Exposure to High Concentrations of Cadmium Which Delay Development of Ostrinia Nubilalis Hbn. Larvae Affected the Balance of Bioelements.

All processes involved in metal homeostasis must be coordinated to provide sufficient, but not toxic, concentrations of important bioelements, and to minimize detrimental effects of toxic metals. Our previous studies dealing with the exposure of O. nubilalis non-diapausing larvae to dietary Cd demonstrated that exposure to higher concentrations of Cd caused delay in the development of larvae, induced oxidative stress and also induced defense mechanisms against the toxic effects of Cd. The aim of the present study was to evaluate how acute and chronic exposure of O. nubilalis larvae to increased concentrations of dietary Cd affected the balance of important bioelements. The concentration of bioelements was analyzed in larvae (after short-term exposure) and pupae (after long-term exposure). The short-term exposure of final instar larvae (L5) to Cd did not affect significantly the concentration of any of the analyzed bioelements, while the long-term exposure of developing larvae to higher concentrations of Cd caused increase in the concentrations of Ca, Mg and Na in pupae. The bioaccumulation factor, calculated for bioelements after long-term exposure to Cd, was higher for the most bioelements in groups fed with diet containing higher concentrations of Cd, except K which displayed the opposite trend. Pearson correlation coefficient showed positive correlations between Cd and Ca, Mg, Na, Fe, Cu and Zn, while negative correlation was observed between Cd and K. The results indicate that impact on the balance of important bioelements might be one of the mechanisms of cadmium toxicity and certainly raise numerous questions for future research.

Effect of Acrylamide Treatment on Cyp2e1 Expression and Redox Status in Rat Hepatocytes.

Acrylamide (AA) toxicity is associated with oxidative stress. During detoxification, AA is either coupled to gluthatione or biotransformed to glycidamide by the enzyme cytochrome P450 2E1 (CYP2E1). The aim of our study was to examine the hepatotoxicity of AA in vivo and in vitro. Thirty male Wistar rats were treated with 25 or 50 mg/kg b.w. of AA for 3 weeks. Qualitative and quantitative immunohistochemical evaluation of inducible nitric oxide synthase (iNOS), CYP2E1, catalase (CAT), superoxide dismutase 1 (SOD1), and SOD2 expression in liver was carried out. Bearing in mind that the liver is consisted mainly of hepatocytes, in a parallel study, we used the rat hepatoma cell line H4IIE to investigate the effects of AA at IC20 and IC50 concentrations on the redox status and the activity of CAT, SOD, and glutathione-S-transferase (GST), their gene expression, and CYP2E1 and iNOS expression. Immunohistochemically stained liver sections showed that treatment with AA25mg induced a significant decrease of CYP2E1 protein expression (p < 0.05), while treatment with AA50mg led to a significant increase of iNOS protein expression (p < 0.05). AA treatment dose-dependently elevated SOD2 protein expression (p < 0.05), while SOD1 protein expression was significantly increased only at AA50mg (p < 0.05). CAT protein expression was not significantly affected by AA treatments (p > 0.05). In AA-treated H4IIE cells, a concentration-dependent significant increase in lipid peroxidation and nitrite levels was observed (p < 0.05), while GSH content and SOD activity significantly decreased in a concentration-dependent manner (p < 0.05). AA IC50 significantly enhanced GST activity (p < 0.05). The level of mRNA significantly increased in a concentration-dependent manner for iNOS, SOD2, and CAT in AA-treated H4IIE cells (p < 0.05). AA IC50 significantly increased the transcription of SOD1, GSTA2, and GSTP1 genes (p < 0.05), while AA IC20 significantly decreased mRNA for CYP2E1 in H4IIE cells (p < 0.05). Obtained results indicate that AA treatments, both in vivo and in vitro, change hepatocytes; drug-metabolizing potential and disturb its redox status.

Modulations of the antioxidants defence system in two maize hybrids during flooding stress.

