Inhibition of DT-diaphorase potentiates the in vivo neurotoxic effect of intranigral injection of salsolinol in rats.

The present study shows that intranigral injection of dicoumarol, a DT-diaphorase inhibitor, potentiates the neurotoxic effect of salsolinol (salsolinol 1.25 nmoles plus dicoumarol 2 nmoles; in 2 microl). Rats treated with dicoumarol plus salsolinol presented a characteristic contralateral rotational behaviour when they were stimulated with apomorphine (0.5 mg/kg, s.c.), similar to rats injected unilaterally with 6-hydroxydopamine (6-OHDA). These rats also exhibited impairment of motor and cognitive behaviours. The results support the hypothesis that DT-diaphorase plays a protective role in the nigrostriatal dopaminergic systems.

MPP(+)-induced degeneration is potentiated by dicoumarol in cultures of the RCSN-3 dopaminergic cell line. Implications of neuromelanin in oxidative metabolism of dopamine neurotoxicity.

We have tested the idea that oxidative metabolism of dopamine may be involved in MPTP toxicity using the RCSN-3 cell line derived from the substantia nigra of an adult rat. Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (10 microM), MPTP combined with 40 microM dicoumarol (an inhibitor of DT-diaphorase) and dicoumarol alone, did not induce toxicity in RCSN-3 cells after 72 h incubation. The lack of toxicity in MPTP-treated RCSN-3 cells may be explained by the fact that they are unable to metabolize MPTP to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion (MPP+ as determined by HPLC. Incubation for 72 h with 100 microM MPP+ induced a 6.6 +/- 1.4% cell death of RCSN-3 cells compared to 3.5 +/- 0.4 observed in control cells. However, when the cells were treated with 100 microM MPP+ and 40 microM dicoumarol, cell death increased 4-fold compared to that of cells treated solely with MPP+ (27 +/- 2%; P<0.001). Under these conditions, a significant increase in DNA fragmentation (3-fold compared to MPP+ alone; P<0.01) and in calpain activation (P<0.05 compared to control) was evident. The inhibition of DT-diaphorase by dicoumarol supports the idea that oxidative metabolism of dopamine is involved in MPP+ toxicity in RCSN-3 cells.

Behavioral effects of aminochrome and dopachrome injected in the rat substantia nigra.

The exact mechanism of cell death in neurodegenerative diseases remains obscure, although there is evidence that their pathogenesis may involve the formation of free radicals originating from the oxidative metabolism of catecholamines. The purpose of this study was to evaluate the degree of neurodegenerative changes and behavioral impairments induced by unilateral injection into the rat substantia nigra of cyclized o-quinones, aminochrome and dopachrome, derived from oxidizing dopamine and L-DOPA, respectively, with Mn(3+)-pyrophosphate complex. The behavioral changes were compared with those induced after selective lesions of dopaminergic neurons with 6-hydroxydopamine (6-OHDA). Intranigral injection of aminochrome and dopachrome produced impairment in motor and cognitive behaviors. The behavioral impairment was also revealed by apomorphine-induced rotational asymmetry. Apomorphine (0.5 mg/kg sc) significantly increased rotational behavior in rats injected with aminochrome and dopachrome. These rats presented a clear motor bias showing a significant contralateral rotation activity, similar but less vigorous that in rats injected with 6-OHDA. The avoidance conditioning was seriously impaired in rats injected with aminochrome and dopachrome although only dopachrome-injected rats showed a similar hypomotility to 6-OHDA-injected rats. The behavioral effects were correlated to the extent of striatal tyrosine hydroxylase (TH)-positive fiber loss. Rats receiving unilateral intranigral aminochrome and dopachrome injections exhibited a 47.9+/-5.1% and a 39.7+/-4.4% reduction in nigrostriatal TH-positive fiber density. In conclusion, this study provided evidence that oxidizing DA and L-DOPA to cytotoxic quinones, aminochrome and dopachrome appears to be an important mediator of oxidative damage in vivo.

Inhibition of DT-diaphorase is a requirement for Mn(III) to produce a 6-OH-dopamine like rotational behaviour.

Intracerebral manganese administration together with the DT-diaphorase inhibitor dicoumarol [Mn(III) 40 nmol + -dicoumarol 2 nmol; in 4 micro l] into the left medial forebrain bundle (MFB) produced a behavioural pattern characterized by contralateral behaviour when the rats were stimulated with apomorphine (0.5 mg/kg s.c.), in a manner similar to that when administered to unilaterally 6-hydroxy-dopamine-lesioned animals. The same animals rotated towards the opposite side (ipsilaterally) when stimulated with d-amphetamine (2 mg/kg s.c.). These results support the idea that DT-diaphorase plays a protective role in the dopaminergic systems.

