Endocrine, Metabolic & Immune Disorders-Drug Targets (Formerly Current Drug Targets - Immune, Endocrine & Metabolic Disorders) - Volume 23, Issue 14, 2023

To maintain the integrity of an organism, a well-functioning immune system is essential. Immunity is dynamic, with constant surveillance needed to determine whether to initiate an immune response or to not respond. Both inappropriate immunostimulation and decreased immune response can be harmful to the host. A reduced immune response can lead to high susceptibility to cancer or infections, whereas an increased immune response can be related to autoimmunity or hypersensitivity reactions. Animal testing has been the gold standard for hazard assessment in immunotoxicity but a lot of efforts are ongoing to develop non-animal-based test systems, and important successes have been achieved. The term “new approach methodologies” (NAMs) refer to the approaches which are not based on animal models. They are applied in hazard and risk assessment of chemicals and include approaches such as defined approaches for data interpretation and integrated approaches to testing and assessment. This review aims to summarize the available NAMs for immunotoxicity assessment, taking into consideration both inappropriate immunostimulation and immunosuppression, including implication for cancer development.

Prostate cancer is one of the devastating diseases characterized by genetic changes leading to uncontrolled growth and metastasis of the cells of the prostate gland and affects men worldwide. Conventional hormonal and chemotherapeutic agents are effective in mitigating the disease if diagnosed at an early stage. All dividing eukaryotic cells require mitotic progression for the maintenance of genomic integrity in progeny populations. The protein kinases, upon activation and de-activation in an ordered fashion, lead to spatial and temporal regulation of the cell division process. The entry into mitosis along with the progression into sub-phases of mitosis is ensured due to the activity of mitotic kinases. These kinases include Polo-Like-Kinase 1 (PLK1), Aurora kinases, and Cyclin-Dependent- Kinase 1 (CDK1), among others. The mitotic kinases, among others, are usually overexpressed in many cancers and can be targeted using small molecule inhibitors to reduce the effects of these regulators on mechanisms, such as regulation of genomic integrity and mitotic fidelity. In this review, we attempted to discuss the appropriate functions of mitotic kinases revealed through cell culture studies and the impact of their respective inhibitors derived in pre-clinical studies. The review is designed to elucidate the growing field of small molecule inhibitors and their functional screening or mode of action at the cellular and molecular level in the context of Prostate Cancer. Therefore, studies performed specifically on cells of Prostatic-origin are narrated in this review, culminating in a comprehensive view of the specific field of mitotic kinases that can be targeted for therapy of Prostate cancer.

The hepatotoxicity of drugs is one of the leading causes of drug withdrawal from the pharmaceutical market and high drug attrition rates. Currently, the commonly used hepatocyte models include conventional hepatic cell lines and animal models, which cannot mimic human drug-induced liver injury (DILI) due to poorly defined dose-response relationships and/or lack of human-specific mechanisms of toxicity. In comparison to 2D culture systems from different cell sources such as primary human hepatocytes and hepatomas, 3D organoids derived from an inducible pluripotent stem cell (iPSC) or adult stem cells are promising accurate models to mimic organ behavior with a higher level of complexity and functionality owing to their ability to self-renewal. Meanwhile, the heterogeneous cell composition of the organoids enables metabolic and functional zonation of hepatic lobule important in drug detoxification and has the ability to mimic idiosyncratic DILI as well. Organoids having higher drug-metabolizing enzyme capacities can culture long-term and be combined with microfluidic-based technologies such as organ-on-chips for a more precise representation of human susceptibility to drug response in a high-throughput manner. However, there are numerous limitations to be considered about this technology, such as enough maturation, differences between protocols and high cost. Herein, we first reviewed the current preclinical DILI assessment tools and looked at the organoid technology with respect to in vitro detoxification capacities. Then we discussed the clinically applicable DILI assessment markers and the importance of liver zonation in the next generation organoid- based DILI models.

