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Drug and Chemical Toxicology
ISSN: 0148-0545 (Print) 1525-6014 (Online) Journal homepage: https://www.tandfonline.com/loi/idct20
Sex-related effects of imidacloprid modulated by
piperonyl butoxide and menadione in rats. Part II:
genotoxic and cytotoxic potential
Mehmet Arslan, Yusuf Sevgiler, Mehmet Buyukleyla, Mustafa Yardimci,
Mehmet Yilmaz & Eyyup Rencuzogullari
To cite this article: Mehmet Arslan, Yusuf Sevgiler, Mehmet Buyukleyla, Mustafa Yardimci, Mehmet Yilmaz & Eyyup Rencuzogullari (2016) Sex-related effects of imidacloprid modulated by piperonyl butoxide and menadione in rats. Part II: genotoxic and cytotoxic potential, Drug and Chemical Toxicology, 39:1, 81-86, DOI: 10.3109/01480545.2015.1029049
To link to this article: https://doi.org/10.3109/01480545.2015.1029049
Published online: 31 Mar 2015.
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ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, 2016; 39(1): 81–86
!2015 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2015.1029049
RESEARCH A RTICL E
Sex-related effects of imidacloprid modulated by piperonyl butoxide
and menadione in rats. Part II: genotoxic and cytotoxic potential
Mehmet Arslan1, Yusuf Sevgiler2, Mehmet Buyukleyla3, Mustafa Yardimci4, Mehmet Yilmaz3, and Eyyup Rencuzogullari2
1Department of Nursing, School of Health Sciences, Ardahan University, Ardahan, Turkey,2Department of Biology, Faculty of Science and Letters,
Adiyaman University, Adiyaman, Turkey,3Department of Biology, Institute of Natural and Applied Sciences, Cukurova University, Balcali, Adana, Turkey, and4Department of Biology, Institute of Natural and Applied Sciences, Adiyaman University, Adiyaman, Turkey
Abstract
Despite its intended use, imidacloprid causes genotoxic and cytotoxic effects in mammals, especially in the presence of metabolic activation systems. The aim of this study was to determine to which extent these effects are sex related and how its metabolism modulators piperonyl butoxide and menadione affect its toxicity. Male and female Sprague-Dawley rats were injected with the intraperitoneal LD50 dose of imidacloprid alone (170 mg/kg) or
pretreated with piperonyl butoxide (100 mg/kg) and menadione (25 mg/kg) for 12 and 24 h. Structural chromosome aberrations, abnormal cells and mitotic index were determined microscopically in bone marrow cells. Male rats showed susceptibility to the genotoxic effects of imidacloprid. Piperonyl butoxide was effective in countering this effect only at 24 h, whereas menadione exacerbated imidacloprid-induced genotoxicity. Piperonyl butoxide and mena-dione pretreatments increased the percentage of structural chromosome aberrations and abnormal cells in females. Imidacloprid decreased the mitotic index, whereas pretreatment with piperonyl butoxide and menadione showed improvement in both sexes. We believe that CYP450-mediated metabolism of imidacloprid is under the hormonal control and therefore that its genotoxicity is sex related. Piperonyl butoxide pretreatment also showed sex-related modulation. The hormonal effects on imidacloprid biotransformation require further investigation.
Keywords
Cytotoxicity, genotoxicity, imidacloprid, menadione, piperonyl butoxide, rats, sex, structural chromosomal aberrations History
Received 8 July 2014 Revised 10 January 2015 Accepted 10 March 2015 Published online 31 March 2015
Introduction
Imidacloprid (IMI; IUPAC name: N-{1-[(6-Chloro-3-pyridyl)methyl]-4,5-dihydroimidazol-2-yl}nitramide; CAS no: 138261-41-3) is a systemic neonicotinoid nitroguanidine insect neurotoxin, first registered in 1994 for the use on food crops, ornamentals, turf, seed treatments, domestic pets and structural pests (EPA, 2008).
