Dioxins are known to be toxic for biota at every dose and their long-term effects are reported for almost 50 years of investigations. Our researches of dioxin accumulation by bank voles living in the vicinity of several landfills near Moscow (landfills “Lesnaya” and “Kouchino”) demonstrated the similarity of dioxin profiles in soil and animal samples, and an increase of highly toxic congeners’ concentrations in the bank voles’ bodies. We investigated also the probable public cancer risk resulting from the comparable lifetime exposure of the voles’ population with dioxins’ low doses, and our evaluation verified the hazard of cancer development supported by alterations in the ahr gene expression in the liver cells. The results of this study determined the usefulness of calculating the probability of cancer development in hypothetical residents of the area around both landfills. For the landfill “Lesnaya,” the values corresponded to the level acceptable for professional groups (3.72E-04), but unacceptable for the population, whereas for residents of the area in the vicinity of the landfill “Kouchino,” the level was considered to be unacceptable (1.91E-03) neither for the population nor for the professional groups. The revealed ecotoxicological situation may be quite hazardous for the population living in the area contaminated with dioxins, and the human health risks should be assessed. Thus, the prospects of the initial public health screening should consider our set of techniques used, as the effects described permit taking into account the mechanisms of interaction between the environmental factors and those determined by the characteristics of population real exposure.

Transposable elements (TEs) increase the frequency of spontaneous mutations in the genome and are capable of altering the gene structure and expression. TE activities and genomic positions are therefore important to study. A combination of two sequencing methods proved advantageous in searching for TE insertions and chromosomal rearrangements, i.e., full-genome nanopore sequencing allowed detection of TE insertions, and transcriptome sequencing on the Illumina platform evaluated their effects on gene expression. Genome sequencing data were obtained for Drosophila melanogaster strains with the SS (w1, flamenco mutant) and MS (w1, flamenco mutant, active gypsy copy) flamenco phenotypes. The wild-type laboratory strain D32 was used as a control. TE insertions and deletions in euchromatin genome regions and gene introns were found in the mutant and wild-type strains as compared with a reference genome (NCBI GCF_000001215.4). The genomes under study were searched for insertions and deletions in RNA interference system genes and genes differentially expressed in the SS and MS strains. TE insertions were detected in various regions of the AGO3, CG17147, Su(var)3-3, Gasz, CG43348, moody, and CG17752 genes. A change in TE position did not correlate with a decrease or an increase in gene transcription in most genes. A chromosomal rearrangement affecting the 3'-untranslated region was observed in the vig gene. A de novo genome assembly was conducted for the MS strain based on the long-read sequencing data. Higher expression of CR45822 and pst in the SS and MS strains was found to be due to a triplication rather than to changes in regulatory sequences or a TE insertion.

Drosophila melanogaster is a common genetic object for research of RNA interference pathways and regulation of mobile elements. At present, taking part in control of retrotransposon expression, the system of piRNA interference is well studied in ovary tissues. It is strongly believed that, in D. melanogaster, the system of piRNA interference is used for retrotransposon suppression only in the gonads, and two distinct pathways of piRNA biogenesis exist. Both mechanisms use transcripts of piRNA clusters (accumulations of truncated and defective copies of mobile elements): from uni-strand clusters in the first case and from dual-strand clusters in the second case, transcribed from one or both DNA chains, respectively. It is well known that proper function of dual-strand clusters depends on the gene rhino, while uni-strand clusters are transcribed and then spliced independently of rhino. In this paper, we show that rhino participates in splicing of uni-strand cluster flamenco transcripts. Moreover, the system of piRNA interference is necessary for regulation of several retrotransposons not only in the gonads but also in other organs.