Neurotoxicity of some MAO inhibitors in adult rat hypothalamic cell culture.

Monoamine oxidase-A (MAO-A) [amiflamine (AMF) and 4-methylthioamphetamine (MTA)] and MAO-B (L-deprenyl) inhibitors were found to be cytotoxic in a concentration-dependent manner for RCHT cells derived from adult rat hypothalamus. The cytotoxic effects were increased when the inhibitors were co-incubated with dicoumarol and especially with 25 micro M AMF+100 micro M dicoumarol (2.5-fold; P <0.001). The treatment of RCHT cells solely with AMF induced a marked decrease in the expression of DT-diaphorase mRNA.

Possible role of salsolinol quinone methide in the decrease of RCSN-3 cell survival.

The endogenous dopamine-derived neurotoxin salsolinol was found to decrease survival in the dopaminergic neuronal cell line RCSN-3, derived from adult rat substantia nigra in a concentration-dependent manner (208 microM salsolinol induced a 50% survival decrease). Incubation of RCSN-3 cells with 100 micro;M dicoumarol and salsolinol significantly decreased cell survival by 2.5-fold (P < 0.001), contrasting with a negligible effect on RCHT cells, which exhibited nearly a 5-fold lower nomifensine-insensitive dopamine uptake. The levels of catalase and glutathione peroxidase mRNA were decreased when RCSN-3 cells were treated with 100 microM salsolinol alone or in the presence of 100 microM dicoumarol. In vitro oxidation of salsolinol to o-quinone catalyzed by lactoperoxidase gave the quinone methide and 1,2-dihydro-1-methyl-6,7-isoquinoline diol as final products of salsolinol oxidation as determined by NMR analysis. Evidence of the formation of salsolinol o-semiquinone radical has been provided by ESR studies during one-electron oxidation of salsolinol catalyzed by lactoperoxidase.

Copper neurotoxicity is dependent on dopamine-mediated copper uptake and one-electron reduction of aminochrome in a rat substantia nigra neuronal cell line.

The mechanism of copper (Cu) neurotoxicity was studied in the RCSN-3 neuronal dopaminergic cell line, derived from substantia nigra of an adult rat. The formation of a Cu-dopamine complex was accompanied by oxidation of dopamine to aminochrome. We found that the Cu-dopamine complex mediates the uptake of (64)CuSO(4) into the Raúl Caviedes substantia nigra-clone 3 (RCSN3) cells, and it is inhibited by the addition of excess dopamine (2 m M) (63%, p < 0.001) and nomifensine (2 microM) (77%, p < 0.001). Copper sulfate (1 m M) alone was not toxic to RCSN-3 cells, but was when combined with dopamine or with dicoumarol (95% toxicity; p < 0.001) which inhibits DPNH and TPNH (DT)-diaphorase. Electron spin resonance (ESR) spectrum of the 5,5-dimethylpyrroline-N-oxide (DMPO) spin trap adducts showed the presence of a C-centered radical when incubating cells with dopamine, CuSO(4) and dicoumarol. A decrease in the expression of CuZn-superoxide dismutase and glutathione peroxidase mRNA was observed when RCSN-3 cells were treated with CuSO(4), dopamine, or CuSO(4) and dopamine. However, the mRNA expression of glutathione peroxidase remained at control levels when the cells were treated with CuSO(4), dopamine and dicoumarol. The regulation of catalase was different since all the treatments with CuSO(4) increased the expression of catalase mRNA. Our results suggest that copper neurotoxicity is dependent on: (i) the formation of Cu-dopamine complexes with concomitant dopamine oxidation to aminochrome; (ii) dopamine-dependent Cu uptake; and (iii) one-electron reduction of aminochrome.

The possible role of one-electron reduction of aminochrome in the neurodegenerative process of the dopaminergic system.

We present for discussion a possible molecular mechanism explaining the formation of reactive oxygen species involved in the neurodegenerative process of dopaminergic system in Parkinson's disease. This new hypothesis involves one-electron reduction of aminochrome to o-semiquinone radical, which seems to be the reaction responsible for neurodegenerative process of dopaminergic system. Leukoaminochrome o-semiquinone is extremely reactive with oxygen, which reoxidizes by reducing oxygen to superoxide radicals. Superoxide radicals enzymatically or spontaneously dismutate to dioxygen and hydrogen peroxide which is a precursor of hydroxyl radicals. ESR-experiments have showed that aminochrome o-semiquinone is extremely reactive in the presence of oxygen compared to dopamine o-semiquinone. In addition, the antioxidant enzymes superoxide dismutase and catalase play a prooxidant role by increasing the autoxidation rate and formation of superoxide radicals. One electron reduction of aminochrome to o-semiquinone can be performed by flavoenzymes which use NADPH and NADH as electron donator. The ability of aminochrome o-semiquinone to autoxidize in the presence of oxygen gives rise to a redox cycling process which will continue until oxygen, NADH and/or NADPH are depleted. Depletion of NADPH will prevent glutathione reductase from reducing glutathione, which is one of the main antioxidants in the cell. In addition depletion of NADH will prevent the formation of ATP in the electron transport chain in the mitochondria. Two antioxidants, probably, neuroprotective reactions are also discussed.

