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1.  Zinc carnosine, a health food supplement that stabilises small bowel integrity and stimulates gut repair processes 
Gut  2006;56(2):168-175.
Zinc carnosine (ZnC) is a health food product claimed to possess health‐promoting and gastrointestinal supportive activity. Scientific evidence underlying these claims is, however, limited.
To examine the effect of ZnC on various models of gut injury and repair, and in a clinical trial.
In vitro studies used pro‐migratory (wounded monolayer) and proliferation ([3H]‐thymidine incorporation) assays of human colonic (HT29), rat intestinal epithelial (RIE) and canine kidney (MDCK) epithelial cells. In vivo studies used a rat model of gastric damage (indomethacin/restraint) and a mouse model of small‐intestinal (indomethacin) damage. Healthy volunteers (n = 10) undertook a randomised crossover trial comparing changes in gut permeability (lactulose:rhamnose ratios) before and after 5 days of indomethacin treatment (50 mg three times a day) with ZnC (37.5 mg twice daily) or placebo coadministration.
ZnC stimulated migration and proliferation of cells in a dose‐dependent manner (maximum effects in both assays at 100 µmol/l using HT29 cells), causing an approximate threefold increase in migration and proliferation (both p<0.01). Oral ZnC decreased gastric (75% reduction at 5 mg/ml) and small‐intestinal injury (50% reduction in villus shortening at 40 mg/ml; both p<0.01). In volunteers, indomethacin caused a threefold increase in gut permeability in the control arm; lactulose:rhamnose ratios were (mean (standard error of mean)) 0.35 (0.035) before indomethacin treatment and 0.88 (0.11) after 5 days of indomethacin treatment (p<0.01), whereas no significant increase in permeability was seen when ZnC was coadministered.
ZnC, at concentrations likely to be found in the gut lumen, stabilises gut mucosa. Further studies are warranted.
PMCID: PMC1856764  PMID: 16777920
2.  Association between Genetic Polymorphisms of DNA Repair Genes and Chromosomal Damage for 1,3-Butadiene-Exposed Workers in a Matched Study in China 
BioMed Research International  2015;2015:234675.
The aim of the study was to examine the association between polymorphisms of DNA repair genes and chromosomal damage of 1,3-butadiene- (BD-) exposed workers. The study was conducted in 45 pairs of occupationally exposed workers in a BD product workshop and matched control workers in an administrative office and a circulatory water workshop in China. Newly developed biomarkers (micronuclei, MNi; nucleoplasmic bridges, NPBs; nuclear buds, NBUDs) in the cytokinesis-blocked micronucleus (CBMN) cytome assay were adopted to detect chromosomal damage. PCR and PCR-restriction fragment length polymorphism (RFLP) are adopted to analyze polymorphisms of DNA repair genes, such as X-ray repair cross-complementing Group 1 (XRCC1), O6-methylguanine-DNA methyltransferase (MGMT), poly (adenosine diphosphate-ribose) polymerases (ADPRT), and apurinic/apyrimidinic endonucleases (APE1). The BD-exposed workers exhibited increased frequencies of MNi and NPBs when compared to subjects in the control group. The results also show that the BD-exposed workers carrying XRCC1 diplotypes TCGA-CCGG (4.25 ± 2.06‰) (FR = 2.10, 95% CI: 1.03–4.28) and TCGG-TCGA (5.80 ± 3.56‰) (FR = 2.75, 95% CI: 0.76–2.65) had statistically higher NBUD frequencies than those who carried diplotype TCGG-TCGG (1.89 ± 1.27‰). Our study suggests that polymorphisms of XRCC1 gene may influence chromosomal damage in BD-exposed workers.
PMCID: PMC4538405  PMID: 26339595
3.  Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica 
PLoS ONE  2013;8(6):e66790.
The dimorphic yeast Yarrowia lipolytica is used as a model to study fungal differentiation because it grows as yeast-like cells or forms hyphal cells in response to changes in environmental conditions. Here, we report the isolation and characterization of a gene, ZNC1, involved in the dimorphic transition in Y. lipolytica. The ZNC1 gene encodes a 782 amino acid protein that contains a Zn(II)2C6 fungal-type zinc finger DNA-binding domain and a leucine zipper domain. ZNC1 transcription is elevated during yeast growth and decreases during the formation of mycelium. Cells in which ZNC1 has been deleted show increased hyphal cell formation. Znc1p-GFP localizes to the nucleus, but mutations within the leucine zipper domain of Znc1p, and to a lesser extent within the Zn(II)2C6 domain, result in a mislocalization of Znc1p to the cytoplasm. Microarrays comparing gene expression between znc1::URA3 and wild-type cells during both exponential growth and the induction of the yeast-to-hypha transition revealed 1,214 genes whose expression was changed by 2-fold or more under at least one of the conditions analyzed. Our results suggest that Znc1p acts as a transcription factor repressing hyphal cell formation and functions as part of a complex network regulating mycelial growth in Y. lipolytica.
PMCID: PMC3691278  PMID: 23826133
4.  Investigation of genome instability in patients with non-alcoholic steatohepatitis 
AIM: To evaluate the occurrence of micronucleus (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in the mitogen-stimulated lymphocytes of patients with non-alcoholic steatohepatitis (NASH).
METHODS: The study was performed in 25 (9 females, 16 males) patients newly diagnosed with NASH, and 25 healthy subjects of similar ages and genders were used as a control group. None of the controls was known to be receiving any drugs for medical or other reasons or using alcohol. Hepatosteatosis was further excluded by abdominal ultrasound imaging in the control group. The numbers of MN, NPBs and NBUDs scored in binucleated (BN) cells were obtained from the mitogen-stimulated lymphocytes of patients and control subjects. Statistical comparisons of the numbers of BN cells with MN, NPBs and NBUDs and ages between the patients with NASH and control subjects were performed.
RESULTS: The mean ages of the patients and the control group were 41.92 ± 13.33 and 41.80 ± 13.09 years (P > 0.05), respectively. The values of the mean body mass index (BMI), HOMA-IR, hemoglobin, creatinin, aspartate aminotransferase, alanine aminotransferase, triglyceride, high density lipoprotein, and low density lipoprotein were 31.19 ± 4.62 kg/m2 vs 25.07 ± 4.14 kg/m2, 6.71 ± 4.68 vs 1.40 ± 0.53, 14.73 ± 1.49 g/dL vs 14.64 ± 1.30 g/dL, 0.74 ± 0.15 mg/dL vs 0.80 ± 0.13 mg/dL, 56.08 ± 29.11 U/L vs 16.88 ± 3.33 U/L, 92.2 ± 41.43 U/L vs 15.88 ± 5.88 U/L, 219.21 ± 141.68 mg/dL vs 102.56 ± 57.98 mg/dL, 16.37 ± 9.65 mg/dL vs 48.72 ± 15.31 mg/dL, and 136.75 ± 30.14 mg/dL vs 114.63 ± 34.13 mg/dL in the patients and control groups, respectively. The total numbers and frequencies of BN cells with MN, NPBs and NBUDs, which were scored using the CBMN cytome assay on PHA-stimulated lymphocytes, were evaluated in the patients with NASH and control group. We found significantly higher numbers of MN, NPBs and NBUDs in the BN cells of patients with NASH than in those of the control subjects (21.60 ± 9.32 vs 6.88 ± 3.91; 29.28 ± 13.31 vs 7.84 ± 3.96; 15.60 ± 5.55 vs 4.20 ± 1.63, respectively, P < 0.0001).
