Catechol-O-methyltransferase inhibition protects against 3,4-dihydroxyphenylalanine (DOPA) toxicity in primary mesencephalic cultures: new insights into levodopa toxicity

Inhibition of catechol-O-methyltransferase (COMT) has protective effects on levodopa (L-DOPA), but not D-DOPA toxicity towards dopamine (DA) neurons in rat primary mesencephalic cultures [Mol. Pharmacol. 57 (2000) 589]. Here, we extend our recent studies to elucidate the mechanisms of these protective effects. Thus, we investigated the effects of all main L-DOPA/DA metabolites on survival of tyrosine hydroxylase immunoreactive (THir) neurons in primary rat mesencephalic cultures. 3-O-Methyldopa, homovanillic acid, dihydroxyphenyl acetate and 3-methoxytyramine had no effects at concentrations up to 300 micro M after 24h, whereas DA was more toxic than L-DOPA with toxicity at concentrations of >or=1 micro M. The coenzyme of COMT, S-adenosyl-L-methionine (SAM), and its demethylated product S-adenosylhomocystein caused no relevant alteration of THir neuron survival or L-DOPA toxicity. In contrast, inhibition of SAM synthesis by selenomethionine showed time- and dose-dependent increase of THir neuron survival, but did not affect L-DOPA toxicity. L-DOPA-induced lipid peroxidation in mesencephalic cultures was not modified by the COMT inhibitor Ro 41-0960 (1 micro M). Increased contamination of the cultures with glial cells attenuated L- and D-DOPA toxicity, but caused significant enhancement of protection by COMT inhibitors against L-DOPA toxicity only. Investigations of L-DOPA uptake in rat striatal cultures using HPLC revealed a significant reduction of extracellular L-DOPA concentrations by Ro 41-0960. Our data confirm that L-DOPA toxicity towards DA neurons is mediated by an autooxidative process, which is attenuated by glial cells. In addition, we demonstrate a second mechanism of L-DOPA toxicity in vitro mediated by a COMT- and glia-dependent pathway, which is blocked by COMT inhibitors, most likely due to enhanced glial uptake of L-DOPA.     

The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity

In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O⁶-methylguanine (O⁶MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O⁶-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O⁶-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O⁶-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O⁶MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O⁶MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.     

Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta) in the brain. Transthyretin (TTR) is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4) and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14) and (15-42) showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI) domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.     

Dexamethasone protects cultured rat hepatocytes against cadmium toxicity: involvement of cellular thiols

In the present study, the effects of dexamethasone on cadmium-induced toxicity were evaluated in isolated rat hepatocytes. Hepatocytes were cultured for 24 h in William’s E medium containing fetal calf serum (10%), insulin (0.1 IU/ml), and glucagon (0.01 μM) in the absence or presence of 0.1 μM dexamethasone. Cadmium chloride, 5 or 10 μM, was added to the medium and the toxicity was evaluated for up to 48 h after treatment. Lactate dehydrogenase (LDH) release, the reduced and oxidized glutathione ratio (GSH/GSSG), protein-SH groups, and lipid peroxidation levels were evaluated. Cadmium induced a dose- and time-dependent LDH release in control hepatocytes at 24 h (Cd 10 μM 42%) while hepatocytes pretreated with dexamethasone showed lower necrosis (Cd 10 μM 12% at 24 h). GSH/GSSH ratio and protein-SH groups were higher while lipid peroxidation was lower in dexamethasone-treated hepatocytes as compared with untreated cells. In conclusion, cadmium toxicity was associated with an increase in intracellular oxidative stress responsible for accelerated cell death. The use of dexamethasone prevented cadmium damage, suggesting that the cytoprotective action of this hormone is related to its effect in preventing changes in thiols such as glutathione and protein-SH groups.     

High-affinity selective inhibitor against phospholipase A2 (PLA2): a computational study

Phospholipase A₂ (PLA₂) is the most abundant protein found in snake venom. PLA₂ induces a variety of pharmacological effects such as neurotoxicity, myotoxicity and cardiotoxicity as well as anticoagulant, hemolytic, anti-platelet, hypertensive, hemorrhagic and edema inducing effects. In this study, the three dimensional structure of PLA₂ of Naja sputatrix (Malayan spitting cobra) was modeled by I-TASSER, SWISS-MODEL, PRIME and MODELLER programs. The best model was selected based on overall stereo-chemical quality. Further, molecular dynamics simulation was performed to know the stability of the modeled protein using Gromacs software. Average structure was generated during the simulation period of 10?ns. High throughput virtual screening was employed through different databases (Asinex, Hit finder, Maybridge, TOSLab and ZINC databases) against PLA₂. The top seven compounds were selected based on the docking score and free energy binding calculations. These compounds were analyzed by quantum polarized ligand docking, induced fit docking and density functional theory calculation. Furthermore, the stability of lead molecules in the active site of PLA₂ was employed by MD simulation. The results show that selected lead molecules were highly stable in the active site of PLA₂.     

Poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, protects against experimental immune ovarian failure in mice

The activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) may play an important role in numerous pathological conditions. The aim of the present study was to clarify the role of PARP in the pathogenesis of immune ovarian failure in mice and to examine the possible protective action of PARP inhibitor, 3-aminobenzamide (3-ABA). An experimental ovarian injury induced in mice by immunization with allogenic ovarian extract impaired the meiotic maturation of oocytes and increased apoptosis and necrosis of follicular cells and cells isolated from the spleen, lymph nodes and thymus. The immunization affected blood leukogram indicating the presence of an inflammatory response. Treatment with 3-ABA (1 h before antigen administrations, 0.02 mg/g intraperitoneally, twice a week during the three-week experiment) was effective to prevent meiotic maturation impairment, cell death and inflammation. The decrease in the necrosis of follicular and immune cells after 3-ABA treatment was more pronounced than that in apoptosis. It is concluded that PARP may contribute to immune-mediated ovary injury and PARP inhibitor, 3-ABA, protects against experimental immune ovarian failure, partially via a decrease in necrotic cell death.     

5'-Adenosinephosphosulphate reductase (CysH) protects Mycobacterium tuberculosis against free radicals during chronic infection phase in mice

A major obstacle to tuberculosis (TB) control is the problem of chronic TB infection (CTBI). Here we report that 5'-adenosinephosphosulphate reductase (CysH), an enzyme essential for the production of reduced-sulphur-containing metabolites, is critical for Mycobacterium tuberculosis (Mtb) survival in chronic infection phase in mice. Disruption of cysH rendered Mtb auxotrophic for cysteine and methionine, and attenuated virulence in BALB/c and C57BL/6 immunocompetent mice. The mutant and wild-type Mtb replicated similarly during the acute phase of infection, but the mutant showed reduced viability during the persistent phase of the infection. The cysH mutant caused disease and death after 4-7 weeks of infection in four different groups of mice - Rag1(-/-), NOS2(-/-), gp91phox(-/-) NOS2(-/-) and gp91phox(-/-) mice given aminoguanidine [to suppress the effects of nitric oxide synthase 2 (NOS2)]- indicating minimal metabolic effect on the cysH mutant survival in these mice. The cysH mutant was also susceptible to peroxynitrite and hydrogen peroxide in vitro. These results show that CysH is important for Mtb protection during the chronic infection phase, and that resistance to nitrosative and oxidative stress may be the mechanism of this protection. Thus, this metabolic gene of an intracellular pathogen could have a secondary role in protection against the host immune response. Finally the lack of an endogenous human orthologue of cysH and its possible role in defence against adaptive immunity renders CysH an attractive enzyme for further studies as a target for therapeutics active against CTBI.     

Overexpression of superoxide dismutase 1 protects against beta-amyloid peptide toxicity: effect of estrogen and copper chelators

Beta-amyloid peptides (Abeta) are major constituents of senile plaques in Alzheimer's disease (AD) brain and contribute to neurodegeneration, operating through activation of apoptotic pathways. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. Estrogen is thought to play a protective role against neurodegeneration through a variety of mechanisms including scavenging of reactive oxygen species (ROS). In this study, we have challenged with Abeta, either in the presence or in the absence of 17beta-estradiol, differentiated human neuroblastoma SH-SY5Y cells (named line SH) and the same line overexpressing anti-oxidant enzyme superoxide dismutase 1 (SOD1; named line WT). We have observed that: (1) WT cells are less susceptible than SH cells to Abeta insult; (2) caspase-3, but not caspase-1, is involved in Abeta-induced apoptosis in this system; (3) estrogen protects both lines, without significantly affecting SOD activity; and (4) copper chelators prevent Abeta-induced toxicity. Our results further support the notion that anti-oxidant therapy might be beneficial in the treatment of AD by preventing activation of selected apoptotic pathways.     

