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Effects of nac_full paper-jkkim et al

VIII Congreso Regional de Seguridad Radiológica y Nuclear,
I Congreso Latinoamericano del IRPA
V Congreso Nacional de Protección Radiológica DSSA
EFFECTS OF N-ACETYL-L-CYSTEINE ON PLHC-1 CELLS AFTER IRRADIATION
Jin Kyu Kim1,♦, Min Han1, Mohammad Nili2 1Korea Atomic Energy Research Institute, Jeongeup 580-185, Korea 2Dawnesh Radiation Research Institute, Barcelona 08007, Spain ABSTRACT
The antioxidant and free-radical scavenger N-acetyl-L-cysteine (NAC) is used extensively as a conditional nutrient. NAC acts as a cysteine donor and maintains or even increases the intracellular levels of glutathione (GSH), a tripeptide which protects cells from toxins such as free-radicals. This study was designed to evaluate the effects of NAC in different doses on the activity levels of GSH and the cell viability in the fish cell line PLHC-1 against ionizing radiation. Effects of NAC were assessed in the cells irradiated with 100 ~ 500 Gy. The data showed that NAC in lower concentrations prevented cell death after irradiation of lower doses. However NAC didn't prevent cells from radiation-induced death at higher doses of radiation (300, 500 Gy). The intracellular GSH levels significantly decreased after treatment with radiation alone while the combined treatment of NAC and radiation alleviated the decrease in GSH levels. These results suggest that radiation-induced oxidative stress and NAC prevents radiation-induced cell damage including cell death by increasing the level of GSH. Based on these results, it is suggested that PLHC-1 cells can serve as rapid screening test tools for detecting the response of radiation. Also, this study may be useful for elucidating the effects of radiation on aquatic organisms. Key Words: radiation, PLHC-1 cell, NAC, mercury chloride, combined effect.
1. Introduccion

