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.
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.
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.
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.
Zinc is an essential trace metal ion for growth, but an excess of Zn is toxic and microorganisms express diverse resistance mechanisms. To understand global bacterial responses to excess Zn, we conducted transcriptome profiling experiments comparing Escherichia coli MG1655 grown under control conditions and cells grown with a toxic, sublethal ZnSO4 concentration (0.2 mM). Cultures were grown in a defined medium lacking inorganic phosphate, permitting maximum Zn bioavailability, and in glycerol-limited chemostats at a constant growth rate and pH. Sixty-four genes were significantly up-regulated by Zn stress, including genes known to be involved in Zn tolerance, particularly zntA, zraP, and hydG. Microarray transcriptome profiling was confirmed by real-time PCR determinations of cusF (involved in Ag and Cu efflux), ais (an Al-inducible gene), asr (encoding an acid shock-inducible periplasmic protein), cpxP (a periplasmic chaperone gene), and basR. Five up-regulated genes, basR and basS [encoding a sensor-regulator implicated in Salmonella in Fe(III) sensing and antibiotic resistance], fliM (flagellar synthesis), and ycdM and yibD (both with unknown functions), are important for growth resistance to zinc, since mutants with mutations in these genes exhibited zinc sensitivity in liquid media and on metal gradient plates. Fifty-eight genes were significantly down-regulated by Zn stress; notably, several of these genes were involved in protection against acid stress. Since the mdt operon (encoding a multidrug resistance pump) was also up-regulated, these findings have important implications for understanding not only Zn homeostasis but also how bacterial antibiotic resistance is modulated by metal ions.
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.
Protease inhibitors; epithelial cells; viability; HAART
A modified zinc sulfate flotation technique using Formalinized fecal specimens (F-ZnSO4) was compared to the Formalin-ether (FE) concentration method for the laboratory diagnosis of intestinal parasites. Many laboratories have difficulty storing, using, and disposing of either and need a procedure for concentrating fecal specimens which does not require ether. Comparative data were obtained for the recovery of protozoan cysts and helminth eggs and larvae from feces preserved in Formalin less than and longer than 1 month. Whereas the FE method was found generally to be more efficient, F-ZnSO4 was apparently more effective for the recovery of some species of parasites. F-ZnSO4 was not satisfactory for recovery of schistosome eggs. We conclude that, except for schistosomes, F-ZnSO4 compares favorably to the FE method for detecting infections of clinical significance.
We studied the contribution of the main olfactory system to mate recognition and sexual behavior in female mice. Female mice received an intranasal irrigation of either a zinc sulfate (ZnSO4) solution to destroy the main olfactory epithelium (MOE) or saline (SAL) to serve as control. ZnSO4-treated female mice were no longer able to reliably distinguish between volatile as well as nonvolatile odors from an intact versus a castrated male. Furthermore, sexual behavior in mating tests with a sexually experienced male was significantly reduced in ZnSO4-treated female mice. Vomeronasal function did not seem to be affected by ZnSO4 treatment: nasal application of male urine induced similar levels of Fos protein in the mitral and granule cells of the accessory olfactory bulb (AOB) of ZnSO4 as well as SAL-treated female mice. Likewise, soybean agglutinin staining, which stains the axons of vomeronasal neurons projecting to the glomerular layer of the AOB was similar in ZnSO4-treated female mice compared to SAL-treated female mice. By contrast, a significant reduction of Fos in the main olfactory bulb was observed in ZnSO4-treated females in comparison to SAL-treated animals, confirming a substantial destruction of the MOE. These results show that the MOE is primarily involved in the detection and processing of odors that are used to localize and identify the sex and endocrine status of conspecifics. By contrast, both the main and accessory olfactory systems contribute to female sexual receptivity in female mice.
