Combined Effects of Fluoride and Arsenic on Mitochondrial Function in the Liver of Rat.

Biochemical and/or molecular mechanisms of arsenic or fluoride toxicity in experimental animals have been widely investigated in the recent past. However, their combined effects on target cells/organelle are poorly understood. The present study was executed to delineate their combined effects on mitochondrial function in the liver of rat. Female Wistar rats (140 ± 20 g) were force fed individually or in combination with sodium arsenate (4 mg/kg body weight) and sodium fluoride (4 mg/kg body weight) for 90 days. Thereafter, established markers of mitochondrial function viz. mitochondrial lipid peroxidation, oxidative phosphorylation, ATPase, succinic dehydrogenase, and caspase-3 activity were determined. Cytochrome C release and oxidative DNA damage were also estimated in the liver of respective groups of rats. The study showed significant differences in these results amongst the three groups. Observations on parameters viz. LPO, cytochrome-C, caspase-3, and 8-OHdG suggested an antagonistic relationship between these two elements. Results on ATPase, SDH, and ADP:O ratio indicated synergism. It is concluded that AsIII + F in combination may express differential effects on signalling pathways and proapoptotic/antiapoptotic proteins/genes that contribute to liver cell death. Interaction of As and F with mitochondria.

Significance of Inflammation and Apoptosis in Hepatocellular Death in Rat, Co-treated with Arsenic and Fluoride.

Health effects elicited by combined environmental exposures to xenobiotics, in many instances, still remain unresolved. One of these examples is the combined toxicity of arsenic and fluoride. The present study was undertaken to delineate the role of inflammation and apoptosis in hepatocellular death caused by co-exposure to arsenic and fluoride in rat. Sodium arsenate (4 mg/kg body weight) and sodium fluoride (4 mg/kg body weight) were administered to female Wistar rats, individually and in combination, for 90 days. Results on tumor necrotic factor-α (TNF-α), interleukin-12 (IL-12), and comet assay showed increased values in comparison to those obtained in arsenic- or fluoride-treated rats. Results on NO, TBARS, and caspase-9 showed higher values than fluoride-treated rats but lower levels than arsenic-treated rats. It is hypothesized that increased generation of nitric oxide induces the release of cytokines that activates caspase-9. Caspase-9 promotes the synthesis of caspase-3 that executes apoptosis. Histopathological observations on apoptotic bodies and Kupffer cells support these observations.

Zinc Oxide Nanoparticles Ameliorate Dimethylnitrosamine-Induced Renal Toxicity in Rat.

Dimethylnitrosamine (DMN) is an established carcinogen. It is toxic to several organs, viz., the liver, kidney, and lungs, and immune system. Several drugs have been used in the past to modulate its toxicity using experimental animal models. The present study was designed to investigate the effect of zinc oxide nanoparticles (ZnONPs) on renal toxicity caused by DMN in laboratory rat. Since oxidative mechanisms are mainly involved in its toxicity, the proposed study focuses on the amelioration of oxidative stress response by ZnONPs, if any. The present results show that administration of ZnONPs (50 mg/kg body weight/rat) to DMN (2 µl/100 g body weight/rat)-treated rats diminuted the concentration of malonaldehyde, H2O2, and NO in the kidney. However, reduced glutathione (GSH) concentration increased after ZnONP treatment. Results on glutathione S-transferase and glutathione peroxidase favored its antioxidative effects. These results are supported by the recovery of oxidative DNA damage and less pronounced histopathological changes in the kidney. It is hypothesized that ZnONPs might be toxic to renal tissue; however, its strong therapeutic/antioxidative potential helps in ameliorating DMN-induced renal toxicity in rat.

Possible Mechanisms of Liver Injury Induced by Cadmium Sulfide Nanoparticles in Rat.

