[Risk assessment and toxicity effects of materials used during additive manufacturing with FDM technology]. / Ocena zagrozen i dzialanie toksyczne materialów stosowanych podczas drukowania przestrzennego w technologii FDM.

This paper discusses the potential of additive printing, the risks it poses to users' health (including 3D printer operators) and the effects of chemical substances released during the printing based on the available in vitro and in vivo studies. It was shown that substances emitted during printing with the commonly used acrylonitrile butadiene styrene (ABS) filament in additive manufacturing might have carcinogenic, hepatotoxic and teratogenic effects, as well as toxic effect on the respiratory system. The latest research on the mechanism of formation of particles and volatile organic compounds (VOCs) during 3D printing, the parameters affecting their potential emission, and trends in reducing these hazards are indicated. The need for the design of more environmentally friendly and less emissive printing materials, as well as strategies for prevention and individual and collective protection measures are emphasized. Users of 3D printers should be familiar with all possible aspects of the threats associated with the printing process. Insufficient data on direct exposure to chemicals and particles released during the use of filaments makes it difficult to build awareness of safe working practices. Of particular concern is the health impact of emitted chemicals and particles from thermally treated materials in one of the most popular technologies for 3D printing, i.e., fused deposition modelling. Exposure of the users to, for example, plasticizers added to filaments occurs through a variety of routes, by absorption through the skin, by inhalation or ingestion. Available epidemiological data, as well as current experimental works, indicate that such exposure is a high risk of cardiovascular diseases, atherosclerosis in adults, and cardiac problems and metabolic disorders in children. This review, by identifying potential risk factors, may contribute to reducing the health loss of printer users and improving working conditions and safety, especially in enterprises where additive manufacturing technology is used. Med Pr Work Health Saf. 2024;75(2).

[The exposure routes of micro- and nanoplastics and their potential toxic effects on human health]. / Drogi ekspozycji mikro- i nanoplastiku oraz potencjalne toksyczne efekty ich dzialania na zdrowie czlowieka.

This article discusses the classification of micro- and nanoplastics (MNP), the routes of their exposure and the effects of MNP on the reproductive, respiratory, digestive and immune systems based on in vitro and in vivo studies, as well as available epidemiological data. The MNP can enter our body through inhalation, food or skin. The presence of microplastics (MP) in tap, bottled and deep sea water, as well as in sea salt, fruit and vegetables has been demonstrated. Due to their small size, MNP can be absorbed and easily distributed through the blood and lymphatic vessel system to tissues and organs. Recent studies have provided evidence of the accumulation of MNP in human lungs and even in the placenta. The accumulation of MNP in the body may have long-term effects and lead to health problems in humans, such as bronchitis, development of asthma, pulmonary fibrosis, inflammation and cancer. The information included in the article gives partial insight into how MNP may affect the human body. However, to fully assess the toxicity of MNP, comprehensive research is necessary, including standardization of MNP detection techniques and determination of the MNP content in food and water. It is also advisable to assess toxicokinetic parameters, as well as to determine the daily dose of exposure and interaction of MNP with various cells. Insufficient data on direct exposure to MNP in the work environment, as well as in other public places, constitutes a factor hindering the establishment of appropriate legal standards. In 2024, work on establishing the first act of EU law enabling the monitoring of MP in drinking water should be completed, which raises great hopes that in the future limit values for MNP in water and food and in workplaces will also be established. Med Pr Work Health Saf. 2024;75(1):81-96.

Purification and Transfection Methods of Chicken Primordial Germ Cells.

Primordial germ cells (PGCs) play a special role in the vertebrate life cycle since they are the precursors of germ cells through which the genome is passed to the next generations. PGCs are found in different locations and variable numbers in the chick embryo, as in other species, depending on the developmental stages. Here, we describe in detail a method based on the Percoll gradient, routinely used in our laboratory, allowing us to obtain from blood and gonad anlages significant numbers of viable PGCs which can be successfully cultured or efficiently genetically modified.

