Dose-Dependent Effect of Mitochondrial Superoxide Dismutase Gene Overexpression on Radioresistance of HEK293T Cells.

Over the last two decades, a multitude of gain-of-function studies have been conducted on genes that encode antioxidative enzymes, including one of the key enzymes, manganese superoxide dismutase (SOD2). The results of such studies are often contradictory, as they strongly depend on many factors, such as the gene overexpression level. In this study, the effect of altering the ectopic expression level of major transcript variants of the SOD2 gene on the radioresistance of HEK293T cells was investigated using CRISPRa technology. A significant increase in cell viability in comparison with the transfection control was detected in cells with moderate SOD2 overexpression after irradiation at 2 Gy, but not at 3 or 5 Gy. A further increase in the level of SOD2 ectopic expression up to 22.5-fold resulted in increased cell viability detectable only after irradiation at 5 Gy. Furthermore, a 15-20-fold increase in SOD2 expression raised the clonogenic survival of cells after irradiation at 5 Gy. Simultaneous overexpression of genes encoding SOD2 and Catalase (CAT) enhanced clonogenic cell survival after irradiation more effectively than separate overexpression of both. In conjunction with the literature data on the suppression of the procarcinogenic effects of superoxide dismutase overexpression by ectopic expression of CAT, the data presented here suggest the potential efficacy of simultaneous overexpression of SOD2 and CAT to reduce oxidative stress occurring in various pathological processes. Moreover, these results illustrate the importance of selecting the degree of SOD2 overexpression to obtain a protective effect.

Soil Microbiome in Conditions of Oil Pollution of Subarctic Ecosystems.

The present study aimed to investigate the recovery of soil quality and the bacterial and fungal communities following various recultivation methods in areas contaminated with oil. Oil spills are known to have severe impacts on ecosystems; thus, the restoration of contaminated soils has become a significant challenge nowadays. The study was conducted in the forest-tundra zone of the European North-East, where 39 soil samples from five oil-contaminated sites and reference sites were subjected to metagenomic analyses. The contaminated sites were treated with different biopreparations, and the recovery of soil quality and microbial communities were analyzed. The analysis of bacteria and fungi communities was carried out using 16S rDNA and ITS metabarcoding. It was found that 68% of bacterial OTUs and 64% of fungal OTUs were unique to the reference plot and not registered in any of the recultivated plots. However, the species diversity of recultivated sites was similar, with 50-80% of bacterial OTUs and 44-60% of fungal OTUs being common to all sites. New data obtained through soil metabarcoding confirm our earlier conclusions about the effectiveness of using biopreparations with indigenous oil-oxidizing micro-organisms also with mineral fertilizers, and herbaceous plant seeds for soil remediation. It is possible that the characteristics of microbial communities will be informative in the bioindication of soils reclaimed after oil pollution.

Effects of Antioxidant Gene Overexpression on Stress Resistance and Malignization In Vitro and In Vivo: A Review.

Reactive oxygen species (ROS) are normal products of a number of biochemical reactions and are important signaling molecules. However, at the same time, they are toxic to cells and have to be strictly regulated by their antioxidant systems. The etiology and pathogenesis of many diseases are associated with increased ROS levels, and many external stress factors directly or indirectly cause oxidative stress in cells. Within this context, the overexpression of genes encoding the proteins in antioxidant systems seems to have become a viable approach to decrease the oxidative stress caused by pathological conditions and to increase cellular stress resistance. However, such manipulations unavoidably lead to side effects, the most dangerous of which is an increased probability of healthy tissue malignization or increased tumor aggression. The aims of the present review were to collect and systematize the results of studies devoted to the effects resulting from the overexpression of antioxidant system genes on stress resistance and carcinogenesis in vitro and in vivo. In most cases, the overexpression of these genes was shown to increase cell and organism resistances to factors that induce oxidative and genotoxic stress but to also have different effects on cancer initiation and promotion. The last fact greatly limits perspectives of such manipulations in practice. The overexpression of GPX3 and SOD3 encoding secreted proteins seems to be the "safest" among the genes that can increase cell resistance to oxidative stress. High efficiency and safety potential can also be found for SOD2 overexpression in combinations with GPX1 or CAT and for similar combinations that lead to no significant changes in H2O2 levels. Accumulation, systematization, and the integral analysis of data on antioxidant gene overexpression effects can help to develop approaches for practical uses in biomedical and agricultural areas. Additionally, a number of factors such as genetic and functional context, cell and tissue type, differences in the function of transcripts of one and the same gene, regulatory interactions, and additional functions should be taken into account.

