Genotoxicity Evaluation of Dimethoate to Experimental Mice by Micronucleus,Chromosome Aberration Tests,and Comet Assay

Abstract

Dimethoate (DM) is an organophosphate insecticide with numerous uses on field and agricultural crops and ornamentals. Data concerning DM-acute genotoxicity are controversial and knowledge on its delayed effect is limited. For this reason, we aimed to further explore DM genotoxicity resulting from subchronic intoxication of experimental mice. Thus, DM was administered to mice at doses ranging from 1 to 30 mg/kg body weight for a period of 30 consecutive days. There was a significant increase (P < .05) in the frequency of micronucleated bone marrow cells following DM administration. Furthermore, the chromosome aberration assay revealed a significant increase in the percentage of chromosome abnormalities in a dose-dependent manner. Dimethoate was also found to induce significant DNA damage in mouse bone marrow cells as assessed by the comet assay. Altogether, our results showed that, after a subchronic exposure, DM was a genotoxic compound in experimental mice.

Keywords

dimethoate genotoxicity micronucleus test chromosome aberration test comet assay mouse bone marrow cells subchronic exposition

Introduction

Pesticides play an important role in modern agriculture, and their use has increased steadily. Because many of them are suspected to have mutagenic and carcinogenic activities,¹ concern for their adverse effects on human health has increased. Several studies have reported the genotoxic potential of organophosphorous (OP) pesticide,² but the results reported so far are not conclusive. Organophosphorous compounds are among the most widely used pesticides, either as insecticides or as herbicides. In acute exposure, the main mechanism of toxicity of OP pesticides is irreversibly binding to the enzyme acetylcholinesterase and inhibiting its activity that results in accumulation and prolonged effect of acetylcholine and consequently follows with acute muscarinic and nicotinic effects.³ In chronic and subchronic exposures, added to cholinesterase inhibition, induction of oxidative stress has been reported as the main mechanism of OP pesticides’ toxicity.^4

–8 In addition, other mechanisms are responsible for their genotoxicity, such as those related to their alkylating^9,10 and phosphorylating properties.¹¹

Dimethoate ([DM], O,O-dimethyl S-methyl carbamoylphosphoro-dithioate) is one of the most important OP insecticides used extensively on a large number of crops against several pests. Dimethoate exerts toxic effects on many tissues and organs including pancreas,¹² ^- ¹⁴ liver,^15–
20 kidney,²¹ and brain.¹⁶ ^- ¹⁹ Dimethoate was also revealed to be immunotoxic^{22

24} and causes several adverse effects in the reproductive system of male and female mice.^25–28 Interestingly, DM was reported to cause benign and malignant neoplasms of the liver, endocrine organs, and lymphatic system²⁹; however, the International Agency for Research on Cancer (IARC) was unable to classify DM with regard to its potential carcinogenicity due to the inadequacies of existing studies.³⁰

The information on the genotoxic properties of DM is limited and inconsistent. It is reported that DM was mutagenic in Escherichia coli, Saccharomyces cerevisiae, and Salmonella typhimurium TA 100 but nonmutagenic in S typhimurium strains ^31

–36 TA 1535, TA 1537, TA 1538, TA 98, and TA 102. In vitro exposure to DM induced a concentration-dependent increase in sister chromatid exchange (SCE) frequency in toadfish lymphocytes³⁷ and in mammalian cell cultures.³⁸ Dimethoate gave positive responses in the Salmonella reversion assay and the SOS chromotest.³⁹ Dimethoate induced a statistically significant elevation of micronuclei (MNs) frequency in human lymphocytes in a nondose-related manner⁴⁰ and in mouse bone marrow after acute intoxication.⁴¹ This pesticide was also found to increase the incidence of numerical but not structural chromosomal aberrations in male Wistar rats.²⁴ On the other hand, it was also reported that DM was nonmutagenic in some other genotoxicity tests.⁴² Recent study of Undeger and Basaran⁴³ showed that DM significantly increased DNA damage in human lymphocytes using the comet assay.

Since data on the DM acute genotoxicity are controversial and knowledge on its delayed effect is much more limited, we aimed in the present study to further explore DM genotoxicity after subchronic intoxication of experimental mice. For this, we utilized the MN and chromosome aberrations tests and the comet assay in mouse bone marrow cells.

