Nuclear Buds Research Articles

Folate metabolism modifies chromosomal damage induced by 1,3-butadiene: results from a match-up study in China and in vitro experiments

ObjectivesTo explore the role of folate metabolism in 1,3-Butadiene (BD)'s genotoxicity, we conducted a match-up study in BD-exposed workers in China to analyze the associations between the polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and the chromosomal damage induced by BD exposure, and culture-based experiments in TK-6 cells to examine the global DNA methylation levels and chromosomal damage when exposed both to BD’s genotoxic metabolite, 1,2:3,4-diepoxybutane (DEB), and MTHFR’s direct catalytic product, 5-methyltetrahydrofolate (5-MTHF).MethodsCytokinesis block micronucleus assay (CBMN) was used to examine the chromosomal damage induced by BD or DEB. Poisson regression models were produced to quantify the relationship of chromosomal damage and genetic polymorphisms in the BD-exposed workers. Global DNA methylation levels in TK6 cells were examined using DNA Methylation Quantification Kit.ResultsWe found that BD-exposed workers carrying MTHFR C677T CC (2.00 ± 2.00‰) (FR = 0.36, 95%CI: 0.20–0.67, P < 0.01) or MTHFR C677T CT (2.87 ± 1.98‰) (FR = 0.49, 95%CI: 0.32–0.77, P < 0.01) genotypes had significantly lower nuclear bud (NBUD) frequencies than those carrying genotype MTHFR 677 TT (5.33 ± 2.60‰), respectively. The results in TK6 cells showed that there was a significant increment in frequencies of micronucleus (MN), nucleoplasmic bridge (NPB) and nuclear bud (NBUD) with exposure to DEB at each 5-MTHF dose (ANOVA, P < 0.01). Additionally, there was a significant decrease in frequencies of MN, NPB and NBUD in DEB-exposed cultures with increasing concentration of 5-MTHF (ANOVA, P < 0.05). The levels of global DNA methylation were significantly decreased by DEB treatment in a dose-dependent manner within each 5-MTHF concentration in TK-6 cells (ANOVA, P < 0.01), and were significantly increased by 5-MTHF supplementation within each DEB concentration (ANOVA, P < 0.01).ConclusionWe reported that folate metabolism could modify the association between BD exposure and chromosomal damage, and such effect may be partially mediated by DNA hypomethylation, and 5-MTHF supplementation could rescue it.

Genotoxic evaluation of the infusion of Baccharis spicata (Lam.) Baill (Asteraceae), a substitute of the medicinal plant known as “carqueja” in Argentina

BackgroundBaccharis spicata (Lam.) Baill (Asteraceae) is employed as a substituent of medicinal species of the Baccharis genus known as “carqueja”, though the scientific evidence available nowadays is scarce regarding its efficacy, safety and quality. The objective of this article was to assess the genotoxic activity of an infusion of B. spicata leaves. MethodsGenotoxicity of the infusions was assessed. Infusions were the extract of choice since its use is widespread among the population of Argentina. Selected assays for this purpose were the Ames test and the micronucleus assay, and metabolic activation was induced with S9-mix reagent. The qualitative and quantitative composition of the extract was determined by high performance liquid chromatography, and the content of total phenols, total hydroxycinnamic derivatives and total flavonoids was measured. ResultsThe infusion contained flavonoids and hydroxycinnamic derivatives such as rutin, 5-caffeoylquinic acid and 3,5-dicaffeoylquinic acid. The frequency of biomarkers related to genotoxic damage such as frequencies of number of colony-forming units in the Ames test and micronuclei, nuclear buds and nucleoplasmic bridges/1000 binucleated cells in the micronuclei test was not significantly different from the respective negative controls. ConclusionsThe absence of mutagenic effects in the in vitro models employed provides evidence of the safety of the infusion of B. spicata for its medicinal use in humans, though further research is needed in order to fully assure this affirmation. The importance of our study lies in the fact that this plant is used as a substitute for another medicinal species and the lack of scientific evidence regarding its safe consumption in commonly employed preparations.

