Shelley Bhattacharya

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.

Lipid induced overexpression of NF-κB in skeletal muscle cells is linked to insulin resistance

Lipid induced NF-kappaB activation is known to be associated with insulin resistance and type2 diabetes. Here we show that incubation of L6 skeletal muscle cells with palmitate significantly increased NF-kappaB p65 and NF-kappaB p50 expression along with their phosphorylation. NF-kappaB p65 siRNA inhibited palmitate induced overexpression of NF-kappaB p65 indicating palmitate effect on transcriptional activation. RT-PCR and real time PCR experiments also showed a significant increase in NF-kappaB p65 gene expression due to palmitate. Overexpression of NF-kappaB p65 by palmitate was linked to impairment of insulin activity. Palmitate effect on NF-kappaB gene and protein expression was found to be mediated by phospho-PKCepsilon as calphostin C (an inhibitor of PKC) and epsilonV1 (PKCepsilon translocation inhibitor) significantly reduced NF-kappaB expression. To understand the underlying mechanism, we purified NF-kappaB and pPKCepsilon from palmitate incubated skeletal muscle cells and their interaction in cell free system demonstrated the transfer of phosphate from PKCepsilon to NF-kappaB. This prompted us to transduct pPKCepsilon to the skeletal muscle cells. These cells showed increased amount of pNF-kappaB and NF-kappaB. Excess of NF-kappaB p65 pool thus created in the cells made them insulin resistant. Addition of NF-kappaB p65 siRNA and SN50 inhibited palmitate induced NF-kappaB p65 expression indicating NF-kappaB regulation of its gene expression. Increase of NF-kappaB did not affect the activation of IKK/IkappaB indicating NF-kappaB p65 expression to be a distinct effect of palmitate. Since NF-kappaB p65 is linked to several diseases, including type2 diabetes, this report may be important in understanding the pathogenicity of these diseases.

Toxic Metals and Autophagy

The earths resources are finite, and it can no longer be considered a source of inexhaustible bounty for the human population. However, this realization has not been able to contain the human desire for rapid industrialization. The collateral to overusing environmental resources is the high-level contamination of undesirable toxic metals, leading to bioaccumulation and cellular damage. Cytopathological features of biological systems represent a key variable in several diseases. A review of the literature revealed that autophagy (PCDII), a high-capacity process, may consist of selective elimination of vital organelles and/or proteins that intiate mechanisms of cytoprotection and homeostasis in different biological systems under normal physiological and stress conditions. However, the biological system does survive under various environmental stressors. Currently, there is no consensus that specifies a particular response as being a dependable biomarker of toxicology. Autophagy has been recorded as the initial response of a cell to a toxic metal in a concentration- and time-dependent manner. Various signaling pathways are triggered through cellular proteins and/or protein kinases that can lead to autophagy, apoptosis (or necroptosis), and necrosis. Although the role of autophagy in tumorigenesis is associated with promoting tumor cell survival and/or acting as a tumor suppressive mechanism, PCDII in metal-induced toxicity has not been extensively studied. The aim of this review is to analyze the comparative cytotoxicity of metals/metalloids and nanoparticles (As, Cd, Cr, Hg, Fe, and metal-NP) in cells enduring autophagy. It is noted that metals/metalloids and nanoparticles prefer ATG8/LC3 as a potent inducer of autophagy in several cell lines or animal cells. MAP kinases, death protein kinases, PI3K, AKT, mTOR, and AMP kinase have been found to be the major components of autophagy induction or inhibition in the context of cellular responses to metals/metalloids and nanoparticles.

Detoxication of industrial pollutants by the glutathione glutathione-S-transferase system in the liver of Anabas testudineus (Bloch)

The interrelationship of reduced glutathione (GSH) and glutathione-S-transferase in the liver of a freshwater climbing perch Anabas testudineus (Bloch) exposed to common industrial pollutants has been studied. In both short- and long-term treatments there was a concomitant decrease in reduced glutathione profile and an increase in glutathione-S-transferase activity. It may be surmised that the majority of xenobiotics of industrial origin are detoxicated by the glutathione glutathione-S-transferase pathways enabling the fish to survive exposure to the additive and/or synergistic toxicity of mixtures of poisons.

Histopathology of kidney of Channa punctatus exposed to chronic nonlethal level of Elsan, mercury, and ammonia

Histopathological changes in the head and trunk kidneys of Channa punctatus induced by chronic nonlethal levels of Elsan (211 ppb), mercuric chloride (16.7 ppb), and aqueous ammonia (15.64 ppm) were studied on 7, 28, 63, and 90 days of exposure. The pathology of the head kidney was characterized by degeneration and dispersion of interrenal and chromaffin tissue and necrosis in the haemopoietic elements. Kidney lesions were observed throughout the entire experimental period in fish exposed to Elsan and mercuric chloride. In contrast, the lesion induced by exposure to aqueous ammonia began to heal during the first phase of treatment. Marked abnormalities in trunk kidney histology were also found. Renal lesions consisted of minimal to mild multifocal, acute tubular epithelial degeneration, karyolysis, and dilation or shrinkage of Bowmans capsule and glomerulus. Elsan treatment resulted in a highly significant decrease in the dimension of Bowmans capsule and glomerulus at all days of sampling, except on Day 28. The response of the fish trunk kidney tissue to mercuric chloride was similar to that observed with Elsan exposure in terms of the alteration in the mean dimensions of Bowmans capsule and glomerulus. The response to ammonia was significant reduction in the size of Bowmans capsule and glomerulus throughout the experimental period except at Day 28. Little dilation of Bowmans capsule and a significant dilation of glomerulus were found at Day 28 of ammonia exposure. This study demonstrated that a chronic nonlethal exposure to Elsan, mercuric chloride affect both endocrine and excretory parts of the kidney while ammonia specifically damages the excretory part of the kidney of C. punctatus.

