Niacin: vitamin and antidyslipidemic drug.

Niacin is defined collectively as nicotinamide and nicotinic acid, both of which fulfill the vitamin functions of niacin carried out by the bioactive forms NAD(P). In the last few decades numerous new enzymes that consume NAD(P) as substrates have been identified. The functions of these enzymes are emerging as exciting paradigm shifts, even though they are in early stages of discovery. The recent identification of the nicotinic acid receptor has allowed distinction of the drug-like roles of nicotinic acid from its vitamin functions, specifically in modulating blood lipid levels and undesirable side effects such as skin vasodilation and the more rare hepatic toxicities. This information has led to a new strategy for drug delivery for niacin, which, if successful, could have a major impact on human health through decreasing risk for cardiovascular disease. Understanding the many other effects of niacin has much broader potential for disease intervention and treatment in numerous diseases including cancer.

Folate in skin cancer prevention.

Skin, the largest, most exposed organ of the body, provides a protective interface between humans and the environment. One of its primary roles is protection against exposure to sunlight, a major source of skin damage where the UV radiation (UVR) component functions as a complete carcinogen. Melanin pigmentation and the evolution of dark skin is an adaptive protective mechanism against high levels of UVR exposure. Recently, the hypothesis that skin pigmentation balances folate preservation and Vitamin D production has emerged. Both micronutrients are essential for reproductive success. Photodegradation of bioactive folates suggests a mechanism for the increased tendency of populations of low melanin pigmentation residing in areas of high UV exposure to develop skin cancers. Folate is proposed as a cancer prevention target for its role in providing precursors for DNA repair and replication, as well as its ability to promote genomic integrity through the generation of methyl groups needed for control of gene expression. The cancer prevention potential of folate has been demonstrated by large-scale epidemiological and nutritional studies indicating that decreased folate status increases the risk of developing certain cancers. While folate deficiency has been extensively documented by analysis of human plasma, folate status within skin has not been widely investigated. Nevertheless, inefficient delivery of micronutrients to skin and photolysis of folate argue that documented folate deficiencies will be present if not exacerbated in skin. Our studies indicate a critical role for folate in skin and the potential to protect sun exposed skin by effective topical delivery as a strategy for cancer prevention.

Synthesis and degradation of cyclic ADP-ribose by NAD glycohydrolases.

Cyclic adenosine diphosphoribose (cADPR), a recently discovered metabolite of nicotinamide adenine dinucleotide (NAD), is a potent calcium-releasing agent postulated to be a new second messenger. An enzyme that catalyzes the synthesis of cADPR from NAD and the hydrolysis of cADPR to ADP-ribose (ADPR) was purified to homogeneity from canine spleen microsomes. The net conversion of NAD to ADPR categorizes this enzyme as an NAD glycohydrolase. NAD glycohydrolases are ubiquitous membrane-bound enzymes that have been known for many years but whose function has not been identified. The results presented here suggest that these enzymes may function in the regulation of calcium homeostasis by the ability to synthesize and degrade cADPR.

trans-dominant inhibition of poly(ADP-ribosyl)ation sensitizes cells against gamma-irradiation and N-methyl-N'-nitro-N-nitrosoguanidine but does not limit DNA replication of a polyomavirus replicon.

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30), with NAD+ serving as the substrate. PARP is strongly activated upon recognition of DNA strand breaks by its DNA-binding domain. Experiments with low-molecular-weight inhibitors of PARP have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evidence for nonspecific inhibitor effects prompted us to develop a molecular genetic system to inhibit PARP in living cells, i.e., to overexpress selectively the DNA-binding domain of PARP as a dominant negative mutant. Here we report on a cell culture system which allows inducible, high-level expression of the DNA-binding domain. Induction of this domain leads to about 90% reduction of poly(ADP-ribose) accumulation after gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In contrast, induction does not affect normal cellular proliferation or the replication of a transfected polyomavirus replicon. Thus, trans-dominant inhibition of the poly(ADP-ribose) accumulation occurring after gamma-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflicted damage.

Mechanism of alteration of poly(adenosine diphosphate-ribose) metabolism by hyperthermia.