Cadmium is primarily utilized in the construction of particles known as quantum dots. Hepatotoxicity caused by microparticles of cadmium is very well known; however, toxicity of nanoparticles of cadmium is not well understood. The present study describes the toxicity of cadmium sulfide nanoparticles (CdSNPs) in the liver of rat. Adult Wistar rats were administered CdSNPs (10 mg/kg) on alternate days for 45 days. Serum enzymes (ALT, AST, ALP), biomarkers of lipid peroxidation (MDA, H2O2, and NO), and metallothionein concentration were determined. Histopathological and TEM observations were also made to record morphological changes. CdSNPs (10 mg/kg) induced significant changes in the structure and function of liver. Values of serum enzymes and reactive species increased significantly in rats treated with CdSNPs in comparison to CdS-treated rats. Histopathological observations showed extensive parenchymal degeneration. Ultrastructural studies exhibited proliferation of endoplasmic reticulum, microsomes, and lysosomes. It is concluded that NP-membrane interaction leads to the generation of reactive species that alter membrane integrity and induce oxidative stress. These events may activate cell death pathways in hepatocytes.

Endoplasmic Reticulum Stress Induced by Toxic Elements-a Review of Recent Developments.

Endoplasmic reticulum of all eukaryotic cells is a membrane-bound organelle. Under electron microscope it appears as parallel arrays of "rough membranes" and a maze of "smooth vesicles" respectively. It performs various functions in cell, i.e., synthesis of proteins to degradation of xenobiotics. Bioaccumulation of drugs/chemicals/xenobiotics in the cytosol can trigger ER stress. It is recognized by the accumulation of unfolded or misfolded proteins in the lumen of ER. Present review summarizes the present status of knowledge on ER stress caused by toxic elements, viz arsenic, cadmium, lead, mercury, copper, chromium, and nickel. While inorganic arsenic may induce various glucose-related proteins, i.e., GRP78, GRP94 and CHOP, XBP1, and calpains, cadmium upregulates GRP78. Antioxidants like ascorbic acid, NAC, and Se inhibit the expression of UPR. Exposure to lead also changes ER stress related genes, i.e., GRP 78, GRP 94, ATF4, and ATF6. Mercury too upregulates these genes. Nickel, a carcinogenic element upregulates the expression of Bak, cytochrome C, caspase-3, caspase-9, caspase-12, and GADD 153. Much is not known on ER stress caused by nanoparticles. The review describes inter-organelle association between mitochondria and ER. It also discusses the interdependence between oxidative stress and ER stress. A cross talk amongst different cellular components appears essential to disturb pathways leading to cell death. However, these molecular switches within the signaling network used by toxic elements need to be identified. Nevertheless, ER stress especially caused by toxic elements still remains to be an engaging issue.

Assessment of genotoxicity amongst smokers, alcoholics, and tobacco chewers of North India using micronucleus assay and urinary 8-hydroxyl-2'-deoxyguanosine, as biomarkers.

The main objective of the present study was to screen the genotoxicity caused by individual and combined habits of smoking, tobacco chewing, and alcohol consumption in human population of North India. Study recruited 67 male subjects aged 25 to 65 years. Buccal mucosal cells were subjected to micronucleus (MN) assay, and 8-hydroxyl-2-deoxyguanosine (8-OHdG) was estimated in their urine samples. Number and shape of the MN cells varied in the buccal epithelium of different groups. Maximum number of MN (0.47%) were found in tobacco chewers followed by smokers (0.45%) and alcoholics (0.44%) (P < 0.05). These results reciprocated the concentration of urinary 8-OHdG. Maximum value for 8-OHdG was also recorded in tobacco chewers (21.07 ± 5.51 mg/mg creatinine) followed by smokers (20.25 ± 3.96 mg/mg creatinine) and alcoholics (19.06 ± 3.41 mg/mg creatinine) (P < 0.05). Combined effects of these agents were found to be statistically different from individual effects. Carcinogenic compounds present in cigarette smoke, nitrosamines found in solid tobacco, and acetaldehyde, a metabolic product of alcohol, induce oxidative stress that manifests into genotoxicity. In conclusion, demographical differences occur in the genotoxicity caused by these three habits. MN assay and urinary 8-OHdG are simple, noninvasive, and reliable biomarkers of genotoxicity.

Perspectives in endocrine toxicity of heavy metals--a review.

