In vitro and in vivo genotoxic activity of miral, an organophosphorus insecticide used in Colombia

Mutation Research 415 Ž1998. 59–67

In vitro and in vivo genotoxic activity of miral, an organophosphorus insecticide used in Colombia C.H. Sierra-Torres a , N. Cajas-Salazar a , L.S. Hoyos b, M. Zuleta c , E.B. Whorton a , W.W. Au a,) a

Department of PreÕentiÕe Medicine and Community Health, The UniÕersity of Texas Medical Branch, GalÕeston, TX 77555-1110, USA b Department of Biology, UniÕersity of Cauca, Popayan, ´ Colombia c Department of Biology, UniÕersity of Antioquia, Medellın, ´ Colombia Received 17 September 1997; revised 6 April 1998; accepted 9 April 1998

Abstract Miral w 500 CS ŽCASa 42509-80-8., an organophosphorus insecticide, has been widely used in Colombia to fumigate coffee plantations. Therefore, there is extensive human exposure to this pesticide. Miral’s mutagenic and genotoxic activities, however, are not known. In this study, such activities of the pesticide were evaluated using the Salmonella TA98rS9 test and the chromosome aberration assay in bone marrow cells of Swiss albino CD1 male mice. All doses tested with Salmonella in the presence of S9 mix Ž3.2, 16, 80, 400 and 2000 m grplate. induced a mutagenic response that was three times the spontaneous mutation frequency. The mutagenic response without S9 was twice the spontaneous frequency. Based on a 4-day treatment Ži.p.. of mice with Miral, the median lethal dose ŽLD50. and the maximum tolerated dose ŽMTD. were 912.5 mgrkg and 730 mgrkg, respectively. A significant dose-dependent cell cycle delay Ž r 2 s 0.85, p - 0.01. was observed in bone marrow cells when mice were treated for 24 h with 73, 146, 219, 292, 365, 438, 511, 584, 657 and 730 mgrkg. Significant increases in mitotic indices Ž p - 0.02. and chromosome aberrations Ž p - 0.05. were induced in bone marrow cells, when mice were treated for 18 h with the highest dose 511 mgrkg. Our results indicate that Miral is a mutagenic compound in Salmonella and is capable of inducing chromosome aberrations at high doses in mice. Additional genotoxicity studies in farmers exposed to Miral should be conducted to determine the potential human health risk resulting from chronic low-dose exposures to this pesticide. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Organophosphorus insecticide; Mutagenicity; Cytotoxicity; Genotoxicity; Salmonella typhimurium; Bone marrow cell

1. Introduction Pesticides represent one of the greatest sources of exposure to toxic chemicals for humans. Several epidemiological studies have shown that farmers who

) Corresponding author. Tel.: q1-409-772-1803; fax: q1-409772-9108; E-mail: [email protected]

are exposed to a variety of pesticides have significantly elevated risks for developing cancers of the blood and of the immune system w1,2x. In particular, organophosphorus insecticide ŽOP. exposure is associated with risk of non-Hodgkin’s lymphoma w3x. OPs, increasingly used by farmers, are responsible for more poisonings than any other class of pesticides w4x, indicating the need for investigating their potential hazard to human health.

1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 5 4 - 0

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C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67

In Colombia, agriculture accounts for 21% of gross national product and employs 40% of the labor force w5,6x. According to the Colombian Agriculture and Livestock Institute ŽICA., 40,000 tons of pesticides were used in 1990 w7x. It is estimated that up to 40% of the entire Colombian population is directly exposed to pesticides w8x. Over the last few years, the use of OP compared to organochlorates has increased in Colombia because organophosphates have a shorter half life in the environment and do not accumulate through the food chain. The OP, trade formulation Miral w 500 CS ŽA.I. Isazophos, CAS Reg. No. 42509-80-8. is widely used on coffee, banana, and potato plantations in Colombia. As an OP, it can inhibit the acetyl-cholinesterase enzyme which is essential for normal neural transmission, leading to respiratory arrest and muscular weakness w4x. Although the general toxicity of this insecticide has been described w9x, based on review of the scientific literature and documents from EPA, and inquiry to the manufacturer, Ciba-Geigy, in Colombia, the genotoxic potential of Miral has not been investigated. Previous studies have demonstrated that OPs have mutagenic and clastogenic activities in several biological test systems w10–15x. These studies have raised public concerns about the potential for adverse genotoxic effects in humans. Therefore, additional well-conducted in vitro and in vivo genotoxic studies are necessary to assess possible health risks associated with the extensive use of OPs w16x. Short-term genotoxic assays for the detection of potential human carcinogens have been used by many investigators and validated in international collaborative programs w17x. The in vitro Salmonella typhimurium assay with the rat-liver microsomal fraction S9 w18,19x is one of the most frequently used tests for assessing the mutagenic potential of both pure compounds w20x and complex mixtures w21x. When the genotoxicity of

