Enhanced Kidney Damage Induced by Increasing Nonylphenol Doses: Impact on Autophagy-Related Proteins and Proinflammatory Cytokines in Rats.

Nonylphenol (NP) is an organic pollutant and endocrine disruptor chemical that has harmful effects on the environment and living organisms. This study looked at whether kidney tissues subjected to increasing doses of nonylphenol generated alterations in histopathologic, pro-inflammatory, and autophagic markers. Fifty rats were divided into five groups of ten each: group I: healthy group, II: control (corn oil), group III: 25 µl/kg NP, group IV: 50 µl/kg NP, group V: 75 µl/kg NP. The kidney tissue samples were obtained for histopathological, immunohistochemical, and biochemical analyses. The histological deteriorations observed in all NP groups included tubular epithelial cell degeneration, inflammation areas, and hemorrhage. The immunohistochemical investigations showed that NP significantly elevated the autophagy markers (Beclin-1, LC3/2, p62), pro-inflammatory cytokines (TNF-α, IL-6), HIF-1α, and eNOS in group III, IV and V compared with group I and II. The biochemical analysis also revealed that pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) increased in correlation with the NP doses, but only IL-1ß reached statistical significance in NP treated rats kidney tissue. The biochemical findings have been confirmed by the histological studies. The damage to renal tissue caused by NP exposure may worsen it by increasing inflammatory and autophagic markers.

Cytotoxicity screening of Thymus vulgaris L. in breast cancer: in vitro study.

Breast cancer is one of the leading causes of cancer-related deaths due to its aggressive course. There is an increasing need for alternative therapy strategies, including herbal medications, to treat the disease because of its high incidence. Medicinal plants, such as Thymus vulgaris L. (T. vulgaris), have recently attracted great interest due to the antitumor properties of their extracts. The purpose of this investigation was to ascertain whether T. vulgaris had any cytotoxic effects on two different breast cancer cell lines. MTT test was applied to evaluate the effect of T. vulgaris on cell viability. TUNEL method was used to determine its apoptotic effect. LC3 and Beclin-1 expression levels were determined by immunofluorescence staining method and its autophagic effect was evaluated. Our findings demonstrate that T. vulgaris greately lowers proliferation, both in terms of concentration and duration. Consistent with decreased proliferation, an increase in apoptotic and autophagic cell death were also observed. The migration capacity of MCF-7 and MDA-MB-231 breast cancer cells was greatly suppressed by T. vulgaris, while significantly reducing colony formation. This study is the first to look into how T. vulgaris methanol extract affects breast cancer cells. All of these findings demonstrate that T. vulgaris prevents breast cancer cells from developing a malignant phenotype. It is possible to say that the methanol extract of T. vulgaris is suitable for the treatment of breast cancer, including aggressive types. However, in vivo research should support these results.

Semicarbazides Carrying Indole Core: Synthesis, Cytotoxicity Evaluation against Human Breast Cancer Cell Lines, and Molecular Modeling Studies.

In this article, we report the synthesis and cytotoxicity evaluation of novel indole-carrying semicarbazide derivatives (IS1-IS15). The target molecules were obtained by the reaction of aryl/alkyl isocyanates with 1H-indole-2-carbohydrazide that was in-house synthesized from 1H-indole-2-carboxylic acid. Following structural characterization by 1 H-NMR, 13 C-NMR, and HR-MS, IS1-IS15 were investigated for their cytotoxic activity against human breast cancer cell lines, MCF-7 and MDA-MB-231. According to the data obtained from the MTT assay, phenyl ring with a lipophilic group at its para-position and alkyl moiety were preferential substituents on the indole-semicarbazide scaffold for antiproliferative activity. The effect of IS12 (N-(4-chloro-3-(trifluoromethyl)phenyl)-2-(1H-indole-2-carbonyl)hydrazine-1-carboxamide), the compound that demonstrated remarkable antiproliferative activity on both cell lines, was also evaluated on the apoptotic pathway. Moreover, the calculation of critical descriptors constituting drug-likeness confirmed the position of the selected compounds in the anticancer drug development process. Finally, molecular docking studies suggested the inhibition of tubulin polymerization as the potential activity mechanism of this class of molecules.