Living organisms are continuously being exposed to harmful factors present in the environment. The combined exposure to various chemical, physical, and even social factors is an inevitable feature of life. The biological effects, due to the combined action of ionizing radiation with another factor, are hard to estimate and predict in advance. The most effective simultaneous exposure to ionizing radiation and another factor may result in four possible modes of interaction: (a) no interaction, the effects observed are based on the most toxic agent; (b) additivity, the effects produced by separate application of each agent are simply summed up; this mode can be subdivided into the additive and independent interaction; (c) antagonism, the effect is less than expected additivity; (d) synergism or supra-additivity, the effect is greater than expected additivity. Ionizing radiation is used for therapy, diagnosis, and sterilising of food. But ionizing radiation is known to cause cell death, mainly due to its ability to produce reactive oxygen species in cells. Exposure of cells to DNA-damaging agents like ionizing radiation results in complex response mechanisms to maintain a genetic integrity after DNA damage. These include a cell cycle delay, repair of DNA damage, transcriptional responses, and a programmed cell death. Mercury chloride is a widespread environmental pollutant that is known to have toxic effects. There are many reports indicating its genotoxic potential in a 11 al 15 de Octubre de 2010,
Medellín Colombia
VIII Congreso Regional de Seguridad Radiológica y Nuclear,
I Congreso Latinoamericano del IRPA
V Congreso Nacional de Protección Radiológica DSSA
variety of aquatic species. Synergistic effects of radiation and HgCl2 on human cells was previously reported [1]. And it is necessary to study bioassay techniques in relation to a specific biological end-point using diverse organisms. Various in vitro cytotoxicity test systems using fish cell lines of different origins have been applied to examine the toxic potency of numerous selected chemicals. The PLHC-1 hepatoma cell line derived from topminnow (Poeciliopsis lucida) is one of the most commonly used cell lines in aquatic toxicology. PLHC-1 cells have metabolic activities necessary to study the metabolism of chemicals acting via metabolic activation and are easy to cultivate. PLHC-1 cells are therefore a promising tool in the toxicity screening and in the toxicity evaluation of chemicals. All aerobically growing organisms suffer from exposure to oxidative stress, caused by partially reduced forms of molecular oxygen, known as reactive oxygen species. These are highly reactive and capable of damaging cellular constituents such as DNA, lipids, and proteins. Consequently, cells from many different organisms have evolved mechanisms to protect their components against reactive oxygen species. Reactive oxygen species can also be formed by exposure of cells either to ionizing radiation or redox-cycling chemicals present in the environment like heavy metals [2]. The antioxidant and free-radical scavenger N-acetyl-L-cysteine (NAC) is used extensively as a conditional nutrient. NAC acts as a cysteine donor and maintains or even increases the intracellular levels of glutathione (GSH), a tripeptide which protects cells from toxins such as free-radicals. With regard to the radioprotective effects of NAC, the majority of studies have been performed in vitro [3-4]. NAC were used to protect the Chinese hamster ovary (CHO) cells from radiation-induced apoptosis by controlling the enzyme that triggers programmed c e l l d e a t h [ 5 ] . S o m e s t u d i e s h a v e s u c c e s s f u l l y d e m o n s t r a t e d s p o r a d i c r a d i o p r o t e c -tion following low-level chronic administration of NAC, though the mode and optimal dose of NAC The present study was designed not only to evaluate the effects of NAC in different doses on the activity levels of GSH and the cell viability in the fish cell line against ionizing radiation, but also to
investigate protective effects of NAC on cell viability in cells against HgCl2.
2. Materials and Methods
PLHC-1 cells are derived from a hepatocellular carcinoma in an adult female (Poeciliopsis Lucida), a topminnow from the Sonoran desert (ATCC® # CRL-2406). PLHC-1 cells are grown at 30°C in a humidified incubator containing 5% CO2 and propagated in Eagle's Minimum Essential Medium (Hyclone, UT, USA) supplemented with 5% foetal bovine serum (Gibco™, Grand Island, NY), L-glutamine (Sigma-Aldrich, MO, USA), sodium pyruvate (Sigma-Aldrich), gentamicin (Hyclone). The cells routinely grow in 75 ㎠ flasks (Costar), and are subcultured every 3 to 5 days at a split ratio of 1:4. Cells were treated γ-rays from a 60Co isotopic source (Korea Atomic Energy Research Institute, Korea) with 100 ∼ 500 Gy in the presence or absence of 0.05 ∼ 10 mM of NAC which were added in the medium 1 hr before. The cytotoxicity of NAC on PLHC-1 cells was assessed. The MTT assay is based on the uptake of thiazolyl blue tetrazolium bromide (MTT) and its following reduction in mitochondria of living cells to MTT formazan, while dead cells are almost completely negative in this cleavage activity. To assess MTT assay, 100 ㎕ of MTT solution was added to each well after removal of 100 ㎕ supernatant and incubated for another 4 hr at 30 °C. The generated formazan crystal was dissolved and the absorbance was detected at 570nm using ELISA reader (MultiskanⓇ EX, Forma Scientific, Inc.). All organisms are continuously being exposed to harmful factors present in the environment. The combined action of various chemical, physical, and even social factors is an inevitable feature of modern life. The biological effects, due to the combined action of ionizing radiation with another factor, are hard 11 al 15 de Octubre de 2010,
Medellín Colombia
VIII Congreso Regional de Seguridad Radiológica y Nuclear,
I Congreso Latinoamericano del IRPA
V Congreso Nacional de Protección Radiológica DSSA
3. Results and Discussions
In the cytotoxicity tests, NAC treatment elicited an increasing of cell viability in PLHC-1 cells in a dose dependent pattern. But the data showed that NAC in higher concentration ( > 21.25 mM of NAC ) decreased cell viability. The results of experiments on GSH levels by NAC were shown in Fig. 1. In the treated PLHC-1 cells GSH levels were higher than the untreated cells. However, 10 mM NAC treatment caused a decrease in the intracellular level of GSH. l)
tro 200

