• Molecular & Cellular Toxicology
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• v.3 no.2
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• pp.113-118
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• 2007

Chlorobenzenes due to their acute toxicity and the capability of bioaccumulating are of great health and environmental concern. Especially, 1, 2-dichlorobenzene (CAS No. 95-50-1) is used for organic synthesis, dye manufacture, as a solvent and for other applications in chemical industry. Adverse effects of 1, 2-dichlorobenzene includes increases in liver and kidney weights and hepatotoxicity. In this study, we evaluated the genetic toxicity of 1, 2-dichlorobenzene with more advanced methods, in vitro mouse lymphoma assay $tk^{+/-}$ gene assay (MLA) and in vitro mouse supravital micronucleus (MN) assay. 1, 2-Dichlorobenzene appeared the significantly positive results and the induction of large mutant colonies only in the presence of metabolic activation system with MLA. But in vitro testing of 1, 2-dichlorobenzene yielded negative results with supravital MN assay. These results suggest that 1, 2-dichlorobenzene may play a mutagen rather than clastogen in vitro mammalian system.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.6
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• pp.705-712
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• 2018

The tube formation assay is a widely used in vitro experiment model to evaluate angiogenic properties by measuring the formation of tubular structures from vascular endothelial cells (ECs). In vitro experimental results are crucial when considered the advisability of moving forward to in vivo studies. Thus, the additional attentions to the in vitro assay is necessary to improve the quality of the pre-clinical data, leading to better decision-making for successful drug discovery. In this study, we improved the tube formation assay system in three aspects. First, we used human endothelial colony forming cells (ECFCs), which are endothelial precursors that have a robust proliferative capacity and more defined angiogenic characteristics compared to mature ECs. Second, we utilized a real-time cell recorder to track the progression of tube formation for 48 hours. Third, to minimize analysis error due to the limited observation area, we used image-stitching software to increase the microscope field of view to a $2{\times}2$ stitched area from the $4{\times}$ object lens. Our advanced tube formation assay system successfully demonstrated the time-dependent dynamic progression of tube formation in the presence and absence of VEGF and FGF-2. Vatalanib, VEGF inhibitor, was tested by our assay system. Of note, $IC_{50}$ values of vatalanib was different at each observation time point. Collectively, these results indicate that our advanced tube formation assay system replicates the dynamic progression of tube formation in response to angiogenic modulators. Therefore, this new system provides a sensitive and versatile assay model for evaluating pro- or anti-angiogenic drugs.

• Molecular & Cellular Toxicology
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• v.3 no.3
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• pp.172-176
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• 2007

Butylated hydroxytoluene (BHT) is widely used antioxidant food additives. It has been extensively studied for potential toxicities. BHT appears adverse effects in liver and thyroid. In this study, we evaluated the genetic toxicity of BHT with more advanced methods, in vitro mouse lymphoma assay $tk^{+/-}$ gene assay (MLA) and in vivo mouse supravital micronucleus (MN) assay. BHT did not appear the significantly results in the absence and presence of metabolic activation system with MLA. Also, in vivo testing of BHT yielded negative results with supravital MN assay. These results suggest that BHT itself was not generally considered genotoxic.

• Korean Journal of Environmental Biology
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• v.28 no.1
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• pp.8-13
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• 2010

This study aimed the role of gold nanoparticles (AuNP) in advanced glycation end-products (AGEs) induced migration and invasion in bovine retinal endothelial cells (BRECs). BRECs were isolated from the retina. Cell viability was confirmed by the MTT assay. In vitro wound migration assay was performed to investigate the migration of BRECs. In vitro tube formation was measured by on-gel system. Apoptosis induced by AuNP was confirmed by caspase-3 assay. AGE-bovine serum albumin (BSA) demonstrated increase of cell migration and proliferation in BRECs. In addition, AuNP regardless of the existence of AGE-BSA suppressed proliferation, migration, and angiogenesis. AuNP suppressed AGE-BSA induced migration and invasion, and induced apoptosis through caspase-3. As a results, AuNP have a potential anti-angiogenic effect for AGE-induced angiogenesis in vitro and offer possibility for the treatment of diabetic retinopathy.

