镉对大鼠睾丸生化毒作用的研究

镉2.5mg/kg腹腔染毒2天后,大鼠血镉和睾丸镉含量明显升高,同时血浆丙二醛含量和乳酸脱氢酶同工酶(LDH-X)活性亦显著增高。在睾丸匀浆及线粒体丙二醛含量明显升高的同时,睾丸匀浆的LDH-X则随之明显降低。光镜检查间质内明显出血,曲细精管生精细胞萎缩性变性,数量减少。推测镉可诱发睾丸生精细胞线粒体膜的脂质过氧化作用。电镜检查也证实,染毒大鼠精原细胞线粒体大量膨胀、破损。     

三氧化二砷对大鼠睾丸细胞酶的作用

目的:探讨药理浓度的三氧化二砷(As2O3)对雄性大鼠血清睾丸酶及精子生成的影响.方法:不同剂量的As2O3(2mg·kg-1·d-1,4 mg·kg-1·d-1和8 mg·kg-1·d-1)给予大鼠腹腔内注射,2周后检测大鼠血清睾丸酶的含量,同时,测定大鼠血清砷的含量、睾丸脏器系数、睾丸精子头计数,并计算每日精子生成量.结果:中剂量组各种酶都有所下降,G-6-PD、LDH及LDH-X的减少有统计学意义(P＜0.01,P＜0.01和P＜0.05);大剂量组ACP、ALP、G-6-PD、LDH及LDH-X均明显下降(P＜0.01).随着给大鼠腹腔注射的As2O3浓度增加,血中砷浓度明显增加(P＜0.01).3种剂量组的睾丸脏器系数与对照组差异均无显著性(P＞0.05),大剂量组睾丸精子头数和每克睾丸每日精子生成量与对照组比较减少明显(P＜0.01),血清砷含量与大鼠精子头数之间呈直线负相关(r=-0.515,P＜0.01).结论:As2O3对睾丸酶的抑制,影响了睾丸精子的生成而导致雄性生殖毒性.     

短期大剂量益母草对大鼠肾脏的影响

目的观察短期大剂量益母草对大鼠肾脏的影响。方法取益母草煎液,以60g/(kg d)剂量给大鼠连续灌胃14天,给药后第8天、15天取大鼠尿、血和肾组织;检测尿N-乙酰-β-葡萄糖苷酶(NAG)、尿β2-微球蛋白(β2-MG)、尿蛋白(Upro)、血Cr、BUN等相关指标,并观察肾脏形态组织学变化。结果给药1周和2周后尿NAG、β2-MG、Upro及血BUN、Cr与对照组比较无显著性差异(P>0.05);大鼠肾组织未见明显病理改变。结论益母草在短时间内大剂量使用对大鼠肾脏未造成显著毒副作用。     

镉致大鼠血液、肝脏、肾脏和睾丸的毒性损伤

目的为研究镉对大鼠血液、肝脏、肾脏和睾丸的毒性损伤。12只SD大鼠分为对照组和镉处理组,每组6只,对照组自由饮用自来水,用0.9%的生理盐水腹腔注射,镉处理组用无菌生理盐水配制的Cd Cl2溶液腹腔注射,剂量为2 mg/kg·bw,注射容量均为10 ml/kg·bw,1次/d,共5周。实验结束,剖杀大鼠,采集血液,检测大鼠WBC、Hb、RBC、MCH、MCV、HCT和MCHC等血液参数;取大鼠肝脏、肾脏和睾丸并称重,计算脏器系数;匀浆组织,检测组织内MDA、GSH、GSH-PX、CAT和SOD含量。结果表明,与对照组相比,镉处理组大鼠RBC升高差异有统计学意义(P0.01),Hb、MCH、MCV和HCT均降低差异有统计学意义(P0.05或P0.01),MCHC差异则无统计学意义。镉组大鼠肝脏和肾脏的脏器系数升高(P0.05或P0.01),但大鼠睾丸的脏器系数与对照组比较降低(P0.01)。镉组肝脏、肾脏和睾丸中MDA均升高(P0.05或P0.01);肝脏、肾脏中GSH升高(P0.01),GSH-PX、SOD和CAT活性(P0.05或P0.01),但睾丸中这些指标与之相反,GSH含量降低(P0.01);GSH-PX、SOD和CAT活性升高(P0.05或P0.01),上述差异均有统计学意义。实验结果表明,镉可引起大鼠血液、肝脏、肾脏和睾丸的毒性损伤。     