AOX1a Expression in Arabidopsis thaliana Affects the State of Chloroplast Photoprotective Systems under Moderately High Light Conditions.

Alternative oxidase (AOX) in the mitochondrial electron transport chain is considered important for sustaining photosynthesis under high light conditions. Here, we examined the effects of the AOX pathway on the state of chloroplast photoprotective systems. Arabidopsis thaliana plants (4 weeks old), comprising three genotypes (wild type [WT], overexpressing [XX-2] and antisense [AS-12] lines for AOX1a), were exposed to moderately high light conditions (MHL, 400 µmol m-2 s-1) in a short-term experiment (8 h). After 8 h of MHL, the WT and XX-2 plants showed stable non-photochemical quenching (qN) and violaxanthin cycle activity. Antisense plants displayed the lowest level of qN and a lower de-epoxidation state (DEPS) relative to plants of the same line after 4-6 h MHL, as well as compared to WT and XX-2 plants after 8 h MHL. The decline in DEPS in AS-12 plants was attributed to an insufficient violaxanthin de-epoxidase activity, which in turn was associated with a decrease in reduced ascorbate levels in the chloroplasts and leaves. Simultaneously, gene expression and the activity of ascorbate peroxidase in the antisense line increased after 8 h of MHL, supporting the compensatory effect of the antioxidant system when AOX1a expression is suppressed. This study emphasizes the role played by AOX in modulating the photoprotection processes and in the maintenance of relationships between mitochondria and chloroplasts by influencing ascorbate content.

Melatonin treatment improves ventricular conduction via upregulation of Nav1.5 channel proteins and sodium current in the normal rat heart.

Melatonin treatment was reported to reduce the risk of cardiac arrhythmias, and crucial for this antiarrhythmic action was the effect of melatonin on activation spread. The aim of the present study was evaluation of the mechanisms of this activation enhancement. Experiments were performed in a total of 123 control and melatonin-treated (10 mg/kg, daily, for 7 days) male Wistar rats. In epicardial mapping studies (64 leads, interlead distance 0.5 mm) in the anesthetized animals, activation times (ATs) were determined in each lead as dV/dt minimum during QRS complex under sinus rhythm. Epicardial pacing was performed to measure conduction velocity (CV) across the mapped area. Average left ventricular ATs were shorter in the treated animals as compared to the controls, whereas the minimal epicardial ATs indicating the duration of activation propagation via the ventricular conduction system did not differ between the groups. CV was higher in the treated groups indicating that melatonin affected conduction via contractile myocardium The area of Cx43-derived fluorescence, as well as the expression of Cx43 protein, was similar in ventricles in the control and melatonin-treated groups. Expression of Gja1 gene transcripts encoding Cx43, was increased in the last group. An uncoupling agent octanol modified myocardial conduction properties (time of activation, action potential upstroke velocity, passive electrotonic phase duration) similarly in both groups. On the other hand, the expression of both Scn5a gene transcripts encoding Nav1.5 proteins, as well as peak density of transmembrane sodium current were increased in the ventricular myocytes from the melatonin-treated animals. Thus, a week-long melatonin treatment caused the increase of conduction velocity via enhancement of sodium channel proteins expression and increase of sodium current in the ventricular myocytes.

Responses of genes of DNA repair, alternative oxidase, and pro-/antioxidant state in Arabidopsis thaliana with altered expression of AOX1a to gamma irradiation.

