DHI) And 5,6-dihydroxyindole-2-carboxylic Acid Research Articles

The Effect of Cosmetic Ingredients of Phenol Type on Immediate Pigment Darkening and Their (Photo)Protective Action in Association with Melanin Pigmentation: A Model In Vitro Study

Immediate pigment darkening, the first response of skin to solar exposure leading to undesired irregular pigmentation and dark spots, is the rapid onset of melanin pigmentation resulting from oxidation of the melanogenic indoles, namely 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) available in epidermal melanocytes. The search for effective sunscreen formulations is nowadays focused on UVA/B filters and additional ingredients that may scavenge the reactive oxygen species generated in these processes. In this work the effects of phenolic cosmetic ingredients (CIs), paradol-6, a ginger CO2 extract, and phenylethyl resorcinol on photosensitized DHI and DHICA oxidation were investigated showing a decrease of their consumption and melanin formation (25–30% decrease with phenylethyl resorcinol). The photoprotective role of CIs was also evaluated in model systems. Paradol-6 and ginger CO2 extract can halve linoleic acid peroxidation in the riboflavin-sensitized reaction, while dienes generation reduction (30% of control) was observed in the Rose-Bengal-sensitized photooxidation with paradol-6. The presence of DHI/DHICA melanin exerted a synergistic effect. The decay of thymine free or as a DNA base was almost completely inhibited by CIs. These results open new perspectives in the design of skin care formulations for ameliorating skin spots and contrasting ageing processes associated with sun exposure.

Pigment molecular composition reveals significant information for visual communication

Abstract Melanins are the most common pigments in vertebrates and, as such, fufill multiple adaptive functions, including honest signalling. This is the case of male pied flycatchers Ficedula hypoleuca, whose dorsal plumage, composed of black and grey feathers pigmented by eumelanin, is sexually selected by females regarding the proportion of black. However, the basis of such mating preferences and other associations with life‐history traits are unknown. Here, I take the advantage of recent advances in Raman spectroscopy analysis to investigate the monomeric composition of eumelanin, constituted by 5,6‐dihydroxyindole (DHI) and 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) subunits, in male pied flycatchers. I found that plumage blackness (percentage of dorsal black feathers) increases with the DHICA:DHI ratio of the constituent black freathers, but not with that of grey feathers. The repeatability of DHICA:DHI measurements in black feathers is two times that in grey feathers. Eumelanin chemistry may thus constitute the basis of signal honesty in pied flycatchers, as females preferentially mate with males with higher relative DHICA feather contents and this may be related to the lower pro‐oxidant effect of DHICA. Given the the ubiquitous nature of melanin‐based pigmentation phenotypes, the monomeric composition of eumelanin should therefore be considered, instead of eumelanin as a whole, for a better understanding of how and why phenotypes are linked to life‐history traits in animals. Read the free Plain Language Summary for this article on the Journal blog.

5,6-Dihydroxyindole eumelanin content in human skin with varying degrees of constitutive pigmentation.

Human skin contains two distinct components: brown to black, insoluble eumelanin and light colored, alkaline-soluble pheomelanin. Eumelanin consists of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) moieties, while pheomelanin consists of benzothiazine (BT) and benzothiazole (BZ) moieties. These melanin monomer units can be quantitatively analyzed through specific degradation products by high-performance liquid chromatography (HPLC). Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin gives rise to pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA) as specific degradation products of the DHICA and DHI moieties, respectively. BZ moiety in pheomelanin can be analyzed as thiazole-2,4,5-tricarboxylic acid (TTCA). By reductive hydrolysis with hydroiodic acid, BT moieties in pheomelanin can be analyzed as 4-amino-3-hydroxyphenylalanine (4-AHP). As a recently improved AHPO-HPLC method enabled a better characterization of PDCA, this prompted us to address the question of DHI to DHICA ratio in human skin samples with varying degrees of constitutive pigmentation ranging from very light to dark. Results showed for the first time the ratio of 4 moieties: DHI 35%, DHICA 41%, BZ 20%, and BT 4%. The ratio is constant regardless of the degree of pigmentation. The high content of DHICA moiety may impart an antioxidant property to the epidermis melanin.