Micronuclei and other nuclear abnormalities in phorate exposed fish, Channa punctatus

Aim: The present study aims to establish morphology-based nuclear abnormalities (NAs) including micronuclei (MN) as effective and sensitive genotoxic endpoint biomarkers in fish against the sub-lethal exposure of phorate. Methodology: Fish, Channa punctatus (35 ± 3.0 g; 14.5 ± 1.0 cm) were randomly exposed in two sets, treated group 1 and 2 (0.0375 mg l-1 and 0.075 mg l-1 of phorate, respectively) along with a simultaneous control (0 mg l-1). The blood was sampled after 30 days. Results: A significant (p &lt; 0.05) induction in reactive oxygen species (ROS) coupled with elevated frequency of blood cells showing micronuclei along with the gross appearance of notched nuclei, curved nuclei, blebbed nuclei, kidney-shaped nuclei, V-shaped nuclei, nuclear buds, nucleoplasmic bridges, dumbbell nuclei, and condensed/rounded nuclei were recorded in a dose-dependent manner. Interpretation: A significant (p &lt; 0.05) induction in reactive oxygen species (ROS) coupled with elevated frequency of blood cells showing micronuclei along with the gross appearance of notched nuclei, curved nuclei, blebbed nuclei, kidney-shaped nuclei, V-shaped nuclei, nuclear buds, nucleoplasmic bridges, dumbbell nuclei, and condensed/rounded nuclei were recorded in a dose-dependent manner.

Assessing genotoxic potential of petroleum refinery wastewater using biomarkers of laboratory exposed and field captured fishes

Petroleum refinery wastes contain mixtures of toxic compounds including Polycyclic Aromatic Hydrocarbons (PAH) which may pose genotoxic threats to the biota. The objective of the present study was to assess the genotoxic potential of wastewaters of the petroleum refinery at Sapugaskanda, Sri Lanka using erythrocytic micronuclei and nuclear abnormalities as genotoxic biomarkers in the fish Oreochromis niloticus exposed to the refinery wastewater under controlled laboratory conditions and in the field captured fishes from the canals close to the refinery. Erythrocytic micronuclei and nuclear abnormalities in the peripheral blood were evaluated using cytogenetic tests. Patterns of fluorescent aromatic compounds in the fish bile were also examined using fixed fluorescence spectrometry to assess potential PAH exposure. Erythrocytic micronuclei, nuclear buds and notched nuclei in the peripheral blood were highly induced (P &lt; 0.05) in O. niloticus exposed to the wastewater for 7 days under laboratory conditions in comparison to the control fish. The field-captured fishes, Trichogaster pectoralis and Dawkinsia singhala inhabiting water canals near the petroleum refinery also demonstrated significant induction (P &lt;0.05) of erythrocytic micronuclei and other nuclear abnormalities. PAH exposure indicative bile fluorescence patterns (naphthalene, phenanthrene, pyrene, and benzo(a)pyrene types) were detected in the fish exposed to the wastewater under laboratory conditions and in the field captured fishes. The results revealed that the wastewater of petroleum refinery contains genotoxic chemicals including PAHs. Hence, incorporation of genotoxicity tests as bioanalytical tools for regulating the discharge of final refinery wastewater to the aquatic ecosystems would be prudent in consideration of sustainable development goals focusing on the ecosystem and human safety.

Effect of Heracleum sosnowskyi extract aqueous solution on the Allium cepa root meristem