Flooding stress nowadays is one of the major stressors for plants under climate change. This kind of stress may cause severe depression of the plant's growth through inhibition of photosynthesis and oxidative cell damage as well as changes in cell respiration. The present work aimed to study the effect of flooding stress on oxidative and antioxidative parameters in leaves of two maize hybrids (ZP 555 and ZP 606). Leaves of maize plants at the stage of three fully developed leaves were harvested after 6, 24, 72, and 144 h of applied flooding stress. Leaves were used for determination of physiological (the content of photosynthetic pigments and soluble proteins), oxidative stress parameters (the content of malondialdehyde (MDA) and H2O2) as well as antioxidants (the total polyphenols content, and activity of antioxidative enzymes [catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), and Class III peroxidases (POX, EC, 1.11.1.7)]). Results indicated that flooding stress-induced time-dependent changes of measured parameters and those hybrids differ in response to stress. The noticeable difference between hybrids was detected in the H2O2 and MDA content. An increase in the activity of SOD, POX and polyphenols content, with the most pronounced changes in POX activity and polyphenols concentration, could minimize the cellular damage caused by flooding. The results of the present study suggest that a more robust antioxidative metabolism is essential under flooding stress and could be a protective strategy against oxidative damage induced by flooding in ZP 606 maize plants compared to ZP 555 plants.

Identification of a metallothionein gene in honey bee Apis mellifera and its expression profile in response to Cd, Cu and Pb exposure.

Metallothioneins are ubiquitous proteins important in metal homeostasis and detoxification. However, they have not previously been identified in honey bees or other Hymenoptera, where metallothioneins could be of ecophysiological and ecotoxicological significance. Better understanding of the molecular responses to stress induced by toxic metals could contribute to honey bee conservation. In addition, honey bee metallothionein could represent a biomarker for monitoring environmental quality. Here we identify and characterize a metallothionein gene in Apis mellifera (AmMT). AmMT is 1,680 bp long and encodes a 48 amino acids protein with 15 cysteines and no aromatic residues. A metal response element upstream of the start codon, coupled with numerous cis-regulatory elements indicate the functional context of AmMT. Molecular modelling predicts several transition metal binding sites, and comparative phylogenetic analysis revealed five putative metallothionein proteins in three other hymenoptera species. AmMT was characterized by cloning the full-length coding sequence of the putative metallothionein. Recombinant AmMT was found to increase metal tolerance upon overexpression in Escherichia coli supplemented with Cd, Cu or Pb. Finally, in laboratory tests on honey bees, gene expression profiles showed a dose-dependant relationship between Cd, Cu and Pb concentrations present in food and AmMT expression, while field experiments showed induction of AmMT in bees from an industrial site compared to those from an urban area. These studies suggest that AmMT has metal binding properties in agreement with a possible role in metal homeostasis. Further functional and structural characterization of metallothionein in honey bees and other Hymenoptera are necessary.

ENVIRONMENTAL EFFECTS ON SUPEROXIDE DISMUTASE AND CATALASE ACTIVITY AND EXPRESSION IN HONEY BEE.

Understanding the cellular stress response in honey bees will significantly contribute to their conservation. The aim of this study was to analyze the response of the antioxidative enzymes superoxide dismutase and catalase in honey bees related to the presence of toxic metals in different habitats. Three locations were selected: (i) Tunovo on the mountain Golija, as control area, without industry and large human impact, (ii) Belgrade as urban area, and (iii) Zajaca, as mining and industrial zone. Our results showed that the concentrations of lead (Pb) in whole body of bees vary according to habitat, but there was very significant increase of Pb in bees from investigated industrial area. Bees from urban and industrial area had increased expression of both Sod1 and Cat genes, suggesting adaptation to increased oxidative stress. However, in spite increased gene expression, the enzyme activity of catalase was lower in bees from industrial area suggesting inhibitory effect of Pb on catalase.

Metabolomic Analysis of Diapausing and Noni-diapausing Larvae of the European Corn Borer Ostrinia nubilalis (Hbn.) (Lepidoptera: Crambidae).

In this study, an (1)H-NMR -based metabolomic approach was used to investigate the biochemical mechanisms of diapause and cold hardiness in diapausing larvae of the European corn borer Ostrinia nubilalis. Metabolomic patterns in polar hemolymph extracts from non-diapausing and diapausing larvae of O. nubilalis were compared. Analysis indicated 13 metabolites: 7 amino acids, glycerol, acetate, citrate, succinate, lactate and putrescine. Results show that diapausing larvae display different metabolomic patterns compared to active non-diapausing larvae, with predominant metabolites identified as glycerol, proline and alanine. In specific diapausing larvae initially kept at 5 °C then gradually chilled to ­3 °C and ­16 °C, alanine , glycerol and acetate were predominant metabolites. (1)H-NMR spectroscopy provides new insight into the metabolomic patterns associated with cold resistance and diapause in O. nubilalis larvae, suggesting distinct metabolomes function in actively developing and diapausing larvae.