It is impossible to reliably evaluate the ecotoxicity of small subtoxic doses of dioxins without duly taking into account how the factors of real exposure conditions affect their properties. The best approach in this regard may be biomonitoring of the initial manifestations of the toxic effects. In the present work, the authors studied bank voles ( Clethrionomys glareolus ) from the natural population living close to a dumpsite, and more specifically the summer-born adults and overwintered functional groups of animals that differed by the dioxin burden. Voles from the vivarium-kept line served as a control. The initial toxic effects were characterized by the expression levels of the following genetic markers: ahr, cyp1a2, keap1 , dnmt1 , dnmt3a , dnmt3b, LINE-1 and B1-SINE. The expression levels of the ahr, keap1, dnmt3a , and dnmt3b genes were statistically higher in the summer-born adult group than in the control voles. The expression of the cyp1a2 and keap1 genes was increased in the overwintered group compared to the control, while no differences were detected for dnmt1 , LINE-1 , and SINE B1 . The increase in the expression of the marker genes observed in the dioxin-exposed voles is in good correspondence with the mechanisms of formation and development of toxicological effects under the conditions of low-dose toxin action on several generations in the population. Our findings will contribute to the development of an evaluation technique that could be used in biomonitoring of the initial ecotoxicity effects in the dioxin-contaminated environment.

Introduction. Landfills of production and consumption waste exist everywhere. Their operation is closely related to unwanted emissions and/or discharges of dioxins. The processes of transfer of these chemicals by air and water flow outside sanitary zones, inclusion in trophic chains, bioaccumulation and overcumulation can contribute to the launch of a toxic process when low subtoxic doses are contained in the medium. It is especially important to take this into account for agro-industrial and residential regions. The study aims to assess the dioxin pollution of the environment outside the sanitary zone of the solid waste landfill of production and consumption "Lesnaya" (landfill "Lesnaya") by initial (screening) studies of the expected risk to public health. Materials and methods. Experts characterized dioxin contamination by concentrations of compounds toxic to humans in specially prepared samples, as well as indicators of general toxicity (WHO-TEQ 05). The researchers obtained samples from the surface layers of soil and animal carcasses representing successive generations (groupings) of the winter and summer seasons of the natural population of the bank vole (Clethrionomys glareolus). Concentrations of related substances were measured by high-resolution chromatography-mass spectrometry. Results. All samples contained dioxins. The good comparability of the profiles of congeners of these substances in soil samples and animal tissues testifies to the general history of their origin, as well as the formation of conditions in the environment for the almost continuous action of small doses of mixtures of these substances on the inhabitants. In soil samples, the values of the WHO-TEQ 05 index were significantly less than the hygienic standard. In animal tissues, concentrations of congeners characterized by pronounced hormone-like toxicity, teratogenic, embryotoxic and carcinogenic properties, primarily 2,3,7,8-tetrachlorodibenzo-p-dioxin, were very high. Conclusions. The consequences of prolonged exposure to the local natural population of the bank vole of small doses of dioxins in conditions of environmental pollution outside the sanitary zone of the Lesnaya landfill were characterized by the accumulation in their tissues of toxicants with hormone-like properties capable of triggering the mechanisms of formation and development of the toxic process at any concentrations. The scientific and practical significance of the obtained result is determined by the prospects for creating a methodological technique based on it to solve the problem of initial (screening) assessment of the risk to public health, namely the danger of low concentrations (doses) of dioxins polluting the environment.

Один из подходов мониторинга загрязнения среды диоксинами – натурно-экспериментальные исследования. Апробирован метод оценки начальных токсических эффектов действия малых субтоксичных доз диоксинов, заключающийся в анализе уровня транскрипции (Т) генов-маркеров с помощью специально введенного статистического показателя Тмакс (максимального значения для каждого гена в контроле). Превышение Тмакс у экспонированных полёвок и высокая частота таких особей рассмотрены как следствие токсического действия.

To assess the ecotoxicity of low doses of dioxins is almost impossible without considering the in uence of real exposure conditions on these substances’ properties. The best approach to take these into account is the biomonitoring of the initial toxic e ects’ manifestation. We studied bank voles from population naturally exposed to dioxins, the summer-born adults and overwintered functional groups of animals di ered by dioxin body burden. Dioxin-free samples of a vivarium bank voles’ line served as a control. Initial e ects of ecotoxicity were characterized by transcriptional levels of genetic markers: ahr, cyp1a2, keap1, dnmt1, dnmt3a, dnmt3b, LINE-1 and B1-SINE. Summer-born functional group had signi cantly higher expression levels of ahr, keap1, dnmt3a and dnmt3b genes versus their control group. Overwintered functional group had elevated expression levels of cyp1a2 and keap1, but no changes were found versus controls for dnmt1, LINE-1 and SINE B1. The increased expression of marker genes in dioxin-exposed voles was quite well associated with toxic process’ mechanisms - their formation and progression under exposure of several generations to low sub-toxic doses. The data obtained will contribute to the development of a biomonitoring method for assessing the initial e ects of dioxin ecotoxicity.

