• Toxicological Research
• /
• v.35 no.4
• /
• pp.343-351
• /
• 2019

Many in vitro developmental toxicity assays have been proposed over several decades. Since the late 1980s, we have made intermittent attempts to introduce in vitro assays as screening tests for developmental toxicity of inhouse candidate products. Two cell-based assays which were developed two decades apart were intensively studied. One was an assay of inhibitory effects on mouse ascites tumor cell attachment to a concanavalin A-coated plastic sheet surface (MOT assay), which we studied in the early days of assay development. The other was an assay of inhibitory effects on the differentiation of mouse embryonic stem cell to beating heart cells (EST assay), which we assessed more recently. We evaluated the suitability of the assays for screening in-house candidates. The concordance rates with in vivo developmental toxicity were at the 60% level. The EST assay classified chemicals that inhibited cell proliferation as embryo-toxic. Both assays had a significant false positive rate. The assays were generally considered unsuitable for screening the developmental toxicity of our candidate compounds. Recent test systems adopt advanced technologies. Despite such evolution of materials and methods, the concordance rates of the EST and MOT systems were similar. This may suggest that the fundamental predictivity of in vitro developmental toxicity assays has remained basically unchanged for decades. To improve their predictivity, in vitro developmental toxicity assays should be strictly based on elucidated pathogenetic mechanisms of developmental toxicity.

• Journal of Life Science
• /
• v.11 no.2
• /
• pp.94-98
• /
• 2001

Type I signal peptidase cleaves the signal sequence from the amino terminus of membrane and secreted proteins afters these protein insert across the membrane. This enzyme serves as a potential target for the development of novel antibacterial agents due to its unique physiological and biochemical properties. Despite considerable research, the signal peptidase assay still remains improvement to provide further understanding of the mechanism and high-throughput inhibitor screening of this enzyme. In this paper, three known signal peptidase assays are tested with an E. coli D276A mutant signal peptidase to distinguish the sensitivity of each assays. In vitro assay using the procoat synthesized by in vitro transcription translation shows that the D276A signal peptidase I was inactive while in vivo processing of pro-OmpA expressed in the temperature-sensitive E. coli strain IT41 as well as in vitro assay using pro-OmpA nuclease A substrate show that D276A signal peptidase I has activity like wild-type signal peptidase. These results suggest that in vitro assay using the pro-OmpA nuclease A and in vivo pro-OmpA processing assay are more sensitive monitors than in vitro assay using the pro-coat. In conculsion, caution should be used when interpreting the in vitro results using the procoat.

• Asian-Australasian Journal of Animal Sciences
• /
• v.9 no.4
• /
• pp.371-374
• /
• 1996

Experiments were conducted to determine the effect of time intervals between collecting and use of cattle faeces as a source of micro-organisms in in vitro digestibility assays of forages. The results suggested that temperature conservation capacity by faeces depended on the size of the sample. There was no significant difference(p>0.05) between the first (T1 or 08:30 h) and second using time (T2 or 10:30 h). In vitro organic matter digestibility was significantly lower when faeces was used 5 h (T3 or 13:30 h) after collection. However, the organic matter digestibility determined at the second using time (T2) and third using time (T3) were highly correlated ($R^2=0.99$) with the first using time. It was concluded that faeces can be used as a source of microorganisms for in vitro digestibility assays of forages even 5 h after being voided.

• The Korean Journal of Pesticide Science
• /
• v.3 no.3
• /
• pp.37-44
• /
• 1999

Two sulphamide (dichlofluanid and tolylfluanid) and three dicarboximide fungicides (iprodione, vinclozolin, procymidone) were used to investigate the correlation between in vitro antifungal activities and in vivo disease controlling activities against Botrytis cinerea, a causal agent of tomato gray mold and to develop efficient in vitro assays. They controlled effectively the development of tomato gray mold disease in vivo and their controlling activities were similar one another. However, several in vitro assays revealed that their in vitro antifungal activities were quite different between sulphamide and dicarboximide fungicides; the formers showed stronger inhibition activities for spore germination than the latters, whereas the formers inhibited mycelial growth less severely than the latters. The results indicate that the fungicides having different modes of action can show different in vitro antifungal activities according to in vitro assays, even if they have similar in vivo disease controlling activities. On the other hand, two rapid and efficient in vitro assays named Microtiter plate methods I (MPM I) and II (MPM II) were developed for the evaluation of fungicides for inhibitory activities against spore germination and mycelial growth of B. cinerea, respectively. The antifungal activities of five fungicides of two chemical groups in MPM I and II were correlated with the inhibitory activities against spore germination and mycelial growth using solid media, respectively.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.40 no.7
• /
• pp.1053-1062
• /
• 2011

Reactive oxygen species (ROS), including singlet oxygen (${O_2}^1$), superoxide anion radical ($O_2{\cdot}^-$), hydroxyl radical ($HO{\cdot}$), peroxyl radical ($ROO{\cdot}$), hydrogen peroxide ($H_2O_2$), and hypochlorous (HOCl), are generated as byproducts of normal cellular metabolism. ROS induce damage to many biological molecules, such as lipids, proteins, carbohydrates, and DNA. It is widely believed that some degenerative diseases caused by ROS can be prevented by the high intake of fruits and vegetables due to their antioxidant activities. Recently, research on natural antioxidants has become increasingly active in various fields. Several assays have been developed to measure the total antioxidant capacity of antioxidants in fruits and vegetables in vitro. These assays include those for DPPH radical scavenging activity, SOD-like activity, total polyphenol content, oxygen radical absorbance capacity, reducing power, trolox equivalent antioxidant capacity (ABTS assay), single-cell gel electrophoresis (comet assay), and a cellular antioxidant activity assay. Because different antioxidant compounds may act through different mechanisms in vitro, no single assay can fully evaluate the total antioxidant capacity of foods. Due to the complexity of the composition of foods, it is important to be able to measure antioxidant activity using biologically relevant assays. In this review, recently used assays were selected for extended discussion, including a comparison of the advantages and disadvantages of each assay and their application to fruits and vegetables.