Background: CHO-K1 cells were used as in vitro model to explore mechanisms of cytotoxicity of the test drugs. Aim: To provide in vitro data on toxicity mechanisms of clozapine, diclofenac and nifedipine. Objective: Cytotoxic mechanisms of clozapine (CLZ), diclofenac (DIC) and nifedipine (NIF) were studied in CHO-K1 cells in vitro. All three drugs induce adverse reactions in some patients with partially unknown mechanisms. Methods: Following the determination of time- and dose-dependency of cytotoxicity by the MTT test, cytoplasmic membrane integrity was explored by the LDH leakage test. Both end-points were further examined in the presence of soft and hard nucleophilic agents, glutathione (GSH) and potassium cyanide (KCN), respectively, and either individual or general cytochrome P450 (CYP) inhibitors, whether CYPcatalysed formation of electrophilic metabolites play a role in the observed cytotoxicity and membrane damage. The generation of reactive metabolites during the incubations was also explored. Formation of malondialdehyde (MDA) and oxidation of dihydrofluorescein (DCFH) were monitored whether peroxidative membrane damage and oxidative stress take place in cytotoxicity. Incubations were also conducted in the presence of chelating agents of EDTA or DTPA to explore any possible role of metals in cytotoxicity by facilitating electron transfer in redox reactions. Finally, mitochondrial membrane oxidative degradation and permeability transition pore (mPTP) induction by the drugs were tested as markers of mitochondrial damage. Results: The presence of an individual or combined nucleophilic agents significantly diminished CLZand NIF-induced cytotoxicities, while the presence of both agents paradoxically increased DIC-induced cytotoxicity by a factor of three with the reason remaining unknown. The presence of GSH significantly increased DIC-induced membrane damage too. Prevention of membrane damage by the hard nucleophile KCN suggests the generation of a hard electrophile upon DIC and GSH interaction. The presence of CYP2C9 inhibitor sulfaphenazole significantly diminished DIC-induced cytotoxicity, probably by preventing the formation of 4-hydroxylated metabolite of DIC, which further converts to an electrophilic reactive intermediate. Among the chelating agents, EDTA caused a marginal decrease in CLZ-induced cytotoxicity, while DIC-induced cytotoxicity was amplified by a factor of five. Both reactive and stable metabolites of CLZ could be detected in the incubation medium of CLZ with CHO-K1 cells, which are known to have low metabolic capacity. All three drugs caused a significant increase in cytoplasmic oxidative stress by means of DCFH oxidation, which was confirmed by increased MDA from cytoplasmic as well as mitochondrial membranes. The addition of GSH paradoxically and significantly increased DICinduced MDA formation, in parallel with the increase in membrane damage when DIC and GSH combined. Conclusion: Our results suggested that the soft electrophilic nitrenium ion of CLZ is not responsible for the observed in vitro toxicities, and this may originate from a relatively low amount of the metabolite due to the low metabolic capacity of CHO-K1. A hard electrophilic intermediate may contribute to cellular membrane damage incubated with DIC, while a soft electrophilic intermediate seems to exacerbate cell death by a mechanism other than membrane damage. A significant decrease in cytotoxicity of NIF by GSH and KCN suggested that both soft and hard electrophiles contribute to NIF-induced cytotoxicity. All three drugs induced peroxidative cytoplasmic membrane damage, while only DIC and NIF induced peroxidative mitochondrial membrane damage, which suggested mitochondrial processes may contribute to adverse effects of these drugs in vivo.

Background: Prenatal period is a critical developmental phase that is sensitive to hormonal disruption by natural and/or exogenous hormones. Some pharmaceuticals frequently prescribed and used safely during pregnancy are shown to interact with the developmental programming of fetus, resulting in endocrine-related adverse effects. Objective: In this research, we aimed to determine the endocrine disrupting potential of paracetamol, indomethacin, alpha-methyldopa and pantoprazole which are frequently prescribed pharmaceuticals during pregnancy. Methods: In vitro aromatase inhibitory, estrogen receptor (ER) agonist/antagonist (E-Screen assay) and hormone biosynthesis modulatory effects (H295R steroidogenesis assay) of the selected pharmaceuticals were evaluated. Furthermore, their effects on viability of MCF-7/BUS and H295R cells were also evaluated by MTT assay. Results: None of the pharmaceuticals affected H295R cell viability. Only indomethacin reduced MCF- 7/BUS cell viability at 100μM and 300μM. Among the tested pharmaceuticals, only paracetamol and indomethacin showed aromatase inhibitory activity with IC50values of 14.7 x 10^-5M and 57.6 x 10^-5M, respectively. Moreover, indomethacin displayed a biphasic ER agonist effect. ER antagonist effects of indomethacin and pantoprazole were confirmed by performing two stepped E-Screen assay. After the partial validation of the H295R steroidogenesis assay with forskolin and prochloraz, the effects of pharmaceuticals on synthesis of testosterone (T) and estradiol (E2) levels were tested. Alpha-methyldopa increased E2 at all tested concentrations and T at 1.48 and 4.4μM. Contrarily other tested pharmaceuticals did not affect steroidogenesis. Conclusion: Present data suggest that all tested pharmaceuticals may have potential endocrine disrupting effect, which should be considered when used in pregnancy.

