Recent advances and current challenges of new approach methodologies in developmental and adult neurotoxicity testing.

Adult neurotoxicity (ANT) and developmental neurotoxicity (DNT) assessments aim to understand the adverse effects and underlying mechanisms of toxicants on the human nervous system. In recent years, there has been an increasing focus on the so-called new approach methodologies (NAMs). The Organization for Economic Co-operation and Development (OECD), together with European and American regulatory agencies, promote the use of validated alternative test systems, but to date, guidelines for regulatory DNT and ANT assessment rely primarily on classical animal testing. Alternative methods include both non-animal approaches and test systems on non-vertebrates (e.g., nematodes) or non-mammals (e.g., fish). Therefore, this review summarizes the recent advances of NAMs focusing on ANT and DNT and highlights the potential and current critical issues for the full implementation of these methods in the future. The status of the DNT in vitro battery (DNT IVB) is also reviewed as a first step of NAMs for the assessment of neurotoxicity in the regulatory context. Critical issues such as (i) the need for test batteries and method integration (from in silico and in vitro to in vivo alternatives, e.g., zebrafish, C. elegans) requiring interdisciplinarity to manage complexity, (ii) interlaboratory transferability, and (iii) the urgent need for method validation are discussed.

Inhibitory effect of silver nanoparticles on proliferation of estrogen-dependent MCF-7/BUS human breast cancer cells induced by butyl paraben or di-n-butyl phthalate.

In this study the effect of silver nanoparticles (AgNPs) on proliferation of estrogen receptor (ER)-positive human breast cancer MCF-7/BUS cells was assessed by means of in vitro assay. The cells were exposed in the absence of estrogens to AgNPs alone or in combination with aluminum chloride (AlCl3), butyl paraben (BPB) and di-n-butyl phthalate (DBPh). The results revealed that AgNPs at the non-cytotoxic concentrations (up to 2µg/mL) and AlCl3 (up to 500µM) did not induce proliferation of MCF-7/BUS cells whereas BPB and DBPh showed strong estrogenic activity with the highest effect at 16µM and 35µM, respectively. AgNPs inhibited the proliferation of the cells induced by DBPh, BPB or even with 17ß-estradiol (E2) during 6-day incubation in the absence of estrogens. ICI 182,780 (10nM), a known estrogen receptor (ER) antagonist, induced strong inhibitory effect. AgNPs also decreased transcription of the estrogen-responsive pS2 and progesterone receptor (PGR) genes but modulated expression neither of ERα nor ERß in MCF-7/BUS cells exposed to BPB, DBPh or E2 for 6h. Our results indicate that AgNPs may inhibit growth of breast cancer cells stimulated by E2 or estrogenic chemicals, i.e. BPB and DBPh.

Gene-occupation interactions: a review of the literature on bladder and prostate cancer.

Bladder cancer (BCa) and prostate cancer (PCa) are genitourinary cancers which constitute significant health problems in men and in which environmental factors play an important role. Understanding the genetic susceptibility to BCa or PCa and occupational exposure is paramount to improving cancer prevention and early detection. The aim of this review article was to address the scientific evidence on the genetic risk factors and occupational exposure associated with the occurrence of BCa and PCa. The authors identified relevant original articles that have been published between 1994 and 2023. Variations of the following search terms: "gene" and "occupational" combined with one of the following terms: "bladder cancer" or "prostate cancer" were applied for the search purpose. The authors found 342 publications of which 50 population studies met their requirements for gene-occupation interactions. In total, 34 full-text manuscripts were about BCa and 16 about PCa. These research examines the genes involved in detoxification processes of xenobiotics (glutathione S-transferase, N-acetyltransferase, cytochrome P450, UDP-glucuronosyltransferase), oxidative stress (glutathione peroxidase 1, manganese superoxide dismutase, catalase), altering DNA repair capacity (X-ray repair cross-complementing 1, base excision repair, nucleotide excision repair), tumour suppression (TP53 gene), and vitamin D pathway (vitamin D receptor gene). The role of genetic factors in the occupational exposure has not been conclusively established, but it appears the possibility of genetic involvement. Determination of environmentally responsive genes provides important mechanistic implications for the etiology of occupational cancers, and valuable input in occupational exposure limits set by taking genetic susceptibility into account. More genetic research is needed to corroborate these findings and assess their significance in the workplace. Med Pr. 2023;74(2):127-44.

