Effects on cell cycle progression and cytoskeleton organization of five Bothrops spp. venoms in cell culture-based assays.

Snake envenomation is a neglected tropical disease. In Brazil, the Bothrops genus is responsible for about 86% of snakebite accidents. Despite extensive evidence of the cytotoxicity of snake venoms, the cellular and molecular mechanisms involved are not fully understood, especially regarding the effects on cell cycle progression and cytoskeleton organization. Traditionally, the effectiveness and quality control tests of venoms and antivenoms are assessed by in vivo assays. Despite this, there is a rising effort to develop surrogate in vitro models according to the 3R principle (Replacement, Reduction, and Refinement). In this study, we treated rat liver cells (BRL-3A) with venoms from five Bothrops species (B. jararaca, B. jararacussu, B. moojeni, B. alternatus, and B. neuwiedi) and analyzed cell viability and IC50 by MTT assay, cell cycle phases distribution by flow cytometry, and morphology and cytoskeleton alterations by immunofluorescence. In addition, we evaluated the correlation between IC50 and the enzymatic and biological activities of each venom. Our results indicated that Bothrops spp. venoms decreased the cell viability of rat liver BRL-3A cells. The rank order of potency was B. jararacussu > B. moojeni > B. alternatus > B. jararaca > B. neuwiedi. The mechanisms of cytotoxicity were related to microtubules and actin network disruption, but not to cell cycle arrest. No clear correlation was found between the IC50 and retrieved literature data of in vitro enzymatic and in vivo biological activities. This work contributed to understanding cellular and molecular mechanisms underlying the Bothrops spp. venom cytotoxicity, which can help to improve envenomation treatment, as well as disclose potential therapeutic properties of snake venoms.

Transcriptome profile analysis reveals putative molecular mechanisms of 5-aminolevulinic acid toxicity.

5-aminolevulinic acid (5-ALA) is the first precursor of the heme biosynthesis pathway, accumulated in acute intermittent porphyria (AIP), an inherited metabolic disease characterized by porphobilinogen deaminase deficiency. An increased incidence of hepatocellular carcinoma (HCC) has been reported as a long-term manifestation in symptomatic AIP patients. 5-ALA is an α-aminoketone prone to oxidation, yielding reactive oxygen species and 4,5-dioxovaleric acid. A high concentration of 5-ALA presents deleterious pro-oxidant potential. It can induce apoptosis, DNA damage, mitochondrial dysfunction, and altered expression of carcinogenesis-related proteins. Several hypotheses of the increased risk of HCC rely on the harmful effect of elevated 5-ALA in the liver of AIP patients, which could promote a pro-carcinogenic environment. We investigated the global transcriptional changes and perturbed molecular pathways in HepG2 cells following exposure to 5-ALA 25 mM for 2 h and 24 h using DNA microarray. Distinct transcriptome profiles were observed. 5-ALA '25 mM-2h' upregulated 10 genes associated with oxidative stress response and carcinogenesis. Enrichment analysis of differentially expressed genes by KEGG, Reactome, MetaCore™, and Gene Ontology, showed that 5-ALA '25 mM-24h' enriched pathways involved in drug detoxification, oxidative stress, DNA damage, cell death/survival, cell cycle, and mitochondria dysfunction corroborating the pro-oxidant properties of 5-ALA. Furthermore, our results disclosed other possible processes such as senescence, immune responses, endoplasmic reticulum stress, and also some putative effectors, such as sequestosome, osteopontin, and lon peptidase 1. This study provided additional knowledge about molecular mechanisms of 5-ALA toxicity which is essential to a deeper understanding of AIP and HCC pathophysiology. Furthermore, our findings can contribute to improving the efficacy of current therapies and the development of novel biomarkers and targets for diagnosis, prognosis, and therapeutic strategies for AHP/AIP and associated HCC.