Spermidine supplementation in honey bees: Autophagy and epigenetic modifications.

Polyamines (PAs), including putrescine (Put), spermidine (Spd), and spermine (Spm), are essential polycations with wide-ranging roles in cellular functions. PA levels decline with age, making exogenous PA supplementation, particularly Spd, an intriguing prospect. Previous research in honey bees demonstrated that millimolar Spd added to their diet increased lifespan and reinforced oxidative resilience. The present study is aimed to assess the anti-aging effects of spermidine supplementation at concentrations of 0.1 and 1 mM in honey bees, focusing on autophagy and associated epigenetic changes. Results showed a more pronounced effect at the lower Spd concentration, primarily in the abdomen. Spd induced site-specific histone 3 hypoacetylation at sites K18 and 27, hyperacetylation at K9, with no change at K14 in the entire body. Additionally, autophagy-related genes (ATG3, 5, 9, 13) and genes associated with epigenetic changes (HDAC1, HDAC3, SIRT1, KAT2A, KAT6B, P300, DNMT1A, DNMT1B) were upregulated in the abdomens of honey bees. In conclusion, our findings highlight profound epigenetic changes and autophagy promotion due to spermidine supplementation, contributing to increased honey bee longevity. Further research is needed to fully understand the precise mechanisms and the interplay between epigenetic alterations and autophagy in honey bees, underscoring the significance of autophagy as a geroprotective mechanism.

Management of inorganic elements by overwintering physiology of cold hardy larvae of European corn borer (Ostrinia nubilalis, Hbn.).

The European corn borer (Ostrinia nubilalis, Hbn.), enters diapause, a strategy characterized by arrest of development and reproduction, reduction of metabolic rate and the emergence of increased resistance to challenging seasonal conditions as low sub-zero winter temperatures. The aim of this study was to investigate the potential role of inorganic elements in the ecophysiology of O. nubilalis, analysing their content in the whole body, hemolymph and fat body, both metabolically active, non-diapausing and overwintering diapausing larvae by ICP-OES spectrometer following the US EPA method 200.7:2001. O nubilalis as many phytophagous lepidopteran species maintain a very low extracellular sodium concentration and has potassium as dominant cation in hemolymph of their larvae. Changes in hemolymph and the whole body sodium content occur already at the onset of diapause (when the mean environmental temperatures are still high above 0 ºC) and remain stable during the time course of diapause when larvae of this species cope with sub-zero temperatures, it seems that sodium content regulation is rather a part of diapausing program than the direct effect of exposure to low temperatures. Compared to non-diapausing O. nubilalis larvae, potassium levels are much higher in the whole body and fat body of diapausing larvae and substantially increase approaching the end of diapause. The concentration of Ca, Mg, P and S differed in the whole body, hemolymph and fat body between non-diapausing and diapausing larvae without a unique trend during diapause, except an increase in their contents at the end of diapause.

Role of Arthrospira Platensis in Preventing and Treating High-Fat Diet-Induced Hypercholesterolemia in Adult Rats.

Hyperlipidaemia is a recognised risk factor for cardiovascular disease. In this study, the antihyperlipidaemic properties of spirulina (Arthrospira platensis, strain S2 from Serbia) were tested in adult Wistar rats before and after induction of hypercholesterolaemia by a high-fat diet (HFD) to compare the preventive with the curative effect. Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine transaminase (ALT) and aspartate transaminase (AST) levels were measured in the blood samples. The chemical composition (lipids, proteins and cholesterol) and the content of bile acids in the faeces of the animals were also analysed. Feeding rats with an atherogenic diet for 10 weeks led to the successful development of hyperlipidaemia, as serum TC and LDL-C levels as well as lipids, cholesterol and bile acids in the animals' faeces were significantly increased. Pre- and post-treatment with spirulina led to a reduction in serum LDL, TC and ALT levels. Administration of spirulina resulted in both a significant increase in primary bile acids excretion and a decrease in bile acids metabolism, with pre-treatment being more effective than post-treatment in some cases. These results suggest that increased excretion of bile acids as well as an effect on the gut microbiota may be the mechanism responsible for the anti-hyperlipidaemic activity of the tested spirulina strain.

Hydrogen peroxide and ecdysone in the cryoprotective dehydration strategy of Megaphorura arctica (Onychiuridae: Collembola).

