N-Acetyl Cysteine Modifies the Action of Disulfiram on Cyclophosphamide Induced Cytotoxicity in Allium cepa Model

Meenakshy E. S., Nandana C. R., Lone Ahtisham UL Farooq, Rita Antony Rozario, Sajna Sabu, Binil Raj S. S.,

doi:10.5281/zenodo.15452458

Research Paper | Open Access
Volume 03 | Issue 05 | Article Id IJPS/250305173

N-Acetyl Cysteine Modifies the Action of Disulfiram on Cyclophosphamide Induced Cytotoxicity in Allium cepa Model
Meenakshy E. S.* Nandana C. R. Lone Ahtisham UL Farooq Rita Antony Rozario Sajna Sabu Binil Raj S. S.
College of Pharmaceutical Sciences, Government Medical College, Thiruvananthapuram, Kerala, India. Kerala University of Health Sciences

Abstract

Disulfiram (DSF), also known as Bis(diethylthiocarbamoyl) disulfide, is widely used in the treatment of alcoholism due to its inhibition of aldehyde dehydrogenase (ALDH), leading to acetaldehyde accumulation and an adversive response to alcohol consumption. Beyond this application, Disulfiram has the potential to act as a pro-oxidant, increasing intracellular reactive oxygen species (ROS) levels, and inducing oxidative stress-mediated apoptosis in cancer cells. It has been shown to upregulate pro-apoptotic proteins, while downregulating antiapoptotic proteins, enhancing its cytotoxic effects. Due to these pro-apoptotic actions of Disulfiram when used with chemotherapeutic agents resulted in adverse drug reaction. So, antioxidant may be useful in mitigating this interaction of Disulfiram’s action with chemotherapeutic agents. In this study we evaluated the effect of antioxidants such as N-Acetyl Cysteine and Oxalic acid in altering Disulfiram’s cytotoxic action on chemotherapeutic agents such as Cyclophosphamide and 5-Fluorouracil by Allium cepa assay. The root length was measured and histopathological studies were done to evaluate the cytotoxicity. Antioxidants such as N-Acetyl Cysteine and Oxalic acid can alter the Disulfiram’s effect on cytotoxic action of Cyclophosphamide. N-Acetyl Cysteine is found to be having more significant action in altering the Disulfiram’s action. Antioxidants can be used in cases where Disulfiram is used in combination with chemotherapeutic agents such as Cyclophosphamide to mitigate the synergistic effect of the cytotoxic agents and thus to avoid the associated toxicities.

Keywords

Disulfiram, Cyclophosphamide, 5-Fluorouracil, N-Acetyl Cysteine, Oxalic Acid, Reactive Oxygen Species, Aldehyde Dehydrogenase Inhibition

Introduction

Disulfiram, a well-known aldehyde dehydrogenase inhibitor used for treating chronic alcoholism.¹ Its aldehyde dehydrogenase inhibition leads to the accumulation of toxic aldehydes that form complexes with proteins and DNA, resulting in increased intracellular reactive oxygen species levels, thereby act as a pro-oxidant inducing oxidative stress and promoting apoptosis. This pro-oxidant and cytotoxic effect of Disulfiram causes interaction with various chemotherapeutic agents. When Disulfiram combined with 5-Fluorouracil, it increased the cytotoxicity in colorectal cells. And also enhanced the potency of various chemotherapeutic drugs such as Cisplatin, Paclitaxel and Temozolomide compared to their individual treatment.²When disulfiram is used in combination with chemotherapeutic agents, there is a reported increased risk of Grade 3 or higher adverse effects compared to chemotherapy alone.³ Gamelli et. al. suggested that neutropenia was observed when Disulfiram given in combination with Cyclophosphamide.⁴ Also, study by Huang et. al. suggested that Disulfiram and Temozolomide combination may produces vomiting, dizziness and drug induced liver injury.⁵ In order to overcome these crises, the interaction should be managed properly. Since the potentiation of the cytotoxicity by Disulfiram is primarily due to reactive oxygen species generation, agents which can reduce the reactive oxygen species level can be useful to manage the interactions. In this regard, we intended to evaluate the effect antioxidants such as N-Acetyl Cysteine and Oxalic acid in altering the Disulfiram’s action on chemotherapeutic agents such as Cyclophosphamide and 5-Fluorouracil in the Allium cepa model.

