Maria Augustyniak

Activity of glutathione S-transferase in Spodoptera exigua larvae exposed to cadmium and zinc in two subsequent generations

The aim of our study was to establish changes in activity of important in detoxification enzyme-glutathione S-transferase (GST): in alimentary tract, fat body and Malpighian tubules of Spodoptera exigua larvae being under cadmium and zinc exposure through the first as well as the second generation. There was registered enhancement of the enzyme activity in the fat body and the Malpighian tubules caused by zinc as well as its decrease in the Malpighian tubules under cadmium action. Amounts of metals in the alimentary tract were either several times higher than in the diet ingested by larvae or than in the fat body. Metal concentration in the fat body correlated with the level of the enzyme activity (positive correlation for zinc and negative for cadmium). The effect of metal action differentiated dependently on time exposition.

Does the grasshopper Chorthippus brunneus adapt to metal polluted habitats? A study of glutathione-dependent enzymes in grasshopper nymphs

Glutathione contents and activity of glutathione‐dependent enzymes in the body of insects inhabiting polluted areas depend on toxin kind, concentration and exposure time. Enzymatic response may be modified by gender, age, developmental stage and state of nutrition. Also, chronic exposure to metals in the environment may cause the selection of individuals resistant to some environmental toxins. To assess the degree of adaptation of Chorthippus brunneus to metal‐polluted habitats, we measured glutathione contents and the activity of selected glutathione‐dependent enzymes in the offspring of aging mothers which differed in time and intensity of exposure to metals in their habitats. We tested whether differences represent temporal shifts in tolerance range or were genetically preserved and inherited by future generations. We investigated insects from three populations. Two live in heavily metal‐burdened areas, exposed to metals for 170 (Szopienice) or 50 years (Olkusz) and the third inhabits an unpolluted reference site (Pilica). The most important findings were age‐by‐site interactions for all biochemical analyses. Nymphs from Szopienice had lower glutathione contents and lower glutathione‐dependent enzyme activity in comparison with nymphs from the reference site. This was especially distinct in nymphs hatched from eggs laid by young females. The offspring of aging females from Olkusz, in terms of glutathione contents and glutathione reductase activities, revealed similar patterns to those from the reference site. For the remaining parameters, enzyme activity patterns in nymphs from Olkusz were similar to those of nymphs from Szopienice.

The Comet assay in insects--Status, prospects and benefits for science.

The Comet assay has been recently adapted to investigate DNA damage in insects. The first reports of its use in Drosophila melanogaster appeared in 2002. Since then, the interest in the application of the Comet assay to studies of insects has been rapidly increasing. Many authors see substantial potential in the use of the Comet assay in D. melanogaster for medical toxicology studies. This application could allow the testing of drugs and result in an understanding of the mechanisms of action of toxins, which could significantly influence the limited research that has been performed on vertebrates. The possible perspectives and benefits for science are considered in this review. In the last decade, the use of the Comet assay has been described in insects other than D. melanogaster. Specifically, methods to prepare a cell suspension from insect tissues, which is a difficult task, were analyzed and compared in detail. Furthermore, attention was paid to any differences and modifications in the research protocols, such as the buffer composition and electrophoresis conditions. Various scientific fields in addition to toxicological and ecotoxicological research were considered. We expect the Comet assay to be used in environmental risk assessments and to improve our understanding of many important phenomena of insect life, such as metamorphosis, molting, diapause and quiescence. The use of this method to study species that are of key importance to humans, such as pests and beneficial insects, appears to be highly probable and very promising. The use of the Comet assay for DNA stability testing in insects will most likely rapidly increase in the future.

Evaluation of in vivo graphene oxide toxicity for Acheta domesticus in relation to nanomaterial purity and time passed from the exposure

Graphene and its oxidized form-graphene oxide (GO) have become exceptionally popular in industry and medicine due to their unique properties. However, there are suspicions that GO can cause adverse effects. Therefore, comprehensive knowledge on its potential toxicity is essential. This research assesses the in vivo toxicity of pure and manganese ion contaminated GO, which were injected into the hemolymph of Acheta domesticus. The activity of catalase (CAT) and gluthiathione peroxidases (GSTPx) as well as heat shock protein (HSP 70) and total antioxidant capacity (TAC) levels were measured at consecutive time points-1h, 24h, 48h and 72h after injection. Neither pure GO nor GO contaminated with manganese were neutral to the organism. The results proved the intensification of oxidative stress after GO injection, which was confirmed by increased enzyme activity. The organism seems to cope with this stress, especially in the first 24h after injection. In the following days, increasing HSP 70 levels were observed, which might suggest the synthesis of new proteins and the removal of old and damaged ones. With that in mind, the potential toxicity of the studied material, which could lead to serious and permanent damage to the organism, should still be taken into consideration.