The effect of different doses of nonylphenol on the blood-testicular barrier integrity, hormone level, and DNA damage in the testes of rats.

Determining the molecular characteristics of the damage caused by NP exposure in the testis is very important for understanding the source of the damage and developing treatment methods accordingly. Therefore, in this study, it is aimed to evaluate the toxic effects that different doses of NP may cause in the testis, including blood-testicular barrier integrity and sperm DNA damage. For this purpose, 50 adult male Wistar albino rats were used in the study. Low, medium, and high-dose NP groups and the corn oil group were formed. After NP administration at determined doses for 15 days, the testis tissue taken under anesthesia was fixed in formaldehyde. Paraffin blocks were embedded using the routine histological tissue follow-up method. Histopathological and immunohistochemical analyses were performed by taking 5 µm thick sections from paraffin blocks. The other testicular tissue was taken for the Western blot, Elisa, and comet methods, and the findings of sperm DNA analysis and the blood-testicular barrier were examined. NP caused the seminiferous epithelium to be disorganized and have significantly fewer cells in the testes of rats in different dose NP-induced groups. Compared with the control group, mTOR, Cx43, SCF, and HSP70 protein levels were decreased, while the expression of MMP-9 levels was increased in the different NP dose groups. Furthermore, tissue testosterone and inhibin B levels and SF-1 immunoreactivity intensity gradually decreased depending on the dose increase of NP. DNA damage of testicular tissues were increased in NP groups depending on NP dose. These results suggest that it is evident that NP, a commonly used industrial chemical, is an endocrine disrupting chemical (EDC) with estrogenic activity exerting adverse effects on health and that urgent measures are needed regarding the use.

Therapeutic potential of hesperidin: Apoptosis induction in breast cancer cell lines.

Hesperidin is a flavonoid commonly found in citrus fruits. Studies have shown that hesperidin has anti-inflammatory, analgesic, and antimicrobial properties, as well as its effectiveness in carcinogenesis. In this paper, we aim to investigate the molecular mechanisms of hesperidin-induced apoptosis in MCF-7 and MDA-MB-231 cancer cells. The inhibitory effect of hesperidin on cellular proliferation was evaluated with the MTT assay. Cell cycle analysis of hesperidin-treated cells was then performed, as well as immunocytochemical analysis of the effect on the apoptosis pathway (TUNEL, Bax, and Bcl-2 expression). Moreover, hesperidin induced cellular apoptosis in MCF-7 breast cancer cells by inhibiting Bcl-2 and enhancing Bax expression at protein levels. On the other hand, hesperidin caused apoptosis in the MDA-MB-231 breast cancer cell line, but it did not activate the Bax/Bcl-2 pathway. Hesperidin also induced cell cycle arrest at the S phase in the MCF-7 and MDA-MB-231 cell lines. These findings showed that hesperidin is a potential therapeutic candidate for preventing the progression of breast cancer. In addition, hesperidin could significantly stimulate the death mechanisms in ER/PR (+) MCF-7 cells by changing the expression balance of Bax and Bcl-2 proteins, but lead ER/PR (-) MDA-MB-231 breast cancer cells to apoptosis in a different way.

The Effect of Cyclosporine A on Proteins Controlling Intracellular Calcium Concentration in Breast Cancer Cells.

Cyclosporine A (CsA) is an immunosuppressive drug commonly used to prevent autoimmune diseases. At the same time, CsA is a calcineurin (CaN) inhibitor. It affects the intracellular calcium signaling pathway. The effect of CsA on breast cancer cells, MDA-MB-231, plasma membrane calcium pump 1 (PMCA1), calmodulin (CaM), calcineurin (CaN), and cMyc, which are proteins that affect calcium signaling, were investigated. CsA inhibited the proliferation of MDA-MB-231 cells but did not affect the migration of the cells. After 24 h of incubation, CsA suppressed the PMCA1 protein, which pumps intracellular calcium out of the cell. At the same time, calcium started to accumulate inside the cell and CaM protein was expressed, while PMCA1 was suppressed. The CaN protein was suppressed 72 h after the administration of CsA, but the cMyc protein was expressed. Interestingly, 24 h incubation when the PMCA1 protein is down-regulated after the duration of time, the cMyc protein is also down-regulated. Although the indirect effect of CaN and cMyc is known, this relationship between PMCA1 and cMyc was not known. As a result, it has been shown that CsA affects the PMCA pump by disrupting the intracellular calcium pathway in breast cancer cells.

