Fluconazole induces genotoxicity in cultured human peripheral blood mononuclear cells via immunomodulation of TNF-α, IL-6, and IL-10: new challenges for safe therapeutic regimens

Context: Fluconazole (FNZ) is a drug used in antifungal therapy. However, the minimum FNZ dose to interfering with immune responses or inducing DNA damage is still unknown.

Objective: This study investigated the toxicological profile of FNZ on cultured human peripheral blood mononuclear cells (PBMCs) treated with different concentrations of this azole.

Materials and methods: Cultured PBMCs were exposed to FNZ (6, 12, 30, 60 and 120?μg/mL) and the toxicological profile was assessed by the following parameters: cytotoxic and nuclear division index (necrotic, apoptotic and viable cells), DNA damage (alkaline comet test), mutagenic potential (micronucleus test), cytokine modulation (IL-1, IL-6, IL-10, TNF-α, IFN-γ), and predictive toxicity (Osiris^® and LAZAR^® programs).

Results: Our results demonstrated that FNZ induced cellular DNA damage and mutagenicity at concentrations above the plasma peak (>30?μg/mL) and 6?μg/mL, respectively, which was associated with increased TNF-α, and decrease IL-6 and IL-10 concentrations. These effects may be related to increased apoptosis and cytotoxic nuclear division index in the cultured PBMCs. In silico results indicated potential mutagenic, tumorigenic, irritant, and carcinogenic effects, which were partially confirmed by the above assays.

Discussion and conclusions: Together, these findings suggest the need to rationalize the use of FNZ, especially if it is used for long periods or with concomitant pathologies requiring azole therapy that may increase FNZ's plasma concentration.     

Evaluation of genome damage and its relation to oxidative stress induced by glyphosate in human lymphocytes in vitro

In the present study we evaluated the genotoxic and oxidative potential of glyphosate on human lymphocytes at concentrations likely to be encountered in residential and occupational exposure. Testing was done with and without metabolic activation (S9). Ferric‐reducing ability of plasma (FRAP), thiobarbituric acid reactive substances (TBARS) and the hOGG1 modified comet assay were used to measure glyphosate's oxidative potential and its impact on DNA. Genotoxicity was evaluated by alkaline comet and analysis of micronuclei and other nuclear instabilities applying centromere probes. The alkaline comet assay showed significantly increased tail length (20.39 μm) and intensity (2.19%) for 580 μg/ml, and increased tail intensity (1.88%) at 92.8 μg/ml, compared to control values of 18.15 μm for tail length and 1.14% for tail intensity. With S9, tail length was significantly increased for all concentrations tested: 3.5, 92.8, and 580 μg/ml. Using the hOGG1 comet assay, a significant increase in tail intensity was observed at 2.91 μg/ml with S9 and 580 μg/ml without S9. Without S9, the frequency of micronuclei, nuclear buds and nucleoplasmic bridges slightly increased at concentrations 3.5 μg/ml and higher. The presence of S9 significantly elevated the frequency of nuclear instabilities only for 580 μg/ml. FRAP values slightly increased only at 580 μg/ml regardless of metabolic activation, while TBARS values increased significantly. Since for any of the assays applied, no clear dose‐dependent effect was observed, it indicates that glyphosate in concentrations relevant to human exposure do not pose significant health risk. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

牡蛎壳的生物相容性评价：细胞毒性和遗传毒性研究

目的：探讨天然牡蛎壳粉末的生物相容性,为其进一步在临床上作为牙齿根管充填材料的研究提供理论依据。方法：按照我国医药行业对口腔材料生物学评价要求,制备牡蛎壳粉末的浸提液。分别采用分子滤过法和鼠伤寒沙门氏菌回复突变试验（Ames试验）的平板掺入法,对牡蛎壳粉末浸提液进行细胞毒性和遗传毒性检测。用测量滤膜褪色面积和计数平皿中细菌回变菌落数的方法判断该材料的毒性。结果：分子滤过法2h实验结果显示细胞毒性为0级,24h试验结果细胞毒性为1级。Ames各试验组菌落回变数均未高于相应菌株自发回变菌落数的2倍,且无剂量反应关系,实验结果为阴性。结论：牡蛎壳粉末具有较好的细胞生物相容性,致突变试验为阴性,对机体没有潜在的遗传毒性作用,符合口腔材料的临床前生物学要求。     