Assessing cytotoxicity and endoplasmic reticulum stress in human blood-brain barrier cells due to silver and copper oxide nanoparticles.

In recent years, it has been generally accepted that metal-based nanoparticles (NPs) may induce stress in the endoplasmic reticulum (ER), a key organelle where protein folding occurs. We examined ER stress in immortalized human cerebral microvascular cells (hCMEC/D3) after exposure to silver-NPs (Ag-NPs)- and copper oxide-NPs (CuO-NPs) induced toxicity at < 10 nm and < 40 nm or < 50 nm diameters, respectively. In cytotoxicity assessments, cells were exposed to different CuO-NPs (5-400 µg/mL) or Ag-NPs (1-10 µg/mL) concentration ranges for 24 h and 72 h, and tetrazole salt reduction assays (EZ4U) were performed. Also, Ag-NP or CuO-NP effects on cell proliferation, apoptosis (caspase 3/7 assays), and ER stress and cell morphology were evaluated. In ER stress assessments, RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1a), and others stress factor mRNA levels were determined after 24 h treatment using Real-Time PCR. Increased stress sensors (IRE1a, PERK, and ATF6) mRNA levels were observed after exposure to Ag-NPs (< 10 and < 40 nm) or CuO-NPs (< 50 nm). We investigated the expression of tight junction (TJ) proteins (barrier junctions) and showed that both types of NP reduced of OCLN gene expression. Morphological changes were observed after Ag-NP or CuO-NP exposure using holotomographic microscopy. Our data suggest that Ag- and CuO-NPs should undergo future in vitro and in vivo toxicology studies, especially for downstream biomedical application and occupational risk assessments.

The effects of co-exposure to methyl paraben and dibutyl phthalate on cell line derived from human skin.

Data on the cumulative effects of chemical substances are necessary for the proper risk assessment, but their availability is still insufficient. The aim of the study was to evaluate the cytotoxic effect of methyl paraben (MePB) and dibutyl phthalate (DBP) on the cells of the skin line (A431) and to compare the cytotoxic effects of the tested substances after single application to A431 cells with the effects of an equimolar/equitoxic (1:1) binary mixture of these compounds as well as their mixtures in ratio 1:3: and 3:1. On the basis of the obtained results, it was found that there were interactions between the tested compounds in terms of cytotoxic effect on A431, assessed on the basis of metabolic activity of cells (MTT test) and integrity of their cell membranes (NRU test). The obtained values of synergy coefficients (SI) and isobolographic analysis indicate that between the tested chemicals in a two-component equimolar mixture (1:1) there is a synergism of action, which, at a high DBP content in the mixture (> 50%) turned into antagonism. Observations using a holotomographic microscope show morphological changes in A431 cells after exposure to both DBP and MePB separately and binary mixtures of these compounds, compared to untreated cells. The observed changes in cell morphology seem to be more pronounced when the cells are exposed to the binary mixtures of DBP and MePB than when exposed to these substances individually, which may confirm the synergy of cytotoxic activity between them (this phenomenon was observed for the higher of the tested concentrations in all tested proportions). It is important to consider such effects when considering the effects of cumulative exposure in the risk assessment in order not to underestimate the risk of adverse effects associated with exposure to chemical mixtures.

The in vitro toxicity evaluation of halloysite nanotubes (HNTs) in human lung cells.

