Comparative behavioral, biochemical and pigmentary effects of MPTP, MPP+ and paraquat in Rana pipiens

We demonstrate that injections of 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP), 1-methyl-4-phenyl-pyridinium ion (MPP+) and Paraquat (PQ+) produce in Rana Pipiens different behavioral, biochemical and skin pigmentation changes. MPTP causes in frogs the main symptoms of Parkinsonism (rigidity, akinesia and tremor) and it darkens the skin of animals. It also decreases brain and, less so, adrenal medulla dopamine. These effects are blocked by Pargyline. MPP+ causes the same symptoms but more rapidly. In contrast, skin pigmentation is clearly lightened. Brain and particularly adrenal dopamine reserves are nearly abolished. Pargyline increases these effects. Paraquat, in a cumulative fashion, eventually causes the same behavioral changes and a slight increase in pigmentation. It initially produces an increase in brain and adrenal dopamine concentrations, but later a significant dopamine concentration decrease. Pargyline potentiates these long term effects, blocks the dopamine increase, but reverses the PQ+ effect upon melanin, producing the same depigmentation as MPP+ alone.     

Comparative toxicity and antioxidant activity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and its monoamine oxidase B-generated metabolites in isolated hepatocytes and liver microsomes

MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is converted by monoamine oxidase B to its putative toxic metabolite MPP+ (1-methyl-4-phenylpyridinium ion) via MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridinium ion). Both the parent compound and these two major metabolites were toxic to isolated rat hepatocytes with MPDP+ being the most toxic and MPP+ the least effective. MPP+ produced a slight increase in lipid peroxidation above control levels in hepatocytes, while both MPTP and MPDP+ showed antioxidant effects. The latter two compounds also protected against chemically and nonchemically induced lipid peroxidation in rat liver microsomes. MPDP+ was effective at much lower concentrations than MPTP. MPDP+ was also markedly more efficient when NADPH was used to induce microsomal lipid peroxidation. Lipid peroxidation as a consequence of oxygen radical generation is therefore unlikely to be involved in MPTP toxicity in vitro and the rationale of using chain-breaking antioxidants as protective agents in vivo needs a more careful evaluation.     

1-Methyl-4-phenylpyridine (MPP+) induces oxidative stress in the rodent

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) produces an irreversible parkinsonism in primates. Recent evidence suggests metabolism of MPTP to 1-methyl-4-phenylpyridine (MPP+) is required for toxicity. We have proposed that MPP+ may play a central role in the toxicity of MPTP, but direct assessment of the effects of MPP+ in brain is difficult. Therefore, we have sought to define the mechanism of peripheral MPP+ toxicity in the rat and mouse. Systemically administered MPP+ produced its major pathology in the lung and was typified by perivascular edema. An increase in plasma glutathione disulfide concentrations also resulted, suggesting that MPP+ in analogy to paraquat produces oxidative stress. In addition, the lethality of MPP+ in the mouse was increased by dietary selenium deficiency. These results define in both pathological and chemical terms the potent systemic toxicity of MPP+ and suggest that MPP+, because of its high concentration in primate brain, has the potential to play an important role in the CNS toxicity of MPTP.     

Tissue concentrations of MPTP and MPP+ in relation to catecholamine depletion after the oral or subcutaneous administration of MPTP to mice

One hour after MPTP was given to mice at a dose of 30 mg/kg s.c., its concentration in tissues varied in the order kidney greater than liver greater than lung greater than brain greater than heart. When the same dose of MPTP was given orally, concentrations in most tissues were much lower at 1 hr than after s.c. administration, although the MPTP concentration in liver was only slightly lower. The concentrations of MPP+ (a metabolite of MPTP) at 1 hr were as high or higher than those of MPTP in all tissues except kidney, and MPP+ disappeared from the various tissues with half-lives from 3-20 hrs. The highest concentrations of MPP+, both absolute and relative to MPTP, were in heart. After oral administration of MPTP, no MPP+ was found in brain, and MPP+ concentrations in other tissues were lower than those after s.c. dosing. The depletion of heart norepinephrine was similar after MPTP administration by either route of administration even though MPTP and MPP+ concentrations in heart were lower after oral administration, suggesting that other metabolites of MPTP might also contribute to heart norepinephrine depletion.     