Environmental exposure to cadmium in breast cancer - association with the Warburg effect and sensitivity to tamoxifen.

The association between cadmium and breast cancer remains unexplained due to inconsistent epidemiological data and unknown underlying mechanisms. This study aimed to assess the relationship between environmental exposure to cadmium and the Warburg effect in breast cancer and, thus, its possible interference with breast cancer treatment. The observational study in two groups of breast cancer patients indicated a positive correlation between urinary cadmium concentration and tumor expression of HIF1A (a master regulator of the Warburg effect). Further explanatory research in MCF-7 cells showed no impact of cadmium exposure on molecular and biochemical markers of the Warburg effect. However, long-term exposure to a low and environmentally relevant concentration of cadmium led to the accumulation of the metal in MCF-7 cells and decreased their sensitivity to tamoxifen. To conclude, the association between cadmium and the Warburg effect was suggested in the observational study, although not confirmed in vitro. Nevertheless, cadmium seems to interfere with tamoxifen treatment which deserves further investigation in terms of its possible implication in intrinsic resistance to hormone therapy.

Metabolism and in vitro assessment of the mutagenic activity of urinary extracts from rats after inhalation exposure to 1-methylnaphthalene.

OBJECTIVES: 1-Methylnaphthalene (1-MN) is composed of 2 benzene rings and belongs to polycyclic aromatic hydrocarbons. The metabolism of 1-MN in laboratory animals and bacteria leads to the formation of 1-naphthoic acid (1-NA). MATERIAL AND METHODS: In this study the distribution of 1-NA in lung, liver, spleen, kidney and urinary excretion of 1-NA in rats after single and repeated inhalation exposure to 1-MN vapors were investigated. The activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and cytochrome were measured of the rats. Genotoxic effects were evaluated with the in vitro micronucleus test on V79 hamster fibroblasts. RESULTS: The concentrations of 1-NA in the tissues of rats after single and repeated exposure to 1-MN were dependent on the exposure dose. High levels of 1-NA were found in kidneys of animals after the single and repeated exposure to 1-MN. With an increase of 1-MN dose, an increase in the activity of cytochrome P450 (CYP1A1 and CYP1A2) was observed in the liver of rats. Compared to control animals, significantly higher ALT activity was noted in serum of rats exposed to 1-MN. The micronuclei frequency in V79 cells exposed to 1-MN (in the range of analyzable concentrations; i.e., 5-25 µg/ml) did not differ significantly from the vehicle control, whereas urine extracts from rats exposed to 1-MN induced a significant increase in the frequency of micronuclei compared to urine extracts from the group of control animals. CONCLUSIONS: Metabolism of 1-MN in rats after the inhalation exposure leading to 1-NA was mainly observed during the first day after the end of exposure. It is likely that 1-MN metabolites present in rat urine can induce the increased micronuclei frequency as was shown in V79 cells. Int J Occup Med Environ Health. 2022;35(6):731-46.

Cadmium and breast cancer - Current state and research gaps in the underlying mechanisms.

Cadmium (Cd), a heavy metal with strong carcinogenic properties has been linked with breast cancer risk. Epidemiological data on the association between Cd exposure and breast cancer are not consistent and suggest that this relationship may be modulated by a number of different factors. The mechanisms of action underlying the molecular effects of Cd, especially in terms of its carcinogenicity, are generally not well understood. Specifically, in the mammary gland, the effects of Cd are considered to be related mainly to its oestrogenic potential, however, several other mechanisms have also been suggested, such as epigenetic alterations, inhibition of DNA repair pathways, induction of oxidative stress, interference with metallothioneins, cadherins and integrins, as well as interactions with xenobiotics. This review summarizes the current state of knowledge in this field, including potential mechanisms of action of Cd in breast cancer initiation and progression, as well as possible ways of protection against its toxicity. Importantly, there are many research gaps in this area since limited evidence is available from experimental studies. Important issues to be further investigated concern exact molecular mechanisms of Cd accumulation in the tissues and Cd-induced activation of eostrogen receptors. Impact on DNA damage and epigenome upon Cd exposure in breast cancer development remains still highly unexplored area and should gain more interest.