An attempt has been made to review the endocrine/hormonal implications of a few environmentally significant metals, viz, lead, mercury, cadmium, copper, arsenic and nickel, in man and animals. Special emphasis has been given to the adrenals, thyroid, testis, ovary and pancreas. Toxic metals can cause structural and functional changes in the adrenal glands. Their effects on steroidogenesis have been reviewed. It has been reported that thyroid hormone kinetics are affected by a number of metallic compounds. Occupational exposure to a few of these metals can cause testicular injury and sex hormone disturbances. Protective effects of a few antioxidants on their reproductive toxicity have also been discussed. Information gathered on female reproductive toxicity of heavy metals shows that exposure to these metals can lead to disturbances in reproductive performance in exposed subjects. Certain metals can cause injury to the endocrine pancreas. Exposure to them can cause diabetes mellitus and disturb insulin homeostasis. The need to develop molecular markers of endocrine toxicity of heavy metals has been suggested. Overall information described in this review is expected to be helpful in planning future studies on endocrine toxicity of heavy metals.

Modulation of phase-II enzyme activities in benzene treated ovariectomized rats.

The aim of the study was to determine the influence of ovariectomy on phase II enzymes viz. glutathione-S-transferase (GST), glutathione peroxidase (GPX) and catalase (CAT) in liver and kidney of female rats treated with benzene. The results showed the significant decrease of the GST and GPX activity in benzene treated rats after ovariectomy. However progesterone supplementation stimulated the activity of GST and GPX in liver and kidney of benzene treated non ovariectomized and ovariectomized rats. Progesterone supplementation to benzene treated ovariectomized rats helps to gain in CAT activity. Our results on DNA damage using single cell gel electrophoresis also confirmed our findings on antioxidant enzymes. The results showed that lack of protective progesterone against benzene toxicity is reflected in alterations in antioxidant enzyme activities. However progesterone therapy to benzene treated ovariectomized rats results in activating the antioxidant defence system. Since female workers are engaged in industrial sector, these results are important from occupational health point of view. Benzene exposure affects their reproductive health. Nevertheless, it could be modulated by suitable hormonal therapy.

Molecular markers of heavy metal toxicity--a new paradigm for health risk assessment.

Last decade has witnessed increased interest in studies dealing with molecular markers of health and disease expression of genes. Specific toxicant "signatures" have been detected using genome base technologies such as microarrays. Further toxins have been classified on the basis of these signatures. Knowledge on these signatures has helped in the identification of novel drug candidates. This review discusses the gene expression studies recently made on arsenic, cadmium, mercury, chromium, lead, copper, nickel, manganese, and other essential elements. Toxicogenomics standards and their organizations have also been briefly described. Although this information can not be considered as complete, recent reports from different laboratories on bacteria, fish, laboratory animals and humans have been summarized. It is expected that toxicogenomics data presented in this review will be helpful in planning and excretion of human health risk assessment programs.

Modulation of CYP4502E1 and oxidative stress by testosterone in liver and kidney of benzene treated rats.

Bilateral castration increased lipid peroxidation and consequently reduced glutathione in both liver and kidney. Testosterone administration reduced lipid peroxidation in the liver of castrated and benzene treated rats, however, reduced glutathione status could not be restored. Benzene depleted CYP4502E1 in castrated rats, however, the enzyme was restored in liver and kidney both after testosterone treatment. The results suggest that testosterone affects the metabolism and disposition of benzene by influencing CYP4502E1. Other hormonal and cellular/molecular factors may also alter the actions of testosterone. Testosterone dependent mechanism of toxicity of benzene in the liver and kidney has been discussed.

Time Dependent Gene Expression Changes in the Liver of Mice Treated with Benzene.

Benzene is used as a general purpose solvent. Benzene metabolism starts from phenol and ends with p-benzoquinone and o-benzoquinone. Liver injury inducted by benzene still remains a toxicologic problem. Tumor related genes and immune responsive genes have been studied in patients suffering from benzene exposure. However, gene expression profiles and pathways related to its hepatotoxicity are not known. This study reports the results obtained in the liver of BALB/C mice (SLC, Inc., Japan) administered 0.05 ml/100 g body weight of 2% benzene for six days. Serum, ALT, AST and ALP were determined using automated analyzer (Fuji., Japan). Histopathological observations were made to support gene expression data. c-DNA microarray analyses were performed using Affymetrix Gene-chip system. After six days of benzene exposure, twenty five genes were down regulated whereas nineteen genes were up-regulated. These gene expression changes were found to be related to pathways of biotransformation, detoxification, apoptosis, oxidative stress and cell cycle. It has been shown for the first time that genes corresponding to circadian rhythms are affected by benzene. Results suggest that gene expression profile might serve as potential biomarkers of hepatotoxicity during benzene exposure.