Short-term in vivo exposure to graphene oxide can cause damage to the gut and testis.

Graphene oxide (GO) has unique physicochemical properties and also has a potentially widespread use in every field of daily life (industry, science, medicine). Demand for nanotechnology is growing every year, and therefore many aspects of its toxicity and biocompatibility still require further clarification. This research assesses the in vivo toxicity of pure and manganese ion-contaminated GO that were administrated to Acheta domesticus with food (at 200mgkg-1 of food) throughout their ten-day adult life. Our results showed that short-term exposure to graphene oxide in food causes an increase in the parameters of oxidative stress of the tested insects (catalase - CAT, total antioxidant capacity - TAC), induces damage to the DNA at a level of approximately 35% and contributes to a disturbance in the stages of the cell cycle and causes an increase of apoptosis. Moreover, upon analyzing histological specimens, we found numerous degenerative changes in the cells of the gut and testis of Acheta domesticus as early as ten days after applying GO. A more complete picture of the GO risk can help to define its future applications and methods for working with the material, which may help us to avoid any adverse effects and damage to the animal.

Oxidative stress and genotoxic effects of diamond nanoparticles

Due to the unique and useful properties of nanodiamonds (ND), their production and use is rapidly increasing. Thus, more of these particles will be released into the environment and organisms will inevitably be exposed to them. The current knowledge about the toxicity of ND, especially in vivo toxicity, is fragmentary. In this study, the toxicity of nanodiamonds was assessed in Acheta domesticus following chronic exposure to different nominal concentrations of ND (20 and 200µgg(-1) food) administrated in food for the entire lifespan. The activity of oxidative stress enzymes (catalase, glutathione peroxidase), total antioxidant capacity, as well as the level of heat shock protein were determined. A significant increase in all of the measured parameters was observed after seven weeks of exposure in individuals exposed to higher concentrations of ND (200µgg(-1) food). In animals exposed to lower concentrations of ND (20µgg(-1) food), there were few significant changes to these parameters. Analysis of DNA damage performed after fourteen weeks using the comet assay revealed DNA instabilities in the insects, especially the ones that had been exposed to the higher doses of ND. These findings may suggest that the toxicity of ND is concentration dependent. While high doses interact in a toxic manner, trace amounts, which are more likely in the environment, might be safe for organisms. Extreme caution should be taken when handling nanodiamonds.

Phenotypic Plasticity, Epigenetic or Genetic Modifications in Relation to the Duration of Cd-Exposure within a Microevolution Time Range in the Beet Armyworm

In the case of the pests inhabiting metal polluted or fields where the use of pesticides is common, a natural selection of resistant individuals can occur. This may pose serious problems for humans, agriculture, as well as the economies of many countries. In this study, the hypothesis that multigenerational (120 generations) exposure to cadmium of a beet armyworm population could be a selecting factor toward a more efficient DNA protection was verified. The hemocytes of individuals from two culture strains (control and Cd-exposed) were treated with H2O2 (a DNA-damaging agent) or PBS (reference). The level of DNA damage was assessed using the Comet assay immediately and 5, 15 and 30 min. after the treatment. The immediate result of the contact with H2O2 was that the level of DNA damage in the hemocytes of the insects from both strains increased significantly. However, in the cells of the Cd-exposed individuals, the level of DNA damage decreased over time, while in the cells from the control insects it remained at the same level with no evidence of repair. These results suggest that efficient defense mechanisms may exist in the cells of insects that have prolonged contact with cadmium. Some evolutionary and trade-off aspects of the phenomenon are discussed. In a wider context, comparing the results obtained in the laboratory with field studies may be beneficial for understanding basic mechanisms of the resistance of an organism. To summarize, the high potential for the repair of DNA damage that was observed in the insects from the cadmium strain may confirm the hypothesis that multigenerational exposure to that metal may possibly contribute to the selection of insects that have a wider tolerance to oxidative stress. However, our investigations of polymorphism using AFLP did not reveal differences between the two main insect strains.

