Does age pay off? Effects of three-generational experiments of nanodiamond exposure and withdrawal in wild and longevity-selected model animals.

Nanodiamonds (NDs) are considered a material with low toxicity. However, no studies describe the effects of ND withdrawal after multigenerational exposure. The aim was to evaluate ND exposure (in the 1st and 2nd generations) effects at low concentrations (0.2 or 2 mg kg-1) and withdrawal (in the 3rd generation) in the wild (H) and longevity-selected (D) model insect Acheta domesticus. We measured selected oxidative stress parameters, immunity, types of cell death, and DNA damage. Most of the results obtained in the 1st generation, e.g., catalase (CAT), total antioxidant capacity (TAC), heat shock proteins (HSP70), defensins, or apoptosis level, confirmed no significant toxicity of low doses of NDs. Interestingly, strain-specific differences were observed. D-strain crickets reduced autophagy, the number of ROS+ cells, and DNA damage. The effect can be a symptom of mobilization of the organism and stimulation of physiological defense mechanisms in long-living organisms. The 2nd-generation D-strain insects fed ND-spiked food at higher concentrations manifested a reduction in CAT, TAC, early apoptosis, and DNA damage, together with an increase in HSP70 and defensins. ROS+ cells and cells with reduced membrane potential and autophagy did not differ significantly from the control. H-strain insects revealed a higher number of ROS+ cells and cells with reduced membrane potential, decreased CAT activity, and early apoptosis. Elimination of NDs from the diet in the 3rd generation did not cause full recovery of the measured parameters. We noticed an increase in the concentration of HSP70 and defensins (H-strain) and a decrease in apoptosis (D-strain). However, the most visible increase was a significant increase in DNA damage, especially in H-strain individuals. The results suggest prolonged adverse effects of NDs on cellular functions, reaching beyond "contact time" with these particles. Unintentional and/or uncontrolled ND pollution of the environment poses a new challenge for all organisms inhabiting it, particularly during multigenerational exposure.

Why Is Longevity Still a Scientific Mystery? Sirtuins-Past, Present and Future.

The sirtuin system consists of seven highly conserved regulatory enzymes responsible for metabolism, antioxidant protection, and cell cycle regulation. The great interest in sirtuins is associated with the potential impact on life extension. This article summarizes the latest research on the activity of sirtuins and their role in the aging process. The effects of compounds that modulate the activity of sirtuins were discussed, and in numerous studies, their effectiveness was demonstrated. Attention was paid to the role of a caloric restriction and the risks associated with the influence of careless sirtuin modulation on the organism. It has been shown that low modulators' bioavailability/retention time is a crucial problem for optimal regulation of the studied pathways. Therefore, a detailed understanding of the modulator structure and potential reactivity with sirtuins in silico studies should precede in vitro and in vivo experiments. The latest achievements in nanobiotechnology make it possible to create promising molecules, but many of them remain in the sphere of plans and concepts. It seems that solving the mystery of longevity will have to wait for new scientific discoveries.

Age- and Lifespan-Dependent Differences in GO Caused DNA Damage in Acheta domesticus.

The rising applicability of graphene oxide (GO) should be preceded by detailed tests confirming its safety and lack of toxicity. Sensitivity to GO of immature, or with different survival strategy, individuals has not been studied so far. Therefore, in the present research, we focused on the GO genotoxic effects, examining selected parameters of DNA damage (total DNA damage, double-strand breaks-DSB, 8-hydroxy-2'-deoxyguanosine-8-OHdG, abasic site-AP sites), DNA damage response parameters, and global methylation in the model organism Acheta domesticus. Special attention was paid to various life stages and lifespans, using wild (H), and selected for longevity (D) strains. DNA damage was significantly affected by stage and/or strain and GO exposure. Larvae and young imago were generally more sensitive than adults, revealing more severe DNA damage. Especially in the earlier life stages, the D strain reacted more intensely/inversely than the H strain. In contrast, DNA damage response parameters were not significantly related to stage and/or strain and GO exposure. Stage-dependent DNA damage, especially DSB and 8-OHdG, with the simultaneous lack or subtle activation of DNA damage response parameters, may result from the general life strategy of insects. Predominantly fast-living and fast-breeding organisms can minimize energy-demanding repair mechanisms.

