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In vivo cytogenetic effects of a commercially formulated mixture of cypermethrin and quinalphos in mice
Mutation Research 587 (2005) 120–125
In vivo cytogenetic effects of a commercially formulated mixture of cypermethrin and quinalphos in mice L.K.S. Chauhan, S. Chandra, P.N. Saxena, S.K. Gupta ∗ Cell Biology Section, Industrial Toxicology Research Centre, P.O. Box No. 80, M.G. Marg, Lucknow 226 001, India Received 10 February 2005; received in revised form 4 August 2005; accepted 12 August 2005 Available online 26 September 2005
Abstract In vivo cytogenetic effects of commercially formulated cypermethrin (CYP, synthetic pyrethroid insecticide) and/or quinalphos (QUI, organophosphate insecticide), generally used in combination, were examined through chromosomal aberrations (CA) and micronucleus test (MT) in mice. Male mice were orally gavaged to a single dose of CYP/QUI commercial mixture (22, 44 or 67 mg/kg b.wt.) for 24 h (CA) or 48 h (MT). Based on the concentrations of active ingredients of CYP and QUI present in the test doses of CYP/QUI mixture, mice were orally exposed to 0.66, 1.32 and 2 mg/kg of CYP or 4.4, 8.8 and 13.4 mg/kg of QUI. For reference, a group of five mice was intraperitoneally administered to cyclophosphamide (20 or 50 mg/kg) or orally gavaged to peanut oil for vehicle control. Exposure of CYP/QUI mixture inhibited the mitotic index (MI) and induced CA in a dose-dependent manner at 24 h; however, significant (p < 0.01 or 0.001) frequencies of CA were observed at 44 mg/kg onwards, whereas inhibition of MI at 67 mg/kg. Independent exposure of QUI at 8.8 mg/kg onwards also significantly (p < 0.01 or 0.001) inhibited MI and induced CA, whereas CYP at 2 mg/kg (highest concentration in CYP/QUI mixture) inhibited MI significantly but failed to induce CA. Chromatid breaks and fragments found to be frequent aberrations in all the test groups. Treatment of CYP/QUI mixture also induced micronucleus formation dose-dependently at 48 h, yet statistically significant (p < 0.001) frequencies of micronucleated polychromatic erythrocytes (MNPCE) were observed at 44 mg/kg onwards. QUI (8.8 and 13.4 mg/kg) alone also induced significant frequencies of MNPCE, whereas frequencies of MNPCE observed with the CYP even at 2 mg/kg were comparable to that of vehicle control. Present findings indicate the genotoxicity potential of CYP/QUI mixture and suggest that the simultaneous presence of the toxic doses of CYP and QUI can lead to synergistic genotoxicity in mice and may pose mutagenic risk in human beings. © 2005 Elsevier B.V. All rights reserved. Keywords: Cypermethrin; Quinalphos; Commercial formulations; Chromosomal aberrations; Micronucleus; Mice
1. Introduction Developing resistance in insects against the individual insecticides encouraged the use of insecticide mixtures for the effective pest control as a result several mixtures of insecticides have come in the market. Though
∗
Corresponding author. E-mail address: [email protected] (S.K. Gupta).
1383-5718/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2005.08.004
large database is available on the toxicity/genotoxicity of individual insecticides, yet information on the insecticide mixtures is meagre. It is indeed possible that the mixture of insecticides may interact synergistically and cause more deleterious effects than that of their individual effects. Combined exposure of non-toxic doses of atrazine and alachlor have been demonstrated to induce significant frequencies of chromatid breaks and fragments in mouse bone marrow cells while independent exposure of similar doses failed to induce any signifi-
L.K.S. Chauhan et al. / Mutation Research 587 (2005) 120–125
cant effect [1]. Similarly, corn seeds pre-soaked in the aqueous concentrations of alachlor or bifenox and the extracts from treated seeds when tested in Salmonella assay showed no mutagenicity, whereas combined treatment of seed extracts found to be mutagenic in the same system [2]. A study carried out on human lymphocytes following exposure of atrazine (0.01 g/ml) or linuron (0.001 g/ml) showed modest frequency of CA, whereas lymphocytes jointly exposed to half of the concentrations of these chemicals induced significant frequency of CA [3]. In fact, it is the reason that prompts us to evaluate the genotoxicity of commercially formulated mixtures of insecticides that can ensure the safe and effective mixtures. Organophosphates are generally used in combination of synthetic pyrethroids. Evidence already exists that mixtures of these insecticides increase the neurotoxicity [4]. A commercially formulated mixture of a synthetic pyrethroid insecticide, cypermethrin, (3%, w/v) and an organophosphate insecticide, quinalphos, (20%, w/v) is adequately used on various agricultural crops. Independent exposure of cypermethrin has been reported to induce chromosomal