农达染毒对30d龄雄性大鼠睾丸及附睾某些指标的影响

目的研究农达染毒对30 d龄雄性大鼠睾丸和附睾毒性、精子毒性及血清睾酮水平的影响,为农达的职业健康危险度评定提供科学依据。方法 30 d龄雄性SD大鼠40只,按体重随机分为4组,染毒低、中、高剂量组分别给250、500和1 000 mg/kg·bw的农达,连续灌胃30 d,对照组给予等量去离子水。观察各组大鼠一般情况,睾丸和附睾的脏器系数、病理学改变;精子总数、精子畸形率;用放免法测定各组大鼠血清睾酮含量。结果 1 000 mg/kg·bw染毒组大鼠睾丸和附睾系数明显下降(P0.01);染毒组大鼠生精小管上皮变薄,细胞层排列紊乱,生精细胞数目明显减少,附睾管腔中线状精子数减少;各染毒组大鼠精子总数均明显降低(P0.05),精子畸形率均明显升高(P0.05),精子畸形率与染毒剂量之间存在剂量-反应关系(P0.01);各染毒组大鼠血清睾酮含量均明显下降(P0.01)。结论 30 d龄雄性大鼠农达染毒可诱导其生殖毒性,表现为精子数量下降、畸形率增加和血清睾酮含量降低。     

实验性汞中毒时肾组织和尿中碱性磷酸酶活性的变化

本研究采用亚急性汞中毒肾损害的大鼠模型,探讨了汞中毒时血、肾和尿中碱性磷酸酶(ALP)活性的变化关系。结果表明,大鼠肾匀浆中 ALP活性明显低于对照组,尿 ALP活性则显著增加。ALP 活性降低的部位在肾近曲小管。体外实验未发现氯化汞对肾、尿ALP 具有直接抑制作用或激活作用。尿中ALP 活性的增高是汞引起的肾小管上皮细胞损伤所致。它可作为汞中毒肾损害的一个观察指标。     

洛伐他汀、辛伐他汀和阿托伐他汀对大鼠肝肾毒性的比较研究CSCD

目的观察洛伐他汀、辛伐他汀和阿托伐他汀连续给药1个月对大鼠肝肾的毒性作用,为临床用药安全性提供数据支持。方法SD大鼠分别灌胃给予洛伐他汀、辛伐他汀和阿托伐他汀(68.5和205.5 mg/kg·bw),给药1次/d,28 d后测血清中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、碱性磷酸酶(ALP)、肌酐(CRE)和尿氮(BUN)。取肝和肾,甲醛固定,苏木素-伊红(HE)染色,光学显微镜观察肝和肾组织的病理结构变化。结果辛伐他汀和阿托伐他汀的主要毒性反应剂量在205.5 mg/kg·bw,给药后大鼠出现了脱毛、腹泻等表现,血液生化检查显示ALP、ALT和AST明显升高。病理结果表明,辛伐他汀高剂量组、阿托伐他汀高剂量组和阿托伐他汀低剂量组对大鼠肝均有一定程度的毒性损伤作用。结论阿托伐他汀和辛伐他汀高剂量组可引起大鼠肝组织的病理损伤,ALP、ALT和AST明显升高;洛伐他汀高剂量组对大鼠肝肾毒性作用不明显。根据肝病理毒性由弱到强排序为:洛伐他汀高剂量<辛伐他汀高剂量组<阿托伐他汀低剂量组<阿托伐他汀高剂量组。     