Multigenerational selection towards longevity changes the protective role of vitamin C against graphene oxide-induced oxidative stress in house crickets.

This research was designed to investigate changes that can arise in an invertebrate organism due to stress caused by a strong prooxidant, graphene oxide (GO), and a potent antioxidant, vitamin C. The study aimed to investigate if vitamin C may support convalescence after chronic GO intoxication. We investigated the toxicity of chronic dietary graphene oxide administration in house cricket (Acheta domesticus) types: wild and selected for longevity (with a better developed antioxidant system, conducive to long life). Vitamin C was applied immediately after cessation of graphene oxide intoxication to check if it can support the remedial effect. The condition of cells, DNA stability, catalase activity, and the reproduction potential, measured as the Vitellogenin (Vg) protein expression level, were investigated in control and GO treated groups, recovery groups (-GO), and recovery groups with Vit. C (-GO + Vit.C). In this study vitamin C had no evident remedial effect on the house crickets exposed to graphene oxide. Most probably, the mechanism of vitamin C action, in case of intoxication with nanoparticles, is much more complicated. In the context of the results obtained, it is worth considering whether Vit. C, applied after GO intoxication, causes further disturbance of homeostasis in terms of the cells' redox potential.

The Structure-Properties-Cytotoxicity Interplay: A Crucial Pathway to Determining Graphene Oxide Biocompatibility.

Interest in graphene oxide nature and potential applications (especially nanocarriers) has resulted in numerous studies, but the results do not lead to clear conclusions. In this paper, graphene oxide is obtained by multiple synthesis methods and generally characterized. The mechanism of GO interaction with the organism is hard to summarize due to its high chemical activity and variability during the synthesis process and in biological buffers' environments. When assessing the biocompatibility of GO, it is necessary to take into account many factors derived from nanoparticles (structure, morphology, chemical composition) and the organism (species, defense mechanisms, adaptation). This research aims to determine and compare the in vivo toxicity potential of GO samples from various manufacturers. Each GO sample is analyzed in two concentrations and applied with food. The physiological reactions of an easy model Acheta domesticus (cell viability, apoptosis, oxidative defense, DNA damage) during ten-day lasting exposure were observed. This study emphasizes the variability of the GO nature and complements the biocompatibility aspect, especially in the context of various GO-based experimental models. Changes in the cell biomarkers are discussed in light of detailed physicochemical analysis.

Multigenerational graphene oxide intoxication results in reproduction disorders at the molecular level of vitellogenin protein expression in Acheta domesticus.

The anthropogenic activities may lead to accumulation of graphene oxide (GO) pollution in the environment. Organisms exposed to chronic or multigenerational GO intoxication can present reproduction depletion. Vitellogenin (Vg) has been used as a parameter for evaluating female fertility due to its importance in embryo nutrition. In this study, we used a promising model organism, Acheta domesticus, which was intoxicated with GO in food for three generations. The aim of the study was to investigate the process of Vg synthesis in crickets depending on the exposure time, GO concentration, and age of the females. The results revealed that chronic GO intoxication had adverse effects on the Vg expression pattern. The 1st generation of insects showing low Vg expression was most affected. The 2nd generation of A. domesticus presented a high Vg expression. The last investigated generation seemed to cope with stress caused by GO, and the Vg expression was balanced. We suggest that the epigenetic mechanisms may play a role in the information transfer to the next generations on how to react to the risk factor and keep reproduction at a high rate. We suspect that chronic GO intoxication can disturb the regular formation of the Vg quaternary structure, resulting in consequences for developing an embryo.