Applications and Biological Activity of Nanoparticles of Manganese and Manganese Oxides in In Vitro and In Vivo Models.

The expanding applications of nanotechnology seem to be a response to many technological, environmental, and medical challenges. The unique properties of nanoparticles allow for developing new technologies and therapies. Among many investigated compounds is manganese and its oxides, which in the form of nanoparticles, could be a promising alternative for gadolinium-based contrast agents used in diagnostic imaging. Manganese, which is essential for living organisms as an enzyme cofactor, under excessive exposure-for example, due to water contamination or as an occupational hazard for welders-can lead to neurological disorders, including manganism-a condition similar to Parkinson's disease. This review attempts to summarise the available literature data on the potential applications of manganese and manganese oxide nanoparticles and their biological activity. Some of the published studies, both in vitro and in vivo, show negative effects of exposure to manganese, mainly on the nervous system, whereas other data suggest that it is possible to develop functionalised nanoparticles with negligible toxicity and novel promising properties.

The Role of Masculinity Threat in Homonegativity and Transphobia.

According to the precarious manhood perspective, masculinity threat triggers various compensatory mechanisms and heightens motivation to restore and reaffirm masculinity via typically masculine attitudes and behaviors. Four experiments aiming to examine the effect of masculinity threat on prejudices toward sexual and gender minorities were undertaken, controlling for adherence to sex role stereotypes. Our studies showed that Polish men representing university and high school students exposed to gender threat experienced increased negative affect (Experiment 1) and expressed higher prejudices toward gay people (Experiment 2) and transgender individuals (Experiments 3a and 3b). Furthermore, Experiment 2 revealed an effect of masculinity threat on modern prejudices predominantly toward gay people but not old-fashioned homonegativity. The results are discussed in terms of the role of masculinity threat as the mechanism responsible for gender differences in the attitudes toward sexual and gender minorities.

Combined effect of silver nanoparticles and aluminium chloride, butylparaben or diethylphthalate on the malignancy of MDA-MB-231 breast cancer cells and tumor-specific immune responses of human macrophages and monocyte-derived dendritic cells.

The aim of this study was to assess whether silver nanoparticles (AgNP) or selected cosmetic ingredients may modify functions of various immunocompetent cell populations. To this end, the effect of two AgNP (size of 15 nm or 45 nm), alone and in combination with aluminium chloride, butyl paraben, di-n-butyl phthalate or diethyl phthalate was assessed on: (1) migration and invasion of MDA-MB-231 human breast cancer cells; (2) M1/M2 polarization of phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages (M0) and (3) activation/maturation of monocyte-derived dendritic cells (DCs). The results of this study showed that neither any of the test chemicals alone nor the mixtures significantly changed the migration or invasion ability of MDA-MB-231 cells following, both 72-h and 21-day exposure. Analysis of the expression of marker genes for both M1 (IL-1B, CXCL9, TNF) and M2 (DCSIGN, MRC1) polarization revealed that the chemicals/mixtures did not activate M1/M2 differentiation of the M0 macrophages. In addition, no significant changes were observed in the expression of CD86, HLA-DR and CD54 surface markers and phagocytic activity of DCs following 48-h exposure to AgNP alone or in combination with test compounds. Our study suggests that AgNP alone or in combination with tested cosmetic ingredients do not alter function of immunocompetent cells studied.

Transcriptomic analysis of the PI3K/Akt signaling pathway reveals the dual role of the c-Jun oncogene in cytotoxicity and the development of resistance in HL-60 leukemia cells in response to arsenic trioxide.