Angiotensin receptor II is present in dopaminergic cell line of rat substantia nigra and it is down regulated by aminochrome.

Angiotensin receptor II mRNA was found to be expressed in dopaminergic neuronal cell line RCSN3 of rat substantia nigra using RT-PCR reaction. Aminochrome (150 microM), a metabolite of the dopamine oxidative pathway, was found to down regulate the expression of angiotensin receptor mRNA in RCSN3 cells by 83% (p < 0.05).

Studies of aminochrome toxicity in a mouse derived neuronal cell line: is this toxicity mediated via glutamate transmission?

Aminochrome was found to be toxic in a mouse-derived neuronal cell line (CNh). The effect was concentration dependent (10-150microM). The issue whether aminochrome toxicity involves glutamate transmission was studied with several glutamate receptors antagonists. Incubation of the cells with aminochrome (150microM) in the presence of 100microM of the AMPA antagonist, NBQX resulted in an increase of cell survival, from 52 to 73%. However, this protective effect did not seem to be related to activation of ionotropic glutamate receptors since incubation of CNh cells with 200microM of glutamate resulted in only 10% decrease of cell survival. However, NBQX was found to inhibit in vitro the autoxidation process. One hundred microM AP-5 did not have any effect on aminochrome toxicity. The toxic effect of aminochrome on CNh cells seems to be dependent of extracellular activation since addition of dicoumarol, a specific inhibitor of DT-diaphorase, did not affect that toxicity, which can be explained perhaps by a lack of a transport system for aminochrome into the CNh cells.

Glutathione transferase M2-2 catalyzes conjugation of dopamine and dopa o-quinones.

Human glutathione transferase M2-2 prevents the formation of neurotoxic aminochrome and dopachrome by catalyzing the conjugation of dopamine and dopa o-quinone with glutathione. NMR analysis of dopamine and dopa o-quinone-glutathione conjugates revealed that the addition of glutathione was at C-5 to form 5-S-glutathionyl-dopamine and 5-S-glutathionyl-dopa, respectively. Both conjugates were found to be resistant to oxidation by biological oxidizing agents such as O(2), H(2)O(2), and O(*-)(2), and the glutathione transferase-catalyzed reaction can therefore serve a neuroprotective antioxidant function.

Reduction of brain antioxidant defense upon treatment with butylated hydroxyanisole (BHA) and Sudan III in Syrian golden hamster.

Treatment with the antioxidant butylated hydroxyanisole (BHA) or the azo dye Sudan III during two weeks led to changes in the brain enzymatic antioxidant defense of Syrian golden hamsters. BHA was able to induce liver superoxide dismutase (SOD) 2-fold but had no effect on the brain SOD activity, whereas SOD activity was reduced to 50% in brain and remained unchanged in liver with Sudan III. These two substances are known inducers of DT-diaphorase and in fact this enzymatic activity was induced 4- and 6-fold in liver with BHA and Sudan III, respectively. However, BHA promoted a significant 40% reduction, whereas no change was observed with Sudan III in brain DT-diaphorase activity. Glutathione(GSH)-related enzymatic activities were also assayed in brain and liver. No induction was observed with BHA or Sudan III for any of the activities tested in hamster brain: GSH S-transferase (GST), GSH peroxidase (GSH-Px) and glutathione disulfide (GSSG) reductase (GR). Only 1.3- and 1.4-fold increases of GST and GR activities were observed in liver and no change in any of these enzymatic activities in brain with BHA; a partial limitation of permeability to BHA of the blood-brain barrier may explain this results. Furthermore, Sudan III promoted reductions in all these GSH-related enzymatic activities in brain and liver. The possible explanations for these results are discussed.

Regioselectivity and reversibility of the glutathione conjugation of quercetin quinone methide.