Locating the bandgap edges of eumelanin thin films for applications in organic electronics

AbstractBACKGROUNDBio‐sourced (natural) organic materials are often chemically and structurally disordered, such that their structure‐property relationships must be explored using model systems. Eumelanin is an interesting candidate among natural organic materials.RESULTSIn this work, the locations of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy levels of 5,6‐dihydroxyindole (DHI) and 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) building blocks (monomers) of the black‐brown biopigment eumelanin, in film form, are studied. The films are fabricated by the spin‐coating technique (i.e., here indicated as DHI‐ and DHICA‐films), which is sometimes followed by ammonia‐induced solid‐state polymerization (i.e., indicated as AISSP‐DHI and AISSP‐DHICA films), as well as by thermal evaporation (i.e., evaporated DHI and DHICA films). From Ultraviolet photoemission spectroscopy (UPS), we deduced the ionization energies (EI) for all DHI‐ and DHICA‐based films to be in the range of 5.34‐5.56 eV and 5.35‐5.80 eV, respectively. The electron affinities (χE) are measurable using inverse photoemission spectroscopy (IPES) for DHI films (3.80 eV) and both evaporated DHI (4.0 eV) and DHICA (3.9 eV) films. On the other hand, the χE values of DHICA, AISSP‐DHI, and AISSP‐DHICA films are estimated with about 0.5 eV of uncertainty. UV‐visible spectroscopy reveals the preferred chromophoric bands for DHICA, AISSP‐DHICA, and DHI are in the range of 300‐330 nm, while AISSP‐DHI exhibits a broadened UV‐visible absorbance.CONCLUSIONOur study paves the way for the design of suitable metal‐eumelanin interfaces for electronic applications. © 2021 Society of Chemical Industry (SCI).

Effects of Aging on Hair Color, Melanosome Morphology, and Melanin Composition in Japanese Females.

In a previous study, we showed that the size of melanosomes isolated from Japanese female hairs enlarges with age, and this affects the hair color. In this study, we analyzed the age-dependent changes in hair melanin in order to further explore the factors related to hair color changing by aging. A significant positive correlation with age was found in the total melanin amount (TM) and the mol% of 5,6-dihydroxyindole (DHI) units, while no correlation was found in pheomelanin mol%. TM showed significant correlations with hair color parameters and the melanosome volume, suggesting that hair color darkening by aging is caused by the increase in TM due to the enlargement of the size of melanosome. From the measurement of absorbance spectra on synthetic eumelanins with different ratios of DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), we found that the increase in DHI mol% also contributes to the darkening of hair color by aging. In addition, the level of pyrrole-2,3-dicarboxylic acid (PDCA), a marker of DHI melanin, showed a significant negative correlation with the aspect ratio of melanosome, suggesting a contribution of DHI melanin to the change in melanosome morphology by aging.

The Late Stages of Melanogenesis: Exploring the Chemical Facets and the Application Opportunities.

In the last decade, the late stages of melanin biosynthesis involving the oxidative polymerization of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) have been extensively investigated. Most of the information derived from a biomimetic approach in which the oxidation of melanogenic indoles was carried out under conditions mimicking those occurring in the biological environment. Characterization of the early oligomers allowed for drawing a structural picture of DHI and DHICA melanins, providing also an interpretative basis for the different properties exhibited by these pigments, e.g., the chromophore and the antioxidant ability. The improved knowledge has opened new perspectives toward the exploitation of the unique chemistry of melanins and its precursors in cosmetic and health care applications. A noticeable example is the development of an innovative hair dyeing system that is based on the marked ease of DHI to give rise to black melanin on air oxidation under slightly alkaline conditions. The advantage of this method for a step-wise coverage of gray hair with a natural shade pigmentation on repeated treatment with a DHI-based formulation with respect to traditional dyes is presented. A variant of DHICA melanin combining solubility in water-miscible organic solvents, an intense chromophore in the UltraViolet-A UV-A region, and a marked antioxidant potency was evaluated as an ingredient for cosmetic formulations.