松花粉对急性汞中毒大鼠肝肾的保护作用及机制研究

目的研究松花粉对急性汞中毒大鼠肝肾的保护作用及机制。方法将大鼠随机均分为正常对照组,染汞模型组,松花粉低、中、高剂量组(2、4、8 g/kg)。通过皮下注射HgCl_2建立急性汞中毒模型,末次染汞后,各组分别灌胃给予相应药物,2次/d,共5 d。通过电感耦合等离子体质谱仪,测定肝汞、肾汞、血汞和尿汞的含量。通过试剂盒,测定尿素氮(BUN)、尿蛋白、尿乳酸脱氢酶(LDH)、尿碱性磷酸酶(ALP),血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、血清总胆红素(TBIL),以及肝、肾中的超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽(GSH)和丙二醛(MDA)的含量或活性。结果与正常对照组比较,染汞模型组大鼠的肝汞、肾汞、血汞、尿汞、BUN、尿蛋白、尿LDH、尿ALP、血清ALT、血清AST、血清TBIL、肾脏MDA和肝脏MDA含量或活性均升高(P<0.01),肾脏和肝脏SOD、GSH-Px、GSH含量或活性降低(P<0.01)。与染汞模型组比较,松花粉低、中、高剂量组大鼠的尿汞含量增加(P<0.05或P<0.01),上述其余指标均能显著地向正常对照组的数值变化(P<0.05或P<0.01)。结论松花粉具有清除体内汞蓄积、修复急性汞中毒诱导的急性肝肾损伤的作用,其机制与抑制脂质过氧化损伤相关。     

镉诱发大鼠肾重吸收钙障碍的机理(英文)

给雄性Wistar大鼠sc不同剂量的镉金属硫蛋白(CdMT),结果表明镉接触组尿镉、尿钙和尿蛋白浓度都高于对照组,并与镉剂量间存在剂量效应关系,而血清游离钙浓度无变化;镉接触组肾皮质钠泵和钙泵活性低于对照组,体外试验结果也显示镉能抑制钙泵活性,谷胱甘肽(GSH)和半胱氨酸对这种抑制有保护作用}镉接触组肾皮质GSH含量低于对照组,而丙二醛(MDA)含量则高于对照组,高剂量组肾皮质cAMP/cGMP比值低于对照组。肾皮质钙泵活性、cAMP/cGMP比值、GSH含量三者都与尿钙浓度间呈负相关,MDA含量则与尿钙呈正相关,揭示镉引起的钙尿症是由肾重吸收钙障碍造成的,可能与镉引起的肾脏钠泵、钙泵、环核苷酸、GSH和脂质过氧化等改变有关。     

三硝基甲苯对雄性生殖毒性的研究

急性、亚急性和亚慢性TNT染毒大鼠睾丸铜锌含量和多种酶活性皆有所变化;睾丸和血清睾酮含量下降。大鼠辜丸游离间质细胞与TNT共同孵育时,睾酮形成量明显下降;间质细胞与TNT孵育后,活性氧与脂质过氧化阳性物质含量明显增加。补锌在一定程度上能拮抗TNT对大鼠的生殖毒性。横断面调查表明,TNT接触男工自觉性功能降低者增加,血清睾酮含量下降,精液常规检查有阳性所见。     

工业品六氯环己烷对小鼠睾丸和精子LDH-X的影响

采用组织化学-显微分光光度法和聚丙烯酰胺凝胶电泳法定量测定了工业品六氯环己烷染毒小鼠的精子、睾丸及附睾乳酸脱氢酶X同工酶(LDH-X)的含量,结果表现出明显下降并呈剂量-效应关系。提示工业品六氯环己烷能抑制小鼠生殖细胞LDH-X进而影响精子的能量代谢及运动能力。并对工业品六氯环己烷影响小鼠生殖细胞LDH-X的机理进行了讨论。     

Responses of testis,epididymis, and sperm of pubertal rats exposed to functionalized multiwalled carbon nanotubes

The present study investigated the response of testes, epididymides and sperm in pubertal Wistar rats following exposure to 0, 0.25, 0.5, 0.75, and 1.0 mg kg^?1 functionalized multi‐walled carbon nanotubes (f‐MWCNTs) for 5 days. The results showed that administration of (f‐MWCNTs) significantly increased the activities of superoxide dismutase, catalase, and glutathione peroxidase in a dose‐dependent manner in both testes and sperm compared with control group. Moreover, the significant decrease in the activity of glutathione‐S‐transferase and glutathione level was accompanied with significant elevation in the levels of hydrogen peroxide and malondialdehyde in both testes and sperm of (f‐MWCNTs)‐treated rats. The spermiogram of (f‐MWCNTs)‐treated rats indicated significant decrease in epididymal sperm number, sperm progressive motility, testicular sperm number and daily sperm production with elevated sperm abnormalities when compared with the control. Exposure to (f‐MWCNTs) decreased plasma testosterone level and produced marked morphological changes including decreased geminal epithelium, edema, congestion, reduced spermatogenic cells and focal areas of tubular degeneration in the testes. The lumen of the epididymides contained reduced sperm cells and there was mild to severe hyperplasia epithelial cells lining the duct of the epididymis. Collectively, pubertal exposure of male rats to (f‐MWCNTs) elicited oxidative stress response resulting in marked testicular and epididymides dysfunction. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 543–551, 2016.     

