Vitamin B3 in Health and Disease: Toward the Second Century of Discovery.

This introductory chapter briefly reviews the history, chemistry, and biochemistry of NAD (the term NAD as it is used here refers to both oxidized and reduced forms of the molecule) consuming ADP-ribose transfer enzymes as components of the involvement of vitamin B3 in health and disease.

Quantification of poly(ADP-ribose) in vitro: determination of the ADP-ribose chain length and branching pattern.

The structural integrity of eukaryotic genomes, to a great extent, depends on highly regulated and -coordinated enzymatic chromosomal poly(ADP-ribosyl)ation cycles that target chromatin proteins for specific covalent epigenetic poly(ADP-ribose) modification. As a result, the accurate determination of poly(ADP-ribosyl)ation amino acid specificity, as well as, a detailed characterization of the structural -complexity of the protein-bound ADP-ribose polymers generated, e.g.

Discovery and structure-activity relationships of modified salicylanilides as cell permeable inhibitors of poly(ADP-ribose) glycohydrolase (PARG).

The metabolism of poly(ADP-ribose) (PAR) in response to DNA strand breaks, which involves the concerted activities of poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG), modulates cell recovery or cell death depending upon the level of DNA damage. While PARP inhibitors show high promise in clinical trials because of their low toxicity and selectivity for BRCA related cancers, evaluation of the therapeutic potential of PARG is limited by the lack of well-validated cell permeable inhibitors. In this study, target-related affinity profiling (TRAP), an alternative to high-throughput screening, was used to identify a number of druglike compounds from several chemical classes that demonstrated PARG inhibition in the low-micromolar range.

Nicotinic acid receptor abnormalities in human skin cancer: implications for a role in epidermal differentiation.

Background: Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through G(i)-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells.

Identification of a regulatory segment of poly(ADP-ribose) glycohydrolase.

Coordinate regulation of PARP-1 and -2 and PARG is required for cellular responses to genotoxic stress. While PARP-1 and -2 are regulated by DNA breaks and covalent modifications, mechanisms of PARG regulation are poorly understood. We report here discovery of a PARG regulatory segment far removed linearly from residues involved in catalysis.

Photobiological implications of folate depletion and repletion in cultured human keratinocytes.

Folate nutrition is critical in humans and a high dietary folate intake is associated with a diminished risk of many types of cancer. Both synthetic folic acid and the most biologically abundant extracellular reduced folate, 5-methyltetrahydrofolate, are degraded under conditions of ultraviolet radiation (UVR) exposure. Skin is a proliferative tissue with increased folate nutrient demands due to a dependence upon continuous epidermal cell proliferation and differentiation to maintain homeostasis.

A specific isoform of poly(ADP-ribose) glycohydrolase is targeted to the mitochondrial matrix by a N-terminal mitochondrial targeting sequence.

Poly(ADP-ribose) polymerases (PARPs) convert NAD to polymers of ADP-ribose that are converted to free ADP-ribose by poly(ADP-ribose) glycohydrolase (PARG). The activation of the nuclear enzyme PARP-1 following genotoxic stress has been linked to release of apoptosis inducing factor from the mitochondria, but the mechanisms by which signals are transmitted between nuclear and mitochondrial compartments are not well understood. The study reported here has examined the relationship between PARG and mitochondria in HeLa cells.

NAD in skin: therapeutic approaches for niacin.

The maintenance and regulation of cellular NAD(P)(H) content and its influence on cell function involves many metabolic pathways, some of which remain poorly understood. Niacin deficiency in humans, which leads to low NAD status, causes sun sensitivity in skin, indicative of deficiencies in responding to UV damage. Animal models of niacin deficiency demonstrate genomic instability and increased cancer development in sensitive tissues including skin.

Validation of a simple and rapid HPLC method for determination of metronidazole in dermatological formulations.

A rapid and simple method using an isocratic high-pressure liquid chromatography (HPLC) and UV detection for the determination of metronidazole in dermatological formulations is presented. Metronidazole samples were extracted with a solution composed of 60% methanol and 40% mobile phase by a procedure that can be completed in less than 10 min. Subsequent separation and quantification was accomplished in less than 20 min using reversed-phase HPLC with isocratic elution with 0.

