James B. Kirkland

Niacin and Carcinogenesis

The dietary status of niacin (vitamin B3) has the potential to influence DNA repair, genomic stability, and the immune system, eventually having an impact on cancer risk, as well as the side effects of chemotherapy in the cancer patient. In addition to its well-known redox functions in energy metabolism, niacin, in the form of NAD, participates in a wide variety of ADP-ribosylation reactions.Poly(ADP-ribose) is a negatively charged polymer synthesized, predominantly on nuclear proteins, by at least seven different enzymes. Poly(ADP-ribose) polymerase-1 (PARP-1) is responsible for the majority of polymer synthesis and plays important roles in DNA damage responses, including repair, maintenance of genomic stability, and signaling events for stress responses such as apoptosis. NAD is also used in the synthesis of mono(ADP-ribose), often on G proteins, with poorly understood roles in signal transduction. Last, NAD and NADP are required for the synthesis of cyclic ADP-ribose and nicotinic acid adenine dinucleotide (NAADP), two mediators of intracellular calcium signaling pathways. Disruption of any of these processes has the potential to impairgenomic stability and deregulate cell division, leading to enhanced cancer risk. There are various sources of evidence that niacin status does have an impact on cancer risk, including animal models of leukemogenesis and skin cancer, as well as epidemiological data from human populations.

IMMUNOLOGICAL DETERMINATION AND SIZE CHARACTERIZATION OF POLY(ADP-RIBOSE) SYNTHESIZED IN VITRO AND IN VIVO

Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from beta-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280-283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells.

Niacin deficiency increases spontaneous and etoposide-induced chromosomal instability in rat bone marrow cells in vivo

Poly(ADP-ribose) polymerase (PARP) binds to DNA single and double strand breaks and uses NAD in the synthesis of poly(ADP-ribose) (pADPr). Niacin deficiency in rats decreases bone marrow NAD(+) and limits pADPr synthesis in response to DNA damage, while pharmacological supplementation with nicotinic acid (NA) increases bone marrow NAD(+) and pADPr. The purpose of this study was to determine if niacin status alters the extent of DNA damage and chromosomal instability before and after treatment with the chemotherapy drug etoposide (ETO). Genotoxicity was evaluated using the comet, micronucleus and sister chromatid exchange (SCE) assays. Male Long-Evans rats were fed niacin deficient (ND), or pair-fed (PF) niacin replete (30mg niacin/kg) or NA supplemented (4g niacin/kg) diets for 3 weeks. Rats were gavaged with ETO (1-25mg/kg) suspended in corn oil or an equal volume of vehicle (CON). Comet analysis demonstrated that ETO-induced DNA damage (mean tail moment (MTM) and proportion of cells with significant damage) was greater in bone marrow cells from ND rats, compared to PF or NA rats. Surprisingly, niacin deficiency alone caused 6.2- and 2.8-fold increases in spontaneous micronucleus formation and SCE frequency, respectively. As expected, ETO treatment increased the level of micronuclei (MN) and SCEs in all diet groups; however, the absolute increases were greater in ND bone marrow. These data show that niacin is required for the maintenance of chromosomal stability and may facilitate DNA repair in vivo, in a tissue that is sensitive to niacin depletion and impaired pADPr metabolism. Pharmacological intakes of niacin do not appear to be further protective compared to adequate intakes. Niacin supplementation may help to protect the bone marrow cells of cancer patients with compromised nutritional status from the side effects of genotoxic chemotherapy drugs.

