A novel method to assess antibody-dependent cell-mediated cytotoxicity against influenza A virus M2 in immunized murine models

The matrix protein 2 (M2) is a preferred target for developing a universal vaccine against the influenza A virus (IAV). This study aimed to develop a method for assessing antibody-dependent cell-mediated cytotoxicity (ADCC) associated with M2-based immunization in mice. We first established a stable cell line derived from mouse lymphoma cells (YAC-1) expressing M2 of H3N2. This cell line, designated as YAC-1-M2, was generated using a second-generation lentiviral tricistronic plasmid system to transduce the M2 gene into YAC-1 cells. The ADCC effect induced by polyclonal antibodies targeting matrix protein 2 ectodomain (M2e) was demonstrated by YAC-1-M2 cell lysis by natural killer cells (NK) derived from mice, in the presence of anti-M2 antibodies obtained from mice immunized with an mRNA vaccine based on M2e. This ADCC effect was found to be stronger compared to the effect induced by monoclonal antibodies (14C2) against M2. Moreover, the ADCC effect was enhanced as the effector-to-target ratio of NK to YAC-1-M2 cells increased. In conclusion, we established a novel method to detect ADCC of M2 of IAV, which paves the way for the development of an M2-based universal vaccine against IAV and an in-depth analysis of its mechanism of broad-spectrum immune protection in mice.

In Vitro Evaluation of Cytotoxicity and Oxidative Stress Induced by Multiwalled Carbon Nanotubes in Murine RAW 264.7 Macrophages and Human A549 Lung Cells

Objective To investigate in vitro cytotoxicity and oxidative stress response induced by multiwalled carbon nanotubes （MWCNTs）. Methods Cultured macrophages （murine RAW264.7 cells） and alveolar epithelium cells type II （human A549 lung cells） were exposed to the blank control, DNA salt control, and the MWCNTs suspensions at 2.5, 10, 25, and 100 ug/mL for 24 h. Each treatment was evaluated by cell viability, cytotoxicity and oxidative stress. Results Overall, both cell lines had similar patterns in response to the cytotoxicity and oxidative stress of MWCNTs. DNA salt treatment showed no change compared to the blank control. In both cell lines, significant changes at the doses of 25 and 100 ug/mL treatments were found in cell viabilities, cytotoxicity, and oxidative stress indexes. The reactive oxygen species （ROS） generation was also found to be significantly higher at the dose of 10 ug/mL treatment, whereas no change was seen in most of the indexes. The ROS generation in both cell lines went up in minutes, reached the climax within an hour and faded down after several hours. Conclusion Exposure to MWCNTs resulted in a dose-dependent cytotoxicity in cultured RAW264.7 cells and A549 cells, that was closely correlated to the increased oxidative stress.

Evaluation of antibody-dependent cell-mediatedcy totoxicity activity and cetuximab response in KRAS wildtype metastatic colorectal cancer patients

AIM:To investigate the prognostic role of invariant natural killer T(iNKT) cells and antibody-dependent cell-mediated cytotoxicity(ADCC) in wild type KRAS metastatic colorectal cancer(mC RC) patients treated with cetuximab.METHODS: Forty-one KRAS wt mC RC patients,treated with cetuximab and irinotecan-based chemotherapy in Ⅱ and Ⅲ lines were analyzed. Genotyping of single nucleotide polymorphism(SNP)s in the FCGR2A,FCGR3A and in the 3' untranslated regions of KRAS and mutational analysis for KRAS,BRAF and NRAS genes was determined either by sequencing or allelic discrimination assays. Enriched NK cells were obtained from lymphoprepperipheral blood mononuclear cell and iN KT cells were defined by co-expression of CD3,TCRVα24,TCRVβ11. ADCC was evaluated as ex vivo NK-dependent activity,measuring lactate dehydrogenase release.RESULTS: At basal,mCRC patients performing ADCC activity above the median level(71%) showed an improved overall survival(OS) compared to patients with ADCC below(median 16 vs 8 mo;P=0.026). We did not find any significant correlation of iN KT cells with OS(P=0.19),albeit we observed a trend to a longer survival after 10 mo in patients with iN KT above median basal level(0.382 cells/microliter). Correlation of OS and progression-free survival(PFS) with interesting SNPs involved in ADCC ability revealed not to be significant. Patients carrying alleles both with A in FCGR2 A and TT in FCGR3A presented a trend of longer PFS(median 9 vs 5 mo;P=0.064). Chemotherapy impacted both iN KT cells and ADCC activity. Their prognostic values get lost when we analysed them after 2 and 4 mo of treatment.CONCLUSION: Our results suggest a link between iN KT cells,basal ADCC activity,genotypes in FCGR2A and FCGR3A,and efficacy of cetuximab in KRAS wt mC RC patients.

