Copper affects steroidogenesis and viability of human adrenocortical carcinoma (NCI-H295R) cell line in vitro.

Copper is an environmental risk factor, which has various effects on reproductive endocrinology. In this study human adrenocortical carcinoma (NCI-H295R) cell line was used as an in vitro biological model to study the effect of copper sulfate (CuSO4.5H2O) on steroidogenesis and cytotoxicity. The cell cultures were exposed to different concentrations (3.90, 62.50, 250, 500, 1000 µM) of CuSO4.5H2O and compared to control group (medium without CuSO4.5H2O). Cell viability was measured by the metabolic activity assay. Quantification of sexual steroid production directly from the medium was performed by ELISA assay. Following 48 h culture of NCI-H295R cell line in the presence of CuSO4.5H2O a dose-dependent depletion of progesterone release was observed even at the lower concentrations of CuSO4.5H2O. The lowest levels of progesterone were detected in groups with the higher doses (≥ 250 µM) of CuSO4.5H2O, which elicited significant cytotoxic action. Testosterone production decreased significantly, and this decline was more prominent in comparison to that of progesterone. The lowest release of testosterone was recorded at 1000 µM of CuSO4.5H2O. The cytotoxic effect of CuSO4.5H2O was evident at all concentrations used in the study. The presented data suggest that copper has detrimental effects on sexual steroid hormones and consecutively on reproductive physiology.

Effects of 4-nonylphenol on the steroidogenesis of human adrenocarcinoma cell line (NCI-H295R).

In this study, the human H295R adrenocarcinoma cell line was exposed to different concentrations (0.04, 0.2, 1.0, 2.5 or 5 µg/mL) of nonylphenol (NP) to investigate its impact on the inhibition or induction of the steroid hormones production during 48 h of in vitro culture. The hormone production was measured using ELISA kits. Results of this in vitro study suggest various effect of nonylphenol in relatively low concentrations on the selected steroid hormones production by the human H295R adrenocarcinoma cell line. The inhibiting impact on progesterone and androstenedione production was observed. The amount of progesterone was significantly decreased at 1.0, 2.5 and 5 µg/mL NP. Equally, the androstenedione production significantly decreased at 5 µg/mL NP. On the other hand, the amount of testosterone and 17ß-estradiol was induced after nonylphenol exposition. The significant increase of testosterone level was found out at treatment with 5 µg/mL NP. 17ß-estradiol production significantly increased at the doses of 2.5 and 5 µg/mL NP.

Endocrine disruptive effects of cadmium on steroidogenesis: human adrenocortical carcinoma cell line NCI-H295R as a cellular model for reproductive toxicity testing.

Cadmium (Cd) is a known endocrine disruptor with the ability to affect the production of hormones involved in the regulation of reproductive processes. In this study human adrenocortical carcinoma cell line NCI-H295R was used as an in vitro biological model to study the effect of cadmium (CdCl2) on steroidogenesis. The cell cultures were exposed to different concentrations of CdCl2 (1.90, 3.90, 7.80, 15.60, 31.20 and 62.50 µM) and compared to control (medium without CdCl2). Cell viability was measured by the metabolic activity (MTT) assay for estimation of mitochondria structural integrity. Quantification of sexual steroid production directly from aliquots of the medium was performed by enzyme linked immunosorbent assay (ELISA). Following 48 h culture of the cells in the presence of CdCl2 a concentration-dependent depletion in progesterone production was observed at the lower concentrations of CdCl2. The lowest amount of progesterone was significantly detected in groups with the higher doses (≥ 31.20 µM) of CdCl2, which elicited significant (P < 0.01) cytotoxic action, too. Cadmium decreased testosterone release in the whole applied range even at the lower concentration of CdCl2. The release of 17ß-estradiol decreased as well, but the decline was less pronounced compared to decrease of progesterone and testosterone. The cytotoxic effect was significantly (P < 0.01) detected at all concentrations of CdCl2 (1.90-62.50 µM) used in the study. However, the cell viability remained relatively high (>75%) up to 7.80 µM of CdCl2 and significantly (P < 0.01) decreased at 15.60 µM and higher concentrations of CdCl2. These results suggest that cadmium has endocrine disruptive effects on sexual steroid synthesis even at very low concentrations.

