Environmentally Relevant Hexavalent Chromium Disrupts Elemental Homeostasis and Induces Apoptosis in Zebrafish Liver.

Although hexavalent chromium Cr [VI] is known as a toxicant in the aquatic environment, its effect in low, environmentally relevant concentration (ERC; 2 mg L-1) is less characterized. Against this backdrop, the effects of Cr [VI] in ERC on zebrafish liver has been investigated in this study. Fluorescence microscopy and gel electrophoresis detected excess DNA damage and cell death via apoptosis in 2 mg L-1 Cr [VI]-treated fish when compared with that of control. Besides, there were transcriptional activations of p53, Bax, Caspase 9 and Caspase 3 genes but downregulation of Bcl2 gene in the treated group, confirming the apoptotic pathway. Energy dispersive X-ray fluorescence (EDXRF) data showed significant (p < 0.05) increase in hepatic content of Cr, selenium, iron, manganese, calcium, sulfur and magnesium but depletion of zinc, copper and cobalt in the treated group. Collectively, the study shows that even a low, ERC of Cr [VI] is toxic to the zebrafish as it elicited marked apoptosis in the hepatocytes and altered the liver elemental profile.

Chronic exposure to environmentally relevant concentration of fluoride alters Ogg1 and Rad51 expressions in mice: Involvement of epigenetic regulation.

Chronic exposure to fluoride (F) beyond the permissible limit (1.5 ppm) is known to cause detrimental health effects by induction of oxidative stress-mediated DNA damage overpowering the DNA repair machinery. In the present study, we assessed F induced oxidative stress through monitoring biochemical parameters and looked into the effect of chronic F exposure on two crucial DNA repair genes Ogg1 and Rad51 having important role against ROS induced DNA damages. To address this issue, we exposed Swiss albino mice to an environmentally relevant concentration of fluoride (15 ppm NaF) for 8 months. Results revealed histoarchitectural damages in liver, brain, kidney and spleen. Depletion of GSH, increase in lipid peroxidation and catalase activity in liver and brain confirmed the generation of oxidative stress. qRT-PCR result showed that expressions of Ogg1 and Rad51 were altered after F exposure in the affected organs. Promoter hypermethylation was associated with the downregulation of Rad51. F-induced DNA damage and the compromised DNA repair machinery triggered intrinsic pathway of apoptosis in liver and brain. The present study indicates the possible association of epigenetic regulation with F induced neurotoxicity.

Calcium and Vitamin D Supplementation Effectively Alleviates Dental and Skeletal Fluorosis and Retain Elemental Homeostasis in Mice.

Fluoride (F) is an essential trace element, but chronic exposure beyond the permissible limit (1.5 ppm) effectuates dental and skeletal fluorosis. Although 200 million people across the world are suffering from toxic manifestations of F, till now proper treatment is not available. In this study, we assessed the effectiveness of calcium and vitamin D supplementation for alleviation of fluorosis. Swiss albino mice were divided into 6 groups; group I-control group (received drinking water ˂ 0.5 ppm F; within the permissible limit), group II-treated with 15 ppm of sodium fluoride (NaF) for 4 months, group III-treated with 15 ppm of NaF for 8 months through drinking water. Group IV-orally treated with 15 ppm NaF for 4 months, thereafter received only drinking water for next 4 months, group V-orally treated with 15 ppm NaF for 4 months, thereafter received drinking water supplemented with calcium and vitamin D (2.5-g calcium kg-1 diet and 1000 IU vitamin D kg-1 diet) for next 4 months, and group VI was treated with 15 ppm of NaF through drinking water as well as supplemented with calcium and vitamin D for 4 months. NaF treatment caused dental fluorosis, skeletal fluorosis, and alteration of bone's metal profile. Substitution of NaF-containing water with normal drinking water reduced the severity of fluorosis but supplementation of calcium and vitamin D effectively alleviated dental and skeletal fluorosis, reduced F deposition, and retained elemental homeostasis of the bone. Our findings strongly support that calcium and vitamin D act as redeemer of fluorosis. Graphical Abstract.

Combined effect of arsenic and fluoride at environmentally relevant concentrations in zebrafish (Danio rerio) brain: Alterations in stress marker and apoptotic gene expression.

Arsenic and fluoride are two naturally occurring toxicants to which various organisms including a major part of the human populations are co-exposed to. However, interactions between them inside body are quite complicated and needs proper evaluation. Inconclusive reports regarding their combined effects on brain prompted us to conduct this study where we investigated their individual as well as combined effects on female zebrafish brain at environmentally relevant concentrations (50 µgL-1 arsenic trioxide and 15 mgL-1 sodium fluoride) after different time intervals (15, 30 and 60 days). Persistent near-basal level of GSH, least increased MDA content and catalase activity portrayed arsenic and fluoride co-exposure as less toxic which was corroborated with far less damage caused in the histoarchitecture of optic tectum region in midbrain. Stress-responsive genes viz., Nrf2 and Hsp70 were overexpressed after individual as well as combined exposures, indicating a common cellular response to combat the formed oxidative stresses. Biphasic response of AChE upon individual exposure confirmed their neurotoxic effects too. Expression profile of p53 (unaltered), Bax (lower or near-basal) and Bcl2 (comparatively higher), along with absence of DNA fragmentation indicated no induction of apoptosis in the co-exposed group. Tissue accumulation of arsenic and fluoride was significantly less in the brain of co-exposed zebrafish when compared to their individual exposures. This preliminary study indicates an antagonistic effect of these two toxicants in zebrafish brain and needs further studies involving oxidative stress independent markers to understand the detailed molecular mechanism.

Mixture effect of arsenic and fluoride at environmentally relevant concentrations in zebrafish (Danio rerio) liver: Expression pattern of Nrf2 and related xenobiotic metabolizing enzymes.

Nrf2 is a crucial transcription factor that regulates the expression of cytoprotective enzymes and controls cellular redox homeostasis. Both arsenic and fluoride are potent toxicants that are known to induce Nrf2. They are reported to coexist in many areas of the world leading to complex mixture effects in exposed organisms. The present study investigated the expression of Nrf2 and related xenobiotic metabolizing enzymes along with other stress markers such as histopathological alterations, catalase activity, reduced glutathione content and lipid peroxidation in zebrafish liver as a function of combined exposure to environmentally relevant concentrations of arsenic (37.87 µgL-1 or 5.05 × 10-7 M) and fluoride (6.8 mg L-1 or 3.57 × 10-4 M) for 60 days. The decrease in the total reduced glutathione level was evident in all treatment conditions. Hyperactivity of catalase along with conspicuous elevation in reactive oxygen species, malondialdehyde content and histo-architectural anomalies signified the presence of oxidative stress in the treatment groups. Nrf2 was seen to be induced at both transcriptional and translational levels in case of both individual and co-exposure. The same pattern was observed in case of its nuclear translocation also. From the results of qRT-PCR it was evident that at each time point co-exposure to arsenic and fluoride seemed to alter the gene expression of Cu/Zn Sod, Mn Sod, Gpx and Nqo1 just like their individual exposure but at a very low magnitude. In conclusion, this study demonstrates for the first time the differential expression and activity of Nrf2 and other stress response genes in the zebrafish liver following individual and combined exposure to arsenic and fluoride.

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.