METHODOLOGY

Allium cepa Assay

A series of equal sized healthy onions were grown in separate cylinders containing water as a medium. Prior to the test, the outer scales of the bulbs and the brownish bottom plate were removed, the ring of root primordia being left intact, onions were kept for sprouting at room temperature for two days and onions with uniform root length were selected for further studies.^6,7

Evaluation of Cytotoxic Agents

After two days of sprouting, the bulbs were transferred to a beaker containing vehicle and test drugs. To check the efficacy of cytotoxic agents (Cyclophosphamide and 5-Fluorouracil), we used three bulbs of A. cepa. Prior to the drug treatment, the root length was measured and considered "Day 0." After 24 hours, the root length was measured and documented and considered "Day 1." After the next 24 hours, the root length was again measured and considered "Day 2." After Day 2, the root tips were excised and used for microscopic evaluation. The experiments were done in triplicate.

Evaluation of Cytotoxic Effect of Chemotherapeutic Agents in Presence Of Disulfiram

After the sprouting period, the bulbs of A. cepa were used for evaluating the combined effect of Disulfiram with cytotoxic agents such Cyclophosphamide and 5-Fluorourcil.⁸ Bulbs of A. cepa were used for each treatment, with each bulb's roots immersed in different combinations. The bulbs were treated with Disulfiram treated, a combination of Disulfiram (100µM) + Cyclophosphamide (500µg/ml) and Disulfiram (100µM) + 5-Fluorouracil (500µg/ml), and one each was treated with Cyclophosphamide (500µg/ml) and 5-Fluorouracil (500µg/ml), respectively. The root length was measured similarly while performing the evaluation of cytotoxic agents. The root length was measured twice, and the average percentage change was calculated to ensure accurate results using the following equation.

% Change in root length=Root length in day 2-Root length in day 0Root length in day 0 ×100

Evaluation of Antioxidant Effect on Altering Disulfiram’s Action on Chemotherapeutic Agents

The antioxidants used for performing this experiment were Oxalic acid (100 µM) and N-Acetyl Cysteine (25mM). Here apart from the above-mentioned set of combinations, a new set of combination therapy has been introduced, consisting of N-Acetyl Cysteine (NAC) and Oxalic acid, alongside dual combinations of Disulfiram (100µM) with either Cyclophosphamide (500µg/ml) or 5-Fluorouracil (100µg/ml).

Histopathological evaluation of Allium cepa root tips

After the drug treatment, two or three roots from each set were cut (about 2mm in length), fixed in glacial acetic acid-ethanol (1:1) for 30 minutes, and preserved in 70% ethanol under refrigeration for further use.^9,10 The fixed root tips were placed in 1N HCl in a watch glass and heated for 5 seconds. After cooling and washing, it was stained with Aceto-Orcein, heated for 3 seconds, and waited for 1 minute before being washed with water. The root tip was cut, and excess stain was removed using blotting paper. After removing excess stain, DPX (Dibutyl Phthalate Xylene) was added, a cover slip was placed. The prepared slides were then examined under a Magnus MX2LI compound microscope and the images were captured using Magvision software.

Statistical analysis

The statistical analysis was done by Student’s t-test

RESULTS

Our preliminary studies revealed that Cyclophosphamide and 5-Fluorouracil at 500µg/ml produce a significant cytotoxicity in Allium cepa. So, we have selected this dose for further studies.

Evaluation of Cytotoxic Effect of Cyclophosphamide and 5 -Fluorouracil in Combination with Disulfiram by Allium cepa Assay

The Fig.1&2 shows the effect of Cyclophosphamide and 5-Fluorouracil (500µg/ml) in combination with Disulfiram (100µg/ml). The combination of cytotoxic agents with Disulfiram shows significant decrease in percentage change in root growth than the cytotoxic drugs alone. In accordance with the previous studies, we could observe a significant potentiation on cytotoxic action of Cyclophosphamide and 5-Fluorouracil (500µg/ml) along with Disulfiram. The same were found in Allium cepa assay too. This indicates that Disulfiram can potentiate the action of the chemotherapeutic agents such as Cyclophosphamide and 5-Fluorouracil.

Fig.1: Graphical representation of % change in root length after 48 hrs when chemotherapeutic agents were used in combination with Disulfiram. *a indicates P<0.05 compared to control group, **a indicates P<0.05 when compared with, ***a indicates P<0.05 and ****a indicates P<0.05 in drug treated groups. CMC- 0.1 % CMC (Vehicle control); CYP- Cyclophosphamide (500µg/ml); DSF- Disulfiram (100µM); 5-FU- 5-Fluorouracil (500µg/ml).