Do nanoparticles cause hormesis? Early physiological compensatory response in house crickets to a dietary admixture of GO, Ag, and GOAg composite.

This study aimed to identify the physiological responses of house cricket females following short-term exposure to relatively low dietary doses of graphene oxide (GO, 20 µg · g-1 food), silver (Ag, 400 µg · g-1 food) nanoparticles (NPs), or graphene oxide­silver nanoparticle composite (GO-AgNPs, 20: 400 µg · g-1 food). Energy intake and distribution were measured on the third, sixth, and tenth day. A semi-quantitative API®ZYM assay of digestive enzyme fingerprints was performed on the third and tenth day of continuous treatment. Physicochemical properties of the NPs were obtained by combining SEM, EDX spectrometry, AFM, and DLS techniques. The obtained results showed decreased energy consumption, particularly assimilation as an early response to dietary NPs followed by compensatory changes in feeding activity leading to the same consumption and assimilation throughout the experimental period (10 days). The increased activities of digestive enzymes in NP-treated females compared to the control on the third day of the experiment suggest the onset of compensatory reactions of the day. Moreover, the insects treated with GO-AgNP composite retained more body water, suggesting increased uptake. The observed changes in the measured physiological parameters after exposure to NPs are discussed in light of hormesis.

The impact of polystyrene consumption by edible insects Tenebrio molitor and Zophobas morio on their nutritional value, cytotoxicity, and oxidative stress parameters.

The objective of this study was to determine of nutritional value, in vitro cytotoxicity, and oxidative stress parameters in cells of selected insect species (Tenebrio molitor and Zophobas morio) after 30 days of Styrofoam consumption. Furthermore, part of our research is also a consumer survey on the willingness to eat insects fed with Styrofoam (EPS 80). Mealworms fed with Styrofoam were determined to have higher protein (48.66 ± 0.92%) and ash content (4.81 ± 0.22%) with reduced fat (24.05 ± 0.55%) and carbohydrate content (2.95 ± 0.15%) than insects with a conventional diet (48.66 ± 0.92, 2.82 ± 0.12, 43.74 ± 0.77, and 4.78 ± 0.18, respectively) while in the case of superworms, no significant difference in nutrient composition was observed. Moreover, Styrofoam has no influence on the health status of gut cells in examined insects. Additionally, in studied concentrations of insects extracts standardized for protein replacement of the traditional insect diet with polystyrene foam did not increase the cytotoxic properties.

DNA damage in Spodoptera exigua after multigenerational cadmium exposure - A trade-off between genome stability and adaptation.

Human activity is a serious cause of extensive changes in the environment and a constant reason for the emergence of new stress factors. Thus, to survive and reproduce, organisms must constantly implement a program of adaptation to continuously changing conditions. The research presented here is focused on tracking slow changes occurring in Spodoptera exigua (Lepidoptera: Noctuidae) caused by multigenerational exposure to sub-lethal cadmium doses. The insects received food containing cadmium at concentrations of 5, 11, 22 and 44 µg per g of dry mass of food. The level of DNA stability was monitored by a comet assay in subsequent generations up to the 36th generation. In the first three generations, the level of DNA damage was high, especially in the groups receiving higher doses of cadmium in the diet. In the fourth generation, a significant reduction in the level of DNA damage was observed, which could indicate that the desired stability of the genome was achieved. Surprisingly, however, in subsequent generations, an alternating increase and decrease was found in DNA stability. The observed cycles of changing DNA stability were longer lasting in insects consuming food with a lower Cd content. Thus, a transient reduction in genome stability can be perceived as an opportunity to increase the number of genotypes that undergo selection. This phenomenon occurs faster if the severity of the stress factor is high but is low enough to allow the population to survive.

Vitellogenin expression, DNA damage, health status of cells and catalase activity in Acheta domesticus selected according to their longevity after graphene oxide treatment.