a chemical is unknown, as in the case of Miral, it is advisable to conduct this in vitro mutation assay using a wide range of doses w16x. In addition, the U.S. Environmental Protection Agency recommends that a positive in vitro response should be confirmed by an in vivo assay, since some chemicals producing positive responses in the Salmonella assay have shown negative results after in vivo evaluations w22,23x. Short-term test batteries, including combinations of the Salmonellarmicrosome assay together with the chromosome aberration test in mammalian cells, were found to improve significantly the sensitivity for detection of potential human carcinogens w24x. The chromosome aberration assay is used extensively in population monitoring w25,26x since it is recognized as a reliable biomarker for documentation of genotoxic effects due to exposure w27–30x. Therefore, we have used both the Salmonella mutation assay and the in vivo chromosome aberration assay in this study to evaluate the genotoxic potential of Miral. Our testing shows that Miral induced a positive mutagenic effect in Salmonella TA98 in the presence of metabolic activation ŽS9 mix.. In addition, Miral was found to be cytotoxic and clastogenic in the bone marrow cells of mice treated with this pesticide.

2. Materials and methods 2.1. Chemicals Miral w 500 CS wchloro-1-Ž1-methylethyl.-1 H1,2,4-triazol-3-yl.-0,0-diethyl phosphorothioatex ŽFig. 1., a yellow liquid, is formulated by Ciba-Geigy in Colombia for use as an insecticide. 2-Aminofluorene Ž2-AF. was obtained from Lancaster Synthesis. Glucose-6-phosphate, NADP, cyclophosphamide and

Fig. 1. Chemical structure and characteristics of Miral 500 CS.

C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67

vinblastine were from Sigma. All other chemicals used were of the highest purity available commercially. 2.2. Mutagenicity testing in Salmonella 2.2.1. Bacterial strain To evaluate the mutagenic activity of Miral, the Ames S. typhimurium strain TA98 Ž hisD3052 . was used. This tester strain Žgenerously provided by Dr. Bruce Ames, Department of Biochemistry, University of California, USA. was maintained as frozen stocks in liquid nitrogen and grown in media from the master plate as described by Maron and Ames w31x. The strain was periodically raised from a single colony to check for the presence of the genetic markers Ž uÕrB, rfa and pKM101.. S9 fraction was prepared from the livers of Aroclor-1254 treated rats. S9 mix was freshly prepared for each experiment according to the method of Maron and Ames w31x. 2.2.2. Ames mutagenicity testing Five doses of Miral Ž3.2, 16, 80, 400 and 2000 m grplate. were plated in duplicate with approximately 1 = 10 8 bacterial cells per plate. A total of 100 m lrplate of distilled water and 10 m grplate of 2-AF Ždissolved in DMSO 0.1 mgrml. were used as negative and positive controls, respectively. The solutions were tested in the absence Ž0.5 mlrplate phosphate buffer 1 M, pH s 7.4. and presence of S9 mix Ž0.5 mlrplate.. The mixture containing chemicals and bacteria with or without S9 was vortexed and pre-incubated at 378C for 30 min as described by Maron and Ames w31x. The mixture was then plated with 2 ml of soft agar on glucose supplemented minimal agar. After 48 h of incubation at 378C, the plates were scored for revertant colonies Ž his q .. 2.3. Genotoxicity assays in mice 2.3.1. Animals and exposure route Swiss albino CD1 mice, 10–12 weeks old Ž35 g, approximately., were kindly provided to us free of charge by Veterinaria de Colombia VECOL. Only male mice were available for the study and were acclimatized in our laboratory animal facility for 2 weeks before use. The mice were distributed in cages

