Jarkko Loikkanen

Interactions of excitatory neurotransmitters and xenobiotics in excitotoxicity and oxidative stress: glutamate and lead.

Increased glutamate release is associated with serious neurological disorders such as epilepsy, stroke, Alzheimers disease and other brain injuries. Excessive glutamate release and subsequent glutamatergic neuronal stimulation increase the production of reactive oxygen species (ROS), which in turn induce oxidative stress, excitotoxicity and neuronal damage. A number of studies have shown that co-exposure of neuronal cells to glutamate, and an environmental toxin, lead, can greatly amplify glutamate excitotoxicity and cell death through apoptosis or necrosis. Even though the mechanisms of excitotoxicity or those of glutamate-lead interactions have not been exhaustively delineated, there is ample evidence to suggest that increased production of ROS may play an important role in both events. Subsequently, increased DNA binding of redox-regulated transcription factors, NF-kappaB and AP-1, seems to be associated with these events. Induction of an immediate early gene, c-fos, is seen in neuronal cells exposed to glutamate or lead. Immediate early genes are important in regulating the expression of other neuronal genes; Elevated expressions of the genes encoding Hsp70 or cyclo-oxygenase-2 seem to be involved in the apoptosis or necrosis induced by glutamate, and may be associated with induction of several of the genes in cells exposed to lead, or to the glutamate-lead combination. Further studies are required to clarify the mechanisms of glutamate-lead neurotoxicity.

Lead amplifies glutamate-induced oxidative stress

Lead markedly amplified L-glutamate-induced oxidative stress, that is, increased L-glutamate-induced production of reactive oxygen species, decreased cellular glutathione, and induced cytotoxicity in human neuroblastoma cells. It was notable that oxidative burst induced by L-glutamate alone was observed only when neuronal glutathione was depleted. A role of protein kinase C (PKC) in glutamate-induced production of reactive oxygen species is likely because it was blocked by a PKC inhibitor. We suggest here that the mechanism whereby lead causes its neurotoxicity may be through the amplification of glutamate-induced oxidative stress, possibly through PKC activation.

Modification of Glutamate-Induced Oxidative Stress by Lead: The Role of Extracellular Calcium

The role of extracellular calcium in glutamate-induced oxidative stress, and the role of glutamatergic neuronal stimulation and oxidative stress in lead neurotoxicity were explored in mouse hypothalamic GT1-7 cells. Glutamate increased the production of reactive oxygen species (ROS) whether or not extracellular calcium was present. Glutamate-induced ROS production was amplified by lead acetate (PbAc), but only in the absence of extracellular calcium. However, PbAc on its own did not increase the production of ROS. A PKC inhibitor (Ro 31-8220) and superoxide dismutase (SOD) abolished the amplification of glutamate-induced production of ROS by PbAc, but did not inhibit ROS production induced by glutamate alone. Both glutamate and PbAc decreased the levels of intracellular glutathione (GSH), and amplified each others effect on GSH depletion. Glutamate did not decrease cell viability, whereas the cytotoxicity of PbAc was amplified by glutamate. Extracellular calcium, a PKC inhibitor, or SOD did not modify the effects of glutamate, PbAc or their combination on the levels of GSH or cell viability. These data indicate that in GT1-7 cells extracellular calcium is not essential for glutamate-induced ROS production, which is amplified by PbAc, but only without extracellular calcium. The joint cytotoxicity of glutamate and PbAc is mainly induced by PbAc, preferentially through mechanisms other than ROS production.