Heracleum sosnowskyi (Apiaceae) contains a lot of useful chemical ingredients that can be used in industry, medicine and other fields as plant component extracts and as chemical compounds that have been extracted in different ways, which requires the last to be tested for chemical safety, including a genotoxic test in vivo. In the present paper, the 96-hour effect of the H. sosnowskyi extract aqueous solution at concentrations of 0.01, 0.05, 0.10, and 0.50 mL/L on the genetic apparatus and mitotic activity of the cells of the Allium cepa (Alliaceae) root meristem is discussed. Distilled water was applied as a negative control, and hydrogen peroxide 1% as a positive one. The extract was prepared from the plant’s fresh leaves by soaking them in acetone. It was then distilled at 57 ºС and diluted with distilled water to obtain the experimental concentrations. As extract content in the aqueous solution increased, a statistically significant decrease in mitotic activity, an increase in aberrant cell percentage and a concentration-dependent inhibition of root growth were observed. In the 0.5 mL/L solution, if compared against the other experimental concentrations, an increase in the metaphase, anaphase and telophase indices along with a decrease in the prophase index were observed. The most common aberrations for all the concentrations were lagging and sticking chromosomes, anaphase bridges, ring chromosomes and nuclear buds. The same solution and the positive control produced membrane damage; giant and ghost cells. The results of the experiment performed have demonstrated the extract’s aneugenic effect that causes spindle disturbance, mitodepression and inhibits the cells of the Allium cepa root meristem, prevails over its clastogenic effect.

Methylglyoxal induces chromosomal instability and mitotic dysfunction in lymphocytes.

Type 2 diabetes is associated with elevated levels of DNA damage, in particular micronuclei (MNi) which are formed by acentric chromosome fragments caused by double-stranded DNA breaks (DSBs), or whole chromosomes which fail to segregate during mitosis. We investigated if methylglyoxal (MGO), a reactive dicarbonyl known to be elevated in type 2 diabetes is capable of increasing chromosomal instability and DNA damage as measured by the cytokinesis block micronucleus cytome (CBMNcyt) assay in B-lymphoblastoid WIL2-NS cells and primary peripheral blood lymphocytes (PBL). We also investigated the level of various dicarbonyl stress biomarkers, including extracellular and intracellular MGO, protein and MGO modifications of DNA. WIL2-NS cells exposed to either MGO or a glyoxalase 1 inhibitor showed increases in MNi and nuclear buds, which were associated with an increase in intracellular MGO. DNA damage in the form of MNi and nucleoplasmic bridges were observed in primary PBL exposed to 10 µM MGO, suggesting low concentrations of MGO may be genotoxic. Furthermore, we showed, using fluorescent in situ hybridisation, that the majority of MNi caused by MGO in WIL2-NS cells were caused by whole chromosome loss events, rather than DSBs. Our data suggest that MGO, a reactive metabolite elevated in type 2 diabetes and other pathologies, can affect genomic integrity by impairing chromosome segregation during mitosis.

Exposure to hypomethylating 5-aza-2′-deoxycytidine (decitabine) causes rapid, severe DNA damage, telomere elongation and mitotic dysfunction in human WIL2-NS cells

Background5-aza-2′-deoxycytidine (5azadC, decitabine) is a DNA hypomethylating agent used in the treatment of myelodysplastic syndromes. Due to cytotoxic side effects dose optimization is essential. The aim of this study was to define and quantify the effects of 5azadC on biomarkers of chromosomal stability, and telomere length, in human lymphoblastoid cell line, WIL2-NS, at clinically relevant dosages. MethodsHuman WIL2-NS cells were maintained in complete medium containing 0, 0.2 or 1.0 μM 5azadC for four days, and analysed daily for telomere length (flow cytometry), chromosomal stability (cytokinesis-block micronucleus cytome (CBMN-cyt) assay), and global methylation (%5me-C). ResultsDNA methylation decreased significantly in 1.0 μM 5azadC, relative to control (p < 0.0001). Exposure to 1.0 μM 5azadC resulted in 1.7-fold increase in telomere length (p < 0.0001), in parallel with rapid increase in biomarkers of DNA damage; (micronuclei (MN, 6-fold increase), nucleoplasmic bridges (NPB, a 12-fold increase), and nuclear buds (NBud, a 13-fold increase) (all p < 0.0001). Fused nuclei (FUS), indicative of mitotic dysfunction, showed a 5- and 13-fold increase in the 0.2 μM and 1.0 μM conditions, respectively (p = 0.001) after 4 days. ConclusionsThese data show that (i) clinically relevant concentrations of 5azadC are highly genotoxic; (ii) hypomethylation was associated with increased TL and DNA damage; and (iii) longer TL was associated with chromosomal instability. These findings suggest that lower doses of 5azdC may be effective as a hypomethylating agent, while potentially reducing DNA damage and risk for secondary disease.