Dose- and time-dependent effects of bisphenol A on bovine spermatozoa in vitro.

The present study was to evaluate the effect of bisphenol A (BPA) at the doses 1, 10, 100 and 200 µg mL(-1) on the bovine spermatozoa motility, viability and production of superoxide radical. The CASA system was used to determine the spermatozoa motility. The initial motility showed the significant differences (P < 0.001) between the groups higher than 100 µg BPA mL(-1) and the control group. Evaluation of the spermatozoa motility after 6 h of cultivation at the doses > 10 µg BPA mL(-1) was found to decrease motility significantly. After 24 h it was observed that the doses < 10 µg BPA mL(-1) statistically increased motility, while the doses > 100 µg BPA mL(-1) significantly decreased motility in comparison to control. The viability of spermatozoa as detected by the MTT assay decreased in all experimental groups, but significant differences were noted only at the highest doses of BPA after 24 h of in vitro cultivation. The intracellular superoxide production was observed by the NBT test after 24 h of BPA exposure. The results indicated that in all experimental groups the amount of superoxide increased as compared to the control group; significant changes were observed at the doses > 100 µg BPA mL(-1). In conclusion, the results from our experiments suggest the negative effects of BPA at the highest doses used on motility and viability of bovine spermatozoa and production of superoxide radical.

Blood Concentration of Macro- and Microelements in Women Who Are Overweight/Obesity and Their Associations with Serum Biochemistry.

Risk elements in blood matrices can affect human health status through associations with biomarkers at multiple levels. The aim of this study was to analyze 15 macro- and microelements in the blood serum of women with overweight (BMI of ≥25 kg/m2) and obesity (BMI of ≥30 kg/m2) and to examine possible associations with biochemical, liver enzymatic parameters, and markers of oxidative stress. Based on the power calculation, the study involved women (in the postmenopausal stage) with overweight (n = 26) and obesity (n = 22), aged between 50-65 years. Multifrequency bioelectrical impedance analysis was used to measure body composition parameters. Concentrations of elements were determined by inductively coupled plasma optical emission spectrometry, and Hg was measured using cold-vapor atomic absorption spectroscopy. Individuals with obesity, as indicated by a higher BMI, percentage of body fat, and visceral fat area, had elevated serum levels of Ca, Mg, Fe, Al, Sr, Pb, and Hg. Concentrations of Al, Cu, K, Sb, Zn, and Pb significantly affected biochemical and liver function markers in women with overweight or obesity. Elements such as Cu and Al were associated with increased total cholesterol. The correlation analysis between total antioxidant status and Cu, Al, and Ni confirmed associations in both groups. Our findings underscore the importance of addressing excess body weight and obesity in relation to risk elements. The results of the research could be beneficial in identifying potential targets for the treatment or prevention of comorbidities in people with obesity.

The effect of nonylphenol on the motility and viability of bovine spermatozoa in vitro.

The objective of this in vitro study was to determine the effect of nonylphenol (NP) as an environmental toxicant on the spermatozoa motility and viability. The dose- and time-dependent effect of nonylphenol (1, 10, 100 and 200 µg/mL) dissolved either in 0.1% dimethyl sulfoxide (DMSO) or 0.1% ethanol (ETOH) on the motility and viability of bovine spermatozoa, as a cell model, during several time periods (0 h, 2 h, 4 h and 6 h) were examined. The motility of spermatozoa was determined by the Sperm Vision(TM) CASA (Computer Assisted Semen Analyzer) system. The results showed decreased spermatozoa motility in all experimental groups with the addition of NP dissolved in 0.1% DMSO and 0.1% ETOH (P < 0.001 and P < 0.05). The lowest spermatozoa motility was found at doses > 100 µg/mL of NP in comparison with the control group. The viability of bovine spermatozoa detected by the MTT cytotoxicity assay was decreased significantly (P < 0.001) in all experimental groups with NP dissolved in 0.1% ETOH. The viability in groups with NP dissolved in 0.1% DMSO was significantly (P < 0.05) decreased at 1 µg/mL of NP and significantly decreased (P < 0.001) at doses > 10 µg/mL of NP. After 6 h of culture the MTT assay proved a negative effect of all NP doses the on cell viability. The obtained data clearly indicate the negative effect of NP as an endocrine disruptor on spermatozoa motility and viability, which should be seriously considered in the case of exposure to NP in animals and humans and as a reason of male reproductive dysfunction.