Neonicotinoids generally have low toxicity to mammals because of the low affinity to mammalian nicotinic acetyl-choline receptors (nAChRs) compared with insect neuron receptors (Tomizawa & Casida, 2005). However, nitroguani-dine and aminoguaninitroguani-dine derivatives are considered bioacti-vation products for mammals (Chao & Casida, 1997; Honda et al., 2006; Tomizawa & Casida, 2000). Mammalian CYP450s, (CYP3A4 in particular) and aldehyde oxidase
(AOX; EC 1.2.3.1) are two important enzymes in IMI metabolism (Costa et al., 2009). CYP450s are responsible for the conversion of IMI to 5-hydroxy, olefin, nitrosoimine, guanidine and urea derivatives (Schulz-Jander & Casida, 2002), whereas AOX converts IMI to nitrosoguanidine and aminoguanidine metabolites (Dick et al., 2005).
In carcinogenic and reproductive toxicology, the genotoxic potential of chemicals is a primary risk factor for long-term effects. The majority of pesticides have been tested in a wide variety of mutagenicity assays covering gene mutation, chromosomal alteration and DNA damage (see Bolognesi (2003) for detailed review). Several studies have reported the genotoxic and cytotoxic effects of IMI in vitro and in vivo (Demsia et al., 2007; Feng et al., 2005; Shah et al., 1997; Zang et al., 2000).
In in vitro studies, biotransformation of IMI by the S9 system produced higher micronucleus (MN) frequency and comet score in human peripheral blood leukocytes (Costa et al., 2009) and the number of revertants in TA98 and TA100 Salmonella strains (Karabay & Oguz, 2005). In vivo, piperonyl butoxide (PBO), CYP450 and esterase inhibitor, showed synergistic effects with IMI in insects (Tomizawa & Casida, 2003).
Address for correspondence: Yusuf Sevgiler, Department of Biology, Faculty of Science and Letters, Adiyaman University, 02040 Adiyaman, Turkey. Tel: +90 416 2233800. Fax: +90 416 2231774. E-mail: [email protected]
All these reports raise the question whether the IMI metabolism also increases its genotoxicity in mammals in vivo. To find this out, we used specific metabolism modulators such as PBO and menadione (MEN). PBO (IUPAC name: 5-[2-(2-butoxyethoxy) ethoxymethyl]-6-propyl-1,3-benzodioxole; CAS no: 51-03-6) is a methylene-dioxyphenyl compound that is used to inhibit CYP450s and esterases in order to enhance the potency of certain pesticides (Bingham et al., 2011; Sivori et al., 1997). MEN (IUPAC name: 2-methylnaphthalene-1,4-dione; CAS no: 58-27-5) is a specific inhibitor of AOX (Cui et al., 2005).
In our earlier study with PBO and MEN modulators (Yardimci et al., 2014), IMI showed sex, tissue and duration-dependent pro-oxidative and neurotoxic effects in the liver and kidney of Sprague-Dawley rats. PBO and MEN pretreat-ment exacerbated the pro-oxidative effects of IMI in the liver of male rats; but they also counteracted IMI’s oxidative effects in the kidney. The aim of this study was to investigate how IMI metabolism affects its genotoxic and cytotoxic potential in vivo by determining structural chromosome aberration (SCA) and abnormal cells (AC) and the mean of mitotic index (MI). We also investigated whether these effects are sex related.
Materials and methods
Chemicals
Analytical grade imidacloprid (purity 498%) was purchased from Supelco (Sigma-Aldrich Chemie GmbH, Schnelldorf, Bavaria, Germany). Pure crystalline MEN, urethane (IUPAC name: ethyl carbamate; CAS no: 51-79-6, purity 99) and colchicine (IUPAC name: N-[(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide; CAS no: 64-86-8; purity95) were purchased from Sigma (Sigma-Aldrich Chemie GmbH). PBO was technical grade (90%) and supplied from Aldrich (Sigma-Aldrich Chemie GmbH). Dimethyl sulfoxide (DMSO), NaCl, KCl, acetic acid, metha-nol, Giemsa stain, KH2PO4, Na2HPO4.12H2O and Entellan
were of analytical grade. Animals and treatment
This study was approved by the ethics committee of the Cukurova University Medical Sciences, Experimental Research, and Application Center (no:11, date: July 02, 2010). Young adult, male and female rats (weighing about 200 g regardless of sex) of the Sprague-Dawley strain (Rattus norvegicus var. albinos) were supplied by the Cukurova University Medical Sciences, Experimental Research and Application Center. They were separated by sex, kept in clean plastic cages and acclimatized to laboratory conditions for 7 d. Wood shavings in cages were changed every day. Laboratory temperature was 22 ± 1C and relative humidity was 65 ± 5%. The rats were maintained under natural photo-period and fed ad libitum.