Aims: Investigate the immunomodulatory effects of bisphenols in the THP-1 cell line and peripheral blood mononuclear cells in response to lipopolysaccharide (LPS) activation or to phorbol 12-myristate 13-acetate (PMA) and ionomycin. Background: We have previously demonstrated the usefulness of the evaluation of RACK1 expression as a link between endocrine disrupting activity and the immunotoxic effect of xenobiotics. We demonstrated that while BPA and BPAF reduced RACK1 expression, BPS was able to increase it. Objective: Bisphenol A (BPA) is one of the most commonly used chemicals in the manufacturing of polycarbonate plastics and plastic consumer products. Its endocrine disrupting (ED) potential and changes in European regulations have led to replacing BPA in many uses with structurally similar chemicals, like bisphenol AF (BPAF) and bisphenol S (BPS). However, emerging data indicated that bisphenol analogues may not be safer than BPA both in toxic effects and ED potential. Methods: THP-1 cell line and peripheral blood mononuclear cells were activated with lipopolysaccharide (LPS) or with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Results: BPA and BPAF decreased LPS-induced expression of surface markers and the release of pro-inflammatory cytokines, while BPS increased LPS-induced expression of CD86 and cytokines. BPA, BPAF, and BPS affected PMA/ionomycin-induced T helper differentiation and cytokine release with gender-related alterations in some parameters investigated. Conclusion: Data confirm that bisphenols can modulate immune cell differentiation and activation, further supporting their immunotoxic effects.

Background: Studies have indicated that the conservative Notch pathway contributes to steroid hormone synthesis in the ovaries; however, its role in hormone synthesis of the testis remains unclear. We have previously reported Notch 1, 2, and 3 to be expressed in murine Leydig cells and that inhibition of Notch signaling caused G0/G1 arrest in TM3 Leydig cells. Methods: In this study, we have further explored the effect of different Notch signal pathways on key steroidogenic enzymes in murine Leydig cells. TM3 cells were treated with Notch signaling pathway inhibitor MK-0752, and different Notch receptors were also overexpressed in TM3 cells. Results: We evaluated the expression of key enzymes of steroid synthesis, including p450 cholesterol side-chain cleavage enzyme (P450Scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and steroidogenic acute regulatory protein (StAR), and key transcriptional factors for steroid synthesis, including steroidogenic factor 1 (SF1), GATA-binding protein 4 (GATA4) and GATA6. Conclusion: We found the level of P450Scc, 3β-HSD, StAR and SF1 to be decreased after treatment with MK-0752, while overexpression of Notch1 up-regulated the expression of 3β-HSD, P450Scc, StAR and SF1. MK-0752 and overexpression of different Notch members had no influence on the expression of GATA4 and GATA6. In conclusion, Notch1 signaling may contribute to the steroid synthesis in Leydig cells through regulating SF1 and downstream steroidogenic enzymes (3β-HSD, StAR and P450Scc).

Background: Gaucher disease (GD) is an autosomal recessive lysosomal storage disease. The disease develops due to glucocerebrosidase enzyme deficiency caused by biallelic pathogenic variants in the glucosylceramidase beta 1 (GBA1) gene, which encodes the glucocerebrosidase enzyme. The GBA1 gene is located at chromosomal location 1q22 and consists of 11 exons. In this article, we report a novel pathogenic variant in the GBA1 gene. Case Presentations: A 32-year-old female patient with no known chronic disease was admitted with complaints of weakness, bone pain, and abdominal pain. Her evaluation included hepatosplenomegaly, thrombocytopenia, osteoporosis, and anemia. The clinical suspicion of Gaucher disease was confirmed by glucocerebrosidase enzyme level and genetic testing. In her family screening, her sister also had hepato-splenomegaly, osteoporosis, thrombocytopenia, and anemia. Both sisters had no neurological symptoms. As a result of GBA1 gene sequence analysis in two of our patients, a missense variant was detected in the c.593C > A homozygous genotype. This variant has not been reported in any previously published case. Conclusion: In this case report, we aimed to contribute to the literature by reporting a new novel pathogenic variant in the GBA1 gene leading to type 1 Gaucher disease that has not been described before.