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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 9  |  Issue : 1  |  Page : 28-32

Evaluation of cyclophosphamide-induced genotoxicity and cytotoxicity in cultured human lymphocytes


1 Department of Research, Bhopal Memorial Hospital and Research Centre (BMHRC); Department of Molecular Biology and Genetics, ICMR-National Institute for Research in Environmental Health, Bhopal, India
2 Department of Biotechnology, Barkatullah University, Bhopal, India
3 Department of Biotechnology, St. Aloysius College, Rani Durgavati University, Jabalpur, Madhya Pradesh, India
4 Department of Molecular Biology and Genetics, ICMR-National Institute for Research in Environmental Health, Bhopal, India

Date of Web Publication22-Jan-2018

Correspondence Address:
Ravindra M Samarth
Department of Research, Bhopal Memorial Hospital and Research Centre (BMHRC); Department of Molecular Biology and Genetics, ICMR-National Institute for Research in Environmental Health, Bhopal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrcr.jrcr_1_18

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  Abstract 

Aim: The present study was aimed to examine and evaluate the genotoxicity and cytotoxicity induced by different doses of cyclophosphamide (CP) in normal healthy cultured human peripheral blood lymphocytes. Materials and Methods: Genotoxicity and cytotoxicity was evaluated through mitotic index (MI), chromosomal aberrations, micronuclei frequency, and colony formation assay (plating efficiency [PE] and survival fraction), respectively. Results: It has been observed that CP (1, 2.5, and 5 μg/ml)) induced a dose-dependent increase in chromosomal aberrations and micronuclei frequencies in cultured human peripheral blood lymphocyte as compared to normal. A significant increase was observed in the percentage of aberrant cells and dicentrics/exchanges at 1 and 2.5 μg/ml CP and aberrant cells, breaks, fragments, and dicentrics/exchanges at 5/μg/ml CP. A dose-dependent decrease in values of MI and nuclear division index was also observed in CP-treated group. The frequency of micronuclei in binucleated cells showed a dose-dependent increase. In colony formation assay, PE and surviving fraction values showed significant (P < 0.001) and dose-dependent decrease in the CP treatment groups. Conclusion: The results of present study suggest that CP has genotoxic and cytotoxic effect on cultured human lymphocytes.

Keywords: Chromosomal aberrations, colony formation assay, cyclophosphamide, human peripheral blood lymphocytes, micronuclei frequency


How to cite this article:
Samarth RM, Khan T, Srivas S, Mishra PK, Tiwari RR. Evaluation of cyclophosphamide-induced genotoxicity and cytotoxicity in cultured human lymphocytes. J Radiat Cancer Res 2018;9:28-32

How to cite this URL:
Samarth RM, Khan T, Srivas S, Mishra PK, Tiwari RR. Evaluation of cyclophosphamide-induced genotoxicity and cytotoxicity in cultured human lymphocytes. J Radiat Cancer Res [serial online] 2018 [cited 2018 Oct 23];9:28-32. Available from: http://www.journalrcr.org/text.asp?2018/9/1/28/223742




  Introduction Top


Cyclophosphamide (CP) popularly known as Cytoxan or Endoxan is an extensively used drug to treat wide range of various neoplastic diseases such as Hodgkin's disease, non-Hodgkin's lymphoma, many types of leukemia, multiple myeloma, retinoblastoma, neuroblastomas, carcinomas of the ovary, breast, and endometrium, and certain malignant neoplasms of the lung.[1],[2],[3] It is also used as an immunosuppressive agent for scleroderma, rheumatoid arthritis, glomerulonephritis, chronic hepatitis, multiple sclerosis, and organ transplantation.[1],[3],[4] CP specifically acts on T-cells and B-cells and causes suppression of cell-mediated and humoral immunity.[5] CP which is a nonactive cyclic phosphamide ester of mechlorethamine causes cross-linking of DNA and RNA strands which leads to an increase in inhibition of DNA polymerase activity and thus prevents cell division. Although CP has proved to be a promising and effective chemotherapeutic agent, the International Agency for Research on Cancer (IARC), 1991, designated it as carcinogenic to humans.[1],[3]