Material and Methods

Chemicals

Dimethoate (analytical standard), supplied by Sigma Aldrich Chemie GmbH (France), was used for the study. Corn oil was used for preparing suspensions of DM (volume of injection was 100 µL per animal). All other chemicals used were of analytical grade.

Animals

This study was conducted on female mice BALBL/c (average body weight 20 ± 0.3 g, age: 6 weeks) purchased from the Central Pharmacy (SIPHAT, Tunis, Tunisia). Before any experience, all animals were maintained 7 days under the same laboratory conditions of temperature (22°C ± 3°C), relative humidity (55% ± 5%), and a 12:12 hours light/dark cycle and received a nutritionally standard diet (SICO, Sfax, Tunisia) and tap water ad libitum. The experimental procedures were carried out according to the American College of Toxicology Statement on the Use of Animals in Toxicology and approved by the local Ethics Committee.

Experimental Design

After an acclimation period, mice were randomly divided into 6 groups (15 mice per group). Animals of groups 2, 3, 4, 5, and 6 were administered intraperitoneally (ip) DM (dissolved in corn oil) at doses of 1, 5, 10, 15, and 30 mg/kg body weight for 30 consecutive days. These doses⁴⁴ correspond, respectively, to 2%, 10%, 20%, 30%, and 60% of the LD₅₀. Group 1, receiving equivalent amount of vehicle alone, served as the control. Animals within different treatment groups were divided into 3 subgroups: A (for the MN assay), B (for the comet assay), and C (for the chromosome aberration assay; 5 animals per subgroup) and received their respective treatment. All animals were sacrificed by cervical dislocation on the 31st day of the study.

Body Weight

Animals were examined daily throughout the experimental period for signs of toxicity. Body weight was recorded in the beginning and at the end of the treatment period.

In Vivo MN Assay

Immediately after the animals were sacrificed, femur and tibia of mice in the subgroup A were freed from adherent tissues and were dissected out. The bone marrow was sampled by injection of filtered fetal calf serum using a syringe. The collected cells were centrifuged at 390g for 5 minutes, a little volume of supernatant was discarded and the cells were resuspended in the remaining fluid. A small drop of the resuspended cell pellet was spread on a glass slide, fixed in absolute methanol for 5 minutes, and air-dried for conservation at room temperature. Air-dried slides were stained for 15 minutes in phosphate-buffered saline ([PBS], 0.15 mol/L, pH 7.4) containing 10 µg/mL of acridine orange (freshly prepared), rinsed in the same buffer for 15 minutes, and allowed to dry in the dark at room temperature.

Scoring Criteria

As a result of acridine orange staining, red fluorescence is observed in polychromatic erythrocytes (PCEs); normochromatic erythrocytes (NCEs) appear slightly green or opaque and polychromatic erythrocyte micronucleated (PCEMN) appear red with one or more yellow fluorescent corpuscles which are MN. Scoring of MN was performed according to the criteria described by Hayashi et al.⁴⁵ These criteria are based essentially on the diameter and the shape of the MN.

Slide Analysis

The slides, randomly coded, were observed under ×1000 magnification using a fluorescence microscope (Nikon Eclipse E 400, New York). The number of PCE among 1000 (NCE + PCE) was determined in order to evaluate any cytotoxic effect of DM. The number of PCEMN among 2000 PCE per mouse was calculated to determine the induction of MN.

In Vivo Comet Assay

After sacrificing the animals of the subgroup B, both the femurs and tibias were removed and the content was directly flushed out with the help of a 24-gauge needle into a microcentrifuge tube. The cell suspension was prepared in PBS. From the suspension, 5 µL of sample containing approximately 2 to 5 × 10⁴ cells/mL was added to 95 µL of 0.5% low melting point agarose (LMA in Ca²⁺- and Mg²⁺-free PBS) to prepare the final cell–agarose suspension. The comet assay was performed as described⁴⁶ with some modifications. From the final cell–agarose suspension, 80 µL was spread over the microscope slide which was precoated with 1% normal melting point agarose (NMA). The cells were then lysed in a buffer containing 2.5 mol/L NaCl, 100 mmol/L EDTA, and 10 mmol/L Tris (pH 10.0) with freshly prepared 1% Triton X-100 and 10% dimethyl sulfoxide (DMSO) for 24 hours at 4°C. After lysis, slides were rinsed 3 times in deionized water to remove salt and detergent. Slides were placed in a horizontal electrophoresis unit and DNA was allowed to unwind for 20 minutes in alkaline solution containing 300 mmol/L NaOH and 1 mmol/L EDTA, pH > 13. The DNA was electrophoresed for 15 minutes at 300 mA and 25 V (0.90 V/cm). The slides were neutralized with 0.4 mol/L Tris (pH 7.5), stained with ethidium bromide (20 µg/mL) before examination with a Nikon Eclipse TE 300 fluorescence microscope (Nikon, Tokyo, Japan). A total of 100 comets on each randomly coded slide were visually scored according to the relative intensity of fluorescence in the tail and classified as belonging to 1 of 5 classes. Each comet class was given a value of 0, 1, 2, 3, or 4 (from undamaged, 0, to maximally damaged, 4) as described previously by Collins et al.⁴⁷ The total score was calculated by the following equation: (percentage of cells in class 0 × 0) + (percentage of cells in class 1 × 1) + (percentage of cells in class 2 × 2) + (percentage of cells in class 3 × 3) + (percentage of cells in class 4 × 4). Consequently, the total score ranges from 0 to 400.

