Antiproliferative, genotoxic and mutagenic potential of synthetic chocolate food flavoring

Flavoring additives are of great technological importance for the food industry. However, there is little information regarding the toxicological properties of these micro-ingredients, especially at the cellular level. The present study used meristematic root cells of Allium cepa L. to evaluate the toxicity of a liquid, aroma and flavor synthetic chocolate additive, manufactured and widely marketed throughout Brazil and exported to other countries in South America. The flavoring concentrations evaluated were 100.00; 50.00; 25.00; 1.00; 0.50 and 0.25 µL/L, where the highest concentration established was one-hundred times lower than that commercially suggested for use. The concentration 100 µL/L substantially reduced cell division of meristems within 24- and 48-hours exposure. Concentrations from 100.00 to 0.50 µL/L resulted in a significant number of prophases to the detriment of the other phases of cell division, indicating an aneugenic activity, and induced a significant number of cellular changes, with emphasis on micronuclei, nuclear buds and chromosomal breaks. Under the established analysis conditions, with the exception of concentration 0.25 µL/L, the flavoring of chocolate caused cytotoxicity, genotoxicity and mutagenicity to root meristems.

PRDX1 is essential for the viability and maintenance of reactive oxygen species in chicken DT40

Background Peroxiredoxin 1 (PRDX1) is a member of a ubiquitous family of thiol peroxidases that catalyze the reduction of peroxides, including hydrogen peroxide. It functions as an antioxidant enzyme, similar to catalase and glutathione peroxidase. PRDX1 was recently shown act as a sensor of reactive oxygen species (ROS) and play a role in ROS-dependent intracellular signaling pathways. To investigate its physiological functions, PRDX1 was conditionally disrupted in chicken DT40 cells in the present study. Results The depletion of PRDX1 resulted in cell death with increased levels of intracellular ROS. PRDX1-depleted cells did not show the accumulation of chromosomal breaks or sister chromatid exchange (SCE). These results suggest that cell death in PRDX1-depleted cells was not due to DNA damage. 2-Mercaptoethanol protected against cell death in PRDX1-depleted cells and also suppressed elevations in ROS. Conclusions PRDX1 is essential in chicken DT40 cells and plays an important role in maintaining intracellular ROS homeostasis (or in the fine-tuning of cellular ROS levels). Cells deficient in PRDX1 may be used as an endogenously deregulated ROS model to elucidate the physiological roles of ROS in maintaining proper cell growth.

Cytogenetic abnormalities in young cattle during vaccination against salmonelloses

The spectrum and the frequencies of cytogenetic abnormalities in young cattle immunized with a vaccine against salmonellosis of calves were investigated. The study was carried out on the farm of Novosibirsk region on 10 clinically healthy Holstein black-and-white calves of 10-17 days of age. A concentrated formol-alum vaccine against salmonellosis (paratyphoid) of calves was used at a dose of 2 ml (reimmunization at a dose of 2 ml) with an interval of 10 days between injections. The vaccine was made from the culture of bacteria of the Salmonella dublin strain № 373, inactivated with formalin with the addition of potassium alum and calcium chloride. Cytogenetic analysis of peripheral blood in calves was carried out before vaccination (control), 2 and 9 days after vaccination, 2 and 9 days after revaccination. It was found that the spectrum of somatic chromosomal instability in peripheral blood lymphocytes of calves after vaccination and revaccination is represented by polyploidy, hypoploidy and hyperploidy, chromatid and chromosomal breaks, single and paired fragments of chromosomes. It was revealed that the spectrum of somatic chromosomal instability after double immunizations with an inactivated vaccine against salmonellosis did not differ from the spectrum of spontaneously occurring mutations in this species. Vaccination and subsequent revaccination of calves in comparison with the pre-vaccination period did not lead to a significant increase in the frequency of aneuploid and polyploid cells. During double immunization of calves, a wave pattern in the variation of genomic mutation frequencies from maximum to minimum values in the lymphocytic blood cells of animals was noted, similar to prolonged mutagenesis. A tendency was found for the frequency of structural chromosome abnormalities to increase 2 and 9 days after vaccination and 2 days after revaccination. There was a credible 2.9-fold increase in the frequency of cells with chromosomal aberrations in the blood lymphocytes of animals 9 days after their repeated immunization due to breaks and paired fragments of chromosomes. After vaccination and revaccination, chromatid breaks were most often recorded in the medial regions of one of the chromatids, and chromosomal breaks in the medial and telomeric regions of both chromatids.