Acute Toxic and Genotoxic Effects of Aluminum and Manganese Using In Vitro Models.

The objective of this study was to use the same concentrations of aluminum (Al) and manganese (Mn) detected previously in groundwater above those permitted by Brazilian law and assess their cytotoxic and genotoxic effects in hamster ovary cell lines and their mutagenic effects through the Salmonella microsome assay. Chinese hamster ovary (CHO) and CHO-XRS5 cells were treated with different concentrations of Al and Mn (0.2 to 2.0 mg/L and 0.1 to 3.0 mg/L, respectively). The Ames test was used to analyze the concentrations of Al and Mn ranging from 0.025 to 1.0 mg/L and 0.0125 to 1.5 mg/L, respectively. Both metals showed cytotoxic effects on both cell lines and two bacterial strains (TA98 and TA100). The genotoxic effects of the highest concentrations of Al and Mn in cell lines showed nuclear buds, micronuclei, and DNA damage; however, none of the concentrations showed a positive mutagenic response in the Ames test. This is one of the few studies to demonstrate the cytotoxic effects of Al and Mn through the Ames test. In addition, the metals caused genomic instability in cell lines. Therefore, this study may help hasten the review of established regulatory standards for human consumption of groundwater.

Cytogenetic damage in native Baltic Sea fish species: environmental risks associated with chemical munition dumping in the Gotland Basin of the Baltic Sea.

This study represents the first attempt to assess genotoxicity and cytotoxicity effects in herring (Clupea harengus membras), flounder (Platichthys flesus), and cod (Gadus morhua callarias) caught at 47 study stations, located close to chemical munition dumpsites in the Gotland Basin, the Baltic Sea. Herring sampled from stations located in the center of chemical munition dumpsites exhibited the highest levels of micronuclei (MN) and total genotoxicity (ΣGentox), which is defined as the sum of frequencies of such nuclear abnormalities as micronuclei, nuclear buds, nuclear buds on the filament, and bi-nucleated erythrocytes with nucleoplasmic bridges. Exceptionally high and high ΣGentox risks were determined for flounder (89.47%), herring (79.31%), and cod (50%) caught at the stations located close to the chemical munition dumpsites.

Assessment of Cytogenetic Damage and Cholinesterases' Activity in Workers Occupationally Exposed to Pesticides in Zamora-Jacona, Michoacan, Mexico.

Pesticides have been considered as potential chemical mutagens; however, little is known about toxic and genotoxic effects during pesticide application in Zamora-Jacona, Michoacan State in Mexico. This study sought to determine DNA damage and cholinesterase activities inhibitions in 54 agricultural workers exposed to complex mixtures of pesticides vs. control group (26 individuals) using Comet assay in peripheral whole blood, micronucleus (MN) test in oral mucosa cells, Cytokinesis-blocked MN assay in lymphocytes (L-CBMNcyt) and measuring AChE and BChE activities in whole blood and plasma samples, respectively. Exposed subjects demonstrated significantly elevated levels of primary (Comet assay: tail intensity, tail length, tail moment, Olive tail moment) and permanent DNA damage (MN assay: in blood/buccal cells; frequencies of nuclear buds, binucleated cells, cells with condensed chromatin, karyorrhexis, pyknosis, and karyolysis). However, inhibition of cholinesterase activities (AChE and BChE) was not observed in the workers. Confounding factors including sex, age, BMI, working exposure period, protection level, smoking habit (cigarettes per day units), alcohol consumption (weekly), medication, were considered in the analysis. These combined techniques demonstrated usefulness in the health hazards risks pesticide exposure assessment and suggested the need for periodic monitoring together with the education and the training of occupational workers for the safe application of potentially harmful pesticides.

Impact of preterm birth on the cellular characteristics of neonatal buccal cells.