The Arctic springtail, Megaphorura arctica, survives sub-zero temperatures in a dehydrated state via trehalose-dependent cryoprotective dehydration. Regulation of trehalose biosynthesis is complex; based in part on studies in yeast and fungi, its connection with oxidative stress caused by exposure of cells to oxidants, such as hydrogen peroxide (H2O2), or dehydration, is well documented. In this respect, we measured the amount of H2O2 and antioxidant enzyme activities (superoxide dismutases: copper, zinc--CuZnSOD and manganese containing--MnSOD, and catalase--CAT), as the regulatory components determining H2O2 concentrations, in Arctic springtails incubated at 5 °C (control) versus -2 °C (threshold temperature for trehalose biosynthesis). Because ecdysone also stimulates trehalose production in insects and regulates the expression of genes involved in redox homeostasis and antioxidant protection in Drosophila, we measured the levels of the active physiological form of ecdysone--20-hydroxyecdysone (20-HE). Significantly elevated H2O2 and 20-HE levels were observed in M. arctica incubated at -2 °C, supporting a link between ecdysone, H2O2, and trehalose levels during cryoprotective dehydration. CAT activity was found to be significantly lower in M. arctica incubated at -2 °C versus 5 °C, suggesting reduced H2O2 breakdown. Furthermore, measurement of the free radical composition in Arctic springtails incubated at 5 °C (controls) versus -2 °C by Electron Paramagnetic Resonance spectroscopy revealed melanin-derived free radicals at -2 °C, perhaps an additional source of H2O2. Our results suggest that H2O2 and ecdysone play important roles in the cryoprotective dehydration process in M. arctica, linked with the regulation of trehalose biosynthesis.

Spermidine dietary supplementation and polyamines level in reference to survival and lifespan of honey bees.

Honey bee health has been an important and ongoing topic in recent years. Honey bee is also an important model organism for aging studies. Polyamines, putrescine, spermidine and spermine, are ubiquitous polycations, involved in a wide range of cellular processes such as cell growth, gene regulation, immunity, and regulation of lifespan. Spermidine, named longevity elixir, has been most analysed in the context of aging. One of the several proposed mechanisms behind spermidine actions is antioxidative activity. In present study we showed that dietary spermidine supplementation: (a) improved survival, (b) increased the average lifespan, (c) influenced the content of endogenous polyamines by increasing the level of putrescine and spermidine and decreasing the level of spermine, (d) reduced oxidative stress (MDA level), (e) increased the antioxidant capacity of the organism (FRAP), (f) increased relative gene expression of five genes involved in polyamine metabolism, and (g) upregulated vitellogenin gene in honey bees. To our knowledge, this is the first study on honey bee polyamine levels in reference to their longevity. These results provide important information on possible strategies for improving honey bee health by introducing spermidine into their diet. Here, we offer spermidine concentrations that could be considered for that purpose.

Identification of a metallothionein gene and the role of biological thiols in stress induced by short-term Cd exposure in Ostrinia nubilalis.

Cadmium (Cd) is a non-essential metal that is highly toxic to all living forms, characterized by an extremely high affinity for thiol (SH) groups. The aim of this work was to identify and experimentally verify metallothionein gene and to analyze the role of biological thiols in stress induced by short-term Cd exposure in Ostrinia nubilalis, one of the most important corn pests. The coding region of a metallothionein (MT) gene in O. nubilalis was identified, encoding protein, OnMT1, which contains 46 amino acids, including 12 cysteine residues, and has no aromatic amino acids. Phylogenetic analysis revealed that OnMT1 clustered together with metallothionein from Bombyx mori. Structural bioinformatics analysis strongly suggests that OnMT1 is a metallothionein with affinity for multiple transition metals. Further, in order to elucidate the role of biological thiols, O. nubilalis L5 larvae were exposed to increasing Cd concentrations in diet (6.85, 41.71, 77.35 mg kg-1) during a 48 h period, after which Cd concentration in larvae was measured (3.50, 12.02, 47.37 mg kg-1, respectively). Due to short-term Cd exposure, concentration of free protein SH groups and relative expression of OnMT1 and thioredoxin (Trx) genes was elevated, while the reduced glutathione content remained unchanged. The presented results provide evidence that OnMT1 plays a role in Cd detoxification and homeostasis, and confirm the importance of biological thiols, especially OnMT1 and Trx, in the early response of O. nubilalis to Cd poisoning, indicating interaction between Cd and thiol-linked redox reactions. Insects provide valuable insight into molecular adaptations to metals.

