Evaluation of Cytotoxic and Mutagenic Effects of the Synthetic Cathinones Mexedrone, α-PVP and α-PHP.

Mexedrone, α-PVP and α-PHP are synthetic cathinones. They can be considered amphetamine-like substances with a stimulating effect. Actually, studies showing their impact on DNA are totally absent. Therefore, in order to fill this gap, aim of the present work was to evaluate their mutagenicity on TK6 cells. On the basis of cytotoxicity and cytostasis results, we selected the concentrations (35-100 µM) to be used in the further analysis. We used the micronucleus (MN) as indicator of genetic damage and analyzed the MNi frequency fold increase by flow cytometry. Mexedrone demonstrated its mutagenic potential contrary to the other two compounds; we then proceeded by repeating the analyzes in the presence of extrinsic metabolic activation in order to check if it was possible to totally exclude the mutagenic capacity for α-PVP and α-PHP. The results demonstrated instead the mutagenicity of their metabolites. We then evaluated reactive oxygen species (ROS) induction as a possible mechanism at the basis of the highlighted effects but the results did not show a statistically significant increase in ROS levels for any of the tested substances. Anyway, our outcomes emphasize the importance of mutagenicity evaluation for a complete assessment of the risk associated with synthetic cathinones exposure.

1-Methylpyrene induces chromosome loss and mitotic apparatus damage in a Chinese hamster V79-derived cell line expressing both human CYP1A2 and sulfotransferase 1A1.

1-Methylpyrene (1-MP) is a ubiquitous environmental pollutant and rodent carcinogen. Its mutagenic activity depends on sequential activation by various CYP and sulfotransferase (SULT) enzymes. Previously we have observed induction of micronuclei and mitotic arrest by 1-MP in a Chinese hamster (V79)-derived cell line expressing both human CYP1A2 and SULT1A1 (V79-hCYP1A2-hSULT1A1), however, the mode of chromosome damage and the involvement of mitotic tubulin structures have not been clarified. In this study, we used immunofluorescent staining of centromere protein B (CENP-B) with the formed micronuclei, and that of ß- and γ-tubulin reflecting the structures of mitotic spindle and centrioles, respectively, in V79-hCYP1A2-hSULT1A1 cells. The results indicated that 1-MP induced micronuclei in V79-hCYP1A2-hSULT1A1 cells from 0.125 to 2 µM under a 24 h/0 h (exposure/recovery) regime, while in the parental V79-Mz cells micronuclei were induced by 1-MP only at concentrations ≥ 8 µM; in both cases, the micronuclei induced by 1-MP were predominantly CENP-B positive. Following 54 h of exposure, 1-MP induced mitotic spindle non-congression and centrosome amplification (multipolar mitosis) in V79-hCYP1A2-hSULT1A1 cells, and anaphase/telophase retardation, at concentrations ≥ 0.125 µM with concentration-dependence; while in V79-Mz cells it was inactive up to 8 µM. This study suggests that in mammalian cells proficient in activating enzymes 1-MP may induce chromosome loss and mitotic disturbance, probably by interfering with the mitotic spindle and centrioles.

Association between genotoxic properties of inhalation anesthetics and oxidative stress biomarkers.

Exposure to inhalation anesthetics (IAs) has been associated with DNA damage as reflected in the increased frequency of micronuclei (MN) and chromosomal aberrations (CAs). The present study was undertaken to ascertain whether there was any correlation between increased MN and CA and the extent of oxidative stress as well as the antioxidant status of a group of operating room personnel exposed to a mixture of IAs, including nitrous oxide, isoflurane, and sevoflurane. In this cross-sectional study, 60 operating room personnel (exposed group) in whom the frequencies of MN and CA had already been shown to be significantly higher than those of a referent group, as well as 60 unexposed nurses, were studied. Venous blood samples were taken from all participants, and malondialdehyde (MDA) levels as an index of oxidative stress (OS) and the activity of superoxide dismutase (SOD) and levels of total antioxidant capacity (TAC) as indices of antioxidant status were measured. The level of TAC (1.76 ± 0.59 mM vs. 2.13 ± 0.64 mM, p = 0.001) and the activity of SOD (11.22 ± 5.11 U/ml vs. 13.36 ± 4.12 U/ml, p = 0.01) were significantly lower, while the mean value of MDA was significantly higher (2.46 ± 0.66 µM vs. 2.19 ± 0.68 µM, p = 0.03) in the exposed group than in the nonexposed group. After adjusting for potential confounders, there were statistically significant associations between exposure to IAs, gender, SOD, and TAC with MN frequency and between exposure to IAs and SOD with numbers of CA. The findings of the present study indicated that exposure to IAs was associated with OS, and this, in turn, may be causally linked with DNA damage.