To demonstrate the impact of preterm birth on the cytological, cytomorphometrical, and nuclear parameters of neonatal buccal smears. This study consisted of Early Preterm Neonates (EPN; ≤34th gestational week [gw]; n=36), Late Preterm Neonates (LPN; 34th to <37th gw; n=46), and Term Neonates (control; ≥37th gw; n=56). Cytological evaluation and buccal cytome assay were performed using Papanicolaou and Feulgen methods, respectively. Cytological evaluation demonstrated that smear background was cleaner (P<.05) and there were less macrophages in the control group (P<.001). Cyto-morphometric analysis showed that the measurements of nuclear diameter, nuclear area, and nucleus-to-cytoplasm ratio were higher in the preterm (EPN and LPN) versus the control groups (P=.016, P<.001, and P<.001, respectively). We also demonstrated that staining intensity of the nucleus and cytoplasm were less intense in the EPN and LPN groups (P<.001). There was no statistically significant difference between the EPN and LPN groups for any parameters (P>.05). Buccal cytome assay showed that nuclear buds were more prevalent in term newborns compared to preterm neonates (P<.001). Morphological and cytological properties of neonatal buccal cells are influenced by preterm birth status, and buccal smears may be used as a tool to detect biological markers of neonatal health problems.

Genotoxic effect induced by dried nicotiana tabacum leaves from tobacco barns (kiln-houses) in chinese hamster lung fibroblast cells (V79)

ABSTRACT Nicotiana tabacum is the most cultivated tobacco species in the state of Rio Grande do Sul, Brazil. Workers who handle the plant are exposed to the leaf components during the harvesting process and when separating and classifying the dried leaves. In addition to nicotine, after the drying process, other components may be found including tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, as well as pesticides residues. The objective of this study was to examine the genotoxicity attributed to the aqueous extract of dried tobacco leaves obtained from tobacco barns using Chinese hamster lung fibroblast cells (V79) as a model system by employing alkaline comet assay, micronucleus (MN) and Ames test. MTT assay was used to assess cytotoxicity and establish concentrations for this study. Data demonstrated cell viability > 85% for concentrations of 0.625–5 mg/ml while the comet assay indicated a significant increase in DNA damage at all concentrations tested. A significant elevation of MN and nuclear buds (NBUD) was found for 5 mg/ml compared to control and other dry tobacco leaves concentrations (0.625–2.5 mg/ml). Mutagenicity was not found using the Salmonella/Microsome test (TA98, TA100, and TA102 strains) with and without metabolic activation. The concentration of inorganic elements was determined employing the PIXE technique, and 13 inorganic elements were detected. Using CG/MS nicotine amounts present were 1.56 mg/g dry tobacco leaf powder. Due to the observed genotoxicity in V79 cells, more investigations are needed to protect the health of tobacco workers exposed daily to this complex mixture of toxic substances present in dry tobacco leaves.

Chromosomal and oxidative DNA damage in non-functioning pituitary adenomas.

Clinically non-functioning pituitary adenomas (NFPA) are common tumours of the pituitary gland and are mainly considered as benign. The primary aim of this study was to research the effects of NFPA on genome instability in patients with non-functioning pituitary adenoma by using the cytokinesis-block micronucleus cytome (CBMN-cyt) assay and 8-hydroxy- 2'-deoxyguanosine (8-OHdG) assay. The second objective of this study was to assess whether there is a relationship between age, pituitary adenoma diameters, 8-OHDG levels, CBMN site assay parameters, and tumour aggressiveness. The study was performed on 30 patients who had been diagnosed with NFPA and were admitted to the Department of Endocrinology and Metabolism, and 20 healthy subjects of similar age and sex. Micronucleus (MN), nucleoplasmic bridges (NPBs), nuclear bud (NBUD) frequencies, and apoptotic and necrotic cell frequencies in patients with NFPA were found to be significantly higher than in control subjects, and plasma 8-OHdG levels in patients with NFPA were statistically significantly lower than control subjects in this study. It is believed that this is the first study to evaluate the aggressiveness of tumour with chromosome/oxidative DNA damage in patients with NFPA. However, further studies are needed in order to understand the cause of NFPA aggression and to evaluate these patients in terms of risk of cancer.

Acute Toxicity and DNA Instability Induced by Exposure to Low Doses of Triclosan and Phthalate DEHP, and Their Combinations, in vitro.