Oxidative Stress Induced by Fumonisin B1 in Continuous Human and Rodent Neural Cell Cultures

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides, which is a common infectant of corn and other cereal grains. Of concern to human health is also a possible airborne exposure to FB1-producing strains of F. verticillioides, which may grow in moisture-damaged buildings. In this study, we have characterized oxidative stress-related parameters induced by FB1 in three different neural cell lines, human SH-SY5Y neuroblastoma, rat C6 glioblastoma and mouse GT1-7 hypothalamic cells. The cells were exposed to graded doses of FB1 between 0.1 and 100 μM for 0-144 h after which the production of reactive oxygen species (ROS), lipid peroxidation, intracellular glutathione (GSH) levels and cell viability were measured. FB1 caused a dose-dependent increase of ROS production in C6 glioblastoma and GT1-7 hypothalamic cells but was without an effect in SH-SY5Y cells. Decreased GSH levels, increased MDA-formation, indicative of lipid peroxidation and necrotic cell death were observed in all cell lines after incubation with FB1. These findings indicate that FB1 induces oxidative stress in human, rat and mouse neural cell cultures.

Amplification of glutamate-induced oxidative stress

Glutamate is a ubiquitous neurotransmitter which causes excess neuronal excitotoxicity and neurodegenerative insults such as stroke, trauma and seizures. A salient feature of the activation of glutamate receptors is the induction of oxidative burst. Moreover, glutamate stimulates Ca2+ influx and translocates protein kinase C (PKC). PKC mediates cellular processes mediated via phosphorylations which may be essential for oxidative burst in many cells. Subsequent oxidative stress may be a causal factor of neurodegenerative diseases. Increased glutamate release and oxidative burst may thus both be essential in the cascade of events leading to neuronal damage. Glutamate may also mediate neurotoxic effects of environmental toxic agents such as lead which amplify glutamate excitotoxicity. In these interactions, excessive activation of glutamate receptors and oxidative burst may converge into a common pathway leading to cell death through a cascade involving PKC or other protein important in oxidative burst in neurons.

Pb2+-induced toxicity is associated with p53-independent apoptosis and enhanced by glutamate in GT1-7 neurons.

Recent studies indicate that the glutamatergic neurotransmitter system is involved in neurotoxicity caused by inorganic lead (Pb2+). We studied the role of apoptosis in the effects induced by Pb2+ (0.01-100 microM) and glutamate (0.1 and 1 mM) in mouse hypothalamic GT1-7 neurons. Although glutamate alone had no effect on cell viability, it enhanced neuronal cell death induced by Pb2+ (1-100 microM) within 72 h. Glutamate alone neither induced caspase-3-like protease activity nor promoted internucleosomal DNA fragmentation, both biochemical hallmarks of apoptosis. However, concurrent exposure to Pb2+ (10 or 100 microM) and glutamate (1 mM) resulted in more prominent cleavage of the fluorogenic caspase-3 substrate (Ac-DEVD-AMC) than caused by the same Pb2+ concentrations alone at 24-72 h. The highest caspase-3-like protease activities were measured at 48 h. Internucleosomal DNA fragmentation caused by Pb2+ (10 or 100 microM) alone or together with glutamate (1 mM) was evident at 96 h, less clear at 72 h and absent at 48 h. Immunoblotting did not reveal any changes in p53 protein levels in cells exposed to Pb2+, glutamate or their combination at any studied time point (3-72 h). Our results suggest that Pb2+-induced neurotoxicity may partially be mediated through p53-independent apoptosis and enhanced by glutamate.

CHOLINERGIC-INDUCED PRODUCTION OF REACTIVE OXYGEN SPECIES IN HUMAN NEUROBLASTOMA CELLS

Stimulation of human SH-SY5Y neuroblastoma cells by a muscarinic receptor agonist, carbachol (CCh; 1 mM), elevated levels of free intracellular calcium and subsequently increased the production of reactive oxygen species (ROS). Quinuclidinylbenzilate (QNB) binding increased at 1 h after CCh, but returned back to the control level at 3 h. Production of ROS increased, however, during the 3 h time period. CCh also increased the translocation of protein kinase C (PKC) to the membrane. ROS production was completely blocked by atropine and a PKC inhibitor, Ro 31-8220. These results show that increased ROS production was a result of muscarinic receptor stimulation, and that PKC had an active role in this cellular stimulation. ROS production upon cellular stimulation by CCh was completely inhibited also by superoxide dismutase, and partially by catalase, indicating that the formation of superoxide anion dominated in cholinergic-induced generation of ROS in human neuroblastoma cells. These results also show that muscarinic stimulation causes sustained ROS production in human neuroblastoma cells. The slow increase in ROS production by CCh suggest a stepwise cascade of events leading to oxidative stress with a triggering role of cholinergic muscarinic receptors in this process.

