• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Identification of cadmium containing metabolites in HepG2 cells after treatment with cadmium-selenium quantum dots

    2023-03-14 06:52:52BeibeiChenLuPengManHeChuanWangBinHu
    Chinese Chemical Letters 2023年1期

    Beibei Chen,Lu Peng,Man He,Chuan Wang,Bin Hu

    Department of Chemistry,Wuhan University,Wuhan 430072,China

    Keywords:CdSe/ZnS quantum dots Cd speciation Metallothionein Cells High performance liquid chromatography Inductively coupled plasma mass spectrometry Electrospray ionization mass spectrometry

    ABSTRACT The transformation of quantum dots (QDs) by organisms has attracted broad attention but remains unclear.Understanding of the metabolites helps to reveal the transformation pathway of QDs.Cd containingmetallothionein (MT) are the main species formed by Cd released from CdSe QDs in HepG2 cells,while speciation analysis of Cd containing MTs remains a challenge because MTs has several subisoforms and can bind with several metals.Herein,we built a hyphenated platform for speciation analysis of QDs in HepG2 cells after treatment with CdSe/ZnS QDs.The Cd-containing MTs were separated in reversed phase high performance liquid chromatography (RP-HPLC) and subsequently online detected by inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) parallelly.Four groups of Cd-containing metabolites were found by detecting Cd in ICP-MS.Their structures were identified in ESI-Q-TOF-MS and further confirmed with standards of four subisoforms of MT,including N-terminal acetylation MT2a,N-terminal acetylation MT1e,N-terminal acetylation MT1g and MT1m.Each group of them contains various stoichiometry of Cd/Zn.The metabolites of QDs remain same while the concentrations of each metabolite and its stoichiometry of Cd/Zn vary for different incubation concentration/time.This work provides a new parallel hyphenation technique of HPLC-ICP-MS/ESI-MS with high separation resolution and powerful detection ability,and the obtained results provide detailed metabolism information of QDs in HepG2 cells after treatment of CdSe/ZnS QDs,contributing to deep exploration of the functional mechanisms of QDs in organisms.

    Nanoparticles (NPs) have been widely used in bioimaging,biosensors,disease diagnosis and drug delivering [1–5],in which quantum dots (QDs) are a special kind of with unique fluorescence advantages [6].However,with the rapid development of QDs-related nanotechnology,the health effect of QDs to human being has become a non-ignorable issue,and the biosafety of QDs remains unclear because little is known about the functional mechanism of QDs.The recent mechanism studies of QDs toxicity found QDs-mediated reactive oxygen species (ROS) induction was an important pathway to generate cytotoxicity [7,8].Cd could be released from the QDs in living cells [9,10],which further induce oxidative stress and mitochondrial damage in cells [11].Thus,understanding the metabolism of QDs could reveal the basis of cytotoxicity of QDs.

    The decomposition of Cd-QDs in living cells or organism was obtained by many researches [12–14].However,little is known about the existing species and the trans-formation of Cd after its release from QDs in cells,which significantly affect the transport,metabolism,accumulation and ultimate toxicity of QDs.In our previous study,the existing forms of CdSe/ZnS QDs in HepG2 cells after co-incubation was explored by hyphenated techniques;two species were found and identified as QD-like NPs and Cd containing-metallothionein fraction (MT-F),respectively [15].For the MT-F,it was assumed to be formed by Cd released from QDs with MTs in HepG2 cells; while more detailed information is required.

    The hyphenation techniques combining high resolution separation tools such as high performance liquid chromatography (HPLC)with high sensitivity detectors such as inductively coupled plasma mass spectrometry (ICP-MS) are effective way for elemental speciation.And utilization of molecular MS is a good choice for identification of unknown species.However,speciation analysis of MTs is still difficult because MTs has several subisoforms with similar retention behavior and can bind with several metals with different molar ratio.Coupling HPLC with ICP-MS and electrospray ionization (ESI)-MS in parallel were shown to be an attractive technique to separate and identify individual MT isoforms [16,17].However,reports on online parallel connection of ICP-MS and ESI-MS as HPLC detectors are few,and the analytical performance should be further improved.

    Fig.1.Schematic diagram of HPLC-ICP-MS/ESI-Q-TOF-MS analysis (operation conditions listed in Table S1 in Supporting information).