CONCLUSION: The increased numbers of MN, NPBs and NBUDs observed in the lymphocytes obtained from patients with NASH may reflect genomic instability.
PMCID: PMC3752563  PMID: 23983432
Non-alcoholic steatohepatitis; Micronucleus; Nucleoplasmic bridges; Nuclear buds
5.  Inhalative Exposure to Vanadium Pentoxide Causes DNA Damage in Workers: Results of a Multiple End Point Study 
Environmental Health Perspectives  2008;116(12):1689-1693.
Inhalative exposure to vanadium pentoxide (V2O5) causes lung cancer in rodents.
The aim of the study was to investigate the impact of V2O5 on DNA stability in workers from a V2O5 factory.
We determined DNA strand breaks in leukocytes of 52 workers and controls using the alkaline comet assay. We also investigated different parameters of chromosomal instability in lymphocytes of 23 workers and 24 controls using the cytokinesis-block micronucleus (MN) cytome method.
Seven of eight biomarkers were increased in blood cells of the workers, and vanadium plasma concentrations in plasma were 7-fold higher than in the controls (0.31 μg/L). We observed no difference in DNA migration under standard conditions, but we found increased tail lengths due to formation of oxidized purines (7%) and pyrimidines (30%) with lesion-specific enzymes (formamidopyrimidine glycosylase and endonuclease III) in the workers. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycin-induced lesions was reduced. Workers had a 2.5-fold higher MN frequency, and nucleoplasmic bridges (NPBs) and nuclear buds (Nbuds) were increased 7-fold and 3-fold, respectively. Also, apoptosis and necrosis rates were higher, but only the latter parameter reached statistical significance.
V2O5 causes oxidation of DNA bases, affects DNA repair, and induces formation of MNs, NPBs, and Nbuds in blood cells, suggesting that the workers are at increased risk for cancer and other diseases that are related to DNA instability.
PMCID: PMC2599764  PMID: 19079721
comet assay; cytokinesis-block micronucleus assay; DNA damage; occupational exposure; vanadium pentoxide
6.  Zinc-chelated Vitamin C Stimulates Adipogenesis of 3T3-L1 Cells 
Adipose tissue development and function play a critical role in the regulation of energy balance, lipid metabolism, and the pathophysiology of metabolic syndromes. Although the effect of zinc ascorbate supplementation in diabetes or glycemic control is known in humans, the underlying mechanism is not well described. Here, we investigated the effect of a zinc-chelated vitamin C (ZnC) compound on the adipogenic differentiation of 3T3-L1 preadipocytes. Treatment with ZnC for 8 d significantly promoted adipogenesis, which was characterized by increased glycerol-3-phosphate dehydrogenase activity and intracellular lipid accumulation in 3T3-L1 cells. Meanwhile, ZnC induced a pronounced up-regulation of the expression of glucose transporter type 4 (GLUT4) and the adipocyte-specific gene adipocyte protein 2 (aP2). Analysis of mRNA and protein levels further showed that ZnC increased the sequential expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα), the key transcription factors of adipogenesis. These results indicate that ZnC could promote adipogenesis through PPARγ and C/EBPα, which act synergistically for the expression of aP2 and GLUT4, leading to the generation of insulin-responsive adipocytes and can thereby be useful as a novel therapeutic agent for the management of diabetes and related metabolic disorders.
PMCID: PMC4093222  PMID: 25049900
Zinc-chelated Vitamin C; Adipogenesis; PPARγ; C/EBPα; GLUT4; 3T3-L1
7.  Biofortification and Bioavailability of Rice Grain Zinc as Affected by Different Forms of Foliar Zinc Fertilization 
PLoS ONE  2012;7(9):e45428.
Zinc (Zn) biofortification through foliar Zn application is an attractive strategy to reduce human Zn deficiency. However, little is known about the biofortification efficiency and bioavailability of rice grain from different forms of foliar Zn fertilizers.
Methodology/Principal Findings
Four different Zn forms were applied as a foliar treatment among three rice cultivars under field trial. Zinc bioavailability was assessed by in vitro digestion/Caco-2 cell model. Foliar Zn fertilization was an effective agronomic practice to promote grain Zn concentration and Zn bioavailability among three rice cultivars, especially, in case of Zn-amino acid and ZnSO4. On average, Zn-amino acid and ZnSO4 increased Zn concentration in polished rice up to 24.04% and 22.47%, respectively. On average, Zn-amino acid and ZnSO4 increased Zn bioavailability in polished rice up to 68.37% and 64.43%, respectively. The effectiveness of foliar applied Zn-amino acid and ZnSO4 were higher than Zn-EDTA and Zn-Citrate on improvement of Zn concentration, and reduction of phytic acid, as a results higher accumulation of bioavailable Zn in polished rice. Moreover, foliar Zn application could maintain grain yield, the protein and minerals (Fe and Ca) quality of the polished rice.
Foliar application of Zn in rice offers a practical and useful approach to improve bioavailable Zn in polished rice. According to current study, Zn-amino acid and ZnSO4 are recommended as excellent foliar Zn forms to ongoing agronomic biofortification.
