Vitamins 696x496 1

vitamin c nac

Rwei-Fen S. Huang, Sheu-Mai Huang, Bo-Shiou Lin, Chien-Ya Hung, Hsing-Te Lu, N-Acetylcysteine, Vitamin C and Vitamin E Diminish Homocysteine Thiolactone-Induced Apoptosis in Human Promyeloid HL-60 Cells, The Journal of Diet, Quantity 132, Difficulty 8, August 2002, Pages 2151–2156,



Homocysteine thiolactone (HcyT)3 is a by-product of homocysteine; it’s a sulfated amino acid product of the demethylation of methionine. HcyT and homocysteine are metabolically interchangeable in human cells (1,2) and serum (3). Elevated ranges of homocysteine in addition to HcyT are thought of to be atherosclerotic and carcinogenic (4–6). The molecular mechanisms of those homocysteine derivatives to trigger mobile harm have been investigated extensively. It was steered that each homocysteine and thiolactone derivatives might elicit oxidative stress in cells and animals (7–10). The ensuing oxidative injury is evidenced by enhanced lipid peroxidation (11–13), elevated 8-hydroxy-2′-deoxyguanosine ranges in DNA (14) and impaired antioxidant enzymatic perform (15). Homocysteine-induced oxidative harm will be prevented by a wide range of antioxidants, suggesting that reactive oxygen species (ROS) act as mediators (16,17).

Apoptosis, a programmed cell dying, will be activated in response to oxidative stress circumstances resembling throughout ROS era (18,19). Apoptosis in cells is characterised by chromatin condensation, nucleosomal DNA fragmentation and membrane disruption attributable to phosphatidylserine (PS) publicity (20–22). As cells are stimulated to bear intra- or extranuclear apoptotic occasions, the frequent signaling pathways for initiation and execution of apoptosis are concerned within the activation of caspases, a household of aspartate-specific cysteine proteases (23,24). One of many alerts for apoptosis execution includes caspase-3, which is a vital apoptotic effector for DNA injury or membrane dysfunction (25–27). Though the mechanisms by which ROS induce apoptosis are usually not totally outlined, it has been steered that the activation of caspases is concerned. Proof has proven that apoptosis induced by ROS was accompanied by caspase-3 activation, whereas inhibition of caspase-3 blocked apoptosis (28). After hydrogen peroxide (H2O2) therapy, caspase-3 was activated throughout apoptosis in HL-60 cells (29).

HcyT was proven beforehand to induce apoptotic injury in vascular endothelial cells (30). We demonstrated that HcyT-induced apoptosis is mediated by H2O2 era and caspase 3-activation in HL-60 cells (31). As a result of oxidative apoptotic injury might play a pivotal function within the etiology of malignancies and ailments (32), it’s of curiosity to review the protecting results of some antioxidants on HcyT-induced apoptosis. N-Acetylcysteine (NAC) is a precursor of lowered glutathione that has the power to quench hydroperoxides. Vitamin C (Vit C) is an efficient scavenger of hydroxyl radicals, and vitamin E (Vit E) is a lipid radical chain breaker that scavenges oxygen radicals and alkyl radicals (33). The power of NAC, Vit C or Vit E to cut back cell injury elicited by varied apoptotic stimuli has additionally been nicely studied (34–37). Folate has lately been proposed to scavenge peroxyl radicals, azide radicals and hydroxyl radicals in an in vitro radical response mannequin system (38). Nevertheless, the antioxidant motion of folate towards apoptotic injury has not been outlined. To find out whether or not and the way these antioxidants would possibly be capable of counteract HcyT-induced apoptosis, results of NAC, Vit C, Vit E and folate, together with catalase, have been investigated utilizing HL-60 cells, a promyeloid-like cell line with particular myeloid traits, which is likely one of the mostly used cell traces within the examine of apoptosis.




l-HcyT (homocysteine thiolactone hydrochloride salt), Vit C (l-ascorbic acid sodium salt), Vit E (α-tocopherol acetate), NAC (N-acetyl-l-cysteine), folate (pteroylmonoglutamic acid), catalase, RNase, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), hydroethidine (HE), propidium iodide (PI) and different reagents have been bought from Sigma Chemical (St. Louis, MO). The caspase-3 colorimetric substrate Ac-benzyloxycarbonyl aspartyl glutamylvalylaspartic acid (DEVD)-para-nitroaniline (pNA) was bought from R & D Programs (Minneapolis, MN). The Annexin-V-Fluos reagent was from Boehringer Mannheim Biochemicals (Mannheim, Germany). Fetal bovine serum (FBS) was from HyClone Laboratories (Logan, UT). RPMI-1640 medium, penicillin, streptomycin, Fungizone, trypsin and trypan blue have been from GIBCO Laboratories (Grand Island, NY).

