Environmental and occupational genotoxins

Assessment of tumor inducing potential of lead using crown gall tumor disc bioassay Abstract The exposure of human beings to the compounds of lead (such as commercially important lead acetate) via different routes (including inhalation, ingestion, skin contact etc.) poses severe health risks. The present study involved the application of crown gall tumor assay using potato discs, to check the tumor inducing potential of lead acetate. Though originally designed for estimation of antitumor activity of various test compounds/plant extracts, this assay has recently been used for analysis of tumor inducing potential of different environmental samples. The experiment was divided into two sets to check the tumor induction by different concentrations of lead acetate alone and in combination with Agrobacterium tumefaciens. The controls used in the present study were A. tumefaciens alone and only discs. The study revealed that number of tumors induced in potato discs treated with different concentrations of lead alone (29.8–49.2) and in combination with A. tumefaciens (36.4–50.8) were significantly higher than the negative control (2.4). The increase in number of tumors was significantly higher in the discs which were treated with lead in combination with culture than the culture alone which indicates that the presence of lead enhanced the expression of Ti-plasmid of A. tumefaciens. Thus, the present study indicates that lead acetate has a significant tumor inducing potential and crown gall tumor disc bioassay can be used as a potential and reliable tool for screening the tumor inducing potential of various test agents.

Cytotoxicity and genotoxicity of silver nanoparticles in Chinese Hamster ovary cell line (CHO-K1) cells Abstract Biomedical and pharmaceutical products comprising silver nanoparticles are attracting interest due to their potent antibacterial activities. For their safe use it has become imperative to test their cyto-genotoxic potential. In the present study the cytotoxicity and genotoxicity of three different sizes of AgNPs ranging from 15 to 22 nm and at concentrations 0.005–500 μg/ml were studied in Chinese Hamster ovary cell line (CHO-K1) cells. Cytoxicity was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and for genotoxicity comet, and micronucleus assays were utilized. AgNPs were able to internalize CHO-K1 cells and cause cytotoxicity at concentrations 0.005–500 μg/ml. AgNP‐induced cyto-genotoxicity in CHO-K1 cells could be attributed to its smaller primary size. AgNP-C of size ~ 15 nm was the most potent among the three AgNPs. The genotoxic response was biphasic that increased at lower concentrations (0.005–0.025 μg/ml) and decreased at higher concentrations (0.05–0.1 μg/ml) after 24 h of exposure. Such potential in vitro genotoxic effect of AgNPs remains to be further confirmed in animal cells in vivo.

Cytotoxic and mutagenic effects of green silver nanoparticles in cancer and normal cells: a brief review Abstract Applications of silver nanoparticles (AgNPs) have revolutionized the medicinal industry. Due to small size (1–100 nm) these nanoparticles are able to cross cell and nuclear membrane and induce cyto/genotoxicity. Green synthesized (plant based) AgNPs (GSAgNPs) have emerged as alternative antimicrobial agent to chemically synthesized nanoparticles assuming their methods of synthesis will be environment friendly and non toxic. In this review we report that the GSAgNPs were found to be cytotoxic and genotoxic to different cancer cell lines. They affected cell proliferation caused apoptosis and cell death of human cancer cells but did not cause such damages to normal human peripheral blood lymphocyte cells. This property can be utilized in cancer therapeutics to overcome the major drawbacks of chemotherapeutics with surface modifications of the nanoparticles by adding functionalized AgNPs.

