Prevalidation trial for a novel in vitro eye irritation test using the reconstructed human cornea-like epithelial model, MCTT HCE™.

Here, we report the results of a prevalidation trial for an in vitro eye irritation test (EIT) using the reconstructed human cornea-like epithelium, MCTT HCE™. The optimal cutoff to determine irritation in the prediction model was established at 35% with the receiver operation characteristics(ROC) curve for 126 substances. Within-lab(WL) and between-lab(BL) reproducibility was tested for 20 reference substances by 3 participating laboratories. Viability data described by mean±SD or ±1/2 difference between duplicate wells, and scatter plots, demonstrated the WL/BL consistency. WL/BL concordance with the binary decision, whether non-irritant or irritant was estimated to be 85-95% and 95%, respectively. WL/BL reproducibility of viability data was further supported by a strong correlation(ICC, r>0.9). WL/BL agreement of binary decisions was also examined by Fleiss' Kappa statistics, which showed a strong level of agreement (>0.78), nevertheless weaker than the reproducibility of the viability. The EIT with MCTT HCE™ exhibited a sensitivity of 82.2% (60/73), a specificity of 81.1% (43/53), and an accuracy of 81.8% (103/126) for 126 reference substances (for liquids; a sensitivity of 100% (47/47), a specificity of 70.6% (24/34), and an accuracy of 87.7% (71/81), and for solids, a sensitivity of 50% (13/26), a specificity of 100% (19/19), and an accuracy of 71.1% (32/45), suggesting that the accuracy is satisfactory but the sensitivity needs improvement, which shall be addressed through correcting the poor sensitivity for solid substances in future full validation trials.

A 90-day study of subchronic oral toxicity of 20 nm, negatively charged zinc oxide nanoparticles in Sprague Dawley rats.

PURPOSE: The widespread use of nanoparticles (NPs) in industrial and biomedical applications has prompted growing concern regarding their potential toxicity and impact on human health. This study therefore investigated the subchronic, systemic oral toxicity and no-observed-adverse-effect level (NOAEL) of 20 nm, negatively charged zinc oxide (ZnO(SM20(-))) NPs in Sprague Dawley rats for 90 days. METHODS: The high-dose NP level was set at 500 mg/kg of bodyweight, and the mid- and low-dose levels were set at 250 and 125 mg/kg, respectively. The rats were observed during a 14-day recovery period after the last NP administration for the persistence or reduction of any adverse effects. Toxicokinetic and distribution studies were also conducted to determine the systemic distribution of the NPs. RESULTS: No rats died during the test period. However, ZnO(SM20(-)) NPs (500 mg/kg) induced changes in the levels of anemia-related factors, prompted acinar cell apoptosis and ductular hyperplasia, stimulated periductular lymphoid cell infiltration and excessive salivation, and increased the numbers of regenerative acinar cells in the pancreas. In addition, stomach lesions were seen at 125, 250, and 500 mg/kg, and retinal atrophy was observed at 250 and 500 mg/kg. The Zn concentration was dose-dependently increased in the liver, kidney, intestines, and plasma, but not in other organs investigated. CONCLUSION: A ZnO(SM20(-)) NP NOAEL could not be established from the current results, but the lowest-observed-adverse-effect level was 125 mg/kg. Furthermore, the NPs were associated with a number of undesirable systemic actions. Thus, their use in humans must be approached with caution.

A 90-day study of sub-chronic oral toxicity of 20 nm positively charged zinc oxide nanoparticles in Sprague Dawley rats.