UVA is the major contributor to the photodegradation of tretinoin and isotretinoin: Implications for development of improved pharmaceutical formulations.

The chemical stability of tretinoin (RA) and isotretinoin (13RA) in ethanol and dermatological cream preparations exposed to solar simulated light (SSL), UVA, and visible light has been studied. Photostability was monitored by an HPLC method that allowed simultaneous analysis of RA and 13RA, thus allowing photodegradation due to isomerization to other retinoids and photolysis to non-retinoid products to be monitored. Both retinoids undergo both isomerization and photolysis following SSL, UVA and visible light exposure but RA is more sensitive to photodegradation than 13RA.

Formulation compatibility of myristyl nicotinate with drugs used to treat dermatological conditions involving an impaired skin barrier.

A number of dermatology conditions including skin photodamage, atopic dermatitis, and rosacea involve skin barrier impairment and first line therapies for these conditions including retinoids and steroids further impair skin barrier function. We have evaluated the compatibility of myristyl nicotinate, an agent that enhances skin barrier function, with drugs used to treat conditions where skin barrier impairment is present including photodamage (retinoic acid), atopic dermatitis (hydrocortisone, triamcinolone acetonide), rosacea (metronidazole), and seborrheic dermatitis (ketoconazole). Myristyl nicotinate was found to be compatible with each of the drugs examined when formulated together and also was shown to be photocompatible with retinoic acid.

Effect of myristyl nicotinate on retinoic acid therapy for facial photodamage.

Based on the hypothesis that skin barrier impairment is a contributor to side-effects associated with retinoic acid therapy, a double-blind, placebo-controlled pilot study examined the combined use of retinoic acid with myristyl nicotinate (MN), a lipophilic derivative of niacin that enhances skin barrier function, in female subjects with mild to moderate facial photodamage. The study involved a 1-month run-in period with placebo or MN prior to initiation of retinoic acid therapy for 3 months. Analysis of skin biopsies revealed that retinoic acid therapy resulted in stratum corneum thinning of approximately 25% (P = 0.

A topical lipophilic niacin derivative increases NAD, epidermal differentiation and barrier function in photodamaged skin.

The effects of myristyl nicotinate (MN), a nicotinic acid derivative designed to deliver nicotinic acid to skin without vasodilatation, on subjects with photodamaged skin have been studied. MN increased skin cell nicotinamide adenine dinucleotide (NAD) by 25% (P = 0.001) demonstrating effective delivery of nicotinic acid to skin.

Two small enzyme isoforms mediate mammalian mitochondrial poly(ADP-ribose) glycohydrolase (PARG) activity.

Poly(ADP-ribose)glycohydrolase (PARG) is the major enzyme capable of rapidly hydrolyzing poly(ADP-ribose) (PAR) formed by the diverse members of the PARP enzyme family. This study presents an alternative splice mechanism by which two novel PARG protein isoforms of 60 kDa and 55 kDa are expressed from the human PARG gene, termed hPARG60 and hPARG55, respectively. Homologous forms were found in the mouse (mPARG63 and mPARG58) supporting the hypothesis that expression of small PARG isoforms is conserved among mammals.

Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase.

Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-Delta2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein.

A pilot study evaluating the efficacy of topically applied niacin derivatives for treatment of female pattern alopecia.

Background: Female pattern alopecia is a common dermatologic condition that manifests after puberty. The only approved drug treatment for this condition is 2% minoxidil for topical application.

Aims: This pilot study examined the effect of topical application of two niacin derivatives, octyl nicotinate and tetradecyl nicotinate, on hair fullness in female alopecia.

Analysis and stability study of myristyl nicotinate in dermatological preparations by high-performance liquid chromatography.

Myristyl nicotinate is an ester prodrug under development for delivery of nicotinic acid to skin for treatment and prevention of conditions that involve skin barrier impairment such as chronic photodamage and atopic dermatitis or for mitigating skin barrier impairment that results from therapy such as retinoids or steroids. The formulation stability of myristyl nicotinate is crucial because even small amounts of free nicotinic acid cause skin flushing, an effect that is not harmful but would severely limit tolerability. We report here reversed-phase HPLC methods for the rapid analysis of myristyl nicotinate and nicotinic acid in dermatological preparations.