The effects of hyperthermia on adenine nucleotide metabolism including NAD and poly(ADP-ribose) have been studied in confluent cultures of C3H10T1/2 cells. Cells replated immediately following hyperthermic treatment showed only 9% survival relative to controls while after a 24-h recovery period at 37 degrees C survival was 87% of control. Hyperthermic treatment caused no detectable effect on total cellular levels of either NAD or ATP but produced a prolonged increase in cellular content of poly(ADP-ribose). Studies of the mechanism of this effect show that a major alteration of poly(ADP-ribose) metabolism caused by hyperthermia involves a decrease in the rate of turnover of polymers of ADP-ribose. Normal polymer turnover rates were restored during recovery at 37 degrees C even in the presence of cyclohexamide. The results argue that poly(ADP-ribose) glycohydrolase activity is reversibly altered by hyperthermia. Inhibition of poly(ADP-ribose) synthesis following hyperthermia delays recovery of normal rates of protein synthesis and recovery of the ability of the cells to plate and form colonies.

Reduction of nicotinamide adenine dinucleotide levels by ultimate carcinogens in human lymphocytes.

The effect of several classes of DNA-damaging chemicals and closely related compounds on cellular nicotinamide adenine dinucleotide (NAD) levels was studied in freshly isolated peripheral human lymphocytes. Of the 21 compounds examined, 7 were direct DNA-damaging agents and 14 were non-DNA-damaging compounds or required metabolic activation to casue DNA damage. Rapid lowering of cellular NAD levels was caused by each of the direct DNA-damaging chemicals examined in this study including N-methyl-N'-nitr-N-nitrosoguanidine, methyl methanesulfonate, N-acetoxy-2-acetylaminofluorene, 7-bromomethylbenz(a)anthracene, and the benzo(a)pyrene derivatives, r-7,t-8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene and benzo(a)pyrene-4,5-epoxide. The indirect-acting carcinogen 2-acetylaminofluorene, 13 polycyclic aromatic hydrocarbons, and derivatives that were non-DNA-damaging did not cause lowering of NAD. The results suggest a general correlation between DNA damage and acute lowering of cellular NAD pools.

Effect of hyperthermia on poly(adenosine diphosphate-ribose) glycohydrolase.

The effects of supranormal temperature on the activity of poly(ADP-ribose) glycohydrolase were studied by assaying the enzyme in cell extracts derived from cells subjected to hyperthermia and comparing with extracts that were heated in vitro. The enzyme activity was reduced by both hyperthermic treatment of cells and by heating of cell extracts; however greater reductions were observed when intact cells were subjected to hyperthermia. The additional reduction observed when intact cells were heated was reversed when cells were allowed to recover at 37 degrees C following hyperthermia. We postulate that hyperthermia alters poly(ADP-ribose) glycohydrolase activity by two mechanisms, an irreversible thermal denaturation of the enzyme and a reversible metabolic alteration. Changes in poly(ADP-ribose) glycohydrolase activity can account in full for the observed alterations of poly(ADP-ribose) metabolism that occur following hyperthermia.

Effect of nicotinamide analogues on recovery from DNA damage in C3H10T1/2 cells.

The effects of nicotinamide analogues on cellular recovery following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment have been characterized in the transformable cell line, C3H10T1/2. The recovery of cell division potential was measured under conditions which allow simultaneous quantification of intracellular levels of poly(adenosine diphosphate ribose), nicotinamide adenine dinucleotide, and rates of RNA, DNA, and protein synthesis. 3- Methoxybenzamide (MBA), 3-aminobenzamide, and benzamide, which are effective inhibitors of adenosine diphosphate ribosyltransferases , blocked recovery of cell division following treatment with 34 microM MNNG, while the noninhibitors , 3- methoxybenzoate and benzoate, had no effect. In the presence of MBA, cells progressively lost the ability to resume cell division during the first 24 to 36 hr following DNA damage. The intracellular levels of poly(adenosine diphosphate ribose) increased approximately 7-fold within 20 min following MNNG treatment, and 1 mM MBA inhibited this increase by approximately 82%. In the presence of MBA, a dramatic decrease in the rate of DNA synthesis occurred approximately 16 hr after MNNG treatment, while RNA and protein synthesis continued at rates similar to those in cells treated with MNNG alone.

Isolation and characterization of yeast nicotinamide adenine dinucleotide kinase.

NAD+ kinase (ATP:NAD+ 2'-phosphotransferase, EC 2.7.1.23) from yeast has been purified utilizing ion-exchange and NAD+-agarose affinity chromatography to give a 2100-fold purification. The apparent homogeneity of the enzyme preparation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a subunit molecular weight of 31,000, and a native molecular weight of 124,000, and is, thus, probably a tetramer. The single form of the enzyme has an apparent isoelectric point of 5.85. Initial velocity studies in the forward direction with both substrates gave intersecting Lineweaver-Burk plots, and this suggests a sequential mechanism in which both substrates are bound before products are released. Replots of these data were linear and gave Km values for NAD+ and ATP of 0.68 mM and 2.3 mM, respectively.