Effects on cell cycle progression and cytoskeleton organization of five Bothrops spp. venoms in cell culture-based assays

Snake envenomation is a neglected tropical disease. In Brazil, the Bothrops genus is responsible for about 86% of snakebite accidents. Despite extensive evidence of the cytotoxicity of snake venoms, the cellular and molecular mechanisms involved are not fully understood, especially regarding the effects on cell cycle progression and cytoskeleton organization. Traditionally, the effectiveness and quality control tests of venoms and antivenoms are assessed by in vivo assays. Despite this, there is a rising effort to develop surrogate in vitro models according to the 3R principle (Replacement, Reduction, and Refinement). In this study, we treated rat liver cells (BRL-3A) with venoms from five Bothrops species (B. jararaca, B. jararacussu, B. moojeni, B. alternatus, and B. neuwiedi) and analyzed cell viability and IC50 by MTT assay, cell cycle phases distribution by flow cytometry, and morphology and cytoskeleton alterations by immunofluorescence. In addition, we evaluated the correlation between IC50 and the enzymatic and biological activities of each venom. Our results indicated that Bothrops spp. venoms decreased the cell viability of rat liver BRL-3A cells. The rank order of potency was B. jararacussu > B. moojeni > B. alternatus > B. jararaca > B. neuwiedi. The mechanisms of cytotoxicity were related to microtubules and actin network disruption, but not to cell cycle arrest. No clear correlation was found between the IC50 and retrieved literature data of in vitro enzymatic and in vivo biological activities. This work contributed to understanding cellular and molecular mechanisms underlying the Bothrops spp. venom cytotoxicity, which can help to improve envenomation treatment, as well as disclose potential therapeutic properties of snake venoms.

Transcriptome profile analysis reveals putative molecular mechanisms of 5-aminolevulinic acid toxicity

5-aminolevulinic acid (5-ALA) is the first precursor of the heme biosynthesis pathway, accumulated in acute intermittent porphyria (AIP), an inherited metabolic disease characterized by porphobilinogen deaminase deficiency. An increased incidence of hepatocellular carcinoma (HCC) has been reported as a long-term manifestation in symptomatic AIP patients. 5-ALA is an α-aminoketone prone to oxidation, yielding reactive oxygen species and 4,5-dioxovaleric acid. A high concentration of 5-ALA presents deleterious pro-oxidant potential. It can induce apoptosis, DNA damage, mitochondrial dysfunction, and altered expression of carcinogenesis-related proteins. Several hypotheses of the increased risk of HCC rely on the harmful effect of elevated 5-ALA in the liver of AIP patients, which could promote a pro-carcinogenic environment. We investigated the global transcriptional changes and perturbed molecular pathways in HepG2 cells following exposure to 5-ALA 25 mM for 2 h and 24 h using DNA microarray. Distinct transcriptome profiles were observed. 5-ALA ’25 mM-2h′ upregulated 10 genes associated with oxidative stress response and carcinogenesis. Enrichment analysis of differentially expressed genes by KEGG, Reactome, MetaCore™, and Gene Ontology, showed that 5-ALA ‘25 mM-24h’ enriched pathways involved in drug detoxification, oxidative stress, DNA damage, cell death/survival, cell cycle, and mitochondria dysfunction corroborating the pro-oxidant properties of 5-ALA. Furthermore, our results disclosed other possible processes such as senescence, immune responses, endoplasmic reticulum stress, and also some putative effectors, such as sequestosome, osteopontin, and lon peptidase 1. This study provided additional knowledge about molecular mechanisms of 5-ALA toxicity which is essential to a deeper understanding of AIP and HCC pathophysiology. Furthermore, our findings can contribute to improving the efficacy of current therapies and the development of novel biomarkers and targets for diagnosis, prognosis, and therapeutic strategies for AHP/AIP and associated HCC.

Biological effects of an oxyphytosterol generated by ß-Sitosterol ozonization.