Biochemical Assessment of Niacin Deficiency Among Carcinoid Cancer Patients

OBJECTIVE:Carcinoid cancer patients often have elevated levels of serotonin or its precursor 5-hydroxytryptophan. Normally, serotonin synthesis accounts for a small fraction of tryptophan catabolism, which should be directed along a pathway that allows partial conversion to niacin; hence, increased diversion of tryptophan toward serotonin could cause variable degrees of niacin deficiency in carcinoid patients. Therefore, the prevalence of niacin deficiency among carcinoid patients was investigated by clinical assessment of pellagra and biochemical assessment of whole blood niacin number, a ratio derived from two biologically active forms of niacin (NAD/NADP × 100).METHODS:Clinical and biochemical niacin status were assessed in a cohort of newly diagnosed carcinoid patients with carcinoid syndrome (CCS, n = 36), carcinoid patients without carcinoid syndrome (CWCS, n = 32) and noncarcinoid controls (n = 24) recruited at two primary care clinics. Other aspects of serotonin metabolism were measured by analyses of plasma serotonin and tryptophan and urinary excretion of 5-hydroxyindoleacetic acid.RESULTS:Biochemical niacin deficiency (niacin number < 130) was significantly more common in CCS patients (10 out of 36) compared to controls (p < 0.05, Fishers exact test), while CWCS patients displayed an incidence that was not significantly elevated (4 out of 32). Only one CCS patient, who was also identified biochemically as niacin deficient, was clinically diagnosed with pellagra.CONCLUSION:Biochemical niacin deficiency is more prevalent among newly diagnosed CCS patients than in controls. Manifestation of pellagra is a less sensitive indicator, and dependence on this endpoint could lead to a lack of appropriate nutritional support for this group of patients.

Niacin status, NAD distribution and ADP-ribose metabolism.

Dietary niacin deficiency, and pharmacological excesses of nicotinic acid or nicotinamide, have dramatic effects on cellular NAD pools, ADP-ribose metabolism, tissue function and health. ADP-ribose metabolism is providing new targets for pharmacological intervention, and it is important to consider how the supply of vitamin B3 may directly influence ADP-ribosylation reactions, or create interactions with other drugs designed to influence these pathways. In addition to its redox roles, NAD+ is used as a substrate for mono-, poly- and cyclic ADP-ribose formation. During niacin deficiency, not all of these processes can be maintained, and dramatic changes in tissue function and clinical condition take place. Conversely, these reactions may be differentially enhanced by pharmacological intakes of vitamin B3, and potentially by changing expression of specific NAD generating enzymes. A wide range of metabolic changes can take place following pharmacological supplementation of nicotinic acid or nicotinamide. As niacin status decreases towards a deficient state, the function of other types of pharmaceutical agents may be modified, including those that target ADP-ribosylation reactions, apoptosis and inflammation. This article will explore what is known and yet to be learned about the response of tissues, cells and subcellular compartments to excessive and limiting supplies of niacin, and will discuss the etiology of the resulting pathologies.

Poly ADP-ribose polymerase-1 and health.

Niacin (vitamin B3) is required to form nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which are involved in scores of anabolic and catabolic redox reactions throughout metabolism. It is now understood that NAD+ is also a substrate for several families of ADP-ribosylation reactions, which control processes like DNA repair, replication and transcription, the activity of G-proteins, chromatin structure and intracellular calcium signalling. Poly(ADP-ribose)polymerase-1 (PARP-1) is the most active of the PARP enzymes, and it has been implicated in both prevention and aggravation of disease processes. Inhibition of poly-ADP-ribose formation will tend to cause genomic instability and tumorigenesis in chronic models of DNA damage, but the same inhibition can prevent many acute disease processes, such as stroke, myocardial infarction and septic shock. In models of acute stress, PARP-1 inhibition may protect cellular NAD pools and prevent nuclear factor-κB-dependent inflammatory signalling, while long-term protective roles for PARP-1 include DNA repair and regulation of chromatin structure. Promising new PARP-1 inhibitors may display interactions with dietary niacin status and may have long-term deleterious effects on genomic stability, but may be extremely valuable for the treatment of acute inflammatory conditions.

Niacin requirements for genomic stability.

Through its involvement in over 400 NAD(P)-dependent reactions, niacin status has the potential to influence every area of metabolism. Niacin deficiency has been linked to genomic instability largely through impaired function of the poly ADP-ribose polymerase (PARP) family of enzymes. In various models, niacin deficiency has been found to cause impaired cell cycle arrest and apoptosis, delayed DNA excision repair, accumulation of single and double strand breaks, chromosomal breakage, telomere erosion and cancer development. Rat models suggest that most aspects of genomic instability are minimized by the recommended levels of niacin found in AIN-93 formulations; however, some beneficial responses do occur in the range from adequate up to pharmacological niacin intakes. Mouse models show a wide range of protection against UV-induced skin cancer well into pharmacological levels of niacin intake. It is currently a challenge to compare animal and human data to estimate the role of niacin status in the risk of genomic instability in human populations. It seems fairly certain that some portion of even affluent populations will benefit from niacin supplementation, and some subpopulations are likely well below an optimal intake of this vitamin. With exposure to stressors, like chemotherapy or excess sunlight, suraphysiological doses of niacin may be beneficial.