Comparative studies on the mechanisms of paraquat and 1-methyl-4-phenylpyridine (MPP+) cytotoxicity

1-methyl-4-phenylpyridine (MPP+) is the putative toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is structurally similar to the herbicide paraquat (PQ++). We have therefore compared the effects of MPP+ and PQ++ on a well characterized experimental model, namely isolated rat hepatocytes. PQ++ generates reactive oxygen species within cells by redox cycling and its toxicity to hepatocytes was potentiated by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. In BCNU-treated cells, PQ++ caused GSH depletion, lipid peroxidation and cell death. These cytotoxic effects were prevented by the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) and the iron-chelating agent desferrioxamine. MPP+ also caused GSH depletion in BCNU-treated hepatocytes but its cytotoxicity was not markedly affected by BCNU, nor was it accompanied by significant lipid peroxidation. DPPD and desferrioxamine also failed to prevent MPP+-induced cell death. We conclude that the production of active oxygen species is likely to play a major role in PQ++ cytotoxicity, while MPP+-induced cell damage may involve additional, more important toxic mechanisms.     

Participation of oxygen activated species in enzymatic lipid peroxidation in biomembranes]

The participation of oxygen activated species in the induction of lipid peroxidation (LPO) in the membrane systems containing cytochrome P-450 (liver microsomes) and in the membrane fragments devoid of this hemoprotein (brain and skeletal muscle microsomes) was studied. It was shown that the rate of NADH-dependent LPO does not depend on the presence of hemoproteins and the activity of NADH-specific flavoprotein in the membranes. On the other hand, the microsomal membranes of the liver with high specific contents of b5 and P-450 cytochromes and NADPH-specific flavoprotein, had the highest rates of NADPH-dependent LPO. It was found that the most effective inhibitors of free oxygen activated species in the case of NADPH- and NADH-dependent LPO in the microsomal fractions of liver, brain and skeletal muscles are the superoxide (O ./2) anion radical inhibitors. The singlet oxygen (1O2) quenchers inhibit only NADPH-dependent LPO in the liver, however, in a far lesser degree. The hydroxyl radical (OH) scavengers had no effect on enzymatic LPO in all systems studied.     

Effects of MPTP, MPP+ and paraquat on mitochondrial potential and oxidative stress

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium (MPP+) and 1,1-dimethyl-4,4-bipyridinium (paraquat) upon the electrical potential across the plasma and mitochondrial membranes within synaptosomes has been investigated. MPTP selectively depressed plasma membrane potential while MPP+ specifically reduced mitochondrial potential. The structurally similar compound paraquat had no effect on either membrane potential. Enhancement of the lipid peroxidative activity with an Fe-ADP complex depressed both potentials. Paraquat effected increased peroxidative activity in brain homogenates that was less pronounced than that due to Fe-ADP. MPTP reduced basal but stimulated Fe-ADP enhanced peroxidation. The mechanisms underlying the toxicity of MPP+ are likely to differ from those of paraquat, primarily involving impaired mitochondrial function rather than increased oxidative stress.     