Persuasive evidence that quinone reductase type 1 (DT diaphorase) protects cells against the toxicity of electrophiles and reactive forms of oxygen

An extensive body of evidence supports the conclusion that by catalyzing obligatory two-electron reductions of quinones to hydroquinones, NAD(P)H:quinone reductase (QR1) protects cells against the deleterious effects of redox cycling of quinones, their ability to deplete glutathione, and to produce neoplasia. The effects of elevation of QR1 levels by various enzyme inducers, inhibition of the enzyme by dicumarol, and genetic deletion of the enzyme (knockout mouse) are all consistent with the proposed protective functions. Measurement of QR1 activity in murine hepatoma cells grown in 96-well microtiter plates has provided a rapid and quantitative method for detecting inducer activity and determining inducer potency. This constitutes a strategy for the identification of potential chemoprotectors against cancer. Epidemiological studies show that humans who are genetically deficient in QR1 are more susceptible to the hematological toxicity and carcinogenicity of benzene exposure, and may be more susceptible to the development of a number of malignant tumors.     

Disruption of poly (ADP-ribose) polymerase (PARP) protects against stress-evoked immunocompromise.

BACKGROUND: Chronic stress, mediated by adrenal hormones, is a major risk factor in the progression and outcome of human disease. While the secretion of adrenal hormones is known to be the primary endocrine mediator of stress-induced immunocompromise, the molecular mechanisms underlying the immunocompromise remain unspecified. Overproduction of the nuclear enzyme, poly (ADP-ribose) polymerase (PARP) has been implicated in the molecular pathway that leads to cell death by energy depletion following stress. MATERIALS AND METHODS: Wild-type (WT) mice and mice with targeted disruption of the gene encoding PARP-1 (PARP-1 -/-) were subjected to 2 wk daily cold-water swim; splenocyte proliferation, anti-KLH IgG, and serum corticosterone concentrations were assessed. Additional mice of each genotype received daily i.p. injections of dexamethasone (DEX) (0.75 mg/kg) for 2 wk, and splenocyte proliferation and anti-KLH IgG were assessed. RESULTS: Splenocyte proliferation and specific antibody concentrations of stressed WT mice were reduced by ~20% of their pre-stress levels. In contrast, PARP-1 -/- mice maintained normal cell-mediated and humoral immune function following enforced cold-water swim stress. PARP-1 -/- mice also failed to compromise immune function following DEX treatment, whereas WT mice displayed significant reductions of immune function following this treatment. CONCLUSIONS: These results provide support for the involvement of PARP activation in immunological damage following physical stress. These results suggest that glucocorticoid-induced immunosuppression may require the activation of PARP in order for apoptosis of immune cells to take place. Taken together, these results suggest that therapies designed to inhibit PARP may prove valuable in the treatment of stress-related diseases.     

The Ginkgo biloba extract (EGb 761) protects and rescues hippocampal cells against nitric oxide-induced toxicity: involvement of its flavonoid constituents and protein kinase C

An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. Numerous studies have shown that the Ginkgo biloba extract EGb 761 is a NO scavenger with neuroprotective properties. However, the mechanisms underlying its neuroprotective ability remain to be fully established. Thus, we investigated the effect of different constituents of EGb 761, i.e., flavonoids and terpenoids, against toxicity induced by NO generators on cells of the hippocampus, a brain area particularly susceptible to neurodegenerative damage. Exposure of rat primary mixed hippocampal cell cultures to either sodium nitroprusside (SNP; 100 microM) or 3-morpholinosydnonimine resulted in both a decrease in cell survival and an increase in free radical accumulation. These SNP-induced events were blocked by either EGb 761 (10-100 microg/ml) or its flavonoid fraction CP 205 (25 microg/ml), as well as by inhibitors of protein kinase C (PKC; chelerythrine) and L-type calcium channels (nitrendipine). In contrast, the terpenoid constituents of EGb 761, known as bilobalide and ginkgolide B, as well as inhibitors of phospholipases A [3-[(4-octadecyl)benzoyl]acrylic acid (OBAA)] and C (U-73122), failed to display any significant effects. Moreover, EGb 761 (50 microm) CP 205 (25 microg/ml), and chelerythrine were also able to rescue hippocampal cells preexposed to SNP (up to 1 mM). Finally, EGb 761 (100 microg/ml) was shown to block the activation of PKC induced by SNP (100 microM). These data suggest that the protective and rescuing abilities of EGb 761 are not only attributable to the antioxidant properties of its flavonoid constituents but also via their ability to inhibit NO-stimulated PKC activity.     