Frequency of micronuclei in population of Bhopal exposed to methyl isocyanate in 1984 Abstract Micronuclei represent whole chromosome or large acentric fragments lagging behind the ana-telophasic separation of cell division. However, it progresses towards completion of karyokinesis and eventually gets included in one of the two daughter cells through cytokinesis. The frequency of micronuclei was estimated in 107 subjects exposed to methyl isocyanate (MIC) gas 30-years post-disaster and compared with 35 unexposed subjects of similar socio-cultural background from the same geographical region. Ethical approval and participants’ informed consent were obtained, and quality issues were validated prior to the proposed investigation. Peripheral blood was collected for each participant from their residence and transported to Mumbai for technical processing and cytogenetic analysis. Replicate cultures with 0.5 ml of whole blood were stimulated in 5 ml of serum supplemented RPMI1640 medium and maintained at 37°C for 72 h. The cells were dropped onto chilled slides following standard harvesting schedule, air dried and stained in Giemsa. Microscopic observation of 1000 cells for each subject has recognized lymphocyte cells (MNC) with micronucleus (MN). The ratio of MN and MNC indicated number of micronuclei per micronucleated cell. Statistical analysis revealed exposure related incidences of MN, MNC and MN/MNC, which was significant (p < 0.05) when compared with unexposed population and/or between moderate and severe exposure status (MN) of the population. Age-related MNC-incidence was significant between adult and childhood exposure to MIC gas. The differences of MN-incidences in the three severely exposure-zones (S1, S2, and S7) were significantly higher than the unexposed zones (C14, C16). The incidence of MN was consistent with our earlier study on chromosome aberrations detected in the same population 30-years post-disaster. However, the MN/CA incidences cannot be directly linked to MIC-exposure of 1984 owing to contribution of several confounding factors over 30 years. Nevertheless, the study could be considered as a low-cost tool but useful for estimation of DNA-damage in risk-assessment.

Cytotoxic effect of graphene oxide-functionalized gold nanoparticles in human breast cancer cell lines Abstract As the global burden of cancer is on the rise it is essential to develop alternate therapeutics that confer zero or minimum side effects. In this experiment, we evaluated the anticancer efficacy of graphene oxide-functionalized gold nanoparticles (GO-AuNPs) in human breast cancer cell lines MCF7 and MDA-MB-231. MTT assay revealed a dose-dependent decline in cancer cell survival. Significant alterations were detected in normal cellular morphology after treatment. Two different cell staining methods viz. acridine orange-ethidium bromide staining and annexin-cy3.18/6-carboxyfluorescein diacetate staining confirmed that the GO-AuNPs rendered their detrimental effect in cancer cells through apoptosis. To decipher the mode of apoptosis Western blotting was performed in which expression pattern of several proteins (PARP1, P53, P21, Bcl2, Bax, Caspase 9 and Caspase 3) established the intrinsic apoptosis pathway in these cell lines after GO-AuNP exposure. Intracellular calcium level, as measured by energy dispersive X-ray fluorescence analysis, was present at non-detectable level following treatment that can be linked to the cell cycle arrest and apoptotic cell death in them. Thus, GO-AuNPs were found effective in both types of human breast cancer cells viz. hormone-responsive MCF7 and chemoresistant MDA-MB-231 and can be considered as a highly potential anticancer agent in breast cancer therapy.

Toxicity assessment of magnesium oxide nano and microparticles on cancer and non-cancer cell lines Abstract Testing of magnesium oxide nanoparticles (MgO NPs) on established cell lines at cellular levels using toxicological endpoints provide valuable information about their adverse effects upon exposure. In vitro toxicity assessment of MgO NPs and their microparticles was carried out at 50, 100, 200 and 400 µg/mL concentrations by using cytotoxicity, genotoxicity, oxidative stress, cellular apoptosis and cellular uptake studies in cancer (HepG2) and non-cancer (NRK 49F) cell lines after 24 h of treatment. IC50 concentration for MgO NPs was found to be > 400 µg/mL in both cell lines after 24 h treatment. A concentration dependent toxicity was noted in genotoxic studies and oxidative stress parameters. A significant increase in the comet tail DNA was recorded at 200 and 400 µg/mL concentrations of MgO NPs when compared with controls in HepG2 and NRK 49F cells. Exposure to MgO NPs led to an increase in the generation of reactive oxygen species (ROS) in both the cell types. Genotoxicity results were further supported by apoptotic analysis. MgO particles were found adhered to the cell membrane when assayed by ICP-OES. The results of this study showed that the MgO NPs were toxic at high concentrations only. Furthermore, MgO NPs are more toxic to cancerous cells compared to non-cancerous cells. ROS mediated genotoxicity was observed when treated with MgO NPs. The current study adds to the information on MgO particles. The results of this investigation may help in advancement of understanding of toxicological nature of MgO NPs and aid in their use.