Halloysite nanotubes (HNTs) have been increasingly used in many industrial and biomedical fields. Therefore, the assessment of risk and consequences of exposure to HNTs is very important to better protect human safety. This study aims to investigate the short- (24 or 72 h) and long-term (7 days) cytotoxic effects of HNTs at doses 10-200 µg/mL on human alveolar carcinoma epithelial cells (A549) and human bronchial epithelial cells (BEAS-2B). The effect of HNTs on cell viability, apoptosis, cell proliferation, oxidative/antioxidative status and cell morphology was evaluated. Our results showed that cytotoxicity of HNTs is dependent on dose, cell model and time of exposure. During the time of exposition toxic effects were intensified. To the best of our knowledge, this is the first study to use holo-tomographic microscopy (HTM) to visualise changes in cell morphology due to exposure from HNTs. We observed cells contraction, changes in the size and shape, cell surface folding and cytoplasmic vacuolization, peripheral arrangement of cell nuclei and even increase number of nucleus, which undoubtedly confirmed cytotoxic effect of HNTs at low doses (5 µg/mL and 25 µg/mL). Our results demonstrated that HTM technique provides a new insight into the assessment of HNTs toxicity. Further studies with different cell models are recommended to assess the toxic effect of HNTs on whole human body.

CRISPR/Cas9 gene editing in a chicken model: current approaches and applications.

Improvements in genome editing technology in birds using primordial germ cells (PGCs) have made the development of innovative era genome-edited avian models possible, including specific chicken bioreactors, production of knock-in/out chickens, low-allergenicity eggs, and disease-resistance models. New strategies, including CRISPR/Cas9, have made gene editing easy and highly efficient in comparison to the well-known process of homologous recombination. The clustered regularly interspaced short palindromic repeats (CRISPR) technique enables us to understand the function of genes and/or to modify the animal phenotype to fit a specific scientific or production target. To facilitate chicken genome engineering applications, we present a concise description of the method and current application of the CRISPR/Cas9 system in chickens. Different strategies for delivering sgRNAs and the Cas9 protein, we also present extensively. Furthermore, we describe a new gesicle technology as a way to deliver Cas9/sgRNA complexes into target cells, and we discuss the advantages and describe basal applications of the CRISPR/Cas9 system in a chicken model.

Efficient Source of Cells in Proximal Oviduct for Testing Non-Viral Expression Constructs in the Chicken Bioreactor Model and for Other in Vitro Studies.

This work shows the usefulness of chicken oviduct epithelial cells (COEC) in evaluating the efficacy of non-viral expression vectors carrying human therapeutic genes. Secondly, an efficient source of progenitor COEC for in vitro studies is described. Within the distal part of the oviduct, weak to moderate expression of a trans membrane glycoprotein (CD44) was observed. Single cells presenting only weak expression of CD44 were found in magnum sections. in vitro cultured oviduct cells originating from the distal oviduct were suitable for subculturing and showed a stable proliferation potential up to the 2nd passage. However, the pavimentous epithelial-like morphology of COEC was progressively lost over time and mainly a fibroblast-like monolayer was established in consecutive passages. Moreover, various commercial transfection agents including FuGENE6 and XtremeGENE9 DNA were used to optimize delivery of human interferon alfa-2a, (IFNα2a) a therapeutic protein gene under an ovalbumin promoter. The transfection efficiency of adherent COEC was estimated for up to 40% at a ratio of 6:1 of transfectant to pOVA5EIFN + GFP plasmid. Expression of IFNα2a was confirmed by western blotting in transformed COEC. In conclusion, the population of epithelial progenitor cells sourced from the distal oviduct can significantly contribute to in vitro culture of COEC, representing an efficient model to develop the production of avian bioreactors and other in vitro studies related to oviduct tissue.

Cryoconservation of Chicken Blastodermal Cells: Effects of Slow Freezing, Vitrification, Cryoprotectant Type and Thawing Method during In Vitro Processing.

Cryoconservation of blastodermal cells (BCs) can preserve genetic material for the future reconstruction of poultry breeds. The aim of our study was to compare the effects of three slow freezing programs and vitrification, different cryoprotectants (5% DMSO, 10% DMSO, or multi-component cryoprotectant (MC) and two thawing methods on the viability of chicken BCs. Significant differences in the survival of slowly frozen BCs using program 3 (2°C/min. to 0.4°C/min.) compared with programs 1 (1°C/min. to 0.3°C/min.) and 2 (4°C/min. to 0.3°C/min.) were observed. The percentage of live BCs was significantly higher after slow freezing in the presence of the MC compared with DMSO. The thawing method did not have a significant effect on the percentage of live BCs. We also observed significant differences in the survival rate of BCs after vitrification (81%) and slow freezing in the presence of 10% DMSO using program 3 (60%). The highest percentage of viable BCs was achieved by slow freezing with the MC using program 2 and thawing with method 1 (94%). The most unfavorable combination for BCs survival was slow freezing in 5% DMSO using program 3 and thawing with method 2 (58.3%). This is the first study to apply MC to the slow freezing of BCs. We also showed successful BCs vitrification.