Formation of a protein-bound pyrazinium free radical cation during glycation of histone H1.

Glycation, the nonenzymatic reaction between protein amino groups and reducing sugars, induces protein damage that has been linked to several pathological conditions, especially diabetes, and general aging. Here we describe the direct identification of a protein-bound free radical formed during early glycation of histone H1 in vitro. Earlier EPR analysis of thermal browning reactions between free amino acids and reducing sugars has implicated the sugar fragmentation product glycolaldehyde in the generation of a 1,4-disubstituted pyrazinium free radical cation. In order to evaluate the potential formation of this radical in vivo, the early glycation of BSA, lysozyme, and histone H1 by several sugars (D-glucose, D-ribose, ADP-ribose, glycolaldehyde) under conditions of physiological pH and temperature was examined by EPR. The pyrazinium free radical cation was identified on histone H1 glycated by glycolaldehyde (g = 2.00539, aN = 8.01 [2N], aH = 5.26 [4H], aH = 2.72 [4H]), or ADP-ribose. Reaction of glycoaldehyde with poly-L-lysine produced an identical signal, whereas reaction with BSA or lysozyme produced only a minor unresolved singlet signal. In the absence of oxygen the signal was stable over several days. Our results raise the possibility that pyrazinium radicals may form during glycation of histone H1 in vivo.

Evaluating the role of niacin in human carcinogenesis.

Our understanding of the role of ADP-ribose polymer metabolism in limiting carcinogenic events and the dependence of this metabolism on cellular NAD levels predicts that niacin deficiency leading to reduced NAD levels may enhance carcinogenesis. This prediction has led us to initiate studies to evaluate the potential of niacin as a preventive factor in human cancer. The first approach involves development of a method to assess biochemically niacin status in humans using intracellular NAD derived from whole blood, primarily erythrocytes, as the relevant marker of niacin status. We have shown that erythrocyte NAD content varies by as much as 12-fold within a population and can be modulated readily by supplementation. A second approach to testing this hypothesis involves understanding the relationship of dietary niacin, circulating levels of NAD precursors (nicotinamide and nicotinic acid) and NAD in target tissues for human cancer. Current analytical methods for quantification of plasma levels of nicotinic acid and nicotinamide following intake in the dietary range are not sufficient. Thus, we have developed a GC-MS method for the rapid, sensitive, and selective determination of both nicotinamide and nicotinic acid in plasma. These methods will now allow assessment of niacin metabolism in humans that could lead to a new understanding of niacin in prevention of cancer.

NAD glycohydrolases and the metabolism of cyclic ADP-ribose.

Cyclic ADP-ribose is a recently discovered metabolite of NAD that functions in cellular calcium signalling. The discovery that NAD glycohydrolases can catalyze the synthesis and hydrolysis of cyclic ADP-ribose has renewed interest in this class of ADP-ribose transferring enzymes that were discovered over 50 years ago.

Mechanism of inhibition of poly(ADP-ribose) glycohydrolase by adenosine diphosphate (hydroxymethyl)pyrrolidinediol.

Adenosine diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD), a nitrogen-in-the-ring analog of ADP-ribose, was recently shown to be a potent and specific inhibitor of poly(ADP-ribose) glycohydrolase. Analysis of the inhibition kinetics of the hydrolase by ADP-HPD using the method of Lineweaver and Burk yields a noncompetitive double-reciprocal plot. Both the intercept (1/V) versus [inhibitor] replot and the slope (Km/V) versus [inhibitor] replot are hyperbolic, indicating partial noncompetitive inhibition. Inhibitor dissociation constants Kii = 52 nM and Kis = 80 nM were determined for ADP-HPD by analysis of the intercept versus [inhibitor] and slope versus [inhibitor] replots. These results show that although ADP-HPD is extremely potent in inhibiting poly(ADP-ribose) glycohydrolase, its effectiveness is limited by its partial inhibition. ADP-HPD was significantly less potent as an inhibitor of the NAD glycohydrolase from Bungarus fasciatus venom. Analysis of the inhibition kinetics using the Lineweaver and Burk method indicated that ADP-HPD was a linear-competitive inhibitor of the NAD glycohydrolase with a Ki of 94 microM. The results indicate that at low concentration ADP-HPD will be a selective inhibitor of poly(ADP-ribose) glycohydrolase; however, complete inactivation of the activity will be difficult to obtain.