Triclosan exhibits a tendency to accumulate in the epididymis and shows sperm toxicity in male sprague‐dawley rats

Triclosan (TCS) is considered a potent endocrine disruptor that causes reproductive toxicity in non‐mammals, but it is still unclear exactly whether TCS has adverse effects on the sperm or reproductive organs in mammals. In this study, we aimed to evaluate the distribution status of TCS in male reproductive organs of rats, and seek the correlation with the TCS‐induced sperm toxicity or reproductive organ damage. Male rats were intragastrically administered with TCS at a dose of 50 mg/kg, the kinetics of TCS in the plasma and reproductive organs were investigated. TCS in testes and prostates both showed a lower‐level distritbution compared to that in the plasma, which indicates it has no tendency to accumulate in those organs. However, TCS in the epididymides showed a longer elimination half‐life (t_1/2z), a longer the mean retention time (MRT), and a lower clearance (CL_Z/F) compared with those in the plasma. Besides, the ratios of mean area under the concentration‐time curve (AUC)_{0–96h(epididymides/plasma)} and AUC_{0–∞(epididymides/plasma)} were 1.13 and 1.51, respectively. These kinetic parameters suggest TCS has an accumulation tendency in the epididymides. Based on this, we investigated the TCS‐induced sperm toxicity and histopathological changes of reproductive organs in rats. TCS was given intragastrically at doses of 10, 50, and 200 mg/kg for 8 weeks. Rats treated with the high dose (200 mg/kg) of TCS showed a significant decrease in daily sperm production (DSP), changes in sperm morphology and epididymal histopathology. Considering the histopathological change in the epididymides, TCS may induce the epididymal damage due to the epididymal accumulation of that. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 83–91, 2015.     

Toxic effects of cypermethrin on the male reproductive system: with emphasis on the androgen receptor

The 15‐day intact adult male assay was used to investigate the reproductive toxicity of cypermethrin. We also evaluated the contributions of the androgen receptor (AR) to cypermethrin‐induced reproductive impairments. Sixty adult male Sprague–Dawley rats were randomly divided into five groups and treated with different doses of cypermethrin (0, 6.25, 12.5, 25 and 50 mg kg^?1 per day) by oral gavage for 15 days. After the rats were sacrificed, the testes, epididymides, seminal vesicles and prostates were excised and weighed. One testis was frozen to be used for daily sperm production. Another testis was processed for AR immunohistochemical analysis and electron microscopic observation. We found that the weights of prostates were significantly decreased in cypermethrin treatment at doses of 25 and 50 mg kg^?1 per day. Rats treated with cypermethrin at 50 mg kg^?1 per day exhibited a significant reduction in testicular daily sperm production. Seminiferous tubule changes were noted, including atrophy and distorted seminiferous tubules, reduction and deformation of spermatogonia, spermatocyte and disordered arrangement of spermatoblasts. Ultrastructural changes were found in cypermethrin‐treated groups with disrupted cellular junctions, abnormal morphology of the nucleus, necrosis of spermatogonia spermatocytes and Sertoli cells. To clarify the possible mechanism, AR expression and the serum levels of testosterone were assayed. AR levels were significantly reduced in the rats treated with cypermethrin and the serum levels of testosterone were reduced in cypermethrin treatment at a dose of 50 mg kg^?1 per day. These data suggested that cypermethrin can induce impairments of the structure of seminiferous tubules and spermatogenesis in the male rats. The impairments can be attributed to the reduced AR expression. Copyright © 2011 John Wiley & Sons, Ltd.     

