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A rapid and sensitive bioassay for detecting cyto-toxicity was developed in this study to be used in evalu-ating many kinds of chemicals. This assay, based on the LDL (low density lipoprotein)-uptake activity of human hepatoblastoma cells, Hep G2, can evaluate cytotoxicity for 48 h with high sensitivity and selectiv-ity using a 96 well plate and a...
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The number of legacy chemicals without toxicity reference values combined with the rate of new chemical development are overwhelming the capacity of the traditional risk assessment paradigm. More efficient approaches are needed to quantitatively estimate chemical risks. In this study, rats were dosed orally with multiple doses of six chemicals for...
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... Liver is considered as one of the most commonly affected organs in pre-clinical toxicity studies. Therefore, primary cultures of rat hepatocytes serve as a suitable model to assess the cytotoxic potentials of poisons and hepato-totoxicity in particular 17,18,24 . The work of Niknahad et al. (1984) refers to screening of various nutrients like glycolytic substrates and keto-acid metabolites against cytotoxicity of cyanide in isolated rat hepatocytes 20 . ...
... Additionally, the interaction of A-KG with cyanide was assessed by the levels of cyanide, A-KG and thiocyanate in vitro and in vivo. Leakage of LDH serves as an index of compromised plasma membrane integrity, while leakage of ALAT and ASAT are also critical to cell viability and indicate hepatotoxic potential of a chemical [24][25][26] . During our preliminary unpublished work, different doses of KCN (1.25-20 mM) were added to primary culture of rat hepatocytes for 0.5-24 h, and cytotoxicity was measured by TBDE and leakage LDH, ALAT, and ASAT. ...
Cyanide is primarily a neurotoxin but its hepatotoxic and nephrotoxic potentials are also known. The present study reports the effect of alpha-ketoglutarate A-KG (2.5–20 mM; 0 min), a potential cyanide antidote on potassium cyanide (KCN; 1.25–20 mM) induced cytotoxicity in primary culture of rat hepatocytes. Cytotoxicity measured at various time points (0.5–24 h), was characterized by increased leakage of intracellular lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase, accompanied by diminished mitochondrial function (MTT assay), mitochondrial membrane potential (Rhodamine 123 assay), and ATP levels. However, lipid peroxidation (malondialdehyde assay) and DNA damage were not observed. In a separate study, levels of cyanide, AKG and thiocyanate were measured in the culture medium of hepatocytes, treated with KCN (5 mM) and/or A-KG (5 or 10 mM; 0 min), and in the serum of rats given oral treatment of KCN (10 mg/kg) and/or A-KG(0.5, 1 or 2 g/kg; 0 min). Cyanide and A-KG interaction was best exhibited when both were added in equimolar dose in vitro. In rats, cyanide levels were significantly reduced by 1 and 2 g/kg A-KG. It can be concluded from the results that, (i) a very high dose of cyanide is required to produce cytotoxicity and other cellular perturbations in rat hepatocytes, (ii) cytotoxicity is independent of lipid peroxidation and DNA damage, (iii) A-KG provides significant protection against cyanide, particularly at equimolar dose in vitro, and (iv) a very high dose of A-KG is required for cyanide detoxification in vivo, suggesting that the dose of A-KG could be reduced by improving its bioavailability.
... The approach adopted here was regarded as fully effective, more easy, less time consuming, more elegant, and more reproducible, and consisted of adding 5 l of a 0.92 g/ml dense silicon oil to each well, which removed all bubbles after some air was gently blown over it after addition. [6][7][8][9][10][11][12][13][14][15][16] It should be noted here that LDH signals related to LDH leakage are lower-range signals mostly; Fig. 2 thus indicates that especially the assessment of cell toxicity may be specifically improved by defoaming. The resulting 500 cells per well sensitivity in 490 nm LDH reading (see above) means that at an illustrative cell density of 10 5 cells per well, cell death may be tracked down to 0.5%. ...