Interaction between nicotine and MPTP/MPP+ in rat brain endothelial cells

To examine the interaction between nicotine and MPTP/MPP+ in the blood-brain barrier, cellular uptake of MPTP and MPP+ was studied in the presence of nicotine and several compounds, including MPTP/MPP+ analogs and a specific inhibitor of organic cation transporter (OCT) in an adult rat brain microvascular endothelial cell line (ARBEC). The kinetic properties of the uptake of MPTP, MPP+, and nicotine were also examined. In addition, a microdialysis study was performed to evaluate the in vivo effect of nicotine (i.p.) on extracellular levels of MPTP and MPP+ in the brain after intravenous administration of MPTP. The results showed that uptake of MPTP, MPP+, and nicotine was partly mediated by a carrier system that was sensitive to decynium22, a specific OCT inhibitor. RT-PCR showed the presence of OCT1 mRNA in ARBEC. Capacity for uptake of MPTP and nicotine was much higher than that for MPP+ (Km and Vm values of 10.94+/-1.44 microM and 0.049+/-0.007 pmol/mg s, respectively, for MPP+, compared to values of 35.75+/-0.85 microM and 40.95+/-3.56 pmol/mg s for MPTP and 25.29+/-6.44 microM and 51.15+/-14.18 pmol/mg s for nicotine). In addition, nicotine competitively inhibited the uptake of both MPTP and MPP+, with inhibition constants (Ki) of 328 microM and 210 microM, respectively. In vivo microdialysis results showed that nicotine significantly reduced brain extracellular levels of MPTP in the first 30 min (507.4+/-8.5 ng/ml vs. 637.9+/-30.8 ng/ml with and without nicotine pre-treatment, respectively), but did not have significant effect on those of MPP+. In conclusion, nicotine can inhibit in vitro cellular uptake and in vivo transfer of MPTP across the blood-brain barrier, which can be mediated by multiple pathways including OCT1.     

Differences in the metabolism of MPTP in the rodent and primate parallel differences in sensitivity to its neurotoxic effects

Primates and rodents show marked differences in sensitivity to the neurotoxic effects of MPTP. We and others have previously shown that the toxic effects of MPTP on nigrostriatal cells are dependent on the oxidative metabolism of MPTP to the quaternary species MPP+. We have therefore compared the distribution and metabolism of MPTP in the monkey and several rodent species. Three major differences have been identified: 1) the primate, but not the rodents, showed a persistently high concentration of MPTP metabolites in the caudate nucleus compared to other brain regions; 2) the rodent brains cleared MPTP and its metabolites much more rapidly than did the monkey, and; 3) the predominant metabolite retained by the monkey brain was MPP+, while MPP+ cannot be detected in rodent brains for more than a few hours after injection. The persistence of MPP+ in the primate brain may explain the heightened toxicity of MPTP in this species.     

Serotonergic conversion of MPTP and dopaminergic accumulation of MPP+

[3H]MPP+ had lower Km and higher Vmax values for its accumulation in rat brain synaptosomes than did [3H]MPTP. The kinetic parameters favored the uptake of [3H]MPP+ in the striatum to that in hypothalamus, whereas they were equally favorable for the uptake of [3H]MPTP in both regions. Hypothalamic uptake of [3H]MPTP and [3H]MPP+ was inhibited by desipramine, imipramine, norepinephrine, and serotonin. Striatal uptake of [3H]MPP+ and [3H]MPTP was blocked by nomifensine and dopamine. These results support the concept that MPTP accumulates in serotonergic neurons where it is oxidized by monoamine oxidase B to MPP+, which is released and then is selectively accumulated in dopaminergic neurons via the dopamine uptake system.     

MPTP, the neurotoxin inducing Parkinson's disease, is a potent competitive inhibitor of human and rat cytochrome P450 isozymes (P450bufI, P450db1) catalyzing debrisoquine 4-hydroxylation

In human liver microsomal preparations the neurotoxic chemical N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and several of its analogs competitively inhibited bufuralol 1'-hydroxylase activity of cytochrome P450bufI. This enzyme is the target of the common genetic polymorphism of drug oxidation known as debrisoquine polymorphism. Bufuralol 1'-hydroxylase activity was detectable in rat brain tissue. The activity was inhibited by antisera raised against a rat liver cytochrome P450 called P450db1. Immunoblotting experiments revealed the presence of a protein in rat and human brain microsomes with the same electrophoretic properties as the liver enzyme. These data suggest that P450bufI may be involved in the metabolism and neurotoxicity of MPTP.     