CP has been shown to produce gene mutations, DNA damage, chromosomal aberrations, micronuclei, and sister chromatid exchange in cultured cells of humans, animals, and microorganisms. In the presence of metabolic activation, some mutations in base-pair substituting strains of Salmonella typhimurium have been produced by CP, but these mutations are shown to be negative in Escherichia coli. Mutations induced by CP have been shown to be positive in D7 strain in Saccharomyces cerevisiae. In somatic cells of rats, mice, and Chinese hamsters, CP has produced chromosomal damage and micronuclei. In peripheral blood lymphocytes of pharmacists, nurses, and workers occupationally exposed to CP, increase in their chromosome damage and gene mutations has been found, and also in somatic cells of patients who are treated therapeutically with CP, some gene mutations, sister chromatid exchanges, and chromosomal aberrations have been observed.[1]

The aim of the present study was to examine and evaluate the genotoxicity and cytotoxicity induced by different doses of CP in cultured human peripheral blood lymphocytes. Parameters such as mitotic index (MI), chromosomal aberrations, and micronuclei frequency were studied for the evaluation of genotoxicity. Colony formation assay or clonogenic assay was performed for evaluation of cell survival based on the reproductive capability of a single cell to grow into a colony.


  Materials and Methods Top


Chromosome aberration assay

The standard method of Rooney and Czepulkowski was followed for preparation of the CA test with minor modifications.[6],[7] Whole blood (0.3 mL) from five healthy donors was added to 4.5 mL PB-MAX (supplemented with phytohemagglutinin) karyotyping medium. Cultures were incubated at 37°C for 72 h. The cells were treated with CP (1, 2.5, and 5 μg/ml) for 48 h after initiating the culture. The cells were exposed to colchicine (0.06 μg/mL) 2 h before harvesting. At the end of the incubation, cells were centrifuged at 1200 rpm for 15 min. Then, the cells were treated with 0.075 M KCl (37°C) as the hypotonic solution and methanol: glacial acetic acid (3:1) as the fixative (at room temperature 22°C ± 1°C); fixative treatments were repeated three times. The cells were centrifuged at 1200 rpm for 15 min after each fixative treatment. The staining of the air-dried slides was performed following the standard methods using 5% Giemsa stain for CA. Totally 500 metaphases per concentration were evaluated for CA and frequency was expressed as percentage (%).

In vitro cytokinesis-block micronucleus assay

The standard method of Fenech was followed for preparation of the cytokinesis-block micronucleus assay with minor modifications.[8] For the analysis of MN in binucleated lymphocytes, 0.3 mL of fresh whole blood was used to establish the cultures which were incubated for 68 h. After 24 h of culture initiation, the cells were treated CP (1, 2.5, and 5 μg/ml). To block cytokinesis, cytochalasin B was added at 44 h of the incubation at a final concentration of 6 μg/mL. After additional 24 h incubation at 37°C, cells were initially harvested by centrifugation at 1200 rpm for 15 min and further processed identically as described for the preparation of CA. The cells were hypotonically treated with 7 ml of cold (4°C) 0.075M KCl to lyse red blood cells and centrifuged immediately 1200 rpm for 8 min. Finally, the slides were stained with 10% Giemsa. In all 5000 binucleated lymphocytes were scored from each concentration for MN frequency, MN frequency expressed as MN/1000 (‰). A total of 1000 cells were scored to calculate the nuclear division index (NDI) for the cytotoxicity of CP using the formula: NDI = ([1 × M1] + [2 × M2] + [3 × M3] + [4 × M4])/N; where M1–M4 represent the number of cells with one to four nuclei and N is the total number of the cells scored.