aberrations, micronucleus formation and SCE in mice and human lymphocytes [5–9] and chromosomal as well as mitotic aberrations in the root meristem cells of Allium cepa [10]. Quinalphos too has been demonstrated to induce chromosome breaks/fragments in mice and human lymphocytes [11]. Since independent exposure of CYP and QUI cause genotoxic effects in mammals, it is important to study the combined effects of these chemicals particularly of commercial formulations of which industrial workers and users are more prone to occupational exposure and consequent risk. In a step further, in the present study, we have examined combined and independent cytogenetic effects of commercially formulated cypermethrin and quinalphos on the mouse bone marrow cells and discussed the possibilities of synergism. 2. Materials and methods 2.1. Chemicals Commercially formulated mixture of CYP and QUI (containing CYP 3% (w/v) and QUI 20% (w/v)), CYP (Cyperkill 25% EC) and QUI (Ekalux 25% EC) were procured from the local market. Stains and fixatives used in this study were bought from BDH, England or Glindia, India. 2.2. Test animals Healthy adult male mice (6–8 weeks, 20 ± 1 g wt.) bred and raised in animal facility of Industrial Toxicology Research Centre Lucknow were used in this study. Before treatment, mice
121
were held for acclimation for 4 days housed in polycarbonate boxes with steel wire tops and bedded with paddy husk. During the experiment, mice were maintained at controlled temperature 22 ± 2 ◦ C, 12 h-dark/light periods and 50–70% humidity with free access to pellet feed (Dayal Industries, Barabanki, India) and fresh water ad libitum. 2.3. Treatment schedule CYP/QUI mixture was diluted with peanut oil to administer a constant volume of 0.2 ml to each mouse. The acute oral LD50 (112 mg/kg) was determined according to Trimmed Spearman–Karber method [12] and mice (five per group) were orally gavaged to single dose of 22, 44 or 67 mg/kg of CYP/QUI mixture corresponding to 20, 40 or 60% of LD50 (112 mg/kg) dose, respectively. To analyze the independent effects, mice were orally gavaged to 0.66, 1.32 and 2 mg/kg of CYP or 4.4, 8.8 and 13.4 mg/kg of QUI. A group of five mice was identically treated to peanut oil 0.2 ml/mice designated as vehicle control or injected to cyclophosphamide (20 or 50 mg/kg, single i.p.) for positive control. For CA, mice were exposed for 24 h, whereas for MT, exposure period was 48 h. 2.4. Chromosome aberration assay CA assay was carried out as described earlier [13]. In brief, mice were injected to aqueous solution of colchicine (2.5 mg/kg) 2 h prior to scheduled killing by cervical dislocation and the bone marrow was flushed out from both the femurs in HBSS (pH 7.2). The cells were centrifuged at 1000 rpm for 5 min and pellet was dispersed in 0.56% KCl to incubate for 30 min at 37 ◦ C. Cells were recentrifuged and dispersed in the fresh and chilled Carnoy’s for fixation. Next day cells were dropped over clean chilled slides, air dried to stain with Giemsa and mounted. 2.5. Micronucleus test Bone marrow sampling, preparations of slide and scoring of MNPCE were done as described before [13]. In brief, bone marrow was gently flushed out in a self-designed medium containing HBSS + BSA (1%) + EDTA (0.15%), pH 7 [14], centrifuged at 1000 rpm for 5 min and the medium was decanted. Pellet obtained was dispersed in 0.25 ml medium, smeared on clean slides, fixed in 70% methanol, air dried and stained with May-Grunwald/Giemsa protocol. 2.6. Statistical analysis The mitotic index was determined by scoring metaphase cells out of approximately 3000 nuclei/mouse (1000/slide). CA were scored from well spread 80–100 metaphase cells/mouse of each group. Cells with chromatid gaps were also scored but not included in percent aberrations or aberrant cells. For MT, a minimum of 2000 polychromatic erythrocytes/mouse were also scored from treated or control group. Data on CA
122
Treatment groups and doses (mg/kg)
Mitotic indexa
Metaphase cells scored
Vehicle control
3.10 ± 0.7
577
4 (4)
CYP/QUI mixture 22(CYP 0.66 + QUI 4.4 mg) 44(CYP 1.32 + QUI 8.8 mg) 67(CYP 2 + QUI 13.4 mg)
3.46 ± 0.13 2.15 ± 0.18* 1.94 ± 0.26**
554 546 462
CYP 2 QUI 8.8 13.4 Cyclophosphamide 20
2.19 ± 0.42* 2.21 ± 0.19* 0.89 ± 0.09** 1.78 ± 0.19**
443 456 402 517
* ** a b c
Fragments
Exchange/cells with multiple breaks
Chromatid gapsb
4 (3)
−/−
32 (19)
8 (7)
11 (8) 18 (15) 13 (11)
7 (6) 12 (9) 25 (14)
−/1 −/− 1/2
49 (27) 67 (41) 63 (36)
18 (15) 30 (24) 39 (27)
3.25 (2.71) 5.49 (4.39)* 8.44 (5.84)**
4 (4) 11 (8) 13 (11) 35 (26)
3 (2) 8 (6) 10 (8) 27 (21)
−/− −/− 2/1 2/6
10 (7) 13 (9) 25 (11) 64 (38)
7 (6) 19 (14) 25 (20) 63 (53)
1.58 (1.35) 4.16 (3.07)* 6.22 (4.79)** 12.37 (10.25)**
P < 0.01. P < 0.001. Expressed as group mean ± S.E. (ANOVA). Chromatid gaps excluded from total aberrations and aberrant cells. Significance determined by Chi-square test. Parentheses represent number of cells.
