Ansuman Chattopadhyay

Low dose of arsenic trioxide triggers oxidative stress in zebrafish brain: expression of antioxidant genes.

Occurrence of arsenic in the aquatic environment of West Bengal (India), Bangladesh and other countries are of immediate environmental concern. In the present study, zebrafish (Danio rerio) was used as a model to investigate oxidative stress related enzyme activities and expression of antioxidant genes in the brain to 50µg/L arsenic trioxide for 90 days. In treated fish, generation of reactive oxygen species (ROS), malondialdehyde (MDA) and conjugated diene (CD) showed a triphasic response attaining a peak at the end of the exposure. In addition, a gradual increase in GSH level was noted until 60 days and at 90 days, a sudden fall was recorded which heightened arsenic toxicity. However, GSH level does not correlate well with the glutathione reductase (GR) activity. Generation of ROS in zebrafish brain due to As2O3 exposure was further evidenced by significant alteration of glutathione peroxidase (GPx) and catalase (CAT) activity, which converts H2O2 to water and helps in detoxication. Moreover, enhanced mRNA level of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in As2O3 exposed zebrafish indicates a protective role of Nrf2. kelch-like ECH-associated protein 1 (Keap1), a negative regulator of Nrf2, inversely correlates with the mRNA expression of Nrf2. As2O3 induced toxicity was also validated by the alteration in NRF2 and NRF2 dependent expression of proteins such as heme oxygenase1 (HO1) and NAD(P)H dehydrogenase quinone1 (NQO1). The mRNA expression of glutathione peroxidase (Gpx1), catalase (Cat), manganese superoxide dismutase (Mn-Sod), copper/zinc superoxide dismutase (Cu/Zn Sod) and cytochrome c oxidase1 (Cox1) were also up regulated. The expression of uncoupling protein 2 (Ucp2), an important mitochondrial enzyme was also subdued in arsenic exposed zebrafish. The oxidative stress induced by arsenic also cause reduced mRNA expression of B-cell lymphoma 2 (Bcl2) present in the inner mitochondrial membrane and thereby indicating onset of apoptosis in treated fish. It is concluded that even a low dose of arsenic trioxide is toxic enough to induce significant oxidative stress in zebrafish brain.

Sodium fluoride generates ROS and alters transcription of genes for xenobiotic metabolizing enzymes in adult zebrafish (Danio rerio) liver: expression pattern of Nrf2/Keap1 (INrf2)

Abstract Anthropogenic activities have resulted in an increase in the level of fluoride (F), a natural pollutant in water, causing great threat to the aquatic organisms including fishes. Earlier we reported that sodium fluoride (NaF) exposure alters histological ultrastructure in zebrafish (Danio rerio) liver evidenced by hyperplasia, cytoplasmic degeneration, heteropycnosis etc. In this study, zebrafish were exposed to 7.5, 15 and 30 mg NaF l−1 for 30 days as well as to 15 mg NaF l−1 for 90 days. In NaF treated fish, generation of reactive oxygen species (ROS), depletion of glutathione (GSH) and increase in malondialdehyde (MDA) content along with enhanced activities of oxyradical-scavenging enzymes like catalase (CAT) and superoxide dismutase (SOD) were recorded. Activity of GSH-metabolizing enzyme, glutathione-S-transferase (GST) was also enhanced. The mRNA levels of genes for xenobiotic metabolizing enzymes (XMEs) like cytochrome P450 1A (Cyp1A), NADPH Q Oxidase 1 (Nqo1) and Heme Oxygenase 1 (Ho-1) increased along with nuclear factor (erythroid-derived 2)-like 2 (Nrf2) whereas Kelch-like ECH-associated protein 1 (Keap1) decreased in the treated groups in comparison to their controls. The increase in Nrf2 protein levels in NaF treated fish confirmed its key regulatory role in F-induced oxidative stress. Chromatin condensation and nuclear fragmentations were evidenced in NaF-treated groups indicating possible induction of apoptosis. The modulation of these toxicological parameters at genetic and biochemical levels may be used as an early warning for the environmental risk assessment of F− toxicity to aquatic organisms including fishes.

A single probe for sensing both acetate and aluminum(III): visible region detection, red fluorescence and human breast cancer cell imaging

A single probe (L) can recognise both AcO− and Al3+ as prepared by coupling 2-hydroxy-1-naphthaldehyde with hydrazine. Both the ions provide visible range emission and absorbance upon interaction with the probe allowing their naked eye detections. AcO− sensing is attributed to the formation of hydrogen-bond while Al3+ recognition is due to the combination of three effects, viz. inhibition of excited-state proton transfer (ESPT), CHN isomerization and chelation-enhanced fluorescence (CHEF).