PMCID: PMC3447881  PMID: 23029003
8.  Cellular Zn depletion by metal ion chelators (TPEN, DTPA and chelex resin) and its application to osteoblastic MC3T3-E1 cells 
Trace mineral studies involving metal ion chelators have been conducted in investigating the response of gene and protein expressions of certain cell lines but a few had really focused on how these metal ion chelators could affect the availability of important trace minerals such as Zn, Mn, Fe and Cu. The aim of the present study was to investigate the availability of Zn for the treatment of MC3T3-E1 osteoblast-like cells and the availability of some trace minerals in the cell culture media components after using chelexing resin in the FBS and the addition of N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN, membrane-permeable chelator) and diethylenetriaminepentaacetic acid (DTPA, membrane-impermeable chelator) in the treatment medium. Components for the preparation of cell culture medium and Zn-treated medium have been tested for Zn, Mn, Fe and Cu contents by atomic absorption spectrophotometer or inductively coupled plasma spectrophotometer. Also, the expression of bone-related genes (ALP, Runx2, PTH-R, ProCOL I, OPN and OC) was measured on the cellular Zn depletion such as chelexing or TPEN treatment. Results have shown that using the chelexing resin in FBS would significantly decrease the available Zn (p<0.05) (39.4 ± 1.5 µM vs 0.61 ± 10.15 µM) and Mn (p<0.05) (0.74 ± 0.01 µM vs 0.12 ± 0.04 µM). However, levels of Fe and Cu in FBS were not changed by chelexing FBS. The use of TPEN and DTPA as Zn-chelators did not show significant difference on the final concentration of Zn in the treatment medium (0, 3, 6, 9, 12 µM) except for in the addition of higher 15 µM ZnCl2 which showed a significant increase of Zn level in DTPA-chelated treatment medium. Results have shown that both chelators gave the same pattern for the expression of the five bone-related genes between Zn- and Zn+, and TPEN-treated experiments, compared to chelex-treated experiment, showed lower bone-related gene expression, which may imply that TPEN would be a stronger chelator than chelex resin. This study showed that TPEN would be a stronger chelator compared to DTPA or chelex resin and TPEN and chelex resin exerted cellular zinc depletion to be enough for cell study for Zn depletion.
PMCID: PMC2882573  PMID: 20535382
Zn depletion; metal ion chelators; DTPA; TPEN; chelexing; MC3T3-E1 cells
9.  Analysis of 7,8-Dihydro-8-oxo-2′-deoxyguanosine in Cellular DNA during Oxidative Stress 
Chemical Research in Toxicology  2009;22(5):788-797.
Analysis of cellular 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dGuo) as a biomarker of oxidative DNA damage has been fraught with numerous methodological problems. This is primarily due to artifactual oxidation of dGuo that occurs during DNA isolation and hydrolysis. Therefore, it has become necessary to rely on using the comet assay, which is not necessarily specific for 8-oxo-dGuo. A highly specific and sensitive method based on immunoaffinity purification and stable isotope dilution liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) that avoids artifact formation has now been developed. Cellular DNA was isolated using cold DNAzol (a proprietary product that contains guanidine thiocyanate) instead of chaotropic- or phenol-based methodology. Chelex-treated buffers were used to prevent Fenton chemistry-mediated generation of reactive oxygen species (ROS) and artifactual oxidation of DNA bases. Deferoxamine was also added to all buffers in order to complex any residual transition metal ions remaining after Chelex treatment. The LC-MRM/MS method was used to determine that the basal 8-oxo-dGuo level in DNA from human bronchoalveolar H358 cells was 2.2 ± 0.4 8-oxo-dGuo/107 dGuo (mean ± standard deviation) or 5.5 ± 1.0 8-oxo-dGuo/108 nucleotides. Similar levels were observed in human lung adenocarcinoma A549 cells, mouse hepatoma Hepa-1c1c7 cells, and human HeLa cervical epithelial adenocarcinoma cells. These values are an order of magnitude lower than is typically reported for basal 8-oxo-dGuo levels in DNA as determined by other MS- or chromatography-based assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO3) as a positive control or with the methylating agent methyl methanesulfonate (MMS) as a negative control. A linear dose−response for 8-oxo-dGuo formation (r2 = 0.962) was obtained with increasing concentrations of KBrO3 in the range of 0.05 mM to 2.50 mM. In contrast, no 8-oxo-dGuo was observed in H358 cell DNA after treatment with MMS. At low levels of oxidative DNA damage, there was an excellent correlation between a comet assay that measured DNA single strand breaks (SSBs) after treatment with human 8-oxo-guanine glycosylase-1 (hOGG1) when compared with 8-oxo-dGuo in the DNA as measured by the stable isotope dilution LC-MRM/MS method. Availability of the new LC-MRM/MS assay made it possible to show that the benzo[a]pyrene (B[a]P)-derived quinone, B[a]P-7,8-dione, could induce 8-oxo-dGuo formation in H358 cells. This most likely occurred through redox cycling between B[a]P-7,8-dione and B[a]P-7,8-catechol with concomitant generation of DNA damaging ROS. In keeping with this concept, inhibition of catechol-O-methyl transferase (COMT)-mediated detoxification of B[a]P-7,8-catechol with Ro 410961 caused increased 8-oxo-dGuo formation in the H358 cell DNA.
PMCID: PMC2684441  PMID: 19309085
10.  DNA-dependent protein kinase (DNA-PK)-deficient human glioblastoma cells are preferentially sensitized by Zebularine 
Carcinogenesis  2009;31(2):184-191.
Brain tumor cells respond poorly to radiotherapy and chemotherapy due to inherently efficient anti-apoptotic and DNA repair mechanisms. This necessitates the development of new strategies for brain cancer therapy. Here, we report that the DNA-demethylating agent Zebularine preferentially sensitizes the killing of human glioblastomas deficient in DNA-dependent protein kinase (DNA-PK). In contrast to DNA-PK-proficient human glioblastoma cells (MO59K), cytotoxicity assay with increasing Zebularine concentrations up to 300 μM resulted in a specific elevation of cell killing in DNA-PK-deficient MO59J cells. Further, an elevated frequency of polyploid cells observed in MO59J cells after Zebularine treatment pointed out a deficiency in mitotic checkpoint control. Existence of mitotic checkpoint deficiency in MO59J cells was confirmed by the abnormal centrosome number observed in Zebularine-treated MO59J cells. Although depletion of DNA methyltransferase 1 by Zebularine occurred at similar levels in both cell lines, MO59J cells displayed increased extent of DNA demethylation detected both at the gene promoter-specific level and at the genome overall level. Consistent with increased sensitivity, deoxy-Zebularine adduct level in the genomic DNA was 3- to 6-fold higher in MO59J than in MO59K cells. Elevated micronuclei frequency observed after Zebularine treatment in MO59J cells indicates the impairment of DNA repair response in MO59J cells. Collectively, our study suggests that DNA-PK is the major determining factor for cellular response to Zebularine.
PMCID: PMC2812572  PMID: 19933707
11.  Uptake epithelia behave in a cell-centric and not systems homeostatic manner in response to zinc depletion and supplementation† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c3mt00212h Click here for additional data file.  
Metallomics  2013;6(1):154-165.
Global transcriptomic analysis, non-invasive real-time flux, nutritional profiling and metallomics reveal cell-centric response to zinc supplementation/depletion in zebrafish uptake epithelia.
