Genotoxicity of Marijuana in Mono-Users

Marijuana (Cannabis sp.) is among the most recurred controlled substances in the world, and there is a growing tendency to legalize its possession and use; however, the genotoxic effects of marijuana remain under debate. A clear definition of marijuana's genotoxic effects remains obscure by the simultaneous consumption of tobacco and other recreational substances. In order to assess the genotoxic effects of marijuana and to prevent the bias caused by the use of substances other than cannabis, we recruited marijuana users that were sub-divided into three categories: (1) users of marijuana-only (M), (2) users of marijuana and tobacco (M+T), and (3) users of marijuana plus other recreative substances or illicit drugs (M+O), all the groups were compared against a non-user control group. We quantified DNA damage by detection of γH2AX levels and quantification of micronuclei (MN), one of the best-established methods for measuring chromosomal DNA damage. We found increased levels of γH2AX in peripheral blood lymphocytes from the M and M+T groups, and increased levels of MNs in cultures from M+T group. Our results suggest a DNA damage increment for M and M+T groups but the extent of chromosomal damage (revealed here by the presence of MNs and NBuds) might be related to the compounds found in tobacco. We also observed an elevated nuclear division index in all marijuana users in comparison to the control group suggesting a cytostatic dysregulation caused by cannabis use. Our study is the first in Mexico to assess the genotoxicity of marijuana in mono-users and in combination with other illicit drugs.

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.

Antiproliferative and Genotoxic Action of an Underexploited Organoteluran Derivative on Sarcoma 180 Cells

Background: The search for novel metallic chemical compounds with toxicogenic effects have been of great importance for more efficient cancer treatment. Objective: The study evaluated the cytotoxic, genotoxic and mutagenic activity of organoteluran RF07 in S-180 cell line. Methods: The bioassays used were cell viability with 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test, evaluation of apoptosis and necrosis using fluorescence and flow cytometry, cytokinesis-block micronucleus test and comet assay. The compound was tested at 1; 2.5 and 5 µM. Results: The results showed the cytotoxicity of RF07 at concentrations of 2.5, 5, 10 and 20 µM when compared to the negative control. For genotoxicity tests, RF07 showed effects in all concentrations assessed by increased index and frequencies of damage and mutagenic alterations. The compound was also cytotoxic due to the significant decrease in nuclear division index, with significant values of apoptosis and necrosis. The results of fluorescence and flow cytometry showed apoptosis as the main type of cell death caused by RF07 at 5 µM, which is thought to avoid an aggressive immune response of the organism. Conclusion: In addition to cytotoxic and genotoxic effects, RF07 creates good perspectives for future antitumor formulations.

Therapeutic effects of statins on chromosomal DNA damage of dyslipidemic patients

Statins are a group of cholesterol lowering drugs and frequently used in the therapy of dyslipidemia. Our knowledge of the impact of statin therapy on DNA damage is as yet rudimentary. In this study, we aimed to assess the possible (1) genotoxic, cytostatic, and cytotoxic effects of statins in peripheral blood lymphocytes by using the cytokinesis-block micronucleus cytome (CBMN-cyt) assay, and (2) oxidative DNA damage by measuring plasma 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in response to statin therapy. Thirty patients with dyslipidemia who had no chronic diseases and did not use any medicines that interfere lipid values and twenty control subjects were included in the study. Statin therapy was initiated at risk-stratified doses. Blood samples were taken before and after treatment with statins and from control subjects, and CBMN-cyt assay parameters and 8-OHdG levels were evaluated. The chromosomal DNA damage (micronuclei and nucleoplasmic bridges [NPBs]), cytostasis (nuclear division index [NDI]), and cytotoxicity (apoptotic and necrotic cell frequencies) were decreased in patients with dyslipidemia after statin treatment. No significant differences were found for 8-OHdG levels between patients with dyslipidemia before or after statin therapy. The total cholesterol and low-density lipoprotein-cholesterol levels showed positive correlations with NPB frequency in patients with dyslipidemia prior to statin treatment. The present study is the first to evaluate CBMN-cyt assay biomarkers and 8-OHdG levels in patients with dyslipidemia before and after treatment with statins. The observed reductions of chromosomal DNA damage and NDI values with statin treatment could represent an important and under-appreciated pleiotropic effect of these agents. Impact statement In literature, it is possible to find some in vitro cytokinesis-block micronucleus (CBMN) assay studies about human lymphocytes and statins. But, there are no data on CBMN-cytome (CBMN-cyt) assay parameters related to statin therapy in patients with dyslipidemia. The present study is the first to evaluate CBMN-cyt assay biomarkers and 8-OHdG levels in patients with dyslipidemia before treatment and after treatment with statins (5–10 mg/day rosuvastatin or 10–20 mg/day atorvastatin). In this study we show that statin therapy decreased chromosomal DNA damage (micronuclei and nucleoplasmic bridges) and nuclear division index (NDI) values in patients with dyslipidemia by possible molecular reasons independent of oxidative DNA damage. In addition, the decrease of chromosomal DNA damage and NDI values with statin treatment could be indicated by the association between statin use and reduced risk of cancer.