ß-Sitosterol (ßSito) is the most abundant phytosterol found in vegetable oils, grains such as wheat, beans, and corn, and in many phytosterol-enriched foods. It is prone to oxidation by reactive oxygen species, such as ozone, leading to the formation of oxyphytosterols. A better understanding regarding the biological effects and mechanism of action of oxyphytosterols is required since the beneficial and adverse side effects of these compounds on human health remain highly controversial. In this work, we investigated the biological effects of ß-Secosterol (ßSec), a new oxyphytosterol generated by the reaction of ßSito with ozone. Treatment of HepG2 cells with ßSito or ßSec (0.1-100 µM) for 24, 48, and 72 h induced a dose-dependent reduction of cell viability in the MTT assay, with ßSec showing higher efficacy than ßSito. However, ßSec presented a lower potency than ßSito, showing IC50 = 37.32 µM, higher than ßSito (IC50 = 0.23 µM) at 48 h. Cell cycle analyses by flow cytometry showed a slight decrease of G0/G1 phase with ßSito 0.5 µM, but a significant cell cycle arrest at the G0/G1 phase in the treatment for 48 h with ßSec 20 µM (62.69 ± 2.15%, p < 0.05) and ßSec 40 µM (66.96 ± 5.39%, p < 0.0001) when compared to control (56.97 ± 2.60%). No suggestion of apoptosis was indicated by flow cytometry data. Also, ßSec (20 and 40 µM) reduced the mitotic index. In the laser scanning confocal microscopy analysis no alterations in cell morphology were observed with ßSito (0.5 µM). Nevertheless, round-shaped cells, abnormal nuclear morphology with shrinkage, and formation of microtubules clusters were observed in the treatment with ßSec, indicating a disruption in the microtubules network organization. N-acetyl-l-cysteine was not able to inhibit any of these cellular effects, indicating a lack of involvement of oxidative stress in the mechanism of action of ßSec. Although not further investigated in this study, it was discussed the hypothesis that covalent adduct formation with lysine residues of proteins, could play an important role in the biological effects elicited by ßSec. Elucidation of the primary cellular processes induced by ßSec provides the essential knowledge to be aware of its potential adverse side effects or therapeutic use of this oxyphytosterol.

Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells.

The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS2 method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS1 data confirms the presence of the protonated alkaloids, and the MS2 fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN's lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue.

Characterization of oxyphytosterols generated by ß-sitosterol ozonization.

ß-Sitosterol (ßSito) is the most abundant phytosterol found in elevated concentrations in vegetable oils, nuts, seeds, cereals, fruits, and in many phytosterol-enriched foods. Although the benefits, there is a concern in terms of food quality and health due to the increasing consumption of phytosterols and the possible adverse side effects of their oxidation products, oxyphytosterols. ßSito has a similar structure to cholesterol, with an unsaturated double bond at C5-C6, which is susceptible to oxidation by reactive oxygen species like ozone, generating oxyphytosterols. In this work we propose a mechanism of formation of three oxyphytosterols 2-[(7aR)-5-[(1R,4S)-4-hydroxy-1-methyl-2-oxocyclohexyl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7- octahydroinden-4-yl] acetaldehyde (ßSec), (2-[(7aR)-5-[(2R,5S)-5-hydroxy-2-methyl-7-oxo-oxepan- 2-yl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7-octahydroinden-4- yl] acetaldehyde (ßLac) and 2-((7aR)-5-((1R,4S)-4-hydroxy-1-methyl-2- oxocyclohexyl)-1,7a-dimethyloctahydro-1Hinden-4-yl) acetic acid (ßCOOH) generated by ozonization of ßSito, through their synthesis and molecular characterization. The cytotoxic effect of ßSito and its main oxyphytosterol ßSec was evaluated and both reduced the HepG2 cell viability.