PURPOSE: The study reported here was conducted to determine the systemic oral toxicity and to find the no-observed-adverse-effect level of 20 nm positively charged zinc oxide (ZnO(SM20(+))) nanoparticles in Sprague Dawley rats for 90 days. METHODS: For the 90-day toxicity study, the high dose was set as 500 mg per kg of body weight (mg/kg) and the middle and low dose were set to 250 mg/kg and 125 mg/kg, respectively. The rats were held for a 14-day recovery period after the last administration, to observe for the persistence or reduction of any toxic effects. A distributional study was also carried out for the systemic distribution of ZnO(SM20(+)) NPs. RESULTS: No rats died during the test period. There were no significant clinical changes due to the test article during the experimental period in functional assessment, body weight, food and water consumption, ophthalmological testing, urine analysis, necropsy findings, or organ weights, but salivation was observed immediately after administration in both sexes. The total red blood cell count was increased, and hematocrit, albumin, mean cell volume, mean cell hemoglobin, and mean cell hemoglobin concentration were decreased significantly compared with control in both 500 mg/kg groups. Total protein and albumin levels were decreased significantly in both sexes in the 250 and 500 mg/kg groups. Histopathological studies revealed acinar cell apoptosis in the pancreas, inflammation and edema in stomach mucosa, and retinal atrophy of the eye in the 500 mg/kg group. CONCLUSION: There were significant parameter changes in terms of anemia in the hematological and blood chemical analyses in the 250 and 500 mg/kg groups. The significant toxic change was observed to be below 125 mg/kg, so the no-observed-adverse-effect level was not determined, but the lowest-observed-adverse-effect level was considered to be 125 mg/kg in both sexes and the target organs were found to be the pancreas, eye, and stomach.

Toxicity of 100 nm zinc oxide nanoparticles: a report of 90-day repeated oral administration in Sprague Dawley rats.

Nanoparticles (NPs) are used commercially in health and fitness fields, but information about the toxicity and mechanisms underlying the toxic effects of NPs is still very limited. The aim of this study is to investigate the toxic effect(s) of 100 nm negatively (ZnO(AE100[-])) or positively (ZnO(AE100[+])) charged zinc oxide (ZnO) NPs administered by gavage in Sprague Dawley rats, to establish a no observed adverse effect level, and to identify target organ(s). After verification of the primary particle size, morphology, hydrodynamic size, and zeta potential of each test article, we performed a 90-day study according to Organisation for Economic Co-operation and Development test guideline 408. For the 90-day study, the high dose was set at 500 mg/kg and the middle and low doses were set at 125 mg/kg and 31.25 mg/kg, respectively. Both ZnO NPs had significant changes in hematological and blood biochemical analysis, which could correlate with anemia-related parameters, in the 500 mg/kg groups of both sexes. Histopathological examination showed significant adverse effects (by both test articles) in the stomach, pancreas, eye, and prostate gland tissues, but the particle charge did not affect the tendency or the degree of the lesions. We speculate that this inflammatory damage might result from continuous irritation caused by both test articles. Therefore, the target organs for both ZnO(AE100(-)) and ZnO(AE100(+)) are considered to be the stomach, pancreas, eye, and prostate gland. Also, the no observed adverse effect level for both test articles was identified as 31.25 mg/kg for both sexes, because the adverse effects were observed at all doses greater than 125 mg/kg.

Neuroprotective effect of Sanguisorbae radix against oxidative stress-induced brain damage: in vitro and in vivo.