Effects of mercury on the steroidogenesis of human adrenocarcinoma (NCI-H295R) cell line.

In this study the NCI-H295R human adrenocortical carcinoma cell line was used as an in vitro biological model to study the effect of mercury (HgCl(2)) on the steroidogenesis. The cells were cultured for 48 h with addition of 1.0; 5.0; 25; 50 or 100 µM of HgCl(2) and compared to control. Cell viability was measured by the MTT (metabolic activity) assay estimation for the mitochondria structural integrity. Quantification of testosterone and progesterone directly from aliquots of the medium was performed by enzyme linked immunosorbent assay (ELISA). Concentration-dependent depression in testosterone production was detected particularly for higher concentration of Hg(2+). Progesterone production was also decreased, but at the lower concentrations (1.0 and 5.0 µM) of Hg(2+) this decline was lower compared to depression of testosterone. The cell viability significantly decreased at 25 µM and higher concentration of Hg(2+). However, at 25 µM Hg(2+) exposure the cell viability remained relatively high (> 80%). Results of the study indicate dose-dependent decreases in both testosterone and progesterone production of H295R cell culture following a 48 h in vitro HgCl(2) exposure. The results suggest that Hg has detrimental effects on steroid hormone synthesis also at very low concentrations and consecutively on reproductive physiology.

The impact of lead and cadmium on selected motility, prooxidant and antioxidant parameters of bovine seminal plasma and spermatozoa.

The aim of this study was to investigate the effects of lead (Pb) and cadmium (Cd) content on basic motility characteristics (motility-MOT, progressive motility-PROG) as well as selected markers of the prooxidant-antioxidant balance (catalase-CAT, glutathione-GSH, malondialdehyde-MDA) in bovine seminal plasma and spermatozoa. Twenty five semen samples were collected from breeding bulls and used in the study. Motility analysis was carried out using the Computer Assisted Sperm Analysis (CASA) system. The samples were centrifuged, fractions of seminal plasma and spermatozoa were separated, lysates were prepared from the sperm cell fractions. Pb and Cd concentrations were determined by the voltametric method (ASV), antioxidants and MDA were analyzed by UV/Vis spectrophotometry. The analysis showed that the average concentration of Pb in the seminal plasma was 0.23 ± 0.02 µg/mL, while its amount in the sperm cells was significantly higher (0.41 ± 0.07 µg/mL; P < 0.05). The average Cd content in bovine seminal fractions was similar and non-significant: 0.09 ± 0.01 µg/mL in the seminal plasma and 0.11 ± 0.01 µg/mL in spermatozoa (P > 0.05). The correlation analysis revealed that both heavy metals were significantly negatively correlated with MOT and PROG (P < 0.05; P < 0.01; P < 0.001), CAT (P < 0.05; P < 0.01) as well as GSH (P < 0.05; P < 0.01) but significantly positively associated with MDA as the marker of lipid peroxidation (P < 0.05; P < 0.01). Moreover the samples were categorized in three quality groups (Excellent, Good, Moderate) according to their motility values. The lowest Pb, Cd and MDA concentrations but the best antioxidant characteristics were found in samples of the best quality, moderate quality samples exhibited the highest Pb, Cd and MDA content together with the worst antioxidant capacity. This study demonstrates that Pb and Cd are serious toxic elements, which are able to increase the risk of seminal oxidative stress development and a subsequent decrease of male fertility.

Impacts of iron on ultrastructural features of NCI-H295R cell line related to steroidogenesis.