Heavy Metals, Resting Metabolism Rates and Breeding Parameters in Two Populations of Black-Headed Gull Larus ridibundus from the Industrially Polluted Areas of Upper Silesia, Poland

Abstract. Black-headed Gulls breeding in the central part of the Upper Silesian Industrial Region (Katowice-Szopienice) in Southern Poland were compared with colony from less polluted area (Świerklaniec) situated 23 km away. Heavy metals: Cd, Pb, Zn and Cu — were determined in various organs of nestlings, fledglings and mature birds as well as egg yolks. Resting metabolic rates, hatching success and eggshell thickness were used as biomarkers of environmental exposure to industrial pollutants. The clutch size (2.97 versus 3.61) and hatching success (81.5% versus 87%) were lower in the colony from the more polluted site. The relatively high metal contents in the yolks indicate that off spring are only partially protected from toxic compounds. Gulls were able to regulate body contents of essential metals Zn and Cu, but Pb and Cd accumulated rapidly in the liver, kidneys and lungs of growing birds. Cd also accumulated in the ovaries at the similar levels as in the kidneys. Cd and Pb may have affected earlier stages of development when parents were foraged in a close vicinity of the heavily polluted area but once the young gulls had reached maturity they then foraged in distant areas. During this period they were exposed to pollutants in the same way as the gulls from the less polluted site. There was no growth impairment identified in gulls from the more polluted area, and their resting metabolism (RMR) calculated per unit of body weight was lower, indicating that energetic costs for detoxification were not as high.

Alteration of carbohydrates metabolism and midgut glucose absorption in Gromphadorhina portentosa after subchronic exposure to imidacloprid and fenitrothion

This study was undertaken to test the hypothesis that following exposure to insecticides, changes take place in the metabolism of carbohydrates and absorption in the midgut of insects. The Madagascar hissing cockroach (Gromphadorhina portentosa) was chosen for the experiment as a model organism, due to it being easy to breed and its relatively large alimentary tract, which was important when preparing the microperfusion midgut bioassay. In each group of cockroaches treated with imidacloprid and fenitrothion, absorption of glucose, expressed as the area under the curve (AUC), was elevated compared to the control group. Glucose in the hemolymph of the examined insects was present in a vestigial amount, often below the threshold of determination, so the determinable carbohydrate indices were: hemolymph trehalose concentration and fat body glycogen content. The level of trehalose found in the hemolymph of insects when exposed to fenitrothion, and irrespective of the level of concentration mixed into food, were significantly lower when comparing to the control samples. Imidacloprid acted analogically with one exception at the concentration of 10 mg·kg−1 dry food where trehalose concentration did not differ from the control values. Coupling with fat body glycogen concentration was less visible and appeared only at the concentrations of 5 and 10 mg imidacloprid·kg−1 dry food. As described in this study changes in the sugar distribution and midgut glucose absorption indicate that insects cover the increased energy needs induced by insecticides; also at the gastrointestinal tract level. The result indicates that the midgut glucose absorption parameters could be considered as a non-specific biomarker of insecticide toxicity.

Lifespan differences between queens and workers are not explained by rates of molecular damage

ABSTRACT The biological processes that underlie senescence are of universal biological importance, yet they remain poorly understood. A popular theory proposes that senescence is the result of limited investment into mechanisms involved in the prevention and repair of molecular damage, leading to an accumulation of molecular damage with age. In ants, queen and worker lifespans differ by an order of magnitude, and this remarkable difference in lifespan has been shown to be associated with differences in the expression of genes involved in DNA and protein repair. Here we use the comet assay and Western Blotting for poly‐ubiquitinated proteins to explore whether these differences in expression lead to differences in the accumulation of DNA damage (comet assay) or protein damage (protein ubiquitination) with age. Surprisingly, there was no difference between queens and workers in the rate of accumulation of DNA damage. We also found that levels of ubiquitinated proteins decreased with age, as previously reported in honeybees. This is in contrast to what has been found in model organisms such as worms and flies. Overall, these results reveal that the link between investment into macromolecular repair, age‐related damage accumulation and lifespan is more complex than usually recognised. HIGHLIGHTSAnt queens can live an order of magnitude longer than workers.Expression of molecular repair genes is also higher in queens than workers.We investigated the accumulation of DNA and protein damage in both castes.Surprisingly, DNA and protein damage did not accumulate more slowly in queens.These results may reflect the different life histories of queens and workers.