    Herein,to reveal the transformation of Cd released from QDs in HepG2 cells,an on-line hyphenated technique of HPLC-ICP-MS/ESI- quadrupole time-of-flight (Q-TOF)-MS parallel measurement was developed for speciation of Cd containing-MTs,including the subisoforms of MTs and the Cd/MTs molar ratio.Four kinds of CdSe/ZnS QDs with different particle sizes (hydrodynamic diameter ofca.17–23 nm) and functional groups (carboxyl or amino functional group) (namely NH2-525,NH2-585,NH2-625,COOH-525)were used to incubated with HepG2 cells as the same in our previous studies [15,18],and the MT-F fraction after size exclusion chromatographic separation was collected for HPLC-ICP-MS/ESI-Q-TOFMS analysis.The analytical strategy was illustrated in Fig.1.After HPLC separation,the effluent with/without acidification was analyzed by ICP-MS and ESI-Q-TOF-MS simultaneously.Parallel measurement enabled monitoring elemental information of Cd by ICPMS and mass information of Cd-MTs in MT-F by ESI-Q-TOF-MS simultaneously.With post-column acidification,MTs subisoforms were demetallated as apo-MTs which facilitates identification of the involved MTs subisoforms by ESI-Q-TOF-MS.Without postcolumn acidification,mass spectra of metallated-metallothioneins(M-MTs) would be provided by ESI-Q-TOF-MS.Compared the mass to charge ratio of M-MTs to corresponding apo-MTs,the molar ratio of Cd binding on MTs could be obtained.

    Taking MT-F of NH2–525 (termed as NH2-525-F) as the representative,the speciation results obtained for NH2-525-F were analyzed and specified as follows.Firstly,result obtained by ICP-MS detection without post-column acidification was analyzed to investigate the Cd containing species in NH2-525-F.As can be seen from the chromatogram for ICP-MS detection of Cd and total ion chromatogram (TIC) of ESI-Q-TOF-MS detection in Figs.2A and B,four chromatographic peaks were observed in NH2-525-F.These peaks with retention time at 1050,1420,1960 and 2240 s were named as Metabolite 1,2,3,and 4,respectively.

    Secondly,result obtained by ESI-Q-TOF-MS detection with postcolumn acidification was analyzed to investigate the MS information of apo-MTs subisoforms in Metabolites 1–4.The mass result of apo-MTs in Metabolites 1–4 is shown in Fig.3A.To verify the MTs subisoforms in Metabolites 1–4,all the apo-MTs in human beings with possible post-modification sites (such as acetylation) from Uniprot database (http://www.uniprot.org/) were selected as candidates.The mass-charge ratio of the measured value(m/zmeasured,z=4) was compared with the mass-charge ratio of the theoretical value (m/ztheoretical,z=4) of the apo-MTs in human beings.Four subisoforms of apo-MTs,N-terminal acetylation MT2a (N Ac-MT2a),N-terminal acetylation MT1e (N Ac-MT1e),Nterminal acetylation MT1g (N Ac-MT1g) and MT1m (detailed properties listed in Table S2 in Supporting information),were obtained in corresponding retention time of Metabolites 1–4,respectively.The measured mass accuracies are all below 20 ppm and the detail results are listed in Table 1.Meanwhile,the extracted ion chromatogram (EIC) of the four apo-MTs was analyzed (Fig.3B).All the retention time of four apo-MTs in EIC were the same to the result of Metabolites 1–4 in ICP-MS,which further confirmed the one-toone correspondence between apo-MTs and Metabolites 1–4.

    Thirdly,result obtained by ESI-Q-TOF-MS detection without post-column acidification was analyzed to investigate the MS information of Cd-MTs in Metabolites 1–4.The mass result of Cd-MTs in Metabolites 1–4 is shown in Figs.4A–C.After identification of the subisoforms of apo-MTs for Metabolites 1–4,corresponding Cd-MTs were selected as candidates for comparison,and the total molar ratio of Cd and Zn to MT was fixed as 7.The mass-charge ratio (m/z) of the measured value (m/zmeasured,z=5) was compared with them/zof the theoretical value (m/ztheoretical,z=5) of corresponding Cd-MTs candidates in human beings.The measured mass accuracies are all below 20 ppm and the detail results are listed in Table 1.As can be seen,Metabolites 1–4 are Cd,Zn-MTs complex which are formed by different molar ratio of Cd/Zn (3:4,4:3,5:2 and 6:1) with N Ac-MT2a,N Ac-MT1e,N Ac-MT1g and MT1m,respectively.Four group peaks with retention time at 1000,1400,2000 and 2200 s were also detected in MT-F for COOH-525,NH2-585 and NH2-625 QDs incubated cells,as shown in Figs.S1–S12(Supporting information).Components of COOH-525-F,NH2-585-F and NH2-625-F are presented in Tables S3–S5 (Supporting information).Similarly to that observed for NH2-525-F,Cd,Zn-MTs complex are formed by different molar ratio of Cd/Zn with N Ac-MT2a,N Ac-MT1e,N Ac-MT1g and MT1m for Metabolites 1–4,respectively.