Anthropogenic influence on seasonal and spatial variation in bioelements and non-essential elements in honeybees and their hemolymph.

Honeybee colony losses have been a focus of research in the last years, due to the importance of managed honeybee colonies for economy and ecology. Different unfavorable conditions from the outside environment have a strong impact on the hive health. The majority of losses occur mainly during winter and the exact reason is not completely understood. Only a small number of studies are dealing with content of bioelements, their function and influence on honeybee physiology. The aim of the present study was to determine seasonal and spatial variations in content of bioelements and non-essential elements, in hemolymph and whole body of honeybees originating from three regions with different degrees of urbanization and industrialization. Concentrations of 16 elements were compared: macroelements (Ca, K, Mg, Na), microelements (Cu, Fe, Mn, Zn) and non-essential elements (Al, Ba, Cd, Co, Cr, Ni, Pb, Sr) in samples collected from 3 different environments: Golija (rural region), Belgrade (urban region) and Zajaca (industrial region). Content of bioelements and non-essential elements in honeybees was under noticeable influence of the surrounding environment, season and degree of honeybee activity. Hemolymph was proven to be helpful in differentiating air pollution from other sources of honeybee exposure. The results of our study demonstrated that bees can be successfully used as biomonitors since we have observed statistically significant differences among observed locations, but unless compared locations are exposed to excessively different pollution pressures, it is essential that all bees should be collected at the same season.

The effects of 6-month hydrogen-rich water intake on molecular and phenotypic biomarkers of aging in older adults aged 70 years and over: A randomized controlled pilot trial.

In this randomized controlled pilot trial, we investigated the effects of a 6-month intake of hydrogen-rich water (HRW) on several molecular and phenotypic biomarkers of aging in older adults aged 70 years and over. Forty older adults (20 women) were randomly allocated in a parallel-group design to receive 0.5 L per day of HRW (15 ppm of hydrogen) or control drink (0 ppm of hydrogen) during a 6-month intervention period. The biomarkers assessed at baseline and 6-month follow up were molecular markers in the blood (DNA and chromosomes, nutrient sensing, protein, and lipid metabolism, oxidative stress and mitochondria, cell senescence, inflammation), brain metabolism, cognitive functioning, physical function and body composition, resting blood pressure, facial skin features, sleep outcomes, and health-related quality of life. The mean age, weight, and height of study participants were 76.0 ± 5.6 years, 78.2 ± 16.1 kg, height 167.5 ± 11.5 cm, respectively. A significant treatment vs. time interaction was found for telomere length (P = 0.049), with the length increased after HRW intervention (from 0.99 ± 0.15 at baseline to 1.02 ± 0.26 at follow up) and decreased after drinking control water (from 0.92 ± 0.27 to 0.79 ± 0.15). A marker of DNA methylation (Tet methylcytosine dioxygenase 2, TET2) expression at 6-month follow-up increased in both groups, yet the degree of elevation was significantly higher in HRW (from 0.81 ± 0.52 at baseline to 1.62 ± 0.66 at follow up) comparing to the control water (from 1.13 ± 0.82 to 1.76 ± 0.87) (P = 0.040). A strong trend for treatment vs. time interaction was found for a degree of DNA methylation (P = 0.166), with the methylation increased in the HRW group (from 120.6 ± 39.8 ng at baseline to 126.6 ± 33.8 ng at follow up) and decreased after taking control water (from 133.6 ± 52.9 ng to 121.2 ± 38.4 ng). HRW was superior to control water to increase brain choline and NAA levels in the left frontal grey matter, brain creatine at the right parietal white matter, and brain NAA at the right parietal mesial grey matter (P < 0.05). No significant differences were found between interventions for other outcomes (P > 0.05), except for a significantly improved chair stand performance after HRW intervention compared to the control water (P = 0.01). Owing to pleiotropic mechanisms of hydrogen action, this simple biomedical gas could be recognized as a possible anti-aging agent that tackles several hallmarks of aging, including loss of function and telomere length shortening. The study was registered at ClinicalTrials.gov (NCT04430803).

The effect of long term exposure to cadmium on Ostrinia nubilalis growth, development, survival rate and oxidative status.