Triclosan (TCS) is an antimicrobial agent widely used in personal care products (PCP) and the di-(2-ethyl hydroxy-phthalate) (DEHP) is a chemical compound derived from phthalic acid, used in medical devices and plastic products with polyvinyl chloride (PVCs). As result of their extensive use, TCS and DEHP have been found in the environment and previous studies demonstrated the association between their exposure and toxic effects, mostly in aquatic organisms, but there is a shortage in the literature concerning the exposure of TCS and DEHP in human cells. The aim of the present study was to assess the impact of exposure to TCS and DEHP, as well as their combinations, on biomarkers related to acute toxicity and DNA instability, in HepG2 cells, by use of cytokinesis-block micronucleus cytome (CBMNCyt) assay. For that, the cultures were exposed to TCS, DEHP and combinations at doses of 0.10, 1.0, and 10 μM for the period of 4 h and the parameters related to DNA damage (i.e., frequencies of micronuclei (MN) and nuclear buds (NBUDs), to cell division (i.e., nuclear division index (NDI) and nuclear division cytotoxic index (NDCI) and to cell death (apoptotic and necrotic cells) were scored. Clear mutagenic effects were seen in cells treated with TCS, DEHP at doses of 1.0 and 10 μM, but no combined effects were observed when the cells were exposed to the combinations of TCS + DEHP. On the other hand, the combination of the toxicants significantly increased the frequencies of apoptotic and necrotic cells, as well as induced alterations of biomarkers related to cell viability (NDI and NDCI), when compared to the groups treated only with TCS or DEHP. Taken together, the results showed that TCS and DEHP are also able to induce acute toxicity and DNA damage in human cells.

Delphinidin, Luteolin and Halogenated Boroxine Modulate CAT Gene Expression in Cultured Lymphocytes

Luteolin and delphinidin are the flavonoids with known protective roles. They inhibit genotoxic effects induced by halogenated boroxine (HB) in vitro. Statistically significant decrease in the frequency of micronuclei and nuclear buds and suppression of the occurrence of aberrant cells were observed before, but mechanism of its anti-genotoxic activity is still not clear. In our experiment we aimed to quantify HB effects on the relative expression of CAT (catalase) gene and explore antioxidative effects of luteolin and delphinidin via restoration of CAT gene activity. Cell cultures from peripheral blood lymphocytes of five healthy donors were established and treated with independent and concomitant treatments of HB with luteolin or delphinidin. Total RNA was isolated from harvested cells and reverse-transcribed. SYBR based Real-Time PCR amplification method was used. Analysis of results included normalization of ratio of target (CAT) and housekeeping (GAPDH) gene and statistical analysis (REST®). Luteolin itself lead to downregulation of relative CAT gene expression as well as HB. But simultaneous treatment of HB and bioflavonoids lead to upregulation. Delphinidin as independent treatment and as simultaneous treatment caused upregulation of relative CAT gene expression. Obtained results may indicate protective role of delphinidin and luteolin to oxidative damage caused by HB, and also that new approaches to the treatment applications of HB should include bioflavonoids and monitoring corresponding antioxidant system. Our findings indicate that there is a quantifiable effect of luteolin and delphinidine on antioxidant genes which could be used in exact monitoring of oxidative stress related events.

OSTEOGENIC, GENOTOXIC AND ANTIMICROBIAL POTENTIAL OF HYDROXYAPATITE AND ZN-HYDROXYAPATITE NANOPARTICLES ADDED TO AH PLUS ELUATES