In Vivo Chromosome Aberration Assay

Twenty-four hours before sacrifice, the animals of subgroup C were given a suspension of yeast powder (100 mg/500 µL) to accelerate mitosis of bone marrow cells. Vinblastine (200 µL; 250 µg/mL) was injected to animals 45 minutes before sacrifice in order to block dividing cells in metaphase.⁴⁸ Bone marrow cells from femurs and tibia were collected, subjected to hypotonic shock (KCl 0.075 mol/L), and fixed 3 times using methanol–acetic acid, according to the technique of Evans et al.⁴⁹ The cells were spread on glass slides that were blazed on a flame for 5 seconds, then air-dried for conservation at room temperature and finally stained with a 4% dilution of Giemsa reagent in water for 15 minutes. After randomly coding the slides, the chromosomes of 100 cells in metaphase were examined for abnormalities at a magnification of ×1000 using an optical microscope (Carl Zeiss, Germany). This was done for each 1 of 5 replicates (500 metaphases per dose level) for the negative control and the treated groups. Chromosome aberrations were identified according to criteria described by Savage.⁵⁰ Metaphases with chromosome breaks, gaps, rings, and centric fusions (Robertsonian translocation) were recorded and expressed as percentage of total metaphases per group.

Statistical Analysis

All values were expressed as mean ± standard deviation (SD) of 5 animals per group. Data were analyzed using 1-way analysis of variance (ANOVA) followed by Fisher post hoc analysis. All statements of significance were based on a probability (P) ≤ .05.

Results

Body Weight

There were no deaths reported during the course of the study in any of the treated groups. There was a significant reduction in body weight and weight gain in the treated groups of 15 and 30 mg/kg per d at the end of the treatment compared with the control and other treated groups (Table 1 ).

Table 1.

Effect of Dimethoate (DM) Treatment on Body Weight (g) of Experimental Mice After Subchronic Exposure of 30 Days^a

	DM, mg/kg body weight
	0	1	5	10	15	30
Starting body weight	21.5 ± 1	20.75 ± 0.96	22.75 ± 2.5	22.5 ± 1.29	21.75 ± 1.26	21.75 ± 1.26
Terminal body weight	25.25 ± 0.5	24 ± 0.82	25.25 ± 1.26	24 ± 1.83	23 ± 0.82^b	20.5 ± l^b
Weight gain	3.75 ± 0.96	3.25 ± 0.96	2.5 ± 1.73	1.5 ± 1	1.25 ± 0.5^b	−1.25 ± 1.5^b

^a Values are expressed as mean ± SD of 15 animals in each group.

^b P < .05 versus the control group.

In Vivo MN Assay

The cytotoxic potential of DM was evaluated by counting the number of PCE among 1000 cells (PCE + NCE). This number showed a mean value of 378.3 ± 7.6 in the control group and decreased first slightly in mice treated with DM at 1, 5, 10 mg/kg body weight to, respectively, 373.67 ± 8.5, 367.67 ± 8.5, and 357.00 ± 9.5 and then significantly in mice treated with DM at 15 and 30 mg/kg body weight to, respectively, 295.0 ± 22.3 and 275.6 ± 24.1 (P ≤ .05; Figure 1 ).

Figure 1.