Sanguisorbae radix (SR), the root of Sanguisorba officinalis L. (Rosaceae), has been traditionally used for its anti-inflammatory, anti-infectious and analgesic activities in Korea. Previous work has shown that SR prevents neuronal cell damage induced by Abeta (25--35) in cultured rat cortical neurons. The present study was carried out to further investigate the neuroprotective effect of SR on oxidative stress-induced toxicity in primary culture of rat cortical neurons, and on ischemia-induced brain damage in rats. SR, over a concentration range of 10--50 microg/ml, inhibited H2O2 (100 microM)-induced neuronal death, which was significantly inhibited by MK-801 (5 microM), an N-methyl-D-aspartate (NMDA) receptor antagonist, and verapamil (20 microM), an L-type Ca2+ channel blocker. Pretreatment of SR (10-50 microg/ml), MK-801 (5 microM), and verapamil (20 microM) inhibited H2O2-induced elevation of intracellular Ca2+ concentration ([Ca2+]i) measured by a fluorescent dye, Fluo-4 AM. SR (10-50 microg/ml) inhibited H2O2-induced glutamate release into medium measured by HPLC, and generation of reactive oxygen species (ROS) measured by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In vivo, SR prevented cerebral ischemic injury induced by 2-h middle cerebral artery occlusion (MCAO) and 24-h reperfusion. The ischemic infarct and edema were significantly reduced in rats that received SR (10, 30 mg/kg, orally), with a corresponding improvement in neurological function. Catechin isolated from SR inhibited H2O2-induced neuronal death in cultures. Taken together, these results suggest that SR inhibits H2O2-induced neuronal death by interfering with the increase of [Ca2+]i, and inhibiting glutamate release and generation of ROS, and that the neuroprotective effect of SR against focal cerebral ischemic injury is due to its anti-oxidative effects. Thus SR might have therapeutic roles in neurodegenerative diseases such as stroke.

Endothelium-dependent vasodilatory and hypotensive effects of Crotalaria sessiliflora L. in rats.

The aim of the present study was to investigate the vasoactive effect of Crotalaria sessiliflora L. extract (CSE) on rats and its mechanism when combining in vivo and in vitro approaches. CSE (0.5-5 mg/ml) induced concentration-dependent relaxation on endothelium-intact thoracic aortic rings precontracted with phenylephrine (PE, 10(-5) M). This effect disappeared with the removal of functional endothelium. Pretreatment of the aortic strips with either N(G)-nitro-L-arginine (L-NNA, 10(-5) M) or methylene blue (10(-5) M) significantly reduced the relaxation induced by CSE. The endothelium-dependent relaxation caused by CSE was associated with production of cGMP. CSE (5 mg/ml) increased the production of cGMP in endothelium-intact aortic rings and this effect was significantly attenuated by L-NNA (10(-5) M) and methylene blue (10(-5) M). Relaxation in response to CSE in strips precontracted with PGF2alpha (3x10(-5) M) was eliminated by removing extracellular Ca2+ and significantly reduced by pretreatment with ruthenium red (10(-5) M). In in vivo tests, injection of 40 mg/kg of CSE induced an increase in plasma NO production, and this effect was blocked by L-NNA. Furthermore, CSE produced dose-dependent and transient decrease in blood pressure in normotensive rats and this effect was blocked by atropine as well as L-NNA. These findings suggest that CSE induces endothelium-dependent relaxation via NO/cGMP signaling by promoting extracellular Ca2+ influx and the release of Ca2+ from intracellular stores of endothelium, probably due to endothelial muscarinic receptor activation.

Protection of amyloid beta protein (25-35)-induced neurotoxicity by methanol extract of Smilacis chinae rhizome in cultured rat cortical neurons.

Smilax has various pharmacological effects including antiinflammatory, anticancer and antioxidant activity. The present study aims to investigate the effect of the methanol extract of Smilacis chinae rhizome (SCR) from Smilax china L. (Liliaceae) on amyloid beta protein (Abeta) (25-35), a synthetic 25-35 amyloid peptide, -induced neurotoxicity in cultured rat cerebral cortical neurons. Abeta (25-35) (10 microM) produced a reduction of cell viability, which was significantly reduced by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), an N-methyl-D-aspartate (NMDA) receptor antagonist, verapamil, an L-type Ca2+ channel blocker, and NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor. SCR, over a concentration range of 10-50 microg/ml, inhibited 10 microM Abeta (25-35)-induced neuronal cell death, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. SCR (50 microg/ml) inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic calcium concentration ([Ca2+]c), which was measured by a fluorescent dye, Fluo-4 AM. Pretreatment of SCR (10 and 50 microg/ml) also inhibited glutamate release into medium induced by 10 microM Abeta (25-35), which was measured by HPLC, generation of reactive oxygen species and activation of caspase-3. These results suggest that SCR prevents Abeta (25-35)-induced neuronal cell damage in vitro.

