Inhibitory effect of distilled water on viability of human lens epithelial cells in vitro / 中华实验眼科杂志

Objective:To investigate the effect of distilled water on the viability of human lens epithelial cells (LECs) cultured in vitro. Methods:A total of 156 anterior capsule specimens were collected from 156 patients (156 eyes) who were diagnosed with age-related cataract during phacoemulsification combined with intraocular lens implantation from May to December 2020 in Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School.The 156 specimens were divided into 312 small pieces.Of the 312 pieces, 157 pieces were divided into normal control group (23 pieces), positive control group (10 pieces), balanced salt solution (BSS) immersion group (61 pieces) and distilled water immersion group (63 pieces) using computer-generated random numbers.Normal control group received no treatment.Positive control group was directly fixed with a mass fraction of 4% histiocytes fixative solution.For the 61 pieces in BSS immersion group, 20 pieces were soaked for 1 minute, 21 pieces for 2 minutes, and 20 pieces for 3 minutes.For the 63 pieces in distilled water immersion group, 20 pieces were soaked for 1 minute, 23 pieces for 2 minutes, and 20 pieces for 3 minutes.Another 125 pieces were selected to simulate the cataract aspiration-irrigation according to the treatment in BSS immersion group and distilled water immersion group respectively, plus rinse in a bottle containing BSS at a height of 70 cm for 1 minute.Cell viability was detected by trypan blue-eosin staining.LECs density, dead cell count, cell death rate and percentage of shedding (%) were calculated.Of the remaining 30 pieces, every 15 pieces were divided into normal control group, BSS immersion group, and distilled water immersion for 1, 2 and 3 minutes groups, with 3 pieces in each group.BSS immersion group was immersed for 3 minutes, and the other four groups were treated as mentioned above, and the LECs structure of the four groups was observed by light microscopy and transmission electron microscopy.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School (No.2019-248-01). Written informed consent was obtained from each subject.Results:The boundaries of LECs in BSS treatment groups were clear, and there was no significant difference in morphology compared with normal control group.With time increasing, LECs in distilled water treatment groups gradually swelled, and the boundaries of dead cells were not clear.There were significant differences in LECs density, dead LECs count and LECs mortality ( F=13.459, 98.918, 130.600; all at P<0.001). The LECs density was lower in 2-minute and 3-minute distilled water treatment groups than in normal control group, showing statistically significant differences (both at P<0.05). The dead LECs count and LECs mortality were higher in 1-minute, 2-minute and 3-minute distilled water treatment groups than in normal control group and BSS treatment groups for the same time, and the differences were statistically significant (all at P<0.05). Only a few shed LECs were seen in normal control group, 1-minute, 2-minute and 3-minute BSS treatment groups, and BSS immersion combined rinse group.After different time of soaking, there were more shed LECs in distilled water immersion combined rinse group, and the range of LECs shedding increased with the extension of distilled water immersion.There was a significant difference in the shedding percentage of LECs among different groups ( F=123.670, P<0.001). The shedding percentages of LECs at different time points were higher in distilled water immersion groups and distilled water immersion combined rinse groups than in normal control group, and the difference was statistically significant (all at P<0.05). The shedding percentage of LECs increased significantly in distilled water immersion groups with the extension of immersion.Light microscopy showed that the cells were destroyed in 1-minute, 2-minute and 3-minute distilled water treatment groups, and some LECs shed in the 2-minute and 3-minute treatment groups.Transmission electron microscopy showed cell lysis and destruction, suborganelles swelling, disruption of intercellular junctions in 1-minute, 2-minute and 3-minute distilled water treatment groups, loose attachment between cells and capsule in the 2-minute and 3-minute treatment groups, and cell detachment from capsule in the 3-minute treatment group. Conclusions:Distilled water immersion leads to LECs death in a time-dependent manner, and distilled water immersion combined with rinse can remove LECs on the lens capsule.