Much remains to be understood about systemic regulation of zinc uptake in vertebrates, and adequate zinc status is far from always achieved in animals or human. In addition to absorbing zinc from the diet, fish are able to take up zinc directly from the water with the gills. This provides an elegant system to study zinc uptake, how it relates to zinc status, and the expression of genes for proteins involved in zinc acquisition. A 21-day experiment was conducted in which zebrafish were acclimated to deficient, control or excess zinc concentrations in the water and feed. Deficient provision of zinc reduced whole body zinc, potassium, sodium and calcium levels whilst zinc concentrations in the uptake epithelia (gills and gut) remained unchanged. Excess levels of zinc caused accumulation of zinc in the gills, intestine and carcass, but impaired whole body iron, sodium and calcium concentrations. Fish subjected to zinc deficiency had, surprisingly, a reduced zinc influx across the gill epithelium, even when tested at a high concentration of zinc in the water. Zinc influx in the excess group was indistinct from the control. Expression of genes for metallothionein-2 (Mt2) and zinc transporters-1, -2, and -8 (Znt1, Znt2, Znt8) in uptake epithelia showed in general a direct relationship with zinc supply, while mRNA for Zip4 was inversely related to zinc supply. Transcripts for the epithelial calcium channel (Ecac/Trpv6) showed time-dependent increased expression in the gills of the deficiency group, and a transient decrease of expression during zinc excess. Transcriptome profiling by microarrays showed that in both gills and intestine, the most markedly affected biological functions were those related to cell growth, proliferation and cancer, closely followed by processes of gene transcription and protein synthesis in general. Whilst changes in zinc supply had profound effects in the intestine on genes associated with uptake and metabolism of macronutrients, many of the unique categories of genes preferentially regulated in the gill could be mapped onto signalling pathways. This included pathways for PPAR/RXR, LXR/RXR, ATM, chemokine, and BMP signalling. Overall, the responses of epithelial tissue to zinc deficiency and excess are best explained by local epithelial homeostasis with no evidence of systemic control.
PMCID: PMC4157650  PMID: 24301558
12.  Prediction of serum IgG concentration by indirect techniques with adjustment for age and clinical and laboratory covariates in critically ill newborn calves 
The objective of this study was to develop prediction models for the serum IgG concentration in critically ill calves based on indirect assays and to assess if the predictive ability of the models could be improved by inclusion of age, clinical covariates, and/or laboratory covariates. Seventy-eight critically ill calves between 1 and 13 days old were selected from 1 farm. Statistical models to predict IgG concentration from the results of the radial immunodiffusion test, the gold standard, were built as a function of indirect assays of serum and plasma protein concentrations, zinc sulfate (ZnSO4) turbidity and transmittance, and serum γ-glutamyl transferase (GGT) activity. For each assay 4 models were built: without covariates, with age, with age and clinical covariates (infection and dehydration status), and with age and laboratory covariates (fibrinogen concentration and packed cell volume). For the protein models, dehydration status (clinical model) and fibrinogen concentration (laboratory model) were selected for inclusion owing to their statistical significance. These variables increased the coefficient of determination (R2) of the models by ≥ 7% but did not significantly improve the sensitivity or specificity of the models to predict passive transfer with a cutoff IgG concentration of 1000 mg/dL. For the GGT assay, including age as a covariate increased the R2 of the model by 3%. For the ZnSO4 turbidity test, none of the covariates were statistically significant. Overall, the R2 of the models ranged from 34% to 62%. This study has provided insight into the importance of adjusting for covariates when using indirect assays to predict IgG concentration in critically ill calves. Results also indicate that ZnSO4 transmittance and turbidity assays could be used advantageously in a field setting.
PMCID: PMC3605935  PMID: 24082398
13.  HIV Protease Inhibitors Block Oral Epithelial Cell DNA Synthesis 
Archives of oral biology  2009;55(2):95.
Anti-retroviral therapy regimens that include HIV protease inhibitors (PIs) are associated with diverse adverse effects including increased prevalence of oral warts, oral sensorial deficits and gastrointestinal toxicities suggesting that PIs may perturb epithelial cell biology. To test the hypothesis that PIs could affect specific biological processes of oral epithelium, the effects of these agents were evaluated in several oral epithelial cell lines.
Primary and immortalized oral keratinocytes and squamous carcinoma cells of oropharyngeal origin were cultured in the presence of pharmacologically relevant concentrations of PIs. Their affects on cell viability, cytotoxicity and DNA synthesis were assessed by enzymatic assays and incorporation of 5-bromo-2’-deoxyuridine (BrdU) into DNA.
Viability of primary and immortalized oral keratinocytes as well as squamous carcinoma cells of oropharyngeal origin was significantly reduced by select PIs at concentrations found in plasma. Of the seven PIs evaluated, nelfinavir was the most potent with a mean 50% inhibitory concentration [IC50] of 4.1 µM. Lopinavir and saquinavir also reduced epithelial cell viability (IC50 of 10–20 µM). Atazanavir and ritonovir caused minor reductions in viability, while amprenavir and indinavir were not significant inhibitors. The reduced cell viability, as shown by BrdU incorporation assays, was due to inhibition of DNA synthesis rather than cell death due to cytotoxicity.
Select PIs retard oral epithelial cell proliferation in a drug and dose dependent manner by blocking DNA synthesis. This could account for some of their adverse effects on oral health.
PMCID: PMC2818864  PMID: 20035926
Protease inhibitors; epithelial cells; viability; HAART
14.  Photobiological Implications of Folate Depletion and Repletion in Cultured Human Keratinocytes 
Folate nutrition is critical in humans and a high dietary folate intake is associated with a diminished risk of many types of cancer. Both synthetic folic acid and the most biologically abundant extracellular reduced folate, 5-methyltetrahydrofolate, are degraded under conditions of ultraviolet radiation (UVR) exposure. Skin is a proliferative tissue with increased folate nutrient demands due to a dependence upon continuous epidermal cell proliferation and differentiation to maintain homeostasis. Regions of skin are also chronically exposed to UVR, which penetrates to the actively dividing basal layer of the epidermis, increasing the folate nutrient demands in order to replace folate species degraded by UVR exposure and to supply the folate cofactors required for repair of photo-damaged DNA. Localized folate deficiencies of skin are a likely consequence of UVR exposure. We report here a cultured keratinocyte model of folate deficiency that has been applied to examine possible effects of folate nutritional deficiencies in skin. Utilizing this model, we were able to quantify the concentrations of key intracellular folate species during folate depletion and repletion. We investigated the hypotheses that the genomic instability observed under conditions of folate deficiency in other cell types extends to skin, adversely effecting cellular capacity to handle UVR insult and that optimizing folate levels in skin is beneficial in preventing or repairing the pro-carcinogenic effects of UVR exposure. Folate restriction leads to rapid depletion of intracellular reduced folates resulting in S-phase growth arrest, increased levels of inherent DNA damage, and increased uracil misincorporation into DNA, without a significant losses in overall cellular viability. Folate depleted keratinocytes were sensitized toward UVR induced apoptosis and displayed a diminished capacity to remove DNA breaks resulting from both photo and oxidative DNA damage. Thus, folate deficiency creates a permissive environment for genomic instability, an early event in the process of skin carcinogenesis. The effects of folate restriction, even in severely depleted, growth-arrested keratinocytes, were reversible by repletion with folic acid. Overall, these results indicate that skin health can be positively influenced by optimal folate nutriture.