Effects of Bisphenol A on Foxl2 gene expression and DNA damage in adult viviparous fish Goodea atripinnis

ABSTRACT

Bisphenol A (BPA) is an emerging pollutant of global concern. Viviparous fish Goodea atripinnis is endemic to the Central Mexican Plateau where BPA was detected; however, few studies examined the influence of this chemical on native viviparous fish. The effects of BPA (sublethal dose) were determined on DNA integrity and Foxl2 expression in G. atripinnis gonads, and interactions of BPA with FOXL2 protein. Genotoxicity analysis revealed that % comets, at 14 and 28 days and comet tail length (at 14 days) were significantly higher in exposed compared to controls. In general, the % DNA tail was not markedly higher in BPA-treated fish; however, tail moment related to tail length exhibited significant increases in DNA damage. RT-qPCR assays showed Foxl2 overexpression after 14 and 28 days of exposure in females; while in males, Foxl2 was overexpressed after 28 days. In silico analysis demonstrated that BPA interacted with seven residues located in FOXL2 homeodomain. In summary, sublethal BPA doses induced DNA damage and changes in Foxl2 expression in gonadal cells of G. atripinnis, which may adversely affect reproduction in BPA-exposed wild populations. Foxl2 overexpression and BPA-FOXL2 interaction suggested alterations in processes involving Foxl2. Viviparous fish may thus serve as potential non-conventional models for assessing pollutants effects.     

Nanoscale zerovalent iron particles induce differential cytotoxicity,genotoxicity, oxidative stress and hemolytic responses in human lymphocytes and erythrocytes in vitro

The growing usage of nanoscale zerovalent iron particles (nZVI) in the remediation of soil, ground/surface water has elicited large‐scale environmental release triggering human exposure. The size of nanomaterials is a key regulator of toxicity. However, the effect of a variable size of nZVI on genotoxicity is unexplored in human cells. To the best of our knowledge, in this study, the cytotoxic, genotoxic and hemolytic potential of nZVI‐1 (15 nm) and nZVI‐2 (50 nm) at concentrations of 5, 10 and 20 μg/mL was evaluated for the first time in human lymphocytes and erythrocytes treated for 3 hours. In erythrocytes, spherocytosis and echinocytosis occurred upon exposure to nZVI‐1 and nZVI‐2, respectively, leading to hemolysis. Lymphocytes treated with 20 μg/mL nZVI‐2 and 10 μg/mL nZVI‐1, incurred maximum DNA damage, although nZVI‐2 induced higher cyto‐genotoxicity than nZVI‐1. This can be attributed to higher Fe ion dissolution and time/concentration‐dependent colloidal destabilization (lower zeta potential) of nZVI‐2. Although nZVI‐1 showed higher uptake, its lower genotoxicity can be due to lesser Fe content, Fe ion dissolution and superior colloidal stability (higher zeta potential) compared with nZVI‐2. Substantial accumulation of Ca²⁺, superoxide anions, hydroxyl radicals and H₂O₂ leading to mitochondrial impairment and altered antioxidant enzyme activity was noted at the same concentrations. Pre‐treatment with N‐acetyl‐cysteine modulated these parameters indicating the indirect action of reactive oxygen species in nZVI‐induced DNA damage. The morphology of diffused nuclei implied the possible onset of apoptotic cell death. These results validate the synergistic role of size, ion dissolution, colloidal stability and reactive oxygen species on cyto‐genotoxicity of nZVI and unlock further prospects in its environmental nano‐safety evaluation.     

Induction of hepatocellular carcinoma by in vivo gene targeting

The distinct phenotypic and prognostic subclasses of human hepatocellular carcinoma (HCC) are difficult to reproduce in animal experiments. Here we have used in vivo gene targeting to insert an enhancer-promoter element at an imprinted chromosome 12 locus in mice, thereby converting ～1 in 20,000 normal hepatocytes into a focus of HCC with a single genetic modification. A 300-kb chromosomal domain containing multiple mRNAs, snoRNAs, and microRNAs was activated surrounding the integration site. An identical domain was activated at the syntenic locus in a specific molecular subclass of spontaneous human HCCs with a similar histological phenotype, which was associated with partial loss of DNA methylation. These findings demonstrate the accuracy of in vivo gene targeting in modeling human cancer and suggest future applications in studying various tumors in diverse animal species. In addition, similar insertion events produced by randomly integrating vectors could be a concern for liver-directed human gene therapy.     