Number of polychromatic erythrocytes (PCEs) in 1000 PCEs and normochromatic erythrocytes (PCE + NCE) in animals treated with DM at the indicated doses for 30 consecutive days. Values are expressed as mean ± SD, n = 5. *Significant difference from the control group (*P < .05). DM indicates dimethoate; SD, standard deviation.

The number of PCEMN among 2000 PCE, indicative of genotoxicity, is presented in Figure 2. Dimethoate induced a significant (P ≤ .05) increase in PCEMN number in a dose-dependent manner. In fact, this number passed from a basal value of 10 ± 1 in the control group to 18 ± 1, 29.6 ± 3, and 49.6 ± 9.8 in groups treated with DM at, respectively, 5, 10, and 15 mg/kg body weight. At the highest dose of DM (30 mg/kg body weight), the PCEMN number significantly increased to reach 63.3 ± 4.6.

Figure 2.

Number of polychromatic erythrocytes micronucleated (PCEMN) in 2000 polychromatic erythrocytes (PCEs) in animals treated with increasing doses of DM for 30 consecutive days. Values are expressed as mean ± SD, n = 5. *Significant difference from the control group (*P < .05). DM indicates dimethoate; SD, standard deviation.

In Vivo Comet Assay

Bone marrow cells were used to assess genotoxicity of DM using the comet assay which is considered a sensitive, rapid, and relatively simple method for detecting DNA damage at the level of individual cells.⁴⁶ Results of the visual scoring of total DNA damage observed in bone marrow cells are illustrated in Figure 3. Significantly higher levels of DNA damage were detected in mice exposed to DM compared with the negative control. In summary, DM induced significantly (P < .05) a dose-dependent increase in DNA damage of exposed mice (Figure 3).

Figure 3.

DNA damage (as evaluated by the comet assay) induced by DM in bone marrow cells after treatment with increasing doses of DM for 30 consecutive days. Five hundred comets were counted in each group (100 comets per animal). Values are expressed as mean ± SD of 5 animals in each group. *Significant difference from the control group (*P < .05). DM indicates dimethoate; SD, standard deviation.

In Vivo Chromosome Aberration Assay

Only structural aberrations induced by the different treatments were enumerated in the present study, with special emphasis on gaps, rings, breaks, and centric fusions. All these types of structural abnormalities and their frequencies for both control and treated groups are presented in Table 2. Numerical chromosome abnormalities such as polyploidy and aneuploidy were not evaluated. Chromosome aberrations induced in mouse bone marrow cells after 30 days of injections of increasing doses of DM are presented in Figure 4 together with the negative control. Chromosome aberrations whatever the type, especially centric fusions and breaks were statistically significantly (P ≤ .05) increased in a dose-dependent manner in mice treated with DM (Table 2, Figure 4).

Table 2.

Different Types of Chromosome Aberrations and Their Percentage in Bone Marrow Cells After Treatment With Increasing Doses of DM for 30 Consecutive Days^a

			DM doses, mg/kg body weight
Control	1	5	10	15	30
Break	Mean	1.67	3.67	4.67^b	12.00^b	21.67^b	20^b
Break	SD	1.53	0.58	0.58	2.00	4.04	1
Centric fusion	Mean	3.00	8.67^b	9.33^b	12.00^b	16.67^b	16^b
Centric fusion	SD	1.00	1.15	1.53	1.00	1.53	1
Ring	Mean	0.33	1.67	2.00	5.33^b	9.00^b	15.67^b
Ring	SD	0.58	0.58	1.00	2.08	1.00	1.53
Gap	Mean	0.00	0.67	1.00	1.00	1.00	3.67^b
Gap	SD	0.00	0.58	1.00	1.00	1.00	0.58
Total	Mean	5	14.67^b	17^b	30.33^b	48.33^b	55.33^b
Total	SD	1	2.08	1	1.53	4.04	3.21

Abbreviations: DM, dimethoate; SD, standard deviation.

^a Five hundred metaphases were examined per group (100 metaphases per animal). Data are presented as mean ± SD.

^b Significant difference from the control group (*P < .05).

Figure 4.

Percentage of bone marrow cells containing chromosome aberrations (total of all types of chromosome aberrations) after treatment with increasing doses of DM for 30 consecutive days. Five hundred metaphases were examined per group (100 metaphases per animal). Data are presented as mean ± SD. *Significant difference from the control group (*P < .05). DM indicates dimethoate; SD, standard deviation.