Specific inhibition of poly(ADP-ribose) glycohydrolase by adenosine diphosphate (hydroxymethyl)pyrrolidinediol.

Adenosine diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD), an NH analog of ADP-ribose, was chemically synthesized and shown to be a potent and specific inhibitor of poly-(ADP-ribose) glycohydrolase. The synthetic starting material was the protected pyrrolidine, (2R,3R,4S)-1-(benzyloxycarbonyl)-2-(hydroxymethyl)pyrrolidine-3,4-diol 3,4-O-isopropylidene acetal. This starting pyrrolidine was phosphorylated, coupled to adenosine 5'-monophosphate, and deprotected, yielding the title inhibitor ADP-HPD. ADP-HDP was shown to inhibit the activity of poly(ADP-ribose) glycohydrolase by 50% (IC50) at 0.12 microM, a value 1000-times lower than the IC50 of the product, ADP-ribose. The NAD glycohydrolase from Bungarus fasciatus venom was less sensitive to inhibition by ADP-HPD, exhibiting an IC50 of 260 microM. ADP-HPD did not inhibit either poly(ADP-ribose) polymerase or NAD:arginine mono(ADP-ribosyl)-transferase A at inhibitor concentrations up to 1 mM. At low ADP-HPD concentration, inhibition was therefore shown to be highly specific for poly(ADP-ribose) glycohydrolase, the hydrolytic enzyme in the metabolism of ADP-ribose polymers.

Measurement of adenosine concentration in aqueous and vitreous.

PURPOSE: The release of adenosine by the ischemic retina may be an initial signal in the development of ischemic macular edema and neovascularization. The levels of adenosine have never been quantified in ocular fluids. In this study, a technique was developed for in vivo measurement of the concentration of adenosine in aqueous and vitreous. METHODS: Aqueous and vitreous samples were obtained from bovine eyes after death and from live porcine eyes with the subject under general anesthesia. Samples from live eyes were immediately incubated in the sampling syringe with pentoxifylline, erythro-9-(2-hydroxy-3-nonyl) adenine, and dipyridamole to prevent synthesis or degradation of adenosine during the collection procedure, filtered, and flash-frozen in liquid nitrogen. All samples were then filtered and purified on phenylboronate agarose columns and incubated with chloroacetaldehyde to convert the adenosine present in the sample to the fluorescent derivative 1,N6-ethenoadenosine. The 1,N6-ethenoadenosine was separated by high-pressure liquid chromatography and then measured by fluorometry. RESULTS: Levels of adenosine as low as 0.5 pmole could be detected with this procedure, compared with 20 pmoles by UV detection. By using this technique to measure adenosine levels in the eyes of normal weanling domestic pigs, it was determined that the adenosine concentration in the aqueous was 321.3 +/- 164.9 nM and in the vitreous was 210.8 +/- 41.5 nM. CONCLUSIONS: The conversion of adenine-containing compounds to fluorescent 1,N6-etheno derivatives offers analytical advantages of selectivity and sensitivity for the quantitative determination of these compounds, with the fluorometric detection providing substantially greater sensitivity than direct detection by UV absorption. The levels obtained in vivo from anesthetized but otherwise healthy pigs presumably reflected basal aqueous and vitreous adenosine levels under the described conditions. This method should be useful in investigating more directly the role of adenosine in models of retinal or ocular ischemia in vivo and in measuring adenosine levels in vitreous or aqueous samples from human patients.

Correlation of carcinogen-induced unscheduled DNA synthesis and NAD reduction in fresh human lymphocytes.

Incorporation of [3H]thymidine into the DNA of fresh human lymphocytes, treated with various chemical mutagens, was measured and correlated with cellular NAD levels before and after treatment. NAD levels in lymphocytes were significantly reduced following treatment with mutagenic chemicals. Reduction of cellular NAD pools was directly correlated with [3H]thymidine incorporation. As NAD levels decreased, [3H]thymidine incorporation increased. Theophylline, a known inhibitor of poly(ADP-ribose)polymerase, inhibited both the NAD reduction in cells treated with DNA damaging agents and the incorporation of [3H]thymidine into DNA. The inhibitory effect of theophylline on NAD depletion and on [3H]thymidine incorporation was dose and cell number dependent. Near normal responses to carcinogen exposure could be restored to theophylline-treated cells following the removal of theophylline. These data suggest that conversion of NAD to poly(ADP-ribose) may be necessary, or at least closely associated with, DNA repair in human lymphocytes.