Colony formation assay

For colony formation assay, standard protocol by Franken et al. with minor modifications was followed.[9],[10] Lymphocytes were isolated from peripheral blood obtained from healthy individuals and cultured for 24 h before the treatment with CP (1, 2.5, and 5 μg/ml). After 4 h of treatment, the cells were counted and used for colony formation assay. Briefly, Agarose (Difco) 0.25 g was taken into 100 ml glass bottle and 4 ml Milli-Q was added and autoclaved at 121°C for 21 min. The 96 ml of RPMI medium supplemented with 15% serum (fetal bovine serum) in sterilized 100 ml bottle was taken and placed the bottle in water bath set at 40°C. The 60 mm culture dishes were prepared and labeled, 3 for each sample. After the cell count, initially approximately 3 × 105 cells were taken for each treatment and diluted appropriately. The 0.5 ml aliquot (i.e., 600 cells) was taken into 50 ml tube. After autoclave was complete, the bottle was taken out and poured the medium (kept at 40°C). The 12 ml of medium was taken, mixed with the cells, and poured on to 3 mm × 60 mm plates (4 mL each). After solidifying, the culture dishes were placed in a fully humidified (>95%) atmosphere at and incubated for 14 days at 37°C before counting colonies. The colonies with >50 cells were scored with naked eye. The plating efficiency (PE) and surviving fraction (SF) were calculated by the following formula: PE = Number of colonies counted/Number of cells plated × 100. SF = PE of treated sample/PE of control × 100.

Statistical analysis

All statistical analyses were performed with GraphPad software. Data were presented as mean ± SEM; Student's “t”-test was used to determine the difference between treated and normal group.

The significance level was set at P < 0.05, P < 0.001, and P < 0.005.


  Results Top


In the present study, cytogenetic changes induced by CP at 1 μg/ml, 2.5 μg/ml, and 5 μg/ml concentrations in human peripheral blood lymphocytes were studied in vitro. After performing chromosomal aberration assay, MI of all four groups was taken out [Table 1]. The highest value of MI was observed in normal group, i.e., 9.2 ± 0.88 and the lowest value was recorded in the group treated with CP 5 μg/ml, i.e., 4.2 ± 0.38, thus showing significant decrease (P< 0.001).
Table 1: Effect of different concentrations of cyclophosphamide in cultured human lymphocytes - chromosomal aberrations and mitotic index

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Increased chromosomal aberrations such as breaks, fragments, exchanges, and rings were observed in chromosomes treated with CP in comparison to normal group. The maximum percentage of aberrant cell was observed in the group treated with CP 5 μg/ml, i.e., 9.88 ± 2.02 followed by group treated with CP 2.5 μg/ml and 1 μg/ml.

The values of NDI and MN frequencies are shown in [Table 2]. The NDI value in normal group was found to be 2.00 ± 0.18, whereas the lowest value of NDI (1.38 ± 0.12) was noted in the group treated with the highest dose of CP (5 μg/ml) showing significant decrease (P< 0.05).
Table 2: Effect of different concentrations of cyclophosphamide in cultured human lymphocytes - micronuclei frequency and nuclear division index

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Micronuclei were scored per 1000 binucleated cells and its frequency was determined. A significant increase in micronuclei frequency in binucleated cells was observed in all groups treated with CP. However, the highest value for micronuclei frequency in binucleated cells was recorded in the group treated with the highest dose of CP (5 μg/ml), i.e., 3.42 ± 1.16 (P< 0.05) followed by 2.5 μg/ml and 1 μg/ml CP-treated group.

