Half Lethal Dose Research Articles

Relative Neurotoxicity of Ivermectin and Moxidectin in Mdr1ab (−/−) Mice and Effects on Mammalian GABA(A) Channel Activity

The anthelmintics ivermectin (IVM) and moxidectin (MOX) display differences in toxicity in several host species. Entrance into the brain is restricted by the P-glycoprotein (P-gp) efflux transporter, while toxicity is mediated through the brain GABA(A) receptors. This study compared the toxicity of IVM and MOX in vivo and their interaction with GABA(A) receptors in vitro. Drug toxicity was assessed in Mdr1ab(−/−) mice P-gp-deficient after subcutaneous administration of increasing doses (0.11–2.0 and 0.23–12.9 µmol/kg for IVM and MOX in P-gp-deficient mice and half lethal doses (LD50) in wild-type mice). Survival was evaluated over 14-days. In Mdr1ab(−/−) mice, LD50 was 0.46 and 2.3 µmol/kg for IVM and MOX, respectively, demonstrating that MOX was less toxic than IVM. In P-gp-deficient mice, MOX had a lower brain-to-plasma concentration ratio and entered into the brain more slowly than IVM. The brain sublethal drug concentrations determined after administration of doses close to LD50 were, in Mdr1ab(−/−) and wild-type mice, respectively, 270 and 210 pmol/g for IVM and 830 and 740–1380 pmol/g for MOX, indicating that higher brain concentrations are required for MOX toxicity than IVM. In rat α1β2γ2 GABA channels expressed in Xenopus oocytes, IVM and MOX were both allosteric activators of the GABA-induced response. The Hill coefficient was 1.52±0.45 for IVM and 0.34±0.56 for MOX (p<0.001), while the maximum potentiation caused by IVM and MOX relative to GABA alone was 413.7±66.1 and 257.4±40.6%, respectively (p<0.05), showing that IVM causes a greater potentiation of GABA action on this receptor. Differences in the accumulation of IVM and MOX in the brain and in the interaction of IVM and MOX with GABA(A) receptors account for differences in neurotoxicity seen in intact and Mdr1-deficient animals. These differences in neurotoxicity of IVM and MOX are important in considering their use in humans.

Photosensitization of phycocyanin extracted from Microcystis in human hepatocellular carcinoma cells: Implication of mitochondria-dependent apoptosis

The aim of this study was to explore the possibility that Microcystis phycocyanin (MC-PC) functions as a photosensitizer and to investigate the mechanism for the apoptosis induced by Microcystis phycocyanin-mediated photodynamic therapy (MC-PC-PDT) in human hepatocellular carcinoma cells (HepG2). After incubation with MC-PC, HepG2 cells were exposed to a He–Ne Laser beam and the cell survival rate was detected by MTT and Colony forming assay. The mechanism of apoptosis was determined by ultrastructural observation, reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) assay, activity detection of caspase-3 and cytosol cytochrome c and flow cytometry (FCM) for cell cycle analysis. Our results demonstrated that MC-PC-PDT effectively inhibits HepG2 proliferation with a half-lethal dose of 100μg/mL and induces apoptosis at 24h with a dose of 200μg/mL MC-PC. MC-PC was found to localized in mitochondria, it could induce a high level of ROS accumulation at 16h after PDT treatment, cause mitochondrial damage and the release of mitochondrial cytochrome c into the cytosol. These cellular changes are accompanied by a reduction of the Δψm, activation of caspase-3 and G2/M phase arrest, finally leading to apoptosis through a mitochondria-dependent pathway after 24h. Meanwhile, necrosis was also contributed to cell death in MC-PC PDT process. The present study also identified a new source of phycocyanin from Microcystis as a safe and effective photosensitizer.

Serological Correlate of Protection in Guinea Pigs for a Recombinant Protective Antigen Anthrax Vaccine Produced from Bacillus brevis

ObjectiveRecombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model. MethodsSerological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminum hydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD50) of virulent Bacillus anthracis spores. Serum antibody titers were determined by anti-PA IgG ELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay. ResultsTo examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD50 of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA. ConclusionNeutralizing-antibody titers can be used as a surrogate marker.

