A molecular biomarker system for assessing the health of gastropods (Ilyanassa obsoleta) exposed to natural and anthropogenic stressors

We developed a Molecular Biomarker System (MBS) to assess the physiological status of mud snails (Ilyanassa obsoleta) challenged by exposure to high temperature, cadmium, atrazine, endosulfan and the water-accommodating fraction of bunker fuel #2. The MBS is used to assay specific cellular parameters of the gastropod cell that are indicative of a non-stressed or stressed condition. The MBS distinguished among responses to each stressor and to non-stressed control conditions. For example, the biomarkers metallothionein and cytochrome P450 2E1 homologue distinguished between metal and non-metal stresses. MBS data from this study corroborate toxicological studies of organismal responses to endosulfan, atrazine, fuel and cadmium stresses. The MBS technology aids in the accurate diagnosis of the snail's health condition because the physiological significance of the changes of each biomarker is well known. This technology is particularly relevant for environmental monitoring because gastropods are used as key indicator species in many estuarine, marine, freshwater and terrestrial ecosystems. Finally, the Molecular Biomarker System technology is relatively inexpensive, easy to implement, precise and can be quickly adapted to an automated, high-throughput system for large sample analysis.     

A Molecular Biomarker System for Assessing the Health of Coral (Montastraea faveolata) During Heat Stress

Using a novel molecular biomaker system (MBS), we assessed the physiological status of coral (Montastraea faveolata) challenged by heat stress by assaying specific cellular and molecular parameters. This technology is particularly relevant for corals because heat stress is thought to be an essential component of coral bleaching. This phenomenon is widely believed to be responsible for coral mortality worldwide, particularly during 1997–1998. Specific parameters of coral cellular physiology were assayed using the MBS that are indicative of a nonstressed or stressed condition. The MBS distinguished the separate and combined effects of heat and light on the 2 coral symbionts, a scleractinian coral and a dinoflagellate algae (zooxanthellae). This technology aids in the accurate diagnosis of coral condition because each parameter is physiologically well understood. Finally, the MBS technology is relatively inexpensive, easy to implement, and precise, and it can be quickly adapted to a high-throughout robotic system for mass sample analysis. Accepted May 25, 2000.     

Effect of the lipid peroxide reaction caused by repeated cold stress on cisplatin-induced nephrotoxicity

The peroxide reaction in mouse kidney was examined in order to determine the relationship between the lipid peroxide reaction caused by SART (specific alternation of rhythm in temperature) stress and that caused by drug administration. After exerting SART stress for one wk on 6-wk-old male ddY mice (stress group), the peroxide reaction generated by the administration of a single dose of cis-diamminedichloroplatinum (cisplatin: CDDP, 10 mg/kg, ip) into SART-stressed mice (stress + CDDP group) was compared with the reaction of CDDP-administered nonstressed mice (CDDP group), saline-administered nonstressed mice (saline group), and saline-administered SART-stressed mice (stress + saline group). Lipid peroxidation in the kidneys was significantly higher in the stress group upon cessation of stress exertion than in the normal group. However, no significant difference in the lipid peroxide level after administration of CDDP was observed between the CDDP groups. The renal glutathione levels were significantly different between the CDDP groups and the saline administered groups. These results indicate that the peroxide reaction is generated in the kidneys by stress, but stress has no effect on the peroxide damage caused by CDDP administration. However, the contribution of stress to renal function impairment requires further evaluation.     

