A human acute promyelocytic leukemia HL-60 cell line was kindly provided by Dr Tzou-Chi Huang (National Pingtung University of Science and Technology, Pingtung, Taiwan). HL-60 cells were grown in Iscove's modified Dulbecco's medium (Hyclone, Logan, UT, USA) supplemented with 4 mM L-glutamine (Gibco; Invitrogen, Carlsbad, CA, USA), 15% fetal bovine serum (Hyclone), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Invitrogen). HL-60 cells from passages 20–40 were used in the experiments. A WEHI-3 mouse myelomonocytic, macrophage-like leukemia cell line purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan) was grown in Iscove's modified Dulbecco's medium with 4 mM L-glutamine, supplemented with 0.05 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and 10% fetal bovine serum. Both cell lines were cultured in a 95% humidity atmosphere under 5% CO₂ in air at 37°C.

Cultured HL-60 cells were treated with different concentrations of H₂O₂ (ranging from 0–100 mM) or NAC (ranging from 0–50 mM) for 24 h. The cytotoxicities of H₂O₂ and NAC towards HL-60 cells were determined by trypan blue exclusion. This method is based on the principle that live cells possess intact plasma membranes that exclude the trypan blue dye. The CC₁₀ indicated the cytotoxic concentration that kills 10% of treated cells (24,25). The 10% cytotoxic concentration (CC₁₀) after 24 h of treatment was calculated using Microsoft Excel software.

In order to evaluate the radical scavenging effect of NAC, the intracellular ROS level was analyzed. A DCF-DA assay was performed to detect the intracellular production of hydroxyl, peroxyl and other ROS within the cells. HL-60 cells were incubated for 30 min with 10 µM DCF-DA and washed with PBS, followed by treatment with H₂O₂ at the CC₁₀ concentration alone or in combination with various concentrations of NAC for 2 h. The fluorescence intensities of DCF were quantified using a microplate reader (Turner Biosystems, Sunnyvale, CA, USA), with an excitation wavelength of 485 nm and an emission wavelength of 525 nm.

To further understand the antioxidant effects of NAC, the levels of MDA, TAC, SOD and GSH/GSSG in cultured HL-60 cells treated with H₂O₂ at the CC₁₀ concentration were measured. Upregulation of the MDA level indicates oxidative degradation of cellular major components; however, enhancement of the total antioxidation capacity (TAC) represents a cellular protective response to oxidative stress (26). HL-60 cells were treated with H₂O₂ alone or in combination with various concentrations of NAC for 8 h and then subjected to MDA assay. For measurement of the TAC, SOD and GSH/GSSG levels, well-grown HL-60 cells were incubated with various concentrations of compounds for 12 h. Cells were washed twice with PBS, harvested and analyzed following the instructions of the kit manufacturers. The OD value was determined using a spectrophotometer (Bio-Rad Model 680).

HL-60 cells were treated with 20 mM H₂O₂ alone or in combination with various concentrations of NAC for 16 h. The assay protocol followed the instruction manual provided with the kit. Briefly, cellular DNA was extracted and converted to single-stranded DNA at 95°C for 5 min, and rapidly chilled on ice, then DNA nucleosides were digested by incubating the denatured DNA with nuclease P1. The unknown sample or 8-OHdG standard was incubated with a 8-OHdG conjugate-coated plate followed by reaction with anti-8-OHdG antibody. After washing and substrate reaction, the absorbance of the microwells was determined using a spectrophotometer (Bio-Rad Model 680) at a wavelength of 450 nm.