Chromosomal and oxidative DNA damage in non-functioning pituitary adenomas.

INTRODUCTION: Clinically non-functioning pituitary adenomas (NFPA) are common tumours of the pituitary gland and are mainly considered as benign. The primary aim of this study was to research the effects of NFPA on genome instability in patients with non-functioning pituitary adenoma by using the cytokinesis-block micronucleus cytome (CBMN-cyt) assay and 8-hydroxy- 2'-deoxyguanosine (8-OHdG) assay. The second objective of this study was to assess whether there is a relationship between age, pituitary adenoma diameters, 8-OHDG levels, CBMN site assay parameters, and tumour aggressiveness. MATERIAL AND METHODS: The study was performed on 30 patients who had been diagnosed with NFPA and were admitted to the Department of Endocrinology and Metabolism, and 20 healthy subjects of similar age and sex. RESULTS: Micronucleus (MN), nucleoplasmic bridges (NPBs), nuclear bud (NBUD) frequencies, and apoptotic and necrotic cell frequencies in patients with NFPA were found to be significantly higher than in control subjects, and plasma 8-OHdG levels in patients with NFPA were statistically significantly lower than control subjects in this study. CONCLUSIONS: It is believed that this is the first study to evaluate the aggressiveness of tumour with chromosome/oxidative DNA damage in patients with NFPA. However, further studies are needed in order to understand the cause of NFPA aggression and to evaluate these patients in terms of risk of cancer.

Evaluation of chromosomal DNA damage, cytotoxicity, cytostasis, oxidative DNA damage and their relationship with endocrine hormones in patients with acute organophosphate poisoning.

Pesticides are commonly used compounds in agriculture. Especially, organophosphates (OPs) are among the extensively used pesticides. Therefore, OPs poisoning is common, especially in underdeveloped and developing countries. Primary aim of this study was to research the effects of acute OPs poisoning on genome instability in the individuals' lymphocytes with acute OPs poisoning both by using the cytokinesis-block micronucleus cytome (CBMN-cyt) assay to examine chromosome/genome damage, cell proliferation index and cell death rate and by using the plasma 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels to determine oxidative DNA damage. Secondary aim of this study was also to assess whether a relation exists between endocrine hormones and the genome damage in acute OPs poisoning. In the study, blood samples were analysed of 13 patients before and after treatment admitted to the Department of Intensive Care Unit with acute OPs poisoning and of 13 healthy subjects of similar age and sex. The present study demonstrates that genome damage (micronucleus; MN and nucleoplasmic bridges; NPBs frequencies), apoptotic and necrotic cell frequencies increased in lymphocytes of patients with acute OPs poisoning before treatment and decreased after treatment. The present study also show that CBMN cyt assay parameters and 8-OHdG levels could be affected by some endocrine hormones such as E2, fT3, fT4, GH, IGF-1, FSH, LH, TSH, PRL, but not be related to ACTH and tT levels in acute OPs poisoning. In conclusion, it is believed that this is the first study to evaluate the chromosomal/oxidative DNA damage, cell proliferation, cell death and their associations with endocrine hormones in acute OPs poisoning. These preliminary findings need to be supported by further studies with larger sample sizes.

Increased Chromosomal and Oxidative DNA Damage in Patients with Multinodular Goiter and Their Association with Cancer.