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under 12-h lightrdark photoperiod at constant conditions of temperature Ž22 " 38C. and with a diet based on Soya chow and purified water. Animals were injected intraperitoneally Ži.p.. with the test solutions which were based on body weight Žb.w.. and never exceeded a volume of approx. 16.7 mlrkg b.w. The choice of the doses was based on a 4-day assay for the determination of the acute median lethal dose ŽLD50. w32x and the maximum tolerated dose ŽMTD. w33x. A value of 912.5 mgrkg b.w. was determined as the LD50. The MTD of 730 mgrkg b.w. was determined, based on 100% survival when exposing the mice to 80% of the LD50. 2.3.2. Proliferation index To study the effect of Miral on the bone marrow cell proliferation kinetics, a total of 42 mice were randomly divided into 10 groups ŽTable 2.. Each group was exposed to either 73, 146, 219, 292, 365, 438, 511, 584, 657 or 730 mgrkg b.w. of Miral, respectively. A negative control group Ž n s 6. was injected with distilled water. A positive control group Ž n s 6. was injected with 50 mgrkg b.w. of cyclophosphamide ŽCPA., diluted in 0.9% sodium chloride physiological solution 3.5 mgrml. A paraffincoated 5-bromo-2X-deoxyuridine tablet Ž50 mg BrdUrtablet. was inserted into the abdominal region of each animal 1 h prior to treatment for differential staining of metaphase cells w34x. Vinblastine Ž7 mgrkg b.w.. was injected into the animals 2 h prior to harvesting as a cytokinesis blocker Ždiluted in saline solution 1 mgrml.. Bone marrow preparations were made 24 h after treatment according to the method of Hsu and Patton w35x. The slides were stained following the FPG ŽFluorescence Plus Giemsa. staining method as previously described w36x. One hundred metaphase cells per animal were analyzed to document the frequencies of these cells in the first, second and third cycles. The data were used to detect perturbation of cell proliferation by changes in the proliferation index ŽPI.. 2.3.3. Chromosome aberrations From the PI study, three doses were chosen for cytogenetic analysis: one dose with no significant effect on PI Ž365 mgrkg b.w.., one with an intermediate effect Ž438 mgrkg b.w.., and one with a significant effect Ž511 mgrkg b.w... Groups of five

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C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67

mice each were exposed to either Miral, distilled water, or CPA, and sacrificed after 18 h. Cytological preparations were made from the femurs of each mouse, and one hundred metaphase cells were scored per animal. Numerical Žaneuploidy. and structural Žchromatid and isochromatid. chromosome aberrations were scored. Complex aberrations Žtranslocations, dicentrics, etc.. and gaps Žchromatid and isochromatid. were also scored. The mitotic index ŽMI. was calculated by scoring 2000 cells per animal. 2.3.4. Statistical analysis The computer software program ABSTAT Release 1.9 ŽAnderson-Bell. was used to analyze the data. Evaluation of the difference in the means of the PI among treated and control groups was conducted using One-way analysis of variance ŽANOVA.. Multiple linear regression analysis was performed for the PI. The frequency of chromosome aberrations in treated animals was compared with the negative control group using the Fisher’s exact test. MI means were compared against the negative control by the t-test. A probability Ž p . value less than 0.05 was used as the criterion of significance.

3. Results 3.1. Mutagenicity testing in Salmonella The observed mutagenic activity of Miral in S. typhimurium is summarized in Table 1. In the pres-

Fig. 2. Dose–response effects of Miral 500 CS in S. typhimurium TA98. Spontaneous revertants have been subtracted. Vertical bars represent the mean"standard error among three independent experiments. Žl. qS9 mix; Ž`. -S9 mix.

ence of S9 mix, all tested doses of Miral demonstrated mutagenic activity. The number of his q revertantsrplate Žranges 55.2 " 10.2 to 63.2 " 1.93. was three times higher than the spontaneous frequency of 19 " 1.58. As illustrated in Fig. 2, an increase in the revertant response over the spontaneous frequency was reached with the lowest dose Ž3.2 m grplate., followed by a slight upward trend towards a dose-dependent increase. In the assays without S9, the bacteria exhibited a weak to moderate response Žranges 36.8 " 2.77 to 33.2 " 2.62., a doubling over the spontaneous frequency Ž16 " 1.08..