els (% 100
Concentration of NAC (mM)
Fig. 1. The effects of NAC on the intracellular level of GSH. Results represent the average of three different experiments. GSH was calculated as µmole of GSH per mg of protein and then was presented as the percentage of that of the control group. All the points showed a statistically significant difference from the control group according to Student’s t-test (p<0.001). The cell viability in the NAC pre-treated groups was higher than that of the 50 and 100 Gy radiation- treated groups without NAC. The results showed that NAC prevented cells from radiation-induced death, but it caused cytotoxicity in the 300 Gy radiation-treated groups as its concentration increases (Fig. 2). Wu et al. (2008) [5] conducted the initial in vitro studies using NAC as a cytoprotective agent for CHO cells exposed to radiation. The data indicated a significant prevention from loss of cell viability. The cell viability of 1 and 5 mM NAC treated groups was even lower than that of the radiation-induced group, indicating that, although NAC can provide some protection at lower concentrations, it is cytotoxic at higher concentrations. The protective effect of NAC was also studied for the combined exposure to HgCl2 and 4-nonylphenol. The result showed that cell death was prevented in the group pre-treated with NAC. According to these results, it is suggested that the potential utility of NAC in lower doses as a protector against radiation is worth considering here. 11 al 15 de Octubre de 2010,
Medellín Colombia
VIII Congreso Regional de Seguridad Radiológica y Nuclear,
I Congreso Latinoamericano del IRPA
V Congreso Nacional de Protección Radiológica DSSA
Control
100 Gy
300 Gy
500 Gy

Concentration of NAC (mM)
Fig. 2. Protective effects of NAC in radiation-induced cytotoxicity. Cytotoxicity was measured by MTT assay 24 hr after irradiation, respectively. All the points showed a statistically significant difference from the control group according to Student’s t-test (p<0.005). The antioxidant property of NAC can be attributed to its ability to provide cysteine and other precursors of glutathione synthesis, as well as its ability to directly scavenge free radicals [6]. According to several studies, NAC on the one hand acts as an antioxidant, but on the other, it can also act as a pro-oxidant, resulting in cytotoxicity and oxidative stress [7]. GSH is critical for cellular functions and cell survival. As the most abundant reducing molecule in the hepatocyte, it maintains the redox state of the sulfhydryl groups of cellular proteins. As a major antioxidant, GSH quenches the endogenous oxidant species and combats exogenous oxidative stress. As an important regulator, GSH modulates the signalling cascades and the susceptibility of cells to different cell death stimuli [8]. The cells were collected in MES buffer. GSH (µmol/mg protein) were assayed with Cayman Kits according to the manufacturer's instructions and determined by the colorimetric method. Protein content was determined by the method of Bradford, using bovine serum albumin as standard. This study showed that the intracellular GSH levels significantly decreased after treatment with radiation alone while the combined treatment of NAC and radiation prevented the decrease in the GSH levels (Fig. 3). These results suggest that NAC prevents radiation-induced cell damage including cell death by increasing the level of GSH. Protective effects of NAC were assessed in PLHC-1 cells against cell viability induced by HgCl2. In the cytotoxicity tests using the MTT assay, the cell viability in the NAC pre-treated groups was higher than that of the HgCl2-treated groups without NAC pre-treatment. The results indicated that NAC also prevented cells from HgCl2-induced death (Fig. 4). On the other hand, in the previous study, simultaneous treatment of the cells with ionizing radiation and HgCl2 resulted in a dramatic increase of cell death, while neither of them showed cytotoxic effects when treated alone [9]. The cytotoxicity of ionizing radiation was enhanced in the presence of HgCl2. An analysis of the extent of synergistic interaction allows to make a quantitative estimation of irreversibly damaged cells after the combined exposure. 11 al 15 de Octubre de 2010,
Medellín Colombia
VIII Congreso Regional de Seguridad Radiológica y Nuclear,
I Congreso Latinoamericano del IRPA
V Congreso Nacional de Protección Radiológica DSSA
300 Gy
500 Gy