    The MT2a,MT1e,MT1g and MT1m related genes are considered to be highly expressed in HepG2 cells [19,20].Moreover,the expression protein of these MT-genes has high affinity for several heavy-metal ions including Cd and Zn.In this work,Metabolites 1–4 was separated by HPLC in order of the hydrophobicity properties of MTs subisoforms as predicted by grand average of hydropathicity (GRAVY).When the value of GRAVY is positive,the larger the value is,the stronger the hydrophobicity is.The GRAVY value of MT2a,MT1e,MT1g and MT1m is increased from 0.131 to 0.269 in turn; it coincides the order in which Metabolites 1–4 was eluted in HPLC.Meanwhile,the information of M-MTs in Metabolites 1–4 obtained by HPLC-ESI-Q-TOF-MS without post-column acidification indicated the molar ratio between metals and MTs was 7:1;Cd and Zn,with the molar ratio of 2:5 to 7:0,were combined together with N Ac-MT2a,N Ac-MT1e,N Ac-MT1g and MT1m in Cd-MTs,respectively.Among them,molar ratio of 4:3 and 5:2 were the largest existing species,followed by molar ratio of 3:4 and 6:1,and the molar ratio of 2:5 and 7:0 were the least.MTs commonly consist of two subunits:α-domain (C-terminal) andβ-domain (Nterminal) [21].MTs can always incorporate with seven divalent metals (Cd,Zn,etc.) per molecule,providing a highly dynamic,regulated,and uniquely biological metal buffer [22].In vitroandin vivo,MTs exist mainly in Zn form or as mixed-metal proteins[21–23].Previous studies have demonstrated that Cd can displace Zn in MTs and form mixed Cd,Zn-MTs [24–27],because the binding constants for Cd with MTs is commonly two orders of magnitude more than that for Zn [21,23].Thus,in this work,the Cd released from QDs was combined with four MTs subisoforms,forming several different kinds of Cd,Zn-N Ac-MT2a,Cd,Zn-N Ac-MT1e,Cd,Zn-N Ac-MT1m and Cd,Zn-MT1m species in HepG2 cells.Meanwhile,the QDs used in this work consisted of ZnS shell and CdSe core,suggesting that a degradation of ZnS shell was a prerequisite for the decomposition of CdSe core.Thus,when Cd was released from QDs,Zn had been released from ZnS shell to a more extent.As a result,Cd,Zn-MTs with median molar ratio of Cd/Zn(4:3 and 5:2) were the main existing species in Metabolites 1–4,which is the evident that Cd(II) binds with stronger affinity than Zn(II) to MT,implying possible toxicity of the degraded Cd from QDs.

    Fig.2.HPLC-ICP-MS/ESI-Q-TOF-MS analysis of NH2-525-F under different incubation concentrations without post-column acidification.(A) ICP-MS detection of Cd; (B) TIC of ESI-Q-TOF-MS detection.1–4 represent for Metabolites 1–4,respectively.

    Fig.3.MS spectra (A) and EIC (B) of 1521.8347 (z=4),1514.8184 (z=4),1546.66086 (z=4) and 1528.3376 (z=4) for Metabolites 1–4 in NH2-525-F (100 nmol/L) with post-column acidification.1–4 represent for Metabolites 1–4,respectively.

    Fig.4.MS spectra of Metabolites 1–4 in NH2-525-F without post-column acidification under different incubation concentrations.(A) 100 nmol/L,(B) 50 nmol/L and (C)10 nmol/L.1–4 represent for Metabolites 1–4.

    Table 1 MS information of NH2-525-F under different incubation concentrations.

    The variation of Cd-MTs components in cells after incubating with different concentration /time and eliminating for different time was investigated (results shown in Figs.S13–S44 and Tables S6–S13 in Supporting information).Four species,Metabolites 1–4,occurred whichever concentration/time was employed; while,the intensity of Metabolites 1–4 is quite different when the incubation conditions was varied.For four QDs incubation,the intensity of Metabolites 1–4 all rose up when QDs incubation concentrations were increased,which was verified by ICP-MS and ESI-Q-TOF-MS measurement simultaneously.Meanwhile,larger numbers of Cd-MTs in Metabolites 1–4 were found with the increase of incubation concentrations.It was demonstrated that the amount of CdSe/ZnS QDs in cells increased with increasing the incubation concentrations and more Cd was released from QDs [15].Since MT-F was the sum of Metabolites 1–4,these results confirmed the variation tendency of MT-F amount under different incubation concentration in our previous study [15].Similar results were found under different incubation time.Metabolites 1–4 all increased and growing numbers of M-MTs species in Metabolites 1–4 were obtained when the incubation time was extended,due to the increasing release of Cd from original QDs in cell matrix.