Cytogenetic alterations induced by flupyradifurone, a new butenolide insecticide, in human lymphocytes

Flupyradifurone (FPD), a member of the new class of butenolide insecticides, acts on nicotinic acetylcholine receptors. Studies on genotoxic and carcinogenic effects of FPD are very limited. This is the first study to investigate the cytotoxic and genotoxic effects of FPD and its metabolites on human lymphocyte cultures with or without a metabolic activation system (S9 mix) using chromosomal aberration (CA) and micronucleus (MN) tests. The cultures were treated with 85, 170, and 340 µg/ml of FPD in the presence (3 h treatment) and absence (48 h treatment) of S9 mix. Dimethyl sulfoxide (DMSO) was used as a solvent control. Statistically significant decreases were detected at the medium and highest concentrations for 48 h treatments while decreases in mitotic index (MI) in the presence of the S9 mix were found statistically significant at all FPD concentrations tested when compared with the solvent control. FPD also decreased the nuclear division index (NDI) at the highest concentration (340 µg/ml) in the absence of S9 mix. When compared with the solvent control, increases in CA frequencies were significant at the medium and highest concentrations. Significantly increased MN frequency was only found at the highest FPD concentration in cultures without S9 mix compared with the solvent control while increases in the MN frequencies in the presence of S9 mix were statistically significant at all FPD concentrations. The results of the present study indicate that FPD and its metabolites can show cytotoxic and genotoxic effects in human lymphocytes. More genotoxicity studies are necessary to make a possible risk assessment in humans.

Lymphocyte Proliferation Kinetics in Inhabitant of Takandeang Village, Mamuju: A High Background Radiation Areas in Indonesia

BACKGROUND: Mamuju area in West Sulawesi considered as the high natural background radiation area in Indonesia. Our previous study showed that the mean mitotic index (MI) and nuclear division index (NDI) in lymphocytes of Botteng Village, Mamuju inhabitants was lower compared to control samples. To validate our previous study results, here in this study the evaluation of cell proliferation markers which were MI and NDI in peripheral blood lymphocytes (PBL) of Takandeang Village inhabitants was conducted.METHODS: A total 60 people were enrolled in this study, consisted of 35 samples from Takandeang Sub-Village and 25 from normal background radiation area. MI was calculated manually and automatically using Metafer 3.11.2 imaging system. The NDI defined as proportion of mononucleated, binucleated, trinucleated and tetranucleated cells were conducted using cytokinesis block micronucleus (CBMN) assay.RESULTS: The results of this study showed that the mean manual MI in Takandeang Sub-Village inhabitants was lower compared to control group (4.96±2.25 vs. 5.93±2.14). In contrast, the mean automatic MI (20.37±10.49 vs. 18.87±7.49) and NDI (1.555±0.174 vs. 1.523±0.112) in Takandeang Sub-Village inhabitants was higher compared to the control group. Statistical analysis revealed that the difference of mean manual MI, automatic MI and NDI in Takandeang Sub-Village inhabitants was not significantly different compared to the control group(p>0.05).CONCLUSION: It can be concluded that based on this study the chronic low radiation dose exposure in Takandeang Sub-Village, Mamuju has no significant effect on the lymphocytes proliferation.KEYWORDS: lymphocytes, mitotic index, nuclear division index, high background radiation

Evaluation of Possible Genotoxic Activity of Dirithromycin in Cultured Human Lymphocytes

Dirithromycin antibiotic is a 14-membered lactone ring macrolide and is widely used in medicine to treat many different types of bacterial infections. In the present study, the possible genotoxicity of dirithromycin was evaluated in cultured human lymphocytes by using sister chromatid exchanges (SCEs), chromosome aberration (CA), and micronucleus (MN) tests and also cell proliferation kinetics such as mitotic index (MI), replication index (RI), and nuclear division index (NDI) were analyzed for cytotoxicity. Cell cultures were treated with four different concentrations of dirithromycin (37.75, 67.50, 125, and 250 µg/mL) for 24 and 48 h periods. Dirithromycin significantly induced SCE and MN frequency at all concentrations in both 24 and 48 h treated cells. In addition, CA level has been markedly increased in the cells treated with almost all concentrations of dirithromycin for 24 (except 37.75 µg/mL) and 48 h treatment periods as compared to control. However, MI, RI, and NDI values were not affected by the dirithromycin treatment (p> 0.05). The results of this study indicated that dirithromycin treatment caused genetic damage by increasing the level of cytogenetic endpoints, suggesting its genotoxic and mutagenic action on human lymphocytesin vitro.