The in vivo test was performed according to Topaktas et al. (1996). Rats were not fed 24 h before and during the toxicity experiments. They were randomly divided into eight groups of two. All animals received study compounds by intraperitoneal (ip) injection. DMSO was used as the solvent for IMI, PBO and
MEN. The solvent control and the physiological saline control groups received DMSO and physiological saline, respectively, in the same volumes as the IMI group of rats. Positive controls received 400 mg/kg bw of urethane. IMI was applied at its LD50dose of 170 mg/kg (WHO, 2001), whereas for PBO and
MEN we selected non-hepatotoxic 100 mg/kg bw ip dose and non-nephrotoxic 25 mg/kg bw intravenous dose, respectively, based on the reports by Smith et al. (1988) and Chiou et al. (1997). PBO alone and MEN alone groups received ip 100 mg/ kg or 25 mg/kg, respectively. The combination groups IMI + PBO and IMI + MEN first received either PBO or MEN 90 min before receiving IMI; the doses were the same as described above for respective compounds.
No animal died during treatment. To arrest mitosis, the animals received 3 mg/kg bw of colchicine ip 2 h before bone marrow collection. They were killed by cervical dislocation after 12 h or 24 h of exposure.
The bone marrow was aspirated with a syringe from proximally stripped femur and placed into a culture tube with 2 mL of 0.9% NaCl solution (at 37C) until the suspension reached 4 mL. Culture tubes were centrifuged at 2000 rpm for 5 min. The pellet was re-suspended in hypotonic 0.4% KCl solution at 37C, and the cell suspension was maintained at 37C for 30 min to swell. Then the culture tubes were centrifuged at 1200 rpm for 10 min. The pellet was fixed in cold glacial acetic acid:methanol (1:3) for 20 min at room temperature. This suspension was centrifuged at 1200 rpm for 10 min. The fixative treatment was repeated three times. The final pellet was spread on cold glass slides and was air-dried. The preparations were stained for 15 min with 5% Giemsa stain that was prepared in Sorensen buffer. One hundred well-spread metaphases per animal in each group (totalling 200 metaphases per group) were microscoped at 1000 magni-fication for SCA and AC. In total, 3000 cells were scored to determine the MI.
Structural chromosome aberrations and AC percentages were analyzed as genotoxic endpoints of pesticide exposure, whereas MI was used to determine cytotoxic potential (Bolognesi, 2003; Galloway, 2000; Mateuca et al., 2012; Mesi & Kopliku, 2013). Chromosomal breaks, chromatid breaks, fragment formations, dicentric chromosomes, sister chromatid conjugations and chromatid conjugations were determined microscopically and counted as SCA. The cell that showed these anomalies and/or has polyploidy at its metaphase was considered AC. MI is defined as the propor-tion of cells undergoing mitosis (cell division) compared with the total number of cells (Moore et al., 2011).
Statistical analyses
Differences between the groups were determined with the t-test using the MINITAB version 14 statistical software (Minitab Inc., PA). Statistical significance was set at p50.05.
Results and discussion
Our study is the first to show sex-specific genotoxicity of IMI in vivo and the modulatory effects of PBO and MEN in the bone marrow cells of Sprague-Dawley rats. As expected, positive control rats treated with urethane showed higher SCA and AC and lower MI.