    Meanwhile,the analytical results,obtained for Cd-MTs components after eliminating for a certain time,showed that the intensity of Metabolites 1–4 in three MT-F groups (NH2-525-F,COOH-525-F,NH2-625-F) all increased firstly (0–12 h) and then decreased(12–48 h); a little difference was obtained for NH2-585-F,the intensity of Metabolites 1–4 decreased over all the time.It is probably ascribed to the relatively slow degradation rate for NH2-585 QDs [15],and the resulting slow Cd releasing from QDs in cells.The difference needs to be further investigated.The number of MMTs species in Metabolites 1–4 decreased with elimination time increasing from 0 h to 48 h.

    In conclusion,we built an on-line parallel hyphenation technique of HPLC-ICP-MS/ESI-Q-TOF-MS for speciation analysis of Cd in MT-F fraction collected from HepG2 cells incubated with CdSe/ZnS QDs.Four group species,Metabolites 1–4,were obtained in MT-F and they were confirmed to be Cd,Zn-MTs complex formed by different molar ratio of Cd/Zn with N Ac-MT2a,N Ac-MT1e,N Ac-MT1g and MT1m,respectively.Meanwhile,Metabolites 1–4 was also observed in MT-F when different incubation concentration/time and elimination time was employed.The intensity of Metabolites 1–4 all increased and more Cd,Zn-MTs species in each peak were obtained with increasing the incubation concentration/time.This work proposed a MS-based hyphenated platform with improved performance for metallomics research,and the obtained results provided worthwhile information for the transformation of CdSe QDs in cells,greatly contributing to the elucidation of metabolism and toxicity of QDs.

    Declaration of competing interest

    The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

    Acknowledgments

    The financial support from the National Natural Science Foundation of China (Nos.21575107,21775113,21575108),the Science Fund for Creative Research Groups of NSFC (No.20921062) and the Fundamental Research Funds for the Central Universities (No.114009) are greatly acknowledged.

    Supplementary materials

    Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.cclet.2022.02.067.