The establishment of the immortalized mouse brain microvascular pericytes model and its preliminary application in screening of cerebrovascular toxicants / 中华预防医学杂志

Objective@#To establish the immortalized mouse brain microvascular pericytes model and to apply to the cerebrovascular toxicants screening study.@*Methods@#Brain pericytes were isolated from 3 weeks of mice by tissue digestion. Immortalized pericyte cell line was constructed by infecting with LT retrovirus. Monoclone was selected to purify the immortalized pericyte cell line. The pericyte characteristics and purity were explored by immunocytochemistry. Cell proliferation was measured by using the Pomega MTS cell Proliferation Colorimetric Assay Kit. Pericytes were treated with 0, 160, 320, 640, 1 280, 2 560 μmol/L lead acetate, 0, 5, 10, 20, 40, 80 μmol/L cadmium chloride and 0, 5, 10, 20, 40, 80 μmol/L sodium arsenite in 24 hours. Cell toxicity of each group was determined by MTS assay, median lethal dose (LD50) was calculated in linear regression.@*Results@#Mouse brain pericytes were successfully isolated by tissue separation and enzyme digestion method. After immortalized by LT retroviruses, monoclone was selected and expanded to establish pericyte cell line. The brain pericytes exhibited typical long spindle morphology and positive staining for α-SMA and Vimentin. The proliferation of brain pericytes cell lines was very slowly, and the doubling time was about 48 hours. The proliferation of immortalized brain pericytes cell lines was very quickly, and the doubling time was about 24 hours. After lead acetate, cadmium chloride and sodium arsenite treatment for 24 hours respectively, gradual declines in cell viability were observed. The LD50 of lead acetate was 2 025.0 μmol/L, the LD50 of cadmium chloride was 36.6 μmol/L, and the LD50 of sodium arsenite was 33.2 μmol/L.@*Conclusion@#The immortalized mouse brain microvascular pericyte model is established successfully by infecting with LT retrovirus, and can be applied to screen cerebrovascular toxicants. The toxicity of these toxicants to immortalized mouse brain microvascular pericyte is in sequence: sodium arsenite,cadmium chloride, lead acetate.

Toxicity of 1,2-dichloroethane and its metabolites on human astrocytes / 中国职业医学

OBJECTIVE: To explore the toxicity of 1,2-dichloroethane( 1,2-DCE) and its metabolites on human astrocytes( HAs). METHODS: Different doses of 1,2-DCE( 5. 00,10. 00,25. 00,50. 00 and 100. 00 mmol/L),2-chlorohydrins( 5. 00,25. 00,50. 00,100. 00 and 200. 00 mmol/L),2-chloroacetaldehyde( 1. 00,5. 00,10. 00,20. 00 and 50. 00 mmol / L) and chloroacetic acid( 0. 01,0. 05,0. 10,0. 50 and 1. 00 mmol / L) were used for treating HAs in vitro during their logarithmic phase. After 24 hours of culture,the morphology of HAs was observed by fluorescent inverted phase contrast microscope. The survival rate and the inhibition ratio of HAs were detected by CCK-8 colorimetry to estimate the50% inhibiting concentration in 24 hours( 24 h-IC50). The apoptosis of HAs was tested by double-labeling and flow cytometry using Annexin Ⅴ-fluorescein isothiocyanate and propidium iodide. RESULTS: The morphology of HAs changed in varying degrees after 24 hours exposure to 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid. The changes included smaller size of cells,pseudopodia tapering,increased intracellular particles and suspension of circular cells and decreased transparency of cells. With the increasing does of 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid exposure,the survival rates of HAs decreased( P < 0. 01),while its inhibition ratios increased( P <0. 01). They all showed dose-effect relationship. 24 h-IC50 of the above 4 chemicals were 56. 25,235. 00,26. 43 and1. 38 mmol / L,respectively. The 1,2-DCE,2-chlorohydrins and chloroacetic acid could induce the apoptosis of HAs and the apoptosis rate of HAs was positively correlated with the 3 kinds of chemicals( P < 0. 01). CONCLUSION: 1,2-DCE and its metabolites 2-chloroacetaldehyde,2-chlorohydrins and chloroacetic acid can lead to toxic damage and induce the apoptosis of HAs. Chloroacetic acid has the strongest toxicity among the metabolites.

The hemostatic property of calcium alginate sponge: An experimental study / 解放军医学杂志

Objective To investigate the hemostatic property of calcium alginate (CA) sponge and its degree of safety on exterior use. Methods The optimal coagulation concentration of CA was determined by Lee-White clotting test, and then the CA was freeze-dried to form a sponge. The coagulation effect in vitro of the CA sponge was determined by Blood Clotting Index (BCI) test. The ear artery bleeding model and full-thickness skin wound model of rabbit were employed to determine the hemostatic property of CA sponge. The degree of safety of CA sponge was evaluated by cell toxicity test. Results The BCI was significantly decreased in CA sponge group (33.08±4.02) than that in gelatin sponge (72.05±10.48, P0.05), though they were all shorter significantly than that in hospital gauze group (101.00±14.71s, P<0.05). The cell toxicity of 100% CA sponge was level 1, and it complied with the safety requirements for biomaterials. Conclusion The CA sponge has a good hemostatic property without obvious cytotoxicity.