ADMET Evaluation in Drug Discovery. 11. PharmacoKinetics Knowledge Base (PKKB): A Comprehensive Database of Pharmacokinetic and Toxic Properties for Drugs

Good and extensive experimental ADMET (absorption, distribution, metabolism, excretion, and toxicity) data is critical for developing reliable in silico ADMET models. Here we develop a PharmacoKinetics Knowledge Base (PKKB) to compile comprehensive information about ADMET properties into a single electronic repository. We incorporate more than 10 000 experimental ADMET measurements of 1685 drugs into the PKKB. The ADMET properties in the PKKB include octanol/water partition coefficient, solubility, dissociation constant, intestinal absorption, Caco-2 permeability, human bioavailability, plasma protein binding, blood-plasma partitioning ratio, volume of distribution, metabolism, half-life, excretion, urinary excretion, clearance, toxicity, half lethal dose in rat or mouse, etc. The PKKB provides the most extensive collection of freely available data for ADMET properties up to date. All these ADMET properties, as well as the pharmacological information and the calculated physiochemical properties are integrated into a web-based information system. Eleven separated data sets for octanol/water partition coefficient, solubility, blood-brain partitioning, intestinal absorption, Caco-2 permeability, human oral bioavailability, and P-glycoprotein inhibitors have been provided for free download and can be used directly for ADMET modeling. The PKKB is available online at http://cadd.suda.edu.cn/admet.

The Effect of Microwave Radiation on Prickly Paddy Melon (Cucumis myriocarpus)

The growing list of herbicide-resistant biotypes and environmental concerns about chemical use has prompted interest in alternative methods of managing weeds. This study explored the effect of microwave energy on paddy melon (Cucumis myriocarpus) plants, fruits, and seeds. Microwave treatment killed paddy melon plants and seeds. Stem rupture due to internal steam explosions often occurred after the first few seconds of microwave treatment when a small aperture antenna was used to apply the microwave energy. The half lethal microwave energy dose for plants was 145 J/cm2; however, a dose of at least 422 J/cm2was needed to kill seeds. This study demonstrated that a strategic burst of intense microwave energy, focused onto the stem of the plant is as effective as applying microwave energy to the whole plant, but uses much less energy.

A comparative study of the mortality rate of rats receiving a half lethal dose of fat intravenously: Under general anaesthesia versus under spinal anaesthesia

BackgroundThere is no data that demonstrates what anaesthesia is suitable for patients who have a high risk of fat embolism syndrome (FES). We investigated the mortality rates of rats that received a half lethal dose (LD50) of fat by intravenous injection after induction of general or spinal anaesthesia. MethodsAn LD50 of fat for rats was determined by using a toxicological method. Three hundred and seventy five rats were randomly assigned to receive general anaesthesia (group GA, n=125), or spinal block (group SA, n=125), or no anaesthesia (group C, n=125). The rats were injected with the LD50 of fat at 20min after anaesthesia induction. The mortality rates were recorded at 2, 8, 12, and 24h after fat injection. ResultsThe LD50 of fat was 0.706ml/kg and its 95% CI was 0.622ml/kg–0.801ml/kg. The mortality rate was lower in the group GA than in the group SA (p<0.01), whilst there was no statistical difference between the group SA and the group C (p=0.442). ConclusionIt is feasible to assess the efficacy of various treatments for FES by comparing the mortality rates of animals after injection of an LD50 of fat. The mortality rate of rats was lower when FES was induced under general anaesthesia than under spinal anaesthesia which implies that general anaesthesia is superior to spinal anaesthesia for patients who have a high risk of FES.