PURPOSE: High doses of gamma (γ) irradiation cause oxidative stress and DNA damage. Alternative oxidase (AOX) catalyzes the energy-dissipating cyanide-resistant alternative pathway in plant mitochondria and is an important part of the cellular defense network under stress conditions. In this study, Arabidopsis thaliana plants with an altered expression of the AOX1a gene were exposed by high dose-rate ionizing radiation to assess the expression of genes of DNA repair and pro-/antioxidant states to elucidate the functional significance of AOX in plant stress response. MATERIALS AND METHODS: Five-week-old A. thaliana plants, either with basal AOX1a gene expression (wild-type Colombia-0 (Col-0)), antisense silencing of AOX1a (AS-12), and overexpression of the gene (XX-2), were γ-irradiated at a dose of 200 Gy. Gene expression and biochemical analyses were performed 12 h after irradiation. RESULTS: Acute γ-irradiation caused different responses between the genotypes. XX-2 plants, either control or irradiated, showed the highest expression of AOX1a gene and AOX protein, and the lowest expression of DNA repair genes. Wild type and AS-12 plants exposed to γ-irradiation upregulated another stress-induced gene, AOX1d, and DNA repair genes. Furthermore, a higher activity of Mn-dependent superoxide dismutase (Mn-SOD) was observed in the irradiated AS-12 plants than in the untreated plants of this line. However, AS-12 plants were less effective than Col-0 plants in controlling the accumulation of the superoxide anion. XX-2 plants had the lowest reactive oxygen species (ROS) levels among the genotypes. CONCLUSIONS: AS-12 plants display a compensatory mechanism by increasing the expression of AOX1d and the synthesis of the AOX protein, as well as by Mn-SOD activation. However, these were insufficient to maintain the background level of embryonic lethal mutations, and thereby the reproductive capacity. These results highlight the importance of AOX in the successful adaptation of plants to acute γ-irradiation, and indicate that AOX1a plays a key role in the regulation of the stress response.

The gene expression profiles of mitochondrial respiratory components in Arabidopsis plants with differing amounts of ALTERNATIVE OXIDASE1a under high intensity light.

We compared the expression of mitochondrial alternative oxidase (AOX) and other non-phosphorylating respiratory components (NPhPs) in wild type and AOX1a transgenic Arabidopsis thaliana following short-term transfer of plants to higher irradiance conditions to gain more insight into the mechanisms of AOX functioning under light. The AOX1a overexpressing line (XX-2) showed the highest amount of AOX1a transcripts and AOX1A synthesis during the entire experiment, and many NPhPs genes were down-regulated after 6-8 h under the higher light conditions. Antisense AS-12 plants displayed a compensatory effect, typically after 8 h of exposure to higher irradiance, by up-regulating their expression of the majority of genes encoding AOX and other respiratory components. In addition, AS-12 plants displayed 'overcompensation effects' prior to their transfer to high light conditions, i.e., they showed a higher expression level of certain genes. As a result, the ROS content in AS-12, as in XX-2, was consistently lower than in the wild type. All NPhPs genes share, in common with AOX1a, light- and stress-related cis-acting regulatory elements (CAREs) in their promoters. However, the expression of respiratory genes does not always depend on the level of AOX1a expression. This suggests the presence of multiple combinations of signaling pathways in gene induction. Based on our results, we outline possible directions for future research.

Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1.

Molecular responses to genotoxic stress, such as ionizing radiation, are intricately complex and involve hundreds of genes. Whether targeted overexpression of an endogenous gene can enhance resistance to ionizing radiation remains to be explored. In the present study we take an advantage of the CRISPR/dCas9 technology to moderately overexpress the RPA1 gene that encodes a key functional subunit of the replication protein A (RPA). RPA is a highly conserved heterotrimeric single-stranded DNA-binding protein complex involved in DNA replication, recombination, and repair. Dysfunction of RPA1 is detrimental for cells and organisms and can lead to diminished resistance to many stress factors. We demonstrate that HEK293T cells overexpressing RPA1 exhibit enhanced resistance to cell killing by gamma-radiation. Using the alkali comet assay, we show a remarkable acceleration of DNA breaks rejoining after gamma-irradiation in RPA1 overexpressing cells. However, the spontaneous rate of DNA damage was also higher in the presence of RPA1 overexpression, suggesting alterations in the processing of replication errors due to elevated activity of the RPA protein. Additionally, the analysis of the distributions of cells with different levels of DNA damage showed a link between the RPA1 overexpression and the kinetics of DNA repair within differentially damaged cell subpopulations. Our results provide knew knowledge on DNA damage stress responses and indicate that the concept of enhancing radioresistance by targeted alteration of the expression of a single gene is feasible, however undesired consequences should be considered and evaluated.