In this study the effect of long term exposure to cadmium (Cd) on Ostrinia nubilalis larval growth, development, survival rate and oxidative status was analyzed. Newly hatched first instar - L1 larvae were reared on a Cd contaminated diet until the larvae reached the final, fifth instar - L5 or developed into pupae. In total, six experimental groups, five treatments (concentrations of Cd in fresh diet: Cd I: 0.73, Cd II: 3.70, Cd III: 6.85, Cd IV: 41.71 and Cd V: 77.53 mg kg-1) and a control group (C) were set up. The results of the experiment showed that exposure to higher concentrations of Cd (41.71 and 77.53 mg kg-1) had a significant influence on development and redox status of O. nubilalis larvae: (1) the development rate was strongly reduced resulting in a prolonged pupation time; (2) the survival rate of larvae was prominently lower; (3) bioaccumulation factor (measured in pupae) was reduced which indicated that larvae could accumulate Cd to a certain level; (4) the level of the lipid peroxidation was significantly higher, which points to oxidative damage; (5) the expression of Mtn was significantly up-regulated while Cat and GPx genes down-regulated. In conclusion, long term exposure to dietary Cd in a concentration of 41.7 mg kg-1 and higher, induced oxidative stress and slowed down growth and development of O. nubilalis larvae.

Effect of fullerenol nanoparticles on oxidative stress induced by paraquat in honey bees.

The enormous progress in nanomaterials development and their use, followed by their inevitable environmental print, has arisen the emerging questions concerning their influence to the living systems. Honey bees are considered to be quite a suitable model system for the risk assessment and prediction of various external influences. To the best of our knowledge, this is the first study dealing with the influence of fullerenol nanoparticles (FNP), a biodegradable carbon nanomaterials' representative, to honey bees. This investigation was conducted with an aim to merge two different open-ended questions: the potential toxic effect of FNP to the bees on the one hand and antioxidative effect of FNP on the other hand. Since FNP antioxidative properties were proved in a number of in vivo models, we hypothesized the similar outcomes, and according to this assumption, we opted for paraquat as a well-known oxidative stress inducer. FNP did not have toxic effect in none of investigated concentrations. The results also confirmed the potential of FNP to reduce oxidative stress through the gene expression of antioxidative enzymes and the change in the redox state of the cells. Additional experiments are needed for a better understanding of the exact mechanism and complex patterns of FNP's activity.

Effect of Cold Acclimation on Selected Metabolic Enzymes During Diapause in The European Corn Borer Ostrinia nubilalis (Hbn.).

The European corn borer, Ostrinia nubilalis Hbn., is a pest Lepidopteran species whose larvae overwinter by entering diapause, gradually becoming cold-hardy. To investigate metabolic changes during cold hardening, activities of four metabolic enzymes - citrate synthase (CS), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in whole-body homogenates of pupae, non-diapausing and diapausing larvae acclimated to 5 °C, -3 °C and -16 °C. The highest CS activity was detected in non-diapausing larvae, reflecting active development, while the highest in vitro LDH activity was recorded in diapausing larvae at temperatures close to 0 °C, evidencing a metabolic switch towards anaerobic metabolism. However, in-gel LDH activity showed that production of pyruvate from lactate is triggered by sub-zero temperatures. The activities of both aminotransferases were highest in non-diapausing larvae. Our findings suggest that during diapause and cold hardening the aminotransferases catalyse production of L-alanine, an important cryoprotectant, and L-aspartate, which is closely tied to both transamination reactions and Krebs cycle. The results of this study indicate that, during diapause, the activity of metabolic enzymes is synchronized with exogenous factors, such as temperatures close to 0 °C. These findings support the notion that diapause is metabolically plastic and vibrant, rather than simply a passive, resting state.

Laboratory bioassays on the response of honey bee (Apis mellifera L.) glutathione S-transferase and acetylcholinesterase to the oral exposure to copper, cadmium, and lead.