PMCID: PMC2862485  PMID: 20211567
Folate; Keratinocytes; Skin Biology; Solar Simulated Light; DNA Damage; Cancer
15.  Effect of Supplementation with Zinc and Other Micronutrients on Malaria in Tanzanian Children: A Randomised Trial 
PLoS Medicine  2011;8(11):e1001125.
Hans Verhoef and colleagues report findings from a randomized trial conducted among Tanzanian children at high risk for malaria. Children in the trial received either daily oral supplementation with either zinc alone, multi-nutrients without zinc, multi-nutrients with zinc, or placebo. The investigators did not find evidence from this study that zinc or multi-nutrients protected against malaria episodes.
It is uncertain to what extent oral supplementation with zinc can reduce episodes of malaria in endemic areas. Protection may depend on other nutrients. We measured the effect of supplementation with zinc and other nutrients on malaria rates.
Methods and Findings
In a 2×2 factorial trial, 612 rural Tanzanian children aged 6–60 months in an area with intense malaria transmission and with height-for-age z-score≤−1.5 SD were randomized to receive daily oral supplementation with either zinc alone (10 mg), multi-nutrients without zinc, multi-nutrients with zinc, or placebo. Intervention group was indicated by colour code, but neither participants, researchers, nor field staff knew who received what intervention. Those with Plasmodium infection at baseline were treated with artemether-lumefantrine. The primary outcome, an episode of malaria, was assessed among children reported sick at a primary care clinic, and pre-defined as current Plasmodium infection with an inflammatory response, shown by axillary temperature ≥37.5°C or whole blood C-reactive protein concentration ≥8 mg/L. Nutritional indicators were assessed at baseline and at 251 days (median; 95% reference range: 191–296 days). In the primary intention-to-treat analysis, we adjusted for pre-specified baseline factors, using Cox regression models that accounted for multiple episodes per child. 592 children completed the study. The primary analysis included 1,572 malaria episodes during 526 child-years of observation (median follow-up: 331 days). Malaria incidence in groups receiving zinc, multi-nutrients without zinc, multi-nutrients with zinc and placebo was 2.89/child-year, 2.95/child-year, 3.26/child-year, and 2.87/child-year, respectively. There was no evidence that multi-nutrients influenced the effect of zinc (or vice versa). Neither zinc nor multi-nutrients influenced malaria rates (marginal analysis; adjusted HR, 95% CI: 1.04, 0.93–1.18 and 1.10, 0.97–1.24 respectively). The prevalence of zinc deficiency (plasma zinc concentration <9.9 µmol/L) was high at baseline (67% overall; 60% in those without inflammation) and strongly reduced by zinc supplementation.
We found no evidence from this trial that zinc supplementation protected against malaria.
Trial Registration NCT00623857
Please see later in the article for the Editors' Summary.
Editors' Summary
Malaria is a serious global public-health problem. Half of the world's population is at risk of this parasitic disease, which kills a million people (mainly children living in sub-Saharan Africa) every year. Malaria is transmitted to people through the bites of infected night-flying mosquitoes. Soon after entering the human body, the parasite begins to replicate in red blood cells, bursting out every 2–3 days and infecting more red blood cells. The presence of the parasite in the blood stream (parasitemia) causes malaria's characteristic recurring fever and can cause life-threatening organ damage and anemia (insufficient quantity of red blood cells). Malaria transmission can be reduced by using insecticide sprays to control the mosquitoes that spread the parasite and by avoiding mosquito bites by sleeping under insecticide-treated bed nets. Effective treatment with antimalarial drugs can also reduce malaria transmission.
Why Was This Study Done?
One reason why malaria kills so many children in Africa is poverty. Many children in Africa are malnourished, and malnutrition—in particular, insufficient micronutrients in the diet—impairs the immune system, which increases the frequency and severity of many childhood diseases. Micronutrients are vitamins and minerals that everyone needs in small quantities for good health. Zinc is one of the micronutrients that helps to maintain a healthy immune system, but zinc deficiency is very common among African children. Zinc supplementation has been shown to reduce the burden of diarrhea in developing countries, so might it also reduce the burden of malaria? Unfortunately, the existing evidence is confusing—some trials show that zinc supplementation protects against malaria but others show no evidence of protection. One possibility for these conflicting results could be that zinc supplementation alone is not sufficient—supplementation with other micronutrients might be needed for zinc to have an effect. In this randomized trial (a study that compares the effects of different interventions in groups that initially are similar in all characteristics except for intervention), the researchers investigate the effect of supplementation with zinc alone and in combination with other micronutrients on the rate of uncomplicated (mild) malaria among children living in Tanzania.
What Did the Researchers Do and Find?
The researchers enrolled 612 children aged 6–60 months who were living in a rural area of Tanzania with intense malaria transmission and randomly assigned them to receive daily oral supplements containing zinc alone, multi-nutrients (including iron) without zinc, multi-nutrients with zinc, or a placebo (no micronutrients). Nutritional indicators (including zinc concentrations in blood plasma) were assessed at baseline and 6–10 months after starting the intervention. During the study period, there were 1,572 malaria episodes. The incidence of malaria in all four intervention groups was very similar (about three episodes per child-year), and there was no evidence that multi-nutrients influenced the effect of zinc (or vice versa). Moreover, none of the supplements had any effect on malaria rates when compared to the placebo, even though the occurrence of zinc deficiency was strongly reduced by zinc supplementation. In a secondary analysis in which they analyzed their data by iron status at baseline, the researchers found that multi-nutrient supplementation increased the overall number of malaria episodes in children with iron deficiency by 41%, whereas multi-nutrient supplementation had no effect on the number of malaria episodes among children who were iron-replete at baseline.
What Do These Findings Mean?
In this study, the researchers found no evidence that zinc supplementation protected against malaria among young children living in Tanzania when given alone or in combination with other multi-nutrients. However, the researchers did find some evidence that multi-nutrient supplementation may increase the risk of malaria in children with iron deficiency. Because this finding came out of a secondary analysis of the data, it needs to be confirmed in a trial specifically designed to assess the effect of multi-nutrient supplements on malaria risk in iron-deficient children. Nevertheless, it is a potentially worrying result because, on the basis of evidence from a single study, the World Health Organization currently recommends that regular iron supplements be given to iron-deficient children in settings where there is adequate access to anti-malarial treatment. This recommendation should be reconsidered, suggest the researchers, and the safety of multi-nutrient mixes that contain iron and that are dispensed in countries affected by malaria should also be carefully evaluated.