Cytotoxicity and genotoxicity assessment of Euphorbia hirta in MCF-7cell line model using comet assay

Objective:To evaluate the cytotoxicity and genotoxicity activity of Euphorbia hirta(E.hirta)in MCF-7 cell line model using comet assay.Methods:The cytotoxicity of E.hirta extract was investigated by employing brine shrimp lethality assay and the genotoxicity of E.hirta was assessed by using Comet assay.Results:Both toxicity tests exhibited significant toxicity result.In the comet assay,the E.hirta extract exhibited genotoxicity effects against MCF-7 DNA in a time-dependent manner by increasing mean percentage of DNA damage.The extract of E.hirta showed significant toxicity against brine shrimp with an LC_(50)value of 620.382μg/mL(24 h).Comparison with positive control potassium dichroniate signifies that cytotoxicity exhibited by the methanol extract might have moderate activity.Conclusion:The present work confirmed the cytotoxicity and genotoxicity of E.hirta.However,the observed toxicity of E.hta extracts needs to be confirmed in additional studies.

Cytotoxicity of Benzo(a)pyrene 7,8-dihydrodiol on Genetically Engineered V79 Chinese Hamster Cells Expressing Rat Liver Cytochrome P450 1A1

To develop well defined in vitro cell system to test cytotoxicity of a number of model toxins, genetically engineered V79 Chinese hamster fibroblasts expressing isoenzymes of cytochrome P4501A1 XEM2 cells and V79 cells (parental), which lack cytochrome P450 enzyme activities, were used as controls. The cytotoxic effect of trans 7,8 dihydrbenzo(a)pyrene (BP 7,8 diol) on the parental cells V79 and V79 derived XEM2 cells were evaluated by two methods for cell viability. The data obtained expressed that BP 7,8 diol ranging from 1.0 μmol·L 1 to 5.0 μmol·L 1 in concentrations incubated for 24 h showed a strong cytotoxic effect in XEM2 cells (expressing rat cytochrome P4501A1) in a concentration dependent manner. Time dependent decrease for survival of XEM2 cells was also observed at 2.5 μmol·L 1 concentration. Likewise, BP 7,8 diol did not alter the survival of the parental cells V79 under the same condition. This study also showed that α naphthoflavone (αNF), a well known inhibitor of cytochrome P4501A1 might alter BP 7,8 diol induced cytotoxicity in the XEM2 cells. Our results suggested that cytochrome P4501A1 is responsible for BP 7, 8 diol induced cytotoxicity.

Mycobacterium smegmatis enhances shikonin-induced immunogenic cell death—an efficient in situ tumor vaccine strategy

Tumor vaccines are a promising avenue in cancer immunotherapy.Despite the progress in targeting specific immune epitopes,tumor cells lacking these epitopes can evade the treatment.Here,we aimed to construct an efficient in situ tumor vaccine called Vac-SM,utilizing shikonin(SKN)to induce immunogenic cell death(ICD)and Mycobacterium smegmatis as an immune adjuvant to enhance in situ tumor vaccine efficacy.SKN showed a dose-dependent and time-dependent cytotoxic effect on the tumor cell line and induced ICD in tumor cells as evidenced by the CCK-8 assay and the detection of the expression of relevant indicators,respectively.Compared with the control group,the in situ Vac-SM injection in mouse subcutaneous metastatic tumors significantly inhibited tumor growth and distant tumor metastasis,while also improving survival rates.Mycobacterium smegmatis effectively induced maturation and activation of bone marrow-derived dendritic cells(DCs),and in vivo tumor-draining lymph nodes showed an increased maturation of DCs and a higher proportion of effector memory T-cell subsets with the Vac-SM treatment,based on flow cytometry analysis results.Collectively,the Vac-SM vaccine effectively induces ICD,improves antigen presentation by DCs,activates a specific systemic antitumor T-cell immune response,exhibits a favorable safety profile,and holds the promise for clinical translation for local tumor immunotherapy.