Biological effects of an oxyphytosterol generated by β-Sitosterol ozonization

β-Sitosterol (βSito) is the most abundant phytosterol found in vegetable oils, grains such as wheat, beans, and corn, and in many phytosterol-enriched foods. It is prone to oxidation by reactive oxygen species, such as ozone, leading to the formation of oxyphytosterols. A better understanding regarding the biological effects and mechanism of action of oxyphytosterols is required since the beneficial and adverse side effects of these compounds on human health remain highly controversial. In this work, we investigated the biological effects of β-Secosterol (βSec), a new oxyphytosterol generated by the reaction of βSito with ozone. Treatment of HepG2 cells with βSito or βSec (0.1–100 μM) for 24, 48, and 72 h induced a dose-dependent reduction of cell viability in the MTT assay, with βSec showing higher efficacy than βSito. However, βSec presented a lower potency than βSito, showing IC50 = 37.32 μM, higher than βSito (IC50 = 0.23 μM) at 48 h. Cell cycle analyses by flow cytometry showed a slight decrease of G1 phase with βSito 0.5 μM, but a significant cell cycle arrest at the G0/G1 phase in the treatment for 48 h with βSec 20 μM (62.69 ± 2.15%, p < 0.05) and βSec 40 μM (66.96 ± 5.39%, p < 0.0001) when compared to control (56.97 ± 2.60%). No suggestion of apoptosis was indicated by flow cytometry data. Also, βSec (20 and 40 μM) reduced the mitotic index. In the analysis with a confocal laser-scanning microscope, no alterations in cell morphology were observed with βSito (0.5 μM). Nevertheless, round-shaped cells, abnormal nuclear morphology with shrinkage, and formation of microtubules clusters were observed in the treatment with βSec, indicating a disruption in the microtubules network organization. N-acetyl-l-cysteine was not able to inhibit any of these cellular effects, indicating a lack of involvement of oxidative stress in the mechanism of action of βSec. Although not further investigated in this study, it was discussed the hypothesis that covalent adduct formation with lysine residues of proteins, could play an important role in the biological effects elicited by βSec. Elucidation of the primary cellular processes induced by βSec provides the essential knowledge to be aware of its potential adverse side effects or therapeutic use of this oxyphytosterol.

Alternative isolation protocol for desulfo and zwitterionic cylindrospermopsin alkaloids and comparison of their toxicity in hepG2 cells

The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS2 method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS1 data confirms the presence of the protonated alkaloids, and the MS2 fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN’s lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue

Characterization of oxyphytosterols generated by ß-sitosterol ozonization

ß-Sitosterol (ßSito) is the most abundant phytosterol found in elevated concentrations in vegetable oils, nuts, seeds, cereals, fruits, and in many phytosterol-enriched foods. Although the benefits, there is a concern in terms of food quality and health due to the increasing consumption of phytosterols and the possible adverse side effects of their oxidation products, oxyphytosterols. ßSito has a similar structure to cholesterol, with an unsaturated double bond at C5–C6, which is susceptible to oxidation by reactive oxygen species like ozone, generating oxyphytosterols. In this work we propose a mechanism of formation of three oxyphytosterols 2-[(7aR)-5-[(1R,4S)-4-hydroxy-1-methyl-2-oxocyclohexyl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7- octahydroinden-4-yl]acetaldehyde (ßSec), (2-[(7aR)-5-[(2R,5S)-5-hydroxy-2-methyl-7-oxo-oxepan- 2-yl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7-octahydroinden-4- yl] acetaldehyde (ßLac) and 2-((7aR)-5-((1R,4S)-4-hydroxy-1-methyl-2- oxocyclohexyl)-1,7a-dimethyloctahydro-1Hinden-4-yl) (ßCOOH) generated by ozonization of ßSito, through their synthesis and molecular characterization. The cytotoxic effect of ßSito and its main oxyphytosterol ßSec was evaluated and both reduced the HepG2 cell viability.