Thyroid nodules are a common clinical problem worldwide. Although thyroid cancer accounts for a small percentage of thyroid nodules, the majority are benign. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) levels are a marker of oxidative stress and play a key role in the initiation and development of a range of diseases and cancer types. This study evaluates cytokinesis-block micronucleus cytome (CBMN-cyt) assay parameters and plasma 8-OHdG levels and their association with thyroid nodule size and thyroid hormones in patients with multinodular goiter. The study included 32 patients with multinodular goiter and 18 age- and sex-matched healthy controls. CBMN-cyt assay parameters in peripheral blood lymphocytes of patients with multinodular goiter and controls were evaluated, and plasma 8-OHdG levels were measured. The micronucleus (MN) frequency (chromosomal DNA damage), apoptotic and necrotic cells (cytotoxicity), and plasma 8-OHdG levels (oxidative DNA damage) were significantly higher among patients with multinodular goiter. Our study is the first report of increased chromosomal and oxidative DNA damage in patients with multinodular goiter, which may predict an increased risk of thyroid cancer in these patients. MN frequency and plasma 8-OHdG levels may be markers of the carcinogenic potential of multinodular goiters and could be used for early detection of different cancer types, including thyroid cancer.

Evaluation of chromosomal damage, cytostasis, cytotoxicity, oxidative DNA damage and their association with body-mass index in obese subjects.

Over-weight and obesity are serious problems that increase the risk not only for chronic diseases like diabetes and heart disease but also of various types of cancer. This study was conducted to evaluate cytokinesis-block micronucleus cytome (CBMN-cyt) assay parameters and plasma concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG), and their relationship with age, body-mass index (BMI) and waist-to-hip ratio (WHR) in 83 obese, 21 over-weight and 21 normal-weight subjects. Frequencies of micronuclei (MN), nucleoplasmic bridges (NPB), nuclear buds (NBUD), and apoptotic and necrotic cells in lymphocytes of obese subjects were found to be significantly higher than those found in normal-weight and over-weight subjects (p<0.01 and p<0.05), whereas plasma concentrations of 8-OHdG in obese subjects were lower than those observed in normal-weight and over-weight subjects (p<0.05 and p<0.01, respectively). There was a negative correlation between age and frequency of necrotic cells and NDI (p<0.05), whereas there was no correlation between BMI, WHR, CBMN cyt assay parameters and plasma 8-OHdG in normal-weight subjects. In over-weight subjects, a negative correlation was observed between age and NDI (p<0.01) and a positive correlation between age and frequency of NPB (p<0.01) and between BMI and frequency of NBUD (p<0.05). In obese subjects, a negative correlation was observed between age and NDI (p<0.01) and between BMI and NDI (p<0.05), whereas no correlation was observed between WHR and CBMN-cyt assay parameters and plasma 8-OHdG. However, frequencies of MN, NPB, NBUD, apoptotic and necrotic cells in total over-weight/obese (p<0.01/p<0.05) and all subjects (p<0.01) increased with increasing BMI. The increase in genomic damage (MN, NPB and NBUD) in obese subjects and the positive correlation between genomic damage and BMI in total over-weight/obese subjects indicate that obesity increases genomic damage and may be associated with an increased risk of cancer, because an increase in MN frequency is a predictor of cancer risk.

Increased genome instability and oxidative DNA damage and their association with IGF-1 levels in patients with active acromegaly.

OBJECTIVE: The objectives of this study were to assess cytokinesis-block micronucleus cytome (CBMN Cyt) assay parameters and also oxidative DNA damage in patients with active acromegaly and controls and to assess the relationship between age, serum insulin-like growth factor 1 (IGF-1) levels, pituitary adenoma diameters, 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and CBMN Cyt assay parameters in patients with active acromegaly. DESIGN: The study population included 30 patients with active acromegaly and 30 age- and sex-matched healthy controls. CBMN Cyt assay parameters in peripheral blood lymphocytes of patients with active acromegaly and controls were evaluated and plasma 8-OHdG levels were measured. RESULTS: Frequencies of micronucleus (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in lymphocytes of patients with acromegaly were found to be significantly higher than those in controls (p<0.001, p<0.001, p<0.001, respectively). The frequencies of apoptotic and necrotic cells in lymphocytes of patients with acromegaly were found to be significantly higher than those in controls (p<0.001 and p<0.001 respectively). No statistically significant differences in the number of cells in metaphase, the number of bi-nucleated cells (M2), the number of tri-nucleated cells (M3), the number of tetra-nucleated cells (M4) and nuclear division index (NDI) values were observed between patients and controls (p>0.05). Plasma 8-OHdG (ng/ml) levels in patients with acromegaly were found to be significantly higher than those in controls (p<0.005). MN frequency in the lymphocytes of patients with acromegaly increased with elevated serum IGF-1 levels (p<0.05), whereas the number of NPBs and the frequency of apoptotic cells decreased with elevated serum IGF-1 levels (p<0.01 and p<0.05 respectively). CONCLUSIONS: Both the increase in chromosomal/oxidative DNA damage and the positive association between MN frequency and serum IGF-1 levels may predict an increased risk of malignancy in acromegalic patients. Long-term follow-up of patients with acromegaly will be necessary to establish the degree of cancer risk in this population.