The current study was intended to evaluate impacts of both iron (Fe) enrichment and overload (in the form of ferrous sulphate heptahydrate, FeSO4.7H2O) on ultrastructural characteristics of human adrenocarcinoma NCI-H295R cell line. Here, the NCI-H295R cells were treated with 0, 3.90, and 1000 µM FeSO4.7H2O, and consequently proceeded for purposes of ultrastructural studies. Micrographs taken under transmission electron microscope (TEM) were investigated from the qualitative and quantitative (unbiased stereological approaches) aspects, and obtained findings were compared among the three groups of the cells. The ultrastructural features related to the steroidogenic process were found to be similar between the untreated and both Fe-exposed cell populations, with conspicuous mitochondria with well-defined lamellar cristae (creating clusters of varying sizes in the regions of increased energy demands) and concentric whorls of smooth endoplasmic reticulum (SER) being the most noticeable characteristics. The precise estimates of the component (volume, surface) fractions of the nucleus, mitochondria, and lipid droplets (LDs), as well as of the nucleus/cytoplasm (N/C) ratio have revealed close similarities (P > 0.05) in all cell groups investigated. Nonetheless, the low concentration of FeSO4.7H2O exhibited beneficial action on ultrastructural organization of the NCI-H295R cells. In effect, these cells were distinguished by mitochondria with smoother surfaces and clearer outlines, higher density of thin, parallel lamellar cristae (deeply extending into the mitochondrial matrix), and more widespread distribution of fine SER tubules as compared to the control ones, all of them suggesting higher level of energy requirements and metabolic activity, and more intensive rate of steroidogenesis. Interestingly, no obvious ultrastructural modifications were observed in the NCI-H295R cells treated with high FeSO4.7H2O concentration. This finding can be linked to either an adaptive ultrastructural machinery of these cells to cope with the adverse effect of the element or to insufficient dose of FeSO4.7H2O (1000 µM) to induce ultrastructural signs of cytotoxicity. Purposefully, the results of the current study complement our previous paper dealing with impacts of FeSO4.7H2O on the NCI-H295R cell viability and steroidogenesis at the molecular level. Hence, they fill a knowledge gap considering structure-function coupling in this cellular model system upon the metal exposure. This integrated approach can enhance our understanding of the cellular responses to Fe enrichment and overload which can be helpful for individuals with reproductive health concerns.

Selected heavy metals versus antioxidant parameters in bull seminal plasma - a comparative study.

To investigate the effects of lead (Pb) and cadmium (Cd) content on basic motility characteristics (motility, progressive motility) and selected antioxidant parameters (total antioxidant status - TAS, superoxide dismutase - SOD, albumin - ALB) in the bovine seminal plasma semen samples were collected from breeding bulls and used in the study. Motility analysis was carried out using the Computer Assisted Sperm Analysis (CASA) system. Subsequently, the samples were centrifuged and fractions of seminal plasma were collected. Pb and Cd concentrations were determined by the voltametric method (ASV), antioxidant parameters were analyzed by UV/VIS spectrophotometry using commercial kits. The analysis showed that the average concentrations of the trace elements were 0.57 ± 0.01 µg/mL for Pb and 0.11 ± 0.01 µg/mL for Cd. The correlation analysis revealed that both heavy metals were negatively correlated with motility (r = -0.777; P < 0.001 for Pb and r = -0.786; P < 0.001 for Cd), progressive motility (r = -0.763; P < 0.001 for Pb and r = -0.792; P < 0.001 for Cd), TAS (r = -0.375; p > 0.05 and r = -0.334; P > 0.05, respectively), SOD (r = -0.746; P < 0.001 and r = -0.537; P < 0.05, respectively) as well as with ALB (r = -0.609; P < 0.01 and r = -0.699; P < 0.001, respectively). Moreover the samples were categorized in three quality groups (Excellent, Good, Medium) according to their motility values. The lowest Pb and Cd concentrations but the best antioxidant characteristics were found in samples of excellent quality, medium quality samples were described by the highest Pb and Cd concentration and the worst antioxidant power. This study demonstrates that Pb and Cd are serious toxic elements, which are able to increase the risk of oxidative stress development and a subsequent decrease of semen quality.

Dose- and time-dependent effect of copper ions on the viability of bull spermatozoa in different media.