Characterization of oxyphytosterols generated by ß-sitosterol ozonization

ß-Sitosterol (ßSito) is the most abundant phytosterol found in elevated concentrations in vegetable oils, nuts, seeds, cereals, fruits, and in many phytosterol-enriched foods. Although the benefits, there is a concern in terms of food quality and health due to the increasing consumption of phytosterols and the possible adverse side effects of their oxidation products, oxyphytosterols. ßSito has a similar structure to cholesterol, with an unsaturated double bond at C5–C6, which is susceptible to oxidation by reactive oxygen species like ozone, generating oxyphytosterols. In this work we propose a mechanism of formation of three oxyphytosterols 2-[(7aR)-5-[(1R,4S)-4-hydroxy-1-methyl-2-oxocyclohexyl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7- octahydroinden-4-yl]acetaldehyde (ßSec), (2-[(7aR)-5-[(2R,5S)-5-hydroxy-2-methyl-7-oxo-oxepan- 2-yl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7-octahydroinden-4- yl] acetaldehyde (ßLac) and 2-((7aR)-5-((1R,4S)-4-hydroxy-1-methyl-2- oxocyclohexyl)-1,7a-dimethyloctahydro-1Hinden-4-yl) (ßCOOH) generated by ozonization of ßSito, through their synthesis and molecular characterization. The cytotoxic effect of ßSito and its main oxyphytosterol ßSec was evaluated and both reduced the HepG2 cell viability.

Effect of 5-aminolevulinic acid on the expression of carcinogenesis-related proteins in cultured primary hepatocytes

Acute intermittent porphyria (AIP) is a heme pathway disorder caused by a decrease in the activity and synthesis of porphobilinogen deaminase. Thus, the first heme precursor 5-aminolevulinic acid (ALA) accumulates in the liver. Reactive oxygen species (ROS) resulting from ALA oxidation may be correlated to a higher incidence of hepatocellular carcinoma (HCC) in AIP patients. However, the molecular mechanisms of this relationship have not been thoroughly elucidated to date. In this study, we investigated the effect of increasing levels of ALA on the expression of proteins related to DNA repair, oxidative stress, apoptosis, proliferation and lipid metabolism. Primary rat hepatocytes were isolated by the collagenase perfusion method, lipoperoxidation was evaluated by a TBA fluorimetric assay and Western blotting was used to assess protein abundance. The data showed that ALA treatment promoted a dose-dependent increase of p53 expression, downregulation of Bcl-2, HMG-CoA reductase and OGG1 and an increase in lipoperoxidation. There was no alteration in the expression of the transcription factor NF-?B, catalase and superoxide dismutase. ALA oxidation products induced protein regulation patterns, suggesting the interconnection of cellular processes, such as the intrinsic pathway of apoptosis, redox homeostasis, cell proliferation, lipid metabolism and DNA repair. This study helps to elucidate the molecular mechanisms of hepatotoxicity mediated by ALA pro-oxidant effects and supports the hypothesis that ALA accumulation correlates with a higher incidence of hepatic carcinogenic events.

Genome-wide analysis identifies a tumor suppressor role for aminoacylase 1 in iron-induced rat renal cell carcinoma.

A growing number of studies indicate a link between oxidative stress and cancer. We previously developed a rat model of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA). Here, we performed a genome-wide analysis to study characteristics of genomic alteration and identify putative genes involved in the development of Fe-NTA-induced RCCs. Array-based comparative genomic hybridization analyses revealed a chromosomal loss spanning chromosome 8 in most of the RCCs studied, with a common deletion at 8q31-32, which was confirmed by loss of heterozygosity (LOH) analysis. Studies of gene expression in RCCs or following Fe-NTA treatment revealed globally decreased transcription levels of 34 genes derived from chromosome 8 that are expressed in the kidney. Among them, the aminoacylase 1 (Acy1) gene, which maps to 8q32 and is highly expressed in the kidney, displayed a significantly decreased level of expression in RCCs. Significant amounts of the Acy1 protein were detected in the cytoplasm as well as in the nuclei of renal proximal tubular cells of untreated rats. Transfection of Acy1 into RCC cell lines inhibited proliferation and colony formation on soft agar. An increased number of apoptotic cells were observed following Acy1 transfection. The rat 8q31-32 chromosomal region corresponds to human 3p21.31-24.1, a hot spot where LOH is frequently found in various human cancers. Thus, Fe-NTA-induced renal tumor model is ideal for studying the link between deletions within this region and tumor formation. Our data demonstrate that Acy1 functions as a tumor suppressor in this rat RCC model.