Actin-Related Protein 6 (Arp6) Influences Double-Strand Break Repair in Yeast

DNA double-strand breaks (DSBs) are the most deleterious form of DNA damage and are repaired through non-homologous end-joining (NHEJ) or homologous recombination (HR). Repair initiation, regulation and communication with signaling pathways require several histone-modifying and chromatin-remodeling complexes. In budding yeast, this involves three primary complexes: INO80-C, which is primarily associated with HR, SWR1-C, which promotes NHEJ, and RSC-C, which is involved in both pathways as well as the general DNA damage response. Here we identify ARP6 as a factor involved in DSB repair through an RSC-C-related pathway. The loss of ARP6 significantly reduces the NHEJ repair efficiency of linearized plasmids with cohesive ends, impairs the repair of chromosomal breaks, and sensitizes cells to DNA-damaging agents. Genetic interaction analysis indicates that ARP6, MRE11 and RSC-C function within the same pathway, and the overexpression of ARP6 rescues rsc2∆ and mre11∆ sensitivity to DNA-damaging agents. Double mutants of ARP6, and members of the INO80 and SWR1 complexes, cause a significant reduction in repair efficiency, suggesting that ARP6 functions independently of SWR1-C and INO80-C. These findings support a novel role for ARP6 in DSB repair that is independent of the SWR1 chromatin remodeling complex, through an apparent RSC-C and MRE11-associated DNA repair pathway.

A review on the public health implications of heavy metals

In the recent years, the pollution of the environment by heavy metals has become a crucial problem across the world. Heavy metals consist of a group of metals and metalloids which have atomic density greater than 4000 kg m-3. Heavy metals such as Nickel (Ni), Cobalt (Co), Copper (Cu), Zinc (Zn), Lead (Pb) are present in the biota. These metals are also known as trace elements which play a very important role in various metabolic processes of plants, animals and microorganisms. Heavy metals may gain entrance into the human body through consumption of contaminated drinking water or ingestion of soil or crops grown on contaminated land. Heavy metals such as lead, mercury, cadmium and copper are collective poisons, which causes environmental hazards and are reported to be exceptionally toxic. These metals are important sources of oxidative stress in the cell and play an important role in a etiology of diverse human pathologies such as carcinogenesis. Exposure to heavy metal toxicity leads to brain damage, mental retardation, cerebral palsy, lung cancer, gastrointestinal abnormalities, dermatitis and death of the unborn fetus. Many metals have been shown to directly modify and/or damage DNA by forming DNA adducts that induce chromosomal breaks. The unrestricted access to the dumpsite means that each day, scavengers search for raw materials, much of which eventually finds its way back to neighborhoods as animal feed and even human food. Stray chicken, pigs, goats, dogs and cats roam the dumpsite eating the toxic matter and becoming vectors of pests and parasites that are eventually transferred to the surrounding home and hence causing diseases to both animals and human beings.

Systematic Chromosome Rearrangement Induced by CRISPR-Cas9 Reshapes the Genome and Transcriptome of Human Cells

Chromosome rearrangement plays important roles in development, carcinogenesis and evolution. However, its mechanism and subsequent effects are not fully understood. At present, large-scale chromosome rearrangement has been performed in the simple eukaryote, wine yeast, but the relative research in mammalian cells remains at the level of individual chromosome rearrangement due to technical limitations. In this study, we used CRISPR-Cas9 to target the highly repetitive human endogenous retrotransposons, LINE-1 (L1) and Alu, resulting in a large number of DNA double-strand breaks in the chromosomes. While this operation killed the majority of the cells, we eventually obtained live cell groups. Karyotype analysis and genome re-sequencing proved that we have achieved systematic chromosome rearrangement (SCR) in human cells. The copy number variations (CNVs) of the SCR genomes showed typical patterns that observed in tumor genomes. For example, the most frequent deleted region Chr9p21 containing p15 and p16 tumor suppressor, and the amplified region Chr8q24 containing MYC in tumors were all identified in both SCR cells. The ATAC-seq and RNA-seq further revealed that the epigenetic and transcriptomic landscapes were deeply reshaped by the SCR. Gene expressions related to p53 pathway, DNA repair, cell cycle and apoptosis were greatly altered to facilitate the cell survival under the severe stress induced by the large-scale chromosomal breaks. In addition, we found that the cells acquired CRISPR-Cas9 resistance after SCR by interfering with the Cas9 mRNA. Our study provided a new application of CRISPR-Cas9 and a practical approach for SCR in complex mammalian genomes.