main olfactory system; mice; sexual behavior; sex recognition; zinc sulfate
Zinc is an essential cofactor for the activity and folding of up to ten percent of mammalian proteins and can modulate the function of many others. Because of the pleiotropic effects of zinc on every aspect of cell physiology, deficits of cellular zinc content, resulting from zinc deficiency or excessive rise in its cellular concentration, can have catastrophic consequences and are linked to major patho-physiologies including diabetes and stroke. Thus, the concentration of cellular zinc requires establishment of discrete, active cellular gradients. The cellular distribution of zinc into organelles is precisely managed to provide the zinc concentration required by each cell compartment. The complexity of zinc homeostasis is reflected by the surprisingly large variety and number of zinc homeostatic proteins found in virtually every cell compartment. Given their ubiquity and importance, it is surprising that many aspects of the function, regulation, and crosstalk by which zinc transporters operate are poorly understood. In this mini-review, we will focus on the mechanisms and players required for generating physiologically appropriate zinc gradients across the plasma membrane and vesicular compartments. We will also highlight some of the unsolved issues regarding their role in cellular zinc homeostasis.
Historically most residential wood preservatives were aqueous soluble metal formulations, but recently metals ground to submicron size and dispersed in water to give particulate formulations have gained importance. In this study, the specific role nano-zinc oxide (ZnO) particle size and leach resistance plays in termite mortality resulting from exposure to particulate ZnO-treated wood was investigated. Southern yellow pine (SYP) sapwood impregnated with three concentrations of two particle sizes (30 and 70 nm) of ZnO were compared to wood treated with soluble zinc sulphate (ZnSO4) preservative for leach resistance and termite resistance. Less than four percent leached from the particulate nano-ZnO-treated specimens, while 13 to 25% of the zinc sulphate leached from the soluble treated wood. Nano-ZnO was essentially non-leachable from wood treated with 5% formulation for the 30-nm particle size. In a no-choice laboratory test, eastern subterranean termites (Reticulitermes flavipes) consumed less than 10% of the leached nano-ZnO-treated wood with 93 to 100% mortality in all treatment concentrations. In contrast, termites consumed 10 to 12% of the leached ZnSO4-treated wood, but with lower mortality: 29% in the 1% treatment group and less than 10% (5 and 8%, respectively) in the group of wood blocks treated with 2.5 and 5.0% ZnSO4. We conclude that termites were repelled from consuming wood treated with nano-ZnO, but when consumed it was more toxic to eastern subterranean termites than wood treated with the soluble metal oxide formulation. There were no differences in leaching or termite mortality between the two particle sizes of nano-ZnO.
Polaprezinc, a chelate compound consisting of zinc and l-carnosine, is clinically used as a medicine for gastric ulcers. It has been shown that induction of heat shock protein (HSP) is involved in protective effects of polaprezinc against gastric mucosal injury. In the present study, we investigated whether polaprezinc and its components could induce HSP70 and prevent acetaminophen (APAP) toxicity in mouse primary cultured hepatocytes. Hepatocytes were treated with polaprezinc, zinc sulfate or l-carnosine at the concentration of 100 µM for 9 h, and then exposed to 10 mM APAP. Polaprezinc or zinc sulfate increased cellular HSP70 expression. However, l-carnosine had no influence on it. Pretreatment of the cells with polaprezinc or zinc sulfate significantly suppressed cell death as well as cellular lipid peroxidation after APAP treatment. In contrast, pretreatment with polaprezinc did not affect decrease in intracellular glutathione after APAP. Furthermore, treatment with KNK437, an HSP inhibitor, attenuated increase in HSP70 expression induced by polaprezinc, and abolished protective effect of polaprezinc on cell death after APAP. These results suggested that polaprezinc, in particular its zinc component, induces HSP70 expression in mouse primary cultured hepatocytes, and inhibits lipid peroxidation after APAP treatment, resulting in protection against APAP toxicity.
acetaminophen; hepatotoxicity; zinc; polaprezinc; heat shock protein
Human matrix metalloproteinase-8 (hMMP-8) plays a important role in the progression of colorectal cancer, metastasis, multiple
sclerosis and rheumetoid arthritis. Extensive MD-simulation of the PDB and solvated structures of hMMP-8 has revealed the
presence of few conserved water molecules around the catalytic and structural zinc (ZnC and ZnS) ions. The coordination of two
conserved water molecules (W and WS) to ZnS and the H-bonding interaction of WS to S151 have indicated the plausible involvement
of that metal ion in the catalytic process. Beside this the coupling of ZnC and ZnS metal ions (ZnC – WH (W1)…..W2 ….H162 - ZnS)
through two conserved hydrophilic centers (occupied by water molecules) may also provide some rational on the recognition of
two zinc ions which were separated by ~13 Å in their X-ray structures. This unique recognition of both the Zn+2 ions in the enzyme
through conserved water molecules may be implemented/ exploited for the design of antiproteolytic agent using water mimic
drug design protocol.