• Molecular & Cellular Toxicology
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• v.2 no.4
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• pp.244-250
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• 2006

The detection of many synthetic chemicals used in industry that may pose a genetic hazard in our environment is of great concern at present. Since these substances are not limited to the original products, and enter the environment, they have become widespread environmental pollutants, thus leading to a variety of chemicals that possibly threaten the public health. In this respect, to regulate and to evaluate the chemical hazard will be important to environment and human health. The genotoxicity of 3 synthetic chemicals was evaluated in L5178Y $tk^{+/-}$ mouse lymphoma cells in vitro. 9H-carbazole (CAS No. 86-74-8) did not induce significant mutation frequencies both in the presence and absence of metabolic activation system. 1, 3-Dichloro-2-propanol (CAS No. 96-23-1) revealed a significant increase of mutation frequencies in the range of $625-373\;{\mu}g/mL$ in the absence of metabolic activation system and $157-79\;{\mu}g/mL$ in the presence of metabolic activation system. And also, fenpropathrin (CAS No. 64257-84-7) appeared the positive results only in the absence of metabolic activation system. Through the results of MLA tk assay with 3 synthetic chemicals in L5178Y cells in vitro, we may provide the important clues on the genotoxic potentials of these 3 chemicals.

• Molecular & Cellular Toxicology
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• v.2 no.3
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• pp.176-184
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• 2006

The controversy on genotoxicity of molinate, an herbicide, has been reported in bacterial system, and in vitro and in vivo mammalian systems. To clarify the genotoxicity of molinate, we performed bacterial gene mutation test, in vitro chromosome aberration and mouse lymphoma $tk^{+/-}$ gene assay, and in vivo micronucleus assay using bone marrow cells and peripheral reticulocytes of mice. In bacterial gene mutation assay, no mutagenicity of molinate ($12-185{\mu}g/plate$) was observed in Salmonella typhimurium TA 98, 100, 1535 and 1537 both in the absence and in the presence of S-9 metabolic activation system. The clastogenicity of molinate was observed in the presence ($102.1-408.2\;{\mu}g/mL$) of metabolic activation system in mammalian cell system using Chinese hamster lung fibroblast. However, no clastogenicity was observed in the absence ($13.6-54.3\;{\mu}g/mL$) of metabolic activation system. It is suggested that the genotoxicity of molinate was derived some metabolites by metabolic activation. Molinate was also subjected to mouse lymphoma L5178Y $tk^{+/-}$ cells using microtiter cloning technique. In the absence of S-9 mixture, mutation frequencies (MFs) were revealed $1.4-1.9{\times}10^{-4}$ with no statistical significance. However, MFs in the presence of metabolic activation system revealed $3.2-3.4{\times}10^{-4}$ with statistical significance (p<0.05). In vivo micronucleus (MN) assay using mouse bone marrow cells, molinate revealed genotoxic potential in the dose ranges of 100-398 mg/kg of molinate when administered orally. Molinate also subjected to acridine orange MN assay with mouse peripheral reticulocytes. The frequency of micronucleated reticulocytes (MNRETs) induced 48 hr after i.p. injection at a single dose of 91, 182 and 363 mg/kg of molinate was dose-dependently increased as $10.2{\pm}4.7,\;14.6{\pm}3.9\;and\;28.6{\pm}6.3\;(mean{\pm}SD\;of\;MNRETs/2,000\;reticulocytes)$ with statistical significance (p<0.05), respectively. Consequently, genotoxic potential of molinate was observed in in vitro mammalian mutagenicity systems only in the presence of metabolic activation system and in vivo MN assay using both bone marrow cells and peripheral reticulocytes in the dose ranges used in this experiment. These results suggest that metabolic activation plays a critical role to express the genotoxicity of molinate in in vitro and in vivo mammalian system.

• Toxicological Research
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• v.24 no.1
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• pp.37-44
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• 2008