    国产精品香港三级国产av潘金莲| 在线av久久热| 国产精品九九99| 999精品在线视频| 99国产精品99久久久久| 日本撒尿小便嘘嘘汇集6| 久久女婷五月综合色啪小说| 三上悠亚av全集在线观看| 美女福利国产在线| av片东京热男人的天堂| 日本a在线网址| avwww免费| 天堂俺去俺来也www色官网| 一本久久精品| 一本—道久久a久久精品蜜桃钙片| 狠狠婷婷综合久久久久久88av| 天天躁狠狠躁夜夜躁狠狠躁| 日韩有码中文字幕| 嫩草影视91久久| 久久 成人 亚洲| 操美女的视频在线观看| 一级片免费观看大全| 国产一区二区三区综合在线观看| tube8黄色片| a 毛片基地| 国产日韩一区二区三区精品不卡| 在线av久久热| 国产成人精品久久二区二区91| 蜜桃在线观看..| 成年av动漫网址| 不卡av一区二区三区| 国产片内射在线| 亚洲人成电影免费在线| 久久99一区二区三区| 亚洲精品久久成人aⅴ小说| 久久久久久久国产电影| 久久影院123| 视频区图区小说| 国产伦人伦偷精品视频| 青春草视频在线免费观看| 国产精品久久久久久精品电影小说| 国产高清videossex| 久久性视频一级片| 日韩熟女老妇一区二区性免费视频| 国产精品麻豆人妻色哟哟久久| 人妻人人澡人人爽人人| 大片电影免费在线观看免费| 桃红色精品国产亚洲av| 国产精品一区二区在线观看99| 天天影视国产精品| 国产一级毛片在线| 不卡av一区二区三区| 精品国产乱子伦一区二区三区 | 搡老熟女国产l中国老女人| 欧美中文综合在线视频| 国产国语露脸激情在线看| 亚洲av日韩在线播放| 精品少妇黑人巨大在线播放| 欧美在线黄色| 蜜桃国产av成人99| 亚洲三区欧美一区| 国产一区二区 视频在线| 中文字幕人妻熟女乱码| 精品福利永久在线观看| 国产精品.久久久| 日韩欧美一区二区三区在线观看 | 日韩欧美免费精品| 老熟女久久久| 丰满饥渴人妻一区二区三| 老司机亚洲免费影院| 亚洲精品久久午夜乱码| 国产亚洲欧美精品永久| 欧美日韩一级在线毛片| 亚洲精华国产精华精| 精品欧美一区二区三区在线| 日韩欧美一区视频在线观看| 国产亚洲av高清不卡| 精品卡一卡二卡四卡免费| 精品一区在线观看国产| 人人妻人人澡人人看| 美女大奶头黄色视频| 美女高潮喷水抽搐中文字幕| 午夜免费成人在线视频| 欧美精品啪啪一区二区三区 | 亚洲欧美一区二区三区久久| 伊人亚洲综合成人网| 91麻豆精品激情在线观看国产 | 国产日韩一区二区三区精品不卡| 国产精品亚洲av一区麻豆| 久久久国产成人免费| 国产精品 国内视频| 日韩熟女老妇一区二区性免费视频| 亚洲第一青青草原| 久久国产精品男人的天堂亚洲| 啦啦啦啦在线视频资源| 一本大道久久a久久精品| 亚洲自偷自拍图片 自拍| 自拍欧美九色日韩亚洲蝌蚪91| 国产成人影院久久av| 亚洲九九香蕉| 亚洲中文av在线| 欧美黑人精品巨大| 狠狠精品人妻久久久久久综合| 又紧又爽又黄一区二区| 一本综合久久免费| 我要看黄色一级片免费的| 久久热在线av| 日本撒尿小便嘘嘘汇集6| 国产成人免费观看mmmm| 亚洲欧美成人综合另类久久久| 99国产综合亚洲精品| 黄片小视频在线播放| 亚洲国产看品久久| 女人高潮潮喷娇喘18禁视频| 国产亚洲av高清不卡| 亚洲国产精品成人久久小说| 丝袜美足系列| 人人澡人人妻人| 伦理电影免费视频| 一本色道久久久久久精品综合| bbb黄色大片| 男女下面插进去视频免费观看| 两个人免费观看高清视频| 美女中出高潮动态图| 丝袜在线中文字幕| 精品久久久精品久久久| 窝窝影院91人妻| 久久99热这里只频精品6学生| av天堂久久9| 久久久精品94久久精品| 国产激情久久老熟女| 91麻豆av在线| 一级,二级,三级黄色视频| 免费高清在线观看日韩| 免费观看av网站的网址| 成在线人永久免费视频| 人人妻人人爽人人添夜夜欢视频| 久久狼人影院| 欧美黑人精品巨大| 80岁老熟妇乱子伦牲交| 欧美老熟妇乱子伦牲交| 久久精品熟女亚洲av麻豆精品| 18禁观看日本| 亚洲中文字幕日韩| 亚洲五月色婷婷综合| 色老头精品视频在线观看| av电影中文网址| 午夜91福利影院| 亚洲国产欧美网| 午夜免费鲁丝| 欧美大码av| 欧美日韩亚洲国产一区二区在线观看 | 黑人操中国人逼视频| 亚洲情色 制服丝袜| 中国国产av一级| 美女扒开内裤让男人捅视频| 