Chromatid breaks
No. of Aberrations (aberrant cells)c
Aberrations (%) (aberrant cells)c 1.39 (1.21)
L.K.S. Chauhan et al. / Mutation Research 587 (2005) 120–125
Table 1 Chromosomal aberrations in the mouse bone marrow cells following 24 h oral exposure of commercially formulated cypermethrin and/or quinalphos
L.K.S. Chauhan et al. / Mutation Research 587 (2005) 120–125
were expressed in percent and prevalence of significance was determined by Chi-square test, whereas data on MI and MT were analyzed by one way analysis of variance (ANOVA). In all the treatment groups, p-values less than 0.05 were considered significant in comparison of vehicle control [15].
3. Results The acute oral LD50 dose of the commercially formulated mixture of CYP/QUI was determined to be 112 mg/kg in mice. Exposure of CYP/QUI mixture (67 mg/kg) or QUI (13.4 mg/kg) showed overt symptoms of toxicity like tremors and diarrhea after 24 h exposure. Table 1 summarizes the results of combined and independent exposure of CYP and QUI on the MI and CA observed at 24 h in mice bone marrow cells. Exposure of single dose of CYP/QUI mixture inhibited the MI and induced CA dose-dependently. However, as compared to vehicle control, significant (p < 0.001) inhibition of MI was observed at 67 mg/kg and induction of CA at 44 mg/kg onwards. Independent exposure of CYP (2 mg/kg) showed significant (P < 0.01) inhibition of MI but did not show CA. Results obtained with other doses of CYP (0.66 and 1.32 mg/kg; data not presented) were comparable to that of vehicle control. Treatment of QUI (8.8 and 13.4 mg/kg), however, significantly (p < 0.001) inhibited the MI and induced CA. The lowest dose of QUI (4.4 mg/kg; data not shown) was found to be statistically insignificant. Fragments and chromatid breaks were found to be frequent aberrations in all the treatment groups. A number of cells with chromatid gaps were also observed in all the treatment groups. The results of micronucleus test observed in mice following combined or independent oral exposure of CYP and QUI are summarized in Table 2. The test doses (22–67 mg/kg) of CYP/QUI mixture induced micronu-
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cleus formation dose-dependently. Though, significant frequencies of MNPCE were observed at 44 mg/kg onwards. The percentage of MNPCE observed in the mice bone marrow exposed to CYP (0.66–2 mg/kg) alone was comparable to vehicle control, whereas QUI (8.8 and 13.4 mg/kg) alone induced significant (p < 0.001) frequencies of MNPCE. Data obtained with 0.66 and 1.32 of CYP or 4.4 mg/kg of QUI were insignificant, and thus not presented. 4. Discussion Marked increase in the frequencies of CA and MNPCE over the vehicle control and independent exposures of CYP or QUI indicates the genotoxic potential of CYP/QUI mixture, which may be the result of synergistic interactions between the two insecticides, however, mild contribution of adjuvant (solvents, potentiators and emulsifiers) cannot be ruled out. Earlier studies have reported induction of CA, SCE and micronucleus formation in mouse bone marrow and human lymphocytes with the independent treatment of CYP [5–9]. Bhunya and Pati [6] observed significant frequencies of CA and micronucleated cells at 30–50 mg/kg dose levels in mice exposed through various routes (p.o., i.p. and s.c.) to CYP. Data obtained with CYP (0.66–2 mg/kg) in the present study imply that CYP alone at these doses is not causative of genotoxicity. Rupa et al. [11] have demonstrated significant induction of CA and micronucleus formation in mouse bone marrow exposed to 5–20 mg/kg of QUI (technical grade); however, as compared to control group, the significance was observed at 10 mg/kg onwards. In the present study also QUI (>8.8 mg/kg) found to be capable of inducing significant chromosome damage. The possibility of synergistic interactions between CYP and QUI is quite possible as
Table 2 Micronucleated cells in mouse bone marrow following 48 h oral exposure of commercially formulated mixture of cypermethrin and/or quinalphos Treatment groups and doses (mg/kg)
No. of PCE
Vehicle control
11633
7
CYP/QUI mixture 22 (CYP 0.66 + QUI 4.4 mg) 44 (CYP 1.32 + QUI 8.8 mg) 67 (CYP 2 + QUI 13.4 mg)
10998 10995 11106
CYP 2 QUI 8.8a 13.4 Cyclophosphamide 50
11710 8187 13112 12219
* a
P < 0.001 significance determined by ANOVA. Data from four mice.