Cytotoxicity Research of Recombinant Human Lactoferrin on Primary Hepatocyte and Nephrocyte Cell

[Objective] The aim was to study whether recombinant Human Lactoferrin has toxic effect on Primary Hepatocyte and Nephrocyte Cell of rat to provide reference for further safety evaluation.[Method] Recombinant Human Lactoferrin and its digested products were taken as tested compound,cow Lactoferrin was used for contrast.Primary Hepatocyte and Nephrocyte Cell of rat were cultured and IC50 values were tested by MTT,and cytotoxic dose-response relationship was tested.[Result]Target toxicity was not found from recombinant Human Lactoferrin on hepatocytes and nephrocytes,in accordance with sub-chronic toxicity test.[Conclusion] This study is of reference value for further safety evaluation of recombinant Human Lactoferrin and safety of evaluation method of GM food.

Cytotoxicity of methanol extracts of Elaeis guineensis on MCF-7 and Vero cell lines

Objective:To investigate the cytotoxic effect of Elaeis guineensis methanol extract on MCF-7and Vero cell.Methods:In vitro cytotoxicity was evaluated in by MTT assay.Cell morphological changes were observed by using light microscope.Results:The MTT assay indicated that methanol extract of the plant exhibited significant cytotoxic effects on MCF-7.Morphological alteration of the cell lines after exposure with lilaeis guineensis extract were observed under phase contrast microscope in the dose dependent manner.Conclusions:The results suggest the probable use of the Elaeis guineensis methanol extract in preparing recipes for cancer-related ailments.Further studies on isolation of metabolites and their in vivo cytotoxicity are under investigation.

Microwave-induced Apoptosis and Cytotoxicity of NK Cells through ERK1/2 Signaling

Objective To investigate microwave-induced morphological and functional injury of natural killer（NK） cells and uncover their mechanisms. Methods NK-92 cells were exposed to 10, 30, and 50 m W/cm^2 microwaves for 5 min. Ultrastructural changes, cellular apoptosis and cell cycle regulation were detected at 1 h and 24 h after exposure. Cytotoxic activity was assayed at 1 h after exposure, while perforin and NKG2 D expression were detected at 1 h, 6 h, and 12 h after exposure. To clarify the mechanisms, phosphorylated ERK（p-ERK） was detected at 1 h after exposure. Moreover, microwave-induced cellular apoptosis and cell cycle regulation were analyzed after blockade of ERK signaling by using U0126. Results Microwave-induced morphological and ultrastructural injury, dose-dependent apoptosis（P 〈 0.001） and cell cycle arrest（P 〈 0.001） were detected at 1 h after microwave exposure. Moreover, significant apoptosis was still detected at 24 h after 50 m W/cm^2 microwave exposure（P 〈 0.01）. In the 30 m W/cm^2 microwave exposure model, microwaves impaired the cytotoxic activity of NK-92 cells at 1 h and down regulated perforin protein both at 1 h and 6 h after exposure（P 〈 0.05）. Furthermore, p-ERK was down regulated at 1 h after exposure（P 〈 0.05）, while ERK blockade significantly promoted microwave-induced apoptosis（P 〈 0.05） and downregulation of perforin（P 〈 0.01）. Conclusion Microwave dose-dependently induced morphological and functional injury in NK-92 cells, possibly through ERK-mediated regulation of apoptosis and perforin expression.

In vitro acute cytotoxicity of the neonicotinoid insecticide acetamiprid to FG cells, the gill cell line of flounder Paralichthy olivaceus

The in vitro acute cytotoxicity of the neonicotinoid insecticide acetamiprid to FG cell line, derived from the gill of flounder Paralichthys olizaceus, was tested by the three widely used endpoint bioassays-neutral red （NR） assay, tetrazolium （MTT） assay and cell protein assay. It was found that acetamiprid was increasingly toxic to FG cells at concentrations of 1 μg/cm^3 or above, and the inhibitory concentration 50% values for NR, MTF, and cell protein assays were 38.38, 36.27 and 32.03 μg/cm^3, respectively. This appeared to be the first report on the in vitro cytotoxicity of acetamiprid to non-mammalian vertebrate cells. Ultrastructural examination revealed that for the cells exposed to 60 μg/cm^3 acetamiprid for 48 h, their mitochondria were severely damaged with the cristae swelled up or disrupted, while their nuclei and rough endoplasmic reticlum （RER） appeared to be still normal. This suggests that mitochondria are possibly the primary target of acetamiprid.