Raman spectroscopy quantification of eumelanin subunits in natural unaltered pigments.

Eumelanins are large polymers composed of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Reactive oxygen species generation is higher during the synthesis of DHI than during the synthesis of DHICA; thus, the production of eumelanins with high relative DHI contents may compromise the viability of cells and organisms. Here, we show that dispersive Raman spectroscopy allows a noninvasive direct detection and quantification of DHI and DHICA in unaltered eumelanins that can be applied to the analysis of natural pigments in biological tissues. Using synthetic eumelanins purely composed of DHI or DHICA, we develop a model to determine the differential contribution of these subunits to the Raman spectra of mixed eumelanins, and exemplify the predictive capacity of the model with natural eumelanins deposited in black and gray feathers of fowl. We found that the wavenumber ranges of 550-1,200 and 1,650-2,300cm-1 are significant to predict the DHI:DHICA ratio in unknown samples.

An electrochemical study of natural and chemically controlled eumelanin

Eumelanin is the most common form of the pigment melanin in the human body, with functions including antioxidant behavior, metal chelation, and free radical scavenging. This biopigment is of interest for biologically derived batteries and supercapacitors. In this work, we characterized the voltammetric properties of chemically controlled eumelanins produced from 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) building blocks, namely, DHI-melanin, DHICA-melanin, and natural eumelanin, extracted from the ink sac of cuttlefish, Sepia melanin. Eumelanin electrodes were studied for their cyclic voltammetric properties in acidic buffers including Na+, K+, NH4+, and Cu2+ ions.

Sequential Proton-Coupled Electron Transfer Mediates Excited-State Deactivation of a Eumelanin Building Block.

Skin photoprotection is commonly believed to rely on the photochemistry of 5,6-dihydroxyindole (DHI)- and 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-based eumelanin building blocks. Attempts to elucidate the underlying excited-state relaxation mechanisms have been partly unsuccessful due to the marked instability to oxidation. We report a study of the excited-state deactivation of DHI using steady-state and time-resolved fluorescence accompanied by high-level quantum-chemistry calculations including solvent effects. Spectroscopic data show that deactivation of the lowest excited state of DHI in aqueous buffer proceeds on the 100 ps time scale and is 20 times faster than in methanol. Quantum-chemical calculations reveal that the excited-state decay mechanism is a sequential proton-coupled electron transfer, which involves the initial formation of a solvated electron from DHI, followed by the transfer of a proton to the solvent. This unexpected finding would prompt a revision of current notions about eumelanin photophysics and photobiology.

Intermolecular π‐Electron Perturbations Generate Extrinsic Visible Contributions to Eumelanin Black Chromophore in Model Polymers with Interrupted Interring Conjugation

The key structural factors underlying the unique black chromophore of eumelanin biopolymers have so far defied elucidation. Capitalizing on the ability of 1% polyvinylalcohol (PVA) to prevent pigment precipitation during melanogenesis in vitro, we have investigated the visible chromophore properties of soluble eumelanin-like polymers produced by biomimetic oxidation of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) in 1% PVA-containing buffer at pH 7. Upon dilution DHI-eumelanin solutions exhibited almost linear visible absorbance changes, whereas DHICA-eumelanin displayed a remarkable deviation from linearity in simple buffer, but not in PVA-containing buffer. It is suggested that in DHICA polymers, exhibiting repeated interruptions of interring conjugation due to lack of planar conformations, the black chromophore is not due to an overlap of static entities defined intrinsically by the conjugation length across the carbon frame, but results largely from aggregation-related intermolecular perturbations of the π-electron systems which are extrinsic in character.