Flow cytometric analysis of apoptosis in cryoconserved chicken primordial germ cells.

Our research aimed to compare the effects of four cryoprotectants and four slow freezing programs on the viability and apoptosis of primordial germ cells (PGCs) in vitro. PGCs were collected from chicken embryonic blood at Hamburger and Hamilton (HH) stages 14-16 and purified by Percoll density gradient centrifugation and then subjected to cryopreservation. We applied microscopy to determine the survival of PGCs after trypan blue staining and flow cytometry to examine apoptosis and viability after annexin V kit staining. We also examined the functionality of cryopreserved PGCs in vivo. Significant differences in viability of PGCs determined via microscopy and flow cytometry were observed. The most unfavorable combination for slow freezing PGCs was program 3 and MIX H (10% DMSO and 5% glycerol in Hank's solution supplemented with 10% FBS) as the cryoprotectant (48.43 and 15.37% live and early apoptotic PGCs, respectively). The highest average percentage of live PGCs (93.1%) and the lowest percentage of early apoptotic PGCs (6.5%) were achieved by slow freezing PGCs in the presence of DMSO F (10% DMSO in FBS) via program 1. Therefore, this method was chosen for the in vivo test. Cryopreserved (group 1) and freshly isolated (group 2) PGCs were transfectedwith a pEGFP-N1 plasmid, cultured under antibiotic selection, and then injected into 3-day-old embryos. After 5 days of incubation, we identified the EGFP marker gene in the gonads of 40 and 45% of recipients in groups 1 and 2, respectively. This is the first study to apply flow cytometry to examine the apoptosis and viability of cryopreserved PGCs. The in vitro and in vivo findings showed that the developed PGC cryoconservation method, depending on slow freezing at the rate of 2°C/min (program 1) in the presence of 10% DMSO F, is an improvement over previous cryoconservation methods and may be a useful tool for the ex situ strategy of poultry biodiversity preservation.

Changes in Quail Blastodermal Cell Status as a Result of Selection.

Genetic selection over many years has significantly improved the growth rate of broilers and increased the number of eggs laid by egg laying chicken breeds. Selection has improved desired parameters, but has caused some negative effects as well. Adverse effects of selection may negatively affect embryonic development. The number of live and apoptotic blastodermal cells (BCs) at the X stage of embryogenesis may be a good indicator of changes in selected individuals. In this paper, a comparison of the number of live and apoptotic BCs was made for three lines of quail: Pharaoh (F33), meat-type line, selected for body weight; egg laying line (S33), selected for egg number; and laying line (S22), additionally selected (for 17 generations) for high yolk cholesterol content. Apoptotic BCs were separated by the magnetic activated cell sorting (MACS) method. The percentage of live and apoptotic BCs was different (P ≤ 0.01) for F33 (35.8% and 64.2%, respectively) and S33 (60.0% and 36.4%). The number of apoptotic BCs for F33 embryos (45,098) was higher (P ≤ 0.01) compared to the number of apoptotic BCs for S33 embryos (26,667). The selection for high yolk cholesterol content caused an increase (P ≤ 0.01) in the total number of BCs from 78,403 (S33) to 140,139 (S22). The percentage of apoptotic BCs was lower (P ≤ 0.01) in the S22 line (17.1%) compared to the S33 line (36.4%). The results showed that it is possible to evaluate the effects of selection in the early stage of embryonic development.

Obtaining chicken primordial germ cells used for gene transfer: in vitro and in vivo results.

