Protection and potentiation of MPTP-induced toxicity by cytochrome P-450 inhibitors and inducer: in vitro studies with brain slices

Exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes loss of dopaminergic neurons in humans, primates and mice. Exposure of sagittal slices of mouse brain to MPTP (100 pM) caused inhibition of mitochondrial NADH-dehydrogenase activity. Leakage of lactate dehydrogenase from the slice into the medium was observed following incubation of slices with 1 nM MPTP. Neurotoxicity induced by MPTP was prevented by prior exposure of the slices to the dopamine uptake inhibitor GBR 12935. Deprenyl and pargyline (inhibitors of monoamine oxidase), also protected the slices from MPTP-induced toxicity. However, both pargyline and deprenyl also inhibited cytochrome P-450 mediated aminopyrine N-demethylase activity in brain slices. Pargyline, when administered in vivo to mice, decreased brain cytochrome-450 levels significantly. Other cytochrome P-450 inhibitors, namely, piperonyl butoxide and SKF 525A were found to offer protection against MPTP induced neurotoxicity in slices without affecting monoamine oxidase activity. MPTP toxicity was potentiated significantly in brain slices prepared from mice pretreated with phenobarbital, an inducer of cytochrome P-450. The present study suggests the possible involvement of cytochrome P-450 in MPTP-induced neurotoxicity, in vitro, in brain slices

The structure and function of the replication terminator protein of Bacillus subtilis: identification of the 'winged helix' DNA-binding domain.

The replication terminator protein (RTP) of Bacillus subtilis impedes replication fork movement in a polar mode upon binding as two interacting dimers to each of the replication termini. The mode of interaction of RTP with the terminus DNA is of considerable mechanistic significance because the DNA-protein complex not only localizes the helicase-blocking activity to the terminus, but also generates functional asymmetry from structurally symmetric protein dimers. The functional asymmetry is manifested in the polar impedance of replication fork movement. Although the crystal structure of the apoprotein has been solved, hitherto there was no direct evidence as to which parts of RTP were in contact with the replication terminus. Here we have used a variety of approaches, including saturation mutagenesis, genetic selection for DNA-binding mutants, photo cross-linking, biochemical and functional characterizations of the mutant proteins, and X-ray crystallography, to identify the regions of RTP that are either in direct contact with or are located within 11 angstroms of the replication terminus. The data show that the unstructured N-terminal arm, the alpha3 helix and the beta2 strand are involved in DNA binding. The mapping of amino acids of RTP in contact with DNA, confirms a 'winged helix' DNA-binding motif

Protection and potentiation of 1-methyl-4-phenylpyridinium-induced toxicity by cytochrome P450 inhibitors and inducer may be due to the altered uptake of the toxin

Earlier studies from our laboratory have demonstrated that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity could be modulated by inhibitors and inducer of cytochrome P450 (P450) in an in vitro model consisting of sagittal slices of mouse brain. To understand the molecular mechanisms underlying the role of P450 on MPTP toxicity, it was undertaken to study the effect of the modulators of P450 on the toxicity of the metabolite of MPTP, namely, 1-methyl-4-phenylpyridinium ion (MPP<SUP>+</SUP>). Incubation of mouse brain slices with various concentrations of MPP<SUP>+</SUP> (1-100 &#956;M) resulted in dose-dependent inhibition of mitochondrial enzyme NADH-dehydrogenase (NADH-DH) and leakage of the cytosolic enzyme lactate dehydrogenase from the slice into the medium. MPP<SUP>+</SUP>-induced toxicity was abolished by pretreatment of the slices with inhibitors of monoamine oxidase (MAO; pargyline and deprenyl) or inhibitors of P450 (piperonyl butoxide or SKF-525A) or dopamine uptake blocker (GBR-12909), as measured by the activity of NADH-DH in slices and leakage of lactate dehydrogenase from the slice into the medium. Slices prepared from mice pretreated with phenobarbital (an inducer of P450) potentiated the toxic effects of MPP+. Pretreatment of slices with MAO-inhibitor, P450 inhibitors, or dopamine uptake blocker attenuated the uptake of MPP<SUP>+</SUP> into the slices. In contrast, MPP<SUP>+</SUP> uptake was significantly increased in slices prepared from phenobarbital-pretreated mice. Thus, both MAO and P450 inhibitors abolish the toxicity of MPP<SUP>+</SUP> in the sagittal slices of mouse brain by altering the uptake of the toxin into the slices

