Epimutation and Cancer: Carcinogenesis Viewed as Error-Prone Inheritance of Epigenetic Information.

The epimutation concept, that is, malignancy is a result of deranged patterns of gene expression due to defective epigenetic control, proposes that in the majority of adult cancers the primary (initiating) lesion adversely affects the mechanism of vertical transmission of the epigenetic pattern existing in the stem cells of differentiated tissue. Such an error-prone mechanism will result in deviant gene expression capable of accumulation at each mitosis of the affected stem cell clone. It is argued that a proportion of these proliferation products will express combinations of genes which endow them with malignant properties, such as the ability to transgress tissue boundaries and migrate to distant locations.

Oxidative calcium release from catechol.

Oxidation of 4-methylcatechol previously exposed to aqueous calcium chloride was shown by ion chromatography to be associated with release of calcium ions. The catechol was oxidised to the corresponding orthoquinone by the use of tyrosinase from Agaricus bisporus. The oxidative release of calcium from the catechol is ascribed to the diminution of the available hydroxyl functions able to act as chelating groups.

Mechanistic aspects of the tyrosinase oxidation of hydroquinone.

Contradictory reports on the behaviour of hydroquinone as a tyrosinase substrate are reconciled in terms of the ability of the initially formed ortho-quinone to tautomerise to the thermodynamically more stable para-quinone isomer. Oxidation of phenols by native tyrosinase requires activation by in situ formation of a catechol formed via an enzyme generated ortho-quinone. In the special case of hydroquinone, catechol formation is precluded by rapid tautomerisation of the ortho-quinone precursor to catechol formation.

Tyrosinase: the four oxidation states of the active site and their relevance to enzymatic activation, oxidation and inactivation.

Tyrosinase is an enzyme widely distributed in the biosphere. It is one of a group of proteins with a strongly conserved bicopper active centre able to bind molecular oxygen. Tyrosinase manifests two catalytic properties; monooxygenase and oxidase activity.

Mechanistic studies of the inactivation of tyrosinase by resorcinol.

The inactivation of tyrosinase by resorcinol (1,3-dihydroxybenzene) and seventeen simple derivatives has been investigated using combined spectrophotometry and oximetry together with hplc/ms examination of the oxidation products. The results are consistent with a Quintox mechanism, analogous to that proposed for catechol inactivation of tyrosinase, in which the resorcinol substrate is oxidised via the monooxygenase route leading to a hydroxy intermediate that undergoes deprotonation and results in irreversible elimination of Cu(0) from the active site. Hplc/ms evidence for formation of the resorcinol monooxygenase product (3-hydroxy-ortho-quinone) is presented and the relationship between the ring position of simple resorcinol substituents (H, Me, F, Cl) and tyrosinase inactivation is rationalised.

The influence of hydroquinone on tyrosinase kinetics.

In vitro studies, using combined spectrophotometry and oximetry together with hplc/ms examination of the products of tyrosinase action demonstrate that hydroquinone is not a primary substrate for the enzyme but is vicariously oxidised by a redox exchange mechanism in the presence of either catechol, L-3,4-dihydroxyphenylalanine or 4-ethylphenol. Secondary addition products formed in the presence of hydroquinone are shown to stimulate, rather than inhibit, the kinetics of substrate oxidation.

Rapid halogen substitution and dibenzodioxin formation during tyrosinase-catalyzed oxidation of 4-halocatechols.

4-Fluoro-1,2-benzoquinone, generated by tyrosinase oxidation of 4-fluorocatechol in aqueous buffer, rapidly undergoes substitution by O-nucleophiles (water or catechols) with release of fluoride. 4-Chloro- and 4-bromocatechol behave similarly. The reactions, which have toxicological implications, have been monitored by spectrophotometry and HPLC/MS, and intermediate and final products, including dibenzodioxins, identified.

A reactive ortho-quinone generated by tyrosinase-catalyzed oxidation of the skin depigmenting agent monobenzone: self-coupling and thiol-conjugation reactions and possible implications for melanocyte toxicity.

Monobenzone (hydroquinone monobenzylether, 1) is a potent skin depigmenting agent that causes irreversible loss of epidermal melanocytes by way of a tyrosinase-dependent mechanism so far little understood. Herein, we show that 1 can be oxidized by mushroom tyrosinase to an unstable o-quinone (1-quinone) that has been characterized by comparison of its properties with those of a synthetic sample obtained by o-iodoxybenzoic acid-mediated oxidation of 1. Preparative scale oxidation of 1 with tyrosinase and catalytic l-DOPA, followed by reductive workup and acetylation, led to the isolation of two main products that were identified as the acetylated catechol derivative 4 and an unusual biphenyl-type dimer of 4, acetylated 5, arising evidently by coupling of 4 with 1-quinone.