Cross clade prophylactic and therapeutic efficacy of polyvalent equine immunoglobulin F(ab′)2 against highly pathogenic avian influenza H5N1 in mice

BackgroundHighly pathogenic avian influenza H5N1 virus (HPAI H5N1) has the potential to cause a new pandemic, which may lead to disasters in the world. However, we cannot predict the HPAI H5N1 strain that might cause the pandemic. Therefore, broad-spectrum prophylactic or therapeutic preparations for containment of a possible future pandemic are urgently needed. Polyvalent equine immunoglobulin F(ab′)2 may be a promising candidate. MethodsWe prepared four pepsin digested immunoglobulin F(ab′)2 from the horses immunized with purified VNH5N1-Puerto Rico/8/34 (PR8)/CDC-RG (VNRG, Clade 1), A/Indonesia/05/2005(H5N1)-PR8-IBCDC-RG2 (INRG, Clade 2.1), and A/Anhui/01/2005(H5N1)-PR8-IBCDC-RG5 (AHRG, Clade 2.3.4) and PBS (negative control), respectively. The protective effect of the monovalent or polyvalent F(ab′)2 against A/Ostrich/SZ/097/04 (clade 0) infection was determined by cytopathic effect (CPE) in cultured Madin–Darby canine kidney (MDCK) cells. The prophylactic and therapeutic efficacy of the polyvalent F(ab′)2 was further evaluated by observing survival, weight loss and viral load when the polyvalent F(ab′)2 was introduced into mice one day prior to-, three days post-lethal challenge with A/Ostrich/SZ/097/04. ResultsThe half neutralization doses (ND50) of purified monovalent equine F(ab′)2 prepared by the VNRG, INRG or AHRG-immunized horses and polyvalent one against A/Ostrich/SZ/097/04 were 320, 1280, 1280 and 2560 in cultured MDCK cells, respectively. 10μg polyvalent F(ab′)2 could completely protect mice infected with 100 half lethal doses (LD50) of A/Ostrich/SZ/097/04 in preventive settings. In therapeutic settings, even when injected three days post lethal infection, mice were still completely protected, although 200μg of polyvalent F(ab′)2 was required. ConclusionsOur work has provided experimental supports for testing the broad-spectrum protective efficacy of polyvalent equine immunoglobulin F(ab′)2 for the future large trials.

A new neutralizing antibody against botulinum neurotoxin B recognizes the protein receptor binding sites for synaptotagmins II

Botulinum neurotoxins (BoNts) pose a biological hazard to humans and a serious potential bioweapon threat. Given the safety concern regarding the currently used equine antitoxin therapy for botulism, it is imperative to develop agents that are effective binding inhibitors. The aim of this study was to identify a novel neutralizing antibody against botulinum neurotoxin B (BoNtb) that recognizes the protein receptor binding sites for synaptotagmins II. This antibody showed significant dose-dependent protection against lethal toxin challenge in vivo at an intraperitoneal (i.p.) dose 10 times the half lethal dose (LD50). We proved that the efficacy of SC12 was based on its counteraction on the recognition and binding of BoNtb to target cells, resulting from the combination of antibody with the high affinity (KD: 1.34 nM) to protein receptor binding sites of BoNt by targeting a 25-mer dominant antigenic site on Hcc region (residues 1253–1277). The structure of the site targeted by this antibody overlaps the pocket-like protein receptor binding sites located at the distal tip of toxin molecule. Information gained from this study will facilitate the development of potent inhibitors that prevent the binding of BoNts with its receptors.

Inhibitory effect of live-attenuated Listeria monocytogenes-based vaccines carrying MART-1 gene on mouse malignant melanoma.