Genetic analysis in earthworm population from area contaminated with radionuclides and heavy metals.

This study assessed the effects of environmental contamination by naturally occurring radionuclides and heavy metals on the genetic structure of a population of the earthworm Aporrectodea caliginosa. A. caliginosa were collected from four sites and characterized by amplified fragment length polymorphism (AFLP) analyses. No differences in genetic structure and diversity were found between sites that differed greatly in soil contamination levels of radionuclides and metals. However, when the genetic structure of the A. caliginosa population was analyzed without considering information about the sampling site, a complex intraspecific genetic structure was identified. At least three highly divergent lineages were found, in unequal proportions, of each genetically isolated group from each study site. No associations were found between the distribution of the detected genetic clusters and the geographical origin of the samples. Thus, no noticeable adaptive changes or signs of directional selection were detected, despite the long history of genotoxic waste disposal at the sampling site. These results suggest a combined effect of three factors on the genetic structure and diversity of A. caliginosa in soils: the complexity of the contaminant composition, the heterogeneous spatial distribution of the pollutants, and the complexity of the intraspecific genetic structures of A. caliginosa.

Altered levels of AOX1a expression result in changes in metabolic pathways in Arabidopsis thaliana plants acclimated to low dose rates of ultraviolet B radiation.

UV-B is a damaging component of solar radiation that inevitably reaches the Earth's surface. Plants have developed response mechanisms to adapt to UVB exposure. The alternative oxidase (AOX) catalyzes the ATP-uncoupling cyanide-resistant alternative pathway (AP) in plant mitochondria and is thought to be an important part of the cellular defense network under stress conditions. This study aimed to unravel the poorly understood functional significance of AOX1a induction in Arabidopsis thaliana leaves exposed to ecologically relevant doses of UVB radiation, by comparing wild-type (WT) plants with plants with modified expression of the AOX1a gene, either downregulated by antisense (AS-12) or overexpressed (XX-2). UVB exposure resulted in a phenotypic difference between lines. AOX1a overexpression resulted in the highest induction of AOX1A synthesis and MnSOD activity, and the lowest ROS level without pronounced changes in the phenotype relative to other genotypes. In AS-12 plants, expression of the majority of the genes encoding AOX was detected, other non-phosphorylating pathway components and antioxidant enzymes increased along with anthocyanin accumulation in leaves, and the ROS content was lower than in the WT. In addition to the expected AOX1 protein size (34 kDa), an AOX1 30 kDa band appeared under UVB exposure in all genotypes. However, in AS-12, the alterations in the transcript level and in the abundance of AOX1 protein isoforms induced by UVB could not fully functionally compensate for the lack of AOX1A. This was confirmed by the observed low AP capacity and increased levels of the oxidized form of ascorbate. These results highlight the importance of AOX in plant response to UVB for the control of a balanced metabolism, and indicate that AOX1a plays a key role in the regulation of the stress response.

Recent Trends in Enhancing the Resistance of Cultivated Plants to Heavy Metal Stress by Transgenesis and Transcriptional Programming.

Normal growth and development of high plants strongly depends on the concentration of microelements, including essential heavy metals, in the substrate. However, an excess of those elements may become harmful. Therefore, micronutrient concentrations in plant tissue should be well-balanced and controlled by homeostatic mechanisms. The advancement of knowledge on the regulation of metal homeostasis in plants is important for phytoremediation of metal-contaminated soil and for micronutrient malnutrition control. Experimental data from loss-of-function and gain-of-function studies, including functional descriptions and classifications have presented new opportunities for multiplex CRISPR/dCas9-driven control of gene expression and have opened up new prospects for the goal-seeking regulation of metal homeostasis in plants. The aim of this review is to help for multiplex transcriptional programming targets search by summarizing and analyzing data on possible ways to handle a plant's ability to maintain metal homeostasis.