The chemical reactivity, isomerization, and glutathione conjugation of quercetin o-quinone were investigated. Tyrosinase was used to generate the unstable quercetin o-quinone derivative which could be observed upon its subsequent scavenging by glutathione. Identification of the products revealed formation of 6-glutathionyl-quercetin and 8-glutathionyl-quercetin adducts. Thus, in particular, glutathione adducts in the A ring of quercetin were formed, a result which was not expected a priori. Quantum mechanical calculations support the possibility that the formation of these glutathione adducts can be explained by an isomerization of quercetin o-quinone to p-quinone methides. Surprisingly, additional experiments of this study reveal the adduct formation to be reversible, leading to interconversion between the two quercetin glutathione adducts and possibilities for release and further electrophilic reactions of the quercetin quinone methide at cellular sites different from those of its generation.

Interplay between CYP3A-mediated metabolism and polarized efflux of terfenadine and its metabolites in intestinal epithelial Caco-2 (TC7) cell monolayers.

PURPOSE: To further characterize cytochrome P450 (CYP) and P-glycoprotein (Pgp) expression in monolayers of the Caco-2 cell clone TC7, a cell culture model of the human intestinal epithelium. To study the interplay between CYP3A and Pgp as barriers to intestinal drug absorption in TC7 cells using terfenadine and its metabolites as substrates. METHODS: mRNA expression of eight CYPs and Pgp was investigated in TC7 and parental Caco-2 (Caco-2p) cell monolayers using RT-PCR. The CYP3A kinetics was determined in microsomes from both cell lines. The transport, metabolism and efflux of terfenadine and its metabolites were investigated in TC7 monolayers. RESULTS: Both TC7 and Caco-2p cells expressed mRNA for Pgp and several important CYPs. However, mRNA for CYP3A4 was detectable anly from TC7 cells. The relative affinity of CYP3A for terfenadine metabolism in the two cell lines was comparable, but the maximum reaction rate in the TC7 cells was 8-fold higher. The rate of transport of terfenadine and its metabolites hydroxy-terfenadine (HO-T) and azacyclonol across TC7 monolayers was 7.1-, 3.5- and 2.1-fold higher, respectively, in the basolateral to apical direction than it was in the apical to basolateral (AP-BL) direction. Inhibition studies indicated that the efflux was mediated by Pgp. Ketoconazole increased the AP-BL transport terfenadine dramatically by inhibiting both terfenadine metabolism and Pgp efflux. CONCLUSIONS: Cell culture models such as TC7 provide qualitative information on drug interactions involving intestinal CYP3A and Pgp.

Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product.

In the last ten years, there has been an important increase in interest in quercetin action as a unique antioxidant, but its putative role in numerous prooxidant effects is also being continually updated. The mechanism underlying this undesirable ability seems to involve its metabolic oxidoreductive activation. Based on the structural properties of quercetin, we have investigated whether its catechol moiety may be the potential tool for revealed toxicity. We demonstrated, with an ESR spin-stabilization technique coupled to conventional spectrophotometry, that o-semiquinone and o-quinone are indeed the products of enzymatically catalyzed oxidative degradation of quercetin. The former radical might serve to facilitate the formation of superoxide and depletion of GSH, which could confer a specificity of its prooxidative action in situ. The observed one-electron reduction of o-quinone may enrich the semiquinone pool, thereby magnifying its effect. The two-electron reduction of quinone can result in constant resupply of quercetin in situ, thereby also modulating another pathway of its known biological activities. We have also tried to see whether the intracellular oxidative degradation of quercetin can be confirmed under the controlled conditions of model monolayer cell cultures. The results are indicative of the intracellular metabolic activation of quercetin to o-quinone, the process which can be partially associated with the observed concentration-dependent cytotoxic effect of quercetin.

Role of redox cycling and activation by DT-diaphorase in the cytotoxicity of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB-1954) and its analogs.

In tumor cell lines with high content of DT-diaphorase (EC 1.6.99.2), the cytotoxicity of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB-1954) and its derivatives is exerted through DT-diaphorase-catalyzed formation of crosslinking species. However, little is known about other possible mechanisms of CB-1954 action. We have examined the toxicity of CB-1954 and its derivatives to bovine leukemia virus-transformed lamb fibroblasts (line FLK), which possessed moderate DT-diaphorase activity, 260 units/mg protein. The action of these compounds was accompanied by lipid peroxidation, their toxicity was decreased by desferrioxamine and antioxidant N,N'-diphenyl-p-phenylene diamine (DPPD), but, in most cases, not by dicumarol, an inhibitor of DT-diaphorase. Using multiparameter regression analysis, we have found that the toxicity of CB-1954 derivatives as well as that of several non-alkylating nitroaromatics, increased upon the increase in their single-electron reduction potential (E(1)7) and octanol/water partition coefficient (P), and almost did not depend on their reactivity towards DT-diaphorase. It seems that in cell lines with a moderate amount of DT-diaphorase, the toxicity of CB- 1954 and its analogs is exerted through their redox cycling.