Cell tradition and viability.

HL-60, a human promyelocytic cell line, have been cultured in RPMI-1640 medium supplemented with 10% warmth inactivated FBS and antibiotics (2000 u/L penicillin and 20 mg/L streptomycin). Cell cultures have been maintained in a humidified 5% CO2 ambiance at 37°C. Cell viability was decided by trypan blue exclusion.

Antioxidant preparation.

Inventory options of Vit C and NAC have been ready at 5 and 200 mmol/L within the tradition medium, respectively. A inventory answer of Vit E was ready at 30 mmol/L in ethanol. The ethanol focus within the tradition medium has no impact on cell viability. A inventory answer of folate was ready at 10 mmol/L in bicarbonate answer. The catalase answer was ready on the closing focus of 106u/L. All of the antioxidant shares and the catalase answer have been freshly ready earlier than experiments.

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Induction of apoptosis and impact of antioxidant supplementation.

Apoptosis was induced with the HcyT therapy (31). Confluent cells (1 × 109 cells/L) have been handled both with or with out 1 mmol/L HcyT for 3, 6, 12 or 24 h and harvested for apoptosis assay. For antioxidant supplementation, the concentrations and the time of antioxidant therapy to suppress HcyT-induced cytotoxicity have been beforehand examined (39,40). The focus chosen to work on this examine was 5 mmol/L for NAC, 100 μmol/L for each Vit C and Vit E, and 10 μmol/L for folate. Cells (1 × 109 cells/L) have been preincubated with NAC, Vit C, Vit E or catalase for two h, or preincubated with folate for 3 d earlier than HcyT therapy. Cells grew usually beneath our preincubation circumstances. Additional incubation with antioxidants or catalase alone didn’t have an effect on mobile viability (39). After 3 or 6 h of incubation, the indicated time factors, HcyT-untreated (Management), HcyT-treated solely (HcyT) and HcyT-treated cells with antioxidant preincubation have been harvested for the next assays.

Evaluation of apoptotic cells with membrane PS publicity.

The annexin-V-Fluos package was used to measure the apoptotic cells with PS publicity. Cells (1 × 106) have been suspended in incubation buffer (10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl, 5 mmol/L CaCl2) containing 1 mg/L PI and a 1:50 dilution of Annexin-V-Fluos labeling answer (catalog quantity, 1828681). The combined answer was incubated for 15 min on ice. Incubation buffer (400 μL) was added, and inexperienced (annexin-V stain: apoptotic cells) and purple (PI stain: necrotic cells) fluorescence intensities have been analyzed on a Coulter EPICS XL-MCL movement cytometer (Coulter, Miami, FL) utilizing 488 nm excitation, a 515 nm bandpass filter and a filter >560 nm for PI detection. Cells with Annexin-V optimistic and PI unfavorable fluorescence have been outlined as apoptotic cells.

Evaluation of apoptotic cells with hypodiploid DNA contents.

Briefly, cells have been fastened in ice-cold 100% ethanol. RNase A (500 mg/L) and 0.5% Triton have been added to samples at 37°C for additional incubation for 60 min. Cells have been then incubated with PI (50 mg/L) for 20 min at 37°C. After centrifugation (300 × g, 5 min), mobile DNA (purple fluorescence) in 10,000 cells was analyzed on a Coulter EPICS XL-MCL movement cytometer with the System II software program program (Coulter). The proportion of cells in several cell cycle phases was estimated from PI histograms utilizing the WinMDI 2.8 program (Coulter). Hypodiploid cells, i.e., with sub-G0/G1 DNA contents, have been outlined as apoptotic cells as described by Endresen et al. (41).

Agarose electrophoresis of DNA fragmentation.

