Effect of tumour promoter iodoacetate on γ-radiation induced chromosomal aberrations in human lymphocytes

KB Anjaria; KB Shirsath; NN Bhat; B Sreedevi

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Year : 2010 | Volume : 33 | Issue : 4 | Page : 199-201

Effect of tumour promoter iodoacetate on γ-radiation induced chromosomal aberrations in human lymphocytes

KB Anjaria, KB Shirsath, NN Bhat, B Sreedevi
Radiological Physics and Advisory Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India

Date of Web Publication

1-Dec-2011

Correspondence Address:
K B Anjaria
Radiological Physics and Advisory Division, Bhabha Atomic Research Centre, Trombay, Mumbai
India

Source of Support: None, Conflict of Interest: None

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Abstract

Iodoacetate (IA) is a known tumour promoter of moderate potency. Present study reports the effect of IA post-treatment on ⁶⁰ Co gamma radiation induced chromosomal aberrations in human peripheral blood lymphocytes. Cells were exposed to 2 or 4 Gy of radiation and were cultured in the presence of 0.25-2.5 μg/ml IA. It was observed that IA reduced radiation induced dicentrics yield significantly at both the radiation doses.

Keywords: Iodoacetate, gamma radiation, lymphocytes, chromosomal aberrations

How to cite this article:
Anjaria K B, Shirsath K B, Bhat N N, Sreedevi B. Effect of tumour promoter iodoacetate on γ-radiation induced chromosomal aberrations in human lymphocytes. Radiat Prot Environ 2010;33:199-201

How to cite this URL:
Anjaria K B, Shirsath K B, Bhat N N, Sreedevi B. Effect of tumour promoter iodoacetate on γ-radiation induced chromosomal aberrations in human lymphocytes. Radiat Prot Environ [serial online] 2010 [cited 2023 Jun 2];33:199-201. Available from: https://www.rpe.org.in/text.asp?2010/33/4/199/90467

1. Introduction

A large amount of information is available in literature indicating that the action of one agent can be altered by another agent, leading to enhancement, reduction or additivity, following combined exposures (UNSCEAR, 1999). Chemical agents, which act as tumour promoters, or those, which inhibit error-free repair pathways, may enhance the effects of a mutagenic/carcinogenic agent (Anjaria and Rao, 2001; 2004). In contrast, antimutagenic/anticarcinogenic agents, or agents which suppress error-prone repair, may reduce the mutagenic effects of a physical or chemical agent in the combined treatments (Anjaria and Rao, 2001).

Carcinogenesis is suggested to be a two-stage process: initiation and promotion. Initiation is generally induced by DNA damaging agents like radiation or chemicals and is suggested to be an irreversible event, whereas promotion is suggested to be a reversible process resulting from repetitive exposures of tumour promoting agents subsequent to the exposure to initiating agents (Yamasaki, 1980). It has been reported that tumour-promoting agents potentiate a number of genetic events induced by initiating agents (e.g. gene mutations, sister chromatid exchanges, gene conversion) in yeast and mammalian cells in vitro (Trosko et al., 1977; Gentil et al., 1980; Kunz et al., 1980; Fahrig, 1984; Anjaria and Rao, 2001)

IA is reported to be a tumour promoter of moderate potency (Connell and Duncan, 1981) and although to the best of our knowledge, tumour promoting ability of IA in animals has not been reported, a large number of studies have reported various types of effects of IA, which may result in tumour promotion (Connell and Duncan, 1981; Kinsella, 1982). In this paper, we have reported the modifying effects of tumour promoter IA on chromosomal aberrations induced by gamma radiation in human peripheral blood lymphocytes.

2. Materials and Methods

2.1 Chemical

Iodoacetate, RPMI 1640 medium, lectin and colcemid were purchased from Sigma Chemical Co., St. Louis, MO, USA. Foetal bovine serum was purchased from ICN Biochemicals.

2.2 Chromosomal Aberration Analysis

Peripheral blood was collected from a 22 year old healthy donor in sterile vacutainer containing heparin as an anticoagulant. The stock solution of IA was prepared in distilled in water and filter sterilized using a Millipore syringe filter (0.22μm). IA was added to the lymphocyte cultures to achieve a final concentration of 0.25-2.5 μg/ml.

Blood samples were exposed to 2 or 4 Gy radiation in a Gamma Chamber having a dose rate of 1 Gy/min Immediately after irradiation cultures were set up with 4 ml of RPMI 1640 medium), 0.1 ml lectin (5mg/10 ml), 0.5 ml serum and 0.5 ml blood and desired concentrations of IA in 15 ml sterile plastic disposable tubes and incubated at 37°C in a CO ₂ incubator. Colcemid was added at 24 h to get a final concentration of 0.04 μg/ml and the incubation was continued for another 28 h. The cells were harvested at 52 h and after centrifugation, were subjected to hypotonic treatment in pre-warmed 0.075 M KCl solution and allowed to stand for 20 mins at 37°C in a water bath. After centrifugation at 800 rpm for 8 mins, cell button was gently mixed and fixed using chilled 1:3 acetic acid and methanol. The cells were washed 3 times by centrifugation using chilled fixative. After the last centrifugation, the supernatant was removed leaving behind about 0.25 ml of fixative. The cells were resuspended by passing the suspension repeatedly through microtip. Next, with the help of a Pasteur pipette about 3-4 drops were dropped on a wet, chilled slide which was dried by keeping it on a slide warmer maintained at 50°C. The slides were stained with 2% Giemsa stain for 10 mins, mounted with deepex and scored at 1000 magnification.