Microsatellite polymorphism of Trifolium pratense population at the conditions of radioactive and chemical contamination of soil (Komi republic, Russia).

There is no clear understanding of microevolutionary changes in natural populations of plants and animals due to anthropogenic contamination of the environment with toxicants and mutagens. But such data are necessary to forecast long-term effects of human activity. In this research, we studied genetic polymorphism in T. pratense sampled from seven sites varying in radioactive and chemical soil contamination in the vicinity of Vodny settlement (Komi, Russia). Analysis of five SSR loci was shown to be similar in a whole (N), mean (Na) and effective (Ne) numbers of alleles, heterozygosity indexes (Ho and He), and the Shannon index (I). Difference in the private allele numbers was registered: the most contaminated site has 5 and others from 0 up 2 private alleles. No difference was found in the genetic structure of T. pratense population growing at the conditions of radioactive and chemical contamination. The Bayesian analysis provided evidence of a single cluster (K = 1) due to a similar genetic structure of samples, while AMOVA results demonstrated a high variability within individuals (75%) and a low variability (1%) among groups of T. pratense from sites that differ in the contamination level. Thus, the long-term radioactive and heavy metal contamination of soil did not result in significant microevolutionary changes in T. pratense population.

Low dose ionizing irradiation suppresses cellular senescence in normal human fibroblasts.

PURPOSE: Exposure to high dose ionizing radiation leads to premature cell senescence and suppression of cell proliferation. In contrast, low dose and low dose-rate gamma-irradiation can lead to stimulation of cell proliferation. We aimed to examine whether the low dose radiation-induced proliferation of normal human fibroblasts can lead to a progressive depletion of proliferation potential and to an early onset of senescence. MATERIALS AND METHODS: Normal human embryonic lung fibroblasts (HELF-104) at passage 22-24 were gamma-irradiated with doses of 0 (sham-irradiation), 10, 30, 50, 90, 120, 150, 200, and 500 mGy as well as 1 and 2 Gy. After irradiation, the fraction of cells positively stained for senescence-associated ß-galactosidase activity was measured weekly until the cell culture completely ceased to proliferate. RESULTS: We show that single irradiation of HELF-104 cells with 30 and 50 mGy resulted in deceleration of senescence. The suppression of senescence was observed during almost the entire length of the study up to a complete arrest of cell growth. CONCLUSIONS: Our data, together with the previously published observation of delayed stimulation of proliferation in HELF-104 cells exposed to 30 mGy, suggest that low dose gamma-irradiation can increase the overall proliferative potential of normal human fibroblasts.

Programming of Cell Resistance to Genotoxic and Oxidative Stress.

Different organisms, cell types, and even similar cell lines can dramatically differ in resistance to genotoxic stress. This testifies to the wide opportunities for genetic and epigenetic regulation of stress resistance. These opportunities could be used to increase the effectiveness of cancer therapy, develop new varieties of plants and animals, and search for new pharmacological targets to enhance human radioresistance, which can be used for manned deep space expeditions. Based on the comparison of transcriptomic studies in cancer cells, in this review, we propose that there is a high diversity of genetic mechanisms of development of genotoxic stress resistance. This review focused on possibilities and limitations of the regulation of the resistance of normal cells and whole organisms to genotoxic and oxidative stress by the overexpressing of stress-response genes. Moreover, the existing experimental data on the effect of such overexpression on the resistance of cells and organisms to various genotoxic agents has been analyzed and systematized. We suggest that the recent advances in the development of multiplex and highly customizable gene overexpression technology that utilizes the mutant Cas9 protein and the abundance of available data on gene functions and their signal networks open new opportunities for research in this field.