Ovarian damage to Channa punctatus after chronic exposure to low concentrations of elsan, mercury, and ammonia

Histopathological changes were induced in the ovary by chronic nonlethal levels of Elsan (211 ppb), mercury (16.7 ppb), and ammonia (15.64 ppm). In the breeding season, treated Channa punctatus revealed a significant decrease in ovarian weight throughout the duration of the experiment. Acute changes were noted in the diameter and percentage occurrence of the different stages of oocytes. In all cases, stage I oocytes revealed no difference in diameter with respect to control although they demonstrated an increase in the percentage occurrence. For stages II and III, a remarkable decrease occurred in both the number and the diameter of these mature oocytes. The preponderance of stage I and destruction of stage II and stage III oocytes in all three treatments indicate that chemically diverse xenobiotics have equal reproductive toxicity in fish, interfering with their breeding.

Vermicomposting of Tea Factory Coal Ash: metal accumulation and metallothionein response in Eisenia fetida (Savigny) and Lampito mauritii (Kinberg).

Earthworms can accumulate heavy metals in their intestines to a great extent. Impact of feed materials and duration of metal exposure on natural activity of earthworms are rather unclear; this investigation therefore addresses the impact of metal rich Tea Factory Coal Ash (TFCA) on reproduction, composting and metal accumulation ability of Eisenia fetida and Lampito mauritii. Earthworm count and cocoon production increased significantly during vermicomposting. pH of the vermicomposted mixtures shifted toward neutrality, total organic C decreased substantially and total N enhanced significantly compared to composting. High heavy metal (Mn, Zn, Cu, As) accumulation was recorded in the intestine of both the earthworm species. Moreover, gradual increase in the metal-inducible metallothionein concentration indicated the causal mechanism of metal accumulation in these species. TFCA+cow dung (CD) (1:1) were most favorable feed mixture for E. fetida and TFCA+CD (1:2) were good for L. mauritii in regard to metal accumulation and compost quality.

Inorganic mercury binding to fish oocyte plasma membrane induces steroidogenesis and translatable messenger RNA synthesis.

Both in vitro and in vivo HgCl treatment demonstrated a remarkably high rate of progesterone synthesis accompanied by a low rate of conversion to 17β-estradiol in the oocyte of Channa punctatus. On depuration, however, there was a reversal of the steroidogenic scenario with a low progesterone and high estradiol level. The accumulation of progesterone was positively correlated with the significant increase in 3β-hydroxysteroid dehydrogenase activity in the Hg-treated fish. Thus, it was clear that at the early stage of intoxication Hg does play a role in the induction of 3β-hydroxysteroid dehydrogenase in the oocyte of fish at the spawning stage. The induction of this enzyme was found to be mediated by specific binding of Hg to the plasma membrane Na-K-ATPase (B: 14 nmoles mg protein; K 1.14 x 108 moles) and increase in the specific messenger RNA translating 3β-hydroxysteroid dehydrogenase. It is concluded that inorganic mercury is able to initiate translatable messenger RNA synthesis in fish oocyte at a low degree of intoxication.

Metallothionein response in earthworms Lampito mauritii (Kinberg) exposed to fly ash.

Among pollutants, the coal fly ash occupies a significant position in industrial wastes. The fly ash matrix is a complex mixture of various organic (polyhalogenated compounds) and inorganic (Si, Al, Fe, As, Cd, Bi, Hg, etc.) chemicals. The application of fly ash for agricultural purposes and as landfills may lead to the contamination of the land with some of the toxic chemical compounds present in fly ash. Thus prior to the application of fly ash for developmental activities, it requires bio-monitoring and risk characterization. In order to achieve this objective adult Lampito mauritii were exposed to different proportions of fly ash in soil for 30 d and the concentrations of metallothionein in earthworm were assessed. The results revealed that up to 50% of fly ash amendment does not apparently harm the earthworm in respect of their survival and growth. A significant increase in tissue metallothionein level was recorded in L. mauritii exposed to fly ash amended soil without tissue metal accumulation indicating that metallothionein is involved in scavenging of free radicals and reactive oxygen species metabolites. It is concluded that this biochemical response observed in L. mauritii exposed to fly ash amended soil could be used in ecotoxicological field monitoring.

In vitro toxicity of mercury, cadmium, and arsenic to platelet aggregation: Influence of adenylate cyclase and phosphodiesterase activity

In vitro effect of mercury (Hg2+), cadmium (Cd2+), and arsenic (As3+) on adenylate cyclase (AC) and phosphodiesterase (PDE) activity in relation to platelet aggregation (PA) was studied in rats. Cd(2+) significantly elevated cAMP (p < 0.005) in a dose-dependent (5, 10 and 20 pmoles) manner while Hg(2+) and As(3+) significantly reduced the cAMP level (p < 0.01 and p < 0.005, respectively). Our studies further reveal that Hg21 and As(3+) inhibit AC and stimulate PDE activity with a concomitant increase in the rate of PA. On the other hand, Cd(2+) stimulates AC and inhibits PDE activity with a decrease in the rate of PA. The present investigation suggests that cellular cAMP is a regulatory molecule in the event of PA and the disruption of its homeostasis is directly correlated to xenobiotic effects on PA. It is concluded that other than divalent heavy metal cations, As(3+) appears to be one of the most toxic xenobiotics to platelet function.