The purpose of this in vitro study was to determine the effect of copper (Cu) on the motility and viability of spermatozoa in the presence of different culture media. Specifically, we examined the dose- and time-dependent effect of copper ions (Cu(2+)) on the motility and viability of spermatozoa during different time periods (Time 0 h, 1 h, 24 h). The percentage of motile spermatozoa and progressive motile spermatozoa was determined after exposure to concentrations of 3.9; 7.8; 15.6; 31.2; 62.5; 125; 250; 500; 1000 µM/L of Cu(2+) using the Sperm Vision(TM) CASA (Computer Assisted Semen Analyzer) system. The cell viability was measured by the MTT (metabolic activity) assay. The initial spermatozoa motility in the presence of Cu(2+) in physiological saline solution (PS) showed slight increased values at doses <31.20 µM/L of Cu(2+) compared to the control group. The long-term cultivation (Time 24 h) reduced the average motility values in all experimental groups (P < 0.001) in comparison to the control group. Identical spermatozoa motility was detected for the percentage of progressive motile spermatozoa during all time periods. The culture medium containing 20 % bovine serum albumin (BSA), triladyl and 5 % glucose increased the overall percentage of spermatozoa motility after 1 h of cultivation. A concurrently maintained motility of spermatozoa at doses <62.50 µM/L of Cu(2+) during the long-term in vitro cultivation confirms the protective effect of albumin. The cell viability was decreased significantly (P < 0.001) in all experimental groups with copper administration. The obtained data point out that Cu(2+) at high doses is a toxic element on the spermatozoa motility, which subsequently disrupts the viability of cells. However, using a suitable culture medium containing an energy component- and protein-rich substrate, the spermatozoa motility could increase.

Human adrenocortical carcinoma cell line (NCI-H295R): An in vitro screening model for the assessment of endocrine disruptors' actions on steroidogenesis with an emphasis on cell ultrastructural features.

Cell lines as an in vitro model for xenobiotic screening and toxicity studies provide a very important tool in the field of scientific research at the level of molecular pathways and gene expression. Good cell culture practice and intracellular characterization, as well as physiological properties of the cell line are of critical importance for in vitro reproductive toxicity testing of various endocrine-disrupting chemicals. The NCI-H295R, human adrenocarcinoma cell line, is the most widely used in vitro cellular system to study the human adrenal steroidogenic pathway at the level of hormone production and gene expression, as it expresses genes that encode for all the key enzymes for steroidogenesis. In this review, we aim to highlight the information considering the origin, development, physiological and ultrastructural characteristics of the NCI-H295R cell line. The review also creates a broad overview of the cell line usage in various range of studies related to the steroidogenesis issues. To our best knowledge, the paper provides the first report of quantitative data (ex novo) from stereological estimates of component (volume, surface) densities of nuclei, mitochondria, and lipid droplets of the NCI-H295R cells. Such ultrastructural measurements can be valuable in the assessment of underlying mechanisms of changes in the cell steroid hormone production induced by the action of diverse endocrine disruptors. Thus, they can significantly contribute to complexity of structure-function relationships in association with steroidogenesis.

In vitro effects of radiofrequency electromagnetic waves on bovine spermatozoa motility.

In this study the effects of 1800 MHz GSM-like radiofrequency electromagnetic waves (RF-EMW) exposure on bovine semen was monitored. The experimental samples were analyzed in vitro in four time periods (0, 30, 120 and 420 min) and compared with unexposed samples (control). Spermatozoa motility was determined by computer assisted semen analyzer (CASA). Evaluation of the percentage of motile spermatozoa showed significant (P < 0.001) decrease in experimental groups after 120 and 420 min of culture when exposed to microwaves, in comparison to control. Similar spermatozoa motility inhibition was detected for the percentage of progressively motile spermatozoa, too. Average path distance decreased significantly (p < 0.001) in experimental groups after 30 and 420 min of culture. Path velocity increased in the experimental groups exposed to RF-EMW after 30 minutes of culture, but subsequently decreased after 420 min of culture, in comparison to control. This indicates a possible initial stimulation and subsequent velocity inhibition of bovine spermatozoa under RF-EMW exposure. Changes in spermatozoa motility were also detected for some fine parameters, too. A significant decrease (P < 0.001) was noted for amplitude of lateral head displacement in the experimental group after 420 minutes of culture. Detailed in vitro motility analysis of bovine spermatozoa exposed to microwave radiation suggested that the parameters of path and velocity at the beginning of the culture significantly increase, but after longer culture (420 minutes) a significant decrease occur in the experimental group as compared to control. In general, results of this experiment indicate a negative time-dependent effect of 1800 MHz RF-EMW radiation on bovine spermatozoa motility.

Impact of oxidative stress on male fertility - a review.