福州市生活饮用水及其水源水有机提取物的体内遗传毒性研究

目的：探讨福州市生活饮用水及其水源水的体内遗传毒性。方法：随机选择福州市5个区县（市）各1家水厂,采集水源水和出厂水各200 L,经XAD-2树脂柱收集其中的有机物,采用小鼠骨髓细胞微核试验和精子畸形试验检测其遗传毒性。结果：长乐市、平潭县水厂出厂水和水源水及福清市水厂出厂水具有致突变性;各出厂水有机提取物的致突变性基本高于同厂水源水。结论：福州市部分生活饮用水水源水和出厂水受到一定程度的有机物污染。     

Evaluation of DNA Damage Induction and Repair Inhibition in Welders Exposed to Hexavalent Chromium

The soluble hexavalent chromium Cr (VI) used in industrial welding is an environmental contaminant widelyrecognized to act as a carcinogen, mutagen and teratogen towards humans and animals. The carcinogenic potentialof metals is a major issue in defining human health risk from exposure. In the present investigation, 93 weldersand 60control subjects with similar mean ages, smoking prevalences and alcohol consumption were enrolled forDNA damage analysis in blood leucocytes by Micronucleus assay (MN) and the Comet assay. DNA repair inhibitionwas also analyzed by assessing XPD gene polymorphism. Welders showed a significant increase in micronucleatedcells compared to controls with respect to their smoking habits and alcohol consumption, age and years ofexposure (P<0.05). Results indicated that the welders had a larger mean comet tail length than that of thecontrols (P<0.05). The current study suggested that chronic occupational exposure to Cr (VI) during weldingcould lead to increased levels of DNA damage and repair inhibition.     

DNA Damage Induction and Repair Inhibition Among Building Construction Workers in South India

Construction industry workers are exposed to many known carcinogens in their complex occupational environment. Since there are no past studies on genotoxicity among this group in the Indian subcontinent, workers engaged in different construction sites at Coimbatore, Tamil Nadu, India, were assessed here. We enrolled 96 workers and 68 control subjects with similar mean age, smoking, tobacco chewing prevalence and alcohol consumption, for analysis of DNA damage in blood leucocytes by micronucleus (MN) and comet assays. DNA repair inhibition was also analyzed by assessing the XPD gene. Construction workers showed a significant increase in MN and comet tail length compared to controls with adjustment for smoking habits, tobacco chewing, alcohol consumption and years of exposure (P<0.05). The results indicated that chronic occupational exposure to cement during construction work could lead to increased levels of DNA damage and repair inhibition.     

Assessment of genotoxicity of waterpipe and cigarette smoking in lymphocytes using the sister‐chromatid exchange assay: A comparative study

Tobacco smoking is a major world health problem. Recently, waterpipe smoking has become more popular in many countries. Although the genotoxicity associated with cigarette smoking has been extensively investigated, studies evaluating such toxicity in waterpipe users are still lacking. In this study, we examined the genotoxicity of waterpipe smoking in lymphocytes compared with the genotoxicity of cigarette smoking. Genotoxicity was evaluated using the sister chromatid exchanges (SCEs) assay. Fifty waterpipe smokers and 18 healthy nonsmokers participated in this study. Additionally, 18 heavy cigarette smokers (CS) were recruited for comparison. The results show that waterpipe smoking and cigarette smoking significantly increase the frequencies of SCEs (P < 0.01) compared with those of nonsmokers, indicating the genotoxic effect of tobacco smoking. In addition, frequencies of SCEs were significantly higher among waterpipe smokers compared with CS (P < 0.01), indicating that waterpipe smoking is more genotoxic than cigarette smoking. Moreover, the frequency of SCEs increased with the extent of waterpipe use. In conclusion, waterpipe smoking is genotoxic to lymphocytes and the magnitude of its genotoxicity is higher than that induced by regular cigarette smoking. Environ. Mol. Mutagen., 2011. © 2010 Wiley‐Liss, Inc.