Concentration of NAC (mM)
Fig. 3. Intracellular GSH levels measured in the cells treated with radiation only and in combination with NAC. All the points showed a statistically significant difference from the control group according to Student’s t-test (p<0.005). 250uM HgCl2
250uM HgCl2 + 5mM NAC
250uM HgCl2 + 10mM NAC
Fig. 4. Protective effects of NAC in HgCl2-induced cytotoxicity. Cytotoxicity was measured by MTT assay 24 hr after treatment. All the points showed a statistically significant difference from the control group according to Student’s t-test (p<0.005). 11 al 15 de Octubre de 2010,
Medellín Colombia
VIII Congreso Regional de Seguridad Radiológica y Nuclear,
I Congreso Latinoamericano del IRPA
V Congreso Nacional de Protección Radiológica DSSA
Effects of NAC were assessed in PLHC-1 cells irradiated with 100 ~ 500 Gy. The data showed that NAC in lower concentrations prevented cell death after irradiation with lower doses. But NAC did not
prevent cells from radiation-induced death in higher doses of radiation (300 and 500 Gy). The intracellular
GSH levels significantly decreased after treatment with radiation alone while the combined treatment of
NAC and radiation alleviated the decrease in GSH levels. These results suggest that NAC prevents
radiation-induced cellular damage including cell death by increasing the level of GSH. NAC prevented
cells from HgCl2-induced death as well. On the basis of these results, it is suggested that PLHC-1 cells can
serve as rapid screening test tools for detecting the response to ionizing radiation and harmful chemicals.
This study may be also useful for elucidating the effects of radiation on aquatic organisms.


ACKNOWLEDGMENT

This study has been supported by the National R&D Program by the Ministry of Education, Science and Technology (MEST) of Korea, and partly supported by Dawnesh Radiation Research Instiotute,
Spain.

REFERENCES
[1] H. J. Woo, J. H. Kim, A. Cebulska-Wasilewska, J. K. Kim (2006) Evaluation of DNA damage by
mercury chloride (II) and ionizing radiation in HeLa cells. Korean J. Environ. Biol. 24, 46-52.
[2] D. J. Jamieson (1998) Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast 14,
1511-1527.
[3] P. Sminia P, A. H. Van der Kracht, W. M. Frederiks, W. Jansen (1996) Hyperthermia, radiation
carcinogennesis and the protective potential of vitamin A and N-acetylcysteine. J. Cancer Res. Clin. Oncol.
122, 343-350.
[4] G. Abt, H. Vaghef, E. Gebhart, C. V. Dahlgren, B. Hellman (1997) The role of N-acetylcysteine as a
putative radioprotective agent on X-ray-induced DNA damage as evaluated by alkaline single-cell gel
electrophoresis. Mutat. Res. 384, 55-64.
[5] W. Wu L. Abraham, J. Ogony, R. Matthews, G. Goldstein, N. Ercal (2008) Effects of N-
acetylcysteine amide (NACA), a thiol antioxidant on radiation-induced cytotoxicity in Chinese hamster
ovary cells. Life Sci. 82, 1122-1130.
[6] G. S. Kelly (1998) Clinical applications of N-acetylcysteine. Alternative Medicine Review 3, 114-127.
[7] K. D. Held, J. E. Biaglow (1994) Mechanisms for the oxygen-radical mediated toxicity of various
thiol-containing compounds in cultured mammalian cells. Radiat. Res. 139, 544-554.
[8] L. Yuan, N. Kaplowitz (2009) Glutatione in liver diseases and hepatotoxicity. Molecular Aspects of
Medicine
30, 29-41.
[9] M. Han, K. M. Hyun, M. Nili, I. Y. Hwang, J. K. Kim (2009) Synergistic effects of ionizing radiation
and mercury chloride on cell viability in fish hepatoma cells. Korean J. Environ. Biol. 27, 140-145.
11 al 15 de Octubre de 2010,
Medellín Colombia

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