Sex-specific genotoxic and cytotoxic potential of IMI Imidacloprid decreased the percentage of SCA and AC after 12 h of exposure compared with negative control and DMSO groups, while these parameters increased after 24 h of exposure only in male rats (Tables 1 and 2). This points to a faster IMI metabolism in males than in females. European Medicines Agency (EMA, 2009) has reported that IMI is metabolized more effectively in male rats than in females, probably due to sex-related hormonal effects of the two important IMI metabolizing enzymes: CYP3A4 and AOX (Sakuma et al., 2009; Ventura & Dachtler, 1981). We believe that IMI metabolites might directly bind to the DNA to create genotoxic action. In other words, our finding of increased AC and SCA after 24 h of exposure could be related to the accumulation of IMI metabolites. This is only an assumption, as we did not determine the metabolite levels in our study. Shah et al. (1997) reported increased DNA-pesticide adduct formation in the thymus of calves treated with a commercial IMI formulation that was metabolized by the rat liver
S9 fraction. The S9 fraction contains CYP450 isozymes, AOX and other biotransformation enzymes (Costa et al., 2009; Morrison et al., 2012). It is therefore important to know which IMI metabolite is able to react directly with DNA, and further studies should look into that direction.
In our study, a transient decrease in MI in female rats was observed after 12 h of exposure compared with negative control and DMSO groups, whereas in male rats the decrease was significant in respect to the negative control group at both durations (Table 3). Lower MI has also been reported in female Mus musculus treated orally with IMI for 24 h (Malik, 2013), and our study has confirmed the cytotoxic effect of IMI in mammals of both sexes found in other studies (Bal et al., 2012; Kapoor et al., 2011).
Effects of PBO on the genotoxic and cytotoxic potential of IMI
Piperonyl butoxide increased SCA and AC only in female rats after 12 h of exposure compared with the negative and DMSO
Table 1. The percentage of structural chromosome aberrations in the bone marrow cells of ip-treated rats with IMI, PBO and MEN.
Structural chromosome aberrations
Male Female
12 h 24 h 12 h 24 h
Control 2.50 ± 0.50 2.50 ± 0.50 2.00 ± 0.00 2.00 ± 0.00 DMSO 2.50 ± 0.50 3.00 ± 0.00 2.50 ± 1.50 2.50 ± 0.50 Urethane 7.00 ± 1.00 9.50 ± 0.50 7.00 ± 2.00 12.00 ± 1.00 IMI 1.00 ± 0.00a,b,c,d 5.00 ± 0.00a,b,c,d 3.00 ± 1.00 7.00 ± 1.00 PBO 1.50 ± 0.50 2.00 ± 1.00 3.00 ± 0.00a,b,d 2.50 ± 0.50 MEN 2.00 ± 0.00a,b,c 4.50 ± 0.50 4.00 ± 3.00 4.00 ± 0.00a,b IMI + PBO 3.50 ± 0.50 4.00 ± 0.00a,b 4.00 ± 1.00a 2.50 ± 0.50a IMI + MEN 2.50 ± 1.50 8.00 ± 1.00b 4.50 ± 1.50a 4.00 ± 3.00a
Data were given as mean ± standard error of mean. In total, 200 cells were scored for each group except IMI and IMI + PBO groups of male rats at 24 h because of excessive toxicity. About 100 cells were scored in these groups.
aSignificant from control group (p50.05).
bSignificant from solvent control group (DMSO) (p50.05). cSignificant from IMI + MEN group (p50.05).
dSignificant from IMI + PBO group (p50.05).
Table 2. The percentage of abnormal cells in the bone marrow of ip-treated rats with IMI, PBO, and MEN. Abnormal cells Male Female 12 h 24 h 12 h 24 h Control 2.50 ± 0.50 2.50 ± 0.50 2.00 ± 0.00 2.00 ± 0.00 DMSO 2.50 ± 0.50 3.00 ± 0.00 2.50 ± 1.50 2.50 ± 0.50 Urethane 7.00 ± 1.00 8.50 ± 0.50 6.00 ± 1.00 10.00 ± 0.50 IMI 1.00 ± 0.00a,b,c,d 5.00 ± 0.00a,b,c,d 3.00 ± 1.00 5.50 ± 0.50 PBO 1.50 ± 0.50 2.00 ± 1.00 3.00 ± 0.00a,b,d 2.50 ± 0.50 MEN 2.00 ± 0.00a,b,c 4.50 ± 0.50 3.00 ± 3.00 4.00 ± 0.00a,b
IMI + PBO 3.50 ± 0.50 4.00 ± 0.00a,b 4.00 ± 1.00a 2.50 ± 0.50a IMI + MEN 2.50 ± 1.50 8.00 ± 1.00b 4.00 ± 1.00a 4.00 ± 3.00a Data were given as mean ± standard error of mean. In total, 200 cells were scored for each group except IMI and
IMI + PBO groups of male rats at 24 h because of excessive toxicity. About 100 cells were scored in these groups.
