Increased dietary vitamin D3 and calcium partially alleviate heat stress symptoms and inflammation in lactating Holstein cows independent of dietary concentrations of vitamin E and selenium.

Twelve multiparous Holstein cows (42.2 ± 5.6 kg of milk/d; 83 ± 27 d in milk) were used in a split-plot design testing the effects of mineral and vitamin supplementation on the time course of animal performance, metabolism, and inflammation markers during heat stress. The main plot was the average concentrations of dietary vitamin E and Se (adequate: 11.1 IU/kg of vitamin E and 0.55 mg/kg of Se, and high: 223 IU/kg of vitamin E and 1.8 mg/kg of Se, respectively). Within each plot, cows were randomly assigned to (1) heat stress (HS) with adequate concentrations of vitamin D3 and Ca (1,012 IU/kg and 0.73%, respectively), (2) HS with high concentrations of vitamin D3 and Ca (HS+D3/Ca; 3,764 IU/kg and 0.97%, respectively), or (3) pair-feeding (PF) in thermoneutrality with adequate concentrations of vitamin D3 and Ca (1,012 IU/kg and 0.73% Ca) in a Latin square design with 14-d periods and 7-d washouts. The highest rectal temperature was recorded at 1700 h for HS (39.4°C; mean of d 1 to 14), being 1.2 and 0.8°C greater than for PF and HS+D3/Ca, respectively. Respiratory rate and water intake were higher in HS (73 breaths/min and 115 L/d, respectively) relative to PF (28 breaths/min and 76 L/d). Heat stress decreased dry matter intake progressively, reaching a nadir on d 5 to 7 (33% reduction) and was not different between treatments. Milk yield decreased progressively in all treatments, but remained greater in PF relative to HS from d 3 to 14 (10%), whereas HS and HS+D3/Ca were not different. Milk fat, protein, and lactose concentrations and yields were lower in HS relative to PF from d 3 to 14, but not different between HS and HS+D3/Ca. Relative to PF, preprandial insulin concentrations were increased in HS, whereas plasma nonesterified fatty acids were decreased on d 7 and 14. Plasma lipopolysaccharide-binding protein concentrations increased in HS cows on d 7 and 14, respectively, relative to PF, whereas they were reduced in HS + D3/Ca on d 14. Plasma C-reactive protein, tumor necrosis factor-α, and fecal calprotectin were increased in HS relative to both PF and HS+D3/Ca on d 7 and 14. Rectal temperature was positively associated with plasma lipopolysaccharide-binding protein (r = 0.72), tumor necrosis factor-α (r = 0.74), C-reactive protein (r = 0.87), and with milk somatic cells (r = 0.75). Plasma 8-hydroxy-2-deoxyguanosine concentrations presented a 3-way interaction, where 8-hydroxy-2-deoxyguanosine was lower in HS than in PF on d 7 and 14, and lower in HS+D3/Ca relative to HS on d 14 in the adequate vitamin E and Se treatment, but no effects were observed in the high vitamin E and Se group. Plasma superoxide dismutase concentrations increased over time, and were higher in HS relative to PF on d 14, whereas HS+D3/Ca was similar to HS. Heat stress markedly reduced milk production and milk components while increasing markers of leaky gut and inflammation. In contrast, vitamin D3 and Ca supplementation reduced hyperthermia (d 7-14), markers of leaky gut, and inflammation independent of dietary concentrations of vitamin E and Se.

5-Iodotubercidin inhibits SARS-CoV-2 RNA synthesis.

Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK).

Photophysical Properties of S, Se and Te-Substituted Deoxyguanosines: Insight into Their Ability To Act as Chemotherapeutic Agents.

Guanine and guanosine derivatives have long been in use as anticancer drugs and recently have been proposed also as photosensitizers in photodynamic therapy. By means of density functional theory and its time-dependent formulation, the potential power as UVA chemotherapeutic agents has been investigated computing the photophysical properties (absorption spectra, excitation energies, and spin-orbit matrix elements) of sulfur, selenium, and tellurium-substituted deoxyguanosines. Different pathways for the population of the lowest triplet state have been considered. Results show that all the examined systems have the lowest triplet state lying above the energy required for the production of the highly cytotoxic excited molecular oxygen 1Δg and that the heavy atom effect ensures an efficient intersystem spin crossing.