AIM: To study the state of the genome by indicators of retrotransposon activity the gene encoding DNA methyltransferase 1 (DNMT1); DNA damage in animals from the natural population of the bank vole (Clethrionomys glareolus) living in the vicinity of a preserved landfill contaminated with low concentrations of dioxins (landfill of production and consumption waste "Salariyevo", Moscow). MATERIAL AND METHODS: The activity of ERV-L, B1, and L1 retrotransposons and the transcription level of the DNMT1 gene were evaluated by real-time PCR. The stability of DNA in liver and bone marrow cells was characterized by the comet assay method. Afterward, the obtained characteristics of the state (stability, reactivity, and damage) of the genome in response to environmental stress factors were compared in groups of animals from the study and conditionally control samples. RESULTS: The effects of a decrease in the activity of retrotransposons of classes B1 and L1, and an increase in DNMT1 gene expression level were revealed in voles from the natural population living under the long-term chronic exposure to low doses of dioxins decrease in the activity of retrotransposons of classes B1 and L1, and an increase in the expression level of the DNMT1 gene were revealed. An increased level of DNA damage (on average up to 56% of the DNA in the tail of the comet) was detected in hepatocytes, with the addition of winter environmental factors to the chronic effect of small subtoxic doses of dioxins. CONCLUSION: Suppression of retrotransposon activity and increased expression of its epigenetic regulator (DNMT1) are regarded as adaptive responses to long-term chronic exposure to low doses of dioxins polluting the environment. The alteration in the reactivity and destabilization of the genome indicates the launch of the initial mechanisms of the toxic process formation. The created and tested methodological base for its study opens up prospects for establishing a threshold level, and as a result, substantiating indicators for screening assessment of local (territorial) risk to public health by biomonitoring.

To study the causes of impaired control of the activity of mobile genetic elements in the strains with the flamenco phenotype SS (w, flamenco mutant) and MS (w, flamenco mutant, active copy of gypsy), sequencing of these transcriptomes was performed. The D32 strain was used as control (laboratory wild type strain). An algorithm was developed for the search for amino acid substitutions in high-throughput RNA sequencing data using a triplet code for analysis. With the help of this algorithm, seven nonsense mutations were detected. The allele-specific PCR method confirmed the presence of five of the seven nonsense mutations found in silico. However, the detected nonsense mutations are not associated with the flamenco phenotype. A search for mutations in 89 genes of the RNA interference system in the SS and MS strains relative to the reference BDGP6 genome and the wild type D32 strain was carried out. No deletions, insertions, nonsense codons, and other disorders that can unambiguously lead to a change in the function of the gene are detected. To identify genes with specific expression for the strains with the flamenco phenotype, the transcriptomes of the SS and MS strains were compared with the control strains D-32, OregonR, and w1118. A set of 25 genes with differential expression was identified, among which two genes, sosie and CR45822, significantly changed the expression in the SS and MS strains. Both genes, directly or indirectly, are involved in oogenesis. Thus, the expression of the sosie and CR45822 genes can be used as a marker of the flamenco phenotype in the SS and MS strains.

The flamenco locus is one of the main components of the piRNA pathway of regulation of mobile genetic elements (MGEs) in Drosophila melanogaster. Mutations at this locus lead to an increase in the transposition activity of MGEs and, as a result, to genetic instability. In this paper, the fertility of a genetically unstable MS strain obtained more than 25 years ago and characterized by a mutation in the flamenco locus and the presence of a functionally active copy of gypsy retrotransposon was investigated. Complex violations of the ovarian morphology were revealed in the MS strain in females: defects in the follicular layer and ring channels, as well as degradation of trophocytes, which in turn led to a decrease in reproductive abilities. Analysis of the MS strain transcriptome showed a decrease in the expression level of 40 genes encoding chorionic proteins and expression specificity at different stages of follicle development. In the F1 and F2 hybrid females from the crosses of MS females with wild type males, restoration of reproductive abilities was observed, despite the fact that half of the F2 females had the flamenco genotype and genetic instability caused by transposition of gypsy (according to the ovo^(D) test). Moreover, the frequency of gypsy transposition in the hybrid F2 females with the flamenco genotype doubled in comparison with the MS strain females. Thus, the MS strain had acquired partial suppression of the flamenco phenotype and accumulated several recessive mutations in the genes that control oogenesis after cultivation for over 25 years.