A rapid HPLC method for simultaneous determination of tretinoin and isotretinoin in dermatological formulations.

A rapid method using an isocratic high-pressure liquid chromatography and UV detection for determination of both all-trans retinoic acid (tretinoin) and 13-cis retinoic acid (isotretinoin) in dermatological preparations is presented. Tretinoin and isotretinoin samples were extracted with acetonitrile by a procedure that can be completed in less than 10 min. Subsequent separation and quantification of amounts as low as 10 pmol was accomplished in less than 15 min using reversed-phase HPLC with isocratic elution with 0.

Endogenous UVA-photosensitizers: mediators of skin photodamage and novel targets for skin photoprotection.

Endogenous chromophores in human skin serve as photosensitizers involved in skin photocarcinogenesis and photoaging. Absorption of solar photons, particularly in the UVA region, induces the formation of photoexcited states of skin photosensitizers with subsequent generation of reactive oxygen species (ROS), organic free radicals and other toxic photoproducts that mediate skin photooxidative stress. The complexity of endogenous skin photosensitizers with regard to molecular structure, pathways of formation, mechanisms of action, and the diversity of relevant skin targets has hampered progress in this area of photobiology and most likely contributed to an underestimation of the importance of endogenous sensitizers in skin photodamage.

Simultaneous determination of myristyl nicotinate, nicotinic acid, and nicotinamide in rabbit plasma by liquid chromatography-tandem mass spectrometry using methyl ethyl ketone as a deproteinization solvent.

Myristyl nicotinate (Nia-114) is an ester prodrug being developed for delivery of nicotinic acid (NIC) into the skin for prevention of actinic keratosis and its progression to skin cancer. To facilitate dermal studies of Nia-114, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using methyl ethyl ketone (MEK) as a deproteinization solvent was developed and validated for the simultaneous determination of Nia-114, NIC, and nicotinamide (NAM) in rabbit plasma. NAM is the principal metabolite of NIC, which is also expected to have chemopreventive properties.

Antimelanoma activity of apoptogenic carbonyl scavengers.

Therapeutic induction of apoptosis is an important goal of anticancer drug design. Cellular carbonyl stress mediated by endogenous reactive carbonyl species (RCS) such as glyoxal and methylglyoxal (MG) affects proliferative signaling and metastasis of human tumor cells. Recent research suggests that RCS produced constitutively during increased tumor cell glycolysis may be antiapoptotic survival factors and thus represent a novel molecular target for anticancer intervention.

Poly(ADP-ribose) polymerases: managing genome stability.

The importance of poly(ADP-ribose) metabolism in the maintenance of genomic integrity following genotoxic stress has long been firmly established. Poly(ADP-ribose) polymerase-1 (PARP-1) and its catabolic counterpart, poly(ADP-ribose) glycohydrolase (PARG) play major roles in the modulation of cell responses to genotoxic stress. Recent discoveries of a number of other enzymes with poly(ADP-ribose) polymerase activity have established poly(ADP-ribosyl)ation as a general biological mechanism in higher eukaryotic cells that not only promotes cellular recovery from genotoxic stress and eliminates severely damaged cells from the organism, but also ensures accurate transmission of genetic information during cell division.

Nuclear proteasome activation and degradation of carboxymethylated histones in human keratinocytes following glyoxal treatment.

Nuclear DNA damage has been studied in detail, but much less is known concerning the occurrence and fate of nuclear protein damage. Glycoxidation, protein damage that results from a combination of protein glycation and oxidation, leads to the formation of protein-advanced glycation end products (AGE) of which N(epsilon)-carboxymethyllysine (CML) is a major AGE. We have used glyoxal, a product of environmental exposures that readily leads to the formation of CML, to study nuclear protein glycoxidation in HaCaT human keratinocytes.

Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain.

PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of alpha(1''-->2') or alpha(1'''-->2'') O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site.

Poly(ADP-ribose) is required for spindle assembly and structure.

The mitotic spindle is typically thought of as an array of microtubules, microtubule-associated proteins and motors that self-organizes to align and segregate chromosomes. The major spindle components consist of proteins and DNA, the primary structural elements of the spindle. Other macromolecules including RNA and lipids also associate with spindles, but their spindle function, if any, is unknown.