As beforehand described by Huang et al. (42), 3 × 106 cells at every indicated time level have been harvested and suspended in ice-cold lysis buffer (100 mmol/L NaCl, 10 mmol/L Tris HCl, 25 mmol/L EDTA, 5 g/L SDS, and 0.3 g/L proteinase Ok) for 15 h in a 50°C water bathtub. The usual phenol/chloroform/isoamyl alcohol methodology (25:24:1) was used to take away protein and extract DNA. RNA was digested with 1 mg/L ribonuclease A for 1 h at 37°C. DNA fragments have been electrophoresed on a 2% agarose minigel at 100 V for 40 min and visualized with ethidium bromide staining beneath UV illumination. Multimers of 100 base pair DNA have been used as DNA markers (Pharmacia, Piscataway, NJ).

Dedication of intracellular ROS ranges.

Intracellular ROS have been assayed utilizing the fluorescent dyes of DCFH-DA and HE (43). DCFH-DA readily diffuses into cells, and the acetate teams of the molecule are cleaved by intracellular esterase to yield DCFH. As DCFH is trapped inside cells, H2O2 or nitric oxide oxidize DCFH to the extremely fluorescent compound 2′,7′-dichlorofluorescein (DCF). HE oxidation is especially delicate to superoxide anions, hydroxyl radicals and peroxynitrite. The intracellular fluorescence depth of DCF or HE is proportional to the quantity of ROS produced by the cells. Thirty minutes earlier than HcyT therapy was terminated, DCFH-DA (5 μmol/L) or HE (5 μmol/L) was added into cultured cells. At every indicated time level, cells have been harvested, washed and the fluorescence depth of intracellular DCF (excitation 488 nm, emission 530 nm) or HE (excitation, 488 nm; emission, 585 nm) was monitored on a Coulter EPICS XL-MCL movement cytometer. PI (10 mg/L) was added to every tube 10 min earlier than movement evaluation to make sure that solely dwelling and early apoptotic cells have been analyzed.

Caspase-3 enzymatic exercise.

Caspase-3 exercise was assayed by a way modified from DiPietrantonio et al. (29). Cells (1 × 106) have been incubated with 25 μL chilly cell lysis buffer for 10 min. Cell lysate containing 50 μg protein was added to 148 μL response buffer (100 mmol/L HEPES, pH 7.5, 20% glycerol, 0.5 mmol/L EDTA, and 5 mmol/L dithiothreitol) and a couple of μL caspase-3 colorimetric substrate DEVD-pNA. Samples have been incubated at 37°C for six h in a 96-well flat-bottomed microplate. The fluorescence was learn utilizing an MRX mannequin ELISA reader (Dynatech Laboratories, West Sussex, UK) at 405 nm wavelength.

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All information have been offered as means ± sd. One-way ANOVA and Duncan’s take a look at have been used for comparisons amongst teams. Vital distinction was indicated when the P-value was <0.05.  


Time-dependent impact of HcyT therapy on apoptosis, caspase 3 activation and ROS era in HL-60 cells.

Determine 1 exhibits the kinetic profile of apoptotic progress and ROS era in HcyT-treated cells throughout a 24-h incubation. In contrast with the HcyT untreated management cells on the corresponding time, the proportion of apoptotic cells (measured by membrane PS publicity) elevated by 50% after 3 h of HcyT incubation. It elevated to 4.3-fold of management at 6 h, and declined to 1.3-fold at 12 and 24 h of HcyT incubation. Maximal apoptotic injury occurred at 6 h HcyT therapy. Caspase-3 exercise was elevated 3.7-fold after 3 h HcyT therapy in contrast with management values and this persevered for six–12 h. Exercise was suppressed at 24 h of HcyT therapy. The intracellular peroxide stage measured by DCF fluorescence after 3 and 6 h of HcyT therapy was 3.9-fold the management values. Ranges progressively diminished after 6 h. Modifications in HE fluorescent depth induced by HcyT resembled the kinetic profile of DCF depth, though the magnitude of modifications was considerably decrease for HE depth.

Results of antioxidants on ROS scavenging capability throughout HcyT-induced apoptosis.