Discussion

In spite of several attempts that have been made to establish the genotoxicity of DM, this matter remains somehow controversial. Furthermore, IARC was unable to classify DM with regard to its potential carcinogenicity.³⁰ Indeed, the mutagenicity of DM was not sufficiently proven in S typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 102.^31

–36 Dimethoate gave negative results in a mutation assay in Schizosaccharomyces pombe ⁴² and in CHO cells.⁵¹ Despite these findings, DM has been shown to cause an increase in SCE frequency in toadfish lymphocytes.³⁷ Besides, DM induced DNA damage and an increase in the frequency of MN in human lymphocytes at doses ranging from 0.1 to 200 µg/mL.⁴³ ^, ⁴⁰ ^,52

Because of these controversial reports on the genotoxicity and mutagenicity of DM and to elucidate the mechanism underlying the genotoxicity of this pesticide, we evaluated, in the present investigation, its ability to induce MN, chromosome aberrations, and DNA damage in bone marrow cells of mice treated with DM for 30 consecutive days.

The MN assay is a test widely used to assess genotoxicity of chemicals.^53,54 Furthermore, it gives indication concerning the cytotoxic potential of the tested compound. Our results showed that the number of PCE per 1000 cells was significantly reduced for DM doses of 15 and 30 mg/kg body weight (Figure 1). Several mechanisms could explain this reduction, among them, either direct cytotoxicity or MN formation and heavy DNA damage leading to cell death or apoptosis. The data obtained show clearly that DM induced MN in bone marrow cells of mice treated in a dose-dependent manner (Figure 2). From these results, we can deduce, on one hand that DM may interact directly with DNA causing DNA damage. On the other hand, DM may cause dysfunction of the mitotic spindle. In this latter case, it might be considered as an aneugenic compound. However, staining bone marrow cells with acridine orange does not discriminate between MN-containing fragments or whole chromosomes. That is why we performed the comet assay and the chromosome aberration test.

The comet assay is increasingly used in industrial genotoxicity testing in vitro^55

–58 and is also becoming an important tool for evaluating the genotoxic potential of compounds in vivo^59
–61 because of its rapidity, simplicity to perform,⁵⁹ and its higher sensitivity and specificity.⁵⁹ Our results clearly showed that DM induced an increase in DNA damage score in a dose-dependent manner (Figure 3).

The chromosome aberration test, recommended by regulatory authorities for the assessment of genotoxicity and mutagenicity of many chemicals and natural compounds, has provided positive data. In fact, DM increased the number of chromosome aberrations in a dose-dependent manner with relatively high frequencies of DNA breaks compared with other types of chromosome abnormalities (Figure 4, Table 2). Centric fusions (Robertsonian translocations) are also frequent, which may be due to the fact that almost all mouse chromosomes are acrocentric. These types of chromosomes have the exceptional facility to merge with each other. Only the structural chromosome aberrations are presented here, the reason being that only very little polyploidy or aneuploidy were detected. This specific aspect needs further investigation.

The design of the present experiments does not allow formation of direct stable mutations since cells were arrested at the first stage of mitosis. It is strongly believed that cells with heavy chromosome abnormalities cannot survive and will rapidly undergo apoptosis. In fact, DM induced apoptosis in spiders from industrially polluted areas.⁶² One may assume that when cells with a small number of chromosome aberrations are allowed to survive (when not arrested at mitosis), they will divide with possible occurrence of mutations that could be transmitted to the next generation through clonal expansion of somatic or even stem cells. This will then give rise to tumor formation. However, DM was not classified by IARC with regard to its potential carcinogenicity.³⁰

Genotoxicity induced by DM could be attributed to the parent compound itself as well as to its oxygen analog omethoate, which was considerably more toxic than DM.⁶³ These effects can also be attributed to active oxygen species generated by DM.¹³ ^- ²⁰ ^, ⁶⁴ Thus, further investigations are needed to elucidate the mechanism/mechanisms by which DM induced DNA damage.

In conclusion, our results showed that DM induced, in a dose-dependent manner, MN formation, DNA damage revealed by the comet assay, and increased chromosome aberration levels in bone marrow cells of mice treated with DM for 30 consecutive days. These results confirmed the genotoxic potential of DM in vivo in subchronic condition and indicated a need for further investigations to evaluate the carcinogenic potency of DM and the potential risks associated with the exposure of humans and wildlife to this pesticide.