Chromosomal Instability at Fragile Sites in Blue Foxes, Silver Foxes, and Their Interspecific Hybrids

A cytogenetic assay based on fragile sites (FS) enables the identification of breaks, chromatid gaps, and deletions. In healthy individuals, the number of these instabilities remains low. Genome stability in these species is affected by Robertsonian translocations in the karyotype of the blue fox and by B chromosomes in the silver fox. The aims of the study were to characterise the karyotype of blue foxes, silver foxes, and their hybrids and to identify chromosomal fragile sites used to evaluate genome stability. The diploid number of A chromosomes in blue foxes ranged from 48 to 50, while the number of B chromosomes in silver foxes varied from one to four, with a constant number of A chromosomes (2n = 34). In interspecific hybrids, both types of karyotypic variation were identified, with the diploid number of A chromosomes ranging from 40 to 44 and the number of B chromosomes varying from 0 to 3. The mean frequency of FS in foxes was 4.06 ± 0.19: 4.61 ± 0.37 in blue foxes, 3.46 ± 0.28 in silver foxes, and 4.12 ± 0.22 in hybrids. A relationship was identified between an increased number of A chromosomes in the karyotype of the hybrids and the frequency of chromosomal breaks. The FS assay was used as a biomarker for the evaluation of genomic stability in the animals in the study.

Peculiarities of karyotype of the Ukrainian aboriginal Hutsul breed of horse

Saving biodiversity and assessment of the genetic diversity of local breeds of domestic animals as priority subjects of protection in agrobiocenoses is one of the global challenges facing humanity in the 21st century. Hutsul horse is one of the oldest horse breeds in Ukraine, which according to the gene pool subject of horses in Ukraine belongs to the group “Local (mountain and ponies)” of the I category, which is already on the verge of extinction, and according to the FAO classification it is considered to be a subject of the gene pool threatened with extinction. Since the breeding chromosomal polymorphism of horses is insufficiently studied, it is timely to carry out a cytogenetic analysis of the characteristics of spontaneous mutagenesis in Hutsul horses. Karyotypic variability of Hutsul breed horses was determined using the methods of cytogenetic analysis and micronucleus test. The paper presents the results of cytogenetic analysis and micronucleus test of karyotypic variability of Hutsul breed horses. Asynchronous divergence of centromere regions of chromosomes occurs as a result of premature replication of centromere regions of heterochromatin associated with centromere activity. In the studied Hutsul horses, the magnitude of this variability corresponds to the spontaneous level for horses as a whole (2.2-9.1 %). No structural chromosomal abnormalities (chromosomal breaks) were revealed in Hutsul breed horses at Krai Neba LLC, and in the animals of the Hutsulshchina National Nature Park, the percentage of metaphase plates with chromosomal breaks was low (1.1 %), which indicates the stability of the karyotype of the studied animals. The results of the micronucleus test showed that the proportion of lymphocytes with micronuclei in the animals at the both farms was practically the same - 4.0-4.2. It can be concluded based on the data obtained that the animals under study were in ecologically clean conditions relative to the level of radionuclide contamination and were characterized by karyotype stability and reduced sensitivity to mutagenic factors of various nature.

Learning Yeast Genetics from Miro Radman

Miroslav Radman’s far-sighted ideas have penetrated many aspects of our study of the repair of broken eukaryotic chromosomes. For over 35 years my lab has studied different aspects of the repair of chromosomal breaks in the budding yeast, Saccharomyces cerevisiae. From the start, we have made what we thought were novel observations that turned out to have been predicted by Miro’s extraordinary work in the bacterium Escherichia coli and then later in the radiation-resistant Dienococcus radiodurans. In some cases, we have been able to extend some of his ideas a bit further.

Evidence of cytogenetic and histological damage in specimens of Astyanax lacustris (Pisces, Characidae) exposed to the hydrogen cyanide-based herbicide Dormex®

The herbicide Dormex®, a solution of hydrogen cyanamide, is a growth regulator capable of breaking the dormancy of fruit plants, and is commonly applied in agriculture. However, the biological effects of this product on non-target organisms are unknown. The present study investigated the biological response of Astyanax lacustris (Lütken, 1875) specimens exposed to Dormex® using a chromosome aberration test, the mitotic index, and the histological analysis of the gills. Forty specimens of Astyanax lacustris were obtained from a local breeding facility and divided into 10 groups (nine experimental and one control) with four fish in each aquarium (group). The control group was maintained for 24 hours in dechlorinated water while the experimental groups were allocated to one of nine different treatments, with three concentrations of Dormex®, 0.05, 0.1 and 0.5 mL L-1, and exposure for 24, 48 and 72 hours. The fish exposed to Dormex® presented chromosomal aberrations of a number of types, including chromosomal breaks, acentric fragments, decondensation, and gaps at the three Dormex® concentrations, at all exposure times. The mitotic index decreased significantly in comparison with the control group. The histological preparations of the gills revealed alterations such as hyperplasia, and lamellar fusion and edema, whereas in the control group the structure of the gills was preserved. The cytogenetic analysis revealed the genotoxic potential of the herbicide Dormex® and the morphological alterations of the gills demonstrated the sensitivity of the fish, which responded rapidly to the stressor. These findings reinforce the need for special care and restrictions on the use of these herbicides in agricultural areas located near aquatic environments.