Objective To investigate the inhibitory effect of live-attenuated Listeria monocytogenes （LM）-based vaccines expressing the gene encoding a melanoma differentiation antigen,MART-1,on malignant melanoma and their mechanism.Methods The constructed plasmid pERL3-MART-1 was used to transform live-attenuated LM by electroporation to construct recombinant LM.i.e.△inlB LM-MART-1 and △actA/△inlB LM-MART-1.The half lethal dose （LD50） of attenuated listeria strains was determined by concentration gradient dilution method.C57BL/6 mice were randomly divided into three groups,namely PBS group,△inlB LM-MART-1 group and △actA/△inlB LM-MART-1 group.Mice were inoculated by intraperitoneal injection of O.1 LD50 of each rLM strain or PBS only.One week later,the mice were injected subcutaneously with 1×105 B16F10 cells（a mouse melanoma cell strain）in 200μl of PBS.Reimmunization was performed on day 14 and 21.Subsequently,the growth of tumor and survival of tumor bearing mice were observed.All mice were killed on day 28,and tumor tissue as well as splenocytes were obtained from these mice for the detection of MART-1 gene expression by real-time quantitative PCR and the percentage of CD4^＋CD25^＋T cell by flow cytometry.Results The recombinant △inlB LM-MART-1 and △actA/△inlB LM-MART-1 were constructed successfully.The LD50 of △inlB LM and △actA/△inlB LM was lower than LM-EGDe by 100 and 10 000 times respectively.Compared with PBS,the tumor growth was inhibited with △inlB LM-MART-1 by 46.95%（F=6.3,P〈0.05）,and by 83.96% with △actA/△inlB LM-MART-1（F=37.8,P〈0.01）.The relative expression level of MART-1 in △inlB LM-MART-1 group and △actA/△inlB LM-MART-1 group was 8.988±0.207 and 11.315±0.445 times that in PBS group （both P〈0.05）.The percentage of CD4^＋CD25^＋T cells in splenocytes was （2.52±0.20）%,（1.14±0.13）% and （0.44±0.15）% in PBS group,△ialB LM-MART-1 group and △actA/△inlB LM-MART-1 group,respectively;the differences were statistically significant between the three groups （all P〈O.01）.Conclusions The attenuated LM carrying MART-1 gene has a lower virulence than LM reference strain,and could efficiently suppress the growth of melanoma and lengthen the survival of melanoma-beating mice. Key words: Melanoma,experimental; Listeria monocytogenes; MART-1

Comparison on the difference of virulence between Hantaan virus and Seoul virus isolated both from Rattus norvegicus

Comparing the difference of virulence between the strain CGRn5310 (HTNV) and the strain HR54 (SEOV) isolated both from Rattus norvegicus. Suckling mice were used to compare the difference of virulence between the two strains. Hantavirus antigens were detected in brain and lung tissues collected from the infected mice. Compared with the control group, all infected mice grew slowly. Furthermore, the mice inoculated intracerebrally with either CGRn5310 or HR45 appeared ruffled fur, and reduced activity, followed by neurological symptoms, such as paralyses and convulsions. The half lethal dose (LD(50)) of CGRn5310 strain was 10(-6.42), whereas the LD(50) of HR54 strain was 10(-4.51). Hantavirus antigens were identified in brain and lung tissues from the mice infected with the strain CGRn5310 and the strain HR54. LD(50) of the strain CGRn5310 was significantly higher than that of the strain HR54. Our results suggested that the virulence of the spillover hantavirus might only slightly be influenced by the non-reservoir rodents.

THE STUDY OF THE IRRADIATION EFFECT ON RUNNER PLANTS OF STRAWBERRY WITH 60CO-GAMMA RAY

The runner plants of five strawberry varieties were irradiated by three different doses of 60 Co-gamma ray, and the irradiation effect of lethality, lesion, growth restraint on runner plants and the production of new runners was studied. The results indicated there was the dead runner plant in five strawberry varieties treated by 60 Co-gamma ray within 30-80 Gy. The death rate was between 22% and 88.7%,which raised with increase in the dose of 60 Co-gamma ray, but there were different death rates from the different varieties. There happened abnormal leaves in all treatment compared with control. The change of the length and width of leaf, the height and size of plant was different with irradiation dosage and strawberry variety. The occurence rate and the quantity of the new runners decreased significantly. The different varieties had different sensitivity to the irradiation of 60 Co-gamma ray, the most sensitive variety was 'Akihime', the second was 'Hokowase', 'Chun Xu' and 'Sheng Xing', 'Shuo Feng' was most insensitive. The half lethal dose of irradiation to 'Akihime' responded to between 30 Gy and 50 Gy, to 'Hokowase', 'Chun Xu' and 'Sheng Xing' between 50 Gy and 80 Gy, to 'Shuo Feng' above 80 Gy.