Billionfold difference in the toxic potencies of two excitatory plant amino acids, L-BOAA and L-BMAA: biochemical and morphological studies using mouse brain slices

Plant amino acids β-N-oxalylamino-L-alanine (L-BOAA, present in Lathyrus sativus) and β-N-methylamino-L-alanine (L-BMAA, present in Cycas circinalis) have been implicated in the pathogenesis of human neurological disorders lathyrism and amyotrophic lateral sclerosis-Parkinson's dementia complex of Guam (ALS-PD), respectively. In view of the conflicting reports that have emerged on the role of L-BMAA in ALS-PD, we reinvestigated the comparative toxicity of L-BMAA and L-BOAA. We report here the potent toxicity of L-BOAA as examined in an in vitro model consisting of sagittal slices of mouse brain. Incubation of sagittal slices of mouse brain with L-BOAA (1 pM) resulted in significant leakage of lactate dehydrogenase (LDH) and potassium from the slices into the medium. Under similar conditions, L-BMAA-induced LDH leakage from the slices into the medium was observed only at very high concentration of the toxin, namely 1 mM. N-Methyl-D-aspartate (NMDA) receptor antagonists ameliorated the toxic effects of L-BMAA, while non-NMDA receptor antagonists (quinoxalinediones) protected against the toxicity of L-BOAA. Incubation of slices with L-BOAA for 1 h resulted in extensive vacuolation and degeneration of neurons in the thalamus and brain stem, and to a lesser extent in the hippocampus and cerebellar nuclei. The large sized neurons appeared to be affected to a greater extent than the smaller ones. The neurons in other areas of the brain also revealed variable degree of degeneration with swelling of axons and dendrites. Thus, the present study demonstrates the potent toxicity of L-BOAA and elucidates for the first time, the billion-fold difference in the concentration of L-BOAA and L-BMAA required to elicit similar toxic response in vitro, using mouse brain slices. The study also demonstrates the selective vulnerability of certain regions of the brain to toxic insult by L-BOAA

Evidence for generation of oxidative stress in brain by MPTP: in vitro and in vivo studies in mice

The role of oxidative stress in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated neurotoxicity is as yet unclear and the evidence for generation of oxygen free radicals as a primary event in the neurotoxicity is yet to be demonstrated. The present study was undertaken to ascertain the potential role of oxidative damage, and the protective role, if any, of the antioxidant, glutathione (GSH), in MPTP-induced neurotoxicity. Exposure of sagittal slices of mouse brain to MPTP resulted in significant increases of reactive oxygen species (ROS) and malondialdehyde (MDA, the product of lipid peroxidation) and decreases in GSH content. Pretreatment of mouse brain slices, in vitro, with GSH or GSH isopropyl ester attenuated MPTP toxicity as assessed by the tissue activity of the mitochondrial enzyme, NADH-dehydrogenase (NADH-DH), and by leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), from the slice into the medium. In vivo administration of MPTP (30 mg/kg body weight, s.c.), to mice resulted in significant lowering of GSH in the striatum and midbrain, 2 h after dosage; ROS levels in the striatum and midbrain increased after 4 and 8 h, respectively. In the striatum significant inhibition of rotenone-sensitive NADH ubiquinone-1 oxido-reductase (Complex 1) was observed transiently 1 h after MPTP administration. The enzyme activity recovered thereafter; significant inhibition of mitochondrial Complex I was observed in the striatum only 18 h after MPTP dose. In the midbrain, mitochondrial Complex I was inhibited only 18 h after MPTP dose; no change was observed at the early time points examined. Thus, the depletion of GSH and increased ROS formation preceded the inhibition of the mitochondrial enzyme in the midbrain. Evidence presented herein from both in vitro and in vivo studies support that MPTP exposure generates ROS resulting in oxidative stress