In the present study, the influence of cadmium, copper, and lead on two enzymes often used as biomarkers in toxicological analysis was investigated. Bees were fed with 1 M sucrose solution containing 10-fold serial dilutions of CuCl2 (1000 mg L-1, 100 mg L-1, and 10 mg L-1), CdCl2 (0.1 mg L-1, 0.01 mg L-1, and 0.001 mg L-1), or PbCl2 (10 mg L-1, 1 mg L-1, and 0.1 mg L-1) during 48 h. Our results showed that the total glutathione S-transferase activity was not changed under the influence of cadmium and lead, and it was decreased with the highest concentration of copper. The level of gene expression of the three analyzed classes of glutathione S-transferase was significantly increased with increasing concentrations of copper and cadmium. Lead did not cause significant changes in glutathione S-transferase activity and gene expression, while it showed biphasic effect on acetylcholinesterase activity: lower concentration of lead, 0.1 mg L-1 inhibited and higher dose, 10 mg L-1 induced acetylcholinesterase activity in honey bees. Furthermore, our results showed a significant decrease of the acetylcholinesterase activity in honey bees treated with 0.001 and 0.01 mg L-1 CdCl2. Our results indicate the influence of cadmium, copper, and lead on GST and AChE in the honey bees. These results form the basis for future research on the impact of metallic trace element pollution on honey bees.

Effects of acrylamide on oxidant/antioxidant parameters and CYP2E1 expression in rat pancreatic endocrine cells.

Oxidative stress is one of the principle mechanism of acrylamide-induced toxicity. Acrylamide is metabolized by cytochrome P450 2E1 (CYP2E1) to glycidamide or by direct conjugation with glutathione. Bearing in mind that up to now the effects of acrylamide on oxidative stress status and CYP2E1 level in endocrine pancreas have not been studied we performed qualitative and quantitative immunohistochemical evaluation of inducible nitric oxide synthase (iNOS), superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), catalase (CAT) and CYP2E1 expression in islets of Langerhans of rats subchronically treated with 25 or 50mg/kg bw of acrylamide. Since the majority of cells (>80%) in rodent islets are beta cells, in parallel studies, we employed the Rin-5F beta cell line to examine effects of acrylamide on redox status and the activity of CAT, SOD and glutathione-S-transferase (GST), their gene expression, and CYP2E1, NF-E2 p45-related factor 2 (Nrf2) and iNOS expression. Immunohistochemically stained pancreatic sections revealed that acrylamide induced increase of iNOS and decrease of CYP2E1 protein expression, while expression of antioxidant enzymes was not significantly affected by acrylamide in islets of Langerhans. Analysis of Mallory-Azan stained pancreatic sections revealed increased diameter of blood vessels lumen in pancreatic islets of acrylamide-treated rats. Increase in the GST activity, lipid peroxidation and nitrite level, and decrease in GSH content, CAT and SOD activities was observed in acrylamide-exposed Rin-5F cells. Level of mRNA was increased for iNOS, SOD1 and SOD2, and decreased for GSTP1, Nrf2 and CYP2E1 in acrylamide-treated Rin-5F cells. This is the first report of the effects of acrylamide on oxidant/antioxidant parameters and CYP2E1 expression in pancreatic endocrine cells.

The influence of low temperature and diapause phase on sugar and polyol content in the European corn borer Ostrinia nubilalis (Hbn.).

The European corn borer (ECB, Ostrinia nubilalis Hbn.) is a major pest in temperate regions of Europe and North America. Fifth instar ECB larvae enter diapause before winter and gradually develop cold hardiness. Here we investigated the combined influence of diapause phase and low temperature on sugar and polyol content in ECB larvae. Larvae in mid-diapause or diapause termination were acclimated at 5 °C, -3°C or -16 °C, and sugar and polyol content was measured using GC-MS. Control GC-MS measurements were conducted on untreated non-diapausing larvae. We detected differences in polyol (glycerol, sorbitol, myo-inositol) and sugar (trehalose, fructose, glucose) levels in diapausing versus non-diapausing larvae. Glycerol and trehalose were the most abundant of all analyzed cryoprotective compounds in diapausing larvae. Exposure of diapausing larvae to decreasing temperatures induced changes in polyol and sugar levels that depended on the phase of diapause. In mid-diapause larvae, decreasing temperatures induced a significant increase in glycerol and a decrease in sorbitol and myo-inositol. In larvae at diapause termination, polyol content was lower and less influenced by decreasing temperatures. In contrast, sugar levels were lower in larvae at mid-diapause versus diapause termination. Exposure of larvae to -16 °C induced a significant increase in the levels of all detected sugars. In particular, glucose levels were significantly higher in larvae at diapause termination following exposure to -16 °C. We propose that this shift toward sugar synthesis following low temperature exposure in larvae at diapause termination is a consequence of NADPH dependent polyol synthesis, and may be a mechanism for preservation of carbon reserves needed for post-diapause development.