No. of Micronuleated PCE
Micronucleated PCE (%)
PCE/NCE ratio
0.06 ± 0.01
1.09
19 61 109
0.17 ± 0.07 0.55 ± 0.04* 0.98 ± 0.03*
1.11 0.95 0.95
11 26 51 106
0.09 ± 0.02 0.31 ± 0.03* 0.39 ± 0.35* 0.86 (±0.11)*
1.19 1.14 0.89 1.10
124
L.K.S. Chauhan et al. / Mutation Research 587 (2005) 120–125
the frequencies of CA or MNPCE with the treatment of CYP/QUI mixture were relatively higher than that of the independent treatment of CYP or QUI. In general, QUI is a neurotoxic insecticide, inhibits acetylcholine esterases as a primary mechanism of action; however, QUI exposure also generates reactive oxygen species in mice, which may be the cause of genetic damage [16]. A study on the potentiation of insecticides demonstrated that esterase inhibitors, particularly organophosphates, delay the metabolism of synthetic pyrethroid insecticide, deltamethrin, by affecting the ester cleavage [17]. The chemical structure of deltamethrin is quite similar to CYP and both undergo similar metabolic pathways in mammals [18]. Further, the primary cause of pyrethroid resistance in different organisms is associated with the overproduction of esterase isoenzymes, which metabolize these compounds through ester cleavage [19]. The organophosphates bind with these enzymes and thus prevent detoxification of synthetic pyrethroid insecticides [19]. It is possible that QUI, an esterase inhibitor, slowed down the metabolism of CYP, which in its parental form induced additional genotoxicity even at low dose levels. Paraxon, a non-toxic metabolite of an organophosphate insecticide parathion, has been demonstrated to synergies mutagenicity when incubated with plant-activated m-phenylenediamine or with direct acting 2-acetoxyacetylaminofluorene resulting 10-fold increase in mutant yield of Salmonella typhimurium [20]. Similar mutagenic synergy also noted in S. typhimurium when paraxon was incubated with mammalian activated m-phenylenediamine [21]. Synergistic interactions between chlorpyrefos and pyrethrum have also been shown to inhibit neurite growth [4]. Further, if LD50 doses are taken into account, it is evident that the mixture increases the toxicity. For example, in this study, LD50 of formulated CYP/QUI mixture was determined to be 112 mg/kg containing 3.36 mg of CYP and 22.4 mg of QUI as active ingredients, whereas the LD50 doses of non-formulated CYP and QUI in mouse have been reported to be 138 and 62 mg/kg, respectively [22]. Evidence also exists that the interactions of organophosphate and formulating pesticidal components synergies the neurotoxicity [4]. The synergism between the insecticide mixture and adjuvant used as emulsifiers, stabilizers and solvents therefore cannot be ruled out; however, it cannot be ascertained too unless each product of formulation is tested separately with the individual insecticide or insecticide mixture. As chromosome aberrations and micronucleus formation are adjunct cytogenetic indicators of genotoxicity, it can be concluded that the co-existence of CYP and QUI can synergies the genotoxicity in mice. Though humans
are seldom exposed to such a high doses of insecticides unless intent-to-suicide, yet assessment of mutagenic synergy between the low doses of these insecticides for longer exposure periods is more appropriate. Acknowledgements The authors are thankful to Prof. Y.K. Gupta, Director, I.T.R.C. for his keen interest in the study. Authors thank Dr. Neeraj Mathur for statistical analysis. The technical assistance of Mr. V.B. Singh and Sitaram is also acknowledged. References [1] F.L. Meisner, A.D. Belluck, D.B. Roloff, Cytogenetic effects of alachlor an/or atarazine in vivo and in vitro, Environ. Mol. Mutagen. 19 (1992) 77–82. [2] M.J. Plewa, E.D. Wagnar, G.J. Gentile, J.M. Gentile, An evaluation of the genotoxic properties of herbicides following plant and animal activation, Mutat. Res. 136 (1984) 233–245. [3] D.B. Roloff, A.D. Bulluck, F.L. Meisner, Cytogenetic studies of herbicide interactions in vitro and in vivo using atrazine and linuron, Arch. Environ. Contam. Toxicol. 22 (1992) 267–271. [4] J.C. Axelred, C.W. Howerd, W.G. McLean, Interactions between pesticides and components of pesticides formulations in an in vitro neurotoxicity test, Toxicology 173 (2002) 259–268. [5] S.M. Amer, E.I. Aboul-Ela, Cytogenetic effects of pesticides: Induction of micronuclei in bone marrow by the insecticides cypermethrin and rotenone, Mutat. Res. 155 (1985) 135–142. [6] S.P. Bhunya, P.C. Pati, Genotoxic effects of synthetic pyrethroid insecticides cypermethrin in mice in vivo, Toxicol. Lett. 41 (1988) 223–230. [7] J. Suralles, N. Xamena, A. Creus, J. Catalan, H. Naroppa, R. Marcos, Induction of micronuclei by five pyrethroid insecticides in whole blood and isolated lymphocyte culture, Mutat. Res. 341 (1995) 169–184. [8] L.K.S. Chauhan, D.K. Agarwal, V. Sundararaman, In vivo induction of sister chromatid exchanges in mouse bone marrow following oral exposure to commercial formulations of alpha-cyano pyrethroids, Toxicol. Lett. 93 (1997) 153–157. [9] M. Puig, E. Carbonell, N. Xamena, A. Creus, R. Marcos, Analysis of cytogenetic damage induced in cultured human lymphocytes by the pyrethroid insecticides cypermethrin and fenvalerate, Mutagenesis 4 (1989) 72–74. [10] L.K.S. Chauhan, P.N. Saxena, S.K. Gupta, Cytogenetic effects of cypermethrin and fenvalerate on the root meristem cells of Allium cepa, Environ. Exp. Bot. 42 (1999) 181–189. [11] D.S. Rupa, P.P. Reddy, O.S. Reddy, Cytogenetic effects of quinalphos in mice, Food Chem. Toxicol. 29 (1991) 115–117. [12] M.A. Hamilton, R.C. Russo, R.V. Thurston, Trimmed Spearman–Karber method for estimating medium lethal concentrations in toxicity bioassays, Environ. Sci. Technol. 11 (1977) 714–719. [13] L.K.S. Chauhan, N. Pant, S.K. Gupta, S.P. Srivastava, Induction of chromosome aberrations, micronucleus formation and sperm abnormalities in mouse following carbofuran exposure, Mutat. Res. 465 (2000) 123–129.
