adulthood while the onset of PDE activity was not chan~ed but its activity was lower from day 14 onwards. Retinal [Ca "l was increased at all ages examined. To model these results, in vitro studies used retinas incubated in buffers with Ca2+ and/or Pb2+. With increasing [Ca2+] or [Pb2+], high molecular weight DNA fragmentation (50--600 kbp), DNA condensation (acridine orange staining) only in rods, and CPP32-like activity were enhanced. With the lowest [Ca2+] and [Pb2+], an additive effect on all apoptotic measures was observed. These results demonstrate that selective rod apoptosis can be produced both in vivo and in vitro by lead exposure and, importantly
from a toxicological and public health viewpoint,suggest that Ca2+ can amplify this effect These in vivo and in vitro findings show that lead-inducedalterations in cGMP PDE gene expression and enzyme activity result in Ca2+ overload that underlies the apoptotic rod cell death seen during developmental lead exposure. Supported by NllI Grant ES03183.
I
S 15/L31INTERACTIONS OF EXCITATORY NEUROTRANSMITTERS AND XENOBIOTICS IN EXCITOTOXICITY AND OXIDATIVE STRESS
K.M. Savolainen*, J. Naarala, S. Eerikainen, J. Loikkanen. Department of Pharmacology and Toxicology, University ofKuopio, and Department ofEnvironmental Medicine , National Public Health Institute, Kuopio, Finland
Both glutamate (Glu) and acetylcholine (ACh) are ubiquitous cerebral excitatory neurotransmitters. Stimulation of Glu receptor subtypes is associated with several brain conditions, e.g. convulsions, trauma and stroke, as well as Parkinson's and Alzheimer's disease. Also alterations in the activity of cerebral cholinergic muscarinic receptors are associated with Alzheimer's and Parkinson's disease, and with epileptic convulsions. Increased stimulation of both glutamatergic and cholinergicmuscarinic receptors also leads to neuronal activation, production of reactive oxygen species (ROS) and subsequent oxidative stress. Oxidativestress is associated with decreased levelsof neuronalglutathione(GSH) and neuronaldamage in several cell types in vitro. Glu alone and together with lead (PbAc) increases the production of ROS, decreases neuronal GSH, and neuronal viability. Also stimulation of cholinergic receptors by carbachol (CCh) causes neuronal stimulation and subsequent oxidative stress. Glu, Glu + PbAc, CCh, and CCh + PbAc increase also binding of transcription factors NFkappaB and/or AP-l to their target sequences in the DNA. H202 and tertbutylhydroperoxide also induced oxidative stress. Compounds that induce oxidative stress seem to induce DNA fragmentation, and thus apoptosis may be involved. These events are most likely associated with alterations in gene expression that have marked effects on neuronal functions and on the regulation of apoptosis. Supported by the Academyof Finland.
IS15/L41
METHAMPHETAMINE TOXICITY AND ITS MOLECULAR MECHANISMS OF ACTION
*,,
A. Stadlin J.L. Cader'. I Dept ofAnatomy, Chinese University of Hong Kong. Shatin, N.T., Hong Kong, China; 2Molecular Neuropsychiatry Section, NIHIN/DAl/Rp, Baltimore , MD 21224, USA Methamphetamine (METH) is a drug of abuse that has long-lasting neurotoxic effects on the brain monoaminergic systems. It was reported that METH would induce the release of dopamine (DA) and glutamate within the striatum and chronic METH exposure will result in DA depletion, striatal terminal degeneration and astrogliosis. The mechanisms of this neurotoxicityare not well defined althoughevidence suggested that free radicals may play an important role in METH-mediated toxicity. For example, it was shown that METH-induced decrease of DA levels and DA terminal toxicity was attenuated in CuZn-superoxide transgenic mice implicating superoxide radicals in METH-induced neurotoxicity. Further evidence suggested that the degenerative changes observed in the DA striatal terminals were due to free radicals-mediated apoptosis and energy depletion resulting from astrogliosis. bcl-2-expressingimmortalized neural cells were shown to ameliorate METH-induced DNA fragmentation and apoptotic cell death and p53-knockout mice were also shown to protect against long-term METH·induced terminal degeneration. This furthersupports the activation of apoptotic factors like bcl-2 and p53 in the neurotoxiceffects ofMETH . Although there