Oxidative stress is a state related to increased cellular damage caused by oxygen and oxygen-derived free radicals known as reactive oxygen species (ROS). It is a serious condition, as ROS and their metabolites attack DNA, lipids and proteins, alter enzymatic systems and cell signalling pathways, producing irreparable alterations, cell death and necrosis. While small amounts of ROS have been shown to be required for several functions of spermatozoa, their excessive levels can negatively impact the quality of spermatozoa and impair their overall fertilising capacity. These questions have recently attracted the attention of the scientific community; however, research aimed at exploring the role of oxidative stress and antioxidants associated with male fertility is still at its initial stages. This review summarises the current facts available in this field and intends to stimulate interest in basic and clinical research, especially in the development of effective methods for the diagnosis and therapy of semen damage caused by oxidative stress.

In vitro effect of ferrous sulphate on bovine spermatozoa motility parameters, viability and Annexin V-labeled membrane changes.

The aim of this study was to assess the dose- and time-dependent in vitro effects of ferrous sulphate (FeSO4.7H2O) on the motility parameters, viability, structural and functional activity of bovine spermatozoa. Spermatozoa motility parameters were determined after exposure to concentrations (3.90, 7.80, 15.60, 31.20, 62.50, 125, 250, 500 and 1000 µM) of FeSO4.7H2O using the SpermVisionTM CASA (Computer Assisted Semen Analyzer) system in different time periods. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, and the Annexin V-Fluos was applied to detect the membrane integrity of spermatozoa. The initial spermatozoa motility showed increased average values at all experimental concentrations compared to the control group (culture medium without FeSO4.7H2O). After 2 h, FeSO4.7H2O stimulated the overall percentage of spermatozoa motility at the concentrations of ≤ 125 µM. However, experimental administration of 250 µM of FeSO4.7H2O significantly (P < 0.001) decreased the spermatozoa motility but had no negative effect on the cell viability (P < 0.05) (Time 2 h). The lowest viability was noted after the addition of ≥ 500 µM of FeSO4.7H2O (P < 0.001). The concentrations of ≤ 62.50 µM of FeSO4.7H2O markedly stimulated (P < 0.001) spermatozoa activity after 24 h of exposure, while at high concentrations of ≥ 500 µM of FeSO4.7H2O the overall percentage of spermatozoa motility was significantly inhibited (P < 0.001) and it elicited cytotoxic action. Fluorescence analysis confirmed that spermatozoa incubated with higher concentrations (≥ 500 µM) of FeSO4.7H2O displayed apoptotic changes, as detected in head membrane (acrosomal part) and mitochondrial portion of spermatozoa. Moreover, the highest concentration and the longest time of exposure (1000 µM of FeSO4.7H2O; Time 6 h) induced even necrotic alterations to spermatozoa. These results suggest that high concentrations of FeSO4.7H2O are able to induce toxic effects on the structure and function of spermatozoa, while low concentrations may have the positive effect on the fertilization potential of spermatozoa.

Mitochondria: A worthwhile object for ultrastructural qualitative characterization and quantification of cells at physiological and pathophysiological states using conventional transmission electron microscopy.

Mitochondria are highly dynamic intracellular organelles with ultrastructural heterogeneity reflecting the behaviour and functions of the cells. The ultrastructural remodelling, performed by the counteracting active processes of mitochondrial fusion and fission, enables the organelles to respond to diverse cellular requirements and cues. It is also an important part of mechanisms underlying adaptation of mitochondria to pathophysiological conditions that challenge the cell homeostasis. However, if the stressor is constantly acting, the adaptive capacity of the cell can be exceeded and defective changes in mitochondrial morphology (indicating the insufficient functionality of mitochondria or development of mitochondrial disorders) may appear. Beside qualitative description of mitochondrial ultrastructure, stereological principles concerning the estimation of alterations in mitochondrial volume density or surface density are invaluable approaches for unbiased quantification of cells under physiological or pathophysiological conditions. In order to improve our understanding of cellular functions and dysfunctions, transmission electron microscopy (TEM) still remains a gold standard for qualitative and quantitative ultrastructural examination of mitochondria from various cell types, as well as from those experienced to different stimuli or toxicity-inducing factors. In the current study, general morphological and functional features of mitochondria, and their ultrastructural heterogeneity related to physiological and pathophysiological states of the cells are reviewed. Moreover, stereological approaches for accurate quantification of mitochondrial ultrastructure from electron micrographs taken from TEM are described in detail.