UV-Absorption Spectra of Melanosomes Containing Varying 5,6-Dihydroxyindole and 5,6-Dihydroxyindole-2-Carboxylic Acid Content

Central to understanding the photochemical properties of melanosomes is a direct measurement of their absorption coefficients. Herein, the absorption spectra of intact melanosomes of varying molecular compositions and embryonic origins were measured and compared over the spectral range from 245 to 310 nm. The absorption spectra of melanosomes predominately comprised of the eumelanin pigment were found to differ significantly from their constituent precursor molecules, 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). This difference was most notable in the UV-A region and indicates that the electronic structures of the monomeric building blocks, DHICA in particular, are significantly modified upon polymerization to the melanin pigment. Furthermore, in comparing embryonic differences, the absorption coefficients of melanosomes isolated from bovine retinal pigment epithelial (RPE) cells (originating from the primitive forebrain) were greater than those for bovine choroid or iris melanosomes (originating from the neural crest). This difference suggests that either the pigment is present in greater density in RPE melanosomes or that there is an underlying difference in molecular structure.

Ultraviolet Absorption Coefficients of Melanosomes Containing Eumelanin As Related to the Relative Content of DHI and DHICA

Central to understanding the photochemical function(s) of melano- somes is the determination of their absorption properties and an understanding of how the absorption varies with the molecular composition of melanin. Herein, the absorption coefficients for melanosomes containing predominantly eumelanin, a polymeric pigment derived from the molecular precursors 5,6-dihydroxyindole (DHI)and5,6-dihydroxyindole-2-carboxylicacid(DHICA),arereportedfor λ=244nm. The absorption coefficient varies with the DHICA/DHI ratio, determined from chemical degradationanalyses.WithincreasingDHICAcontent,theabsorptioncoefficientof the melanosome increases. This observation is consistent with reported extinction coefficients, which reveal that at 244 nm, the extinction coefficient of DHICA is a factor of ∼2.1 greater than that of DHI. The melanosome absorption coefficients are compared to predicted values based on a linear combination of the absorption of the constituent monomers.

Expedient routes to valuable bromo-5,6-dimethoxyindole building blocks

The synthesis of 3-, 4-, 7-bromo and 4,7-dibrominated 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derivatives is reported. Hemetsberger and Bartoli indole syntheses were investigated and expedient routes to the desired compounds were developed. These indoles are valuable substrates for elaboration using transition metal-mediated cross-coupling chemistry.

An oxygen transporter hemocyanin can act on the late pathway of melanin synthesis

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) are precursors of eumelanin. The effects of crustacean hemolymph proteins on these eumelanin-related metabolites were investigated. Zymogram analysis indicated that polymers of hemocyanin (Hc) subunits converted DHI into black pigment while no effects were observed using DHICA as a substrate. Spectrum changes for mixtures of purified Hc and DHI showed a profile similar to oxidized DHI by mushroom tyrosinase while Hc had only slight effects on DHICA. Typical inhibitors of tyrosinase and phenoloxidase severely hampered the production of oxidized DHI. Taken together with previous results, these data indicate that Hc plays a crucial role in the conversion of DHI in the hemolymph of crustaceans, which promotes late reactions in the melanin synthetic pathway as well as early reactions (oxidation of tyrosine and DOPA to dopaquinone).