Preincubation of cells with catalase (106u/L) for two h utterly diminished the rise of intracellular DCF depth induced by 3- and 6-h HcyT therapy, suggesting that the ROS in these cells have been largely H2O2 species (Desk 1). Preincubation of cells with NAC for two h considerably lowered the elevated H2O2 era at 3 and 6 h of HcyT therapy. Vit C and Vit E additionally have been potent scavengers of intracellular H2O2 in HcyT-treated cells, however their impact was not evident till after 6 h of HcyT therapy. In distinction, preincubation of HcyT-treated cells with folate didn’t cut back the intracellular H2O2 ranges after both 3 or 6 h HcyT therapy.

Preincubation of HcyT-treated cells with NAC, Vit C, Vit E or folate considerably lowered the proportion of HE optimistic cells at 3 h of HcyT therapy (Desk 1). At 6 h of HcyT therapy, NAC preincubation didn’t have an effect on HE depth ranges of HcyT-treated cells, whereas folate, Vit C and Vit E considerably lowered the rise of HE depth in HcyT-treated cells. HE fluorescent depth in HcyT-treated cells preincubated with folate was lowered to 73% of HcyT-untreated controls.

Results of antioxidants on caspase-3 enzymatic exercise throughout HcyT-induced apoptosis.

Catalase pretreatment utterly inhibited caspase-3 activation at 3 and 6 h HcyT incubation, indicating that H2O2 scavenging performed an important function in caspase-3 activation (Desk 1). At 3 h HcyT incubation, all antioxidants examined, NAC, Vit C, Vit E and folate, partially suppressed caspase-3 exercise. The inhibitory impact of NAC, Vit C and Vit E on caspase-3 activation persevered at 6 h HcyT therapy. In distinction, folate didn’t suppress caspase-3 activation in HcyT-treated cells at 6 h.

Results of antioxidants on the apoptosis discount in HcyT-treated cells.

As a result of our experiments revealed that the share of apoptotic cells was maximal after 6 h of HcyT therapy (Fig. 1), this time level was chosen to look at the consequences of antioxidants on apoptosis discount. Preincubation of HL-60 cells with NAC, Vit C or Vit E considerably lowered the share of cells with membrane PS publicity handled with HcyT for six h (Fig. 2). Preincubation with folate didn’t shield cells from apoptotic membrane injury, whereas catalase preincubation utterly protected HcyT-treated cells.

The chances of apoptotic cells with hypodiploid DNA content material have been considerably lowered when HcyT-treated cells have been preincubated with Vit C and Vit E for two h (Fig. 3). These two antioxidants markedly lowered the share of cells with hypodiploid DNA content material to almost half of that of HcyT-treated cells. Preincubation of cells with NAC and folate didn’t shield HcyT-treated cells from DNA breakage. Catalase preincubation utterly protected HcyT-treated cells from chromosome breakage.

The inhibitory impact of the 4 antioxidants on apoptotic DNA injury was additional confirmed by electrophoretic evaluation of DNA laddering (Fig. 4). Preincubation with Vit C or Vit E inhibited the DNA fragmentation induced by 6 h HcyT therapy (lanes 2, 5). NAC or folate didn’t diminish HcyT-induced DNA laddering (lanes 1, 3), whereas catalase (lane 4) or the mixture of NAC, Vit C and Vit E pretreatment (lane 6) utterly protected HcyT-treated cells from DNA fragmentation.



The info on this examine confirmed our earlier discovering that HcyT-induced apoptosis was fast, and additional established that ROS era and caspase-3 activation preceded the maximal apoptotic injury in HcyT-treated cells (Fig. 1). Catalase pretreatment totally quenched the rise of HcyT-induced DCF depth (Desk 1) and utterly prevented HL-60 cells from caspase-3 activation in addition to apoptotic injury (Desk 1, Figs. 234). In distinction, the rise of HE depth in HL-60 cells was considerably decrease than the rise of DCF throughout HcyT therapy (Fig. 1, Desk 1), and the addition of superoxide dismutase didn’t shield cells from caspase-3 activation or from apoptotic DNA injury (31). These observations recommend that the era of superoxide anions isn’t the mediator of apoptosis induced by HcyT, whereas H2O2 era serves as a vital messenger within the signaling transmission of HcyT-induced apoptosis. Equally, the ROS-elicited injury attributable to homocysteine therapy was related to DNA strand breaks and apoptosis in rat hippocampal neurons (44). The prevention of homocysteine-induced toxicity by catalase signifies that H2O2 acts because the mediator of oxidative harm (6,14,16, 17). Our findings with these of others recommend that ROS era, significantly H2O2, performs a key function within the apoptotic injury induced by homocysteine derivatives.