L.K.S. Chauhan et al. / Mutation Research 587 (2005) 120–125 [14] D.K. Agarwal, L.K.S. Chauhan, An improved chemical substitute for fetal calf serum for the micronucleus test, Biotech. Histochem. 68 (1993) 187–188. [15] J.H. Zar, Multiple comparisons, in: Biostatistical Analysis, second ed., Prentice Hall, Eaglewod Cliffs, 1984, pp. 194–195. [16] P.D. Dwivedi, M. Das, S.K. Khanna, Role of Cytochrome P-450 in quinalphos toxicity: effect on hepatic and brain antioxidant enzymes in rats, Food Chem.Toxicol. 36 (1998) 437–444. [17] WHO (1990) Deltamethrin, Environmental Health Criteria, vol. 97, Geneva. [18] J.P. Leahey, The Pyrethroid Insecticides, Taylor & Francis, London, 1985. [19] R. Gunning, D.G. Moores, D.A. Devanshire, Esterase inhibitors synergies the toxicity of pyrethroids in Australian Helicoverpa
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armigera (Hubner), Pestic. Biochem. Physiol. 63 (1999) 50– 62. [20] T. Gichner, J. Veleminsky, E.D. Wagenar, M.J. Plewa, Mutagenic synergy between paraxon and plant-activated mphenylenediamine or 2-acetoxyacetylaminofluorine, Environ. Mol. Mutagen. 27 (1996) 59–66. [21] E.D. Wagner, K. Repenty, J.S. Tan, T. Gichner, M. Plewa, Mutagenic synergy between paraxon and mammalian or plantactivated aromatic amines, Environ. Mol. Mutagen. 30 (1997) 312–330. [22] C.D.S. Tomlin, Quinalphos, in: The Pesticide Manual, A World Compendium, British Crop Protection Council, Surrey, UK, 1994, pp. 890–892.
In vivo cytogenetic effects of a commercially formulated mixture of cypermethrin and quinalphos in mice L.K.S. Chauhan, S. Chandra, P.N. Saxena, S.K. Gupta ∗ Cell Biology Section, Industrial Toxicology Research Centre, P.O. Box No. 80, M.G. Marg, Lucknow 226 001, India Received 10 February 2005; received in revised form 4 August 2005; accepted 12 August 2005 Available online 26 September 2005
Abstract In vivo cytogenetic effects of commercially formulated cypermethrin (CYP, synthetic pyrethroid insecticide) and/or quinalphos (QUI, organophosphate insecticide), generally used in combination, were examined through chromosomal aberrations (CA) and micronucleus test (MT) in mice. Male mice were orally gavaged to a single dose of CYP/QUI commercial mixture (22, 44 or 67 mg/kg b.wt.) for 24 h (CA) or 48 h (MT). Based on the concentrations of active ingredients of CYP and QUI present in the test doses of CYP/QUI mixture, mice were orally exposed to 0.66, 1.32 and 2 mg/kg of CYP or 4.4, 8.8 and 13.4 mg/kg of QUI. For reference, a group of five mice was intraperitoneally administered to cyclophosphamide (20 or 50 mg/kg) or orally gavaged to peanut oil for vehicle control. Exposure of CYP/QUI mixture inhibited the mitotic index (MI) and induced CA in a dose-dependent manner at 24 h; however, significant (p < 0.01 or 0.001) frequencies of CA were observed at 44 mg/kg onwards, whereas inhibition of MI at 67 mg/kg. Independent exposure of QUI at 8.8 mg/kg onwards also significantly (p < 0.01 or 0.001) inhibited MI and induced CA, whereas CYP at 2 mg/kg (highest concentration in CYP/QUI mixture) inhibited MI significantly but failed to induce CA. Chromatid breaks and fragments found to be frequent aberrations in all the test groups. Treatment of CYP/QUI mixture also induced micronucleus formation dose-dependently at 48 h, yet statistically significant (p < 0.001) frequencies of micronucleated polychromatic erythrocytes (MNPCE) were observed at 44 mg/kg onwards. QUI (8.8 and 13.4 mg/kg) alone also induced significant frequencies of MNPCE, whereas frequencies of MNPCE observed with the CYP even at 2 mg/kg were comparable to that of vehicle control. Present findings indicate the genotoxicity potential of CYP/QUI mixture and suggest that the simultaneous presence of the toxic doses of CYP and QUI can lead to synergistic genotoxicity in mice and may pose mutagenic risk in human beings. © 2005 Elsevier B.V. All rights reserved. Keywords: Cypermethrin; Quinalphos; Commercial formulations; Chromosomal aberrations; Micronucleus; Mice
1. Introduction Developing resistance in insects against the individual insecticides encouraged the use of insecticide mixtures for the effective pest control as a result several mixtures of insecticides have come in the market. Though
∗
Corresponding author. E-mail address: [email protected] (S.K. Gupta).