Additional Information
Please access these Web sites via the online version of this summary at
Information is available from the World Health Organization on malaria (in several languages), on micronutrients, and on zinc deficiency; the 2010 World Malaria Report provides details of the current global malaria situation
The US Centers for Disease Control and Prevention provide information on malaria (in English and Spanish), including a selection of personal stories about malaria
Information is available from the Roll Back Malaria Partnership on the global control of malaria and on malaria in Africa
The Malaria Centre at the UK London School of Hygiene & Tropical Medicine develops tools, techniques, and knowledge about malaria, and has a strong emphasis on teaching, training, and translating research outcomes into practice
The Micronutrient Initiative, the Global Alliance for Improved Nutrition, and the Flour Fortification Initiative are not-for-profit organizations dedicated to ensuring that people in developing countries get the minerals and vitamins they need to survive and thrive
The International Zinc Nutrition Consultative Group (iZiNCG) is a non-profit organization that aims to promote and assist efforts to reduce zinc deficiency worldwide, through advocacy efforts, education, and technical assistance
MedlinePlus provides links to additional information on malaria (in English and Spanish)
PMCID: PMC3222646  PMID: 22131908
16.  A comparative study for selectivity of micronuclei in oral exfoliated epithelial cells 
Micronucleus (MN) represents small, additional nuclei formed by the exclusion of chromosome fragments or whole chromosomes lagging at mitosis. MN rates, therefore, indirectly reflect chromosome breakage or impairment of the mitotic apparatus. During the last few decades, micronuclei (“MNi”) in oral exfoliated epithelial cells are widely used as biomarkers of chromosomal damage, genome instability and cancer risk in humans. However, until now only little attention has been given to the effect of different staining procedures on the results of these MN assays.
To compare the MNi frequencies in oral exfoliated epithelial cells using three different stains, i.e.,Feulgen stain, Papanicolaou stain (Pap) and hemotoxylin and eosin stain (H and E).
Materials and Methods:
Oral exfoliated cells from 45 cases of potentially malignant disorders (15 oral submucous fibrosis, 15 lichen planus and 15 leukoplakia) and 15 controls with healthy mucosa, were taken and MNi frequencies (No. of MNi/1000 cells) were compared using three different stains.
Mean MNi frequency in cases was found to be 3.8 with Feulgen stain, 16.8 with PAP and 25.9 with H and E. In controls, mean MNi frequency was 1.6 with Feulgen stain, 7.7 with PAP and 9.6 with H and E stain. Statistically significant value (P < 0.01) were observed when the three stains were compared together using Kruskal Walli's ANOVA test.
Feulgen being a DNA-specific stain gave the least counts, although statistically significant results from the comparison of MNi frequency between cases and controls were obtained with all the three stains.
PMCID: PMC3543590  PMID: 23326025
Feulgen stain; hematoxylin and eosin stain; micronuclei; papanicolaou stain; potentially malignant disorders
17.  Increased frequency of micronuclei in the lymphocytes of patients chronically infected with hepatitis B or hepatitis C virus 
In this study, we analysed the frequency of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) and evaluated mutagen-induced sensitivity in the lymphocytes of patients chronically infected with hepatitis B virus (HBV) or hepatitis C virus (HCV). In total, 49 patients with chronic viral hepatitis (28 HBV-infected and 21 HCV-infected patients) and 33 healthy, non-infected blood donor controls were investigated. The frequencies (‰) of MN, NPBs and NBUDs in the controls were 4.41 ± 2.15, 1.15 ± 0.97 and 2.98 ± 1.31, respectively. The frequencies of MN and NPBs were significantly increased (p < 0.0001) in the patient group (7.01 ± 3.23 and 2.76 ± 2.08, respectively) compared with the control group. When considered separately, the HBV-infected patients (7.18 ± 3.57) and HCV-infected patients (3.27 ± 2.40) each had greater numbers of MN than did the controls (p < 0.0001). The HCV-infected patients displayed high numbers of NPBs (2.09 ± 1.33) and NBUDs (4.38 ± 3.28), but only the HBV-infected patients exhibited a significant difference (NPBs = 3.27 ± 2.40, p < 0.0001 and NBUDs = 4.71 ± 2.79, p = 0.03) in comparison with the controls. Similar results were obtained for males, but not for females, when all patients or the HBV-infected group was compared with the controls. The lymphocytes of the infected patients did not exhibit sensitivity to mutagen in comparison with the lymphocytes of the controls (p = 0.06). These results showed that the lymphocytes of patients who were chronically infected with HBV or HCV presented greater chromosomal instability.
PMCID: PMC4005534  PMID: 24626305
hepatitis B; hepatitis C; lymphocytes; micronucleus
18.  Pharmacokinetics and Biodistribution of Zinc-Enriched Yeast in Rats 
The Scientific World Journal  2014;2014:217142.
Zinc-enriched yeast (ZnY) and zinc sulfate (ZnSO4) are considered zinc (Zn) supplements currently available. The purpose of the investigation was to compare and evaluate pharmacokinetics and biodistribution of ZnY and ZnSO4 in rats. ZnY or ZnSO4 were orally administered to rats at a single dose of 4 mg Zn/kg and Zn levels in plasma and various tissues were determined using inductively coupled plasma-optical emission spectrometry. Maximum plasma concentration values were 3.87 and 2.81 μg/mL for ZnY and ZnSO4, respectively. Both ZnY and ZnSO4 were slowly eliminated with a half-life of over 7 h and bone had the highest Zn level in all tissues. Compared to ZnSO4, the relative bioavailability of ZnY was 138.4%, indicating that ZnY had a significantly higher bioavailability than ZnSO4.
PMCID: PMC4151581  PMID: 25215316
19.  Genomic Instability in Human Lymphocytes from Male Users of Crack Cocaine 
Recent research suggests that crack cocaine use alters systemic biochemical markers, like oxidative damage and inflammation markers, but very few studies have assessed the potential effects of crack cocaine at the cellular level. We assessed genome instability by means of the comet assay and the cytokinesis-block micronucleus technique in crack cocaine users at the time of admission to a rehabilitation clinic and at two times after the beginning of withdrawal. Thirty one active users of crack cocaine and forty control subjects were evaluated. Comparison between controls and crack cocaine users at the first analysis showed significant differences in the rates of DNA damage (p = 0.037). The frequency of micronuclei (MN) (p < 0.001) and nuclear buds (NBUDs) (p < 0.001) was increased, but not the frequency of nucleoplasmic bridges (NPBs) (p = 0.089). DNA damage decreased only after the end of treatment (p < 0.001). Micronuclei frequency did not decrease after treatment, and nuclear buds increased substantially. The results of this study reveal the genotoxic and mutagenic effects of crack cocaine use in human lymphocytes and pave the way for further research on cellular responses and the possible consequences of DNA damage, such as induction of irreversible neurological disease and cancer.
