ROS-dependent genotoxicity, cell cycle perturbations and apoptosis in mouse bone marrow cells exposed to formulated mixture of cypermethrin and chlorpyrifos.

Swiss albino mice were exposed to formulated cypermethrin (CMR) and/or or chlorpyrifos (CPF) through oral gavages for 60 days. Test doses of CMR (0.69, 1.38 or 2.76mg/kg/day) or CPF (0.5, 1.0 or 2.0mg/kg/day) or CMR + CPF (0.69 + 0.5, 1.38 + 1.0 or 2.76 + 2.0mg/kg/day) were based on the acute oral median lethal doses of CMR or CPF. Chromosome aberrations (CA), micronucleus (MN) induction, cell cycle perturbations, apoptosis and reactive oxygen species (ROS) generation were analysed in bone marrow cells. To explore the involvement of ROS induction, HaCat cells were exposed in vitro to arbitrary concentrations of CMR and/or CPF. Exposure of CMR (2.76mg/kg/day) induced significant inhibition of mitotic index. Significant (P < 0.01) frequencies of CA and MN were observed with the CMR at 1.38mg/kg/day, whereas CPF or its mixture CMR + CPF showed at highest doses. Chromosome/chromatid breaks and fragments were found to be major aberrations in all the treatment groups. Highest doses of CMR or CMR + CPF revealed significant (P < 0.01 or 0.001) elevation of G0/G1 peak, while CPF-exposed cells revealed significant (P < 0.01) declined in G1 phase. Decline in S phase was observed with highest dose of CMR only. Apoptosis induction measured by gating cell population beside G1 peak showed 3- to 4-fold increase in apoptotic cells in CPF-exposed mice as compared to control or CMR or CMR + CPF-treated mice. Further, all the treatment groups in vivo as well as in vitro revealed significant generation of ROS in comparison with the control group. Present results, together with the earlier reports, which substantiate ROS generation may be major cause of genotoxicity, cell cycle perturbations and apoptosis, nonetheless co-exposure of low doses of CMR and CPF mixture does not potentiate genotoxicity.

UVB exposure enhanced the dermal penetration of zinc oxide nanoparticles and induced inflammatory responses through oxidative stress mediated by MAPKs and NF-κB signaling in SKH-1 hairless mouse skin.

Besides titanium dioxide (TiO2), zinc oxide (ZnO) nanoparticles (NPs) are commonly used in sunscreen formulations as protective agents against exposure to ultraviolet radiation. Although the majority of prior studies have concluded that NPs do not penetrate healthy skin, compromised skin slightly enhanced metal oxide NP penetration. However, a question arises regarding the possible toxic consequences if consumers who had applied sunscreens containing ZnO-NPs were exposed to environmentally relevant doses of UVB. Considering this, we planned a study where SKH-1 hairless mice were topically exposed to a 5% and/or 10% dose of ZnO-NPs (<50 nm and <100 nm) either alone or along with UVB (50 mJ cm-2). In two additional groups, mice were treated with either bulk ZnO-NPs (<5 µm) or with ZnO-NPs (<5 µm) and subsequently UVB (50 mJ cm-2). Animals of all groups were sacrificed after 6, 24 and 48 h and the Zn ion content in the skin was measured. In addition, estimation of ROS generation, histopathology, myeloperoxidase (MPO) activity, immunohistochemistry for COX-2 and western blot analysis for MAPKs, p-IκBα, p-NF-κB, and COX-2 were also carried out. Significant increases in the Zn ion in exposed skin were seen. Enhanced ROS generation and MPO activity were also found in ZnO-NPs followed by UVB exposed groups at all three time points. Similarly, hyperplasia and over-expression of COX-2 were also greater in ZnO-NPs and UVB exposed groups than in the ZnO-NPs and UVB only groups. The expression of MAPKs, and transcription factors NF-κB along with COX-2 were also enhanced significantly in ZnO-NPs and the UVB treated group. Collectively, our findings suggest that UVB exposure enhanced ZnO-NP penetration in mouse skin and possibly dissolution of these ZnO-NPs takes place during this process, causing significant Zn ion generation leading to oxidative stress by ROS generation which subsequently activates MAPK-NF-κB signaling and increases COX-2 and inflammation.

Unraveling the mechanism of neuroprotection of curcumin in arsenic induced cholinergic dysfunctions in rats.