Oxidation resistance 1 prevents genome instability through maintenance of G2/M arrest in gamma-ray-irradiated cells.

Human oxidation resistance 1 (OXR1) was identified as a protein that decreases genomic mutations in Escherichia coli caused by oxidative DNA damage. However, the mechanism by which OXR1 defends against genome instability has not been elucidated. To clarify how OXR1 maintains genome stability, the effects of OXR1-depletion on genome stability were investigated in OXR1-depleted HeLa cells using gamma-rays (γ-rays). The OXR1-depleted cells had higher levels of superoxide and micronucleus (MN) formation than control cells after irradiation. OXR1-overexpression alleviated the increases in reactive oxygen species (ROS) level and MN formation after irradiation. The increased MN formation in irradiated OXR1-depleted cells was partially attenuated by the ROS inhibitor N-acetyl-L-cysteine, suggesting that OXR1-depeletion increases ROS-dependent genome instability. We also found that OXR1-depletion shortened the duration of γ-ray-induced G2/M arrest. In the presence of the cell cycle checkpoint inhibitor caffeine, the level of MN formed after irradiation was similar between control and OXR1-depleted cells, demonstrating that OXR1-depletion accelerates MN formation through abrogation of G2/M arrest. In OXR1-depleted cells, the level of cyclin D1 protein expression was increased. Here we report that OXR1 prevents genome instability by cell cycle regulation as well as oxidative stress defense.

Elevated H2AX Phosphorylation Observed with kINPen Plasma Treatment Is Not Caused by ROS-Mediated DNA Damage but Is the Consequence of Apoptosis.

Phosphorylated histone 2AX (γH2AX) is a long-standing marker for DNA double-strand breaks (DSBs) from ionizing radiation in the field of radiobiology. This led to the perception of γH2AX being a general marker of direct DNA damage with the treatment of other agents such as low-dose exogenous ROS that unlikely act on cellular DNA directly. Cold physical plasma confers biomedical effects majorly via release of reactive oxygen and nitrogen species (ROS). In vitro, increase of γH2AX has often been observed with plasma treatment, leading to the conclusion that DNA damage is a direct consequence of plasma exposure. However, increase in γH2AX also occurs during apoptosis, which is often observed with plasma treatment as well. Moreover, it must be questioned if plasma-derived ROS can reach into the nucleus and still be reactive enough to damage DNA directly. We investigated γH2AX induction in a lymphocyte cell line upon ROS exposure (plasma, hydrogen peroxide, or hypochlorous acid) or UV-B light. Cytotoxicity and γH2AX induction was abrogated by the use of antioxidants with all types of ROS treatment but not UV radiation. H2AX phosphorylation levels were overall independent of analyzing either all nucleated cells or segmenting γH2AX phosphorylation for each cell cycle phase. SB202190 (p38-MAPK inhibitor) and Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited γH2AX induction upon ROS but not UV treatment. Finally, and despite γH2AX induction, UV but not plasma treatment led to significantly increased micronucleus formation, which is a functional read-out of genotoxic DNA DSBs. We conclude that plasma-mediated and low-ROS γH2AX induction depends on caspase activation and hence is not the cause but consequence of apoptosis induction. Moreover, we could not identify lasting mutagenic effects with plasma treatment despite phosphorylation of H2AX.