PMCID: PMC4210963  PMID: 25264678
crack cocaine; drug withdrawal; DNA damage; comet assay; micronucleus
20.  The topical antimicrobial zinc pyrithione is a heat shock response inducer that causes DNA damage and PARP-dependent energy crisis in human skin cells 
Cell Stress & Chaperones  2009;15(3):309-322.
The differentiated epidermis of human skin serves as an essential barrier against environmental insults from physical, chemical, and biological sources. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in clinical antiseptic products, over-the-counter topical antimicrobials, and cosmetic consumer products including antidandruff shampoos. Here we demonstrate for the first time that cultured primary human skin keratinocytes and melanocytes display an exquisite vulnerability to nanomolar concentrations of ZnPT resulting in pronounced induction of heat shock response gene expression and impaired genomic integrity. In keratinocytes treated with nanomolar concentrations of ZnPT, expression array analysis revealed massive upregulation of genes encoding heat shock proteins (HSPA6, HSPA1A, HSPB5, HMOX1, HSPA1L, and DNAJA1) further confirmed by immunodetection. Moreover, ZnPT treatment induced rapid depletion of cellular ATP levels and formation of poly(ADP-ribose) polymers. Consistent with an involvement of poly(ADP-ribose) polymerase (PARP) in ZnPT-induced energy crisis, ATP depletion could be antagonized by pharmacological inhibition of PARP. This result was independently confirmed using PARP-1 knockout mouse embryonic fibroblasts that were resistant to ATP depletion and cytotoxicity resulting from ZnPT exposure. In keratinocytes and melanocytes, single-cell gel electrophoresis and flow cytometric detection of γ-H2A.X revealed rapid induction of DNA damage in response to ZnPT detectable before general loss of cell viability occurred through caspase-independent pathways. Combined with earlier experimental evidence that documents penetration of ZnPT through mammalian skin, our findings raise the possibility that this topical antimicrobial may target and compromise keratinocytes and melanocytes in intact human skin.
PMCID: PMC2866994  PMID: 19809895
Zinc pyrithione; Keratinocyte; Melanocyte; Comet assay; Heat shock response; PARP-dependent ATP depletion
21.  Molecular Interactions of Human Immunodeficiency Virus Type 1 with Primary Human Oral Keratinocytes 
Journal of Virology  2005;79(13):8440-8453.
Infection of the oral mucosa of human immunodeficiency virus type 1 (HIV-1)-infected individuals remains an under-evaluated and somewhat enigmatic process. Nonetheless, it is of profound importance in the ongoing AIDS pandemic, based on its potential as a site of person-to-person transmission of the virus as well as a location of HIV-1 pathogenesis and potential reservoir of disease in the setting of virally suppressive highly active antiretroviral therapy. We utilized molecular and virological techniques to analyze HIV-1 infection of primary human mucosal cells and also evaluated the proapoptotic potential of selected HIV-1 proteins in primary isolated human oral keratinocytes. Primary isolated human oral keratinocytes were plated on 0.4 μM polyethylenetetraphthalate cell culture inserts to form an in vitro oral mucosal layer. The strength of this layer in forming a barrier was determined by measuring trans-epithelial electrical current passage across the monolayer. The oral keratinocyte monolayers had trans-epithelial electrical resistance of approximately 176 to 208 Ω. For viral infectivity assays, the macrophage-tropic (R5) HIV-1 strains, YU-2 and ADA, and T-cell-line-tropic (X4), NL4-3 virions, incubated with or without deoxynucleoside triphosphates (dNTPs) and/or the polyamines spermine and spermidine, were used to infect oral keratinocytes. Of importance, polyamines and dNTPs have been shown to enhance natural endogenous reverse transcription (NERT), a step essential for early lentiviral infection, and are abundantly present in human semen. The infectivities of HIV-1 strains YU-2, ADA, and NL4-3 for these primary keratinocytes were dramatically increased by the addition of physiological concentrations of dNTPs, spermine, and spermidine. Binding and viral internalization assay studies showed no differences in these oral mucosal cells, with or without NERT-altering agents. It was also observed that the recombinant, cell-free HIV-1 proteins Nef, Tat, and gp120 (R5) induced apoptosis in primary oral keratinocytes compared with the results seen with nontreated cells or cells treated with glutathione S-transferase protein as a control under similar conditions. Microarray analyses suggested that HIV-1 gp120 and Tat induce apoptosis in primary human oral keratinocytes via the Fas/FasL apoptotic pathway, whereas induction of apoptosis by Nef occurs through both Fas/FasL and mitochondrial apoptotic pathways. Thus, these findings suggest molecular mechanisms by which semen in particular, as well as other bodily fluids such as cervicovaginal secretions, could increase oral transmission of HIV-1 via increasing infectivity in confluent and low-replicating oral keratinocytes. As well, the induction of apoptosis in human oral keratinocytes with relevant HIV-1-specific proteins suggests another potential complementary mechanism by which the oral mucosa barrier may be disrupted during HIV-1 infection in vivo.
PMCID: PMC1143773  PMID: 15956588
22.  Carvacrol and rosemary oil at higher concentrations induce apoptosis in human hepatoma HepG2 cells 
Interdisciplinary Toxicology  2015;7(4):189-194.
Natural essential oils are volatile herbal complex compounds which manifest cytotoxic effects on living cells depending on their type and concentration but usually they are not genotoxic. Our previous studies showed that carvacrol (CA) and rosemary essential oil (RO) induced growth inhibition of both human cell lines HepG2 and BHNF-1, with hepatoma HepG2 cells being more sensitive to either compound tested. Cytotoxic concentrations of CA and RO induced the formation of DNA strand breaks. Further ex vivo studies showed that extracts prepared from hepatocytes of CA- and RO-supplemented rats did not increase incision repair activity compared to extracts from liver cells of control animals. Therefore, the aim of this work was to determine the effect of cytotoxic concentrations of CA and RO on the cell cycle and the ability of both natural volatiles to induce DNA fragmentation and apoptotic death of human hepatoma HepG2 cells. These effects were measured after 24 h incubation of HepG2 cells with CA and RO using three independent methods – flow cytometry, internucleosomal DNA fragmentation (electrophoresis) and micronucleus assay. Evaluation of morphological changes and formation of micronuclei in HepG2 cells showed no increase in the number of micronuclei in cells treated by CA and RO compared to control cells. On the other hand, CA and RO induced morphological changes typical for apoptosis in concentration-dependent manner. The presence of necrosis was negligible. Both natural compounds caused shrinking of cytoplasmic membrane and formation of apoptotic bodies. In addition, the highest concentrations of CA and RO induced internucleosomal DNA fragmentation (formation of DNA ladder) in HepG2 cells. Cell cycle analysis revealed the accumulation of cells in the G1 phase, which was accompanied by a reduction in the number of cells in the S phase after 24 h exposure to the substances tested. The cell division was thus slowed down or stopped and this process resulted in cell death.