Table 1 Mutagenicity of Miral 500 CS in S. typhimurium TA98 Treatment

DW Ž m l. Miral 500 CS

2-AF a

Concentration per plate Ž m g.

100 3.2 16 80 400 2000 10

Mean his q revertantsrplate Ž"S.E.. a q S9 mix

yS9 mix

19.0 " 1.58 55.2 " 10.2 55.3 " 3.70 57.2 " 3.63 61.3 " 7.72 63.2 " 1.93 395.5 " 77.78

16.0 " 1.08 36.8 " 2.77 41.6 " 5.41 40.8 " 5.58 37.8 " 4.08 33.2 " 2.62 38.8 " 4.50

The values are means" standard error of three separate experiments, each one in duplicate. DW: distilled water.

C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67

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However, the number of revertantsrplate showed an initial increase followed by a slight decrease at higher doses ŽFig. 2.. For comparison, the response with 2-AF Ž10 m grplate., the positive control, was 395.5 " 77.78 with S9 and 38.8 " 4.50 without S9 ŽTable 1..

Miral ŽTable 2.. However, this problem did not occur in the genotoxicity study, where all the animals survived the selected doses Ž365 to 511 mgrkg b.w.. ŽTable 3..

3.2. Cytotoxicity assay in mice

The frequency of chromosome aberrations and the percentage of aberrant cells per treatment are summarized in Table 3. The observed aberrations were chromatid breaks and chromatid gaps. The tested doses Ž365, 438 and 511 mgrkg. induced a 3-, 4and 7-fold increase in the number of gaps, respectively, compared to the negative control. Based on the observation of chromatid and isochromatid breaks Žexcluding gaps., only the highest dose Ž511 mgrkg b.w.. induced a significant number of chromosome aberrations Ž p - 0.05.. In addition, a few polyploid metaphase cells Ž1%. were observed at this concentration ŽTable 3.. No significant differences in mitotic indexes were found between the 365 or the 438 mgrkg b.w. doses of Miral and the negative control. The dose 511 mgrkg b.w., however, produced a significant increase in the MI Ž p - 0.02. compared to the negative control ŽTable 3..

3.3. Genotoxicity assays in mice

The cytotoxic effect of Miral in bone marrow cells of treated animals is summarized in Table 2. There were not significant differences in PI from mice treated with 73 through 365 mgrkg b.w. Miral Žranges 1.82 " 0.04 to 1.98 " 0.17. when compared to the negative control Ž1.88 " 0.12, p ) 0.05.. Increasing the dose to 438 mgrkg b.w. induced a significant reduction in PI Ž1.48 " 0.02, p - 0.05.. At the four highest doses Ž511, 584, 657 or 730 mgrkg b.w.., Miral caused a significant delay in cell proliferation Ž p - 0.01., stopping the cells in the first cell cycle. Multiple linear regression analysis of the PI data indicates that the effect of Miral in reducing the proliferation of bone marrow cells was dose-dependent Ž r 2 s 0.85, p s 0.02.. Some of the mice died in the groups treated with high doses of

Table 2 Effect of miral on bone marrow proliferation index of mice ŽPI. Miral 500 CS Žmgrkg.

DW Ž16.7 mlrkg. 73 146 219 292 365 438 511 584 657 730 CPA Ž50 mgrkg. a

Mice Treated

Dead

MI

M II

M III

6 3 3 3 3 5 5 4 5 5 6 6

0 0 0 0 0 2 2 1 2 2 4 0

16 15 15 17 21 14 50 100 100 100 100 56

80 72 76 79 76 8 48 0 0 0 0 43

4 13 9 4 3 6 2 0 0 0 0 1

The values are means" standard error between animals. p - 0.05 ŽANOVA.. c p - 0.01 ŽANOVA.. DW: distilled water; CPA: cyclophosphamide PI: proliferation index s ŽMI = 1 q MII = 2 q MIII = 3.rtotal metaphases. b

PI " S.E.a

Cells on replication cycle Ž%.