Earlier, we found that arsenic induced cholinergic deficits in rat brain could be protected by curcumin. In continuation to this, the present study is focused to unravel the molecular mechanisms associated with the protective efficacy of curcumin in arsenic induced cholinergic deficits. Exposure to arsenic (20mg/kg body weight, p.o) for 28 days in rats resulted to decrease the expression of CHRM2 receptor gene associated with mitochondrial dysfunctions as evident by decrease in the mitochondrial membrane potential, activity of mitochondrial complexes and enhanced apoptosis both in the frontal cortex and hippocampus in comparison to controls. The ultrastructural images of arsenic exposed rats, assessed by transmission electron microscope, exhibited loss of myelin sheath and distorted cristae in the mitochondria both in the frontal cortex and hippocampus as compared to controls. Simultaneous treatment with arsenic (20mg/kg body weight, p.o) and curcumin (100mg/kg body weight, p.o) for 28 days in rats was found to protect arsenic induced changes in the mitochondrial membrane potential and activity of mitochondrial complexes both in frontal cortex and hippocampus. Alterations in the expression of pro- and anti-apoptotic proteins and ultrastructural damage in the frontal cortex and hippocampus following arsenic exposure were also protected in rats simultaneously treated with arsenic and curcumin. The data of the present study reveal that curcumin could protect arsenic induced cholinergic deficits by modulating the expression of pro- and anti-apoptotic proteins in the brain. More interestingly, arsenic induced functional and ultrastructural changes in the brain mitochondria were also protected by curcumin.

Status of Stat3 in an ovalbumin-induced mouse model of asthma: analysis of the role of Socs3 and IL-6.

BACKGROUND: Stat3, Socs3 and cytokines play an integral role in the coordination and persistence of inflammation. However, a clear understanding of the role played by the Stat3/IL-6 and Socs3 pathway in airway inflammation is lacking. We report the alteration in the status of expression and activation of Stat3 by ovalbumin (OVA), and establish its relationship with Socs3 and IL-6 in the lungs of mice with eosinophilic pulmonary inflammation and airway hyperresponsiveness. METHODS: Alterations in the expression of Stat3, Socs3 and IL-6 were determined in a murine model of asthma, where Balb/c mice were sensitized and challenged with OVA (OVA/OVA) and compared with control mice sensitized and challenged with saline (SAL) (SAL/SAL) mice. The OVA/OVA mice were characterized by a moderate increase in methacholine-induced specific airway resistance, the presence of 150 microg/ml of OVA-specific IgG and 8.93 microg/ml OVA-specific IgE antibody and elevated levels of eosinophils and Th2 cytokines (IL-4 and IL-5) in the bronchoalveolar lavage fluid. In contrast SAL/SAL mice had low eosinophils, IL-4 and IL-5 and no OVA-specific IgG and IgE antibodies in the BALF. Stat3 and Socs3 expression profiles were monitored in OVA/OVA and Stat3- and Socs3-silenced OVA/OVA mice. Furthermore, expression of IL-6 in Stat3- and Socs3-silenced mice and the exogenous effect of IL-6 on Stat3 were studied. RESULTS: The results show that expression and activation of Stat3 mRNA and proteins are significantly low in lung of OVA/OVA mice in comparison to SAL/SAL mice following OVA challenge. An increased pool of Socs3 mRNA is observed in OVA/OVA mice with or without OVA challenge and in SAL/SAL mice 24 h after OVA challenge. Transient in vivo blocking of Socs3 gene by Socs3 siRNA restores the expression of IL-6 mRNA and protein in OVA/OVA mice, and nasal administration of recombinant IL-6 to OVA/OVA mice enhanced Stat3 mRNA expression. CONCLUSIONS: Our data suggest that airway inflammation is associated with low expression of Stat3 and IL-6 and overexpression of Socs3 genes in a mouse model of asthma. Furthermore, IL-6 is under the influence of the Socs3 gene and may contribute to the negative regulation of Stat3 via IL-6 following a challenge with an allergen during the development of asthma.

Cytogenetic effects of commercial formulations of deltamethrin and/or isoproturon on human peripheral lymphocytes and mouse bone marrow cells.

The cytogenetic effects of deltamethrin (DEL) and/or isoproturon (ISO) were examined in human lymphocytes and mouse bone marrow cells. Peripheral lymphocytes were exposed to DEL (2.5, 5, 10, or 20 microM), ISO (25, 50, 100, or 200 microM), or DEL + ISO (2.5 + 25, 5 + 50, 10 + 100, or 20 + 200 microM) and cytogenic effects were evaluated via chromosomal aberrations (CA) and the cytokinesis-block micronucleus assay (CBMN). Mice were orally gavaged to single dose of DEL (6.6 mg/kg), ISO (670 mg/kg), or DEL+ISO (6.6 + 670 mg/kg) for 24 hr or to DEL (3.3 mg/kg/day), ISO (330 mg/kg/day), or DEL + ISO (3.3 + 330 mg/kg/day) for 30 days and analyzed for CA. DEL induced a significant frequency of CA at 10 microM whereas ISO (25-100 microM) alone, or in combination with DEL, did not show any significant effect. Micronucleus (MN) induction was observed to be concentration-dependent though significant frequencies were observed at 5 microM DEL, 100 microM ISO, or 5 + 50 microM DEL + ISO. In mice, DEL inhibited the mitotic index (MI) significantly (P < 0.001) at 24 hr while ISO alone, or in combination with DEL, did not cause any statistically significant effect. Following a 24 hr exposure, DEL and ISO alone induced significant (P < 0.01) frequencies of CA, whereas DEL + ISO in combination did not. Furthermore, 30 days exposure of ISO significantly inhibited the MI (P < 0.02 or < 0.01) and induced CA while DEL alone, or in combination with ISO, resulted in no significant effect on CA or the MI. The present findings indicate that the in vitro and in vivo exposure of a commercial formulation of DEL can cause genotoxic effects in mammals. However, the coexposure of DEL and ISO did not show additive effects, but instead demonstrated somewhat reduced genotoxicity.