Carnosol protects against spinal cord injury through Nrf-2 upregulation

Background: Carnosol is an ortho-diphenolic diterpene with excellent antioxidant potential. The present study was designed to identify the protective role of carnosol against spinal cord injury (SCI)-induced oxidative stress and inflammation in Wistar rats. Methods: In the present study, oxidative stress status was determined through estimating total antioxidant capacity, total oxidant status, lipid peroxide content, protein carbonyl and sulfhydryl levels, reactive oxygen species (ROS), antioxidant status (superoxide-dismutase, catalase, glutathione, glutathione peroxidase, glutathione-S-transferase). Inflammatory effects were determined by analyzing the expression of NF-κB and COX-2 through Western blot analysis. Further, carnosol-mediated redox homeostasis was analyzed by determining p-AKT and Nrf-2 levels. Results: SCI resulted in a significant increase in oxidative stress status through increased ROS generation, total oxidant levels, lipid peroxide content, protein carbonyl and sulfhydryl levels. The antioxidant status in SCI rats was significantly reduced, indicating imbalance in redox status. In addition, the expression of NF-κB and COX-2 was significantly upregulated, while p-AKT and Nrf-2 levels were downregulated in SCI rats. However, treatment with carnosol showed a significant enhancement in the antioxidant status with concomitant decline in oxidative stress parameters. Further, carnosol treatment regulated the key proteins in inflammation and redox status through significant downregulation of NF-κB and COX-2 levels and upregulation of p-AKT and Nrf-2 expression. Conclusion: Thus, the present study shows for the first time on the protective role of carnosol against SCI-induced oxidative stress and inflammation through modulating NF-κB, COX-2 and Nrf-2 levels in Wistar rats.     

Radical initiated alpha-tocopherol depletion and lipid peroxidation in mitochondrial membranes

The relationships between antioxidant status, lipid peroxidation and membrane protein integrity have been studied in an isolated mitochondrial membrane system. Tocopherol was shown to be present in both the outer and inner membrane of normal rat liver mitochondria; 77.3 and 22.3% of the total alpha-tocopherol was present in the outer and inner membranes, respectively. The endogenous alpha-tocopherol was depleted in a time-dependent manner by low levels of ferrous iron and by irradiation in the presence or absence of ferrous iron. This antioxidant depletion was followed by the appearance of lipid hydroperoxides. Fragmentation of monoamine oxidase, an integral outer membrane protein, was observed at irradiation doses that caused by antioxidant depletion and peroxide generation.     

The influence of glutathione upon light-induced high-amplitude swelling and lipid peroxideformation of spinach chloroplasts

Light-induced high-amplitude swelling and lipid peroxide formationof isolated spinach chloroplasts were studied with regard tothe supposed analogy to lipid peroxide dependent disintegrationof mitochondria. Addition of glutathione to chloroplasts exposedto light results in excessive stimulation of swelling, whereasthe rate of lipid peroxide accumulation is significantly decreased.In the dark, glutathione does not show any substantial effect.Glutathione apparently serves as a donor substrate for the reductionof lipid peroxides in chloroplasts. The increased water uptakeof chloroplasts in the presence of glutathione cannot be conditionedby peroxidation of membrane lipids, as could be demonstratedfor mitochondria in similar experiments. Evidence is presentedthat a glutathione peroxidase activity is present in spinachhomogenate. The enzyme system, however, behaves quite differentlyfrom glutathione peroxidase of animal sources in respect tostability and pH dependency of the reaction rate (Received October 24, 1970; )     

Effect of flooding on the activities of some enzymes of activated oxygen metabolism,the levels of antioxidants,and lipid peroxidation in senescing tobacco leaves

Effects of flooding on the activities of some enzymes of activated oxygen metabolism, the levels of antioxidants, and lipid peroxidation in senescing leaves of tobacco were investigated. As judged by the decrease in chlorophyll and protein levels, flooding accelerated the senescence of tobacco leaves. Total peroxide and the lipid peroxidation product, malondialdehyde, increased in both control and flooding-treated leaves with increasing duration of the experiment. Throughout the duration of the experiment, flooded leaves had higher levels of total peroxide and malondialdehyde than did control leaves. Flooding resulted in an increase in peroxidase and ascorbate peroxidase activities and a reduction of superoxide dismutase activity in the senescing leaves. Glycolate oxidase, catalase, and glutathione reductase activities were not affected by flooding. Flooding increased the levels of total ascorbate and dehydroascorbate. Total glutathione, reduced form glutathione, or oxidized glutathione levels in flooded leaves were lower than in control leaves during the first two days of the experiment, but were higher than in control leaves at the later stage of the experiment. Our work suggests that senescence of tobacco induced by flooding may be a consequence of lipid peroxidation possibly controlled by superoxide dismutase activity. Our results also suggest that increased rates of hydrogen peroxide in leaves of flooded plants could lead to increased capacities of the scavenging system of hydrogen peroxide.Abbreviations GSH reduced form glutathione - GSSG oxidized form glutathione - GSSG reductase glutathione reductase - MDA malondialdehyde - SOD superoxide dismutase     