Regional difference of glutamate-induced swelling in cultured rat brain astrocytes.

L-glutamate (glutamate) is an important neurotoxin as well as the major excitatory neurotransmitter. Extracellular glutamate levels are elevated following ischemia, hypoglycemia, and trauma. One consequence of elevated glutamate levels is cell swelling. Such swelling occurs primarily in astroglial cells. We characterized the regional difference in glutamate-induced swelling response of cultured astrocytes from rat cerebral cortex, hippocampus and cerebellum. Glutamate produced dose-dependent astrocytic swelling in both cerebral cortex and hippocampus, showing a maximal effect in 0.5 mM concentration, as measured by 3-O-methyl-D-[1-3H]glucose uptake. However, in cerebellum, glutamate did not produce astrocytic swelling. It has been suggested that Na+ -dependent glutamate uptake is a possible mechanism of glutamate-induced swelling. The Vmax for glutamate uptake into cerebellum astrocytes was significantly lower (6.7 nmol/mg protein/min) than those for cerebral cortex and hippocampus astrocytes (13.0 and 12.0 nmol/mg protein/min, respectively). In three regions, more than 90% of the cultured cells showed glial fibrillary acidic protein (GFAP) immunoreactivity. Immunoreactivity of GLT, one of the markers of glutamate transporters, which is expressed at low levels in cultured astrocytes, did not show any differences in three regions. However, immunoreactivities of GLAST, the other astroglial glutamate transporter, and aquaporin4 (APQ4), a water transporter, were significantly higher in cerebral cortex and hippocampus than in cerebellum. These results may explain the regional difference of glutamate-induced astrocytic swelling.

Polygalae radix extract protects cultured rat granule cells against damage induced by NMDA.

Polygalae Radix (PR) from Polygala tenuifolia (Polygalaceae) is traditionally used in China and Korea, as this herb has a sedative, anti-inflammatory and antibacterial agent. To extend our understanding of the pharmacological actions of PR in the CNS on the basis of its CNS inhibitory effect, the present study examined whether PR has the neuroprotective action against N-methyl-D-aspartate (NMDA)-induced cell death in primarily cultured rat cerebellar granule neurons. PR, over a concentration range of 0.05 to 5 microg/ml, inhibited NMDA (1 mM)-induced neuronal cell death, which was measured by a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. PR (0.5 microg/ml) inhibited glutamate release into medium induced by NMDA (1 mM), which was measured by HPLC. Pre-treatment of PR (0.5 microg/ml) inhibited NMDA (1 mM)-induced elevation of intracellular Ca2+ concentration ([Ca2+]i), which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). These results suggest that PR prevents NMDA-induced neuronal cell damage in vitro.

Protection of NMDA-induced neuronal cell damage by methanol extract of zizyphi spinosi semen in cultured rat cerebellar granule cells.

Zizypus is one of the herbs widely used in Korea and China due to the CNS calming effect. The present study aims to investigate the effect of the methanol extract of Zizyphi Spinosi Semen (ZSS), the seeds of Zizyphus jujuba Mill var. spinosa, on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in cultured rat cerebellar granule neuron. ZSS, over a concentration range of 0.05-5 microg/ml, inhibited NMDA (1 mM)-induced neuronal cell death, which was measured by a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. ZSS (0.5 microg/ml) inhibited glutamate release into medium induced by NMDA (1mM), which was measured by HPLC. Pretreatment of ZSS (0.5 microg/ml) inhibited NMDA (1mM)-induced elevation of cytosolic calcium concentration ([Ca(2+)](c)), which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). These results suggest that ZSS prevents NMDA-induced neuronal cell damage in vitro.