Induction of in vivo Pig-a gene mutation but not micronuclei by 5-(2-chloroethyl)-2'-deoxyuridine, an antiviral pyrimidine nucleoside analogue.

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) was developed as an antiviral drug. It has been studied in a number of in vitro and in vivo genotoxicity assays and is considered an unusual nucleoside analogue owing to its potent mutagenic potential, with little to no measurable clastogenic activity. Given this atypical profile, CEDU represented an interesting compound for evaluating the in vivo Pig-a gene mutation assay, a test that is undergoing extensive validation for regulatory safety applications. The current report describes two studies with 7-week-old male Wistar Han rats, one that exposed animals to several dose levels of CEDU for 5 consecutive days, the other for 28 consecutive days. Blood samples were collected at several time points and analysed for Pig-a mutant cell frequencies via flow cytometry. These Pig-a analyses were accompanied by micronucleated reticulocyte (MN-RET) measurements performed with blood samples collected 1 day after cessation of treatment. Both studies showed robust CEDU dose-related increases in Pig-a mutant reticulocytes and mutant erythrocytes. Conversely, neither experiment showed evidence of a CEDU-related MN-RET-inducing effect. These rat haematopoietic cell results were in good agreement with those of earlier mouse studies where in vivo mutagenesis was observed, without clastogenicity/aneuploidy. Taken together, these data add further support to the concept that the Pig-a assay represents an important complement to the widely used in vivo micronucleus assay, as it expands the range of important DNA lesions that can be detected in short-term as well as protracted exposure study designs.

Measurement of micronuclei and internal dose in mice demonstrates that 3-monochloropropane-1,2-diol (3-MCPD) has no genotoxic potency in vivo.

In this study 3-monochloropropane-1,2-diol (3-MCPD), a compound that appears as contaminant in refined cooking oils, has been studied with regard to genotoxicity in vivo (mice) with simultaneous measurement of internal dose using state-of-the-art methodologies. Genotoxicity (chromosomal aberrations) was measured by flow cytometry with dual lasers as the frequency of micronuclei in erythrocytes in peripheral blood from BalbC mice intraperitoneally exposed to 3-MCPD (0, 50, 75, 100, 125 mg/kg). The internal doses of 3-MCPD in the mice were calculated from N-(2,3-dihydroxypropyl)-valine adducts to hemoglobin (Hb), quantified at very low levels by high-resolution mass spectrometry. Convincing evidence for absence of genotoxic potency in correlation to measured internal doses in the mice was demonstrated, despite relatively high administered doses of 3-MCPD. The results are discussed in relation to another food contaminant that is formed as ester in parallel to 3-MCPD esters in oil processing, i.e. glycidol, which has been studied previously by us in a similar experimental setup. Glycidol has been shown to be genotoxic, and in addition to have ca. 1000 times higher rate of adduct formation compared to that observed for 3-MCPD. The conclusion is that at simultaneous exposure to 3-MCPD and glycidol the concern about genotoxicity would be glycidol.

Interferon ß improves the efficacy of low dose cisplatin by inhibiting NF-κB/p-Akt signaling on HeLa cells.

The purpose of this study was to evaluate the anticancer efficacy of interferon ß in combination with low dose of cisplatin on human cervical cancer progression, as well as its principal action mechanism. The combination treatment synergistically potentiated the effect of interferon ß on cell growth inhibition and DNA damage on HeLa cells by repressing NF-κB/p-Akt signaling. Synergistic targeting of these pathways has a therapeutic potential. Further, the combination treatment ameliorated the expression of pro-apoptotic Bax, and decreased the expression of anti-apoptotic protein Bcl-2. Additionally, the expression of active PARP was significantly increased and MMP-9 level was decreased in combination group as compared to the expression seen for the treatment with interferon ß or cisplatin alone. Results demonstrate that the synergistic inhibitory effects of interferon ß and low dose of cisplatin on human cervical cancer cells and also suggest that the inhibition of NF-κB/p-Akt signaling pathway plays a critical role in the anticancer effects of combination treatment along with the induction of PARP. Therefore, the combination of interferon ß and cisplatin may be a useful treatment for human cervical cancer, with a greater effectiveness than other treatments.