Complete inhibition of poly(ADP-ribose) polymerase activity prevents the recovery of C3H1OT1/2 cells from oxidative stress

Activation of the poly(ADP-ribose) polymerase after oxidative damage is implicated in different responses of the cells, for example, cell recovery after sublethal damage or cell death after lethal damage. However, the extent and mechanism of involvement of the enzyme in these two processes appear to be different. Inhibitors of this polymerase, such as benzamides, which do not completely inhibit PARP have been shown to protect the cells from killing by massive oxidant damage, could neither reduce the cellular recovery after mild oxidant damage nor completely inhibit DNA repair in vitro. We report here that 1,5-dihydroxyisoquinoline, which was earlier shown to be a strong inhibitor of this polymerase in vitro, is also its potent inhibitor in vivo. Using sensitive techniques for measuring low levels of cellular poly(ADP-ribose) polymer, we show that this inhibitor can completely abolish oxidant-induced activation of the polymerase in C3H10T1/2 cells. We show that only a minor fraction of the poly(ADP-ribose) polymerase activity is sufficient in cellular recovery after sublethal oxidant damage. We also demonstrate that cells are unable to recover from oxidant damage in the complete absence of polymerase activity.

Niacin Status Impacts Chromatin Structure

Niacin is required to form NAD and NADP, which are involved in many essential redox reactions in cellular metabolism. In addition, NAD(+) acts as a substrate for a variety of ADP-ribosylation reactions, including poly- and mono-ADP-ribosylation of proteins, formation of cyclic ADP-ribose, and the generation of O-acetyl-ADP-ribose in deacetylation reactions. These nonredox reactions are critical in the regulation of cellular metabolism, and they are sensitive to dietary niacin status. There are 4 known mechanisms by which ADP-ribosylation reactions directly regulate chromatin structure. These include the covalent modification of histones with poly(ADP-ribose), the extraction of histones from chromatin by noncovalent binding to poly(ADP-ribose) on poly(ADP-ribose) polymerase-1, poly ADP-ribosylation of telomeric repeat-binding factor-1 within telomeres, and deacetylation of histones by the sirtuins. These reactions produce a variety of localized effects in chromatin structure, and altered function in response to changes in niacin status may have dramatic effects on genomic stability, cell division and differentiation, and apoptosis.

Niacin Deficiency Alters p53 Expression and Impairs Etoposide-Induced Cell Cycle Arrest and Apoptosis in Rat Bone Marrow Cells

Abstract One focus of chemoprevention research is the interaction of nutrients with specific molecular targets associated with the maintenance of genomic stability. This study tested the impact of dietary niacin status on bone marrow NAD+ and poly(ADP-ribose) (pADPr) levels, p53 expression, and etoposide (ETO)-induced apoptosis and cell cycle arrest. After 3 wk on niacin-deficient (ND), pair-fed niacin-replete (PF), or nicotinic acid–supplemented (4 g/kg diet) (NA) diets, Long-Evans rats were gavaged with ETO (25 mg/kg) or vehicle. ND and NA diets caused a 72% decrease and a 240% increase in bone marrow NAD+, respectively. Basal and ETO-induced pADPr levels differed dramatically among ND, PF, and NA diets (undetectable, 42 and 216 fmol/million cells, respectively; basal and undetectable, 119 and 484 fmol/million cells, respectively, following ETO). ND diet alone caused overexpression of two distinct isoforms of p53. Levels of p53 in PF and NA marrow increased in response to ETO treatment, but this did not occur in ND bone marrow. Quantitative polymerase chain reaction of regular and alternative spliced variants of p53 mRNA revealed that niacin deficiency actually decreased both forms of p53 message, implicating protein stability in the accumulation of p53 in ND marrow. ETO-induced apoptosis (TUNEL) was suppressed during niacin deficiency and enhanced by supplementation. G1 arrest was also impaired in ND bone marrow relative to PF and NA. Despite a poor G1 arrest, p21 waf1 was overexpressed in the ND bone marrow and dramatically induced following ETO treatment. In conclusion, dietary niacin deficiency causes changes in NAD+ and pADPr metabolism, alters p53 expression, and impairs cellular responses to DNA damage.