1.88 " 0.12 1.98 " 0.17 1.94 " 0.02 1.88 " 0.04 1.82 " 0.04 1.92 " 0.01 1.48 " 0.02 b 1.00 " 0.00 c 1.00 " 0.00 c 1.00 " 0.00 c 1.00 " 0.00 1.45 " 0.04

C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67

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0 0.2 0.2 1 1.2 97 95.8 98.6 94.4 92.8 3 3.4 0.6 3.2 5.4 0 0.6 0.6 1.4 0.6 b

a

0.4"0.40 0.8"0.37 0 2.6"0.75d 22.8"3.56 d 0.4 0.8 0 2.6 d 34.6 d 0 0 0 0 1 0.4 0.8 0 2.6 33.6 0 0 0 0 0 5 5 5 5 5 DW 365 438 511 CPA

Total Dead

Isochromatid Chromatid Treated

Results of five mice treated for each concentration Ž100 cellsranimal.; Total values of register in each concentration Ž500 cellsrdose.; c 2000 cells per animal scored; d p- 0.05 ŽFisher’s exact test.; e p- 0.02 Ž t-test.; DW: distilled water Ž16.7 mlrkg.; CPA: cyclophosphamide Ž50 mgrkg.; MI: mitotic index Ž% metaphase cells..

0 0 0 0 3 0 0 0 2 3

1 3 4 7 31

Polyploid 40 39 38 Chromatid

Aberrant cells Ž%"S.E.. Chromosome aberrations per 100 cells a Mice Miral 500 CS Žmgrkg.

Table 3 Clastogenic and cytotoxic effects of Miral on mouse bone marrow cells

Complex aberrations

Gapsb

Isochromatid

Cells with indicated chromosome number Ž%.

MI"S.E.c Ž%.

2.27"0.18 2.32"0.59 2.96"0.60 3.82"0.44 e 1.87"0.18

4. Discussion In the present study, we show that Miral 500 CS was able to induce G–C base pair mutations w31x causing a frameshift reversion of the histidine dependent tester strain ŽTA98. to the wild type Ž his q .. The moderate number of revertantsrplate induced by Miral without S9 activation is considered to be a mutagenic response, since this effect was reproducible according to the criteria employed by Ames et al. w19x. This response also indicates that Miral by itself is a direct-acting mutagen. The mutagenic activity was enhanced in the presence of metabolic activation with S9, suggesting that Miral may also contain indirect-acting mutagenic metabolites. In both conditions Žwith and without S9., the dose–response curves were non-linear ŽFig. 2.. One explanation for this response is that Miral may be cytotoxic at high doses. In general, mutagens at high concentrations can be toxic to bacteria, thus, decreasing the number of revertants on the plate w31x. Another explanation may be due to hydrolysis of Miral Žwater solubility 69 ppm. w37x in the aqueous media. Hydrolytic reactions of organophosphorus compounds eventually lead to inorganic phosphate which is non-mutagenic w38x. Consequently, Miral may not have been sufficiently persistent in the plate to increase the number of revertantsrplate, a phenomenon which was also reported by Ivanovic et al. using other organophosphorus compound w39x. Our observed mutagenic effect was also accompanied by wider standard errors for some doses, which may have been due to the accumulation of Miral in some regions of the plate causing cell death. This effect has been observed in other studies using the Ames test w40x. The mutagenic activity of Miral may be due to the existence of electrophilic sites in the Miral molecule or its intermediates, which are capable of binding to nucleophilic sites in the bacterial DNA. This is consistent with previous reports showing the ability of some OPs to bind DNA w37,38x and cause mutation w41–44x. For example, chloracetophone induced base pair substitution mutations in TA100 both with and without metabolic activation w41x. According to RTECS w42x, Carbofuran was reported to be mutagenic in Salmonella and in other organisms. Vlckova et al. w43x detected direct mutagenicity of Phosmet Žthe active ingredient of Decemtione EK20., which