<h3>Background</h3> Endodontic sealers are continuously being improved to enhance performance after root canal treatment. Despite their clinical success, commercial sealers do not meet the all the requirements of an ideal endodontic sealer, namely the induction capacity of the periradicular regenerative process that requires the recruitment of mesenchymal stem cells (MSC)/osteoblastic precursor cells and their differentiation into osteoblasts for synthesis of the mineralized matrix. Hydroxyapatite nanoparticles (NHAp) present similarities in size, crystallography, and chemical composition to the bone and teeth enamel. <h3>Methods</h3> The biological profile of NHAp and zinc-containing NHAp (NHAp-Zn), tested alone or added to the eluates of the endodontic sealer AH Plus, was characterized taking advantage of the biocompatibility of NHAp, the osteogenic effect of NHAp and zinc, together with the excellent profile of AH Plus. For this purpose, different cells were used: human mesenchymal stem cells derived from Wharton's jelly (hMSC-WJ) for cytotoxicity and osteogenesis assays; CHO-K1 and FMM1 cells for genotoxic and oxidate stress analysis; four oral bacterial and four fungal strains for antimicrobial studies by the minimum inhibitory concentration method. <h3>Results</h3> MTT results showed that AH Plus eluates were toxic, however, there was an excellent recovery of cell viability in the presence of NHAp, with and without zinc, with the most diluted eluate. The mineralization assay showed improved osteogenesis with NHAp, especially in the osteogenic medium, with NHAp-Zn3 in the lowest concentration more effective than NHAp-Zn5. Regarding genotoxicity through micronucleus assay, the results showed no differences between the cell lineages used, with an increase in the frequency of MN only for the most diluted eluate alone and in conjunction with NHAp-Zn3 at the highest concentration. The combination with the best osteogenic potential (most diluted eluate of AH Plus with NHAp-Zn3 at the lowest concentration) and the one recommended in the study did not lead to changes in the frequency of micronuclei, nuclear buds and nucleoplasmic bridges. Antimicrobial assays revealed some of the combinations were bacteriostatic and fungistatic. <h3>Conclusion</h3> All the aforementioned results suggest that NHAp improves osteogenesis in in osteogenic medium without showing genotoxicity indicating that they may be an alternative for bone regeneration therapies in endodontic treatment.

Biomonitoring of workers employed in a titanium dioxide production plant: Use of buccal micronucleus cytome assay as noninvasive biomarker to evaluate genotoxic and cytotoxic effects.

We aimed to evaluate whether TiO2 production process induces genotoxic and cytotoxic effects on the first target organ of inhalable particles by a sensitive and noninvasive biomarker of effect. Final aim was to find a useful and suitable tool to assess and manage the risk of TiO2 occupational exposure. We enrolled 40 workers employed in TiO2 production, 5 office workers, and 18 external controls. Buccal micronucleus cytome assay (BMCyt assay) was applied because it allows to evaluate micronucleus (MN), nuclear buds (NB), and broken eggs (BE) indicating the presence of chromosomal instability and gene amplification and binucleated cells (BIN), karyolytic cells (KL), and condensed chromatin (CC) indicating cytokinesis defect or arrest, cell death and apoptosis respectively. We characterized the exposure measuring inhalable and respirable particles by personal monitoring. BMCyt-assay showed in exposed workers compared with external controls a higher value of MN frequency (2.57 vs. 0.05‰, p < .001) and MN positivity, evaluated as percentage of subjects with MN frequency higher than a 1.5‰ cut-off value (52.5 vs. 0%). We also found in exposed workers higher frequency of BE + NB (2.41 vs. 0.22‰, p = .002), BIN (9.45 vs. 8.44‰, p = .047) and CC (1.80 vs. 0.21, p = .001) than in controls. Moreover, we found a relationship between personal monitoring results and presence of MN and other cellular anomalies. This study demonstrates induction of genotoxic and cytotoxic effects on buccal cells of workers involved in TiO2 production, suggesting the suitability of BMCyt assay as tool for risk assessment and management of TiO2 exposure.

Giant Toads (Rhinella marina) From the Industrial Zones of Low Basin of the Coatzacoalcos River (Veracruz, MX) Presents Genotoxicity in Erythrocytes.