Footnotes

Guidelines for Ethical Publications

Authors declare that this manuscript was consistent with the guidelines and principles of American College of Toxicology Statement on the Use of Animals in Toxicology.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Ministère Tunisien de l’Enseignement Supérieur et de la Recherche Scientifique et de la Technologie (Laboratoire de Recherche sur les Substances Biologiquement Compatibles: LRSBC).

References

IARC.Occupational Exposures in Insecticide Application, and Some Pesticides. IARC monographs on the evaluation of carcinogenic risks to humans. Vol 53. Lyon, France: IARC. 1991:5–586.

Bolognesi

. Genotoxicity of pesticides: a review of human biomonitoring studies. Mutat Res. 2003;543(3):251–272.

Costa

. Current issues in organophosphate toxicology. Clin Chim Acta. 2006;366(1-3):1–13.

Abdollahi

Ranjbar

Shadnia

Nikfar

Rezaiee

. Pesticides and oxidative stress: a review. Med Sci Monit. 2004;10(6):141–147.

Abdollahi

Donyavi

Pournourmohammadi

Saadat

. Hyperglycemia associated with increased hepatic glycogen phosphorylase and phosphenol pyruvate carboxykinase in rats following sub-chronic exposure to malathion. Comp Biochem Physiol C: Toxicol Pharmacol. 2004;137(4):343–347.

Akhgari

Abdollahi

Kebryaeezadeh

Hosseini

Sabzevari

. Biochemical evidence for free radical-induced lipid peroxidation as a mechanism for sub chronic toxicity of malathion in blood and liver of rats. Hum Exp Toxicol. 2003;22(4):205–211.

Banerjee

Seth

Ahmed

. Pesticide-induced oxidative stress: perspective and trends. Rev Environ Health. 2001;16(1):1–40.

Ranjbar

Pasalar

Abdollahi

. Induction of oxidative stress and acetylcholinesterase inhibition in organophosphorous pesticide manufacturing workers. Hum Exp Toxicol. 2002;21(4):179–182.

Dedek

Grahl

Schmidt

. A comparative study of guanine N7-alkylation in mice in vivo by the organophosphorus insecticides trichlorphon, dimethoate, phosmet and bromophos. Acta Pharmacol Toxicol. (Copenh). 1984;55(2):104–109.

10.

Mehl

Rolseth

Gordon

Bjøraas

Seeberg

Fonnum

. Brain hypoplasia caused by exposure to trichlorfon and dichlorvos during development can be ascribed to DNA alkylation damage and inhibition of DNA alkyltransferase repair. Neurotoxicology. 2000;1(1-2):165–173.

11.

Piña-Guzmán

Solis-Heredia

Quintanilla-Vega

. Diazinon alters sperm chromatin structure in mice by phosphorylating nuclear protamines. Toxicol Appl Pharmacol. 2005;202(2):189–198.

12.

Hagar

Fahmy

. A biochemical, histochemical, and ultrastructural evaluation of the effect of dimethoate intoxication on rat pancreas. Toxicol Lett. 2002;133(2-3):161–170.

13.

Kamath

Rajini

. Altered glucose homeostasis and oxidative impairment in pancreas of rats subjected to dimethoate intoxication. Toxicology. 2007;231(2-3):137–146.

14.

Kamath

Joshi

AKR

Rajini

. Dimethoate induced biochemical perturbations in rat pancreas and its attenuation by cashew nut skin extract. Pestic Biochem Physiol. 2008;90:58–62.

15.

Gomes

Dawodu

Lloyd

Revitt

Anilal

. Hepatic injury and disturbed amino acid metabolism in mice following prolonged exposure to organophosphorus pesticides. Hum Exp Toxicol. 1999;18(1):33–37.

16.

Sharma

Bashir

Irshad

Gupta

Dogra

. Effects of acute dimethoate administration on antioxidant status of liver and brain of experimental rats. Toxicology. 2005;206(1):49–57.

17.

Sharma

Bashir

Irshad

Nagc

Dogra

. Dimethoate-induced effects on antioxidant status of liver and brain of rats following subchronic exposure. Toxicology. 2005;215(3):173–81.

18.

Sayim

. Dimethoate-induced biochemical and histopathological changes in the liver of rats. Exp Toxicol Pathol. 2007;59(3-4):237–243.

19.

Astiz

de Alaniz

Marra

. Effect of pesticides on cell survival in liver and brain rat tissues. Ecotoxicol Environ Saf. 2009;72(7):2025–2032.