Fluoride‐induced genotoxicity in mouse bone marrow cells: effect of buthionine sulfoximine and N‐acetyl‐ l‐cysteine

A significant level of reactive oxygen species generation was observed in sodium fluoride (NaF) treated mouse bone marrow cells (BMCs). Reduced glutathione (GSH) as a free radical scavenger could be an important determining factor in F‐induced genotoxicity. We therefore attempted to monitor GSH to understand the mechanism of NaF‐induced genotoxicity. NaF was injected intra‐peritoneally in normal, buthionine sulfoximine (BSO) or N‐acetyl‐ l‐cysteine (NAC) treated mice (n = 5). After 13 h of NaF‐treatment BMCs were collected to harvest them at the same divisional cycle and processed for analysis of cell cycle, induction of apoptosis and chromosomal aberrations (CAs). Level of GSH was also measured concomitantly. NaF induced significant CAs in all treatment groups except at 2.5 mg NaF kg−1 body weight. BSO‐treatment alone induced significantly high frequency of CAs. BSO treatment prior to injection of 2.5–7.5 mg NaF kg−1 b.w. was found to increase the frequency of CAs, significantly when compared with the positive control group, but the level was not significant in case of higher doses of NaF treatment (15 and 30 mg kg−1 b.w.). NaF‐treated cells also showed a higher population of Annexin‐V positive cells. NAC pre‐treatment significantly reduced the extent of NaF‐induced CAs, which clearly indicates the involvement of GSH in the NaF response. However, further study is warranted to evaluate the low synergistic effect of BSO on higher doses of NaF‐induced CAs. Copyright

In Vivo Effect of Arsenic Trioxide on Keap1-p62-Nrf2 Signaling Pathway in Mouse Liver: Expression of Antioxidant Responsive Element-Driven Genes Related to Glutathione Metabolism

Arsenic is a Group I human carcinogen, and chronic arsenic exposure through drinking water is a major threat to human population. Liver is one of the major organs for the detoxification of arsenic. The present study was carried out in mice in vivo after arsenic treatment through drinking water at different doses and time of exposure. Arsenic toxicity is found to be mediated by reactive oxygen species. Nuclear factor (erythroid-2 related) factor 2 (Nrf2)/Keap1 (Kelch-like ECH-associated protein 1)/ARE (antioxidant response element)—driven target gene system protects cells against oxidative stress and maintains cellular oxidative homeostasis. Our result showed 0.4 ppm, 2 ppm, and 4 ppm arsenic trioxide treatment through drinking water for 30 days and 90 days induced damages in the liver of Swiss albino mice as evidenced by histopathology, disturbances in liver function, induction of heat shock protein 70, modulation of trace elements, alteration in reduced glutathione level, glutathione-s-transferase and catalase activity, malondialdehyde production, and induction of apoptosis. Cellular Nrf2 protein level and mRNA level increased in all treatment groups. Keap1 protein as well as mRNA level decreased concomitantly in arsenic treated mice. Our study clearly indicates the important role of Nrf2 in activating ARE driven genes related to GSH metabolic pathway and also the adaptive response mechanisms in arsenic induced hepatotoxicity.

Differential modulation of cellular antioxidant status in zebrafish liver and kidney exposed to low dose arsenic trioxide

Zebrafish were exposed to a nonlethal dose (1/350LC50; 50µg/L) of As2O3 and sampled at 7, 15, 30, 60 and 90 days of treatment. The oxidative stress response was assessed in terms of time-dependent histopathological changes, lipid peroxidation, GSH status, activities of detoxification enzymes and expression of antioxidant genes in liver and kidney. As2O3 treatment enhanced lipid peroxidation except at day 90 in liver and day 30 in kidney. Glutathione depleted significantly in the liver except on day 30; whereas in kidney, it increased initially but thereafter depleted significantly. The liver GST activity was high until day 30, low on day 60 and high on day 90. On the other hand, activity of GST in kidney remained high throughout the exposure. GR activity in liver decreased initially but augmented from 30 days onwards whereas in kidney it remained high until 30 days of exposure. Significant increase in GPx and CAT activities in liver and kidney confirmed oxidative stress in zebrafish which correlated with mRNA expression of antioxidant genes. Upregulation in mRNA level of Cu-Zn Sod in liver and kidney was prominent. Gpx1 upregulation was more conspicuous in kidney as compared to liver while the pattern of Cat expression was almost similar in both the organs. Among the mitochondrial genes, expression of Cox1 was significantly high only after 90 days in liver, while in kidney it enhanced at 7, 30 and 60 days of arsenic exposure. Ucp2 was upregulated in liver after 15 days of exposure but significantly downregulated at day 90; in kidney it remained unchanged at other time points except at day 90. An overall increased expression of Bcl2 further confirmed As2O3 induced oxidative stress in zebrafish liver and kidney. The pattern of mRNA expression of Nrf2 was not uniform and was in accordance to its downstream antioxidant genes. Present findings elucidate that low dose of As2O3 exposure induces a time dependent differential modulation of antioxidant status in liver and kidney of zebrafish in a tissue-specific manner.