1383-5718/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2005.08.004
large database is available on the toxicity/genotoxicity of individual insecticides, yet information on the insecticide mixtures is meagre. It is indeed possible that the mixture of insecticides may interact synergistically and cause more deleterious effects than that of their individual effects. Combined exposure of non-toxic doses of atrazine and alachlor have been demonstrated to induce significant frequencies of chromatid breaks and fragments in mouse bone marrow cells while independent exposure of similar doses failed to induce any signifi-
L.K.S. Chauhan et al. / Mutation Research 587 (2005) 120–125
cant effect [1]. Similarly, corn seeds pre-soaked in the aqueous concentrations of alachlor or bifenox and the extracts from treated seeds when tested in Salmonella assay showed no mutagenicity, whereas combined treatment of seed extracts found to be mutagenic in the same system [2]. A study carried out on human lymphocytes following exposure of atrazine (0.01 g/ml) or linuron (0.001 g/ml) showed modest frequency of CA, whereas lymphocytes jointly exposed to half of the concentrations of these chemicals induced significant frequency of CA [3]. In fact, it is the reason that prompts us to evaluate the genotoxicity of commercially formulated mixtures of insecticides that can ensure the safe and effective mixtures. Organophosphates are generally used in combination of synthetic pyrethroids. Evidence already exists that mixtures of these insecticides increase the neurotoxicity [4]. A commercially formulated mixture of a synthetic pyrethroid insecticide, cypermethrin, (3%, w/v) and an organophosphate insecticide, quinalphos, (20%, w/v) is adequately used on various agricultural crops. Independent exposure of cypermethrin has been reported to induce chromosomal aberrations, micronucleus formation and SCE in mice and human lymphocytes [5–9] and chromosomal as well as mitotic aberrations in the root meristem cells of Allium cepa [10]. Quinalphos too has been demonstrated to induce chromosome breaks/fragments in mice and human lymphocytes [11]. Since independent exposure of CYP and QUI cause genotoxic effects in mammals, it is important to study the combined effects of these chemicals particularly of commercial formulations of which industrial workers and users are more prone to occupational exposure and consequent risk. In a step further, in the present study, we have examined combined and independent cytogenetic effects of commercially formulated cypermethrin and quinalphos on the mouse bone marrow cells and discussed the possibilities of synergism. 2. Materials and methods 2.1. Chemicals Commercially formulated mixture of CYP and QUI (containing CYP 3% (w/v) and QUI 20% (w/v)), CYP (Cyperkill 25% EC) and QUI (Ekalux 25% EC) were procured from the local market. Stains and fixatives used in this study were bought from BDH, England or Glindia, India. 2.2. Test animals Healthy adult male mice (6–8 weeks, 20 ± 1 g wt.) bred and raised in animal facility of Industrial Toxicology Research Centre Lucknow were used in this study. Before treatment, mice
121
were held for acclimation for 4 days housed in polycarbonate boxes with steel wire tops and bedded with paddy husk. During the experiment, mice were maintained at controlled temperature 22 ± 2 ◦ C, 12 h-dark/light periods and 50–70% humidity with free access to pellet feed (Dayal Industries, Barabanki, India) and fresh water ad libitum. 2.3. Treatment schedule CYP/QUI mixture was diluted with peanut oil to administer a constant volume of 0.2 ml to each mouse. The acute oral LD50 (112 mg/kg) was determined according to Trimmed Spearman–Karber method [12] and mice (five per group) were orally gavaged to single dose of 22, 44 or 67 mg/kg of CYP/QUI mixture corresponding to 20, 40 or 60% of LD50 (112 mg/kg) dose, respectively. To analyze the independent effects, mice were orally gavaged to 0.66, 1.32 and 2 mg/kg of CYP or 4.4, 8.8 and 13.4 mg/kg of QUI. A group of five mice was identically treated to peanut oil 0.2 ml/mice designated as vehicle control or injected to cyclophosphamide (20 or 50 mg/kg, single i.p.) for positive control. For CA, mice were exposed for 24 h, whereas for MT, exposure period was 48 h. 2.4. Chromosome aberration assay CA assay was carried out as described earlier [13]. In brief, mice were injected to aqueous solution of colchicine (2.5 mg/kg) 2 h prior to scheduled killing by cervical dislocation and the bone marrow was flushed out from both the femurs in HBSS (pH 7.2). The cells were centrifuged at 1000 rpm for 5 min and pellet was dispersed in 0.56% KCl to incubate for 30 min at 37 ◦ C. Cells were recentrifuged and dispersed in the fresh and chilled Carnoy’s for fixation. Next day cells were dropped over clean chilled slides, air dried to stain with Giemsa and mounted. 