Inhibitor of Aurora Kinase B Induces Differentially Cell Death and Polyploidy via DNA Damage Response Pathways in Neurological Malignancy: Shedding New Light on the Challenge of Resistance to AZD1152-HQPA.

Aurora kinase B (AURKB), a crucial regulator of malignant mitosis, is involved in chromosome segregation and cytokinesis. AZD1152-HQPA is a selective inhibitor for AURKB activity and currently bears clinical assessment for several malignancies. However, the effect of this drug still needs to be elucidated in neurological malignancies. In this study, we investigated the restrictive potentials of AZD1152-HQPA in U87MG and SK-N-MC. AZD1152-HQPA treatment resulted in growth arrest, modification of cell cycle, and inhibition of colony formation in both cell lines. Furthermore, lower concentrations of AZD1152-HQPA profoundly induced apoptosis in U87GM (p53/p73 wild type) cells in parallel with an upregulation of p53 and its target genes BAX, BAD, APAF1, and PUMA. But remarkably, SK-N-MC (p53/p73 double null) responded to AZD1152-HQPA at much higher concentrations with an upregulation of genes involved in cell cycle progression, induction of excessive endoreduplication, and polyploidy rather than apoptosis. Although SK-N-MC was resistant to AZD1152-HQPA, we did not find a mutation in the coding sequence of Aurora B gene or overexpressions of ABCG2 and ABCB1 as reported previously to be resistance mechanisms. However, our results suggest that p53/p73 status could be an important mechanism for the type of response and resistance of the tumor cells to AZD1152-HQPA. Collectively, inhibition of Aurora kinase B differentially induced cell death and polyploidy via DNA damage response pathways, depending on the status of p53/p73. We suggest p53/p73 could be a key regulator of sensitivity to AZD1152-HQPA and their status should be explored in clinical response to this ongoing drug in clinical trials.

Biological plausibility as a tool to associate analytical data for micropollutants and effect potentials in wastewater, surface water, and sediments with effects in fishes.

Discharge of substances like pesticides, pharmaceuticals, flame retardants, and chelating agents in surface waters has increased over the last decades due to the rising numbers of chemicals used by humans and because many WWTPs do not eliminate these substances entirely. The study, results of which are presented here, focused on associations of (1) concentrations of micropollutants in wastewater treatment plant (WWTP) effluents, surface waters, sediments, and tissues of fishes; (2) results of laboratory biotests indicating potentials for effects in these samples and (3) effects either in feral chub (Leuciscus cephalus) from two German rivers (Schussen, Argen) or in brown trout (Salmo trutta f. fario) and rainbow trout (Oncorhynchus mykiss) exposed in bypass systems to streamwater of these rivers or in cages directly in the rivers. The Schussen and Argen Rivers flow into Lake Constance. The Schussen River is polluted by a great number of chemicals, while the Argen River is less influenced by micropollutants. Pesticides, chelating agents, flame retardants, pharmaceuticals, heavy metals, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs) were detected in effluents of a WWTP discharging to the Schussen as well as in surface water, and/or fishes from downstream of the WWTP. Results obtained by biotests conducted in the laboratory (genotoxicity, dioxin-like toxicity, and embryotoxicity) were linked to effects in feral fish collected in the vicinity of the WWTP or in fishes exposed in cages or at the bypass systems downstream of the WWTP. Dioxin-like effect potentials detected by reporter gene assays were associated with activation of CYP1A1 enzymes in fishes which are inducible by dioxin-like chemicals. Abundances of several PCBs in tissues of fishes from cages and bypass systems were not associated with these effects but other factors can influence EROD activity. Genotoxic potentials obtained by in vitro tests were associated with the presence of micronuclei in erythrocytes of chub from the river. Chemicals potentially responsible for effects on DNA were identified. Embryotoxic effects on zebrafish (Danio rerio), investigated in the laboratory, were associated with embryotoxic effects in trout exposed in streamwater bypass systems at the two rivers. In general, responses at all levels of organization were more pronounced in samples from the Schussen than in those from the Argen. These results are consistent with the magnitudes of chemical pollution in these two streams. Plausibility chains to establish causality between exposures and effects and to predict effects in biota in the river from studies in the laboratory are discussed.