Matrix Metalloproteinase; MD simulation; Zn ions; Catalytic mechanism
D-myo-inositol-1,2,6-triphosphate (α-trinositol, AT) is a polyanionic molecule capable of chelating divalent metal ions with anti-tumour and anti-cachectic activity in a murine model.
To investigate the role of zinc in this process, mice bearing cachexia-inducing MAC16 tumour were treated with AT, with or without concomitant administration of ZnSO4.
At a dose of 40 mg kg−1, AT effectively attenuated both weight loss and growth of the MAC16 tumour, and both effects were attenuated by co-administration of Zn2+. The concentration of zinc in gastrocnemius muscle increased with increasing weight loss, whereas administration of AT decreased the levels of zinc in plasma, skeletal muscle and tumour, which were restored back to control values after administration of ZnSO4.
These results suggest that zinc is important in both tumour growth and cachexia in this animal model.
cachexia; tumour growth; zinc; α-trinositol
Human diploid fibroblasts growth normally in medium containing physiological concentrations of the naturally occurring dipeptide carnosine (beta-alanyl-L-histidine). These concentrations are cytotoxic to transformed and neoplastic cells lines in modified Eagle medium (MEM), whereas these cells grow vigorously in Dulbecco's modified Eagle medium (DMEM) containing carnosine. This difference is due to the presence of 1 mM sodium pyruvate in DMEM. Seven human cell lines and two rodent cell lines were tested and all are strongly inhibited by carnosine in the absence of pyruvate. Experiments with HeLa cells show that anserine is similar to carnosine, but D-carnosine and homocarnosine are without effect. Also, the non-essential amino acids alanine and glutamic acid contribute to the effect of pyruvate in preventing carnosine toxicity, and oxaloacetate and alpha-ketoglutarate can substitute for pyruvate. We have used mixtures of normal MRC-5 fibroblasts and HeLa cells to demonstrate that 20 mM carnosine can selectively eliminate the tumour cells. This has obvious implications which might be exploited in in vivo and in vitro studies. Carnosine is known to react strongly with aldehyde and keto groups of sugars by Amadori reaction, and we propose that it depletes certain glycolysis intermediates. It is well known that tumour cells are more dependent on glycolysis than normal cells. A reduction of glycolysis intermediates by carnosine may deplete their energy supply, but this effect is totally reversed by pyruvate.
A large body of evidence supports an opinion that adequate dietary zinc is essential for prenatal and postnatal brain development. Behavioural effects of maternal supplementation with ZnSO4 were analysed in rat pups with the Morris water task performance, a hole board and a T-maze. Wistar females during pregnancy and lactation received a drinking water solution of ZnSO4 at doses of 16 mg/kg (group Zn16) or 32 mg/kg (group Zn32). Behavioural tests were conducted on the 4-week-old male rat pups. Zinc concentration in the serum, hippocampus and prefrontal cortex of offsprings was determined by means of atomic absorption techniques. The Newman–Keuls multiple comparison test revealed an increase of climbing in the Zn16 group in comparison to the control group (Con) and the Zn32 group during the hole board test. ANOVA for repeated measures showed a significant memory improvement in both supplemented groups compared to the control in the probe trial on day 5 of the water maze test. ZnSO4 treatment significantly elevated zinc levels in the rat serum. Follow-up data on brain content of zinc in the hippocampus revealed significant differences between the groups and in supplemented groups correlated with crossings above the original platform position. These findings suggest that pre- and postnatal zinc supplementation may improve cognitive development in rats.
Zinc; Zinc supplementation; Spatial memory; Behavioural tests; Behaviour; Rat
Included among the quantitative high throughput screens (qHTS) conducted in support of the U.S. Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in 7 isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis.