MPTP, MPP+ and mitochondrial function

1-Methyl-4-phenylpyridinium (MPP+), the putative toxic metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inhibited NAD(H)-linked mitochondrial oxidation at the level of Complex I of the electron transport system. MPTP and MPP+ inhibited aerobic glycolysis in mouse striatal slices, as measured by increased lactate production; MPTP-induced effects were prevented by inhibition of monoamine oxidase B activity. Several neurotoxic analogs of MPTP also form pyridinium metabolites via MAO; these MPP+ analogs were all inhibitors of NAD(H)-linked oxidation by isolated mitochondria. 2'-Methyl-MPTP, a more potent neurotoxin in mice than MPTP, was also more potent than MPTP in inducing lactate accumulation in mouse brain striatal slices. Overall, the studies support the hypothesis that compromise of mitochondrial oxidative capacity is an important factor in the mechanisms underlying the toxicity of MPTP and similar compounds.     

N-methyltetrahydro-beta-carboline analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin are oxidized to neurotoxic beta-carbolinium cations by heme peroxidases

2-Methyl-1,2,3,4-tetrahydro-beta-carboline (2-Me-THbetaC) and 2,9-dimethyl-1,2,3,4-tetrahydro-beta-carboline (2,9-diMe-THbetaC) are naturally occurring analogs of the Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), whereas their corresponding aromatic 2-methyl-beta-carbolinium cations resemble 1-methyl-4-phenylpyridinium (MPP(+)) and are considered potential toxins involved in Parkinson's disease (PD). To become toxicants, 2-methyltetrahydro-beta-carbolines need to be oxidized (aromatized) by human metabolic enzymes to pyridinium-like (beta-carbolinium) cations as occur with MPTP/MPP(+) model. In contrast to MPTP, human MAO-A or -B were not able to oxidize 2-Me-THbetaC to pyridinium-like cations. Neither, cytochrome P-450 2D6 or a mixture of six P450 enzymes carried out this oxidation in a significant manner. However, 2-Me-THbetaC and 2,9-diMe-THbetaC were efficiently oxidized by horseradish peroxidase (HRP), lactoperoxidase (LPO), and myeloperoxidase (MPO) to 2-methyl-3,4-dihydro-beta-carbolinium cations (2-Me-DHbetaC(+), 2,9-diMe-DHbetaC(+)) as the main products, and detectable amount of 2-methyl-beta-carbolinium cations (2-Me-betaC(+), 2,9-diMe-betaC(+)). The apparent kinetic parameters (k(cat), k(4)) were similar for HRP and LPO and higher for MPO. Peroxidase inhibitors (hydroxylamine, sodium azide, and ascorbic acid) highly reduced or abolished this oxidation. Although MPTP was not oxidized by peroxidases; its intermediate metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium cation (MPDP(+)) was efficiently oxidized to MPP(+) by heme peroxidases. It is concluded that heme peroxidases could be key catalysts responsible for the aromatization (bioactivation) of endogenous and naturally occurring N-methyltetrahydro-beta-carbolines and related protoxins to toxic pyridinium-like cations resembling MPP(+), suggesting a role for these enzymes in toxicological and neurotoxicological processes.     

Model study on the bioreduction of paraquat, MPP+, and analogs. Evidence against a "redox cycling" mechanism in MPTP neurotoxicity

The ability of paraquat, MPP+, and analogs to be reduced by chemical reductants and by NADPH, as catalyzed by liver microsomes or purified NADPH cytochrome P-450 reductase, is reported. The analogs span a range of electrochemical potential, including values in-between that of paraquat and MPP+. Analogs with an Eo below -.55 V (vs. NHE) are not reduced by either the NADPH-microsomes or NADPH-reductase systems. The inability of MPP+ to be bio-reduced or to stimulate the production of superoxide during aerobic reduction is evidence against a redox-cycling (oxidant stress) role of MPP+ in MPTP neurotoxicity.     

Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolites on the physicochemical property of the liposomal membrane in relation to their cytochrome P-450 inhibition.

The effects of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its toxic metabolites MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridinium) and MPP+ (1-methyl-4-phenylpyridinium) on liposomal membrane were assessed using fluorescence-polarization and carboxyfluorescein leakage studies as well as in biological membrane preparations. Of the three compounds, MPTP was found to cause the greatest perturbation of membrane followed by MPDP+ and then MPP+. The ability of the three toxins to inhibit cytochrome P-450 enzyme activity (a microsomal membrane-bound enzyme system) was also studied and their relative potency was again found to be MPTP > MPDP+ > MPP+. The changes in the physicochemical property of the liposomal membrane can be related to the ability of the neurotoxin's ability to inhibit cytochrome P-450 activity.     

Trace dosages of the neurotoxins MPTP and MPP+ may affect brain dopamine in vivo.

The present study has examined the effects of systemically administered MPTP and MPP+ upon striatal DA and Dopac of C57 mice, also treated concurrently with either saline or reserpine. MPTP followed by saline did not affect DA level but decreased that of Dopac only at 5.0 mg/kg and higher dosages. The potency of MPTP affecting DA increased greatly when the neurotoxicant was followed by either 5.0 or 10.0 mg/kg reserpine; MPTP at 0.10 mg/kg and higher dosages significantly reversed the DA depleting effects of reserpine. MPP+ (1.0 or 10.0 mg/kg) with saline did not affect either DA or Dopac. In contrast, MPP+ at 0.10 mg/kg and higher dosages, when followed by 10.0 mg/kg reserpine, dose-dependently enhanced the DA depleting effects of reserpine. In agreement with the earlier results obtained in vitro, the present study indicates that MPTP administration at trace level dosages may lead to an inhibition of MAO in vivo. The effect of systemically given MPP+ on DA, however, appears to be more complex in nature, conceivably comprised of actions at the striatal neurones including the intraneuronal vesicles and, possibly, at the substantia nigra which may affect striatum in turn. That MPP+ may have reached brain areas in these experiments is also indicated by the observation of a significant striatal level of 3H-MPP+ after its systemic administration. In conclusion, irrespective of MPTP and MPP+ action mechanisms, trace levels of these neurotoxicants appear to affect brain dopamine neurons.     

Effects of 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine and Its Metabolite 1-Methyl-4-Phenylpyridine on Acetylcholine Synthesis in Synaptosomes from Rat Forebrain

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) and its metabolite, 1-methyl-4-phenylpyridine (MPP+), have been shown to cause a number of lesions in dopaminergic pathways of the nigro-striatal region of the brain. However, data on the effects of these neurotoxins on other aspects of brain metabolism are scarce. The data presented here show that MPTP and MPP+ inhibit glucose oxidation via the tricarboxylic acid cycle, and acetylcholine synthesis in synaptosomal preparations from rat forebrain. Monoamine oxidase B inhibitors (e.g., pargyline, MDL 72145) relieve the inhibition caused by MPTP but not MPP+. The inhibitory effects of MPP+ on glucose oxidation and acetylcholine synthesis are a consequence of the decreased glucose metabolism in synaptosomes and are consistent with its role as an inhibitor of the Complex I (NADH-CoQ reductase) of the mitochondrial respiratory chain.     

Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine

1-methyl-4-phenylpyridine (MPP+), a major metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) inhibited the ADP-stimulated and uncoupled oxidation of NADH-linked substrates by brain mitochondrial preparations. MPTP itself was ineffective. The apparent Ki's for MPP+ inhibition of pyruvate or glutamate oxidation by purified rat brain mitochondria were approximately 300 and 400 microM, respectively; with mouse brain mitochondria the values were lower, 60 and 150 microM, respectively. Succinate oxidation was unaffected by either compound. Compromise of mitochondrial oxidative capacity by MPP+ could be an important factor in mechanisms underlying the toxicity of MPTP.     