国产男人的电影天堂91| 国产日韩一区二区三区精品不卡| 中文字幕色久视频| 一级毛片电影观看| 各种免费的搞黄视频| 操出白浆在线播放| 制服诱惑二区| 久久香蕉激情| 国产成人一区二区三区免费视频网站| 精品一区二区三区四区五区乱码| 精品国产一区二区久久| 在线观看免费午夜福利视频| 中文欧美无线码| 精品一区二区三区四区五区乱码| 日韩一卡2卡3卡4卡2021年| 99久久精品国产亚洲精品| 欧美日韩福利视频一区二区| 日韩有码中文字幕| 99久久国产精品久久久| 日韩有码中文字幕| 精品国产一区二区久久| 日本wwww免费看| bbb黄色大片| 一区二区av电影网| av片东京热男人的天堂| 欧美黄色淫秽网站| 在线av久久热| 一本—道久久a久久精品蜜桃钙片| 精品一区二区三区四区五区乱码| 99热国产这里只有精品6| 亚洲精华国产精华精| 亚洲精品久久久久久婷婷小说| av又黄又爽大尺度在线免费看| 美女主播在线视频| 国产男女内射视频| 一级毛片电影观看| 欧美国产精品一级二级三级| 久久国产精品人妻蜜桃| 两个人看的免费小视频| 黄色怎么调成土黄色| 午夜免费鲁丝| 91九色精品人成在线观看| 淫妇啪啪啪对白视频 | 欧美少妇被猛烈插入视频| 精品熟女少妇八av免费久了| 久久久久久亚洲精品国产蜜桃av| 亚洲精品乱久久久久久| 国产精品亚洲av一区麻豆| 91麻豆av在线| 午夜成年电影在线免费观看| 男女高潮啪啪啪动态图| 欧美另类一区| 正在播放国产对白刺激| 欧美精品av麻豆av| 两个人看的免费小视频| 精品少妇久久久久久888优播| 黄色毛片三级朝国网站| 美女福利国产在线| 搡老熟女国产l中国老女人| 免费一级毛片在线播放高清视频 | 亚洲国产看品久久| 午夜激情av网站| 一级毛片精品| 亚洲成人手机| 久久99热这里只频精品6学生| 制服人妻中文乱码| 少妇的丰满在线观看| 极品人妻少妇av视频| bbb黄色大片| 后天国语完整版免费观看| 久久免费观看电影| 亚洲国产精品一区二区三区在线| 欧美日韩中文字幕国产精品一区二区三区 | 亚洲视频免费观看视频| 精品亚洲成国产av| 无遮挡黄片免费观看| 国产精品久久久av美女十八| avwww免费| 少妇精品久久久久久久| 伊人久久大香线蕉亚洲五| 国产精品一二三区在线看| 亚洲精品成人av观看孕妇| 国产精品99久久99久久久不卡| 欧美激情久久久久久爽电影 | 国产一级毛片在线| 777久久人妻少妇嫩草av网站| 亚洲欧美一区二区三区久久| 下体分泌物呈黄色| 久久精品国产综合久久久| 我的亚洲天堂| 两个人看的免费小视频| 我的亚洲天堂| 久久国产亚洲av麻豆专区| 国产成人a∨麻豆精品| 男男h啪啪无遮挡| 国产成+人综合+亚洲专区| 中文字幕av电影在线播放| 9色porny在线观看| 丰满少妇做爰视频| 黄色视频在线播放观看不卡| 国产精品久久久久久精品电影小说| 五月天丁香电影| 欧美精品啪啪一区二区三区 | 国产高清国产精品国产三级| 午夜久久久在线观看| 黑人猛操日本美女一级片| 黑人猛操日本美女一级片| 久久久久精品国产欧美久久久 | 天堂8中文在线网| 久久久久久久精品精品| 久久人人爽av亚洲精品天堂| 免费在线观看日本一区| 99香蕉大伊视频| 免费在线观看黄色视频的| 精品国产乱码久久久久久小说| 日本av免费视频播放| 色精品久久人妻99蜜桃| 久久免费观看电影| 90打野战视频偷拍视频| 久久久久精品人妻al黑| 亚洲第一av免费看| 三上悠亚av全集在线观看| 久热爱精品视频在线9| 又紧又爽又黄一区二区| 亚洲成人免费电影在线观看| 久久久久久免费高清国产稀缺| 少妇粗大呻吟视频| 日韩 欧美 亚洲 中文字幕| 亚洲国产看品久久| 一区福利在线观看| 国产在线一区二区三区精| 一级黄色大片毛片| 不卡一级毛片| 丰满饥渴人妻一区二区三| 亚洲专区国产一区二区| 水蜜桃什么品种好| 欧美成人午夜精品| 国产一级毛片在线| 日本五十路高清| 久久精品国产亚洲av香蕉五月 | 亚洲成av片中文字幕在线观看| 国产精品二区激情视频| 亚洲精品一二三| 性高湖久久久久久久久免费观看| 欧美xxⅹ黑人| 大香蕉久久成人网| 69精品国产乱码久久久| 国产区一区二久久| 国产高清videossex| 在线观看免费高清a一片| 成年人午夜在线观看视频| 99久久99久久久精品蜜桃| 建设人人有责人人尽责人人享有的| 