• Toxicological Research
• /
• v.16 no.2
• /
• pp.109-116
• /
• 2000

The purposes of our study were to optimize the conditions of the screening and testing methods for endocrine disruptors, to characterize these assays using several compounds with well-defined endocrine activity, and to compare the sensitivity between these assays currently undergoing validation. Two in vitro test systems, MCF-7 cells proliferation (E-screen assay) and competitive binding to estrogen receptors (ER) were selected to evaluate the estrogenic effects. 17$\beta$-Estradiol (E2) and diethylstilbestrol (DES) were used as a positive control in vitro test. Also, E2 and ethinyl estradiol (EE) were used as a positive control in vivo uterotrophic assay. In in vitro test, E2 and DES showed a strong estrogenic response at concentration of 1.0 nM. In uterotrophic assay, E2 (0.3 $\mu\textrm{g}$/kg) and EE (0.3 $\mu\textrm{g}$/kg) produced a significant increase in uterus and vagina weight in both immature and ovariectomized rats. Although we did not com-pared the specificity between in vivo and in vitro assays, these assay systems may serve as a good tool for endocrine disruptors screening methods. Our data indicate that these assay systems exhibit some difference in their sensitivity to the same estrogenic compounds. Therefore, as a first rapid screening assay for estrogenic activity qf unknown chemicals, at least two assay systems should probably be carried out with a view of high sensitivity and standardization conditions. Also, a careful validation tests are necessary to obtain a reasonable degree of reproducibility.

• Molecular & Cellular Toxicology
• /
• v.2 no.2
• /
• pp.106-113
• /
• 2006

Phthalate analogues are a plasticizer and solvent used in industry. Phthalates were classified in the category of "suspected" endocrine disruptors. The purpose of our study was to screen and elucidate the endocrine disrupting activity of seven phthalate analogues. E-screen assay was performed in MCF-7 human breast cancer cells with seven phthalate analogues. In this cell proliferation assay, benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) showed high estrogenic activity. Their relative proliferation efficiencies (RPE) were 109 and 106%, respectively. In vitro estrogen receptor (ER) binding assay, BBP, di-n-octyl phthalate (DOP) and dinonyl phthalate (DNP) showed weak relative binding affinity (RBA: 0.02%) compared to $17{\beta}-estradiol\;(E2)$ (RBA: 100%). In uterotrophic assay, E2 produced a significant increase, whereas four tested phthalate analogues had potential estrogenic effects in vitro did not increased in uterus weight in immature rats. From these results, we demonstrated that phthalate analogues exhibit weak estrogenic activity in vitro assays at high concentrations. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce a uterus weight increase in vivo. From these, we may suggest that these phthalate analogues are easily metabolized to inactive forms in vivo. Further investigation in other in vitro and in vivo experimental systems might be required.

• Journal of Embryo Transfer
• /
• v.10 no.1
• /
• pp.15-22
• /
• 1995

Since the turn of the century, scientists have earnestly sought to develop a single laboratory assay or combination of laboratory assays which accurately predict the fertility of a semen sample. Most of these assays have focused on evaluating physical characteristics of sperm such as motility, viability, acrosomal integrity and morphology. In recent years new approches have been used to assess the functional aspects of a sperm that are needed to reach the oocyte, fertilize it and contribute to successful embryo development. Among these techniques are the ability of sperm to undergo a heparin induced acrosome reaction and in vitro fertilization, and the affinity of sperm to bind heparin binding protein. Intensification of research efforts in the area of control of sperm fertilizing ability should be a high priority, in view of undoubted benifits both to our basic understanding of sperm fertilizing ability and to our ability to modify it for Al industry.

• Toxicological Research
• /
• v.25 no.1
• /
• pp.51-58
• /
• 2009

Carrageenan is a naturally-occurring sulfated polygalactan which has been widely used in the dairy industry and a gelling agent in non-dairy products. In this study, four short-term in vitro genotoxicity assays were investigated to evaluate the potential genotoxic effects of carrageenan. The mutagenicity of carrageenan was evaluated up to a maximum dose of 5 mg/plate in Ames test. There was no increase in the number of revertant colonies compared to its negative control at any dose in all of strains tested. To assess clastogenic effect, the in vitro chromosomal aberration assay was performed using Chinese hamster lung cells. Carrageenan was not considered to be clastogenic in this assay at up to the highest feasible concentration which could be evaluated. The in vitro comet assay and micronucleus test results obtained on L5178Y cells also revealed that carrageenan has no genotoxicity potential, although there was a marginal increase in micronuclei frequencies and DNA damage in the respective micronucleus and comet assays. Taken together, our results indicate that carrageenan was not genotoxic based on four in vitro genotoxicity results.

• Asian-Australasian Journal of Animal Sciences
• /
• v.11 no.1
• /
• pp.51-54
• /
• 1998

The present study investigated the use of cow faeces as a source of micro-organisms instead of sheep rumen liquor in in vitro digestibility assays of forages. Initially 40 forage samples comprising ryegrass, wheat, maize and barley were screened to select a wide range of digestibility (327-794 g/kg) of forages. Finally 8 forage samples were assessed using rumen liquor as well as faecal liquor. The absolute organic matter digestibility (OMD) values using faecal liquor were lower than those with rumen liquor. However, the relationship between OMD using rumen liquor and faecal liquor was highly significant (p < 0.001). The $R^2$ value exceed 0.90. The results suggest that micro-organisms from faecal liquor are capable of digesting forage samples.