Investigation of genome instability in patients with non-alcoholic steatohepatitis.

AIM: To evaluate the occurrence of micronucleus (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in the mitogen-stimulated lymphocytes of patients with non-alcoholic steatohepatitis (NASH). METHODS: The study was performed in 25 (9 females, 16 males) patients newly diagnosed with NASH, and 25 healthy subjects of similar ages and genders were used as a control group. None of the controls was known to be receiving any drugs for medical or other reasons or using alcohol. Hepatosteatosis was further excluded by abdominal ultrasound imaging in the control group. The numbers of MN, NPBs and NBUDs scored in binucleated (BN) cells were obtained from the mitogen-stimulated lymphocytes of patients and control subjects. Statistical comparisons of the numbers of BN cells with MN, NPBs and NBUDs and ages between the patients with NASH and control subjects were performed. RESULTS: The mean ages of the patients and the control group were 41.92 ± 13.33 and 41.80 ± 13.09 years (P > 0.05), respectively. The values of the mean body mass index (BMI), HOMA-IR, hemoglobin, creatinin, aspartate aminotransferase, alanine aminotransferase, triglyceride, high density lipoprotein, and low density lipoprotein were 31.19 ± 4.62 kg/m(2) vs 25.07 ± 4.14 kg/m(2), 6.71 ± 4.68 vs 1.40 ± 0.53, 14.73 ± 1.49 g/dL vs 14.64 ± 1.30 g/dL, 0.74 ± 0.15 mg/dL vs 0.80 ± 0.13 mg/dL, 56.08 ± 29.11 U/L vs 16.88 ± 3.33 U/L, 92.2 ± 41.43 U/L vs 15.88 ± 5.88 U/L, 219.21 ± 141.68 mg/dL vs 102.56 ± 57.98 mg/dL, 16.37 ± 9.65 mg/dL vs 48.72 ± 15.31 mg/dL, and 136.75 ± 30.14 mg/dL vs 114.63 ± 34.13 mg/dL in the patients and control groups, respectively. The total numbers and frequencies of BN cells with MN, NPBs and NBUDs, which were scored using the CBMN cytome assay on PHA-stimulated lymphocytes, were evaluated in the patients with NASH and control group. We found significantly higher numbers of MN, NPBs and NBUDs in the BN cells of patients with NASH than in those of the control subjects (21.60 ± 9.32 vs 6.88 ± 3.91; 29.28 ± 13.31 vs 7.84 ± 3.96; 15.60 ± 5.55 vs 4.20 ± 1.63, respectively, P < 0.0001). CONCLUSION: The increased numbers of MN, NPBs and NBUDs observed in the lymphocytes obtained from patients with NASH may reflect genomic instability.

Evaluation of genotoxicity, cytotoxicity and cytostasis in human lymphocytes exposed to patulin by using the cytokinesis-block micronucleus cytome (CBMN cyt) assay.