Amelioration of arsenic toxicity by L-Ascorbic acid in laboratory rat.

A study so as to confirm the protective effects of L-ascorbic acid against inorganic arsenic (As23) toxicity was made in male Wistar rats. Multiphase observations made on iAs concentration in target organs viz. liver and kidney, liver function, histopathological changes, ultrastructural alterations, lipid peroxidation, oxidative stress and iAs-DNA interaction strongly favoured its ameliorative effects. These effects could mainly be attributed to its antioxidative property. It offers help in regeneration of GSH and alpha-tocopherol. The chelaticn of iAs by ascorbic acid has also been hypothesized. Inhibition of DNA damage by ascorbic acid in liver and kidney appears to be the most significant part of this study On the basis of these results, we conclude that administration of L-ascorbic acid to arsenic affected population may prevent the occurrence of fatal human diseases.

Protective effect of ascorbic acid against oxidative stress induced by inorganic arsenic in liver and kidney of rat.

Ascorbic acid treatment in arsenic trioxide treated rats increased arsenic excretion, inhibited lipid peroxidation, improved GSH status, regulated GSSG turnover and also restored glutathione-S-transferases activity in liver and kidney. Suitable mechanisms leading to ascorbic acid protection have been discussed. Upregulation of GSH dependent enzymes was found to be necessary for a protective effect. Protection is finally attributed to higher GSH levels observed in the liver and kidney of ascorbic acid and inorganic arsenic treated rats. It is also concluded that ascorbic acid protection is influenced by gender dependent factors. Arsenic poisoning is a global problem now. Gender differences need to be considered while applying therapeutic measures.

Circadian variation in lipid peroxidation induced by benzene in rats.

Time-dependent effect of benzene, a potent carcinogenic industrial solvent, on lipid peroxidaiton and associated mechanisms has been studied in liver and kidney of rats. Significant differences were observed in the values of urinary phenol, microsomal malondialdehyde, reduced glutathione (GSH) and cytochrome P4502E1 in rats treated with benzene in morning and evening hours. Higher were the values for urinary phenol and hepatic microsomal malondialdehyde in rats administered benzene in evening hours. Contrarily, higher were the values for GSH and cytochrome P4502E1 in rats treated with benzene in morning hours. Increased microsomal lipid peroxidation has been attributed to low GSH status, whereas increased phenol concentration could be related to low activity of cytochrome P4502E1 in the liver of rats in evening hours. It is concluded that circadian rhythmicity in hepatic drug metabolizing enzyme system and GSH contributes in toxicity of benzene. The results are important from occupational health point of view.

Influence of thyroxine and n-propylthiouracil on nephro-toxicity of inorganic arsenic in rat.

The effect of hyper or hypoactive thyroid on the renal toxicity of arsenic trioxide has been studied in rats. It was observed that pre-treatment of rats with thyroxine stimulates arsenic excretion in urine. The anti-thyroid drug n-propylthiouracil (PTU), inhibits the accumulation of arsenic in renal tissue. Both treatments affect the renal pathology. Histopathological lesions are less severe in PTU and arsenic-treated rats in comparison to thyroxine and arsenic-treated rats. Ultrastructural studies support light microscopical observations. An adaptive response was noticed against arsenic in PTU pre-treated rats. We attribute this response to decreased glutathione-S-transferase (GSH) activity and increased GSH synthesis in the kidney. A relationship between thyroidal activity and arsenic toxicity is suggested by present observations.

Biochemical toxicity of benzene.