Different effects of monoamine oxidase inhibition on MPTP depletion of heart and brain catecholamines in mice

Pargyline, an inhibitor of monoamine oxidase type B (MAO-B), did not prevent the depletion of heart norepinephrine 24 hr after a single dose of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in mice. In mice killed 24 hr after the last of 4 daily doses of MPTP, the depletion of dopamine in the striatum and of norepinephrine in the frontal cortex was completely prevented by pargyline, but the depletion of heart norepinephrine was not prevented. These results with pargyline are the same as results obtained earlier with deprenyl, another selective inhibitor of MAO-B. The doses of pargyline and of deprenyl that were used resulted in almost complete inhibition of MAO-B activity (phenylethylamine as substrate) in brain, heart and liver of mice. Deprenyl did not inhibit MAO-A activity (serotonin as substrate) in brain, but pargyline caused some inhibition of MAO-A in brain. In heart and liver, serotonin was oxidized only at about 1/10 the rate of phenylethylamine oxidation, suggesting that MAO-B predominates in these tissues. Both pargyline and deprenyl caused some inhibition of serotonin deamination in heart and liver, suggesting that the oxidation may have been due partly to MAO-B. Experiments with selective MAO inhibitors in vitro showed that only about 20% of the oxidation of serotonin was occurring via MAO-B in heart and liver. The in vitro oxidation of MPTP by MAO in mouse brain, heart and liver was almost completely inhibited by pretreatment with either pargyline or deprenyl. Neither pargyline nor deprenyl had any significant effect on the concentrations of MPTP in brain or heart one-half hr after injection of MPTP into mice. The concentrations of the metabolite, MPP+ (1-methyl-4-phenyl-pyridinium), were markedly reduced in brain and in heart by pretreatment with either pargyline or deprenyl. The data suggest that MPP+ formation, which is necessary for the depletion of brain catecholamines after MPTP injection, may not be necessary for depletion of norepinephrine in heart. Since the oxidation of MPTP in vitro was inhibited more by pargyline or deprenyl pretreatment than was the appearance of MPP+ in vivo, the possibility exists that some MPP+ formation might occur by an enzyme other than MAO.     

Pulmonary phosphatidic acid phosphohydrolase: further studies on the activities in rat lung responsible for the hydrolysis of membrane-bound and aqueously dispersed phosphatidate

Rat lung cytosol and microsomal fractions both contain phosphohydrolase activity towards membrane-bound phosphatidic acid (PAmb) and aqueously dispersed phosphatidic acid (PAaq) which cannot be explained through contamination with the other fraction. The phosphohydrolase activities with PAaq demonstrated Km and Vmax values which were more than an order of magnitude greater than those observed with PAmb and with vesicles prepared from the lipids extracted from [32P]PA-labelled microsomes. The PAaq-dependent activities in both fractions were stimulated by preparing mixed liposomes with phosphatidylcholine. The PAmb-dependent activities in rat lung microsomes and cytosol were markedly stimulated by high concentrations of Triton X-100 and Nonidet P-40. The PAmb- and PAaq-dependent activities in the microsomes were stimulated by deoxycholate. Although no difference was observed in the inhibition profiles of the PAmb- and PAaq-dependent activities of the cytosol in the presence of various mercurials, the PAmb-dependent activity in the microsomes was somewhat more susceptible than the PAaq-dependent activity. The PAmb-dependent activities in both fractions were more susceptible to inhibition by iodoacetamide. These results support the view that separate rat lung enzymes were involved in the hydrolysis of PAmb and PAaq. The relative abilities of rat lung cytosol and microsomes to hydrolyse PA endogenously generated on the microsomes were compared using relative concentrations of cytosol corresponding to the levels in intact rat lung. During the initial period (5-10 min) the cytosol phosphohydrolase activity was more effective than the microsomal activity. At later stages (10-20 min), the rates were comparable.