Protective effect of vitamin E on sperm motility and oxidative stress in valproic acid treated rats

Long-term administration of valproic acid (VPA) is known to promote reproductive impairment mediated by increase in testicular oxidative stress. Vitamin E (VitE) is a lipophilic antioxidant known to be essential for mammalian spermatogenesis. However, the capacity of this vitamin to abrogate the VPA-mediated oxidative stress has not yet been assessed. In the current study, we evaluated the protective effect of VitE on functional abnormalities related to VPA-induced oxidative stress in the male reproductive system. VPA (400 mg kg⁻¹) was administered by gavage and VitE (50 mg kg⁻¹) intraperitoneally to male Wistar rats for 28 days. Analysis of spermatozoa from the cauda epididymides was performed. The testes and epididymides were collected for measurement of oxidative stress biomarkers. Treatment with VPA induced a decrease in sperm motility accompanied by an increase in oxidative damage to lipids and proteins, depletion of reduced glutathione and a decrease in total reactive antioxidant potential on testes and epididymides. Co-administration of VitE restored the antioxidant potential and prevented oxidative damage on testes and epididymides, restoring sperm motility. Thus, VitE protects the reproductive system from the VPA-induced damage, suggesting that it may be a useful compound to minimize the reproductive impairment in patients requiring long-term treatment with VPA.     

A new approach for assessment of narcotic physical dependence using urinary sex-dependent low molecular weight proteins in male rats

Attempts have been made to examine the relationship between urinary excretion of sex-dependent low molecular weight proteins found only in male rats (LMWP) and morphine physical dependence. Chronic administration of morphine produced a dose-related decrease in urinary LMWP excretion, which was correlated to the intensity of withdrawal signs including body weight loss and abnormal behaviors recognized after naloxone challenge. Furthermore, a statistically high correlation was obtained between the decrease in urinary LMWP excretion and the loss of body weight precipitated by naloxone challenge. LMWP was identified immunologically in the livers, kidneys, and sera using an antibody against purified LMWP. The serum level of LMWP was increased rapidly following bilateral nephrectomy. After chronic treatment with morphine, the LMWP content in the livers, kidneys, and sera were decreased. These findings indicate that the decrease in urinary LMWP excretion induced by chronic administration of morphine can be a useful parameter to assess the development of physical dependence on narcotics on the peripheral level without requiring drug withdrawal and naloxone challenge. This decrease in urinary LMWP may be caused by the inhibition of LMWP synthesis in the liver.     

Toxic effects of Microcystis cell extracts on the reproductive system of male mice

In this paper, the reproductive toxicity of male mice treated with Microcystis aeruginosa cell extracts containing microcystins was examined. In contrast to the control group, male mice exposed intraperitoneally to 3.33 or 6.67 μg microcystins/kg body weight for 14 days had decreased mean body weight, and the mean absolute weight of the testes and epididymides was decreased. However, the mean relative weight of the testes increased compared to the controls. In addition, histological examination of microcystin-treated mice indicated that the testes were damaged and the space between the seminiferous tubules was more pronounced compared to control mice. The quality of mature sperm in the seminiferous tubules was also decreased in treated mice compared with the control group. Further studies showed that motility and viability of the sperm from microcystin-treated mice were reduced, but no significant difference was found in the concentration and abnormality of the sperm from treated mice compared to the control. This study indicated that microcystins had numerous toxic effects on the reproductive system of male mice.     

Age-related dose response of selected reproductive parameters to acute cadmium chloride exposure in the male Long-Evans rat