Genotoxic and cytotoxic assessment of individual and composite mixture of cadmium, lead and manganese in Clarias gariepinus (Burchell 1822) using micronucleus assay Abstract Heavy metals are ubiquitous environmental and occupational genotoxicants with different absorbability and toxicokinetics towards increasing genetic damage, neoplasm and cell death. Aquatic organisms are exposed to individuals and/or mixtures of these metals. This study investigated the potentials for cadmium(II) (Cd), lead(II) (Pb), manganese(II) (Mn), and their mixture (CPM) to induce cytogenotoxicity in Clarias gariepinus (Family: Clariidae). Clarias gariepinus was exposed to six concentrations ranging from 0 to 1600 mg/L, selected from range finding tests, to evaluate the 96 h acute toxicity for the individual metals and CPM. Fish were exposed to sub-lethal concentrations (6.25, 12.5 25, 50, and 100% of the 96 h LC50) of each metal and their mixture for 7 days to investigate micronucleus (MN) and abnormal nuclei (NAs) in the peripheral blood erythrocytes. Fish were exposed to borehole tap water and 0.01 mL/L of Benzene as negative and positive controls respectively. The tested metals induced toxicity in the order CMP > Cd > Pb > Mn, with CPM (LC50 = 40.6 mg/L) being 11.5 times more toxic than Mn (LC50 = 478.2 mg/L), the least toxic metal, to juvenile catfish. All the tested metals induced significant increase in frequencies of MN and NAs. The induced MN and NAs were in the order CMP > Cd > Mn > Pb. Fragmented and necrotic cells were common NAs in fish treated with 50 and 100% of Cd and CPM, suggesting that severely damaged cells were eliminated by programmed cell death (apoptosis) and/or accidental cell death (necrosis). Antagonistic interaction among the composite mixture of CPM provoked greater genomic instability and cytotoxicity in fish. Significant increase in MN and NAs in exposed fish suggest increased genomic instability which may lead to increase health defects including neoplasms and genetic related disorders, cell dysfunction and/or cell death.

Selenium nanoparticles are less toxic than inorganic and organic selenium to mice in vivo Abstract Nanoparticles (NPs) provide versatile means to reduce the toxicity, enhance bioactivity and improve targeting of cells. The antioxidant and pro-oxidant effects, or bioavailability and toxicity, of selenium depend on its chemical form. In the present study the effects of nano-selenium (Nano-Se) was compared with inorganic and organic selenium on the basis of their antioxidative activities and hematological parameters in Swiss albino mice. At an oral dose of 2 mg Se/kg b.w. per day administered for consecutive 28 days, both forms of selenium suppressed mice growth rather than Nano-Se. Abnormal liver and kidney function were more pronounced with selenite treatment than Nano-Se as indicated by the increase of hepatotoxic and renal toxic marker in serum and also confirmed by histological examination. After being treated with different forms of selenium it can be seen that the activity of enzymes have increased considerably in case of Nano-Se. Synthesized selenium nanoparticles, caused less bone marrow cell death and prevented DNA damage, compared to other forms of selenium. Our results suggest that Nano-Se as an antioxidant can serve as a potential chemopreventive agent with reduced risk of selenium toxicity.

Genotoxicity of nanoscale zerovalent iron particles in tobacco BY-2 cells Abstract Nanoremediation by direct injection of nZVI into contaminated water or soil has engendered their liberation into the environment facilitating interaction with plant systems. Available reports display a scarcity of information on the genotoxicity of nZVI in plants. In this study, the cyto-genotoxic potential of nZVI was evaluated in tobacco BY-2 cells treated for 24 h. A wide range of concentrations from 5 to 500 µg/mL of nZVI were tested for cytotoxicity and for other assays a maximum concentration of 20 µg/mL was used. Cytotoxicity assessed by TTC and Evans Blue tests showed maximum cell death in cells treated with the lowest concentration of nZVI (5 µg/mL). Comet assay confirmed high DNA damage at low concentrations, which corroborated with high accumulation of nZVI as observed by Prussian blue staining. Qualitative and quantitative estimation of ROS confirmed the accumulation of superoxide anions, hydroxyl, peroxyl radicals and hydrogen peroxide upon treatment with low concentrations of nZVI. Taken together, this study confirmed ROS mediated cytotoxicity and genotoxicity of nZVI and unravels further prospects in the safety evaluation of nZVI as well as the toxicity testing of other nanomaterials of ecotoxicological relevance.