The lower basin of Coatzacoalcos River is one of the most polluted regions of the southern Gulf of Mexico. Organochlorine compounds, polybrominated diphenyl ethers, polycyclic aromatic hydrocarbons, and heavy metals have been registered in this region. In the present study, genotoxicity was evaluated in the blood of giant toads (Rhinella marina) from Coatzacoalcos’ rural and industrial zones, and compared with laboratory toads. Determination of the frequency of micronucleus and erythrocyte nuclear abnormalities by the light microscope and cell cycle and apoptosis by flow cytometry were used as biomarkers of genotoxicity. We found more variability in micronucleus and more nuclear buds in toads from industrial zones. Also, cell cycle alterations and an increase of apoptosis in erythrocytes were found in toads from rural and industrial zones. Multivariate statistics show that the toads from the industrial zone were more affected than toads from laboratory and rural zones.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00128-021-03162-2.

The micronucleus cytome assay – A fast tool for DNA damage screening in human conjunctival epithelial cells

PurposeTo assess whether the micronucleus cytome assay (MCyt) reliably detects DNA damage occurring in control and pathological superficial epithelial cells from human conjunctiva. MethodsImpression cytology samples from the bulbar conjunctiva of 33 healthy controls, eight patients with conjunctival intraepithelial neoplasia (CIN) and eight with mucous membrane pemphigoid (MMP) were examined using the MCyt modified for the ocular surface. ResultsThe mean number of micronuclei (MNi) in control samples was 0.94 MNi/1000 epithelial cells, with no significant difference between conjunctival quadrants and independent of sex and age. The MCyt assay applied to CIN-affected eyes showed a significantly higher frequency of MNi (18.63/1000 cells), apoptotic cells, nuclear enlargement, multinucleated cells, and keratolysis compared with the corresponding unaffected paired eyes and with the control value. Although the mean MNi frequency in MMP eyes was also higher (1.73 MNi/1000 cells), it did not prove to be statistically different from the control samples. On the other hand, the MMP-affected eyes revealed significantly elevated percentages of cells with snake-like chromatin, multinucleated cells, apoptotic cells, and nuclear buds compared with controls. ConclusionsMicronucleus cytome assay was adapted as a rapid screening test for genomic instability on the ocular surface. We have determined reference levels for MNi and other nuclear alterations on healthy conjunctiva and demonstrated that particularly frequencies of MNi are significantly elevated in conjunctiva affected by CIN. We demonstrate that MNi are more specific than other nuclear abnormalities and thus can be used for screening of ocular surface neoplasia.

Genomic instability in people living with HIV

The increased life expectancy of people living with HIV (PLWH) receiving antiretroviral treatment (ART) has transformed HIV infection into a chronic disease. However, patients may be at risk of accelerated aging and the accumulation of cellular damage, which may trigger the development of cancer. We evaluated genomic instability in HIV-positive individuals with different viral loads receiving antiretroviral treatment (ART) and in HIV ART-naïve individuals. We included 67 participants divided into four groups: group 1 (n = 24) HIV patients receiving reverse-transcriptase inhibitors (tenofovir/ emtricitabine/ efavirenz and abacavir/ lamivudine/ efavirenz), group 2 (n = 22) HIV patients receiving protease inhibitors combined with other antiretroviral drugs (tenofovir/ emtricitabine with ritonavir/ atazanavir or lopinavir/ ritonavir, and darunavir/ ritonavir/ raltegravir), group 3 (n = 13) HIV ART-naïve patients, and group 4 (n = 8) healthy individuals (controls). Nuclear abnormalities in buccal mucosal samples (micronuclei, binucleated cells, nuclear buds, karyorrhexis, karyolysis, and pyknosis) were quantified. Simultaneously, blood samples were taken to quantify CD4+, CD8+, and HIV viral load. There was a significant age difference between HIV ART-naïve patients and receiving ART groups. Infection time was longer in HIV patients with ART than in ART-naïve patients. There were no differences in sex, smoking, alcohol consumption, or number of micronucleated cells between the study groups. We found higher frequencies of binucleated cells and nuclear buds in HIV patients, HIV ART-naïve, and HIV ART patients compared to the control group. We found a positive correlation between nuclear buds and CD4/CD8 ratio in the HIV ART-naïve group. In conclusion, PLWH showed increased genomic instability. The CD4/CD8 ratio affects the numbers of nuclear buds and binucleated cells. These findings are pertinent to mechanisms of damage and possible strategies to mitigate carcinogenesis in PLWH.