Photoprotection by dietary phenolics against melanogenesis induced by UVA through Nrf2-dependent antioxidant responses.

Dietary phenolics may play a protective role in UV-mediated skin pigmentation through their antioxidant and UV-absorbing actions. In this study, we investigated whether genetic silencing of Nrf2, regulating the transcription of antioxidant genes, affected melanogenesis in primary human epidermal melanocytes (HEMn) and B16F10 melanoma cells subjected to UVA (8J/cm(2)) exposure. Then, we explored the antimelanogenic actions of phenolics; caffeic acid (CA) and ferulic acid (FA) providing partial UVA protection; quercetin (QU) and rutin (RU) providing strong UVA protection and; avobenzone (AV), an efficient UVA filter, in association with modulation of Nrf2-mediated antioxidant defenses in response to UVA insults in B16F10 cells. Upon oxidative insults, Nrf2 silencing promoted melanogenesis in both HEMn and B16F10 cells irradiated with UVA. Stimulation of melanogenesis by UVA correlated with increased ROS and oxidative DNA damage (8-OHdG), GSH depletion as well as a transient downregulation of Nrf2 nuclear translocation and of Nrf2-ARE signaling in B16F10 cells. All test compounds exerted antimelanogenic effects with respect to their abilities to reverse UVA-mediated oxidative damage as well as downregulation of Nrf2 activity and its target antioxidants (GCLC, GST and NQO1) in B16F10 cells. In conclusion, defective Nrf2 may promote melanogenesis under UVA irradiation through oxidative stress mechanisms. Compounds with antioxidant and/or UVA absorption properties could protect against UVA-induced melanogenesis through indirect regulatory effect on Nrf2-ARE pathway.

The cytotoxic and anti-proliferative effects of 3-hydrogenkwadaphnin in K562 and jurkat cells is reduced by guanosine.

3-hydrogenwadaphnin (3-HK) is a new daphnane-type diterpene ester isolated from Dendrostellera lessertii with strong anti-tumoral activity in animal models and in cultures. Here, prolonged effects of this new agent on proliferation and viability of several different cancerous cell lines were evaluated. Using [(3)H]thymidine incorporation, it was found that the drug inhibited cell proliferation and induced G1/S cell cycle arrest in leukemic cells 24 h after a single dose treatment. The cell viability of Jurkat cells was also decreased by almost 10 %, 31 % and 40 % after a single dose treatment (7.5 nM) at 24, 48 and 72 h, respectively. The drug-treated cells were stained with acridine orange/ ethidium bromide to document the chromatin condensation and DNA fragmentation. These observations were further confirmed by detection of DNA laddering pattern in the agarose gel electrophoresis of the extracted DNA from the treated cells. Treatment of K562 cells with the drug at 7.5, 15 and 30 nM caused apoptosis in 25 %, 45 % and 65 % of the cells, respectively. Exogenous addition of 25-50 microM guanosine and/or deoxyguanosine to the cell culture of the drug-treated cells restored DNA synthesis, released cell arrest at G1/S checkpoint and decreased the apoptotic cell death caused by the drug. These observations were not made using adenosine. However, the drug effects on K562 cells were potentiated by hypoxanthine. Based on these observations, perturbation of GTP metabolism is considered as one of the main reasons for apoptotic cell death by 3-HK.

Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

Effect of supplementation of n-3 polyunsaturated fatty acids on oxidative stress-induced DNA damage of rat hepatocytes.