aSignificant from control group (p50.05).
bSignificant from solvent control group (DMSO) (p50.05). cSignificant from IMI + MEN group (p50.05).
control (Tables 1 and 2). However, Butler et al. (1996) reported that PBO was not genotoxic in many test systems, including rat liver primary cell culture. In our previous study (Yardimci et al., 2014), PBO alone had no effect on oxidative stress parameters in male rats, whereas total glutathione content decreased in the liver of female rats after 12 h of exposure. Based on a study on F344/N Slc rat liver, Muguruma et al. (2007) suggest that PBO can damage DNA via oxidative stress in males. Therefore, the genotoxic effects of PBO in our study could be related to oxidative stress. However, further molecular studies should be con-ducted to confirm this hypothesis.
In our study, the 90-min pretreatment with PBO exacer-bated the IMI-induced genotoxic effects after 12 h of exposure in male rats (Tables 1 and 2). It lowered SCA and AC frequency compared with the IMI alone group, but SCA and AC remained higher than in negative and DMSO controls after 24 h of exposure. This points out the contribution of CYP450-mediated metabolites in IMI-induced genotoxicity. Vega et al. (2009) found inhibited genotoxicity of diethylthio-phosphate and diethyldithiodiethylthio-phosphate by sulconazole, a CYP450 inhibitor in WRL68 and HepG2 cell lines. The authors have suggested that the genotoxicity of these compounds depends on the activity of CYP450, probably CYP2D6 and CYP3A4. Similarly, methapyrilene-induced DNA damage in Han Wistar rat hepatocytes was suppressed by the presence of the CYP450 inhibitor aminobenzotriazole (Priestley et al., 2011).
Piperonyl butoxide pretreatment increased SCA and AC in IMI-treated female rats at both durations compared with negative control (Tables 1 and 2). This increase was also higher compared with the PBO alone group after 12 h of exposure. Yamazoe et al. (1987) reported that phenobarbital-inducible CYP450 isoform content was decreased by the growth hormone which male rats release episodically, whereas the secretion is fairly constant in the female rats. They also found that the induction of CYP450s by phenobar-bital was much higher in the liver of male Sprague-Dawley rats compared with females. We believe that as an inhibitor PBO affects male and female enzymes at different rates and
that PBO pretreatment therefore affected IMI-genotoxicity in a sex-specific manner.
Piperonyl butoxide pre-treatment countered the decreasing MI effects of IMI after 12 h of exposure in both sexes (Table 3), but these beneficial effects weakened after 24 h of exposure. This points to CYP450-mediated metabolism of IMI. The case in point is the study by Adams et al. (1993), who have found that CYP450-mediated biotransformation is inhibited by PBO metabolite–CYP450 complex formation, but that CYP450 activity subsequently increases via protein synthesis.
Effects of MEN on the genotoxic and cytotoxic potential of IMI
Menadione alone lowered the SCA and AC percentage after 12 h of exposure in male rats, whereas it increased these parameters in females after 24 h of exposure compared with the negative and DMSO control (Tables 1 and 2). Similarly, Seager et al. (2012) reported that MEN induced chromosomal damage in human lymphoblastoid cells. Cojocel et al. (2006) found that MEN leads to the formation of reactive oxygen species that caused DNA damage in mammalian A-549 cells. Furthermore, in a study by Nutter et al. (1992), MEN-induced DNA damage was attenuated by catalase, an antioxidant enzyme. This suggests that the observed changes in SCA and AC were due to the genotoxic nature of MEN.