The increased use of graphene oxide (GO) raises worrisome questions regarding its possible threat to various ecosystems. Invertebrates represent valuable organisms for environmental studies. The lifespan can influence the ability to cope with toxins, especially those that act via oxidative stress. Two strains of Acheta domesticus, which are selected for longevity, were tested. The main aim was to investigate how GO, when administrated in food, affects: the condition of cells, DNA stability, ROS generation and the reproduction potential (the Vitellogenin (Vg) protein expression). The "recovery effect" - after removing GO from the diet for 15 days - was also measured. The results revealed different responses to GO in the wild (H) and long-living (D) strains. The D strain had a higher catalase activity compared to the H strain on the 25th day of the imago stage. Removing GO from the food resulted in a decrease in the catalase activity to the level of the control. On the 5th day of the imago stage, the H strain had a higher cell mortality than the D strain in the GO-intoxicated groups. There was more DNA damage in the H strain compared to the long-living strain. A remedial effect was seen after the GO was removed from the diet. The total Vg protein expression was higher in the H strain and lower in the D strain. The results indicated a GO concentration-dependent outcome. In both strains, removing the GO from the food led to a high Vg expression. The Vg expression after GO treatment, particularly translation and post-translational processing, should be studied in detail in the future. The D strain of crickets had more specialized mechanisms for maintaining homeostasis than the H strain. Organisms can fight off negative effects of GO, especially when they have systems that are well developed against oxidative stress.

Graphene oxide as a new anthropogenic stress factor - multigenerational study at the molecular, cellular, individual and population level of Acheta domesticus.

Although interest in transgenerational phenomena is constantly growing, little is known about the long-term toxicity of nanoparticles. In this study we investigate the multigenerational effects of graphene oxide (GO) which was given to Acheta domesticus in low doses (0.2, 2 and 20 µg·g-1 of food) for three subsequent generations. We assessed the influence of GO nanoparticles in many contexts, basing on parameters which represented different levels of biological organization: activity of antioxidant enzymes, level of apoptosis, DNA damage, histological analysis, hatching abilities, body mass and body length of insects, as well as their survival rate. The results have shown that exposing insects to nanoparticles over an extended period of time causes surprising intergenerational effects, based on significant differences in the life cycle and reproductive processes, which are not always dose-dependent. The second generation of insects appeared as the most unstable among the parameters that were studied, and did not match trends and patterns in the first and third generation categories. An increase of DNA damage was observed, but only in the third generation. This reduction of genome stability can be perceived as an essential element of adaptation, leading to an increase of genotype variants, which then undergo selection.

Protective role of zinc in Spodoptera exigua larvae under 135-generational cadmium exposure.

The aim of this study was to investigate whether zinc supplementation modulates cadmium toxicity in the beet armyworm Spodoptera exigua selected for 135 generations towards cadmium tolerance. To achieve this, larvae originating from three laboratory populations of S. exigua (control strain - C; cadmium-intoxicated for 135 generations strain - Cd, and control strain intoxicated with Cd for 1 generation - CCd) were additionally exposed to zinc in three concentrations (Zn1, 400 µg Zn·g-1 dry mass of food; Zn2; 200 µg Zn·g-1 dry mass of food; Zn3, 100 µg Zn·g-1 dry mass of food). As the markers of toxicity, a life history traits (the duration of L4 and L5 stages), cellular (DNA damage indices) and biochemical parameters (ADP/ATP ratio and ATP and HSP70 concentrations) were chosen. The duration of larval stages of Zn supplemented larvae was prolonged, while cellular and biochemical indicators, in general, appeared to be lower in comparison to the insects from respective reference groups in each laboratory populations. Moreover, the range of the differences depended on zinc concentration in food. We can suspect that zinc supplementation contributed to the protection of S. exigua individuals against negative effects of cadmium intoxication, probably at the cost of growth rate. Significant differences in the response pattern between insects from different laboratory populations indicate that the influence of additional stress factors is dependent on the overall condition of animals and their previous adaptation to other stressors.