Cytotoxicity study of ethanol extract of the stem bark of asam kandis (Garcinia cowa Roxb.) on T47D breast cancer cell line

Objective:To investigate the cytotoxic effect of ethanol extract of the stem bark of asam kandis[Garcinia cowa Roxb.(G.cowa)]on T47 D breast cancer cell line.Methods:The cytotoxicity of ethanol extract was carried out against human breast cancer cell line(T47D) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay.The extract was added at various concentrations(0.1.1,10 and 100 μg/mL).The level of cytotoxicity was determined by calculating the level of IC_(50),that was based on the percentage of the cell death after 24 h treatment with the extract.Cell morphological changes were observed by using inverted microscope.Results:The 3-(4.5-dimelhylthiazol-2-yl)-2.5-diphenyltelrazolium bromide assay showed that ethanol extract of G.cowa exhibited significant cytotoxic effect on T47 D with IC_(50) value of(5.10+1.68) μg/mL.Morphological alteration of the cell lines after exposure to ethanol extract of G.cowa was observed under phase contrast microscope in a dosc-dependent manner.ConclusionsThe results suggest the possible use of ethanol extract of asam kandis for preparing herbal medicine for cancer-related ailments.

In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines

Objective:To screen crude extracts of propolis,bee pollen and honey from four stingless bee species[Trigona incisa(T.incisa)],Timia apicalis,Trigona fuso-baltata and Trigona filscibasis)native to East Kalimantan.Indonesia for cytotoxic activity against five human cancer cell lines(HepG2,SW620,ChaGo-1,KATO-Ⅲand BT474).Methods:All samples were extracted with methanol,and then subpartitioned with n-hexane and ethyl acetate.Each crude extract was screened at 20μg/mL for in vitro cytotoxicity against the cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Tn addition,four previously shown bioactive components from propolis(apigenin,cafieic acid phenyl ester,kaempferol and naringenin)and two chemotherapeutic drugs(doxorubicin and 5-fluorouracil)were used to evaluate the sensitivity of the cell lines.Results:Overall,crude extracts from propolis and honey had higher cytotoxic activities than bee pollen,but the activity was dependent upon the extraction solvent,bee species and cell line.Propolis extracts from T.incisa and Tarda apicalis showed the highest and lowest cytotoxic activity,respectively.Only the HepG2 cell line was broadly sensitive to the honey extracts.For pure compounds,doxorubicin was the most cytotoxic,the four propolis compounds the least,but the ChaGo-I cell line was sensitive to kaempferol at 10μg/mL and KATO-Ⅲwas sensitive to kaempferol and apigenin at 10μg/mL,.All pure compounds were effective against the BT474 cell line.Conclusions:Propolis from f,incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines.Further study is required,including the isolation and characterization of the active antiproliferative agent(s).

Bio-compatibility and cytotoxicity studies of water-soluble CuInS_2-ZnS-AFP fluorescence probe in liver cancer cells

BACKGROUND： The oncogenesis of hepatocellular carcinoma（HCC） is not clear. The current methods of the pertinent studies are not precise and sensitive. The present study was to use liver cancer cell line to explore the bio-compatibility and cytotoxicity of ternary quantum dots（QDs） probe and to evaluate the possible application of QDs in HCC.METHODS： CuInS_2-ZnS-AFP fluorescence probe was designed and synthesized to label the liver cancer cell HepG 2. The cytotoxicity of CuInS_2-ZnS-AFP probe was evaluated by MTT experiments and flow cytometry. RESULTS： The labeling experiments indicated that CuInS_2-ZnS QDs conjugated with AFP antibody could enter HepG 2 cells effectively and emit intensive yellow fluorescence by ultraviolet excitation without changing cellular morphology. Toxicity tests suggested that the cytotoxicity of CuInS_2-ZnS-AFP probe was significantly lower than that of CdT e-ZnS-AFP probe（t test, F=0.8, T=-69.326, P〈0.001）. For CuInS_2-ZnS-AFP probe, timeeffect relationship was presented in intermediate concentration（〉20%） groups（P〈0.05） and dose-effect relationship was presented in almost all of the groups（P〈0.05）. CONCLUSION： CuInS_2-ZnS-AFP QDs probe had better biocompatibility and lower cytotoxicity compared with CdT e-ZnS-AFP probe, and could be used for imaging the living cells in vitro.