DT-diaphorase catalyzes N-denitration and redox cycling of tetryl.

Rat liver DT-diaphorase (EC 1.6.99.2) catalyzed reductive N-denitration of tetryl (2,4,6-tri-nitrophenyl-N-methylnitramine) and 2,4-dinitrophenyl-N-methylnitramine, oxidizing the excess of NADPH. The reactions were accompanied by oxygen consumption and superoxide dismutase-sensitive reduction of added cytochrome c and reductive release of Fe2+ from ferritin. Quantitatively, the reactions of DT-diaphorase proceeded like single-electron reductive N-denitration of tetryl by ferredoxin:NADP+ reductase (EC 1.18.1.2) (Shah, M.M. and Spain, J.C. (1996) Biochem. Biophys. Res. Commun. 220, 563-568), which was additionally checked up in this work. Thus, although reductive N-denitration of nitrophenyl-N-nitramines is a net two-electron (hydride) transfer process, DT-diaphorase catalyzed the reaction in a single-electron way. These data point out the possibility of single-electron transfer steps during obligatory two-electron (hydride) reduction of quinones and nitroaromatics by DT-diaphorase.

Metabolic activation of dopamine o-quinones to o-semiquinones by NADPH cytochrome P450 reductase may play an important role in oxidative stress and apoptotic effects.

In this study, it is shown that considerable evidence for the possible pathway by which dopamine o-quinone, o-quinone and aminochrome can be activated metabolically by NADPH cytochrome P450 reductase to high reactive semiquinones. These findings were discussed from a mechanistic standpoint as well as in terms of potential physiological implications of dopamine o-quinones and o-semiquinones' concerted action in oxidative stress and apoptotic events.

Quantitative structure activity relationships for the conversion of nitrobenzimidazolones and nitrobenzimidazoles by DT-diaphorase: implications for the kinetic mechanism.

Quantitative structure activity relationships (QSARs) for the conversion of nitrobenzimidazolones and nitrobenzimidazoles by rat liver DT-diaphorase (EC 1.6.99.2) are described. The parameter used for description of the QSARs is the energy of the lowest unoccupied molecular orbital (E(LUMO)) of the nitroaromatic compounds. Interestingly, correlations with E(LUMO) were observed for both the natural logarithm of kcat, but also for the natural logarithm of kcat/Km. The minimal kinetic model in line with these QSARs is a ping-pong mechanism that includes a substrate binding equilibrium in the second half reaction.

Nitrobenzimidazoles as substrates for DT-diaphorase and redox cycling compounds: their enzymatic reactions and cytotoxicity.

We have synthesized a number of nitrobenzimidazoles containing nitro groups in the benzene ring and found that they acted as relatively efficient substrates for rat liver DT-diaphorase (EC 1.6.99.2), their reactivity exceeding reactivities of nitrofurans and nitrobenzenes. Nitrobenzimidazoles were competitive with NADPH inhibitors of DT-diaphorase in menadione reductase reactions, their inhibition constant being unchanged in the presence of dicumarol and being increased in the presence of 2',5'-ADP. These data indicate that the poor reactivity of nitrobenzimidazoles and other nitroaromatics in comparison to quinones could be determined by their binding in the adenosine-phosphate binding region of the NADPH-binding site, whereas quinones bind at the nicotinamide-binding pocket at the vicinity of FAD of DT-diaphorase. The reduction of 4,5,6-trinitrobenzimidazol-2-one by DT-diaphorase most probably involves reduction of 5-nitro group to 5-nitroso or 5-hydroxylamine derivative at the initial step. A certain parallelism existed between reactivities of nitrobenzimidazoles toward DT-diaphorase and their reactivities in single-electron reduction by Anabaena ferredoxin:NADP+ reductase (EC 1.18.1.2) and Saccharomyces cerevisiae flavocytochrome b2 (EC 1.1.2.3), the latter being determined by electronic factors. However, we suppose that the relatively high reactivity of polinitrobenzimidazoles toward DT-diaphorase was due not only to electronic effects, but also to a sterical crowding of nitrogroups by each other. The toxicity of nitrobenzimidazoles to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) with a moderate amount of DT-diaphorase (260 U/mg protein) is partly prevented by dicumarol. That points out to partial determination of nitrobenzimidazole cytotoxicity by their reduction by DT-diaphorase. Another important factor of nitrobenzimidazole toxicity to this cell line was oxidative stress, catalyzed by single-electron transferring enzymes.