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Our outcomes demonstrated that the NAC, Vit C or Vit E pretreatment considerably inhibited HcyT-induced apoptosis. The robust antioxidant impact on H2O2 scavenging might largely account for his or her inhibition of HcyT-mediated cytotoxicity. The capability of Vit C and Vit E to suppress caspase-3 activation coincided with their effectivity in inhibiting HcyT-induced apoptotic occasions together with membrane PS publicity, DNA loss and DNA fragmentation. It was reported that VitE can restore mitochondria perform to inhibit cytochrome c launch (45), which in flip modulates caspase-3 activation to have an effect on apoptotic finish level injury (46). NAC was additionally a potent inhibitor of hydrogen peroxide manufacturing and caspase-3 activation in HcyT-treated cells. Nevertheless, NAC pretreatment lowered solely HcyT-promoted apoptotic membrane PS publicity, not DNA breakage. The explanations are presently unknown. The antioxidant exercise of NAC utterly prevented the induction of varied DNA alterations in rat lung cells (47). Conversely, it was reported that NAC therapy induced DNA injury in HL-60 cells (48). The authors proposed that radicals apart from hydrogen peroxide is likely to be elicited throughout NAC therapy within the presence of metallic ions, which could promote extra DNA injury (48). This risk is additional supported by our discovering that preincubation of HcyT-treated cells with the mixture of NAC, Vit C and Vit E can offset the DNA-damaging impact of NAC beneath HcyT-treated circumstances, and utterly stop cells from apoptotic DNA fragmentation (Fig. 4).

Within the current examine, preincubation of HcyT-treated cells with folate didn’t ameliorate the apoptotic injury. The failure of folate to guard cells from apoptotic DNA fragmentation or membrane dysfunction might consequence from its incapability to scavenge HcyT-elicited intracellular H2O2 and subsequently to suppress caspase-3 activation (Figs. 234). This discovering is in settlement with that of Duthie and Hawdon (49) who confirmed that the folate provide had no impact on the discount of oxidative DNA injury as indicated by oxidized pyrimidine ranges in lymphocytes. Though folate had no H2O2 scavenging capability and didn’t suppress caspase-3 exercise, it demonstrated antioxidant habits. Our information supplied the direct proof to assist the speculation that folic acid is an efficient free radical scavenger, as demonstrated by Joshie et al. (38) in an in vitro radical response system. Our information additionally confirmed the discovering of Doshi et al. (50) that folate exhibited an antioxidant impact and lowered intracellular superoxide. The concentrations of folate vital to cut back intracellular ROS was reported by Doshi et al. (50) to be within the 500 micromolar vary; we reported 10 μmol/L on this current examine, whereas regular human plasma folate ranges are on the nanomolar vary (12,51). At micromolar concentrations, folate (10 μmol/L), Vit C (100 μmol/L) and Vit E (100 μmol/L) had related antioxidant capacities to scavenge superoxide, hydroxyl radicals and peroxynitrite. To our data, this phenomenon has not been noticed beforehand in intact cells. In circumstances by which ROS resembling superoxide anions, hydroxyl radicals or peroxynitrite are the key contributors towards the etiology of ailments, folate supplementation could also be protecting. Additional research are required to elucidate folate’s antioxidant capabilities beneath varied physiologic and pathologic circumstances.

In conclusion, the current work demonstrated that preincubation of HL-60 cells with NAC, Vit C or Vit E considerably attenuated HcyT-induced apoptotic injury. The capability of NAC, Vit C and Vit E to scavenge HcyT-induced hydrogen peroxide and to suppress caspase-3 activation correlates nicely with their effectivity to guard towards HcyT-promoted apoptotic injury. Folate had no hydrogen peroxide-scavenging capability and didn’t counteract HcyT-induced apoptosis, though it did exhibit antioxidant habits towards superoxide anions, hydroxyl radicals and peroxynitrite.

The authors acknowledge Y.-A. Lee within the Division of Life Science for offering UV illumination gear.


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