Comparison of cytotoxicity and wound healing effect of carboxymethylcellulose and hyaluronic acid on human corneal epithelial cells

AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs were exposed to 0.5% CMC(Refresh plus, Allergan, Irvine, California, USA) and 0.1% and 0.3%HA(Kynex , Alcon, Seoul, Korea, and Hyalein mini,Santen, Osaka, Japan) for the period of 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase(LDH) leakage assay to assess the cytotoxicity. apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24 h after confluent HCECs were scratch wounded.RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells weredemonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC.CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.

Cytotoxicity of nonylphenol on spermatogonial stem cells via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway

BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stable degradation product widely used in daily life and production and has been proven to affect male fertility.However,the underlying mechanisms therein are unclear.Thus,it is necessary to study the effect and mechanism of NP on spermatogonial stem cells(SSCs).AIM To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway.METHODS SSCs were treated with NP at 0,10,20 or 30μmol.MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs.Flow cytometry was conducted to measure SSC apoptosis.The expression of Bad,Bcl-2,cytochrome-c,pro-Caspase 9,SOX-2,OCT-4,Nanog,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot,and the mRNA expression of SOX-2,OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction.RESULTS Compared with untreated cells(0μmol NP),SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2,Nanog,OCT-4,SOX-2,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,and PLZF(P<0.05),whereas the expression of Bad,cytochrome-c,and pro-Caspase 9 increased significantly(P<0.05).We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K,AKT,mTORC1,and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs(P<0.05).NP exerted the greatest effect at 30μmol among all NP concentrations.CONCLUSION NP attenuated the proliferation,differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress.The associated mechanism may be related to the PI3K/AKT/mTOR pathway.

Evaluation of Cytotoxicity and Genotoxicity of Insecticide Carbaryl to Flounder Gill Cells and Its Teratogenicity to Zebrafish Embryos

In this study, we determined the cytotoxicity and genotoxicity of carbamate insecticide carbaryl to flounder gill （FG） cells and its teratogenicity to zebrafish embryos. The cytotoxicity of carbaryl to FG cells was determined with methods including MTT and neutral red uptaking （NRU）, lactate dehydrogenase （LDH） releasing and Hoechst 33342 and propidiurn idodide （PI） double staining. Moderate cytotoxicity in a concentration-dependent manner was observed. The 24h-IC50 value of 53.48 ± 1.21, 59.13 ± 1.19 and 46.21 ± 1.24 mgL 1 carbaryl was obtained through MTT, NRU and LDH assays, respectively. Double fluorescence staining dem- onstrated that carbaryl induced the death of FG cells mainly through necrosis. There was no significant genotoxicity found in the FG cells exposed to the highest testing concentration of carbaryl （20mgL-1, P〉0.05） as was demonstrated by Comet assay. Zebrafish embryos exposed to carbaryl at concentrations 〉10 mg L-1 displayed moderate toxic effects on the survival, spontaneous movement, hatching, heart rates of the embryos and their development, which were evidenced by yolk and pericardial sac edemas, body length reduction and tail flexure in time- and concentration-dependent manners at specific stages. The 24h-, 48h- and 96h-LC50 values of carbaryl to zebra fish embryos were 41.80 ± 1.10, 17.80 ± 1.04 and 14.46 ± 1.05 mg L-1, respectively. These results suggested that car- baryl is moderately toxic to FG ceils cultured in vitro and zebrafish embryos, and the FG cells were similar to zebrafish embryos in their sensitivity to carbaryl as 24h-IC50 and LC50 indicated.

Cytotoxicity of acrylamide and its epoxide glycidamide in CHO cells expressing human cytochrome P450 2E1

Objective： To investigate whether CYP2E1 is responsible for the acrylamide metabolic activation in FIp-In CHO cell system. Methods： CYP2E1 cDNA was subcloned from the human liver full-length cDNA library and subsequently transfected into the FIp-In CHO cells to generate the stable transfectant of CYP2E1. The CYP2E1 mRNA expression was determined by RT-PCR. Acrylamide and its epoxide glycidamide induced cytotoxicity and cell cycle arrest in G2/M were conducted using MTS assay and flow cytometry, respectively. Results： In the CHO cell stably expressing CYP2E1 （CHO-2E1）, a -1.5 kb size of band was detected from the mRNA in the cells while no corresponding band in the CHO-vector cells, which indicated that CYP2E1 was successfully transfected in the CHO cells. Compared with the CHO-vector cells, acrylamide showed a concentration dependent loss of viability in the CHO-2E1 cells but no significant change of G2/M arrest was found. As expected, glycidamide induced similar profile of cytotoxicity in both of the cells, and G2/M arrest presented a concentration-dependent increased in the CHO-2E1 cells. Conclusion： The result suggested that CYP2E1 might be responsible for the acrylamide metabolism, and its metabolite glycidamide was a direct cytotoxic and genotoxic agent. It should be further considered whether acrylamide-induced toxicity is through its epoxide glycidamide in the presence of CYP2E1.