Differential expression of LvHSP60 in shrimp in response to environmental stress

Previous studies showed that heat-shock protein 60 (HSP60) was known to function as a molecular chaperone and is an important factor in the innate immune system in mammals. However, little was known about the physiological relevance of HSP60 in marine invertebrates. This study focuses on long-term monitoring of the differential expression of LvHSP60 in shrimp Litopenaeus vannamei in response to environmental stress. The thermal aggregation assay elucidated that LvHSP60 was an effective chaperone. It also suggested that LvHSP60 may employ the cell's intrinsic mechanism to start the immunizing process. Using quantitative real-time PCR to monitor gene expression showed that LvHSP60 was variable under different stresses including environmental stress and pathogenic infection. LvHSP60 was speculated to regulate the adaptive responses to overcome environmental stresses. In conclusion, our study proved that LvHSP60 plays an important role in the intrinsic immune system and stress responses of shrimp.     

Hazelnut oil administration reduces aortic cholesterol accumulation and lipid peroxides in the plasma, liver, and aorta of rabbits fed a high-cholesterol diet

Hazelnut oil (HO) is rich in monounsaturated fatty acids and antioxidants. We wanted to investigate the effect of HO on lipid levels and prooxidant-antioxidant status in rabbits fed a high-cholesterol (HC) diet. An HC diet caused significant increases in lipids and lipid peroxide levels in the plasma, liver, and aorta together with histopathological atherosclerotic changes in the aorta. Glutathione levels, glutathione peroxidase, and glutathione transferase activities decreased significantly, but superoxide dismutase activity and vitamin E and C levels remained unchanged in the livers of rabbits following HC diet. HO supplementation reduced plasma, liver, and aorta lipid peroxide levels and aorta cholesterol levels together with amelioration in atherosclerotic lesions in the aortas of rabbits fed an HC diet, without any decreasing effect on cholesterol levels in the plasma or liver. HO did not alter the antioxidant system in the liver in the HC group. Our findings indicate that HO reduced oxidative stress and cholesterol accumulation in the aortas of rabbits fed an HC diet.     

Comparative antioxidant enzyme study in freshwater fish with different types of feeding behaviour

Comparative studies were made of the activities of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in organ homogenates from two herbivorous (grass carp and silver carp) and three omnivorous fish (barbel, crucian carp and common carp). The protein contents and lipid peroxidation of the organ homogenates were also compared. These comparative measurements provided control values for subsequent toxicological examinations. For the herbivorous fish, the highest total superoxide dismutase activity was observed in the kidney, followed in turn by the liver or spleen. In contrast, the highest total superoxide dismutase activity in the barbel was found in the roe, in the crucian carp in the liver, and in the common carp in the liver too. The quantitative distributions of the two peroxide metabolism enzymes glutathione peroxidase and catalase in the organ homogenates present very varied pictures. The results of the lipid peroxidation are similarly difficult to fit into one framework, even for fish following the same diet. It appears that the enzyme activities mentioned in points 4 and 5, together with the lipid peroxidation, depend not on the species, but on the variety.     