DT40 DNA repair-deficient cell lines; quantitative high throughput screens; cytotoxicity; genotoxicity; chromosomal aberrations; γH2AX positive foci
Herpes genitalis is one of the commonest sexually transmitted infections in the world caused by herpes simplex virus (HSV) 1 and 2. All herpes viruses show latency. Herpes genitalis caused by HSV-1 and HSV-2 is recurrent in 55 and 90% of case respectively.
To comparatively evaluate the therapeutic efficacy of topical zinc sulfate (ZnSO4) in varying concentrations in herpes genitalis.
Materials and Methods:
Ninety patients clinically diagnosed as herpes genitalis and confirmed by Tzanck test were taken up for study and divided into 3 groups of 30 patients each which were applied topical ZnSO4 in concentrations of 1%, 2% and 4% respectively for 3 months. Ten patients of herpes genitalis were kept as control in whom only distilled water was applied. Patients were followed up for a total period of 6 months for any recurrences.
Ten patients of group 1 (1% ZnSO4) showed recurrence, 6 patients in group 2 (2% ZnSO4) and only one patient in group 3 (4% ZnSO4) showed recurrence. In the control group, 8 out of 10 patients showed recurrence. No serious side effects were seen in all 3 groups.
Topical ZnSO4 has been found to be an effective therapeutic modality not only for treatment but also for prolonging remissions in herpes genitalis. Topical 4% ZnSO4 has been found to be most efficacious out of the three concentrations, without any side effects.
Herpes genitalis; new approach; zinc sulfate
Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not. The growth of Chlamydia psittaci was strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+ or Zn2+. The inhibition correlated with the level of INDO activity induced and could be reversed by the addition of excess tryptophan to the medium. The growth of Toxoplasma gondii was also strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+. Expression of Cd(2+)-induced INDO activity also inhibited thymidine incorporation and led to cytotoxicity in INDO +ve cells but not in INDO -ve cells. Thus, the induction of INDO activity by IFN-gamma may be an important factor in the antimicrobial and antiproliferative effects of IFN-gamma in human fibroblasts.
β-alanine is an amino acid that, when combined with histidine, forms the dipeptide carnosine within skeletal muscle. Carnosine and β-alanine each have multiple purposes within the human body; this review focuses on their roles as ergogenic aids to exercise performance and suggests how to best quantify the former’s merits as a buffer. Carnosine normally makes a small contribution to a cell’s total buffer capacity; yet β-alanine supplementation raises intracellular carnosine concentrations that in turn improve a muscle’s ability to buffer protons. Numerous studies assessed the impact of oral β-alanine intake on muscle carnosine levels and exercise performance. β-alanine may best act as an ergogenic aid when metabolic acidosis is the primary factor for compromised exercise performance. Blood lactate kinetics, whereby the concentration of the metabolite is measured as it enters and leaves the vasculature over time, affords the best opportunity to assess the merits of β-alanine supplementation’s ergogenic effect. Optimal β-alanine dosages have not been determined for persons of different ages, genders and nutritional/health conditions. Doses as high as 6.4 g day−1, for ten weeks have been administered to healthy subjects. Paraesthesia is to date the only side effect from oral β-alanine ingestion. The severity and duration of paraesthesia episodes are dose-dependent. It may be unwise for persons with a history of paraesthesia to ingest β-alanine. As for any supplement, caution should be exercised with β-alanine supplementation.
carnosine; dietary supplement; amino acid; paraesthesia
Abnormal zinc homeostasis is involved in β-amyloid (Aβ) plaque formation and, therefore, the zinc load is a contributing factor in Alzheimer's disease (AD). However, the involvement of zinc in amyloid precursor protein (APP) processing and Aβ deposition has not been well established in AD animal models in vivo.
In the present study, APP and presenilin 1 (PS1) double transgenic mice were treated with a high dose of zinc (20 mg/ml ZnSO4 in drinking water). This zinc treatment increased APP expression, enhanced amyloidogenic APP cleavage and Aβ deposition, and impaired spatial learning and memory in the transgenic mice. We further examined the effects of zinc overload on APP processing in SHSY-5Y cells overexpressing human APPsw. The zinc enhancement of APP expression and cleavage was further confirmed in vitro.