In vivo genotoxic effects of industrial waste leachates in mice following oral exposure.

Contamination of ground water by industrial waste poses potential health hazards for man and his environment. The improper disposal of toxic wastes could allow genotoxic chemicals to percolate into ground waters, and these contaminated ground waters may produce toxicity, including mutation and eventually cancer, in exposed individuals. In the present study, we evaluated the in vivo genotoxic potential of leachates made from three different kinds of industrial waste (tannery waste, metal-based waste, and waste containing dyes and pigments) that are disposed of in areas adjoining human habitation. Three different doses of test leachates were administered by oral gavage for 15 consecutive days to Swiss albino mice; their bone marrow cells were examined for chromosome aberrations (CAs), micronucleated polychromatic erythrocytes (MNPCEs), and DNA damage using the alkaline Comet assay. Exposure to the leachates resulted in significant (P < 0.05 or P < 0.001) dose-dependent increases in chromosome and DNA damage. Fragmented chromosomes and chromatid breaks were the major CAs observed. Chemical analysis of the leachates indicated that chromium and nickel were elevated above the limits established by health organizations. The highest levels of genotoxicity were produced by the metal-based leachate and the tannery-waste leachate, while the dye-waste leachate produced weaker genotoxic responses. The cytogenetic abnormalities and DNA damage produced by the leachates indicate that humans consuming water contaminated with these materials are at increased risk of developing adverse health consequences.

Ecotoxicological evaluation of municipal sludge.

Municipal wastes originating from urban and industrial areas have become a major source of soil, ground and surface water pollution. These undesirable agents in our environment significantly interact with our flora and fauna. The aim of this study was to test samples of municipal sludge (MS) for their ecotoxicological potential by using sensitive bioassays involving a plant, Vicia faba, and the earthworm, Eisenia foetida. A 10% leachate of MS was prepared for the experiments, and V. faba seedlings were exposed to three leachate concentrations (2.5%, 5% and 10%) for 5 days. The findings revealed chromosome aberrations during the metaphase as well as the anaphase of cell division, and inhibition of the mitotic index, which reflects that MS originating from domestic and other human activities may be genotoxic to the living organisms of the ecosystem. Abnormalities in chlorophyll content, plant growth, root length, shoot length and root/shoot length ratio in V. faba clearly indicated the toxicity of the sludge. Behavioural and reproduction studies with E. foetida also provided evidence for the toxic nature of the MS.

Unequivocal evidence of genotoxic potential of argemone oil in mice.

Consumption of mustard oil adulterated with argemone oil leads to a clinical condition, commonly referred to as "Epidemic Dropsy." Since in vitro studies have shown that sanguinarine, an active benzophenanthridine alkaloid of argemone oil, intercalates DNA molecule, the in vivo clastogenic and DNA damaging potential of argemone oil was investigated in mice. Swiss albino mice were intraperitoneally administered 0.5, 1.0, 2.0 and 4.0 ml/kg body wt. of argemone oil to analyze chromosome aberrations and micronucleus test, while 0.25, 0.5, 1.0 and 2.0 ml/kg body wt. were given for alkaline comet assay. The frequencies of chromosomal aberrations and micronucleated erythrocytes formation in mouse bone marrow cells increased in a dose-dependent manner following argemone oil treatment. However, significant induction in chromosomal aberrations (83%) and micronucleated erythrocytes formation (261%) were observed at a minimum dose of 1.0 ml/kg. The results of comet assay revealed DNA damage in blood, bone marrow and liver cells following argemone oil treatment. Olive tail moment (OTM) and tail DNA showed significant increase in bone marrow (35-44%) and blood cells (25-40%) even at a dose of 0.25 ml/kg body wt. of argemone oil. In liver cells, OTM was significantly increased (20%) at a dose of 0.25 ml/kg, while all the comet parameters including OTM, tail length and tail DNA showed significant increase (31-101%) at a dose of 0.5 ml/kg. These results clearly suggest that single exposure of argemone oil even at low doses produces genotoxic effects in mice.