2.5. Micronucleus test Bone marrow sampling, preparations of slide and scoring of MNPCE were done as described before [13]. In brief, bone marrow was gently flushed out in a self-designed medium containing HBSS + BSA (1%) + EDTA (0.15%), pH 7 [14], centrifuged at 1000 rpm for 5 min and the medium was decanted. Pellet obtained was dispersed in 0.25 ml medium, smeared on clean slides, fixed in 70% methanol, air dried and stained with May-Grunwald/Giemsa protocol. 2.6. Statistical analysis The mitotic index was determined by scoring metaphase cells out of approximately 3000 nuclei/mouse (1000/slide). CA were scored from well spread 80–100 metaphase cells/mouse of each group. Cells with chromatid gaps were also scored but not included in percent aberrations or aberrant cells. For MT, a minimum of 2000 polychromatic erythrocytes/mouse were also scored from treated or control group. Data on CA
122
Treatment groups and doses (mg/kg)
Mitotic indexa
Metaphase cells scored
Vehicle control
3.10 ± 0.7
577
4 (4)
CYP/QUI mixture 22(CYP 0.66 + QUI 4.4 mg) 44(CYP 1.32 + QUI 8.8 mg) 67(CYP 2 + QUI 13.4 mg)
3.46 ± 0.13 2.15 ± 0.18* 1.94 ± 0.26**
554 546 462
CYP 2 QUI 8.8 13.4 Cyclophosphamide 20
2.19 ± 0.42* 2.21 ± 0.19* 0.89 ± 0.09** 1.78 ± 0.19**
443 456 402 517
* ** a b c
Fragments
Exchange/cells with multiple breaks
Chromatid gapsb
4 (3)
−/−
32 (19)
8 (7)
11 (8) 18 (15) 13 (11)
7 (6) 12 (9) 25 (14)
−/1 −/− 1/2
49 (27) 67 (41) 63 (36)
18 (15) 30 (24) 39 (27)
3.25 (2.71) 5.49 (4.39)* 8.44 (5.84)**
4 (4) 11 (8) 13 (11) 35 (26)
3 (2) 8 (6) 10 (8) 27 (21)
−/− −/− 2/1 2/6
10 (7) 13 (9) 25 (11) 64 (38)
7 (6) 19 (14) 25 (20) 63 (53)
1.58 (1.35) 4.16 (3.07)* 6.22 (4.79)** 12.37 (10.25)**
P < 0.01. P < 0.001. Expressed as group mean ± S.E. (ANOVA). Chromatid gaps excluded from total aberrations and aberrant cells. Significance determined by Chi-square test. Parentheses represent number of cells.
Chromatid breaks
No. of Aberrations (aberrant cells)c
Aberrations (%) (aberrant cells)c 1.39 (1.21)
L.K.S. Chauhan et al. / Mutation Research 587 (2005) 120–125
Table 1 Chromosomal aberrations in the mouse bone marrow cells following 24 h oral exposure of commercially formulated cypermethrin and/or quinalphos
L.K.S. Chauhan et al. / Mutation Research 587 (2005) 120–125
were expressed in percent and prevalence of significance was determined by Chi-square test, whereas data on MI and MT were analyzed by one way analysis of variance (ANOVA). In all the treatment groups, p-values less than 0.05 were considered significant in comparison of vehicle control [15].
3. Results The acute oral LD50 dose of the commercially formulated mixture of CYP/QUI was determined to be 112 mg/kg in mice. Exposure of CYP/QUI mixture (67 mg/kg) or QUI (13.4 mg/kg) showed overt symptoms of toxicity like tremors and diarrhea after 24 h exposure. Table 1 summarizes the results of combined and independent exposure of CYP and QUI on the MI and CA observed at 24 h in mice bone marrow cells. Exposure of single dose of CYP/QUI mixture inhibited the MI and induced CA dose-dependently. However, as compared to vehicle control, significant (p < 0.001) inhibition of MI was observed at 67 mg/kg and induction of CA at 44 mg/kg onwards. Independent exposure of CYP (2 mg/kg) showed significant (P < 0.01) inhibition of MI but did not show CA. Results obtained with other doses of CYP (0.66 and 1.32 mg/kg; data not presented) were comparable to that of vehicle control. Treatment of QUI (8.8 and 13.4 mg/kg), however, significantly (p < 0.001) inhibited the MI and induced CA. The lowest dose of QUI (4.4 mg/kg; data not shown) was found to be statistically insignificant. Fragments and chromatid breaks were found to be frequent aberrations in all the treatment groups. A number of cells with chromatid gaps were also observed in all the treatment groups. The results of micronucleus test observed in mice following combined or independent oral exposure of CYP and QUI are summarized in Table 2. The test doses (22–67 mg/kg) of CYP/QUI mixture induced micronu-