Kinetics of micronucleus induction and cytotoxicity caused by distinct antineoplastics and alkylating agents in vivo.

This mini-review aims to compare the differences in the kinetics of the induction of micronucleated polychromatic erythrocytes (MN-PCE) and cytotoxicity by distinct antineoplastic and genotoxic agents in murine peripheral blood in vivo and to correlate these kinetics with the underlying processes. Comparisons were carried out using our previously obtained data with nominal doses causing similar levels of cytotoxicity, as measured in terms reduction of PCE. The aneuploidogens caused the most rapid induction of MN-PCEs and had the highest rates of cytotoxicity and genotoxicity. The promutagens cyclophosphamide and dimethylnitrosamine showed the most delayed responses and had the lowest genotoxic and cytotoxic efficiencies. DNA crosslinking agents had a similar delay of 4-5 h, greater than those of aneuploidogens, but differed in their cytotoxic and genotoxic efficiencies. Methylnitrosourea and 5-aza-cytidine caused greater delays than crosslinking agents. These delays can be due to the methylnitrosourea-mediated induction of formation of mono alkyl adducts which are interpreted as mismatches during DNA duplication, whereas 5-aza-cytidine requires incorporation into the DNA to induce breakage. This review allows us to conclude that the requirement for metabolic activation and the mechanisms of DNA breakage and of micronucleus induction are the main factors that affect the time of maximal MN-PCE induction.

Cobalt chloride induces metaphase when topically applied to larvae and pupae of the stingless bee Melipona scutellaris (Hymenoptera, Apidae, Meliponini).

In order to optimize preparations of bee metaphases, we tested cobalt chloride, which has been used as a metaphase inducer in other organisms, such as hamsters and fish. Four microliters of 65 mM cobalt chloride aqueous solution was topically applied to larval and pupal stages of the stingless bee Melipona scutellaris. The cerebral ganglion was removed after treatment and prepared for cytogenetic analysis. Identically manipulated untreated individuals were used as controls. The number of metaphases was increased 3-fold in treated individuals compared to controls. The micronucleus test showed no mutagenic effects of cobalt chloride on M. scutellaris cells. We concluded that cobalt chloride is a metaphase-inducing agent in M. scutellaris, thus being useful for cytogenetic analyses.

Sidestream tobacco smoke is a male germ cell mutagen.

Active cigarette smoking increases oxidative damage, DNA adducts, DNA strand breaks, chromosomal aberrations, and heritable mutations in sperm. However, little is known regarding the effects of second-hand smoke on the male germ line. We show here that short-term exposure to mainstream tobacco smoke or sidestream tobacco smoke (STS), the main component of second-hand smoke, induces mutations at an expanded simple tandem repeat locus (Ms6-hm) in mouse sperm. We further show that the response to STS is not linear and that, for both mainstream tobacco smoke and STS, doses that induced significant increases in expanded simple tandem repeat mutations in sperm did not increase the frequencies of micronucleated reticulocytes and erythrocytes in the bone marrow and blood of exposed mice. These data show that passive exposure to cigarette smoke can cause tandem repeat mutations in sperm under conditions that may not induce genetic damage in somatic cells. Although the relationship between noncoding tandem repeat instability and mutations in functional regions of the genome is unclear, our data suggest that paternal exposure to second-hand smoke may have reproductive consequences that go beyond the passive smoker.

Tetrahymena meiotic nuclear reorganization is induced by a checkpoint kinase-dependent response to DNA damage.