Groups of male Long-Evans rats 30, 50, or 70 d old were injected subcutaneously (sc) with a single dose of 0, 5.5, 11.5, or 24.6 mumol Cd/kg as cadmium chloride. All animals were killed 60 d after treatment. At 2 h prior to sacrifice, the rats were injected sc with 100 IU human chorionic gonadotrophin (hCG) to maximally stimulate serum testosterone concentrations. After sacrifice the testes, epididymides and seminal vesicles were removed and weighed. Cardiac blood was taken, and serum concentrations of testosterone (sT) and follicle-stimulating hormone (sFSH) were determined. Sperm concentration in luminal fluid collected from the vas deferens was determined. Significant (p less than 0.01) dose-dependent effects for all measured reproductive parameters were noted in the 70-d-old animals, while no effects were seen in the 30- or 50-d-old rats in either seminal vesicles weight or hCG-stimulated sT concentration. In the absence of significant (p greater than 0.05) changes in body weight gain, effects were seen in testes and epididymides weight, sperm concentration, and sFSH in the 70-d-old rats at Cd doses that were lower than those necessary to bring about similar changes in the 30- or 50-d-old animals. The sensitive indicators of Cd exposure in all age groups were testicular weight greater than epididymal weight greater than vas deferens sperm concentration greater than sFSH concentration. Seminal vesicle weight and sT concentration were found to be the least sensitive. Regression analyses indicated a significant interaction of age with dose; the 70-d-old rats required 30-61% less Cd/kg to cause a 50% change in a measured parameter than did the 30-d-old animals, while the 50-d-old rats required 15-47% less.     

Reproductive and cytogenetic toxicity of metronidazole in male mice

The purpose of this study was to examine the effects of metronidazole (500 mg/kg b.wt. daily by gavage for 14 consecutive days) on male fertility, haematopoiesis and genotoxic affinity. Mature male Swiss mice were treated with metronidazole and divided into 3 groups each with 10 animals, examined after 2 weeks, 1 and 2 months from the onset of drug administration. The results demonstrated that metronidazole significantly (P<0.05) decreased the weight of the testes, epididymides and accessory sexual organs (seminal vesicles and prostates) after one month from the onset of treatment. While accessory sexual organ weights were restored after 2 months from onset of treatment, the decrease in testes and epididymides weights persisted until 2 months later. The deleterious effects of metronidazole on reproductive organ weights might be due to a decrease in testosterone level after 2 weeks, and 1 and 2 months from the onset of treatment. Metronidazole induced a significant decrease in motile sperm and an increase in abnormal sperm after 1 month. The viability of sperm was normal after 2 months. Metronidazole induced anaemia characterized by decreased erythrocyte and leukocytic counts, haemoglobin content and haematocrit %. The ability of oral metronidazole administration to induce genotoxic damage in somatic cells of mice was evaluated using mitotic index, micronuclei and chromosomal aberration. A significant reduction in mitotic activity was observed two weeks from the onset of drug administration, restoration occurred after one month. A significant and persistence increase in the frequency of chromosomal aberration and micronucleus was observed at all periods of the experiment. In conclusion, the results of this study indicate that 1) metronidazole (500 mg/kg by gavage) for 14 days caused a harmful effect on male fertility in mice after one and two months from start of administration, 2) metronidazole induced anaemia after one month from start of administration, 3) metronidazole at this high dose level (3 times the therapeutic dose in mice) has the ability to induce genotoxic effects in somatic cells.     

Reproductive effects of low acute doses of cadmium chloride in adult male rats

Adult male Sprague-Dawley rats were injected sc with cadmium (Cd, as cadmium chloride) in doses ranging from 1.6 to 152 mumol Cd/kg body weight (body wt). Fourteen days after dosing, animals were evaluated for reproductive damage. Evaluations for each animal included testes, seminal vesicles, and epididymides weights, vas deferens sperm concentration, and human chorionic gonadotropin (hCG)-stimulated serum testosterone concentration. Since 10 to 60% mortality occurred in the two highest dose groups (74 and 152 mumol/kg), no additional evaluations were conducted in these groups. The weights of the testes, seminal vesicles, and epididymides were reduced at least 40 to 50% in groups receiving 16 or 33 mumol Cd/kg while vas deferens sperm concentrations and hCG-stimulated serum testosterone concentrations were essentially zero. Significant depressions in the sperm concentrations and in the hCG-stimulated serum testosterone concentrations were found in animals receiving the two lowest doses (1.6 and 7.4 mumol Cd/kg) although no changes in tissue weights were observed in these animals. Curve-linear regression analyses for the dose responsiveness of these parameters demonstrated that serum testosterone concentration initially decreased at a rate of 19%/mumol Cd/kg, respectively, and was the most sensitive to Cd exposure. The initial rates of decrease for sperm concentrations and for seminal vesicles, testes, and epididymides weight were 6.45, 5.30, 4.19, and 2.45%/mumol Cd/kg, respectively, and were less responsive to Cd exposure than serum testosterone levels.