Prostaglandin E2 is critical for the development of niacin-deficiency-induced photosensitivity via ROS production.

Pellagra is a photosensitivity syndrome characterized by three "D's": diarrhea, dermatitis, and dementia as a result of niacin deficiency. However, the molecular mechanisms of photosensitivity dermatitis, the hallmark abnormality of this syndrome, remain unclear. We prepared niacin deficient mice in order to develop a murine model of pellagra. Niacin deficiency induced photosensitivity and severe diarrhea with weight loss. In addition, niacin deficient mice exhibited elevated expressions of COX-2 and PGE syntheses (Ptges) mRNA. Consistently, photosensitivity was alleviated by a COX inhibitor, deficiency of Ptges, or blockade of EP4 receptor signaling. Moreover, enhanced PGE2 production in niacin deficiency was mediated via ROS production in keratinocytes. In line with the above murine findings, human skin lesions of pellagra patients confirmed the enhanced expression of Ptges. Niacin deficiency-induced photosensitivity was mediated through EP4 signaling in response to increased PGE2 production via induction of ROS formation.

Structural and kinetic isotope effect studies of nicotinamidase (Pnc1) from Saccharomyces cerevisiae.

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD(+) salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from Saccharomyces cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction in which ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogues acting as inhibitors or substrates were examined, revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary (15)N and (13)C kinetic isotope effects (KIEs) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased, indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was indicative of a stronger commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid, is discussed. These results will aid in the design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.

Nicotinic acid induces secretion of prostaglandin D2 in human macrophages: an in vitro model of the niacin flush.

Nicotinic acid is a safe, broad-spectrum lipid agent shown to prevent cardiovascular disease, yet its widespread use is limited by the prostaglandin D2 (PGD2) mediated niacin flush. Previous research suggests that nicotinic acid-induced PGD2 secretion is mediated by the skin, but the exact cell type remains unclear. We hypothesized that macrophages are a source of nicotinic acid-induced PGD2 secretion and performed a series of experiments to confirm this. Nicotinic acid (0.1-3 mM) induced PGD2 secretion in cultured human macrophages, but not monocytes or endothelial cells. The PGD2 secretion was dependent on the concentration of nicotinic acid and the time of exposure. Nicotinuric acid, but not nicotinamide, also induced PGD2 secretion. Pre-incubation of the cells with aspirin (100 microM) entirely prevented the nicotinic acid effects on PGD2 secretion. The PGD2 secreting effects of nicotinic acid were additive to the effects of the calcium ionophore A23187 (6 microM), but were independent of extra cellular calcium. These findings, combined with recent in vivo work, provide evidence that macrophages play a significant role in mediating the niacin flush and may lead to better strategies to eliminate this limiting side effect.

M-CSF deficiency leads to reduced metallothioneins I and II expression and increased tissue damage in the brain stem after 6-aminonicotinamide treatment.

6-Aminonicotinamide (6-AN) is a niacin antagonist, which leads to degeneration of gray-matter astrocytes followed by a vigorous inflammatory response. Macrophage colony stimulating factor (M-CSF) is important during inflammation, and in order to further clarify the roles for M-CSF in neurodegeneration and brain cell death, we have examined the effect of 6-AN on osteopetrotic mice with genetic M-CSF deficiency (op/op mice). The 6-AN-induced degeneration of gray-matter areas was comparable in control and op/op mice, but the numbers of reactive astrocytes, macrophages, and lymphocytes in the damaged areas were significantly decreased in op/op mice relative to controls. The levels of oxidative stress (as determined by using immunoreactivity for inducible nitric oxide synthase, nitrotyrosine, and malondialdehyde) and apoptotic cell death (as determined by using TUNEL and immunoreactivity for caspases and cytochrome c) were significantly increased in 6-AN-injected op/op mice relative to controls. From a number of antioxidant factors assayed, only metallothioneins I and II (MT-I+II) were decreased in op/op mice in comparison to controls. Thus, the present results indicate that M-CSF is an important growth factor for coping with 6-AN-induced central nervous system damage and suggest that MT-I+II are likely to have a significant role.

Impaired inflammatory response to glial cell death in genetically metallothionein-I- and -II-deficient mice.