Preparation of the phycoerythrin subunit liposome in a photodynamic experiment on liver cancer cells

Efforts are underway to establish a preparation method for the phycoerythrin subunit (PE-sub) liposome, and enhance the cellular uptake and photodynamic therapy (PDT) effect on cancer cells. A film dispersion method was used to prepare the PE-sub liposome, an orthogonal analysis was conducted to optimize the PE-sub liposome preparation condition and determine the effects of liposomes as carriers on cell uptake in vitro. Under a fluorescence microscope, the cell survival rate of normal liver cell line HL7702 and liver cancer cell line HepG2 was assessed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay. Cell apoptosis was determined with flow cytometry and acridine orange staining after PDT treatment. The optimum preparation conditions of the PE-sub liposome were found: a phosphatidylcholine-to-cholesterin ratio of 1:2, a PE-sub-to-lipid ratio of 1:30, 20 mL buffer volume, 10 min sonication time, and an average encapsulation rate of up to 47.2%. The particle size ranged from 80 to 200 nm, and the average particle diameter was 136 nm. At a concentration of 100 microg/mL, the transfection rate of the PE-sub liposome reached 18% at 2 h and 24% at 4 h, and remained steady at 5-6 h. The half lethal dose of PDT on HepG2 was 75 microg/mL, whereas the cell survival rate of HL7702 reached 80% at the same dosage. The PDT-treated cells showed characteristics of apoptosis. The film dispersion method was found to maintain the biological characteristics of the PE-sub. The use of the liposome carrier increased the PE-sub accumulation in the cells and enhanced its PDT effect on HepG2 compared to the PE-sub. HL7702 cell toxicity on had less apparent change after PDT treatment. The PE-sub liposome demonstrated good tumor-targeting characteristics in the in vitro experiment.

Effects of high-energy-pulse-electron beam radiation on biomacromolecules

To study the molecular mechanism of high mutation frequency induced by high-energy-pulse-electron (HEPE) beam radiation, the effects of HEPE radiation on yeast cells, plasma membrane, plasmid DNA, and protein activity were investigated by means of cell counting, gel electrophoresis, AO/EB double fluorescent staining, etc. The results showed that the viability of yeast cells declined statistically with increase of absorbed doses. The half lethal dose (LD50) was 134 Gy. HEPE beam radiation had little influence on the function of plasma membrane and protein, while it could induce much DNA damage of single strand breaks (SSB) and double strand breaks (DSB) that were required for gene mutation. The G-value for DSB formation of HEPE beam radiation in aqueous solution was 5.7 times higher than that caused by 60Co gamma rays. HEPE can be a new effective method for induced mutation breeding and deserves further research in the future.

Synthesis and antitumor activity of 5-(5′,6′-benzocoumaro-3′-yl)methylaminouracil hydrobromide and its liposomal medicinal form

The antitumor activity of 5-(5′,6′-benzocoumaro-3′-yl)methylaminouracil (BCMU) and its liposomal medicinal form was studied in comparison to 5-fluorouracil (5-FU), a well-known antitumor drug widely used in oncological practice. The half-lethal dose of BCMU is 14.8 ± 4.2 mg/kg, while the optimum effective dose of the drug is 6 mg/kg. In this dose, BCMU combines low toxicity with significant antitumor activity, which is manifested by increased tumor growth inhibition (TGI) at a 19% increase in the lifetime (LT) of experimental animals. The antitumor activity of the liposomal form of BCMU is quantitatively and qualitatively superior to that of the nonmodified compound and 5-FU, which is manifested by the most pronounced TGI value and by a significant LT increase.