Chronic toxicity of nanodiamonds can disturb development and reproduction of Acheta domesticus L.

The use of nanodiamonds in numerous materials designed for industry and medicine is growing rapidly. Consequently health and environmental risks associated with the exposure of humans and other biota to nanodiamonds-based materials are of the utmost importance. Scarcity of toxicological data for these particles led us to examine the potentially deleterious effects of nanodiamonds in model insect species, Acheta domesticus (Orthoptera) chronically exposed to ND in its diet. Organism-level end-point indices (lifespan, body weight, consumption, caloric value of faeces, reproduction) revealed adverse changes in the treated crickets in comparison with the control. Preliminary studies of oxidative stress level in the offspring of ND-treated crickets suggest toxicity of these particles limited to the exposed individuals. EPR analysis showing increase of radical signal in the faeces of ND-fed crickets led us to propose novel mechanism of nanodiamonds toxicity that is discussed in the light of literature data. CAPSULE: Development and reproduction of Acheta domesticus can be disturbed by the chronic exposure to nanodiamonds.

Reduced fecundity and cellular changes in Acheta domesticus after multigenerational exposure to graphene oxide nanoparticles in food.

Despite the fact that the demand for graphene and its derivatives in commercial applications is still growing, many aspects of its toxicity and biocompatibility are still poorly understood. Graphene oxide, which is released into the environment (air, soil and water) as so-called nanowaste or nanopollution, is able to penetrate living organisms. It is highly probable that, due to its specific nature, it can migrate along food chains thereby causing negative consequences. Our previous studies reported that short-term exposure to graphene oxide may increase the antioxidative defense parameters, level of DNA damage, which results in numerous degenerative changes in the gut and gonads. The presented research focuses on reproductive dysfunction and cellular changes in Acheta domesticus after exposure to GO nanoparticles in food (concentrations of 20 and 200 µg·g-1 of food) throughout their entire life cycle. The results showed that long-term exposure to GO caused a significant decrease in the reproductive capabilities of the animals. Moreover, the next generation of A. domesticus had a lower cell vitality compared to their parental generation. It is possible that graphene oxide can cause multigenerational harmful effects.

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.

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.

The level of DNA damage in adult grasshoppers Chorthippus biguttulus (Orthoptera, Acrididae) following dimethoate exposure is dependent on the insects' habitat.

The comet assay was used to study the DNA damage that was induced by dimethoate in the hemocyte cells of adult Chorthippus biguttulus grasshoppers (Insecta: Orthoptera) that originated from two sites with varying levels of pollution. The primary focus of the study was to examine whether continuous exposure to environmental stress can modify the effect of pesticides on genome stability. After three days of acclimation to laboratory conditions, the level of DNA damage in the hemocytes of Bow-winged grasshoppers was within a similar range in the insects from both areas. However, the level of DNA damage following dimethoate treatment was significantly higher in the insects from the reference area (Pogoria) than in the individuals from the heavily polluted location (Szopienice). Four hours after pesticide treatment, the Tail DNA (TDNA) in the hemocytes of the male and female specimens from Pogoria was as high as 75% and 50% respectively, whereas the values in males and females from Szopienice only reached 30% and 20%, respectively. A rapid decrease in DNA damage was observed in both populations 24 h after the pesticide application. The habitat of an insect (site), the administration of the dimethoate (treatment), and the period following the application of the pesticide (time), all significantly influenced the levels of DNA damage. No interactions related to TDNA were observed between the variables 'sex' and 'treatment'. Similarly, the variable 'sex', when analyzed alongside 'treatment' and 'site' (the area from which the insects were collected), or 'treatment' and 'time' had no influence on TL. Exposure to dimethoate undoubtedly contributed to the formation of DNA damage in the hemocytes of adult C. biguttulus. However, the level of damage was clearly dependent on the place where the insects were captured.

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.

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.

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.