Metallothionein I+II (MT-I+II) are acute-phase proteins which are upregulated during pathological conditions in the brain. To elucidate the neuropathological importance of MT-I+II, we have examined MT-I+II-deficient mice following ip injection with 6-aminonicotinamide (6-AN). 6-AN is antimetabolic and toxic for bone marrow cells and grey matter astrocytes. In MT+/+ mice, injection with 6-AN resulted in breakdown of the blood-brain barrier (BBB) and absence of GFAP-positive astrocytes in specific grey matter areas of the brain stem. Reactive astrocytosis encircled the damaged grey matter areas, which were heavily infiltrated by microglia/macrophages. The recruitment of hematogenous macrophages was accompanied by leakage of the BBB. The immunoreactivity (ir) of granulocyte-macrophage-colony-stimulating factor (GM-CSF) and the receptor for GM-CSF (GM-CSFrec) was significantly upregulated in astrocytes and microglia/macrophages, respectively. MT-I+IIir was also clearly increased in astrocytes surrounding the damaged areas, while that of the CNS-specific MT isoform, MT-III, was mildly increased in both astrocytes and microglia/macrophages. In MT-/- mice injected with 6-AN, the BBB remained almost intact. The damage to specific grey matter areas was similar to that observed in MT+/+ mice, but reactive astrocytosis, microglia/macrophages infiltration, and GM-CSFir and GM-CSFrecir were clearly reduced in MT-/- mice. In contrast, MT-IIIir was dramatically increased in MT-/- mice. Total zinc decreased and histochemically detectable zinc increased in the brain stem after 6-AN similarly in MT+/+ and MT-/- mice. Bone marrow myeloid monocytes and macrophages were increased as a reaction to 6-AN only in MT+/+ mice. The results demonstrate that the capability of MT-/- mice to mount a normal inflammatory response in the brain is severely attenuated, at least in part because of 6-AN-induced bone marrow affectation, involving MT-I+II for the first time as major factors during CNS tissue damage.

The effect of 6-aminonicotinamide on testicular development in the rat.

6-Aminonicotinamide (6-AN), a niacin antagonist, was administered sc to pregnant female (1.0, 3.0, or 6.0 mg 6-AN/kg body weight) and neonatal male (1.5, 3.0, 6.0 or 12.0 mg 6-AN/kg body weight) Sprague-Dawley rats on the 15th, 17th and 19th days of gestation or the 5th, 7th and 9th days of life, respectively, to determine the effects of the antimetabolite on testicular morphology and development. In prenatal males, microscopic alterations were present in testes of fetuses from females treated with 6.0 mg 6-AN/kg and consisted of necrosis and loss of gonocytes, and vacuolation of interstitial cells. Histologic changes in testes of neonatal rats treated with 3.0, 6.0 or 12.0 mg 6-AN/kg were qualitatively similar with necrosis and loss of spermatogonia and supporting cells, and increased cross-sectional areas of affected tubules. Quantitation of the number of nuclei/cm2 of seminiferous tubule indicated 6-AN caused a significant reduction in the numbers of supporting cells and spermatogonia/tubular cross-section.

Inhibitory effects of niacin and its analogues on induction of ornithine decarboxylase activity by diethylnitrosamine in rat liver.

Pharmacological doses of niacin and its analogues were given intraperitoneally to rats with and without coadministration of a hepatocarcinogenic dose of diethylnitrosamine (DEN), and their effects on the induction of ornithine decarboxylase (ODC, EC 4.1.1.17) activity in the rat liver were studied. The induction of ODC activity by DEN was inhibited by 74.3, 85.5, 94.6, 97.6, 72.6 and 55.2% by nicotinamide, nicotinic acid, 3-hydroxymethylpyridine, beta-picoline, pyridine-3-aldehyde and ethylnicotinate respectively. When given alone, these analogues did not induce ODC activity. All these compounds are known to have a niacin effect. DEN-induced ODC activity was also inhibited by 84.0, 93.3, 52.8 and 75.9% by 6-aminonicotinamide, picolinic acid, pyridine-3-sulfonic acid and thionicotinamide, respectively, but, peculiarly, they induced ODC activity by their administration alone. These niacin analogues are known to have anti-niacin effects. Tryptophan, N'-methylnicotinamide and isonicotinic acid hydrazide did not affect the DEN-induced ODC activity but could induce ODC by themselves. Tryptophan belongs to the former group and isonicotinic acid hydrazide to the latter group. The reason for these discrepancies is discussed.

Lesions in the skin, intestine, and central nervous system induced by an antimetabolite of niacin.

Pellagra in the human is characterized by the clinical "three D's," namely, dermatitis, diarrhea, and dementia. Newborn mice that received a single intraperitoneal injection of 6-aminonicotinamide (6-AN) (50 mg/kg body weight), an antagonist of niacin, consistently developed lesions in the skin, intestinal tract, and central nervous system. Anterior horn cells in the spinal cord as well as motor neurons in the brain showed the ultrastructural features of neuronal chromatolysis, while glial and ependymal cells showed postinjection (PI) edematous changes on Day 5. In the skin, correlating with clinical delay of hair growth, the first discernible microscopic abnormality was vacuolar change in the hair follicles on PI Day 3. By PI Day 5, hyperkeratosis and irregular acanthosis were noted. Edematous swelling of the enteric glial cells was observed in the myenteric plexus of the descending colon on PI Day 5. Although the pathologic features of these 6-AN-treated mice may not be exactly identical to those of human pellagra, possible contributory mechanisms in the development of pellagra lesions may be elucidated by this experimental model.