Human exposure to benzene in work environment is a global occupational health problem. After inhalation or absorption, benzene targets organs viz. liver, kidney, lung, heart and brain etc. It is metabolized mainly in the liver by cytochrome P450 multifunctional oxygenase system. Benzene causes haematotoxicity through its phenolic metabolites that act in concert to produce DNA strand breaks, chromosomal damage, sister chromatid exchange, inhibition of topoisomerase II and damage to mitotic spindle. The carcinogenic and myelotoxic effects of benzene are associated with free radical formation either as benzene metabolites or lipid peroxidation products. Benzene oxide and phenol have been considered as proheptons. Liver microsomes play an important role in biotransformation of benzene whereas in kidney, it produces degenerative intracellular changes. Cohort studies made in different countries suggest that benzene induces multiple myeloma in petrochemical workers. Though extensive studies have been performed on its toxicity, endocrinal disruption caused by benzene remains poorly known. Transgenic cytochrome P450 IIE1 mice may help in understanding further toxic manifestations of benzene.

Sex differences in oxidative stress induced by benzene in rats.

Role of sex differences on oxidative stress induced by benzene has been studied in liver, kidney and lungs of rat. It was observed that benzene administration enhanced lipid peroxidation in liver, kidney and lungs of rat, nevertheless, significant variations were recorded in male and female rats. Decrease of GSH and CYTP(450)2E1 was higher in female rats than male rats except lungs. The results suggest that oxidative stress induced by benzene is higher in female rats.

Effect of arsenic (AsIII) on glutathione-dependent enzymes in liver and kidney of the freshwater fish Channa punctatus.

The possible role of glutathione-dependent enzymes in the liver and kidney of the freshwater fish Channa punctatus has been studied after exposure to arsenic trioxide for different durations. Activities of glutathione-S-transferases, glutathione peroxidase, glutathione reductase, and catalase decreased in the liver and kidney as a result of the initial increase in arsenic concentration in the liver and kidney. However, during longer exposures, a decline in arsenic concentration corresponded with improved enzyme activity. Because arsenic manifests its toxicity by inducing oxidative stress, the antioxidant enzymes, especially the glutathione-dependent enzymes, play a protective role in arsenic toxicity.

Resistance to oxidative stress in a freshwater fish Channa punctatus after exposure to inorganic arsenic.

The biochemical toxicity of arsenic trioxide (AsIII) in a freshwater edible fish Channa punctatus has been studied on exposures ranging from 7 to 90 d. The arsenic concentration increased exponentially in liver, kidney, gills, and muscles of fish up to 60 d of exposure to arsenic. However, arsenic concentration in these tissues declined at 90 d of exposure. This relationship between period of exposure and concentration of arsenic in selected tissues suggests an adaptive response of fish to arsenic. Furthermore, exposure to arsenic-induced lipid peroxidation in these organs increased initially at 7 d of exposure; however, it decreased up to 60 d of exposure but increased again at 90 d of treatment. Values of reduced glutathione (GSH) reflected the observations of lipid peroxidation. The role of GSH in this adaptive response has been discussed.

Enhancement of radiation-induced apoptosis by Podophyllum hexandrum.

The aqueous extract of Podophyllum hexandrum (RP-1), which has been recently reported to manifest radioprotective and anti-tumour properties, has been investigated for its mode of action. RP-1, under in-vitro conditions dose-dependently chelated metal ions, inhibited radiation or metal ion-induced hydroxyl radicals and lipid peroxidation and scavenged superoxide anions. Intraperitoneal administration of RP-1 to mice pre-irradiation (10 Gy) induced more DNA fragmentation and lipid peroxidation in thymocytes maximally at 4 and 8 h, respectively, in comparison with RP-1 treatment or irradiation. Flow-cytometric quantification of sub-diploid peak, oligonucleosomal cleavage assay (ladder) and depletion of total thiols also corroborated the ability of RP-1 to enhance radiation-induced apoptosis. RP-1 in presence of 100 microM CuSO(4) induced strand breaks in plasmid DNA and addition of metal chelators (EDTA and deferoxamine) inhibited the strand scission. Treatment with a major constituent of RP-1, podophyllin, did not cause strand breaks, but isolated constituents of RP-1, quercetin or podophyllotoxin, induced strand breaks. Depending on its concentration in the milieu, RP-1 acted as a pro- or antioxidant modifying the radiation-induced apoptosis and therefore could be exploited for cancer management.