BACKGROUND: Arsenic trioxide (ATO) is a well-recognized antileukemic drug used for the treatment of newly diagnosed and relapsed acute promyelocytic leukemia (APL). A major drawback of therapy with ATO is the development of APL cell resistance, the mechanisms of which are still not clear. OBJECTIVES: The aim of this study was to investigate the role of the PI3K/Akt signaling pathway in ATOtreated human acute myeloid leukemia (HL-60) cells and in ATO-resistant clones. MATERIAL AND METHODS: The cytotoxicity of ATO was assessed using Trypan blue staining or a WST-1 reduction assay. The Akt phosphorylation level was measured by immunofluorescent staining and flow cytometry. Gene expression analysis was performed using real-time polymerase chain reaction (PCR). RESULTS: The clones derived by culturing for 8-12 weeks in the presence of 1.75, 2.5, and 5 µM ATO were characterized by high viability but a slower growth rate compared to the parental HL-60 cells. The flow cytometry analysis showed that in the parental cells the levels of p-Akt were undetectable or very low, and that ATO had no effect on the level of p-Akt in either the ATO-treated parental cells or the clones. The gene expression analysis revealed that some of the genes involved in the Akt pathway may play a key role in the induction of resistance to ATO, e.g., genes encoding cyclin D1 (CCND1), fork head box O1 (FOXO1), Jun oncogene (JUN), protein kinase C isoform B1 (PRKCB1), because their expression profiles were predominantly changed in the clones and/or the ATO-treated parental HL-60 cells. CONCLUSIONS: The overall results indicate that CCND1, FOXO1, and JUN may contribute to the induction of resistance to ATO, and that the C-Jun N-terminal kinase (JNK) signaling pathway may have greater significance than the phosphoinositide 3-kinase (PI3K)/Akt pathway in mediating the cytotoxic effects of ATO and the development of resistance to ATO in the HL-60 cell line.

A study on the in vitro percutaneous absorption of silver nanoparticles in combination with aluminum chloride, methyl paraben or di-n-butyl phthalate.

Some reports indicate that the silver released from dermally applied products containing silver nanoparticles (AgNP) (e.g. wound dressings or cosmetics) can penetrate the skin, particularly if damaged. AgNP were also shown to have cytotoxic and genotoxic activity. In the present study percutaneous absorption of AgNP of two different nominal sizes (Ag15nm or Ag45nm by STEM) and surface modification, i.e. citrate or PEG stabilized nanoparticles, in combination with cosmetic ingredients, i.e. aluminum chloride (AlCl3), methyl paraben (MPB), or di-n-butyl phthalate (DBPH) was assessed using in vitro model based on dermatomed pig skin. The inductively coupled plasma mass spectrometry (ICP-MS) measurements after 24h in receptor fluid indicated low, but detectable silver absorption and no statistically significant differences in the penetration between the 4 types of AgNP studied at 47, 470 or 750µg/ml. Similarly, no significant differences were observed for silver penetration when the AgNP were used in combinations with AlCl3 (500µM), MPB (1250µM) or DBPH (35µM). The measured highest amount of Ag that penetrated was 0.45ng/cm2 (0.365-0.974ng/cm2) for PEG stabilized Ag15nm+MPB.

Effect of particle size and dispersion status on cytotoxicity and genotoxicity of zinc oxide in human bronchial epithelial cells.

Data available on the genotoxicity of zinc oxide (ZnO) nanoparticles (NPs) are controversial. Here, we examined the effects of particle size and dispersion status on the cytotoxicity and genotoxicity of nanosized and fine ZnO, in the presence and absence of bovine serum albumin (BSA; 0.06%) in human bronchial epithelial BEAS-2B cells. Dynamic light scattering analysis showed the most homogenous dispersions in water alone for nanosized ZnO and in water with BSA for fine ZnO. After a 48-h treatment, both types of ZnO were cytotoxic within a similar, narrow dose range (1.5-3.0µg/cm(2)) and induced micronuclei at a near toxic dose range (1.25-1.75µg/cm(2)), both with and without BSA. In the comet assay, nanosized ZnO (1.25-1.5µg/cm(2)), in the absence of BSA, caused a statistically significant increase in DNA damage after 3-h and 6-h treatments, while fine ZnO did not. Our findings may be explained by better uptake or faster intracellular dissolution of nanosized ZnO without BSA during short treatments (3-6h; the comet assay), with less differences between the two ZnO forms after longer treatments (>48h; the in vitro micronucleus test). As ZnO is genotoxic within a narrow dose range partly overlapping with cytotoxic doses, small experimental differences e.g. in the dispersion of ZnO particles may have a substantial effect on the genotoxicity of the nominal doses added to the cell culture.