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cleus formation dose-dependently. Though, significant frequencies of MNPCE were observed at 44 mg/kg onwards. The percentage of MNPCE observed in the mice bone marrow exposed to CYP (0.66–2 mg/kg) alone was comparable to vehicle control, whereas QUI (8.8 and 13.4 mg/kg) alone induced significant (p < 0.001) frequencies of MNPCE. Data obtained with 0.66 and 1.32 of CYP or 4.4 mg/kg of QUI were insignificant, and thus not presented. 4. Discussion Marked increase in the frequencies of CA and MNPCE over the vehicle control and independent exposures of CYP or QUI indicates the genotoxic potential of CYP/QUI mixture, which may be the result of synergistic interactions between the two insecticides, however, mild contribution of adjuvant (solvents, potentiators and emulsifiers) cannot be ruled out. Earlier studies have reported induction of CA, SCE and micronucleus formation in mouse bone marrow and human lymphocytes with the independent treatment of CYP [5–9]. Bhunya and Pati [6] observed significant frequencies of CA and micronucleated cells at 30–50 mg/kg dose levels in mice exposed through various routes (p.o., i.p. and s.c.) to CYP. Data obtained with CYP (0.66–2 mg/kg) in the present study imply that CYP alone at these doses is not causative of genotoxicity. Rupa et al. [11] have demonstrated significant induction of CA and micronucleus formation in mouse bone marrow exposed to 5–20 mg/kg of QUI (technical grade); however, as compared to control group, the significance was observed at 10 mg/kg onwards. In the present study also QUI (>8.8 mg/kg) found to be capable of inducing significant chromosome damage. The possibility of synergistic interactions between CYP and QUI is quite possible as
Table 2 Micronucleated cells in mouse bone marrow following 48 h oral exposure of commercially formulated mixture of cypermethrin and/or quinalphos Treatment groups and doses (mg/kg)
No. of PCE
Vehicle control
11633
7
CYP/QUI mixture 22 (CYP 0.66 + QUI 4.4 mg) 44 (CYP 1.32 + QUI 8.8 mg) 67 (CYP 2 + QUI 13.4 mg)
10998 10995 11106
CYP 2 QUI 8.8a 13.4 Cyclophosphamide 50
11710 8187 13112 12219
* a
P < 0.001 significance determined by ANOVA. Data from four mice.
No. of Micronuleated PCE
Micronucleated PCE (%)
PCE/NCE ratio
0.06 ± 0.01
1.09
19 61 109
0.17 ± 0.07 0.55 ± 0.04* 0.98 ± 0.03*
1.11 0.95 0.95
11 26 51 106
0.09 ± 0.02 0.31 ± 0.03* 0.39 ± 0.35* 0.86 (±0.11)*
1.19 1.14 0.89 1.10
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L.K.S. Chauhan et al. / Mutation Research 587 (2005) 120–125
the frequencies of CA or MNPCE with the treatment of CYP/QUI mixture were relatively higher than that of the independent treatment of CYP or QUI. In general, QUI is a neurotoxic insecticide, inhibits acetylcholine esterases as a primary mechanism of action; however, QUI exposure also generates reactive oxygen species in mice, which may be the cause of genetic damage [16]. A study on the potentiation of insecticides demonstrated that esterase inhibitors, particularly organophosphates, delay the metabolism of synthetic pyrethroid insecticide, deltamethrin, by affecting the ester cleavage [17]. The chemical structure of deltamethrin is quite similar to CYP and both undergo similar metabolic pathways in mammals [18]. Further, the primary cause of pyrethroid resistance in different organisms is associated with the overproduction of esterase isoenzymes, which metabolize these compounds through ester cleavage [19]. The organophosphates bind with these enzymes and thus prevent detoxification of synthetic pyrethroid insecticides [19]. It is possible that QUI, an esterase inhibitor, slowed down the metabolism of CYP, which in its parental form induced additional genotoxicity even at low dose levels. Paraxon, a non-toxic metabolite of an organophosphate insecticide parathion, has been demonstrated to synergies mutagenicity when incubated with plant-activated m-phenylenediamine or with direct acting 2-acetoxyacetylaminofluorene resulting 10-fold increase in mutant yield of Salmonella typhimurium [20]. Similar mutagenic synergy also noted in S. typhimurium when paraxon was incubated with mammalian activated m-phenylenediamine [21]. Synergistic interactions between chlorpyrefos and pyrethrum have also been shown to inhibit neurite growth [4]. Further, if LD50 doses are taken into account, it is evident that the mixture increases the toxicity. For example, in this study, LD50 of formulated CYP/QUI mixture was determined to be 112 mg/kg containing 3.36 mg of CYP and 22.4 mg of QUI as active ingredients, whereas the LD50 doses of non-formulated CYP and QUI in mouse have been reported to be 138 and 62 mg/kg, respectively [22]. Evidence also exists that the interactions of organophosphate and formulating pesticidal components synergies the neurotoxicity [4]. The synergism between the insecticide mixture and adjuvant used as emulsifiers, stabilizers and solvents therefore cannot be ruled out; however, it cannot be ascertained too unless each product of formulation is tested separately with the individual insecticide or insecticide mixture. As chromosome aberrations and micronucleus formation are adjunct cytogenetic indicators of genotoxicity, it can be concluded that the co-existence of CYP and QUI can synergies the genotoxicity in mice. Though humans
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