The PE and SF were calculated in colony formation assay [Table 3]. The PE and SF were highest in normal group, i.e., 86.42 ± 1.82 and 1.00 ± 0.00, respectively, whereas there was a significant decrease (P< 0.001) in these values in all CP-treated groups.
Table 3: Effect of different concentrations of cyclophosphamide in cultured human lymphocytes - plating efficiency and surviving fraction

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  Discussion Top


The results of the present study indicated that CP is able to induce cytotoxic effects in human peripheral lymphocytes in vitro. CP, a known anticancer drug, belonging to a class of oxazaphosphorines, is used as an immunosuppressive agent before organ transplantation and also in the treatment of wide range of diseases for over 40 years. It is metabolically activated by the help of hepatic mixed-function oxidases. It is an alkylating agent and shows its cytostatic effects by forming covalent DNA adducts.[1],[3],[11],[12] CP cytotoxicity is mediated by alkylation of DNA at the N7 position of guanine and by the formation of DNA–DNA cross-links, DNA–protein cross-links, and single-strand breaks, and double-stranded breaks.[11],[13]

CP is designated as carcinogenic to humans by IARC as it possesses a wide spectrum of cytotoxicity to normal cells. CP has been tested extensively for genotoxicity in vivo and in vitro on microorganisms, plants, insects, animals, and humans and it is known to induce dominant lethal mutation, micronuclei, DNA damage and generation of free radicals or reactive oxygen species (ROS), chromosomal aberrations, sister chromatid exchanges, and gene mutations, which can lead to a number of pathological conditions including cancer.[14]

By metabolic activation of CP, induction of gene mutation has been reported in bacteria, i.e., S. typhimurium and E. coli, yeast, i.e., S. cerevisiae, and mammalian cells, i.e., mouse lymphoma L5178Y.[1] In Chinese hamster ovary cells, Syrian hamster cells, and human lymphocytes, S9-activated CP is found to be clastogenic.[3] In low-dose CP-treated male rats, decrease in weight of reproductive organ and impaired fertility has been reported. CP with low-dose treatment caused temporary interference of normal male reproductive system but might cause permanent dysfunction in high-dose treatment.[11] Many in vivo studies have been conducted to test the genotoxicity produced by CP. It was clearly indicated that genotoxicity may increase with repeated dosage or chronic administration of CP.[15] It was revealed that CP was able to cause numerical chromosome abnormalities – aneuploidy in preimplantation mouse embryos.[12]

Many adverse side effects and toxicity are associated with intake of CP including mutagenicity, carcinogenicity, teratogenicity, myelosuppression, immunosuppression, cardiac toxicity, lung toxicity, and urotoxicity, which are mainly mediated by ROS and lipid peroxide formation. A common consequence of long-term CP chemotherapy in cancer patients is immunosuppression, particularly of humoral immunity. Secondary tumors are also induced by CP in different animal species as well as in humans.[16] It was noticed that CP-based therapy increased the risk of secondary malignant neoplasms in non-Hodgkin lymphoma and leukemia risk was also linked with high-dose CP.[17] The formation of CP-specific DNA adducts in hematological diseases such as Fanconi anemia (FA) was also observed; it was noticed that CP-related toxicity was higher in FA patients.[18] The mutagenicity of CP was found to be increased by encapsulation due interaction of liposomes with cells that results in accumulation of drug inside the cells thus causing damage in chromosomes.[14]

Since the 1970s, handling of chemotherapeutic drugs has been considered as a potential health hazard to workers.[19] One such drug is CP which is the most commonly used anticancer drug.[20],[21],[22] Studies have shown that occupational exposure to cytotoxic drugs can result in adverse health effects including genetic damage which could also lead to cancer or can cause serious reproductive effects such as miscarriages.[23],[24] The primary route of exposure of health-care workers to cytostatic drug is through dermal contact. It can occur directly by handling vials, preparing drug solutions, infusion bags, etc., or through indirect contact such as touching of drug-contaminated surfaces.[25] Exposure can also occur through ingestion or inhalation of these drugs resulting from aerosolization of powder or liquid during reconstitution or spillage during preparation or administration to patients.[26] Recently, environmental contamination by CP from patient's excretory products and hospital waste products has also raised serious concern.[27]


  Conclusion Top


The results of the present study indicated that CP is able to induce genotoxic and cytotoxic effects in human peripheral lymphocytes in vitro.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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