Fig 2: Representative images showing the root length of A. cepa treated with Disulfiram in combination with cytotoxic drugs. (a) 0.1 % CMC (Vehicle control) (b) Cyclophosphamide (500µg/ml) (c) 5-Fluorouracil (500µg/ml) (d) Disulfiram (100 µg/ml) & Cyclophosphamide (500µg/ml) (e) Disulfiram (100µM) & 5-Fluorouracil (500µg/ml). The Fig.3 shows that in combination samples, there are more apoptotic cells and less cellular integrity compared to that of individual standard treatments.

Fig.3: Photographs of A. cepa root cell using Aceto-Orcein. Magnification- 100x. Photographs showing (a) Vehicle control, (b)Cyclophosphamide 500µg/ml(c) 5-Fluorouracil 500µg/ml (d) Cyclophosphamide 500µg/ml and Disulfiram 100µM (e) 5-Fluorouracil 500µg/ml and Disulfiram 100µM.

Evaluation of Antioxidant Effect on Altering Disulfiram’s Action on Cyclophosphamide in Allium cepa Assay

The Disulfiram and Cyclophosphamide combination shows significant decrease in % change in root length than Cyclophosphamide alone. While adding antioxidants (N-Acetyl Cysteine and Oxalic acid) to the above combination, the % change in root length was found to be significantly increased. The Disulfiram and 5-Fluorouracil combination shows significant decrease in % change in root length than 5-Fluorouracil alone. While adding antioxidants (N-Acetyl Cysteine and Oxalic acid) to the above combination, the % change in root length was found to be decreased significantly. This indicates that N-Acetyl Cysteine and Oxalic acid can inhibit the potentiation of cytotoxic activity of Cyclophosphamide by Disulfiram but no significant effect with 5-Fluorouracil. And while comparing the action N-Acetyl Cysteine and Oxalic acid, we found that N-Acetyl Cysteine is more significant.

Fig.4: Graphical representation of % change in root length after 48 hrs when Disulfiram and Cyclophosphamide combination was altered with Oxalic acid and N-Acetyl Cysteine. Results were expressed as Mean ±SEM for statistical analysis done by one tail two sample t-test. * Indicates P<0.05 compared to control group, ** indicates P<0.05 when compared with, *** indicates P<0.05 and **** indicates P<0.05 in drug treated groups. DSF-Disulfiram (100µM), CYP-Cyclophosphamide (500µg/ml), NAC- N acetyl cysteine(25mM), OA-Oxalic acid (100 µM).

Fig 5: Graphical representation of the % change in root length after 48 hrs when Disulfiram and 5-Fluorouracil combination was altered with Oxalic acid and N-Acetyl Cysteine. Results were expressed as Mean ±SEM for statistical analysis done by one tail two sample t-test. # indicates P<0.05 compared to control group, ## indicates P<0.05 when compared with, ### indicates P<0.05 and #### indicates P<0.05 in drug treated groups. DSF-Disulfiram (100mM),5-FU- 5-Fluorouracil (500µg/ml), NAC- N acetyl cysteine (25mM), Oxalic acid (100µg/ml). Fig.6 shows that when antioxidants such as N-Acetyl Cysteine and Oxalic acid were added to the Cyclophosphamide and DSF combination, they tend to regain cell membrane integrity and shows less apoptotic cells. But in Fig.7 When antioxidants were added to the 5-Fluorouracil and DSF combination, it shows indistinct cell membrane and cell lysis.

Fig.6: Photographs of A. cepa root cells treated with Cyclophosphamide and its combination with Disulfiram and Antioxidants. The cells were stained using Aceto-Orcein. Magnification- 100x. Photographs showing (a)Cyclophosphamide 500µg/ml (b)Cyclophosphamide 500µg/ml and Disulfiram (c) Cyclophosphamide 500µg/ml, Disulfiram and N-Acetyl Cysteine (d) Cyclophosphamide 500µg/ml, Disulfiram and Oxalic acid.

Fig.7: Photographs of A. cepa root cells treated with 5-Fluorouracil and its combination with Disulfiram and Antioxidants. The cells were stained using Aceto-Orcein. Magnification- 100x. Photographs showing (a) 5-Fluorouracil (b) 5-Fluorouracil and Disulfiram (c) 5-Fluorouracil, Disulfiram and N-Acetyl Cysteine (d) 5-Fluorouracil, Disulfiram and Oxalic acid.