Protective effects of fangchinoline and tetrandrine on hydrogen peroxide-induced oxidative neuronal cell damage in cultured rat cerebellar granule cells.

The present study was performed to examine the neuroprotective effects of fangchinoline (FAN) and tetrandrine (TET), bis-benzylisoquinoline alkaloids, which exhibit the characteristics of Ca 2+ channel blockers, on H2O2 -induced neurotoxicity using cultured rat cerebellar granule neurons. H2O2 produced a concentration-dependent reduction of cell viability, which was blocked by (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d]cyclohepten-5,10-imine (MK-801), an N-methyl- D-aspartate (NMDA) receptor antagonist, verapamil, an L-type Ca 2+ channel blocker, and NG-nitro- L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. Pretreatment with FAN and TET over a concentration range of 0.1 to 10 microM significantly decreased the H2O2 -induced neuronal cell death as assessed by a trypan blue exclusion test, a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and the number of apoptotic nuclei. In addition, FAN and TET inhibited the H2O2 -induced elevation of glutamate release into the medium, elevation of the cytosolic free Ca 2+ concentration ([Ca 2+] c ), and generation of reactive oxygen species (ROS). These results suggest that FAN and TET may mitigate the harmful effects of H2O2 -induced neuronal cell death by interfering with the increase of [Ca 2+] c, and then by inhibiting glutamate release and generation of ROS. Abbreviations. AP5:D(-)-2-amino-5-phosphonopentanoic acid DMSO:dimethyl sulfoxide FAN:fangchinoline H 2 DCF-DA:2',7'-dichlorodihydrofluorescin diacetate MK-801:(5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d]cyclohepten-5,20-imine MTT:3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide L-NAME: NG-Nitro- L-arginine methyl ester NMDA: N-methyl- D-aspartate TET:tetrandrine

Efffect of recombinant human FSH on ovulation, pregnancy and in vitro fertilization in androgen-sterilized mice.

The effect of a new rhFSH, PG-0801, on oocyte quality, ovulation and in vitro fertilization (IVF) was examined in androgen-sterilized mice. Experimental sterility was induced by a single subcutaneous injection of testosterone propionate (TP, 1 mg/head) into 5 day old female mice. Ovulation was generated in the 10 to 13-week old TP-injected mice by a subcutaneous rhFSH injection (1, 5 or 10 IU/head) followed 48 hours later by a second rhFSH injection (1, 5 or 10 IU/head). For comparison, a subcutaneous PMSG (5 IU/head) injection was used for folliculogenesis and a hCG (5 IU/head) injection was used for ovulation. These were administered using the same protocol. The eggs were harvested from the oviducts and counted 17 to 20 hours after the second injection. IVF was performed by adding sperms (2 x 10(5)/ml to 2 x 106/ ml) to determine the functional activity of the eggs, and the fertilization rate was measured. In addition, the pregnancy rate and fetal development were examined after 15-17 days of gestation. The number of oocytes recovered from the rhFSH/rhFSH group increased dose-dependently and was slightly higher than that of the PMSG/hCG group. The pregnancy rates of the group receiving 1, 5, and 10 IU of rhFSH/rhFSH were 50%, 66.7%, and 75%, respectively, which were significantly higher than that of the control (untreated) group (0%). The numbers of viable fetuses in the 1, 5, and 10 IU/head of the rhFSH/rhFSH group (8.0 +/- 1.50, 8.9 +/- 1.02, and 8.9 +/- 1.12 fetuses/dam, respectively) were comparable to that of the 5 IU/head PMSG/ hCG group (9.4 +/- 0.94). The mice receiving rhFSH/rhFSH and PMSG/hCG showed similar fertilization rates (around 65%) via the IVF procedure. These results demonstrate that a new rhFSH, PG-0801, may be useful for inducing ovulation in functionally infertile patients and for superovulation in ovulatory patients participating in assisted reproductive technology (ART) programs.