欧美亚洲日本最大视频资源| 伊人久久大香线蕉亚洲五| 午夜福利视频在线观看免费| 午夜福利一区二区在线看| av有码第一页| 大型av网站在线播放| 一区二区av电影网| 国产男人的电影天堂91| 久久国产亚洲av麻豆专区| 91精品伊人久久大香线蕉| 天天躁日日躁夜夜躁夜夜| 亚洲自偷自拍图片 自拍| 女性被躁到高潮视频| 黄色 视频免费看| 亚洲精品国产av蜜桃| 国产精品麻豆人妻色哟哟久久| 国产精品久久久av美女十八| 国产伦理片在线播放av一区| 五月开心婷婷网| 成年动漫av网址| 欧美黄色片欧美黄色片| videosex国产| 视频在线观看一区二区三区| 中文字幕人妻熟女乱码| 在线观看免费午夜福利视频| 国产精品一区二区在线不卡| 日日夜夜操网爽| 色老头精品视频在线观看| 欧美精品一区二区大全| 免费在线观看完整版高清| 久久久国产精品麻豆| 交换朋友夫妻互换小说| 十八禁人妻一区二区| 欧美日韩亚洲国产一区二区在线观看 | 韩国精品一区二区三区| 亚洲第一av免费看| 啦啦啦 在线观看视频| av超薄肉色丝袜交足视频| 国产黄频视频在线观看| 欧美日韩视频精品一区| bbb黄色大片| 黑人猛操日本美女一级片| 成年人午夜在线观看视频| 亚洲成人免费电影在线观看| www.自偷自拍.com| 成年人免费黄色播放视频| 狠狠婷婷综合久久久久久88av| 亚洲国产欧美一区二区综合| 久久精品久久久久久噜噜老黄| 制服人妻中文乱码| 国产成+人综合+亚洲专区| 秋霞在线观看毛片| 777久久人妻少妇嫩草av网站| 久久狼人影院| 亚洲精品中文字幕一二三四区 | 久久精品国产亚洲av香蕉五月 | h视频一区二区三区| 中文字幕另类日韩欧美亚洲嫩草| 视频在线观看一区二区三区| 精品一区二区三区四区五区乱码| 国产片内射在线| 建设人人有责人人尽责人人享有的| 久久狼人影院| 国产男女超爽视频在线观看| 日韩人妻精品一区2区三区| √禁漫天堂资源中文www| 王馨瑶露胸无遮挡在线观看| 性色av一级| 午夜影院在线不卡| 大型av网站在线播放| e午夜精品久久久久久久| 9色porny在线观看| 天堂俺去俺来也www色官网| 免费黄频网站在线观看国产| 91精品三级在线观看| 欧美在线一区亚洲| 欧美日韩亚洲国产一区二区在线观看 | 欧美精品一区二区大全| 亚洲伊人久久精品综合| 国产日韩一区二区三区精品不卡| 午夜91福利影院| 亚洲精品在线美女| 国产亚洲av片在线观看秒播厂| 欧美黄色片欧美黄色片| 亚洲一码二码三码区别大吗| 日本vs欧美在线观看视频| 久久综合国产亚洲精品| 午夜两性在线视频| 欧美 日韩 精品 国产| 99国产精品免费福利视频| 国产成人精品无人区| 午夜老司机福利片| 熟女少妇亚洲综合色aaa.| 狂野欧美激情性bbbbbb| 亚洲精品一区蜜桃| 亚洲精品美女久久久久99蜜臀| 亚洲精品久久久久久婷婷小说| 一级片'在线观看视频| 欧美人与性动交α欧美软件| www.自偷自拍.com| videosex国产| 欧美日韩一级在线毛片| 欧美久久黑人一区二区| 悠悠久久av| 18禁国产床啪视频网站| 啪啪无遮挡十八禁网站| 国产精品 欧美亚洲| 欧美乱码精品一区二区三区| 狠狠精品人妻久久久久久综合| 1024香蕉在线观看| 最新的欧美精品一区二区| 日本猛色少妇xxxxx猛交久久| 精品久久久精品久久久| 悠悠久久av| 国产成人精品在线电影| 欧美日韩一级在线毛片| 黑人欧美特级aaaaaa片| 中文字幕制服av| 91精品三级在线观看| 久久九九热精品免费| 一级片'在线观看视频| 久久国产精品影院| 久久精品熟女亚洲av麻豆精品| 丝袜美腿诱惑在线| 亚洲精品国产一区二区精华液| 无限看片的www在线观看| av天堂在线播放| 一二三四社区在线视频社区8| 一进一出抽搐动态| 国产99久久九九免费精品| 九色亚洲精品在线播放| 91字幕亚洲| 国产精品九九99| 午夜激情久久久久久久| 欧美亚洲 丝袜 人妻 在线| 免费不卡黄色视频| 一本久久精品| 女人被躁到高潮嗷嗷叫费观| 日韩欧美一区二区三区在线观看 | 两个人看的免费小视频| 咕卡用的链子| 亚洲精品成人av观看孕妇| 欧美av亚洲av综合av国产av| 国产精品一区二区在线观看99| 日韩有码中文字幕| 一级毛片女人18水好多| 波多野结衣一区麻豆| av网站免费在线观看视频| 亚洲免费av在线视频| 国产在视频线精品| 