Mutagenicity of the non-carcinogenic dibenzylnitrosamine and an alpha-acetoxy derivative.

The mutagenicity of a non-carcinogenic nitrosamine, N,N-dibenzylnitrosamine (I), and a chemically synthesized alpha-acetoxy derivative, N-(alpha-acetoxy-benzyl)-N-benzylnitrosamine (II), has been examined in Salmonella typhimurium TA100 and TA1535. Compound (I) was non-mutagenic when tested directly or in the presence of a metabolic activation system while (II) was highly mutagenic when tested directly. This is the first report on the conversion of a non-mutagenic N-nitrosamine to a mutagen by the formation of an alpha-acetoxy derivative.

Increased sensitivity to DNA-alkylating agents in CHO mutants with decreased poly(ADP-ribose) polymerase activity.

Using a replica-plating procedure and a 32P-NAD+ permeable cell-screening assay, we have isolated a CHO mutant, PADR-9, which displays approximately 17% of the wild-type level of poly(ADP-ribose) polymerase activity. Biochemical analysis of the mutant using activity, Western, and Northern blot techniques indicate that relative to its parent cell, the mutant's enzyme activity, antibody recognition, and mRNA levels have been reduced to approximately the same extent. These results are consistent with a mutation in the PADR-9 cell which has resulted in a reduction in enzyme synthesis due to reduced mRNA synthesis and/or stability. Relative to wild-type CHO cells, the PADR-9 mutant has increased sensitivity to killing by DNA-alkylating agents but has normal gamma-ray sensitivity. Correlation between a decrease in poly(ADP-ribose) polymerase activity and an increased sensitivity to DNA-alkylating agents suggests that poly(ADP-ribose) synthesis may be important in the repair and/or induction of DNA damage produced by these agents.

Optimizing the energy status of skin cells during solar radiation.

Ionizing- and ultraviolet-radiation cause cell damage or death by directly altering DNA and protein structures and by production of reactive oxygen species (ROS) and reactive carbonyl species (RCS). These processes disrupt cellular energy metabolism at multiple levels. The formation of DNA strand breaks activates signaling pathways that consume NAD, which can lead to the depletion of cellular ATP. Poly(ADP)-ribose polymerase (PARP-1) is the enzyme responsible for much of the NAD degradation following DNA damage, although numerous other PARPs have been discovered recently that await functional characterization. Studies on mouse epidermis in vivo and on human cells in culture have shown that UV-B radiation provokes the transient degradation of NAD and the synthesis of ADP-ribose polymers by PARP-1. This enzyme functions as a component of a DNA damage surveillance network in eukaryotic cells to determine the fate of cells following genotoxic stress. Additionally, the activation of PARP-1 results in the activation of a nuclear proteasome that degrades damaged nuclear proteins including histones. Identifying approaches to optimize these responses while maintaining the energy status of cells is likely to be very important in minimizing the deleterious effects of solar radiation on skin.

ADP-ribose in glycation and glycoxidation reactions.

Glycation is initiated by reaction of a reducing sugar with a protein amino group to generate a Schiff base adduct. Following an Amadori rearrangement to form a ketoamine adduct, a complex chemistry involving oxidation often leads to protein glycoxidation products referred to as advanced glycosylation end products (AGE). The AGE include protein carboxymethyllysine (CML) residues and a heterogeneous group of complex modifications characterized by high fluorescence and protein-protein cross links. The sugar sources for the glycoxidation of intracellular proteins are not well defined but pentoses have been implicated because they are efficient precursors for the formation of the fluorescent AGE, pentosidine. ADP-ribose, generated from NAD by ADP-ribose transfer reactions, is a likely intracellular source of a reducing pentose moiety. Incubation of ADP-ribose with histones results in the formation of ketoamine glycation conjugates and also leads to the rapid formation of protein CML residues, histone H1 dimers, and highly fluorescent products with properties similar to the AGE. ADP-ribose is much more efficient than other possible pentose donors for glycation and glycoxidation of protein amino groups. Recently developed methods that differentiate nonenzymic modifications of proteins by ADP-ribose from enzymic modifications now allow investigations to establish whether some protein modifications by monomers of ADP-ribose in vivo represent glycation and glycoxidation.