Always higher or similar SCA and AC values compared with IMI and MEN alone groups were observed in IMI + MEN group in males at both durations but not in females at 24 h (Tables 1 and 2). The highest SCA and AC at 24 h were observed in IMI-treated female rats, but the difference was not significant, whereas MEN pretreatment increased these parameters compared with negative control. The most pronounced effect of MEN pretreatment occurred after 24 h of exposure in male rats. We believe that MEN as an inhibitor of AOX increased the substrate supply for CYP450 and that the increased genotoxicity is therefore due to CYP450-mediated IMI metabolism. Sidorova and Grishanova (2004) also reported that MEN elevated a variety
Table 3. The percentage of mitotic index in the bone marrow of ip-treated rats with IMI, PBO and MEN. Mitotic index Male Female 12 h 24 h 12 h 24 h Control 2.61 ± 0.08 2.61 ± 0.08 2.31 ± 0.21 2.31 ± 0.21 DMSO 1.91 ± 0.01 1.56 ± 0.36 2.10 ± 0.10 1.50 ± 0.10 Urethane 1.49 ± 0.06 1.23 ± 0.43 1.31 ± 0.02 0.87 ± 0.18 IMI 1.67 ± 0.02a,c,d 1.62 ± 0.00a,c 1.47 ± 0.03a,b,c,d 1.81 ± 0.28
PBO 3.21 ± 0.23 2.55 ± 0.22 2.98 ± 0.12 2.64 ± 0.51 MEN 1.76 ± 0.36 2.49 ± 0.01b,c 2.20 ± 0.10 2.26 ± 0.06 IMI + PBO 2.38 ± 0.05b 1.66 ± 0.00a,b 2.95 ± 0.35 1.43 ± 0.20 IMI + MEN 2.61 ± 0.15b 2.00 ± 0.14 2.49 ± 0.09 2.21 ± 0.15 Data were given as mean ± standard error of mean. In total, 200 cells were scored for each group except IMI and
IMI + PBO groups of male rats at 24 h because of excessive toxicity. About 100 cells were scored in these groups.
aSignificant from control group (p50.05).
bSignificant from solvent control group (DMSO) (p50.05). cSignificant from IMI + MEN group (p50.05).
dSignificant from IMI + PBO group (p50.05).
of CYP450 enzyme activities, including CYP1A2, which catalyzed the nitroimine reduction of IMI in the liver of male Wistar rats. However, in vivo metabolite formation needs further investigation in the same conditions as in our study.
Menadione increased MI after 24 h compared with the solvent control. It also increased in the IMI + MEN-treated male rats after the 12-h exposure. The induction of mitotic activity by MEN was also reported in human HeLa cell cultures by Acharya et al. (2009). In contrast to the observed genotoxic effects, MEN pretreatment attenuated IMI-induced cytotoxic effects in male rats compared with negative control and in female rats compared with negative and solvent control groups at both durations (Table 3). The increase in MI could be related to MEN’s ability to increase mitotic activity. Moreover, MEN pre-treatment decreased lipid peroxidation levels in the liver of female rats after 12 h, whereas the decreased protein content by MEN alone was improved by IMI + MEN exposure, as it elevated the total glutathione content in the liver of male rats after 24 h of exposure (Yardimci et al., 2014). MEN was used in our study to inhibit AOX activity, so that we can see whether AOX-mediated IMI metabolism contributes the IMI-induced cytotoxicity. Our findings confirmed as we suspected. Similarly, Yoshihara and Tatsumi (1997) found that AOX purified from male and female Std:ddY mice liver showed similar inhibitory potential against MEN and b-estradiol. However, CYP450 induction by MEN cannot be excluded, as found by Sidorova and Grishanova (2004). This requires further investigation into CYP450 and AOX activities and the formation of corres-ponding metabolites to make a sound conclusion.
Conclusion
Imidacloprid alone and in combination with its metabolic modulator PBO showed sex-specific genotoxic and cytotoxic effects. This specificity is most likely due to different IMI biotransformation between the sexes that could be under hormonal control. Male Sprague-Dawley rats were more prone to IMI genotoxicity. We believe that CYP450-mediated metabolites of IMI have much greater genotoxic potential than the parent compound. However, further investigation should reveal which exact metabolites that bind to DNA are formed by CYP450 and AOX mediation using the same IMI, PBO and MEN doses.
Acknowledgements
We wish to thank Dado Cakalo for having edited the manuscript to read better.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. We wish to thank the Adiyaman University Scientific Research Commission for supporting this study (project grant no. FEFBAP2011/007).
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