In the ciliate Tetrahymena, meiotic micronuclei (MICs) undergo extreme elongation, and meiotic pairing and recombination take place within these elongated nuclei (the "crescents"). We have previously shown that elongation does not occur in the absence of Spo11p-induced DNA double-strand breaks (DSBs). Here we show that elongation is restored in spo11Delta mutants by various DNA-damaging agents including ones that may not cause DSBs to a notable extent. MIC elongation following Spo11p-induced DSBs or artificially induced DNA lesions is probably a DNA-damage response mediated by a phosphokinase signal transduction pathway, since it is suppressed by the ATM/ATR kinase inhibitors caffeine and wortmannin and by knocking out Tetrahymena's ATR orthologue. MIC elongation occurs concomitantly with the movement of centromeres away from the telomeric pole of the MIC. This DNA damage-dependent reorganization of the MIC helps to arrange homologous chromosomes alongside each other but is not sufficient for exact pairing. Thus, Spo11p contributes to bivalent formation in two ways: by creating a favorable spatial disposition of homologues and by stabilizing pairing by crossovers. The polarized chromosome orientation inside the crescent resembles the conserved meiotic bouquet, and crescent and bouquet also share the putative function of aiding meiotic pairing. However, they are regulated differently because in Tetrahymena, DSBs are required for entering rather than exiting this stage.

Selenoprotein deficiency enhances radiation-induced micronuclei formation.

The availability of selenium and the levels of specific selenoproteins might affect cancer risk by influencing the ability of DNA damaging agents to cause genomic instability and mutations. Transgenic mice that express reduced levels of selenoproteins and previously shown to be more susceptible to pathology associated with cancer development were used to study this possibility. These mice were exposed to X-rays and DNA damage assessed in the erythrocytes, where micronuclei formation was higher compared to the same cells obtained from irradiated wild-type controls. To determine whether the selenoprotein glutathione peroxidase-1 (GPx-1) might be involved in this protection, its levels were reduced by siRNA targeting in LNCaP human prostate cells. UV-induced micronuclei frequency was higher in these cells compared to control-transfected cells. These results indicate a role for selenoproteins in protecting DNA from damage and support human data implicating GPx-1 as a possible target of the chemoprotective effect of selenium.

TIF1 activates the intra-S-phase checkpoint response in the diploid micronucleus and amitotic polyploid macronucleus of Tetrahymena.

The ribosomal DNA origin binding protein Tif1p regulates the timing of rDNA replication and is required globally for proper S-phase progression and division of the Tetrahymena thermophila macronucleus. Here, we show that Tif1p safeguards chromosomes from DNA damage in the mitotic micronucleus and amitotic macronucleus. TIF1p localization is dynamically regulated as it moves into the micro- and macronucleus during the respective S phases. TIF1 disruption mutants are hypersensitive to hydroxyurea and methylmethanesulfonate, inducers of DNA damage and intra-S-phase checkpoint arrest in all examined eukaryotes. TIF1 mutants incur double-strand breaks in the absence of exogenous genotoxic stress, destabilizing all five micronuclear chromosomes. Wild-type Tetrahymena elicits an intra-S-phase checkpoint response that is induced by hydroxyurea and suppressed by caffeine, an inhibitor of the apical checkpoint kinase ATR/MEC1. In contrast, hydroxyurea-challenged TIF1 mutants fail to arrest in S phase or exhibit caffeine-sensitive Rad51 overexpression, indicating the involvement of TIF1 in checkpoint activation. Although aberrant micro- and macronuclear division occurs in TIF1 mutants and caffeine-treated wild-type cells, TIF1p bears no similarity to ATR or its substrates. We propose that TIF1 and ATR function in the same epistatic pathway to regulate checkpoint responses in the diploid mitotic micronucleus and polyploid amitotic macronucleus.

Induction of blocks in nuclear divisions and overcondensation of meiotic chromosomes with cycloheximide during conjugation of Tetrahymena thermophila.

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I-III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).