3. Results

[Figure 1] shows the effect of IA post-treatment on dicentric aberrations induced by 2 Gy of radiation. For each point, 500 metaphases were scored to estimate dicentrics yield per cell. The results indicate that IA at all the three concentrations significantly reduced radiation induced dicentrics.

Figure 1: Effect of 0.25/2.5 μg/ml IA on 2 Gy gamma radiation induced dicentric yield. SD was calculated as √X/N where X is the number of dicentrics scored in N metaphases. *p<0.05, δ p<0.005 using Student's t-test

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[Figure 2] shows the effect of IA post-treatment on dicentrics induced by 4 Gy of radiation. For each point (of radiation control and combined treatments), scoring was continued till 200 dicentrics were obtained. Dicentric yield was calculated by dividing number of dicentrics (200) by total number of metaphases scored. The results once again indicate that IA post-treatment reduces radiation induced dicentric aberrations significantly.

Figure 2: Effect of 0.25/2.5 μg/ml IA on 4 Gy gamma radiation induced dicentric yield. SD was calculated as √X/N where X is the number of dicentrics scored in N metaphases. * p<0.01, δ p<0.025 Γ p<0.005 using Student's t-test

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4. Discussion

It has been observed in a number of in vitro test systems that tumour promoters potentiate the genotoxic effects induced by carcinogens in vitro. Trosko et al. (1977) have demonstrated that the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced UV induced mutations in mammalian cells and also chemical carcinogens induced mutations in Salmonella ^{More Details} typhimurium and CHO cells. TPA also enhanced the frequency of spontaneous and X-ray induced sister chromatid exchanges in mammalian cells (Kinsella and Radman, 1974; Ohno, 1974). In addition, our earlier study demonstrated that tumour promoter anthralin enhanced 4-Nitroquinoline 1-oxide (4-NQO) induced mutations in diploid yeast (Anjaria and Rao, 2004).

Very little information is available on the cytotoxic and/or potentiating effects of IA. Kiffe et al. (2003) has studied the characterization of cytotoxic and genotoxic effects of different compounds in CHOK5 cells with the comet assay. They observed that IA does not induce direct DNA damage as was reported earlier by others although it induced statistically significant alterations in DNA migration (Hilliard et al., 1998). Even though it was clearly observed that IA induced genotoxic effects in standard comet assay, it did not, however, increase the tail length or moment when lysed cells were used indicating that the compound did not directly damage DNA. IA induced low numbers of SCE in V79 cells (Connell and Duncan, 1981) and also chromosomal aberrations in CHO cells which was reported to be due to secondary effects occurring due to it's cytotoxicity (Hilliard et al., 1998). However, Kleijer et al. (1973) have reported that IA induced a small number of breaks in the DNA of T cells and also inhibited rejoining of single strand breaks induced by X-rays by inhibiting enzymes directly.

It has been shown that IA potentiates UV induced back mutation, mitotic crossing over and aberrant colony formation in yeast (Kunz et al. 1980). However, in contrast, in our earlier study, IA post-treatment resulted in reduction in gamma radiation and 4-NQO induced back mutation and aberrant colony formation in the same yeast system (Anjaria et al., 2008) Further, in our another earlier study, IA post-treatment significantly reduced gamma radiation induced micronuclei in human lymphocytes (Anjaria et al., 2006). Even in our present study, IA reduced gamma radiation induced dicentric aberrations in human lymphocytes at both the radiation doses. Thus, in spite of being known as a tumour promoter, instead of enhancing, IA is found to reduce various genetic end-points in our studies with yeast and human lymphocytes.

It should be noted that there is an absence/scarcity of data pertaining to the tumour promoting ability of IA in animal systems and also, most of the studies describe only other characteristics related to it's tumour promoting ability. On the basis of our above observations, wherein IA reduces radiation and 4-NQO induced genetic endpoints in yeast, and also radiation induced micronuclei and chromosomal aberrations in human lymphocytes, it is important to carry out extensive studies with IA in combination with various other carcinogens to understand the mechanism by which IA may bring about it's tumour promoting effects in animals. Further, it is necessary to demonstrate it's potentiating effects towards various genetic end-points in different in vitro test systems in order to justify it's classification as a tumour promoter of moderate potency.

5. References

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