Blunted epidermal L-tryptophan metabolism in vitiligo affects immune response and ROS scavenging by Fenton chemistry, part 2: Epidermal H2O2/ONOO(-)-mediated stress in vitiligo hampers indoleamine 2,3-dioxygenase and aryl hydrocarbon receptor-mediated immune response signaling.

Vitiligo is characterized by a mostly progressive loss of the inherited skin color. The cause of the disease is still unknown, despite accumulating in vivo and in vitro evidence of massive oxidative stress via hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)) in the skin of affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Since depletion of the essential amino acid L-tryptophan (Trp) severely affects various immune responses, we here looked at Trp metabolism and signaling in these patients. Our in vivo and in vitro data revealed total absence of epidermal Trp hydroxylase activities and the presence of H(2)O(2)/ONOO(-) deactivated indoleamine 2,3-dioxygenase. Aryl hydrocarbon receptor signaling is severely impaired despite the ligand (Trp dimer) being formed, as shown by mass spectrometry. Loss of this signal is supported by the absence of downstream signals (COX-2 and CYP1A1) as well as regulatory T-lymphocytes and by computer modeling. In vivo Fourier transform Raman spectroscopy confirmed the presence of Trp metabolites together with H(2)O(2) supporting deprivation of the epidermal Trp pool by Fenton chemistry. Taken together, our data support a long-expressed role for in loco redox balance and a distinct immune response. These insights could open novel treatment strategies for this disease.

New anthraquinone dimer from the root bark of Cassia artemisioides (Gaudich. Ex. DC) Randell.

The phytochemical investigation of the root bark of Cassia artemisioides (Gaudich. Ex. DC) Randell resulted in the isolation of one new anthraquinone 1,1'-dihydroxy-3,3'-dimethyl-8,8'-dimethoxy-6,6'-O-bianthraquinone (1) along with four known anthraquinones 1,6-dihydroxy-8-methoxy-3-methylanthraquinone (2), 1-hydroxy-8-methoxy-3-methylanthraquinone (3), 1,8-dihydroxy-6-methoxy-3-methylanthraquinone (4), and 1,6,8-trihydroxy-3-methylanthraquinone (5). The structures of the compounds were elucidated using spectroscopic techniques including 1D and 2D NMR. The compounds were evaluated for antioxidant activity. 1,6,8-Trihydroxy-3-methyl anthraquinone (5) showed good activity among the tested compounds.

Evidence of structure-selective fragmentations in the tandem mass spectra of protonated hydroxyalkylamino-1,4-naphthoquinones formed by electrospray ionisation.

The collision-induced dissociation of protonated hydroxyalkylamino-1,4-naphthoquinones depends strongly on the structure of the substituent [NHCH(2)(CH(2))(n)OH, n = 1-5; or NHCH(2)CH(CH(3))OH] on the quinone ring. Protonated naphthoquinones with an unbranched hydroxypropylamino side chain (n = 3) undergo facile and characteristic CH(2)O loss, whereas isomeric [M + H](+) ions with a branched hydroxypropylamino side chain do not. When n = 1, CH(2)O elimination occurs less readily, accompanied by CH(3)N loss, thus allowing this shorter side chain to be identified. Higher homologous species (n = 3-5) do not expel CH(2)O, but instead eliminate C(n + 1)H(2n)O, C(n + 1)H(2n + 2)O and (for n = 5) C(2n + 1)H(2n + 1)O.

Presence of epidermal allantoin further supports oxidative stress in vitiligo.

Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the hydroxylation of hypoxanthine to xanthine and finally to uric acid in purine degradation. These reactions generate H(2)O(2) yielding allantoin from uric acid when reactive oxygen species accumulates. The presence of XO in the human epidermis has not been shown so far. As patients with vitiligo accumulate H(2)O(2) up to mm levels in their epidermis, it was tempting to examine whether this enzyme and consequently allantoin contribute to the oxidative stress theory in this disease. To address this question, reverse transcription-polymerase chain reaction, immunoreactivity, western blot, enzyme kinetics, computer modelling and high performance liquid chromatography/mass spectrometry analysis were carried out. Our results identified the presence of XDH/XO in epidermal keratinocytes and melanocytes. The enzyme is regulated by H(2)O(2) in a concentration-dependent manner, where concentrations of 10(-6 )m upregulates the activity. Moreover, we demonstrate the presence of epidermal allantoin in acute vitiligo, while this metabolite is absent in healthy controls. H(2)O(2)-mediated oxidation of Trp and Met in XO yields only subtle alterations in the enzyme active site, which is in agreement with the enzyme kinetics in the presence of 10(-3 )m H(2)O(2). Systemic XO activities are not affected. Taken together, our results provide evidence that epidermal XO contributes to H(2)O(2)-mediated oxidative stress in vitiligo via H(2)O(2)-production and allantoin formation in the epidermal compartment.

Methionine sulfoxide reductases A and B are deactivated by hydrogen peroxide (H2O2) in the epidermis of patients with vitiligo.