Glutathione level and its relation to radiation therapy in patients with cancer of uterine cervix

Glutathione functions as an important antioxidant in the destruction of hydrogen peroxide and lipid peroxides by providing substrate for the glutathione peroxidase and also promotes the ascorbic acid. Glutathione plays a vital role in detoxification of xenobiotics, carcinogens, free radicals and maintenance of immune functions. The study was aimed to determine plasma glutathione as well as erythrocyte glutathione and glutathione peroxidase in patients with invasive cervical carcinoma (n = 30) before initiation and after completion of radiotherapy and subsequently, at the time of first three monthly follow-up visit. The levels of plasma glutathione, erythrocyte glutathione and glutathione peroxidase activity were found to be lower in all cervical cancer patients as compared to age matched normal control women. The study indicates a change in antioxidant status in relation with the glutathione system among patients with invasive carcinoma of the uterine cervix. This study also demonstrates the effect of radiation therapy on this antioxidant system.     

Role of calcium in AMF-mediated alleviation of the adverse impacts of cadmium stress in Bassia indica [Wight] A.J. Scott

The aim of this study was to evaluate cadmium stress induced changes in the growth, lipid peroxidation and antioxidant activity of Bassia indica associated with arbuscular mycorrhizal fungi (AMF) and their amelioration by calcium application. Cadmium stress can cause alterations in the physiological and biochemical processes in plants. A calcium application combined with an AMF treatment resulted in the reduction of lipid peroxidation and the production of hydrogen peroxide, thereby mediating the mitigation of cadmium induced oxidative stress. The activity of antioxidant enzymes increased with cadmium application, whereas AMF inoculation combined with a calcium application further enhanced their activity. An increase in the content of non-enzymatic antioxidants such as ascorbate, reduced glutathione (GSH), oxidized glutathione (GSSG) and S-nitrosoglutathione (GSNO) in AMF-inoculated and calcium-treated plants further suggests their role in strengthening the antioxidant defense system that results in maintained growth. The application of calcium combined with the AMF treatment caused a significant reduction in lipid peroxidation and in the production of hydrogen peroxide, thereby mediating the mitigation of the cadmium induced oxidative stress. Increased proline accumulation was clearly evident in stressed plants, and the calcium application as well as the AMF inoculation further induced proline synthesis, thereby providing efficient protection against cadmium stress by increasing the maintenance of the systemic resistance criteria.     

Stage-structured interactions between seasonal and permanent residents of an estuarine nekton community

Estuarine assemblages of fishes and natant decapod crustaceans (i.e. nekton) comprise both permanent resident species and juveniles of coastal marine species that use estuaries primarily as nurseries. In an attempt to understand how the young of marine species successfully invade communities of permanent estuarine residents we studied potential interactions between two of the most abundant decapod crustaceans in nekton assemblages of the southeastern United States. Three years of quantitative samples from an intertidal marsh on Sapelo Island, Georgia showed that densities of the resident daggerblade grass shrimp Palaemonetes pugio were reduced during the time that juvenile white shrimp Penaeus setiferus used the estuary as a nursery. Results of a field enclosure experiment showed that white shrimp had no significant lethal or sublethal effects on adult grass shrimp. However, they did reduce survival of both juvenile and larval grass shrimp in laboratory experiments, suggesting the potential importance of a stage-dependent predatorprey interaction between the two shrimp species. The mortality rate of young grass shrimp in the presence of white shrimp was unaffected by grass shrimp density, but larvae (2.6–3.0 mm) suffered higher mortalities than did juveniles (5.0–15.0 mm). We suggest that the vulnerability of grass shrimp to predation by white shrimp is related to their molting cycle. The window of vulnerability opens more often for younger grass shrimp because they molt more frequently. When combined with losses due to other predators and competitors, the impact of early white shrimp cohorts on grass shrimp larvae and juveniles may prevent the resident species from maintaining its population at high densities, thereby freeing resources in the nursery for subsequent cohorts of juvenile white shrimp.     