Induction of PUMA-α and down-regulation of PUMA-β expression is associated with benzo(a)pyrene-induced apoptosis in MCF-7 cells

Benzo(a)pyrene (BP) forms benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts in human breast adenocarcinoma MCF-7 cells, leading to p53 protein induction and phosphorylation. Although BP-induced apoptosis in rodent cells is known, it is still unclear in human cells. Here we have analyzed the effects of BP on p53 related apoptotic proteins, cell cycle and cell death in MCF-7 cells. PUMA-protein (p53 up-regulated modulator of apoptosis) levels were changed after BP exposure so that PUMA-alpha protein was statistically significantly increased whereas PUMA-beta protein was statistically significantly decreased. PUMA-protein levels were also investigated in ZR-75-1 cells, where PUMA-alpha protein was statistically significantly increased. Cytochrome c, which is released from mitochondria during apoptosis to form the apoptosome, was increased in cytoplasmic fraction after BP exposure in MCF-7 cells. Increased apoptosis was also seen after 48 and 72 h BP exposure (2.5 and 5 microM). In addition, BP decreased dose dependently cell viability (2.5 and 5 microM) and increased ROS formation (1 and 10 microM). Our results suggest that PUMA-alpha protein is involved in BP-induced cell death most likely through a p53 dependent apoptotic pathway.

The significance of ABC transporters in human placenta for the exposure of the fetus to xenobiotics

The placenta has an important role in transporting nutrients and oxygen to the fetus. Transplacental transport is, however, not restricted to physiological exchange of agents, but includes the majority, if not all, of xenobiotics in maternal blood. Although passive diffusion is the most common transfer mechanism, transporter proteins play a significant role in transplacental transfer. The largest superfamily of transporters and one of the most interesting groups regarding xenobiotics is the ATP-binding cassette (ABC) transporters. The efflux ABC transporters from families ABCB, ABCC and ABCG play a role in transporting xenobiotics and their conjugates in addition to endogenous compounds. Many of them probably play a role in the protection of the fetus, as is implicated in animal studies. Both genetic polymorphisms, endogenous regulation, e.g. by hormones, and xenobiotics can modify the expression and function of transporters. Interference in the function of transporters (e.g., inhibition) may significantly increase the fetal exposure to xenobiotics or drugs. Many different models can be used to study human placental ABC transporters. These include human placental perfusion, tissue preparations, tissue explants, primary cell cultures, immortalized cell lines, cancer cell lines, placental membrane vesicles and cloning. When estimating the contribution of an ABC transporter for fetal exposure localization has to be taken into account in addition to the level of expression, functional status and substrate specificity.

Biomarkers of toxicity in human placenta

Nutrient and gas exchange between mother and fetus and production of hormones sustaining fetal development are important placental functions carried out by a rich selection of transporter proteins and a variety of metabolizing enzymes. The same proteins handle many xenobiotics which in placenta may pass, accumulate, and change placental functions, causing toxicity. Because of structural and functional variation between species, human placental tissue and human trophoblastic cell lines are the most used systems in the search for placental biomarkers of toxicity. They would be helpful in evaluation of drug toxicity in placenta, and in toxicological risk assessment. Only a few exist so far, e.g. changes in CYP19A1/aromatase by hormonally active compounds, metallothioneins in metal exposure, and level of PAH-DNA adducts associated with fetotoxicity. New possibilities are provided, e.g. by the emerging field of placental epigenetics. Use of placenta and placental biomarkers in regulatory toxicology also awaits further data.