咕卡用的链子| 大码成人一级视频| 久久99热这里只频精品6学生| 涩涩av久久男人的天堂| 日本a在线网址| 久久 成人 亚洲| 91字幕亚洲| 日韩熟女老妇一区二区性免费视频| 国产黄频视频在线观看| 久久久久久久久免费视频了| 亚洲熟女毛片儿| 亚洲午夜精品一区,二区,三区| 亚洲精品一二三| 国产精品偷伦视频观看了| 日日爽夜夜爽网站| 美女视频免费永久观看网站| 欧美+亚洲+日韩+国产| 国产av一区二区精品久久| 男女之事视频高清在线观看| 99久久精品国产亚洲精品| 精品少妇内射三级| 韩国精品一区二区三区| 午夜福利在线免费观看网站| 男女边摸边吃奶| 自拍欧美九色日韩亚洲蝌蚪91| 超碰成人久久| 天天影视国产精品| 国产有黄有色有爽视频| 亚洲免费av在线视频| 国产熟女午夜一区二区三区| 一级片'在线观看视频| 在线观看免费高清a一片| 欧美日韩精品网址| 国产成人系列免费观看| 天天添夜夜摸| 国产精品秋霞免费鲁丝片| 精品少妇久久久久久888优播| 一区二区三区四区激情视频| 悠悠久久av| 日韩,欧美,国产一区二区三区| 中文字幕人妻熟女乱码| 国产精品一区二区免费欧美 | 免费av中文字幕在线| 亚洲欧美精品综合一区二区三区| avwww免费| 亚洲av日韩在线播放| 亚洲国产欧美网| 一区二区三区激情视频| 国产xxxxx性猛交| 黑人巨大精品欧美一区二区蜜桃| 波多野结衣av一区二区av| 亚洲性夜色夜夜综合| 日韩中文字幕视频在线看片| 亚洲专区字幕在线| 久久国产精品男人的天堂亚洲| 纵有疾风起免费观看全集完整版| av又黄又爽大尺度在线免费看| 老司机亚洲免费影院| 97在线人人人人妻| a 毛片基地| 精品人妻一区二区三区麻豆| 亚洲av国产av综合av卡| 制服人妻中文乱码| 秋霞在线观看毛片| www.999成人在线观看| 国产欧美日韩一区二区精品| 最近中文字幕2019免费版| av超薄肉色丝袜交足视频| 亚洲伊人久久精品综合| 欧美人与性动交α欧美软件| 国产精品久久久久久精品古装| 亚洲国产日韩一区二区| 性少妇av在线| 丝袜脚勾引网站| 另类亚洲欧美激情| 国产色视频综合| 成人手机av| 我的亚洲天堂| 国产日韩欧美亚洲二区| 欧美在线一区亚洲| 久久99热这里只频精品6学生| 老熟女久久久| 一级毛片精品| 欧美亚洲日本最大视频资源| 国产成人精品久久二区二区91| 色综合欧美亚洲国产小说| 777久久人妻少妇嫩草av网站| 男女无遮挡免费网站观看| 亚洲美女黄色视频免费看| 一级毛片精品| 欧美在线黄色| 亚洲美女黄色视频免费看| 国产99久久九九免费精品| av天堂在线播放| 久久99一区二区三区| 精品少妇久久久久久888优播| 久久久久久久大尺度免费视频| 大陆偷拍与自拍| 少妇人妻久久综合中文| 日日夜夜操网爽| 亚洲欧美一区二区三区久久| 国产日韩欧美亚洲二区| 人成视频在线观看免费观看| 久久国产精品人妻蜜桃| 每晚都被弄得嗷嗷叫到高潮| 亚洲国产日韩一区二区| 免费不卡黄色视频| 美国免费a级毛片| 亚洲精品美女久久久久99蜜臀| 久久精品久久久久久噜噜老黄| www日本在线高清视频| 天堂俺去俺来也www色官网| 欧美日本中文国产一区发布| 热99re8久久精品国产| 亚洲色图综合在线观看| 国产精品一二三区在线看| 大香蕉久久网| 日韩大片免费观看网站| 久久99热这里只频精品6学生| 精品亚洲成a人片在线观看| 国产成人精品久久二区二区91| 国产精品 欧美亚洲| 欧美 日韩 精品 国产| 国产有黄有色有爽视频| 成人18禁高潮啪啪吃奶动态图| 欧美 日韩 精品 国产| 亚洲黑人精品在线| 啦啦啦视频在线资源免费观看| 男女午夜视频在线观看| 国产三级黄色录像| 国产麻豆69| 亚洲专区中文字幕在线| 最近最新免费中文字幕在线| 丝袜脚勾引网站| 久久女婷五月综合色啪小说| 两人在一起打扑克的视频| 亚洲 欧美一区二区三区| 亚洲精品第二区| 国产又色又爽无遮挡免| av天堂久久9| 男女午夜视频在线观看| 麻豆av在线久日| 女人爽到高潮嗷嗷叫在线视频| 999久久久国产精品视频| 美女大奶头黄色视频| 在线观看免费高清a一片| 女性生殖器流出的白浆| 国产男人的电影天堂91| av一本久久久久| 久久久欧美国产精品| 极品人妻少妇av视频| 亚洲伊人色综图| 桃红色精品国产亚洲av| 女警被强在线播放| 超碰成人久久| 精品乱码久久久久久99久播|