Synthesis and hepatoprotector activity of water-soluble derivatives of aminoalkylphenols

A series of 2(4)-hydroxybenzyl-and 3-(4-hydroxyaryl)propylamines and their hydrochlorides were synthesized based on 2,4-and 2,6-alkylsubstituted phenols. The synthesized hydrophilic derivatives are characterized by average half-lethal doses (LD50), which fall within 50–170 mg/kg for intraperitoneal administration in mice. Some of the synthesized compounds in a dose of 0.1LD50 show pronounced hepatoprotector activity with respect to tetrachloromethane-induced model toxic hepatitis in mice.

Pharmacokinetic and cytotoxic studies of pegylated liposomal daunorubicin

Pegylated liposomes have been studied for nearly two decades. However, fewer pharmacological studies about its application in daunorubicin (DNR) than those in doxorubicin have been reported. In order to conduct a complete pharmacokinetic study, radiolabeled DNR was encapsulated in pegylated liposomes. Its in vitro drug release kinetics was determined to be in a slow manner, which was reflected in its cytotoxic effect on four cell lines. The lethal dose, plasma pharmacokinetics as well as tissue distribution of the formulation were evaluated in comparison with free DNR. The results revealed that liposomal daunorubicin significantly reduced the toxicity of the drug, with a half lethal dose of 29.35 mg/kg, compared with 5.45 mg/kg for free drug. Pharmacokinetic study of liposomal DNR demonstrated a slower clearance rate, an elevated area under the concentration-time curve, as well as increased half-lives compared to free drug. In addition, an altered tissue distribution of liposomal DNR was observed, with lower cardiac accumulation. Taken together, pegylated liposome-loaded DNR may be a promising anticancer drug and worth further therapeutic study.

Characterisation of surface-modified solid lipid nanoparticles (SLN): Influence of lecithin and nonionic emulsifier

Solid lipid nanoparticles (SLN), an alternative colloidal drug delivery system to polymer nanoparticles, emulsions and liposomes, are generally produced by high pressure melt-emulsification. However, the harsh production process is not applicable for formulations containing shear and temperature sensitive compounds. For that reason, subsequent adsorptive SLN loading might be a promising alternative. The aim of the present study was the development and characterisation of surface-modified SLN for adsorptive protein loading by variation of both the lipid matrix and the emulsifier concentration in the continuous phase. Variations in SLN composition resulted in particle sizes between 674 and 61 nm corresponding to specific surfaces of 4.5 m 2/g and 48.9 m 2/g and zeta potentials between −23.4 mV and −0.9 mV. In dependence of SLN surface properties, albumin payload ranged from 2.5 to 15%. Thermoanalysis, X-ray diffraction and electron microscopy revealed anisometrical and crystalline particles. In vitro cytotoxicity was low in terms of both haemolysis, which was between 1 and 2%, and neutral red test (NRT) showing a half lethal dose between 1.1 and 4.6%.

In vitro mutation and selection of doubled-haploid Brassica napus lines with improved resistance to Sclerotinia sclerotiorum

This paper describes a new protocol to develop doubled-haploid (DH) Brassica napus lines with improved resistance to Sclerotinia sclerotiorum. In this protocol, haploid seedlings derived from microspore cultures of B. napus were used to produce haploid calli for in vitro mutation-selection. For routine screening, mutation was induced by EMS (ethylmethane sulfonate) or occurred spontaneously, and screening for resistant mutants occurred on media with added oxalic acid (OA) as a selection agent. In tests with selected lines, the optimal concentration of EMS for mutation was determined to be 0.15%, and the optimal concentration of OA for in vitro screening was 3 mmol/l (half lethal dose was 3.1 mmol/l) for the first cycle of screening. There was an accumulated effect of OA toxicity on calli over two cycles of screening, but the growth and capacity of the surviving calli for regenerating seedlings were not affected by OA. Of the 54 DH lines produced from the in vitro mutation-selection, two DH lines of resistant mutants, named M083 and M004, were selected following seedling and glasshouse tests. The resistance of M083 and M004 to S. sclerotiorum following tests with both mycelial inoculum and OA was greater than that of their donor lines and the resistant control Zhongyou 821. In both glasshouse and field disease nurseries, disease indices on M083 and M004 were less than 50% of those of the control. The time required for M083 and M004 to mature was 14 days and 10 days shorter, respectively, than that of their donor lines. Furthermore, M083 had more pods per inflorescence, a greater 1,000 seed weight and higher yield than its donor line. Random amplified polymorphic DNA characterisation showed that M083 had DNA band patterns that differed from its donor line.