The lactate dehydrogenase (LDH) assay was addressed for its sensitivity, disturbances by foaming, and cell number and size. Cells were from a U-251 MG grade IV human glioblastoma brain tumor cell line used in 100-microl well volumes. Cells were counted by microscopy and Coulter counting; assays were LDH or trypan blue. The results indicate increased 490 nm signals (level, variance) by using phenol red or by increasing fetal bovine serum from 5% to 10%. The data also indicate that defoaming results in reduced variances ranging from a factor of 2 at 1-3 units of absorption, up to a factor of 4-5 at <1 units of absorption. Coulter counting indicated a decrease in cell volume with increasing end-point cell density, attributed to general shrinking at increasing density. In comparisons, total LDH was considered relative to both cell total volume and cell numbers. The result suggests that total LDH should be regarded as reflecting cell total volume rather than cell numbers. In a comparative Cu exposure test, signals of both LDH and a sodium salt of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) decreased with increasing Cu supply, while bromodeoxyuridine signals remained largely unaffected. The data show the differences in responses in cell viability and proliferation, but, above all, indicate that LDH should be expressed on a per cell volume basis rather than per cell, to avoid the problem that mere density effects contribute to signals on compound or metal toxicity.
A Toxicity Reduction Evaluation (TRE) conducted on various water treatments was successful in optimizing the acute toxicity of the final effluent. In particular, we found that the acute toxicity of the sample water treated by a water treatment process should not be correlated with the concentrations of the parent compounds still remaining in the water. Therefore, it was concluded that an acute toxicity test based on a bioassay must be performed in order to evaluate the efficiency of the water treatment process for various effluents containing environmental pollutants. In addition, the treatability and the resultant toxicity was evaluated as a simple scoring system. The scoring system ranked the treated water in three classes of toxicity with the calculation of several factors for the concerned toxicity. Using an impact evaluation based on the score of the characteristics, different water treatment processes could be directly compared. The new methodology presented here is specifically effective in listing possible unit operations for a water treatment process based on the impact, and thereby enables targeted and cost-effective water treatment.
The sources of tap water have recently become polluted with a number of chemicals discharged from various industries. Among them, pesticides from golf links and agricultural areas can be considered as priority chemicals for water quality management. In water treatment processes, activated carbon is generally used to remove pollutants. However, humic substances also contaminate environmental water, such as river water. In this study, we examined adsorption isotherms and rates on activated carbon at various temperatures, in order to examine the basic characteristics of adsorption using activated carbon for removal of 2,4-dichlorophenoxy acetic acid (2,4-D), humic substances, and their combination. The adsorption isotherms were correlated with Freundlich-type equation in the concentration ranges tested. Surface diffusion was considered to be dominant. For the selective adsorption treatment for 2,4-D in solution with humic substances, activated carbon with small pores was more effective than that with large pores.
Although bioassays are considered to be a rational method for environmental management, the procedure is generally too complicated to be applied to daily water quality management. In this study, the feasibility of using for application of a conventional QSAR (Quantitative Structure-Activity Relationship) method was examined to estimate the cytotoxicity of various pollutants found in environmental water. logP, pKa, and molecular weight were chosen as the physico/chemical properties of the pollutants, and defined equations for estimating cytotoxicity based on multiple linear regression analysis between these properties and in vitro cytotoxicity data from our previous results. As a result, a method for estimating cytotoxicity of environmental pollutants that had a certain probability (R>0.8) for the 255 chemicals was successfully developed. Considerably high reliability was shown in the leave-one-out prediction of multi-regression analysis. In addition, the cytotoxicity of environmental water samples was estimated based on multi-regression analysis, using as our samples leachates from 25 landfill sites in Japan. The method developed in this study estimated quantitatively the cytotoxicity of the environmental water from chemical analysis data without conducting a cytotoxicity test.
A 90-min assay using 24-well plates was developed for screening overall metabolic effects of toxic chemicals by measuring extracellular acidification rate of mammalian cells. During the 90-min test, the pH of each well of a 24-well plate is monitored by measuring the absorbance of phenol red using a spectrophotometric plate reader. The acidification rate is then calculated from the change in pH divided by the time interval and cell density, which is also estimated non-invasively by spectroscopy. The assay was verified by testing the effects of five well-characterized chemical toxins on fibroblast cell cultures. As expected, the responses of the fibroblasts were dependent on the dose and type of toxin. They also corresponded with the established mechanisms of the toxins and with measured lactate production rates. The set-up is simple, inexpensive, and amenable to being automated. The method is easy to perform and rapid. Uses include screening compounds for gross metabolic effects in mammalian cell lines, determining preliminary metabolic dose-response curves for guiding further research, and designing and optimizing media for in vitro systems utilizing cell cultures.