The modulating effect of ATM, ATR, DNA-PK inhibitors on the cytotoxicity and genotoxicity of benzo[a]pyrene in human hepatocellular cancer cell line HepG2.

The effect of inhibitors of phosphatidylinositol-3-kinase-related kinases (PIKK): ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) on response of HepG2 human liver cancer cells to benzo[a]pyrene (BaP) was investigated. PIKK inhibitors: KU55933 (5 µM), NU7026 (10 µM) or caffeine (1 and 2mM) when used as single agents or in combinations (KU55933/NU7026 and caffeine/NU7026) did not significantly influence the BaP (3 µM) cytotoxicity (MTT reduction test). BaP induced a weak proapoptotic effect which was moderately enhanced by both inhibitor combinations. HepG2 cells exposed to BaP showed a strong S-phase arrest which was considerably diminished by both inhibitor combinations. The DNA damage (comet assay) induced after continuous 24h exposure to BaP was significantly diminished by both inhibitor combinations. Weak induction of reactive oxygen species by BaP was observed, which was not modulated by the inhibitor combinations. Similarly, no modulation of the glutathione levels was observed.

The influence of ATM, ATR, DNA-PK inhibitors on the cytotoxic and genotoxic effects of dibenzo[def,p]chrysene on human hepatocellular cancer cell line HepG2.

The effect of inhibitors of phosphatidylinositol-3-kinase related kinases (PIKK): ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) on the response of HepG2 human liver cancer cells to dibenzo[def,p]chrysene (DBC) was investigated. High cytotoxicity of DBC (IC50=0.1µM) was observed after 72h incubation. PIKK inhibitors: KU55933 (5µM), NU7026 (10µM) or caffeine (1 and 2mM) when used alone did not significantly influence the cytotoxicity. However, two combinations: KU55933/NU7026 and caffeine/NU7026 significantly increased HepG2 viability (by 25%) after treatment with DBC at 0.5µM. The cytoprotective effect was confirmed by cell cycle and apoptosis/necrosis analysis. DNA damage level after exposure to DBC assessed by comet assay (single strand breaks) showed a long persistence and significant decrease after incubation of the cells in the presence the inhibitors (the combination of KU55933+NU7026 showed the strongest effect). Weak induction of reactive oxygen species (ROS) by DBC (0.5µM) was observed. Although, KU55933 and NU7026 when used alone did not increase ROS levels in the cells, their combination induced the ROS increase and moderately enhanced ROS generation by DBC. We propose a mechanism how cells with damaged DNA after exposure to DBC and under the condition of PIKK inhibition, may be at higher risk of undergoing malignant transformation.

Genotoxicity of synthetic amorphous silica nanoparticles in rats following short-term exposure. Part 2: intratracheal instillation and intravenous injection.

Synthetic amorphous silica nanomaterials (SAS) are extensively used in food and tire industries. In many industrial processes, SAS may become aerosolized and lead to occupational exposure of workers through inhalation in particular. However, little is known about the in vivo genotoxicity of these particulate materials. To gain insight into the toxicological properties of four SAS (NM-200, NM-201, NM-202, and NM-203), rats are treated with three consecutive intratracheal instillations of 3, 6, or 12 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection (cumulative doses of 9, 18, and 36 mg/kg). Deoxyribonucleic acid (DNA) damage was assessed using erythrocyte micronucleus test and the standard and Fpg-modified comet assays on cells from bronchoalveolar lavage fluid (BALF), lung, blood, spleen, liver, bone marrow, and kidney. Although all of the SAS caused increased dose-dependent changes in lung inflammation as demonstrated by BALF neutrophilia, they did not induce any significant DNA damage. As the amount of SAS reaching the blood stream and subsequently the internal organs is probably to be low following intratracheal instillation, an additional experiment was performed with NM-203. Rats received three consecutive intravenous injections of 5, 10, or 20 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection. Despite the hepatotoxicity, thrombocytopenia, and even animal death induced by this nanomaterial, no significant increase in DNA damage or micronucleus frequency was observed in SAS-exposed animals. It was concluded that under experimental conditions, SAS induced obvious toxic effects but did cause any genotoxicity following intratracheal instillation and intravenous injection.