Preparation and acute toxicology of nano-magnetic ferrofluid.

The nano-magnetic ferrofluid was prepared by chemical coprecipitation and its acute toxicology was investigated. The effective diameter (Eff. Diam. ) of the magnetic particles was about 19.9 nm, and the concentration of the ferrofluid was 17. 54 mg/ml. The acute toxic reaction and the main viscera pathological morphology of mice were evaluated after oral, intravenous and intraperitoneal administration of the nano-magnetic ferrofluid of different doses respectively. Half lethal dose (LD50) > 2104. 8 mg/kg,maximum non-effect dose (ED0) = 320. 10mg/kg with oral; LDs,> 438. 50 mg/kg, EDo = 160. 05 mg/kg with intravenous route; and LDso >1578. 6 mg/kg, ED0 = 320. 10 mg/kg with intraperitoneal administration. Degeneration and necrosis of viscera were not found. So the nano-magnetic ferrofluid, of which toxicity is very low, may be used as a drug carrier.

Do there exist synergistic antitumor effects by coexpression of herpes simplex virus thymidine kinase with cytokine genes on human gastric cancer cell line SGC7901?

To evaluate the synergistic antitumor effects of herpes simplex virus thymidine kinase (HSV-TK) together with tumor necrosis factor alpha (TNF-alpha) or interleukin-2 (IL-2) gene expression on gastric cancer cell line SGC7901. Recombinant vectors pL(TT)SN and pL(TI)SN, which express TK-IRES-TNF-alpha and TK-IRES-IL-2 genes separately, as well as the control plasmids pL(TK)SN and pLXSN were employed to transfect PA317 cells respectively to generate the viruses that can stably express the objective genes through G418 selection. The gastric cancer cells were then transfected by the retroviral serum from the package cells and maintained in culture to determine the cell growth and apoptosis. The cytotoxic effects of HSV-TK together with TNF-alpha or IL-2 gene expression on the transfected cancer cells were evaluated by the cell viability and bystander effects in the presence of GCV supplemented in the cultural medium. Expression of recombinant proteins including TNF-alpha and IL-2 by stable transfectants was confirmed by Western blotting. The percentage of cell apoptosis in the SGC/0, SGC/TK-TNF-alpha, SGC/TK-IL-2 and SGC/TK clone was 2.3%, 12.3%, 11.1% and 10.9% respectively at 24 h post-transfection. Cell growth status among all the experimental groups as judged by cell absorbance (A) at 570nm did not exhibit any significant difference (P>0.05); although it was noted to be slightly lower in the SGC/TT group. Cell survival rate in SGC/TI, SGC/TT and SGC/TK group was significantly decreased in a dose-dependent manner of GCV compared with that of the SGC/0 group (P<0.05-0.01). Among all studied cells, the SGC/TT was shown most sensitive to GCV with a half lethal dose of 0.5 mg/L(-1). In contrast, the survival rate of SGC/0 cells was not affected by the presence of GCV with the doses less than 10 mg/L(-1). The half lethal dose of GCV for SGC/0 cells was more than 100 mg/L(-1). Marked bystander effect induced by SGC/TI, SGC/TT and SGC/TK cells was confirmed by the fact that 20% of these stable transfectants could kill 50% of the co-cultured cells, in which the most prominent bystander effect was found in the circumstance of SGC/TT presence. However, no significant difference of these variables was found among SGC/TI, SGC/TT and SGC/TK cells (P>0.05). The synergistic antitumor effects produced by the co-expression of HSV-TK with TNF-alpha or IL-2 genes were not present in the transfected SGC7901 cells. The mechanism underlying these phenomena was not known.