Association of microRNA-34a overexpression with proliferation is cell type-dependent.

Recently Welch et al. reported that microRNA (miRNA)-34a functions as a potential tumor suppressor in neuroblastoma cells (Oncogene 26: 5017-22, 2007). Here, we conversely show that miRNA-34a supports cell proliferation in rat oxidative stress-induced renal carcinogenesis and is overexpressed in various types of human cancers. While searching for genetically unstable chromosomal areas in rat renal carcinogenesis, we found the miRNA-34 family reciprocally overexpressed in chromosomal areas with frequent allelic loss. By in situ hybridization and reverse transcription-polymerase chain reaction, cerebral neurons and Purkinje cells showed the highest expression of a major type, miRNA-34a, followed by a variety of endocrine cells and proliferating cells including germinal center lymphocytes and mouse embryonic fibroblasts and stem cells. In contrast, normal renal tubules, hepatocytes and myocardial cells showed faint expression. After 3 weeks of ferric nitrilotriacetate (Fe-NTA)-induced oxidative stress, regenerating renal proximal tubular cells showed high miRNA-34a expression. All of the Fe-NTA-induced rat renal carcinomas and an array of human cancers (151 positive cases of 177) showed high expression of miRNA-34a. Furthermore, knockdown of miRNA-34a with small interfering RNA significantly suppressed proliferation not only of renal carcinoma cells but also of HeLa and MCF7 cells. These results indicate that miRNA-34a overexpression, an acquired trait during carcinogenesis, supports cell proliferation in the majority of cancers suggesting an unexpected link in the cellular metabolism between cancer and neuronal and/or endocrine cells, which warrants further investigation.

Oxidative stress in Perna perna and other bivalves as indicators of environmental stress in the Brazilian marine environment: antioxidants, lipid peroxidation and DNA damage.

Oxidative stress can take place in marine bivalves under a series of environmental adverse conditions. The study of different systems related to oxidative stress in these organisms can give important information about their physiological status and also about environmental health. Bivalves have been proposed as good sentinel organisms in pollution monitoring studies through the analysis of biochemical biomarkers, and most of the biomarkers analyzed are those related to oxidative stress. However, it is very important to know how other environmental factors not associated to the presence of pollutants might affect these parameters. We have studied a series of mechanisms related to oxidative stress in mussels which inhabit the Brazilian coast, especially in Perna perna species, subjected to different stress conditions, such as the exposure to different contaminants in the laboratory and in the field, the exposure of mussels to air and re-submersion, simulating the tidal oscillations, and in mussels collected at different seasons. Both oxidative damage levels and antioxidant defense systems were strongly affected by the different environmental stress. This review summarizes the data obtained in some studies carried out in bivalves from the Brazilian coast.

Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis.

Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress.

Alpha-tocopherol induces calnexin in renal tubular cells: another protective mechanism against free radical-induced cellular damage.

Pre-administration of alpha-tocopherol is protective against oxidative renal tubular damage and subsequent carcinogenesis by ferric nitrilotriacetate (Fe-NTA) in rats. We searched for mechanisms other than the scavenging effect of alpha-tocopherol with microarray analyses, which implicated calnexin, a chaperone for glycoproteins. Renal mRNA levels of calnexin significantly increased 3h after an injection of Fe-NTA in rats fed a standard diet whereas those fed an alpha-tocopherol-supplemented diet showed an increase prior to injection, but after injection showed a decrease in renal calnexin mRNA levels, with unaltered protein levels. In experiments using LLC-PK1 cells, addition of alpha-tocopherol was protective against oxidative stress by H2O2, concomitant with calnexin induction. Knockdown of calnexin by siRNA significantly reduced this protection. Furthermore, COS-7 cells transfected with the calnexin gene were more resistant to H2O2. Together with the fact that alpha-tocopherol induced N-acetylglucosaminyltransferase 3, our data suggest that alpha-tocopherol modifies glycoprotein metabolism partially by conferring mild ER stress. This adds another molecular mechanism of alpha-tocopherol toward cancer prevention.