C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67

induced frameshift mutations in TA97 and base pair substitutions in TA100. Water samples contaminated with the organophosphates dimethoate and methyl parathion exhibited a significant degree of mutagenicity with TA102, TA100 and TA98 strains w44x. It is well established that mutation plays an important role in the initiation phase of the multi-stage carcinogenic process. There is increasing awareness that not only DNA alterations, but also cytotoxicity may also play an important role in carcinogenesis w45–48x. The results from our study indicate that Miral can be considered a cytotoxic agent since it induced dose-dependent cell proliferation delay ŽTable 2. and mitotic arrest ŽTable 3. in mouse bone marrow cells. The latter observation indicates blockage of cells in mitosis, therefore, Miral may act as a mitotic poison Že.g., as do colcemid and vinblastine.. Such cellular toxicity may induce heteroploid cells, resulting from karyotypic instability, that can later on progress to cancer cells w49x. The observed lethality in the cytotoxicity assay was not expected ŽTable 2.. However, this could be attributed to a synergistic toxic effect from the multiple chemical exposure Žether, BrdU implantation and vinblastine., the prolonged exposure to high doses of Miral Ž24 h., or simply dosing accidents Žrupture of liver, spleen or digestive tract by i.p. injection.. Lethality was not observed in the chromosome aberration studies ŽTable 3., where mice were exposed to Miral for a shorter period of time Ž18 h.. In the bone marrow assay, Miral induced a significant clastogenic effect only at the highest dose Ž511 mgrkg b.w... Among mice treated with this dose, 1% of the metaphase cells Ž n s 5. were polyploid ŽTable 3.. This observation is associated with a significantly high mitotic index ŽTable 3, p - 0.02. due to cell accumulation in the first cell division cycle ŽTable 2.. It is apparent that the registering of polyploid cells was caused by an overlapping of metaphase cells which were blocked in mitosis. Lower concentrations of Miral did not induce a sufficient number of chromosome aberrations to show detectable clastogenicity. Clastogenic effects induced by OPs have been documented by other investigators. For instance, the available evidence indicates that technical grade malathion has the potential to produce genotoxic effects in several mammalian systems, including humans w50x. The total percentage of cells with

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structural aberrations in curacron-treated mice increased significantly over controls w51x. Significantly increased chromosomal aberrations, micronuclei and sperm abnormalities were observed for Hinosan w15x. Studies conducted in lymphocytes of OP-exposed farm workers have shown an increase in chromosome aberrations compared to control subjects w52– 55x. Kaufmann w56x proposed that gaps are the consequence of the production of lesions in the DNA template, which inhibit DNA polymerase during DNA replication. Formation of chromatid-type aberrations has been suggested to be a consequence of failure of post-replication repair pathways to eliminate the gaps w56x. Although in our study gaps were not considered in the statistical analysis as chromosome aberrations, we detected a substantial dose-dependent increase in gap frequency when mice were exposed to Miral ŽTable 3.. Previous cytogenetic studies have revealed a significant increase in chromatid breaks and gaps in blood lymphocytes among workers exposed to pesticides w53,57x. The close similarity of gaps and breaks, despite the difference in their frequency, is interesting, and might indicate a closer relationship between these two end-points than we have, perhaps, allowed w58x. The Ames test was performed in S. typhimurium TA98 as this was the only bacterial strain available to us in Colombia. Based on the results of this study, the formulation Miral 500 CS can be characterized as a compound having both direct and indirect in vitro mutagenic activities. However, testing with other Salmonella strains may provide additional information about the mechanism of mutagenesis of this organophosphorus insecticide. In vivo, Miral was clastogenic at the highest dose tested Ž511 mgrkg b.w... At this concentration, Miral also caused a delay in cell proliferation and an increase in mitotic arrest. From our review of the literature and U.S. EPA documents, this compound has not yet been tested in a standard cancer bioassay. However, our data suggests that this compound may be potentially carcinogenic after exposure to relatively high doses. Therefore, it is advisable for farmers to follow the Product Label instructions and use excellent safety measures when applying Miral and to reduce environmental contamination as much as possible. In addition, the potential genotoxic hazard in humans

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C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67

after exposure to low concentrations of Miral for a long period of time, or exposure in combination with other pesticides, should be investigated.

w12x

w13x

Acknowledgements The authors wish to thank Dr. Sherif Abdel-Rahman for his helpful review of the manuscript and critical comments. We also wish to thank professor Jaime Calle and the staff at the Laboratory of Mutagenesis and Carcinogenesis at the University of Antioquia, and professor Silvio Carvajal and the staff in the Genetic Toxicology and Cytogenetics Unit at the University of Cauca for their invaluable assistance in this study.

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