The effect of supplementation of n-3 polyunsaturated fatty acids (PUFA) on oxidative stress-induced DNA damage of rat hepatocytes was examined. Male Wistar rats were fed a diet containing safflower oil (control n-6 PUFA diet) or fish oil (n-3 PUFA diet) in 50 g/kg of dried diet and an equal amount of vitamin E in 59 mg/kg of dried diet for 6 weeks. The liver of rats fed safflower oil was rich in n-6 PUFA, whereas that of rats fed fish oil was rich in n-3 PUFA. Isolated hepatocytes were treated in vitro with ADP/Fe (II) ion or hydrogen peroxide at 37 degrees C for 30 min to induce oxidative stress. The degree of lipid peroxidation was assessed by the levels of phospholipid hydroperoxides and thiobarbituric acid-reactive substances. The degree of oxidative DNA damage was assessed based on comet-type characterization in alkaline single-cell gel electrophoresis and 8-hydroxy-deoxyguanosine levels. In both ADP/Fe(II) ion and hydrogen peroxide oxidation, the degree of lipid peroxidation of hepatocytes increased in both diet groups, and the level of increase in the fish oil diet group was slightly higher than that in the safflower oil diet group. In ADP/Fe(II) ion oxidation, the degree of DNA damage increased in both diet groups, but there were no significant differences in the level of increase. In contrast, in hydrogen peroxide oxidation, the degree of DNA damage increased in both diet, and the increase in the fish oil diet group was significantly lower than that in the safflower oil diet group. It is unlikely that an n-3 PUFA-rich diet enhances oxidative stress-induced hepatocyte DNA damage as compared with the control n-6 PUFA-rich diet.

Astaxanthin limits exercise-induced skeletal and cardiac muscle damage in mice.

Dietary antioxidants may attenuate oxidative damage from strenuous exercise in various tissues. Beneficial effects of the antioxidant astaxanthin have been demonstrated in vitro, but not yet in vivo. We investigated the effect of dietary supplementation with astaxanthin on oxidative damage induced by strenuous exercise in mouse gastrocnemius and heart. C57BL/6 mice (7 weeks old) were divided into groups: rested control, intense exercise, and exercise with astaxanthin supplementation. After 3 weeks of exercise acclimation, both exercise groups ran on a treadmill at 28 m/min until exhaustion. Exercise-increased 4-hydroxy-2-nonenal-modified protein and 8-hydroxy-2'-deoxyguanosine in gastrocnemius and heart were blunted in the astaxanthin group. Increases in plasma creatine kinase activity, and in myeloperoxidase activity in gastrocnemius and heart, also were lessened by astaxanthin. Astaxanthin showed accumulation in gastrocnemius and heart from the 3 week supplementation. Astaxanthin can attenuate exercise-induced damage in mouse skeletal muscle and heart, including an associated neutrophil infiltration that induces further damage.

Antioxidant role of N-acetyl cysteine isomers following high dose irradiation.

High dose, acute radiation exposure, as in radiation accidents, induces three clinical syndromes that reflect consequences of oxidative protein, lipid, and DNA damage to tissues such as intestine, lung, and liver. In the present study, we irradiated C57BL/6 mice with 18 Gy whole-body radiation (XRT) and evaluated N-acetyl cysteine (NAC) isomers LNAC and DNAC as potential radioprotectors under conditions that would model the gastrointestinal syndrome. We focused on tissues thought not immediately involved in the gastrointestinal syndrome. Both LNAC and DNAC protected the lung and red blood cells (RBC) from glutathione (GSH) depletion following radiation exposure. However, only LNAC also supplemented the spleen GSH levels following XRT. Protection from increased malondialdehyde (MDA) levels (lung) and increased 8-hydroxy-deoxyguanosine (8-oxo-dG) presence (liver) following XRT was observed with treatment by either isomer of NAC. These results imply that either NAC isomer can act as a radioprotectant against many aspects of oxidative damage; chirality is only important for certain aspects. This pattern would be consistent with direct action of NAC in many radioprotection and repair processes, with a delimited role for NAC in GSH synthesis in some aspects of the problem.

Inhibition of the replication of a hepatitis C virus-like RNA template by interferon and 3'-deoxycytidine.

The development of low molecular weight inhibitors of hepatitis C virus (HCV) replication has been hindered by the lack of a good cell-based system that models the entire HCV replication cycle. To date the only two therapies approved for the treatment of HCV infection are interferon (IFN)-alpha and the nucleoside analogue, ribavirin. We have created a cell-based system that allows for the accurate quantification of the replication of an HCV-like RNA template by proteins that are encoded for by the HCV genome. The system consists of a cell line that constitutively produces luciferase in response to the production of functional HCV replicative proteins. The 293B4alpha cell line has been formatted into a semi-high throughput, cell-based screen for inhibitors of HCV replication. When these cells were treated with either IFN-alpha or -beta, luciferase production decreased in a dose-responsive manner. Counterscreening these molecules in another cell line, 293SVLuc, in which luciferase production in not dependent the presence of functional HCV proteins, showed that the inhibition of luciferase in the 293B4alpha cell line was due to inhibition of the replication of the HCV-like RNA template and not anti-cellular or -luciferase activity. Moreover, when the 293B4alpha cell line was treated with the ribonucleoside analogue, 3'-deoxycytidine, luciferase decreased in a dose-responsive manner. 3'-deoxyguanosine and 3'-deoxyuridine did not inhibit luciferase production and 3'-deoxyadenosine was too cytotoxic to determine if it had any anti-HCV activity.

Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus.

BMS-200475 is a novel carbocyclic 2'-deoxyguanosine analog found to possess potent and selective anti-hepatitis B virus (anti-HBV) activity. BMS-200475 is distinguished from guanosine by replacement of the natural furanose oxygen on the sugar moiety with an exo carbon-carbon double bond. In the HepG2 stably transfected cell line 2.2.15, BMS-200475 had a 50% effective concentration (EC50) of 3.75 nM against HBV, as determined by analysis of secreted HBV DNA. Structurally related compounds with adenine, iodouracil, or thymine base substitutions were significantly less potent or were inactive. Direct comparison of the antiviral activities of BMS-200475 with those of a variety of other nucleoside analogs, including lamivudine (EC50 = 116.26 nM), demonstrated the clearly superior in vitro potency of BMS-200475 in 2.2.15 cells. Intracellular HBV replicative intermediates were uniformly reduced when cells were treated with BMS-200475, but rebounded after treatment was terminated. The concentration of BMS-200475 causing 50% cytotoxicity in 2.2.15 cell cultures was 30 microM, approximately 8,000-fold greater than the concentration required to inhibit HBV replication in the same cell line. Treatment with BMS-200475 resulted in no apparent inhibitory effects on mitochondrial DNA content.

Inhibitory activity of 3'-fluoro-2' deoxythymidine and related nucleoside analogues against adenoviruses in vitro.

Antiviral effects of nucleoside analogues against human adenoviruses (ADV) belonging to subgroup B (ADV3) and C (ADV2) were comparatively analysed using focus reduction assay on Fogh and Lund (FL) cells. 3'-Fluoro-2'-deoxythymidine (FTdR), 3'-fluoro-2'-deoxyuridine (FUdR), 2',3'-dideoxycytidine (ddC) and 3'-fluoro-2'-deoxyguanosine (FGdR) emerged as potent and selective inhibitors. They were nontoxic for the FL cells at the tested doses. FTdR was proved to be the most effective inhibitor against both serotypes ADV2 and ADV3 (0.05 microM/0.02 microM). The inhibitory effect of FTdR was also analyzed on the level of viral proteins and viral DNA synthesis using radioimmunoprecipitation and PCR, respectively. Neither the main structural protein of ADV, the hexon, nor viral DNA could be detected in ADV-infected FL cells that had been exposed to FTdR.

Deoxyadenosine reverses hydroxyurea inhibition of vaccinia virus growth.

Hydroxyurea, an inhibitor of ribonucleotide reductase, blocks replication of vaccinia virus. However, when medium containing hydroxyurea and dialyzed serum was supplemented with deoxyadenosine, the block to viral reproduction was circumvented, provided that an inhibitor of adenosine deaminase was also present. Deoxyguanosine, deoxycytidine, and deoxythymidine were ineffective alone and did not augment the deoxyadenosine effect. In fact, increasing concentrations of deoxyguanosine and deoxythymidine, but not deoxycytidine, eliminated the deoxyadenosine rescue, an effect that was reversed by the addition of low concentrations of deoxycytidine. These results suggested that the inhibition of viral replication by hydroxyurea was primarily due to a deficiency of dATP. Deoxyribonucleoside triphosphate pools in vaccinia virus-infected cells were measured at the height of viral DNA synthesis after a synchronous infection. With 0.5 mM hydroxyurea, the dATP pool was greater than 90% depleted, the dCTP and dGTP pools were 40 to 50% reduced, and the dTTP pool was increased. Assay of ribonucleotide reductase activity in intact virus-infected cells suggested that hydroxyurea may differentially affect reduction of the various substrates of the enzyme.