The present data indicate that excess zinc exposure could be a risk factor for AD pathological processes, and alteration of zinc homeostasis is a potential strategy for the prevention and treatment of AD.
Fanconi anemia (FA) is a recessive genome instability syndrome characterized by heightened cellular sensitivity to DNA damage, aplastic anemia and cancer susceptibility. Leukemias and squamous cell carcinomas (SCC) are the most predominant FA associated cancers, with the latter exhibiting markedly early disease onset and aggressiveness. While studies of hematopoietic cells derived from FA patients have provided much insight into bone marrow deficiencies and leukemogenesis, molecular transforming events in FA deficient keratinocytes, which are the cell type of origin for SCC, are poorly understood. We describe here the growth and molecular properties of FANCA-deficient versus FANCA-corrected, HPV E6/E7 immortalized keratinocytes in monolayer and organotypic epithelial raft culture. In response to DNA damage, FANCA-deficient patient-derived keratinocyte cultures displayed a G2/M phase arrest, senescence and apoptosis. Organotypic raft cultures exhibited DNA repair associated defects with more 53BP1 foci and TUNEL positive cells over their corrected counterparts. Interestingly, together with reduced rates of DNA damage, FA correction resulted in a marked decrease in epithelial thickness and the presence of fewer cell layers. The observed FANCA mediated suppression of hyperplasia correlated with the detection of fewer cells transiting through the cell cycle in the absence of gross differentiation abnormalities or apoptotic differences. Importantly, the knockdown of either FANCA or FANCD2 in HPV positive keratinocytes was sufficient for increasing epithelial hyperplasia. Our findings support a new role for FA pathways in the maintenance of differentiation-dependent cell cycle exit, with the implication that FA deficiencies may contribute to the high risk of FA patients for developing HPV-associated SCC.
Fanconi anemia; human papillomavirus
Inorganic arsenic is a known environmental toxicant and carcinogen of global public health concern. Arsenic is genotoxic and cytotoxic to human keratinocytes. However, the biological pathways perturbed in keratinocytes by low chronic dose inorganic arsenic are not completely understood. The objective of the investigation was to discover the mechanism of arsenic carcinogenicity in human epidermal keratinocytes. We hypothesize that a combined strategy of DNA microarray, qRT-PCR and gene function annotation will identify aberrantly expressed genes in HaCaT keratinocyte cell line after chronic treatment with arsenic trioxide. Microarray data analysis identified 14 up-regulated genes and 21 down-regulated genes in response to arsenic trioxide. The expression of 4 up-regulated genes and 1 down-regulated gene were confirmed by qRT-PCR. The up-regulated genes were AKR1C3 (Aldo-Keto Reductase family 1, member C3), IGFL1 (Insulin Growth Factor-Like family member 1), IL1R2 (Interleukin 1 Receptor, type 2), and TNFSF18 (Tumor Necrosis Factor [ligand] SuperFamily, member 18) and down-regulated gene was RGS2 (Regulator of G-protein Signaling 2). The observed over expression of TNFSF18 (167 fold) coupled with moderate expression of IGFL1 (3.1 fold), IL1R2 (5.9 fold) and AKR1C3 (9.2 fold) with a decreased RGS2 (2.0 fold) suggests that chronic arsenic exposure could produce sustained levels of TNF with modulation by an IL-1 analogue resulting in chronic immunologic insult. A concomitant decrease in growth inhibiting gene (RGS2) and increase in AKR1C3 may contribute to chronic inflammation leading to metaplasia, which may eventually lead to carcinogenicity in the skin keratinocytes. Also, increased expression of IGFL1 may trigger cancer development and progression in HaCaT keratinocytes.
arsenic trioxide; chronic exposure; HaCaT cell; keratinocytes; anti-apoptosis; anti-differentiation
Zinc is involved in virtually all aspects of cellular and molecular biology as a catalytic, structural, and regulatory cofactor in over 1000 proteins. Zinc binding to proteins requires an adequate supply of zinc and intact molecular mechanisms for redistributing zinc ions to make them available at the right time and location. Several dozen gene products participate in this process, in which interactions between zinc and sulfur donors determine the mobility of zinc and establish coupling between cellular redox state and zinc availability. Specifically, the redox properties of metallothionein and its apoprotein thionein are critical for buffering zinc ions and for controlling fluctuations in the range of picomolar concentrations of “free” zinc ions in cellular signaling. Metallothionein and other proteins with sulfur coordination environments are sensitive to redox perturbations and can render cells susceptible to injury when oxidative stress compromises the cellular redox and zinc buffering capacity in chronic diseases. The implications of these fundamental principles for zinc metabolism in type 2 diabetes are briefly discussed.