In vitro ZnCl2 cytotoxicity and genotoxicity in human leukocytes: Zero-order kinetic cellular zinc influx / Citotoxicidade e genotoxicidade do ZnCl2 em leucócitos humanos in vitro: influx cellular de zinco com cinética de ordem zero

Zinc (Zn) is an essential trace element for cellular viability, but concentrations above physiologic level may lead to cellular damage. The purpose of the present study was to evaluate the in vitro ZnCl2 genotoxicity and cytotoxicity in human leukocyte cells. This was assessed in an unprecedented way that correlated the level of intracellular Zn after cell exposition with the cellular damage. The exposure to increased Zn concentrations (2.5-20 µg mL-1), showed significantly reduced cellular leukocyte viability. However, significant DNA damages were observed only when the Zn exposure concentrations were from 10-20 µg mL-1. The Zn intracellular levels found in leukocytes was from 72.25-268.9 ρ g cell-1, starting to induce cytotoxicity and genotoxicity at concentrations of 95.68 and 126.2 ρg cell-1, respectively. The relationship between the exposure concentration and intracellular levels of Zn suggests that the influx of Zn, in the form of ZnCl2, occurs in human leukocytes under zero-order kinetics.

O Zinco (Zn) é um elemento traço essencial para a viabilidade celular, mas em concentrações acima dos níveis fisiológicos pode conduzir a danos celulares. A proposta do presente estudo foi avaliar a citotoxicidade e genotoxicidade do ZnCl2 em leucócitos humanos in vitro. De maneira sem precedentes, foi acessado o nível de Zn intracelular após exposição e relacionado com o nível de dano celular. A exposição a crescentes concentrações de Zn (2,5-20 µg mL-1), mostraram significante redução da viabilidade celular dos leucócitos. Entretanto, danos significativos ao DNA foram encontrados somente a partir das concentrações de exposição ao Zn de 10-20 µg mL-1. Os níveis intracelulares de Zn encontrados nos leucócitos foram de 72,25-268,9 ρg célula-1, começando a induzir citotoxicidade e genotoxicidade nas concentrações de 95,68 and 126,2 ρg célula-1, respectivamente. A relação entre a concentração de exposição e os níveis intracelulares de Zn sugerem que o influxo de Zn, sob a forma de ZnCl2, ocorre em cinética de ordem zero em leucócitos humanos.

Research on Stability and Bio-safety of Warming Moxibustion Gel-unguentum / 中国药师

Objective:To evaluate the stability and bio-safety of warming moxibustion gel-unguentums( WMG) . Methods: With the commercially available package and at 40℃ ± 2℃, RH (75% ± 5%) for six months,the stability of WMG was inspected. Guinea pig skin sensitization test, rabbit skin irritation test and cell toxicity test were used to investigate the safety. Results:Compared to the results of 0 month, there was no difference in the sample character, containing paste percentage, adhesion, shape, weight difference and all theindices in TLC identification. Cell toxicity test showed that WMG had slight cytotoxicity. Guinea pig skin sensitization test and rabbit skin irritation test indicated that the grade of skin reaction was zero and the primary stimulus index of skin tissues was also zero. Conclusion:The quality of WMG is stable and the production preparation technology is feasible. Moreover, it is safe in transder-mal administration.

Exposure and Toxicity Assessment of Ultrafine Particles from Nearby Traffic in Urban Air in Seoul, Korea

OBJECTIVES: We investigated the particle mass size distribution and chemical properties of air pollution particulate matter (PM) in the urban area and its capacity to induce cytotoxicity in human bronchial epithelial (BEAS-2B) cells. METHODS: To characterize the mass size distributions and chemical concentrations associated with urban PM, PM samples were collected by a 10-stage Micro-Orifice Uniform Deposit Impactor close to nearby traffic in an urban area from December 2007 to December 2009. PM samples for in vitro cytotoxicity testing were collected by a mini-volume air sampler with PM10 and PM2.5 inlets. RESULTS: The PM size distributions were bi-modal, peaking at 0.18 to 0.32 and 1.8 to 3.2 microm. The mass concentrations of the metals in fine particles (0.1 to 1.8 microm) accounted for 45.6 to 80.4% of the mass concentrations of metals in PM10. The mass proportions of fine particles of the pollutants related to traffic emission, lead (80.4%), cadmium (69.0%), and chromium (63.8%) were higher than those of other metals. Iron was the dominant transition metal in the particles, accounting for 64.3% of the PM10 mass in all the samples. We observed PM concentration-dependent cytotoxic effects on BEAS-2B cells. CONCLUSIONS: We found that exposure to PM2.5 and PM10 from a nearby traffic area induced significant increases in protein expression of inflammatory cytokines (IL-6 and IL-8). The cell death rate and release of cytokines in response to the PM2.5 treatment were higher than those with PM10. The combined results support the hypothesis that ultrafine particles from vehicular sources can induce inflammatory responses related to environmental respiratory injury.