Recently, several attempts have been made to create a generation of transgenic chickens via chimeric intermediates produced by primordial germ cells (PGCs) transfer. This study aimed to compare the influences of different chicken PGCs isolated from circulating blood (bPGCs) or gonads (gPGCs), purification (ACK, Percoll or trypsin) and transfection methods (electroporation or lipofection) on the expression of transgenes in vitro and the migration of modified donor cells to the recipient gonads. The highest average frequency of pEGFP-N1 plasmid-transfected bPGCs (75.8%) was achieved with Percoll density gradient centrifugation and electroporation. After ammonium chloride-potassium (ACK) treatment and lipofection, in vitro transgene expression was only detected in 35.2% of bPGCs. Chimeric chickens were produced from these purified, transfected and cultured cells, and the transgene was detected in the gonads of 44 and 42% of the recipient embryos that had been injected with bPGCs and gPGCs, respectively. These data confirmed that the combination of PGC purification via Percoll centrifugation and electroporation was an effective method for producing transgenic chickens. Subsequently, we used this method with expression vectors for gene hIFNα 2a/hepatitis B virus surface antigen (HBsAg) under the control of the ovalbumin promoter to generate G0 transgenic chickens. Consequently, we observed that 4.9% of the hens and 3.5% of the roosters carried the hIFNα 2a gene, whereas 16.7% of the hens and 2.4% of the roosters carried the HBsAg gene, thus undisputedly confirming the exceptional effectiveness of the applied methods.

Use of Ubp1 protease analog to produce recombinant human growth hormone in Escherichia coli.

BACKGROUND: Numerous bacterial human growth hormone (hGH) expression methods under conventional fermentation and induction conditions have been described. Despite significant progress made in this area over the past several years, production of recombinant hGH by using cellular expression systems still requires further optimization. Fusion of the ubiquitin (Ub) tag to the hGH protein allowed to increase of the overall efficiency of the biosynthesis and improve the protein stability. Ub is a protein composed of 76 amino acid residues with a molecular mass of 8.6 kDa, expressed in all eukaryotes. This protein is an element of the universal protein modification system, which does not occur in bacteria, and is a useful carrier for heterologous proteins obtained through expression in Escherichia coli. Purification of Ub-fusion proteins is easier than that of unconjugated recombinant proteins, and Ub can be removed by deubiquitinating proteases (DUBs or UBPs). RESULTS AND CONCLUSION: In the present study the UBPD2C protease, a stable UBP1 analog, was produced as a recombinant protein in E. coli and used for production of recombinant human growth hormone (rhGH). hGH was expressed as a fusion protein with Ub as a tag. Our findings show that the UBPD2C protease is very effective in removing the Ub moiety from recombinant Ub-fused hGH. The described approach enables obtaining a considerable yield of rhGH in a purity required for pharmaceutical products.

New cloning and expression vector derived from Escherichia coli plasmid pIGWZ12; a potential vector for a two-plasmid expression system.

We constructed pIGPZ, a new cloning and expression vector derived from Escherichia coli plasmid pIGWZ12::Kan. pIGPZ contains a kanamycin resistance marker, a multiple-cloning-site (MCS) region, and a promoter for constitutive expression of cloned genes. pIGPZ has the same high level of stability as the original plasmid, even in the absence of antibiotic selection. Furthermore, we show that pIGPZ is compatible with ColE1-based plasmids and a pSC101-like plasmid. All the characteristic elements of theta-replicating plasmids were found in the pIGPZ putative origin of replication. Finally, we demonstrate that pIGPZ can be used in a double-plasmid expression system by co-expressing UBP1 protease from pIGPZ with ubi-interferon alpha (IFNA13; GenBank Accession No. NM_006900.3) or ubi-human growth hormone (ubi-hGH; patent No. WO 2005/066208 A2) cloned in another plasmid. In this system, both ubi-interferon alpha and ubi-human growth hormone were deubiquitinated efficiently in E. coli cells.