The animal test procedure examined and approved by the Institutional Animal Care and Use Committee (IACUC) of our University (NPUST-103-052). Co-author Ching-Dong Chang is a veterinary pathologists and in charge of the animal monitoring and minimize the animal distress. A leukemia mice model was established in our laboratory (27). Thirty male BALB/c mice, 6 weeks of age, were obtained from BioLASCO (Taipei, Taiwan). After accommodation for one week, the mice were randomly divided into 5 groups: Group 1 was the negative control with no treatment; group 2 received intraperitoneal (i.p.) injection with NAC 200 mg/kg for 7 days; group 3 was treated with vehicle PBS through the i.p. route for 7 days then injected with WEHI-3 cells via the tail vein; groups 4 and 5 received NAC 50 or 200 mg/kg through i.p. injection for 7 days, following which leukemia was induced by injection of WEHI-3 cells into the tail vein on day 8. The NAC administration dose was cousulted and referred to the literatures (28,29). Following death, blood and major organs were collected. All resting living mice were euthanized by carbon dioxide on day 7 post WEHI-3 injection. The mortality rate and organ weights were recorded. Blood samples were analyzed to assess liver function markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as antioxidant parameters, including total antioxidant capacity (TAC) and levels of SOD, glutathione and lysozyme. Furthermore, blood samples were also subjected to total acid phosphatase (ACP) analysis, a general diagnostic marker of disease condition. Considering the animal welfare, animals inoculated with tumor cells should be euthanized if following conditions is observed and diagnosed by the co-author Ching-Dong Chang veterinarian (1) Animal is unable to present normal activities due to the tumor (2) Animal abdomen appears dark-gray/green or ascites exceed 20% of the animal weight (3). Lethargy, anorexia, dehydration, or other sign of obvious stress or pain (6). Animal is unable to feed or drink normally due to the tumor.

2′,7′-Dichlorofluorescin diacetate (DCF-DA), N-acetyl-L-cysteine (NAC) and H₂O₂ were purchased from Sigma-Aldrich. A Lipid Peroxidation (MDA) Colorimetric Assay Kit, Total Antioxidant Capacity (TAC) Colorimetric Assay Kit, Superoxide Dismutase (SOD) Activity Assay Kit, Glutathione (GSH/GSSG/Total) Fluorometric Assay Kit, Aspartate aminotransferase (AST) Assay Kit, Alanine aminotransferase (ALT) Assay Kit, Total Acid Phosphatase (ACP) and Lysozyme Activity Assay Kit were purchased from Biovision (Mountain View, CA, USA). An 8-OHdG DNA Damage ELISA kit was obtained from Cell Biolabs, Inc. (San Diego, CA, USA).

All cell-based experiments were performed at least 3 times, and data are presented as mean ± standard deviation (SD). Statistical significance was determined between groups using Student's t-test. In the animal experiment, the data were analyzed by one-way analysis of variance (ANOVA), with the post-hoc t-test. A value of P<0.05 was considered to be significant.

First of all, the non-toxic maximum concentration based on our experimental treatment duration needed to be determined. In order to determine the cytotoxicity response, the initial testing concentration range of NAC from 0–50 mM. As the duration of compound treatment in all cell culture assays did not exceed 16 h, we needed to identify the concentration that did not cause the death of more than 10% of cells after 24 h of incubation. Our data indicated that the CC₁₀ values of H₂O₂ and NAC were 11.16 and 12.34 mM, respectively. The growth curve showed the higher NAC concentration, the cell viability decreased gradually (data not shown). The working concentration of the next experiments will not over the CC₁₀ dose. Thus, we can then investigate the benefit effects under the enough safety prerequisites.

Next, we analyzed the oxidative response induced by H₂O₂ in our system, and characterized the protective dose-response in the presence of NAC. Several experiments were performed. A fluorescent probe, DCF-DA, has been widely-used to measure intracellular oxidant levels. As shown in Fig. 1A, H₂O₂ produced significantly higher levels of ROS. Critically, at the CC₁₀, NAC efficiently blocked the H₂O₂-generated ROS; lower dosages of NAC also exerted significant activity in terms of reducing the ROS level in a dose-response manner. Additionally, MDA is one of the most critical byproducts of lipid peroxidation during oxidative stress, and therefore analysis of lipid peroxidation is essential in the study of pathophysiological processes. The results for ROS and MDA showed an identical trend with very similar response patterns. Furthermore, in order to demonstrate that H₂O₂-elicited ROS affected DNA integrity, we measured the concentration of 8-hydroxydeoxyguanosine (8-OHdG), a critical oxidative DNA damage byproduct. As shown in Fig. 1B, NAC exerted an antioxidant protective activity against H₂O₂-mediated oxidative stress in HL-60 cells.