20.

Saafi

Louedi

Elfeki

Protective effect of date palm fruit extract (Phoenix dactylifera L.) on dimethoate induced-oxidative stress in rat liver. Exp Toxicol Pathol. 2010;63(5):433–441; In press.

21.

Mahjoubi-Samet

Fetoui

Zeghal

. Nephrotoxicity induced by dimethoate in adult rats and their suckling pups. Pestic Biochem Physiol. 2008;91(3):96–103.

22.

Institóris

Siroki

Dési

. Immunotoxicity study of repeated small doses of dimethoate and methylparathion administered to rats over three generations. Hum Exp Toxicol. 1995;14(11):879–883.

23.

Institóris

Siroki

Dési

Undeger

. Immunotoxicological examination of repeated dose combined exposure by dimethoate and two heavy metals in rats. Hum Exp Toxicol. 1999;18(2):88–94.

24.

Undeger

Institoris

Siroki

Nehez

Desi

. Simultaneous geno- and immunotoxicological investigations for early detection of organophosphate toxicity in rats. Ecotoxicol Environ Saf. 2000;45(1):43–48.

25.

Mahadevaswami

Kaliwal

. Effect of dimethoate administration schedules on compensatory ovarian hypertrophy, follicular dynamics, and estrous cycle in hemicastrated mice. J Basic Clin Physiol Pharmacol. 2002;13(3):225–248.

26.

Mahadevaswami

Kaliwal

. Evaluation of dimethoate toxicity on pregnancy in albino mice. J Basic Clin Physiol Pharmacol. 2004;15(3-4):211–221.

27.

Farag

El-Aswad

Shaaban

. Assessment of reproductive toxicity of orally administered technical dimethoate in male mice. Reprod Toxicol. 2007;23(2):232–238.

28.

Astiz

Hurtado de Catalfo

de Alaniz

Marra

. Involvement of lipids in dimethoate-induced inhibition of testosterone biosynthesis in rat interstitial cells. Lipids. 2009;44(8):703–718.

29.

Reuber

. Carcinogenicity of dimethoate. Environ Res. 1984;34(2):193.

30.

IARC, Miscellaneous pesticides. IARC Monogr Eval Carcinog Risk Chem Hum. 1983;30:1–424.

31.

Mohn

. 5-Methyl tryptophan resistance mutations in Escherichia coli K-12. Mutagenic activity of monofunctional alkylating agents including organophosphorus insecticides. Mutat Res. 1973;20(1):7–15.

32.

Fahrig

. Comparative mutagenicity studies with pesticides. IARC Sci Publ. 1974;10:161–181.

33.

Moriya

Ohta

Watanabe

Miyazawa

Kato

Shirasu

. Further mutagenicity studies on pesticides in bacterial reversion assay system. Mutat Res. 1983;113(3-4):173–180.

34.

Haworth

Lawlor

Mortelmans

Speck

Zeiger

. Salmonella mutagenesis test results for 250 chemicals. Environ Mutagen. 1983;5(suppl 1):3–142.

35.

Ladhar

Grover

Radhawa

. Mutagenicity of systemic organophosphate pesticides metasystox and rogor. Ind J Exp Biol. 1990;28(4):390–391.

36.

Bianchi

Zannoli

Pizzala

Stivala

Chiesara

. Genotoxicity assay of five pesticides and their mixtures in Saccharomyces cerevisiae D7. Mutat Res. 1994;321(4):203–211.

37.

Ellingham

Christensen

Maddock

. In vitro induction of sister chromatid exchanges and chromosomal aberrations in peripheral lymphocytes of the oyster toadfish and American eel. Environ Mutagen. 1986;8(4):555–569.

38.

Chen

Hsueh

Sirianni

Huang

. Induction of sister-chromatid exchanges and cell cycle delay in cultured mammalian cells treated with eight organophosphorus pesticides. Mutat Res. 1981;88(3):307–316.

39.

. Ruiz

Marzin

Genotoxicity of six pesticides by Salmonella mutagenicity tests and SOS chromotest. Mutat Res. 1997;309(3):245–255.

40.

Santamaria

Gobbi

Cembran

Arnaboldi

. Human lymphocyte micronucleus genotoxicity test with mixtures of phytochemicals in environmental concentrations. Mutat Res. 1997;388(1):27–32.

41.