The in vitro cell transformation assays (CTA) were performed using BALB/3T3 murine fibroblasts and HaCaT human keratinocytes in order to evaluate concordance between both in vitro CTAs and carcinogenicity with compounds differing in their genotoxic and carcinogenic potential. Six test articles were evaluated, two each from three classes of compounds: genotoxic carcinogens (2-amino-5-nitrophenol and 4-nitroquinoline-N-oxide), genotoxic noncarcinogens (8-hydroxyquinoline and benzyl alcohol), and nongenotoxic carcinogens (methyl carbamate and N-nitrosodiphenylamine). Any foci of size $\geq$2 mm regardless of invasiveness and piling was scored as positive in CTA with BALB/3T3. As expected, four carcinogens regardless of their genotoxicity had positive outcomes in two-stage CTA using BALB/3T3 cells. However, of the two genotoxic noncarcinogens, benzyl alcohol was positive CTA finding. We concluded that, of the 6 chemicals tested, the sensitivity for BALB/3T3 system was reasonably high, being 100%. The respective specificity for BALB/3T3 assay was 50%. We also investigated the correlation between results of BALB/3T3 assay and results from HaCaT assay in order to develop a reliable human cell transformation assay. However, evaluation of staining at later time points beyond the confluency stage did not yield further assessable data because most of HaCaT cells were detached after $2{\sim}3$ days of confluency. Thus, after test article treatment, HaCaT cells were split before massive cell death began. In this modified protocol for this HaCaT system, growing attached colonies were counted instead of transformed foci 3 weeks since last subculture. Compared to BALB/3T3 assay, HaCaT assay showed moderate low sensitivity and high specificity. Despite these differences in specificity and sensitivity, both cell systems did exhibit same good concordance between in vitro CTA and rodent carcinogenicity findings (overall 83% concordant results). At present the major weakness of these in vitro CTA is lack of validation for regulatory acceptance and use. Thus, more controlled studies will be needed in order to be better able to assess and quantitatively estimate in vitro CTA data.

• Toxicological Research
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• v.14 no.3
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• pp.427-433
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• 1998

The acute and genetic toxicity of DK1002 was subjected in this study. DK1002 which is a morphine-like new drug candidate synthesized by Dong-Kook Pharmaceutical Co. Ltd. is now under developing as a analgesics that have better drug efficacy and least addictive property. In acute toxicity study, the 50% lethal doses ($LD_{50}$) of DK1002 were determined as>2000mg/kg (p.o.), 237.0mg/kg(i.p.), 57.5mg/kg(i.v.), and 1266.9mg/kg (s.c.). And also, to study the genotoxicity of DK1002, we performed bacterial reversion assay with Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and in vitro chromosomal aberration assay with Chinese hamster lung cells in the presence and absence of S-9 metabolic activation system. In vivo micronucleus assay using mouse bone marrow cells was also performed. From these results, DK1002 was revealed nonmutagenic potential in S. typhimurium TA98, TA100, TA1535, and TA537 both in the absence and presecne of metablic activation system. No clastogenicity of DK1002 was observed in chromosomal aberration assay in vitro as well as in micronucleus assay in vivo.

• Biomolecules & Therapeutics
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• v.11 no.3
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• pp.190-195
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• 2003

To assess the genotoxicity of AS6, several classical toxicological tests were performed. In Ames test, AS6 did not show any transformation of revertant with or without S-9 metabolic activating system, indicating the lack of mutagenic effect of the compound. To assess clastogenic effect, in vivo micronucleus and in vitro chromosomal aberration assays were performed using male ICR mice and Chinese hamster lung (CHL) fibroblast cells, respectively. Chromosomal aberration was not induced regardless of the presence of S-9 metabolic activating system. In addition, AS6 did not cause any increase in the incidence of micronucleated polychromatic erythrocytes at any of the dose levels, suggesting little clastogenicity in vitro or in vivo. Taken together, these results demonstrate that AS-6 has no mutagenic effect in our test system.

• The Korean Journal of Zoology
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• v.20 no.4
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• pp.159-168
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• 1977

In order to evaluate the mutagenic potentential in mammalian system for those pesticides which were proved to be mutagenic in Salmonella microsome assay system, we have studied drug-resistant mutagenesis in cultured L5178Y cells and unscheduled DNA synthesis in human lymphocytes in vitro. We have tested five pesticides: insecticides DDVP and trichlorfon, fungicide TMTD and herbicides MO and NIP. Of these pesticides, TMTD induced weak mutation to MTX-resistance in L5178Y cells in vitro and gave positive responses in DNA repair assay system. Therefore, its potential genetic risks in human beings should be re-evaluated. DDVP and trichlorfon gave negative response in L5178Y mutagenesis test system but stimulated incorporation of $^{3}H$-TdR in DNA repar assay. MO and NIP gave also negative responses both in L5178Y mutagenesis test systemand in DNA repair assay system.