Patulin (PAT) is a fungal secondary metabolite commonly present in apples and apple products. In the present study, PAT was evaluated for its genotoxic, cytotoxic and cytostatic effects to human peripheral blood lymphocytes by using the cytokinesis-block micronucleus cytome (CBMN Cyt) assay. Lymphocyte cultures were treated with PAT at the following concentrations, 0.1, 0.3, 0.5, 1.0, 2.5, 5.0, and 7.5 µM, as well as 0.5 µM mitomycin c (MMC) as a positive control and dimethyl sulfoxide (DMSO) as a vehicle control. PAT was found to induce nucleoplasmic bridges (NPBs) at 5.0 and 7.5 µM concentrations (P < 0.05), apoptotic cells at 0.1, 1.0, 5.0 µM (P < 0.05), 7.5 µM concentrations (P < 0.01) and necrotic cells at 0.3 and 2.5 µM (P < 0.05), 5.0 and 7.5 µM (P < 0.01) concentrations in human lymphocytes. The 2.5, 5.0, and 7.5 µM PAT concentrations also led to a clear decrease in the nuclear division index (NDI) (P < 0.05). PAT caused a significant dose-dependent increase in the number cells of NPBs, in the frequency of apoptotic and necrotic cells, and a significant dose-dependent decrease in the NDI values in lymphocytes. These results indicate that PAT at high concentrations is genotoxic, cytotoxic and cytostatic in cultured human lymphocytes.

Evaluation of the genotoxicity and cytotoxicity in the general population in Turkey by use of the cytokinesis-block micronucleus cytome assay.

The cytokinesis-block micronucleus cytome (CBMN-Cyt) assay was originally developed as an ideal system for measuring DNA damage, cytostasis and cytotoxicity. The objective of the present study is to simultaneously evaluate the background levels of micronuclei (MN), nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (necrosis or apoptosis) and nuclear division index (NDI) in the peripheral blood lymphocytes of non-occupationally exposed, healthy subjects living in the city of Kayseri in Turkey. We used the CBMN-Cyt assay, taking into account factors - age, gender, and smoking habits - that might affect MN frequency and also other CBMN-Cyt assay parameters. Ninety-six healthy subjects (48 female and 48 male) were selected with ages varying between 21 and 60 years. The parameters, except for the number of binucleated (BN) cells with NPBs, showed no statistically significant difference between smokers and non-smokers. There were significant differences between female and male groups in MN frequency (higher in females) and in the number of NPBs (lower in females), while the other parameters were not significantly different between genders. The correlations between years of age and MN frequency, number of NPBs and the frequency of necrotic cells were statistically significant, while the correlations between the years of age and the other parameters were not. The results of the correlation analysis between years of smoking and MN frequency were positive, although no statistically significant correlation was found between the years of smoking and the other parameters. Among the smokers, no correlation was found either between the pack-years of smoking and the parameters assessed in this group. The results of the present study provide evidence of increasing MN frequency, number of NPBs and frequency of necrotic cells with increasing age in the peripheral blood lymphocytes of healthy individuals and influencing MN frequency and number of NPBs by gender.

Micronucleus evaluation in mitogen-stimulated lymphocytes of patients with acromegaly.

Acromegaly is a syndrome characterized by a sustained elevation of circulating growth hormone and insulin-like growth factor-1 (IGF-1). Insulin-like growth factor-1 is a potent mitogen and has a role in the transformation of normal cells to malignant cells. This study aims to evaluate the spontaneous micronucleus (MN) frequency by using the cytokinesis-block MN assay to determine genetic damage in the lymphocytes of patients with acromegaly. The study was carried out in 20 patients who had active acromegaly and in 20 age- and sex-matched healthy controls. The MN values were measured in binucleated cells obtained from mitogen-stimulated lymphocytes of patients and control subjects. The distribution of binucleated cells with 1, 2, 3, or more MNs was also measured. We found significantly higher MN frequency values in the lymphocytes of acromegalic patients than in those of the control subjects (2.23 ± 0.68 vs 1.03 ± 0.54, P = .001). The MN frequency increased with increasing IGF-1 levels of acromegalic patients (P = .036, R = 0.47). We observed that the number of binucleated cells with 2 MNs was higher for the majority of patients with acromegaly than for control subjects. Furthermore, the receiver operating characteristic curve (area under the curve = 0.914, P < .0001) was calculated to assess the discriminative power of the MN frequency. Our results indicate that increased MN frequency in the lymphocytes of patients with acromegaly may reflect genomic instability and this increased MN frequency may be associated with elevated levels of circulating growth hormone and IGF-1.