PMCID: PMC4436207  PMID: 26109899
carvacrol; rosemary oil; apoptosis; HepG2 cells
23.  Antitumor Effects of Synthetic 6,7-Annulated-4-substituted Indole Compounds in L1210 Leukemic Cells In Vitro 
Anticancer research  2012;32(11):4671-4684.
Because annulated indoles have almost no representation in the PubChem or MLSMR databases, an unprecedented class of an indole-based library was constructed, using the indole aryne methodology, and screened for antitumor activity. Sixty-six novel 6,7-annulated-4-substituted indole compounds were synthesized, using a strategic combination of 6,7-indolyne cycloaddition and cross-coupling reactions under both Suzuki-Miyaura and Buchwald-Hartwig conditions, and tested for their effectiveness against murine L1210 tumor cell proliferation in vitro.
Materials and Methods
Various markers of tumor cell metabolism, DNA degradation, mitotic disruption, cytokinesis and apoptosis were assayed in vitro to evaluate drug cytotoxicity.
Most compounds inhibited the metabolic activity of leukemic cells in a time- and concentration-dependent manner but only 9 of them were sufficiently potent to inhibit L1210 tumor cell proliferation by 50% in the low-μM range after 2 (IC50: 4.5–20.4 μM) and 4 days (0.5–4.0 μM) in culture. However, the antiproliferative compounds that were the most effective at day 4 were not necessarily the most potent at day 2, suggesting different speeds of action. A 3-h treatment with antiproliferative annulated indole was sufficient to inhibit, in a concentration-dependent manner, the rate of DNA synthesis measured in L1210 cells over a 0.5-h period of pulse-labeling with 3H-thymidine. Four of the antiproliferative compounds had weak DNA-binding activities but one compound reduced the fluorescence of the ethidium bromide-DNA complex by up to 53%, suggesting that some annulated indoles might directly interact with double-stranded DNA to disrupt its integrity and prevent the dye from intercalating into DNA base pairs. However, all 9 antiproliferative compounds induced DNA cleavage at 24 h in L1210 cells, containing 3H-thymidine-prelabeled DNA, suggesting that these antitumor annulated indoles might trigger an apoptotic pathway of DNA fragmentation. Indeed the antiproliferative annulated indoles caused a time-dependent increase of caspase-3 activity with a peak at 6 h. Interestingly, the compounds with the most potent antiproliferative IC50 values at day 2 were consistently the most effective at inhibiting DNA synthesis at 3 h and inducing DNA fragmentation at 24 h. After 24–48 h, antiproliferative concentrations of annulated indoles increased the mitotic index of L1210 cells and stimulated the formation of many bi-nucleated cells, multi-nucleated cells, apoptotic cells and micronuclei, suggesting that these antitumor compounds might enhance mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis to induce apoptosis.
Although annulated indoles may have interesting bioactivity, novel derivatives with different substitutions must be synthesized to elucidate structure-activity relationships, identify more potent antitumor lead compounds, and investigate their molecular targets and mechanisms of action.
PMCID: PMC4175989  PMID: 23155229
Annulated indoles; tumor cell proliferation; DNA synthesis; interaction and fragmentation; cells with mitotic figures; several nuclei and micronuclei; cytokinesis; apoptosis
24.  Undetectable role of oxidative DNA damage in cell cycle, cytotoxic and clastogenic effects of Cr(VI) in human lung cells with restored ascorbate levels 
Mutagenesis  2012;27(4):437-443.
Cultured human cells are invaluable biological models for mechanistic studies of genotoxic chemicals and drugs. Continuing replacement of animals in toxicity testing will further increase the importance of in vitro cell systems, which should accurately reproduce key in vivo characteristics of toxicants such as their profiles of metabolites and DNA lesions. In this work, we examined how a common severe deficiency of cultured cells in ascorbate (Asc) impacts the formation of oxidative DNA damage by hexavalent chromium (chromate). Cr(VI) is reductively activated inside the cells by both Asc and small thiols but with different rates and spectra of intermediates and DNA adducts. We found that Cr(VI) exposure of H460 human lung epithelial cells in standard culture (<0.01 mM cellular Asc) induced biologically significant amounts of oxidative DNA damage. Inhibition of oxidative damage repair in these cells by stable XRCC1 knockdown strongly enhanced cytotoxic effects of Cr(VI) and led to depletion of cells from G1 and accumulation in S and G2 phases. However, restoration of physiological levels of Asc (∼1 mM) completely eliminated Cr(VI) hypersensitivity of XRCC1 knockdown. The induction of chromosomal breaks assayed by the micronucleus test in Asc-restored H460, primary human lung fibroblasts, and CHO cells was also unaffected by the XRCC1 status. Centromere-negative (clastogenic) micronuclei accounted for 80–90% of all Cr(VI)-induced micronuclei. Consistent with the micronuclei results, Asc-restored cells also showed no increase in the levels of poly(ADP-ribose), which is a biochemical marker of single-stranded breaks. Asc had no effect on cytotoxicity of O6-methylguanine, a lesion produced by direct DNA alkylation. Overall, our results indicate that the presence of physiological levels of Asc strongly suppresses pro-oxidant pathways in Cr(VI) metabolism and that the use of standard cell cultures creates a distorted profile of its genotoxic properties.
PMCID: PMC3382305  PMID: 22241526
25.  The application of the cytokinesis-block micronucleus assay on peripheral blood lymphocytes for the assessment of genome damage in long-term residents of areas with high radon concentration 
Journal of Radiation Research  2013;55(1):61-66.
Estimating the effects of small doses of ionising radiation on DNA is one of the most important problems in modern biology. Different cytogenetic methods exist to analyse DNA damage; the cytokinesis-block micronucleus assay (CBMN) for human peripheral blood lymphocytes is a simple, cheap and informative cytogenetic method that can be used to detect genotoxic-related markers. With respect to previous studies on radiation-induced genotoxicity, children are a poorly studied group, as evidenced by the few publications in this area. In this study, we assessed radon genotoxic effects by counting micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in the lymphocytes of children who are long-term residents from areas with high radon concentrations. In the exposed group, radon was found to cause significant cytogenetic alterations. We propose that this method can be employed for biomonitoring to screen for a variety of measures.
PMCID: PMC3885124  PMID: 23908554
micronucleus assay; micronuclei; genotoxicity; cytochalasin B; ionising radiation; radon; children

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