Relationship of Melanin Degradation Products to Actual Melanin Content: Application to Human Hair

Methods not only for characterizing but also for quantitating melanin subtypes from the two types of melanin found in hair—eumelanin and pheomelanin—have been established. In relation to testing for drugs of abuse in hair, these methods will allow for correction of drug binding to specific melanin subtypes and will serve to improve drug measurement in hair. 5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) make up the majority of the eumelanin polymer while benzothiazene units derived from 2-cysteinyl-S-Dopa (2-CysDopa) and 5-cysteinyl-S-Dopa (5-CysDopa) compose the majority of the pheomelanin polymer. Our results show that: (1) pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), markers for DHI and DHICA units, respectively, are produced in 0.37 and 4.8% yields, respectively, when melanins are subjected to alkaline hydrogen peroxide degradation, (2) 3-aminotyrosine (3AT) and 4-amino-3-hydroxyphenylalanine (AHP), markers for 2-CysDopa and 5-CysDopa, respectively, are produced in 16 and 23% yield, respectively, when subjected to hydriodic acid hydrolysis, and (3) that black human hair contains approximately 99% eumelanin and 1% pheomelanin, brown and blond hair contain 95% eumelanin and 5% pheomelanin; and red hair contains 67% eumelanin and 33% pheomelanin. These data will allow deeper investigation into the relationship between melanin composition and drug incorporation into hair.

Isolation and characterization of mammalian eumelanins from hair and irides

A new enzymatic procedure was developed for isolation of eumelanin from black human hair which might provide a substantially intact pigment for structural characterization. Sequential digestion with protease, proteinase K and papaine in the presence of dithiothreitol afforded a pigment with a 6% w/w protein content. HPLC analysis of pyrrole acids resulting from alkaline H 2O 2 degradation, carboxyl content determination, and ferricyanide titration showed that the isolated pigment is made up of 5,6-dihydroxyindole (DHI)- and 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units at a 6:1 ratio, exhibiting a significant degree of oxidative degradation. For comparison, a different eumelanin isolated from black bovine irides by a similar enzymatic procedure was analyzed. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the final pigment provided evidence for homologous series of DHICA oligomers, while chemical analysis allowed an estimate of 2:1 DHICA/DHI-derived units in the polymer, with a substantial proportion of intact o-diphenolic functions. Iris melanin proved able to promote the Fenton oxidation of deoxyribose while hair melanin was ineffective. Overall, these results provide, for the first time, unambiguous evidence for marked structural differences of mammalian eumelanins which may be directly related to the diversity of the sites of biosynthesis and storage, as well as to functional role of these pigments.

Absolute rate constants for the quenching of reactive excited states by melanin and related 5,6-dihydroxyindole metabolites: implications for their antioxidant activity.

The triplet-excited state of benzophenone and the singlet-excited state of 2,3-diazabicyclo[2.2.2]oct-2-ene (Fluorazophore-P) have been employed as kinetic probes to obtain information on the antioxidant activity of the skin and eye pigment melanin and its biogenetic precursors 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The excited states were generated by the laser-flash photolysis technique and their reaction kinetics was examined by time-resolved transient absorption or fluorescence spectroscopy, respectively. The reaction between triplet benzophenone and DHI produced with unit efficiency the corresponding 6O-centered semiquinone radical, which was characterized by its characteristic transient absorption. The quenching rate constants for DHI (3.1-8.4 x 10(9) M-1 s-1) and DHICA (3.3-5.5 x 10(9) M-1 s-1) were near the diffusion-controlled limit, indicating excellent antioxidant properties. Kinetic solvent effects were observed. The reactivity of synthetic melanin, assessed through the quenching rate constant of Fluorazophore-P and normalized to the number of monomer units, was more than one order of magnitude lower (2.7 x 10(8) M-1 s-1) than that of its precursors. The trend of the quenching rate constants, i.e. DHI > DHICA approximately alpha-tocopherol > melanin, along with the preferential solubility of DHICA in aqueous environments, serves to account for several experimental results from biochemical studies on the inhibition of lipid peroxidation by these natural antioxidants.