Regulation of autophagy in rat hepatocytes treated in vitro with low concentration of mercury

Several proteins are implicated in the regulation of autophagy in cells under various physiological and pathological conditions. Recently it was found that in vitro initiation of autophagy in 5 μM mercuric chloride (HgCl2) treated rat hepatocytes occurred within 30 min of incubation. The aim of this study was to monitor the autophagy–ubiquitination link to determine the regulatory mechanisms underlying autophagy at a concentration of 5 μM HgCl2. Autophagic cell death was evidenced by the presence of LC3B positive hepatocytes throughout the incubation period of 4 hr. Autophagosome maturation occurred following 1 hr of metal treatment accompanied by an enhanced expression of several regulatory proteins such as p62, Keap1, and caspase-8, which drive the hepatocytes toward autophagy. In addition, the rate of increase in down-regulation in a p38 expression was significantly higher than that of extracellular signal-regulated kinase (ERK) at 1 hr incubation, followed by a significant decline in the p38 level at 2 and 4 hr following metal treatment. In contrast, the expression of ERK remained higher than that of p38 at 2 and 4 hr. Data indicate that intracellular activation of the major regulatory proteins such as LC3B, caspase-8, Keap1, p62, ERK, and p38 modulates autophagy through ubiquitination in rat hepatocytes exposed to low concentrations of Hg.

Reduction in fluoride-induced genotoxicity in mouse bone marrow cells after substituting high fluoride-containing water with safe drinking water

Treatment of mice with 15 mg l−1 sodium fluoride (NaF) for 30 days increased the number of cell death, chromosomal aberrations (CAs) and ‘cells with chromatid breaks’ (aberrant cells) compared with control. The present study was intended to determine whether the fluoride (F)‐induced genotoxicity could be reduced by substituting high F‐containing water after 30 days with safe drinking water, containing 0.1 mg F ions l−1. A significant fall in percentage of CAs and aberrant cells after withdrawal of F‐treatment following 30 days of safe water treatment in mice was observed which was highest after 90 days, although their levels still remained significantly high compared with the control group. This observation suggests that F‐induced genotoxicity could be reduced by substituting high F‐containing water with safe drinking water. Further study is warranted with different doses and extended treatment of safe water to determine whether the induced damages could be completely reduced or not. Copyright

Sodium fluoride affects zebrafish behaviour and alters mRNA expressions of biomarker genes in the brain: Role of Nrf2/Keap1

Sodium fluoride (NaF), used as pesticides and for industrial purposes are deposited in the water bodies and therefore affects its biota. Zebrafish exposed to NaF in laboratory condition showed hyperactivity and frequent surfacing activity, somersaulting and vertical swimming pattern as compared to the control group. Reactive oxygen species level was elevated and glutathione level was depleted along with increased malondialdehyde content in the brain. Levels of glutathione-s-transferase (GST), catalase (CAT) and superoxide dismutase were also elevated in the treatment groups. Expression of mRNA of nuclear factor erythroid 2 related factor 2 (Nrf2) and its inhibitor Kelch-like ECH-associated protein 1 (Keap1) during stress condition were observed along with Gst, Cat, NADPH: quinone oxidoreductase 1(Nqo1) and p38. Except Keap1, all other genes exhibited elevated expression. Nrf2/Keap1 proteins had similar expression pattern as their corresponding mRNA. The findings in this study might help to understand the molecular mechanism of fluoride induced neurotoxicity in fish.

Rhodamine derived colorimetric and fluorescence mercury(II) chemodosimeter for human breast cancer cell (MCF7) imaging

Condensation of rhodaminehydrazone with naphthalene-1-carboxaldehyde generates a colourless probe, RDHDNAP that can selectively detect Hg2+ through generation of a pink color along with significant fluorescence enhancement. The binding constant and lowest detection limit for Hg2+ are 2.0 × 105 M−1 and 3 × 10−7 M respectively. Hg2+ imaging in human breast cancer cells (MCF7) under a fluorescence microscope is achieved.