Antioxidative Responses and Morpho-anatomical Alterations for Coping with Flood-Induced Hypoxic Stress in Grass Pea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.) in Comparison with Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

Flooding stress constrains crop growth and yield because most agricultural species are flood-sensitive. However, many of the plant species that live in permanently or temporarily flooded habitats have evolved specific traits to cope with these harsh conditions. Grass pea (Lathyrus sativus L.) is a legume that tolerates stresses such as drought, diseases, and pests; however, it is unclear whether grass pea has a tolerance mechanism for flooding stress. To understand if grass pea tolerates hypoxia and how it deals with hypoxic stress, the effects of hypoxia on root tip death, physiological, and morpho-anatomical alterations in grass pea and pea (Pisum sativum), which is sensitive to hypoxia, were compared. The results showed that activities of antioxidant enzymes, namely superoxide dismutase, catalase, ascorbate peroxidase, and glutathione content in grass pea were greater than in pea during hypoxia, which protected the root tip from oxidative damage and reduced ion leakage, which helped maintain membrane integrity. Furthermore, aerenchyma and lateral root development accompanied by ethylene production, moderate ROS accumulation-mediated cell death, and Ca²⁺ spatial-temporal heterogeneity developed well in grass pea compared to pea, which may not only facilitate internal gas diffusion but also promote removal of toxic by-products under hypoxic conditions. These results demonstrate that grass pea is more tolerant to hypoxic stress induced by flooding than garden pea seedlings. This discovery not only provides significant information for understanding the hypoxia-tolerant mechanisms in plants, but also promotes the usability of grass pea in flood-prone areas.     

Mitochondrial catalase and oxidative injury

Mitochondria dysfunction induced by reactive oxygen species (ROS) is related to many human diseases and aging. In physiological conditions, the mitochondrial respiratory chain is the major source of ROS. ROS could be reduced by intracellular antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase as well as some antioxidant molecules like glutathione and vitamin E. However, in pathological conditions, these antioxidants are often unable to deal with the large amount of ROS produced. This inefficiency of antioxidants is even more serious in mitochondria, because mitochondria in most cells lack catalase. Therefore, the excessive production of hydrogen peroxide in mitochondria will damage lipid, proteins and mDNA, which can then cause cells to die of necrosis or apoptosis. In order to study the important role of mitochondrial catalase in protecting cells from oxidative injury, a HepG2 cell line overexpressing catalase in mitochondria was developed by stable transfection of a plasmid containing catalase cDNA linked with a mitochondria leader sequence which would encode a signal peptide to lead catalase into the mitochondria. Mitochondria catalase was shown to protect cells from oxidative injury induced by hydrogen peroxide and antimycin A. However, it increased the sensitivity of cells to tumor necrosis factor-alpha-induced apoptosis by changing the redox-oxidative status in the mitochondria. Therefore, the antioxidative effectiveness of catalase when expressed in the mitochondrial compartment is dependent upon the oxidant and the locus of ROS production.     

Avocado oil induces long-term alleviation of oxidative damage in kidney mitochondria from type 2 diabetic rats by improving glutathione status

Hyperglycemia and mitochondrial ROS overproduction have been identified as key factors involved in the development of diabetic nephropathy. This has encouraged the search for strategies decreasing glucose levels and long-term improvement of redox status of glutathione, the main antioxidant counteracting mitochondrial damage. Previously, we have shown that avocado oil improves redox status of glutathione in liver and brain mitochondria from streptozotocin-induced diabetic rats; however, the long-term effects of avocado oil and its hypoglycemic effect cannot be evaluated because this model displays low survival and insulin depletion. Therefore, we tested during 1 year the effects of avocado oil on glycemia, ROS levels, lipid peroxidation and glutathione status in kidney mitochondria from type 2 diabetic Goto-Kakizaki rats. Diabetic rats exhibited glycemia of 120–186 mg/dL the first 9 months with a further increase to 250–300 mg/dL. Avocado oil decreased hyperglycemia at intermediate levels between diabetic and control rats. Diabetic rats displayed augmented lipid peroxidation and depletion of reduced glutathione throughout the study, while increased ROS generation was observed at the 3rd and 12th months along with diminished content of total glutathione at the 6th and 12th months. Avocado oil ameliorated all these defects and augmented the mitochondrial content of oleic acid. The beneficial effects of avocado oil are discussed in terms of the hypoglycemic effect of oleic acid and the probable dependence of glutathione transport on lipid peroxidation and thiol oxidation of mitochondrial carriers.     