Fig.8: Representative images showing the root length of A. cepa treated with the antioxidants, Disulfiram and chemotherapeutic drugs (a) 0.1 % CMC (Vehicle control) (b) 5-Fluorouracil (500µg/ml), (c) Cyclophosphamide (500µg/ml) (d) N-Acetyl Cysteine (25mM) (e) Disulfiram (100µg/ml) (f) Disulfiram (100µM) & 5-Fluorouracil (500µg/ml) (g) Disulfiram (100µM) & Cyclophosphamide (500 µg/ml) (h) Disulfiram (100µM), Cyclophosphamide (500 µg/ml) & N-Acetyl Cysteine (25mM) (i) Disulfiram (100µM), 5-Fluorouracil (500 µg/ml) & N-Acetyl Cysteine (25mM).

DISCUSSION

On combining Disulfiram with cytotoxic agents, Disulfiram enhanced cytotoxicity of Cyclophosphamide and 5-Fluorouracil in A. cepa assay. This enhancement of cytotoxicity might be due to the underlying action of Disulfiram: Aldehyde dehydrogenase inhibition-Disulfiram as an established inhibitor of aldehyde dehydrogenase enzyme could sensitize the cells to the drugs and it can also increase the intracellular accumulation of drugs.^11-14 Disulfiram and its metabolite, diethyldithiocarbamate (DDC), can inhibit the activity of NF-?B,p38 MAPK activation and causes G1/S arrest in cancer cells and also cause an increase in oxidized glutathione (GSSG) level which are intended to elevate the oxidative stress in cells and lead to cell apoptosis. Disulfiram could also prevent P-glycoprotein (P-gp) from maturing and make P-gp-transfected cells more susceptible to cytotoxic medications and its metabolites can shield healthy tissues from chemotherapy and radiation.^15-17Antioxidants are the agents which can reduce the ROS level in the cell thereby modulate the interaction between Disulfiram and chemotherapeutic agents. Zafarullah et. al. suggested that N-Acetyl Cysteine is an antioxidant molecule that act primarily by promoting glutathione levels and have ROS scavenger property.¹⁸Li Q et. al. suggested that Oxalic acid was found to accelerate the decomposition of ROS and completed the scavenging of H₂O₂mainly by regulating the activity of antioxidant enzymes.¹⁹ Since these antioxidants have directly proven action on ROS levels, the same were selected for the study. When antioxidants such as N-Acetyl Cysteine and Oxalic acid were given with Disulfiram in combination with Cyclophosphamide, the cytotoxicity was found to be counteractive. But When N-Acetyl Cysteine and Oxalic acid were used along with Disulfiram in combination with 5-Fluorouracil, we couldn’t observe any significant change in cytotoxic action of 5-Fluorouracil.Among the antioxidants we screened, N-Acetyl Cysteine was found to be more active. The alteration of interaction may be resulted from N-Acetyl Cysteine's antioxidant property. This might help to reduce oxidative stress, allowing Disulfiram and chemotherapeutic drugs to target cancer cells more effectively, as they have higher level of aldehyde dehydrogenase. N-Acetyl Cysteine can activate protein kinase C which can phosphorylate and activate NF-?B pathway thereby it can reduce the toxic effects of chemotherapeutic drugs on healthy cells. When N-Acetyl Cysteine is combined with Disulfiram it is reported that the levels of oxidized glutathione were reduced.²⁰ This in turn leads to reduction in ROS level and increase in cellular integrity. When Oxalic acid is treated with this combination, the cytotoxic effect observed may be due to the ability of Oxalic acid to chelate the metal ions and inhibit the metal ion induced oxidative stress produced by Disulfiram, thereby reduces the ROS levels, which can oppose the apoptotic effects of Cyclophosphamide.

CONCLUSION

Disulfiram is a well-known anti-alcoholic drug and a pro-oxidant. When Disulfiram combined with cytotoxic drugs, it synergistically enhances its cytotoxicity. Antioxidants such as N-Acetyl Cysteine and Oxalic acid was found to alter the synergistic action of Disulfiram and Cyclophosphamide. But antioxidants could not alter the synergism between Disulfiram and 5-Fluorouracil. N-Acetyl Cysteine is found to be much effective antioxidant than Oxalic acid. Antioxidants can be used in cases where Disulfiram is used in combination with chemotherapeutic agents such as Cyclophosphamide to mitigate the synergistic effect of the cytotoxic agents and thus to avoid the associated toxicities.

ACKNOWLEDGEMENT

We would like to acknowledge the Head of Department, College of Pharmaceutical Sciences, Govt Medical College, Thiruvananthapuram, Kerala for providing necessary facilities in conducting the work. We also thank the Kerala University of Health Science, Thrissur Kerala for reviewing and approving our project.

CONFLICT OF INTEREST

All the authors have no conflict of interest.

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