Dopamine quinone chemistry: a study of the influence of amide, amidine and guanidine substituents [-NH-CX-Y] on the mode of reaction.

The influence of N-substituents on the mode of reaction of ortho-quinones generated by oxidation of N-substituted dopamine derivatives has been studied. Ortho-quinones with amide, urea or guanidine side chains are relatively stable, with evidence of rearrangement to para-quinomethanes. The N-methylthiourea derivative rapidly cyclises giving a bicyclic product .

Mechanistic studies of melanogenesis: the influence of N-substitution on dopamine quinone cyclization.

The influence of side-chain structure on the mode of reaction of ortho-quinone amines has been investigated with a view, ultimately, to developing potential methods of therapeutic intervention by manipulating the early stages of melanogenesis. Four N-substituted dopamine derivatives have been prepared and quinone formation studied using pulse radiolysis and tyrosinase-oximetry. Ortho-quinones with an amide or urea side chain were relatively stable, although evidence for slow formation of isomeric para-quinomethanes was observed.

Oxidation of N-substituted dopamine derivatives: irreversible formation of a spirocyclic product.

Oxidation of amide, urea and guanidinium derivatives of dopamine gives relatively stable ortho-quinones whereas oxidation of corresponding thioamide and amidinium derivatives rapidly and quantitatively gives novel bicyclic and spirocyclic products formed via the corresponding ortho-quinone.

Melanoma and the problem of malignancy.

Of the overt biological properties exhibited by malignant cells two appear to command particular attention; these are (1) the transmigratory ability which empowers these cells to invade surrounding tissues and results in their metastatic and destructive potential, and (2) their ability to evade detection by the immune system of the host. Both of these characteristics may well involve several disparate mechanisms. However, it may be that there are some metabolic features that are common to malignant neoplasms which could go some way to explaining one of these behavioural anomalies.

Formation of para-quinomethanes via 4-aminobutylcatechol oxidation and ortho-quinone tautomerism.

Enzymatic and chemical oxidation of 4-(4-N,N-dialkylaminobutyl)catechols leads to formation of 1,1-dialkyl-pyrrolidinium salts in good yield. It is proposed that these products are formed by tautomerism of the initially formed ortho-quinones to para-quinomethanes. The corresponding secondary amines do not form para-quinomethanes but cyclise giving tetrahydro-1H-benzo[b]azepine-7,8-diones.

Melanogenesis and melanoma.

Melanins are the principal surface pigments in vertebrates and, in humans, play a major role in photoprotection. Although the product (melanin) has a mainly protective function in the skin, the process of melanogenesis represents a potential cellular hazard and is confined to special membrane-limited organelles (melanosomes) in a set of specialized dendritic cells (melanocytes) which synthesize the pigment and transfer it to recipient cells. Malignant melanocytes tend to exhibit up-regulated melanogenesis and defective melanosomes.

Mechanistic studies of catechol generation from secondary quinone amines relevant to indole formation and tyrosinase activation.

The biological significance of the spontaneous cyclization and redox reactions of ortho-quinone amines is that these appear to be the mechanism of formation of the indolic components of melanin and are also involved in the autoactivation of tyrosinase. We have previously shown that activation of tyrosinase is prevented by the formation of a cyclic betaine from a tertiary amine analogue. Evidence is presented to show that cyclization of ortho-quinones by Michael addition also occurs in the oxidation of secondary catecholamines.

Tyrosinase autoactivation and the chemistry of ortho-quinone amines.

Tyrosinase oxidizes tyrosine to dopaquinone, which undergoes nonenzymatic reactions leading to precursors of melanin pigments. Cyclization of dopaquinone gives cyclodopa, which participates in redox exchange with dopaquinone to give the eumelanin precursor dopachrome plus dopa. The indirect formation of the catechol (dopa) from the phenol (tyrosine) leads to unusual enzyme kinetics.

Du temps perdu a la recherche.

An attempt has been made to justify an academic career by outlining the approximate history of the development of ideas and tracing their origins and arborization. Because there are multifarious influences and formative factors involved in their genesis it has proved almost impossible to acknowledge all the important contributors to the patchwork of investigations that are summarized. However, whilst the review is incomplete and necessarily brief, it is hoped that a glimpse of some twentieth century preoccupations with biological science is afforded.