Activities of biomarkers in multiple life stages of the model crustacean, Palaemonetes pugio

Increased urbanization of coastal areas has led to increased contaminant levels in adjacent sediments and waters. Consequently, many studies have been conducted to determine the potential impacts on estuarine organisms, including the grass shrimp, Palaemonetes pugio. This study investigated baseline levels of four cellular biomarkers (glutathione (GSH), lipid peroxidation (LPx), acetylcholinesterase (AChE), and cholesterol (CHL)) in multiple life stages (stage IV, V, VI, and VII embryos, newly-hatched larvae, 18-day-old larvae, juveniles, and adults) of P. pugio to determine which biomarkers may potentially be useful as indicators of contaminant exposure. There was a similar pattern in both LPx and AChE levels, with a clear increase occurring from the embryonic to the adult stages. Detectable levels of AChE did not occur until embryo stage V. Glutathione shared a similar pattern to LPx and AChE from the newly-hatched larvae through the adult stage, however, it did not exhibit any distinguishable pattern overall with highly variable levels among all life stages. Likewise, CHL did not exhibit any distinguishable pattern, but in contrast, CHL levels were similar throughout all life stages. This research provides valuable background information that may be used in future assessments of grass shrimp population health.     

Extender Supplementation with Antioxidants Selected after the Evaluation of Sperm Susceptibility to Oxidative Challenges in Goats

This study aimed to detect the most deleterious ROS for goat sperm and then supplemented the extender with a proper antioxidant. For this, 12 adult goats (aged 1–7) were used. Fresh samples were submitted to challenge with different ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) and malondialdehyde (MDA—toxic product of lipid peroxidation). After experiment 1, sperms were cryopreserved in extenders supplemented to glutathione peroxidase (Control: 0?UI/mL; GPx1: 1?UI/mL; GPx5: 5?UI/mL, and GPx10: 10?UI/mL) and catalase (Control: 0?UI/mL; CAT60: 60?UI/mL; CAT120: 120?UI/mL, and CAT240: 240?UI/mL). Each sample was evaluated by motility, plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, assay of the sperm chromatin structure, mitochondrial activity (3,3-diaminobenzidine), and measurement of lipid peroxidation (thiobarbituric acid reactive substances [TBARS]). It was possible to observe a mitochondrial dysfunction (DAB—Class IV) and low membrane integrity after hydrogen peroxide action. However, the high rates of TBARS were observed on hydroxyl radical. CAT240 presents the lower percentage of plasma membrane integrity. It was possible to attest that hydrogen peroxide and hydroxyl radical are the more harmful for goat sperm. Antioxidant therapy must be improving perhaps using combination between antioxidants.     

The effect of 2,4-dichlorophenol and pentachlorophenol on antioxidant system in the leaves of Phalaris arudinacea

The purpose of this work was to evaluate the effect of 2,4-dichlorophenol (2,4-DCP) and pentachlorophenol (PCP) on the activity of antioxidative system and lipid peroxidation in the leaves of reed canary grass (Phalaris arudinacea). The activity of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR) and glutathione S-transferase (GST) as well as the content of glutathione, ascorbate and phenolic compounds were determined. An induced-increase in the APX, CAT, GPX and GR activities was stronger for PCP, while a significant increase in the GST activity was noted only for 2,4-DCP. Both compounds increased the content of phenolic compounds, oxidized and reduced glutathione as well as the content of ascorbic acid. PCP induced stronger increase in lipid peroxidation than 2,4-DCP. The observed changes revealed that chlorophenols induce oxidative stress and oxidative damage in the leaves of reed canary grass.