To further confirm the comprehensive antioxidant function of NAC in a HL-60 leukemia cell line, we examined whether NAC elevated cellular endogenous antioxidant mechanisms. There are three different types of antioxidant species, including enzyme systems, small molecules and proteins. The total antioxidant capacity (TAC) assay kit used in this study provided a method by which to measure the combined nonenzymatic antioxidant capacity from culture medium. Thus, both small-molecule antioxidants and protein antioxidants were determined in the NAC-treated HL-60 cell system. Another antioxidant enzyme, superoxide dismutase (SOD), which is considered one of the body's most powerful enzymes, was also evaluated. As shown in Fig. 2A, NAC upregulated the endogenous cellular antioxidant activity, and increased TAC and SOD in a dose-response manner. Furthermore, glutathione (GSH) is critical in terms of protecting cells against free-radical damage, and an increased ratio of GSSG to GSH indicates oxidative stress. As clearly demonstrated in Fig. 2B, H₂O₂ reduced the GSH/GSSG ratio, and NAC upregulated the GSH/GSSG ratio by itself; furthermore, NAC reversed the H₂O₂-mediated oxidative effects in HL-60 cells dramatically. Taken together, these results demonstrated that NAC is a good antioxidant for use in this leukemia cell line, not only upregulating the endogenous cellular antioxidant machinery, but also scavenging free-radicals in cells facing oxidative stress.

To compare and confirm the antioxidant protective activity of NAC against leukemia in vivo, a circulated leukemia mouse model was utilized, created by WEHI-3 injection via the tail vein and resulting in mouse death within one week (30,31). Based on our previous publication, the non-treated-mice died of leukemia by histopathological examination (27). The mortality rates of the individual mouse groups are shown in Fig. 3. Additionally, the spleen size of each group was showed in Fig. 4. The result clearly illustrated the larger spleen in mice received WEHI-3 cells. All mice had died by day 5 after WEHI-3 cell injection; however, in mice that received 50 mg/kg NAC, survival was significantly increased, and in mice receiving 200 mg/kg NAC, all mice were alive on day 7 after WEHI-3 administration (Fig. 3). There are nine mice died in our total thirty mice. These nine died mice occurs during the daytime and we isolate the blood and organs immediately.

Mouse sera and organs were collected and analyzed. First, we evaluated the safety of the dosage of NAC we used. Based on organ weights/10 g body weight (Table I) and serum levels of AST, ALT and ACP markers (Table II), a comparison of group 2 with corn oil-treated healthy mice was performed, and no significant difference was observed in any of these parameters. Thus, 200 mg/kg NAC was sufficiently safe for use in our mouse model. In the group 3 mice, WEHI-3 cells were used to establish leukemia, and the liver, spleen and lung weights were significantly higher than those of the group 1 mice. The food consumption of the WEHI-3-injected mice was significantly lower than that of the control mice. The leukemia mice in group 3 also exhibited higher levels of AST, ALT and ACP, as well as lower antioxidant serum parameters of TAC, SOD, and GSH/GSSG. In the 50 mg/kg NAC administration group, some parameter reversed as compared with group 3, indicated the in vivo protective effects of NAC. In the 200 mg/kg NAC treatment groups, the levels of AST, ALT and ACP were not significantly different to those of the control mice. The levels of TAC, SOD and GSH/GSSG indicated that NAC promoted good health. Finally, administration of both 50 and 200 mg/kg NAC reduced mouse death (Fig. 3).