Characterization of arsenic trioxide resistant clones derived from Jurkat leukemia T cell line: focus on PI3K/Akt signaling pathway.

In this study the role of PI3K/Akt signaling pathway in arsenic trioxide (ATO)-treated parental Jurkat cells and also in derived ATO-resistant clones grown in the presence of given ATO concentration was investigated. ATO-resistant clones (cultured for 8-12weeks in the presence of 1, 2.5 and 5µM ATO) were characterized by high viability in the presence of ATO but slower growth rate compared to the parental cells. Morphological and functional characterization of derived ATO-resistant clones revealed that they did not differ fundamentally from parental Jurkat cells in terms of cell size, level of GSH, the lysosomal fluorescence or CD95/Fas surface antigen expression. However, a slight increase in the mitochondrial potential (JC-1 staining) was detected in the clones compared to parental Jurkat cells. Side population analysis (Vybrant DyeCycle Violet™ staining) in ATO resistant clones did not indicate any enrichment withcancer stem cells. Akt1/2, AktV or wortmannin inhibitors decreased viability of ATO-resistant clones grown in the presence of ATO, with no effect on ATO-treated parental cells. Flow cytometry analysis showed that ATO decreased the level of p-Akt in ATO-treated parental cells, while the resistant clones exhibited higher levels of p-Akt immunostaining than parental Jurkat cells. Expression analysis of 84 genes involved in the PI3K/Akt pathway revealed that this pathway was predominantly active in ATO-resistant clones. c-JUN seems to play a key role in the induction of cell death in ATO-treated parental Jurkat cells, as dose-dependent strong up-regulation of JUN was specific for the ATO-treated parental Jurkat cells. On the other hand, changes in expression of cyclin D1 (CCND1), insulin receptor substrate 1 (IRS1) and protein kinase C isoforms (PRKCZ,PRKCB and PRKCA) may be responsible for the induction of resistance to ATO. The changes in expression of growth factor receptor-bound protein 10 (GRB10) observed in ATO-resistant clones suggest a possibility of induction of different mechanisms in development of resistance to ATO depending on the drug concentration and thus involvement of different signaling mediators.

A strategy for in vitro safety testing of nanotitania-modified textile products.

Titanium dioxide nanomaterials are extensively used in many applications, also for modification of textile materials. Toxicological assessment of such textile materials is currently seldom performed, mainly because of lack of appropriate guidelines. The aim of the study was to assess cytotoxic and genotoxic potential of commercially available TiO2 and TiO2/Ag NMs in pristine form as well as polypropylene fibers modified with the NMs. Both titania NMs showed a cytotoxic effect on BALB/3T3 clone A31 and V79 fibroblasts after 72-h exposure. Both NMs induced a weak genotoxic effect in comet assay, with TiO2/Ag being more active. In vitro micronucleus test on human lymphocytes revealed a weak mutagenic effect of both materials after 24h of exposure. In contrast, no significant increase in micronuclei frequency was observed in the in vitro micronucleus test on V79 fibroblasts. The 24-h extracts prepared from polypropylene fibers modified with TiO2/Ag induced a cytotoxic effect on BALB/3T3 cells which strongly depended on the mode of the fibers manufacturing. The study presents a comprehensive approach to toxicity assessment of textile fibers modified with NMs. Proposed approach may form a good "starting point" for improved future testing strategies.