Deficiency of the bone mineralization inhibitor NPP1 protects mice against obesity and diabetes

The emergence of bone as an endocrine regulator has prompted a re-evaluation of the role of bone mineralization factors in the development of metabolic disease. Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) controls bone mineralization through the generation of pyrophosphate, and levels of NPP1 are elevated both in dermal fibroblast cultures and muscle of individuals with insulin resistance. We investigated the metabolic phenotype associated with impaired bone metabolism in mice lacking the gene that encodes NPP1 (Enpp1^−/− mice). Enpp1^−/− mice exhibited mildly improved glucose homeostasis on a normal diet but showed a pronounced resistance to obesity and insulin resistance in response to chronic high-fat feeding. Enpp1^−/− mice had increased levels of the insulin-sensitizing bone-derived hormone osteocalcin but unchanged insulin signalling within osteoblasts. A fuller understanding of the pathways of NPP1 could inform the development of novel therapeutic strategies for treating insulin resistance.KEY WORDS: NPP1, Mineralization, Obesity, Diabetes     

A mimic of superoxide dismutase activity protects Chlorella sorokiniana against the toxicity of sulfite

The spin-trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) has been used to demonstrate the light-dependent production of O2- by Chlorella sorokiniana. In the presence of SO3= a light-dependent production of the sulfur trioxy anion radical (SO3-.) could also be seen. A complex prepared by reacting desferrioxamine with MnO2, which catalyzes the dismutation of O2-, protected the alga against the toxicity of sulfite. The data suggest that SO2 toxicity is at least partially due to the effects of sulfoxy-free radicals generated by the oxidation of SO3= by O2-.     

Temperature-dependent variation in toxicity of insecticides against Earias vitella (Lepidoptera: Noctuidae)

The toxicity of synthetic pyrethroids was found to be negatively correlated with temperature, whereas contrasting correlation was observed with the toxicity of organophosphorous compounds chlorpyriphos and quinalphos, which was most toxic at higher temperature. A similar phenomenon was observed in endosulfan at higher temperature and humidity combination. The insecticide molecules indoxacarb and spinosad were effective among the insecticides tested. Indoxacarb was effective at lower temperature, and spinosad was effective at all the temperature and relative humidity combinations with minor difference in LD50 values. During both the years, however, the levels of resistance were higher in second year compared with previous year.     

Fumigant toxicity of plant essential oils against Camptomyia corticalis (Diptera: Cecidomyiidae)

The toxicity of 98 plant essential oils against third instars of cecidomyiid gall midge Camptomyia corticalis (Loew) (Diptera: Cecidomyiidae) was examined using a vapor-phase mortality bioassay. Results were compared with that of a conventional insecticide dichlorvos. Based on 24-h LC50 values, all essential oils were less toxic than dichlorvos (LC50, 0.027 mg/cm3). The LC50 of caraway (Carum carvi L.) seed, armoise (Artemisia vulgaris L.), clary sage (Salvia sclarea L.), oregano (Origanum vulgare L.), lemongrass [Cymbopogon citratus (DC.) Stapf], niaouli (Melaleuca viridiflora Gaertner), spearmint (Mentha spicata L.), cassia especial (Cinnamomum cassia Nees ex Blume), Dalmatian sage (Salvia offcinalis L.), red thyme (Thymus vulgaris L.), bay [Pimenta racemosa (P. Mill.) J.W. Moore], garlic (Allium sativum L.), and pennyroyal (Mentha pulegium L.) oils is between 0.55 and 0.60 mg/cm3. The LC50 of cassia (C. cassia, pure and redistilled), white thyme (T. vulgaris), star anise (Illicium verum Hook.f.), peppermint (Mentha X piperita L.), wintergreen (Gaultheria procumbens L.), cinnamon (Cinnamomum zeylanicum Blume) bark, sweet marjoram (Origanum majorana L.), Roman chamomile [Chamaemelum nobile (L.) All.], eucalyptus (Eucalyptus globulus Labill.), rosemary (Rosmarinus officinalis L.),Virginian cedarwood (Juniperus virginiana L.), pimento berry [Pimenta dioica (L.) Merr.], summer savory (Satureja hortensis L.), lavender (Lavandula angustifolia Mill.), and coriander (Coriandrum sativum L.) oils is between 0.61 and 0.99 mg/cm3. All other essential oils tested exhibited low toxicity to the cecidomyiid larvae (LC50, >0.99 mg/cm3). Global efforts to reduce the level of highly toxic synthetic insecticides in the agricultural environment justify further studies on the active essential oils as potential larvicides for the control of C. corticalis populations as fumigants with contact action.