Geetanjali

Rita

Reddy

. Effect of ascorbic acid in the detoxification of the insecticide dimethoate in the bone marrow erythrocytes of mice. Food Chem Toxicol. 1993;31(6):435–437.

42.

Gillot-Delhalle

Coluzzi

Moutschen

Moutschen-Dahmen

. Mutagenicity of some organophosphorus compounds at the ade-6 locus of Scizosaccharomyces pombe. Mutat Res. 1983;117(1-2):139–148.

43.

Undeger

Basaran

. Effects of pesticides on human peripheral lymphocytes in vitro: induction of DNA damage. Arch Toxicol. 2005;79(3):169–176.

44.

Sanderson

Edson

. Toxicological properties of the organophosphorus insecticide dimethoate. Brit J industr Med. 1964;21:52-64.

45.

Hayashi

Sofuni

Ishidate

Jr M

. An application of acridine orange fluorescent staining to micronucleus test. Mutat Res. 1983;120(4):241–247.

46.

Singh

McCoy

Tice

Schneider

. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175(1):184–191.

47.

Collins

Dusinska

Gedik

Stetina

. Oxidative damage to DNA: do we have a reliable biomarker? Environ Health Perspect. 1996;104(suppl 4):465–469.

48.

Ouanes

Abid

Ayed

Induction of micronuclei by Zearalenone in Vero monkey kidney cells and in bone marrow cells of mice: protective effect of Vitamin E. Mutat Res. 2003;538(1-2):63–70.

49.

Evans

Breckon

Ford

. An air drying method for meiotic preparation from mammalian tests. Cytogenetics. 1964;15:289–94.

50.

Savage

JRK

. Classification and relationship of induced chromosomal structural changes. J Med Genet. 1975;12(2):103–122.

51.

Johnson

Caterson

Allen

. Mutagenicity testing of dimethoate in the in vitro CHO/HGPRT mutation assay. Unpublished report project no. 0423 , Princeton, NJ: American Cyanamide Comp; 1985.

52.

Jamil

Shaik

Mahboob

Krishna

. Effect of organophosphorus and organochlorine pesticides (monochrotophos, chlorpyriphos, dimethoate, and endosulfan) on human lymphocytes in-vitro. Drug Chem Toxicol. 2004;27(2):133–144.

53.

Fenech

. The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human postulations. Mutat Res. 1993;285(1):35–44.

54.

Kirsch-Volders

El Hajaji

Cundari

Van Hummelen

. The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction. Mutat Res. 1997;392(1-2):19–30.

55.

Hartmann

Elhajouji

Kiskinis

Use of the alkaline comet assay for industrial genotoxicity screening: comparative investigation with the micronucleus test. Food Chem Toxicol. 2001;39(8):103–118.

56.

Hartmann

Kiskinis

Fjaellman

Suter

. Influence of cytotoxicity and compound precipitation on test results in the alkaline comet assay. Mutat Res. 2001;497(1-2):199–212.

57.

Kiskinis

Suter

Hartmann

. High-throughput comet assay using 96-well plates. Mutagenesis. 2002;17(1):37–43.

58.

Giannotti

Vandin

Repeto

Comelli

. A comparison of the in vitro comet assay with the in vitro chromosome aberration assay using whole human blood or Chinese hamster lung cells: validation study using a range of novel pharmaceuticals. Mutagenesis. 2002;17(2):163–170.

59.

Tice

Agurell

Anderson

The single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35(3):206–221.

60.

Sasaki

Sekihashi

Izumiyama

The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and US NTP Carcinogenicity Database. Crit Rev Toxicol. 2000;30(6):629–799.

61.

Hartmann

Agurell

Beevers

Recommendations for conducting the in vivo alkaline comet assay. Mutagenesis. 2003;18(1):45–51.

62.

Wilczek

. Apoptosis and biochemical biomarkers of stress in spiders from industrially polluted areas exposed to high temperature and dimethoate. Comp Biochem Physiol C Toxicol Pharmacol. 2005;141(2):194–206.

63.

IPCS/WHO, Classification of Pesticides by Hazard and Guidelines to Classification. Switzerland, Geneva; 2001.

64.

Sivapiriya

Jayanthisakthisekaran

Venkatraman

. Effects of dimethoate (O, O,-dimethyl S-methyl carbamoyl methyl phosphorodithioate) and ethanol in antioxidant status of liver and kidney of experimental mice. Pest Biochem Physiol. 2006;85(2):115–21.