Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells.

Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2⁻4 mM) for 3 days markedly stimulated osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1⁻4 mM) may stimulate ALP, osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.

A role for trypanosomatid aldo-keto reductases in methylglyoxal, prostaglandin and isoprostane metabolism.

Trypanosomatid parasites are the infectious agents causing Chagas disease, visceral and cutaneous leishmaniasis and human African trypanosomiasis. Recent work of others has implicated an aldo-keto reductase (AKR) in the susceptibility and resistance of Trypanosoma cruzi to benznidazole, a drug used to treat Chagas disease. Here, we show that TcAKR and homologues in the related parasites Trypanosoma brucei and Leishmania donovani do not reductively activate monocyclic (benznidazole, nifurtimox and fexinidazole) or bicyclic nitro-drugs such as PA-824. Rather, these enzymes metabolise a variety of toxic ketoaldehydes, such as glyoxal and methylglyoxal, suggesting a role in cellular defence against chemical stress. UPLC-QToF/MS analysis of benznidazole bioactivation by T. cruzi cell lysates confirms previous reports identifying numerous drug metabolites, including a dihydro-dihydroxy intermediate that can dissociate to form N-benzyl-2-guanidinoacetamide and glyoxal, a toxic DNA-glycating and cross-linking agent. Thus, we propose that TcAKR contributes to benznidazole resistance by the removal of toxic glyoxal. In addition, three of the four enzymes studied here display activity as prostaglandin F2α synthases, despite the fact that there are no credible cyclooxygenases in these parasites to account for formation of the precursor PGH2 from arachidonic acid. Our studies suggest that arachidonic acid is first converted non-enzymatically in parasite lysates to (PGH2-like) regioisomers by free radical-mediated peroxidation and that AKRs convert these lipid peroxides into isoprostanes, including prostaglandin F2α and 8-iso-prostaglandin F2α.

Isoprostane, an "intermediate phenotype" for oxidative stress heritability, risk trait associations, and the influence of chromogranin B polymorphism.

OBJECTIVES: The purpose of this study is to understand whether isoprostane, a biomarker of oxidative stress, is subject to heritable control; whether it shares heritability with other cardiometabolic risk traits; and finally whether genetic variation at a specific candidate locus contributes to isoprostane variability. BACKGROUND: Isoprostane marks oxidative stress, and elevated isoprostane excretion might be involved in cardiovascular target organ damage. Here we used the classical twin pair method to probe the role of heredity in generating the isoprostane trait. METHODS: Trait heritability (h(2)) and shared genetic determination among traits (pleiotropy, genetic covariance, ρ(G)) were estimated by variance components in twin pairs. Because the isoprostane and Chromogranin B (CHGB) traits shared ρ(G), we examined the CHGB locus for effects on the traits. RESULTS: Urinary isoprostane excretion was substantially heritable (h(2) = 65.8 ± 4.3%), and the isoprostane trait aggregated with multiple traits (CHGB, catecholamines, autonomic/baroreceptor, and renal function), including several features of the metabolic syndrome (body mass index, insulin resistance, dyslipidemia). Isoprostane excretion also aggregated with systemic hypertension. Twin studies demonstrated genetic covariance (pleiotropy) for the isoprostane and CHGB traits (ρ(G) = 0.27), and therefore we investigated the CHGB locus for trait effects. A common variant in the 3'-UTR of CHGB (C+84A) associated with plasma CHGB as well as isoprostane excretion. The C+84A disrupted an A/U-rich messenger ribonucleic acid stability element, and in transfected luciferase/3'-UTR plasmids, the C+84 and +84A alleles differed markedly in reporter expression in chromaffin and neuroblastoma cells, whereas site-directed mutagenesis confirmed the importance of this variant within the context of the A/U-rich motif. CONCLUSIONS: Isoprostane excretion is substantially heritable and shares joint genetic determination with CHGB as well as multiple features of the metabolic syndrome. A common polymorphism in the 3'-UTR (C+84A) of CHGB, which disrupts an A/U-rich messenger ribonucleic acid stability element, associates with not only CHGB secretion but also excretion of isoprostane. We propose a chain of events whereby CHGB genetic variation results in oxidative stress, with isoprostane formation. The results suggest novel links among the catecholaminergic system, oxidative pathways, and systemic hypertension.