Expression profiles of genes for mitochondrial respiratory energy-dissipating systems and antioxidant enzymes in wheat leaves during de-etiolation.

Mitochondrial respiratory components participate in the maintenance of chloroplast functional activity. This study investigates the effects 48h de-etiolation of spring wheat seedlings (Triticum aestivum L., var. Irgina) on the expression of genes that encode energy-dissipating respiratory components and antioxidant enzymes under continuous light conditions. The expression of AOX1a following the prolonged darkness exhibited a pattern indicating a prominent dependence on light. The expression of other respiratory genes, including NDA2, NDB2, and UCP1b, increased during de-etiolation and dark-to-light transition; however, changes in the expression of these genes occurred later than those in AOX1a expression. A high expression of NDA1 was detected after 12h of de-etiolation. The suppression of AOX1a, NDA2, NDB2, and UCP1b was observed 24h after de-etiolation when the photosynthetic apparatus and its defence systems against excess light were completely developed. The expression patterns of the respiratory genes and several genes encoding antioxidant enzymes (MnSOD, Cu-ZnSOD, t-APX, GR, and GRX) were quite similar. Our data indicate that the induction of nuclear genes encoding respiratory and antioxidant enzymes allow the plants to control reactive oxygen species (ROS) production and avoid oxidative stress during de-etiolation.

Dark and Photoinduced Cytotoxic Activity of the New Chlorophyll-a Derivatives with Oligoethylene Glycol Substituents on the Periphery of Their Macrocycles.

In the present work, we investigated the dark and photoinduced cytotoxic activity of the new chlorophyll-a derivatives which contain the substituents of oligoethylene glycol on the periphery of their macrocycles. These compounds were tested using human cell lines to estimate their potential as photosensitizers for photodynamic therapy of cancer. It was shown that all the tested compounds have expressed photoinduced cytotoxic activity in vitro. Detailed study of the biological activity of one of the most perspective compound in this series-pyropheophorbide-a 17-diethylene glycol ester (Compound 21) was performed. This new compound is characterized by lower dark cytotoxicity and higher photoinduced cytotoxicity than previously described in a similar compound (DH-I-180-3) and clinically used PhotolonTM. Using fluorescent microscopy, it was shown that Compound 21 quickly penetrates the cells. Analysis of caspase-3 activity indicated an apoptosis induction 40 min after exposure to red light (λ = 660 nm). The induction of DNA damages and apoptosis was shown using Comet assay. The results of expression analysis of the stress-response genes indicate an activation of the genes which control the cell cycle and detoxification of the free radicals after an exposure of HeLa cells to Compound 21 and to red light. High photodynamic activity of this compound and the ability to oxidize biomolecules was demonstrated on nuclear-free mice erythrocytes. In addition, it was shown that Compound 21 is effectively activated with low energy 700 nm light, which can penetrate deep into the tissue. Thus, Compound 21 is a prospective substance for development of the new drugs for photodynamic therapy of cancer.

Differential Molecular Stress Responses to Low Compared to High Doses of Ionizing Radiation in Normal Human Fibroblasts.

Understanding the mechanisms producing low dose ionizing radiation specific biological effects represents one of the major challenges of radiation biology. Although experimental evidence does suggest that various molecular stress response pathways may be involved in the production of low dose effects, much of the detail of those mechanisms remains elusive. We hypothesized that the regulation of various stress response pathways upon irradiation may differ from one another in complex dose-response manners, causing the specific and subtle low dose radiation effects. In the present study, the transcription level of 22 genes involved in stress responses were analyzed using RT-qPCR in normal human fibroblasts exposed to a range of gamma-doses from 1 to 200 cGy. Using the alkali comet assay, we also measured the level of DNA damages in dose-response and time-course experiments. We found non-linear dose responses for the repair of DNA damage after exposure to gamma-radiation. Alterations in gene expression were also not linear with dose for several of the genes examined and did not follow a single pattern. Rather, several patterns could be seen. Our results suggest a complex interplay of various stress response pathways triggered by low radiation doses, with various low dose thresholds for different genes.