In vitro interaction between recombinant protein paralogs (HP1a, HP1b, and HP1c) of the HP1 family and 5′-untranslated regulatory regions of the gypsy retrotransposon group in Drosophila melanogaster—gypsy, Springer, Tirant, ZAM, Rover, and 17.6—was studied. Using competitive DNA, the conditions that enable specific binding with a matrix were identified. It was found that HP1 family proteins efficiently bind to the 5′-untranslated regulatory region of retrotransposons with tandem repeats. It was found that repeats are absolutely necessary for HP1a to bind to the 5′-untranslated region of the Tirant and ZAM mobile genetic elements. The absence of repeats (ZAM) or the presence of fewer than two repeats (Tirant) makes such interaction impossible. Thus, the presence of tandem repeats in the 5′-untranslated region of the gypsy retrotransposons is an important tool for regulating their transposition by heterochromatin proteins.

It is believed that the heterochromatic locus flamenco, controlling transposition of retrotransposon/retrovirus gypsy in Drosophila melanogaster, is a source of Piwi-interacting RNA. Piwi is the primary component of the RNA interference machinery. Mutations in piwi and flamenco have the same phenotype – an enhanced transcription and frequency of transposition of the retrotransposon/retrovirus gypsy. This paper discusses the results of the complementation test, which involves crossing strains MS and SS, mutant for the flamenco locus, with the piwi strain 3, heterozygous for a mutation in the piwi gene, followed by study of the transcription level of gypsy in the ovaries and testes of hybrids derived from these crosses. Genetic (hybridological) and molecular analysis of the interaction of the flamenco and piwi loci has been conducted. It has been revealed that transcription of gypsy is differently regulated in testis and ovaries of strains SS and MS, apparently owing to the different activity of piRNA clusters in these tissues. It has been also shown that the piwi and flamenco genes interact complementarily in testis tissue.

Results of expression analysis of transcription of the flamenco locus that controls transposition of the mobile genetic element gypsy, RNA interference system genes ago3, zuc, aub, and HP1 heterochromatin protein family genes hp1a, hp1b, hp1c, hp1d (rhino), and hp1e in D. melanogaster SS strain mutant on the flamenco gene are presented. We show that the number of transcripts in the SS strain that are formed in the flamenco locus is unchanged in some freely chosen points, and this is different from the wild-type strain where a decreased number of transcripts is observed, which clearly is a result of processing of the flamenco locus primary transcript, a predecessor of piRNA. At the same time, expression of genes of the RNA interference system is not affected, but there is a reduced level of hp1d gene expression in ovary tissue. We suggest that the hp1d gene product is directly or indirectly involved in the flamenco locus primary transcript processing.

Previous studies indicate that the heterochromatic flamenco locus, which regulates transposition of mobile genetic element gypsy, is the source of antisense RNA interacting with the piwi locus. The Piwi protein in Drosophila melanogaster is an essential component in the RNA interference mechanism. Interestingly, mutations in piwi and flamenco produce the same phenotype: enhanced gypsy transcription and frequency of transposition. To clarify the relationship between piwi and flamenco, we performed a genetic complementation screen between MS and SS drosophila strains, mutant for flamenco, and piwi3 strain, heterozygous for piwi gene. Measurements of gypsy transcript levels in gonads of hybrid offspring uncovered complementation between the flamenco and piwi alleles in the testes. In addition, we found that gypsy transcription is regulated differently in the testis and ovaries in the SS and MS parental strains. We propose that this effect is due to a difference in activity of Piwi-interacting RNA clusters in the male and female gonads.