Tumor necrosis factor (TNF) is a proinflammatory cytokine, which is centrally involved in several inflammatory disorders. Administration of TNF leads to a potentially lethal systemic inflammatory response syndrome (SIRS). We observed that (a) mice lacking functional genes for metallothionein 1 and 2 (MT-null) were protected compared with wild-type controls (P = 0.0078), and (b) mice overexpressing MT-1 (MT-TG) were more sensitized for the lethal effect of TNF than control mice (P = 0.0003), indicating a mediating role for MT in TNF induced SIRS. As MT is involved in the body zinc homeostasis, we tested whether zinc-deprivation or -supplementation alters the response to TNF. Although zinc-depletion strongly sensitized (P = 0.036), and pretreatment with zinc sulfate (ZnSO4) conferred protection against the deleterious effects of TNF (P < 0.0002), it was also found that the protection provided by zinc is independent of MT. Our observation that hsp70 is strongly induced in jejunum after ZnSO4 treatment, suggests a contribution of hsp70 in the protection against TNF. In addition, ZnSO4 cotreatment allowed complete regression of inoculated tumors with TNF and interferon γ, leading to a significantly better survival (P = 0.0045).
systemic inflammatory response syndrome; zinc; acute-phase reaction; interferon; leptin
1,4-Benzoquinone is cytotoxic in V79 Chinese hamster cells and induces gene mutations and micronuclei. The cell-damaging effects of quinones are usually attributed to thiol depletion, oxidation of NAD(P)H, and redox-cycling involving the formation of semiquinone radicals and reactive oxygen species. To elucidate the role of these mechanisms in the genotoxicity of 1,4-benzoquinone, we measured various genotoxic effects, cytotoxicity, and the levels of glutathione, NADPH, NADH, and their oxidized forms all in the same experiment. 1,4-Naphthoquinone, which does not induce gene mutations in V79 cells, was investigated for comparative reasons. The quinones had a similar effect on the levels of cofactors. Total glutathione was depleted, but levels of oxidized glutathione were slightly increased. The levels of NADPH and NADH were reduced at high concentrations of the quinones with a simultaneous increase in the levels of NADP+ and NAD+. Both compounds induced micronuclei, but neither increased the frequency of sister chromatid exchange. Only 1,4-benzoquinone induced gene mutations. This effect was observed at low concentrations, where none of the other parameters studied was affected. When the cells were depleted of glutathione prior to treatment with the quinones, the induction of gene mutations and micronuclei remained virtually unchanged. We conclude that a) induction of micronuclei and glutathione depletion by the two quinones are not linked causally, b) 1,4-benzoquinone induces gene mutations by a mechanism different from oxidative stress and glutathione depletion, and c) glutathione does not fully protect the cells against the genotoxicity of quinones.
This review critically summarizes the literature on the spectrum of health effects of zinc status, ranging from symptoms of zinc deficiency to excess exposure. Studies on zinc intake are reviewed in relation to optimum requirements as a function of age and sex. Current knowledge on the biochemical properties of zinc which are critical to the essential role of this metal in biological systems is summarized. Dietary and physiological factors influencing the bioavailability and utilization of zinc are considered with special attention to interactions with iron and copper status. The effects of zinc deficiency and toxicity are reviewed with respect to specific organs, immunological and reproductive function, and genotoxicity and carcinogenicity. Finally, key questions are identified where research is needed, such as the risks to human health of altered environmental distribution of zinc, assessment of zinc status in humans, effects of zinc status in relation to other essential metals on immune function, reproduction, neurological function, and the cardiovascular system, and mechanistic studies to further elucidate the biological effects of zinc at the molecular level.