Effects of ultraviolet irradiation on melanogenesis from tyrosine, Dopa and dopamine: a matrix-assisted laser desorption/ionization mass spectrometric study.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry experiments were applied to study the influence of ultraviolet (UV) irradiation in melanogenesis. Samples were prepared starting from three different precursors, tyrosine, Dopa and dopamine, in the presence or absence of tyrosinase, the enzyme responsible for the synthesis of melanin. Enzymatic reactions were carried out for 10, 30, 60 and 120 min under UV irradiation at 365 nm, and aliquots were then immediately ultrafiltered and lyophilized. Samples obtained by irradiation of tyrosine solution revealed the formation of 5,6-dihydroxyindole (DHI) oligomers up to pentamers at 120 min; the reaction kinetics were markedly enhanced in the presence of tyrosinase. In the case of Dopa, UV irradiation favored melanogenesis only in the presence of the enzyme; in this case, many reaction pathways were activated, originating various oligomeric species of Dopa, DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Conversely, when dopamine was used as tyrosinase substrate under UV light, mechanisms of melanogenesis different from those generated by simple enzymatic reaction without irradiation were not activated, as the same oligomeric species were present.

5,6-Dihydroxyindoles in the fenton reaction: a model study of the role of melanin precursors in oxidative stress and hyperpigmentary processes.

Increasing evidence supports the view that diffusible melanin-related metabolites do not serve merely as pigment precursors, but may also act as modulators of the responses of the pigmentary cell melanocyte to external stimuli, especially to inflammation. In this study, the effect of melanin precursors 5, 6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) on the Fenton-induced oxidation of deoxyribose was investigated as a model of the oxidative stress processes triggered by the release of iron during inflammation. DHICA caused a powerful inhibition of the H(2)O(2)-Fe(II)/EDTA oxidation under both aerobic and anaerobic conditions, proving to be more efficient than typical hydroxyl radical (HO(*)) scavengers even at low concentrations with respect to deoxyribose. Conversely, DHI in air was a prooxidant at low indole:Fe(II) ratios, but shifted to an antioxidant at higher ratios (>6). The magnitude of the prooxidant effect increased by lowering the pH of the medium or by replacing Fe(II) with Fe(III), but was suppressed by exclusion of oxygen. Both the indoles retained their effects on the Fenton reaction in the absence of EDTA, as a result of their ability to chelate iron ions as evidenced by spectrophotometric experiments. Investigation of the reaction of DHI and DHICA with the Fenton reagent led to the conclusion that the indoles interact efficiently with HO(*), yielding indolesemiquinone species which are then converted to melanin pigments by self-coupling or disproportionation. At low DHI:iron molar ratios, the ability of semiquinones, generated by autoxidation of indoles, to recycle Fe(II) ions prevails, accounting for the observed prooxidant effect. Collectively, the results of this study provide new evidence for melanogenic 5,6-dihydroxyindoles as a novel class of biological antioxidants and point to these compounds as the key to interpreting the response of melanocytes to oxidative injuries. Moreover, the rapid formation of melanin following the exposure of 5, 6-dihydroxyindoles to the Fenton oxidation suggests new mechanisms of skin hyperpigmentation associated with inflammation.

Lipoxygenase/H 2O 2-catalyzed oxidation of dihydroxyindoles: synthesis of melanin pigments and study of their antioxidant properties

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), which are important intermediates in melanogenesis, can be converted into the corresponding melanin pigments by the action of the lipoxygenase/H 2O 2 system. Kinetic and HPLC analyses indicate that both DHI and DHICA are good substrates for this enzymatic system. Enzyme activity on both substrates was measured in comparison with peroxidase and tyrosinase; the oxidizing behaviour of lipoxygenase is more similar to that of peroxidase rather than that of tyrosinase. The antioxidant properties of DHI- and DHICA-melanins have been investigated in comparison with other kinds of melanins. DHICA-melanin shows a more pronounced antioxidant effect than that of DHI-melanin and this behaviour can be ascribed to the different structure and solubility of the two pigments. The mixed polymer synthesized from DHI and DHICA is the most effective one. Some implications about the possible explanation of the above mentioned behaviour are discussed.