Effects of Calf Serum on Human Corneal Epithelial Cells in Vitro

PURPOSE: To investigate the biologic effects of topical calf serum on corneal epithelial cells in vitro. METHODS: The effects of calf serum on the corneal epithelial cells were evaluated using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, and the concentration of IL-1alpha, TGF-beta1 and MMP-9 in the cells was measured. Cell damage was determined using lactate dehydrogenase (LDH), and cellular morphologies were examined by transmission electromicroscopy. RESULTS: Metabolic activity of the corneal epithelial cells decreased at higher concentrations and longer exposure durations. IL-1alpha, TGF-beta1 and MMP-9 titers were lower in calf serum-treated cells than in the control. LDH and cellular damage to the corneal epithelial cells, such as chromatin margination and cytoplasmic organelle swelling, were prominent in cells treated with 30% calf serum. CONCLUSIONS: Cellular metabolic activity was higher and cellular toxicity was lower in cells treated with 10% calf serum compared to those treated with the 20% and 30% concentrations. Furthermore, inflammatory cytokines were sufficiently inhibited in cells treated with the 10% solution. These results indicate that 10% calf serum could be used clinically.

Comparison of the Efficacy of Topical Antihistamine/Mast Cell Stabilizers in vitro

PURPOSE: To investigate the biologic effects of topical anti-allergic agents with H1-receptor antagonism and inhibition of histamine release from mast cells in the cultured conjunctival cells of patients with vernal keratoconjunctivitis in vitro. METHODS: Conjunctival cells of vernal keratoconjunctivitis were exposed to the anti-allergic agents SCD-P101 (Fexofenadine, Samchundang, Korea), Patanol(R) (Alcon, USA), Zaditen(R) (Novartis, USA), and Azelan(R) (Taejoon, Korea). Efficacy of the topical antihistamine/mast cell stabilizers was evaluated using the MTT assay, measuring the concentration of procollagen and inflammatory cytokines. Cell damage was determined using the lactate dehydrogenase (LDH) assay with dilution rates of 10, 20, and 30% and compared with the balanced salt solution-treated group. Cellular morphologic results were examined by inverted light microscopy and transmission electromicroscopy. RESULTS: Metabolic activity of conjunctival cells decreased at higher concentrations and longer exposure durations, except for the SCD-P101 agent. The procollagen, laminin, IL-6 and IL-8 titers tended to be lower than that of the control in the eyes exposed to all the anti-allergic drugs tested in this study, but the concentration of TNF-beta was similar to that of the control group. Zaditen(R) and Azelan(R) tended to show a greater LDH titer and edema, as well as cytoplasmic and nuclear degeneration of the conjunctival cells than did SCD-P101 or Patanol(R). CONCLUSIONS: Cellular metabolic activity was the highest in the new anti-allergic agent SCD-P101. SCD-P101 and Patanol(R) caused marginally less damage to cultured conjunctival cells than did Zaditen(R) and Azelan(R).

Effect of Artificial Tears Used in Contact Lens-wearing Eyes on Human Corneal Epithelial Cells in Vitro

PURPOSE: To investigate the biologic effects of topical ocular artificial tears used in patients wearing contact lens on in vitro corneal epithelial cells. METHODS: The efficacies of the topical artificial tears Iris(R), Irisplus(R), Eyemiru contact pure(R), and Eye2O(R) were evaluated using the MTT and wound healing assays. Cell damage was determined using lactate dehydrogenase (LDH), and the solution ingredients were analyzed. Cellular morphologies were examined by inverted light microscopy and transmission electromicroscopy. RESULTS: Metabolic activity of corneal epithelial cells, as determined by the MTT assay, decreased in the Iris(R) eye drop group, but those of the other groups were similar to that of the control. The LDH titers increased up to one hour after Iris(R) eye drop use, and the increased level was maintained for 24 hours. The other three artificial tears showed similar low LDH titers to that of the control. Cellular migration was not observed, although cellular damage to the corneal epithelial cells, such as chromatin margination and cytoplasmic organelle swelling, was prominent with Iris(R) use. CONCLUSIONS: Among four brands of topical artificial tear drops used among patients wearing contact lens, Iris(R) caused markedly more severe damage to cultured human corneal epithelial cells than did Irisplus(R), Eyemiru contact pure(R), or Eye2O(R).