CYTOTOXICITY AND GENOTOXICITY OF POLYETHYLENIMINE AND NICKEL CHLORIDE IN RED SEA BREAM (Pagrosomus major) FIN CELL LINE RSBF

A continuous marine fish cell line RSBF (i.e. Red Sea Bream Fin) was utilized to screen the cytotoxicity and genotoxicity of polyethylenimine (PEI) and nickel chloride (NiCl 2) in this study on the deleterious effects of aquatic genotoxins on fish. At the 0.01 to 1 μg/ml concentration tested, PEI had acute toxicity to the treated RSBF cells (IC 50 =1.12, 0.92, 0.88 and 0.64 μg/ml PEI for time 0 h, 24 h, 48 h and 72 h after treatment, respectively) and markedly inhibited their proliferation in a dose dependent manner. At the 0.001 to 5 μmol/L concentration tested, NiCl 2 posed no acute toxicity but significantly stimulated their growth (107%-214% of control). Random amplified polymorphic DNA (RAPD) technique was used to detect the genotoxic effects of PEI and NiCl 2 by comparing the RAPD banding patterns of the control and treated cells. RAPD analysis indicated that at the concentrations tested, PEI was more genotoxic than NiCl 2 to RSBF cells; that there was a slight dose dependent response in the genotoxic effect of PEI but not NiCl 2; and that RAPD technique might provide a sensitive, non specific genotoxic endpoint. And the potent cytotoxicity and genotoxicity of PEI on fish cells showed that we should be cautious in utilizing it as gene vector in fish gene transfer and human gene therapy.

Cytotoxicity and genome-wide microarray analysis of intestinal smooth muscle cells in response to hexavalent chromium induction

Chronic ingestion of high concentrations of hexavalent chromium[Cr(VI)]in drinking water induces intestinal tumors in mice;however,information on its toxicity on intestinal smooth muscle cells is limited.The present study aimed to assess the in vitro and in vivo toxicological effects of Cr(VI)on intestinal smooth muscle cells.Human intestinal smooth muscle cells(HISM cells)were cultured with different concentrations of Cr(VI)to evaluate effects on cell proliferation ability,oxidative stress levels,and antioxidant system.Furthermore,tissue sections in Cr(VI)exposed rabbits were analyzed to evaluate toxicity on intestinal muscle cells in vivo.Gene chips were utilized to assess differential gene expression profiles at the genome-wide level in 1μmol/L Cr(VI)treated cells.Intestinal tissue biopsy results showed that Cr(VI)increased the incidences of diffuse epithelial hyperplasia in intestinal jejunum but caused no obvious damage to the structure of the muscularis.Cell proliferation analysis revealed that high concentrations(≥64μmol/L)but not low concentrations of Cr(VI)(≤16μmol/L)significantly inhibited the growth of HISM cells.For oxidative stress levels,the expression of reactive oxygen species(ROS)and nitric oxide(NO)was elevated at high concentrations(≥64μmol/L)but not at low concentrations of Cr(VI)(≤16μmol/L).In addition,dose-dependent increases in the activity of oxidized glutathione(GSSH)/total-glutathione(T-GSH)were also observed.Gene chip screened 491 differentially expressed genes including genes associated with cell apoptosis,oxidations,and cytoskeletons.Some of these differentially expressed genes may be unique to smooth muscle cells in response to Cr(VI)induction.

Artemyrianins A-G from Artemisia myriantha and Their Cytotoxicity Against HepG2 Cells

Four new sesquiterpenoids,artemyrianins A-D(1-4),and three new norlignans,artemyrianins E-G(5-7),together with five known compounds(8-12),were isolated from the aerial parts of Artemisia myriantha(Asteraceae).The new compounds were established by spectroscopic data analyses(HRMS,IR,1D and 2D NMR),and their absolute configurations were confirmed by the single-crystal X-ray diffraction or ECD calculations.The isolates showed cytotoxicity against HepG2 cells with IC50 values ranging from 33.3 to 145.2μM.