Patients with the depigmentation disorder vitiligo have low catalase expression/activities and constantly accumulate 10(-3) M hydrogen peroxide (H(2)O(2)) in their skin. Such high concentrations of H(2)O(2) oxidize L-methionine residues in proteins and peptides to (R and S)-methionine sulfoxide diasteriomers. In vivo FT-Raman Spectroscopy revealed the presence of methionine sulfoxide in the depigmented skin of patients with active vitiligo. In normal healthy human skin, methionine sulfoxide reductases A and B specifically reduce methionine sulfoxides (S) and (R), respectively, back to L-methionine consequently repairing oxidatively damaged proteins and peptides. In this report, we show that the expression/activities of MSRA and MSRB are significantly decreased in the epidermis of patients with vitiligo compared to healthy controls. Also, we used recombinant human MSRA and MSRB1 to show that both enzymes are deactivated by 10(-3) M H(2)O(2) by 85 and 40%, respectively. Structural modelling based on the crystal structure of human MSRA revealed that the active site of this enzyme is significantly altered after H(2)O(2)-mediated oxidation of L-methionine, L-tryptophan, and L-cysteine residues in its active site. Taken together, our results confirm that very important anti-oxidant enzymes are seriously affected in acute vitiligo.

Specific interaction of the diastereomers 7(R)- and 7(S)-tetrahydrobiopterin with phenylalanine hydroxylase: implications for understanding primapterinuria and vitiligo.

Pterin-4a-carbinolamine dehydratase (PCD) is an essential component of the phenylalanine hydroxylase (PAH) system, catalyzing the regeneration of the essential cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin [6(R)BH4]. Mutations in PCD or its deactivation by hydrogen peroxide result in the generation of 7(R,S)BH4, which is a potent inhibitor of PAH that has been implicated in primapterinuria, a variant form of phenylketonuria, and in the skin depigmentation disorder vitiligo. We have synthesized and separated the 7(R) and 7(S) diastereomers confirming their structure by NMR. Both 7(R)- and 7(S)BH4 function as poor cofactors for PAH, whereas only 7(S)BH4 acts as a potent competitive inhibitor vs. 6(R)BH4 (Ki=2.3-4.9 microM). Kinetic and binding studies, as well as characterization of the pterin-enzyme complexes by fluorescence spectroscopy, revealed that the inhibitory effects of 7(R,S)BH4 on PAH are in fact specifically based on 7(S)BH4 binding. The molecular dynamics simulated structures of the pterin-PAH complexes indicate that 7(S)BH4 inhibition is due to its interaction with the polar region at the pterin binding site close to Ser-251, whereas its low efficiency as cofactor is related to a suboptimal positioning toward the catalytic iron. 7(S)BH4 is not an inhibitor for tyrosine hydroxylase (TH) in the physiological range, presumably due to the replacement of Ser-251 by the corresponding Ala297. Taken together, our results identified structural determinants for the specific regulation of PAH and TH by 7(S)BH4, which in turn aid in the understanding of primapterinuria and acute vitiligo.

Pharmacological and biological evaluation of a series of substituted 1,4-naphthoquinone bioreductive drugs.

The indolequinone compound EO9 has good pharmacodynamic properties in terms of bioreductive activation and selectivity for either NAD(P)H:quinone oxidoreductase-1 (NQO1)-rich aerobic or NQO1-deficient hypoxic cells. However, its pharmacokinetic properties are poor and this fact is believed to be a major reason for EO9's lack of clinical efficacy. The purpose of this study was to develop quinone-based bioreductive drugs that retained EO9's good properties, in terms of bioreductive activation, but have improved pharmacokinetic properties. Out of 11 naphthoquinone compounds evaluated, 2-aziridinyl-5-hydroxy-1,4-naphthoquinone (compound 2), 2,3-bis(aziridinyl)-5-hydroxy-1,4-naphthoquinone (compound 3), and 2-aziridinyl-6-hydroxymethyl-1,4-naphthoquinone (compound 11) were selected for further evaluation based on good substrate specificity for NQO1 and selectivity towards NQO1-rich cells in vitro. Compound 3 was of particular interest as it also demonstrated selectivity for NQO1-rich cells under hypoxic conditions. Compound 3 was not metabolised by murine whole blood in vitro (in contrast to compounds 2, 11 and EO9) and pharmacokinetic studies in non-tumour-bearing mice in vivo (at the maximum soluble dose of 60 mg kg(-1) administered intraperitoneally) demonstrated significant improvements in plasma half-life (16.2 min) and AUC values (22.5 microM h) compared to EO9 (T(1/2) = 1.8 min, AUC = 0.184 microM h). Compound 3 also demonstrated significant anti-tumour activity against H460 and HCT-116 human tumour xenografts in vivo, whereas EO9 was inactive against these tumours. In conclusion, compound 3 is a promising lead compound that may target both aerobic and hypoxic fractions of NQO1-rich tumours and further studies to elucidate its mechanism of action and improve solubility are warranted.