Effect of Preservative-free Artificial Eye Drop on Human Corneal Epithelial Cell in vitro

PURPOSE: To investigate the biologic effects of preservative-free artificial tear drops on cultured human corneal epithelial cells in vitro. METHODS: Efficacies of the preservative-free artificial tear drops-Kynex(R), Hyalein Mini 0.3%(R), and Refresh Plus(R)-were evaluated using the MTT assay. Cell damage was determined using the lactate dehydrogenase (LDH) assay. Cellular proliferation was determined using a migration and wound-healing assay. The ingredients of the drugs were analyzed. Apoptotic response was evaluated with flow cytometric analysis. RESULTS: Metabolic activity of the corneal epithelial cells showed similar activity to that of the control. Cellular migration and proliferation also were not significantly different between the preservative-free artificial tear drop groups and the control. The LDH titers tended to increase for up to 24 hours after exposure to the preservative-free artificial tear drops, but there was no significant difference in LDH titers between the control groups and the artificial tear drop-treated groups. Apoptosis and necrosis were observed using flow cytometry at 24 hours in all groups. The electrolyte levels, pHs and osmolarities of the three drugs were not significantly different. CONCLUSIONS: The clinically available preservative-free artificial tear drops Kynex(R), Hyalein Mini 0.3%(R), and Refresh Plus(R) had no significant toxic effects on corneal epithelial cells and thus can be used safely.

Anti-tumor activity of extracts from cultured cells of Taxus chinensis on cancer cell lines SMMC-7721 and HEp-2 / 中国药理学通报

AIM To analyze the toxicity and inhibitory mechanism of extracts from cultured cells (F 4 cell line) of Taxus chinensis on cancer cell lines SMMC 7721 and HEp 2. METHODS MTT assay for cell viability and flow cytometry for cell cycle analysis. RESULTS IC 50 of SMMC 7721 and HEp 2 were 0 161 4 g DCW?L -1 and 0 275 6 g DCW?L -1 respectively,tumor cells in G 2～M stage all increased with higher concentration and longer incubation of extracts from Taxus chinensis cells. CONCLUSION Extracts from cultured cells of Taxus chinensis could have cytotoxic effect on SMMC 7721 and HEp 2 and could induce apoptosis of both two cancer cells.

Advances in the Toxicity of Pentacholrophenol on Organism / 环境与健康杂志

As a kind of pesticide, fungicide and preservative, PCP had been used extensively in industry, agriculture and domesticity throughout the world. PCP contamination is generally associated with sediments or soil, it can also concentrate in organism. Regarding the character of high toxicity, long persistence and difficult to degrade, PCP has become a kind of conspicuous environmental pollutant because of widely use and inappropriate disposal. In the contaminated area, PCP can be detected in the water, soil and the body of organisms. PCP can affect human health through directly exposure or through food chain. The absorbed PCP can be stored in liver, kidney and fat,it can also increase the incidence rate of tumork, disturb the endocrine system, affect immune function,inhibit reproduction and development. PCP not only has a direct impairment on human body but also shows a potential impact in genetics.

Elementary Evaluation of the Safety of Chitosan DCX-16 / 中国药房

OBJECTIVE:To evaluate the safety of Chitosan DCX-16elementarily.METHODS:DCX-16was injected i.p.to observe the acute toxicity in mice.The local irritating effects were observed on tolerance test,cerato-conjunctiva and muscles stimulus test in rabbits.The cell shape and proliferative rate of3T3cells were determinated by MTT in cell culture with DCX-16.RESULTS:It showed that DCX-16had no irritation on the eyes and muscles in rabbits.Tolerance dose of DCX-16in mice was as high as3.0g/kg per day.Cell culture with DCX-16demonstrated that3T3cell's shape and growth were normal.The relative growth rate of3T3cells had no statistical difference between control and DCX-16groups on the2nd,3rd,4th day.CONCLUSION:DCX-16is safe.