Transplacental genotoxicity evaluation of cypermethrin using alkaline comet assay

Abstract

Transplacental genotoxic effect of cypermethrin technical was investigated. Three doses (25, 50 and 75 mg/kg body weight) were administered to groups of pregnant Wistar rats during 6–15 days of gestation. Animals were killed on gestation day 20. Fetal blood and liver samples were evaluated for DNA damage using alkaline comet assay. A marginal increase in the mean percentage of DNA damage was recorded in both blood and liver samples of fetuses from cypermethrin-treated dams, but the values were not statistically significant. No skeletal or visceral fetal abnormalities were recorded in treated groups. Nevertheless, the results lead to an understanding that transplacental exposure to cypermethrin can induce low levels of DNA damage in fetuses. This observation could be an explanation for the teratogenic effect exhibited by this chemical in many other studies. The results indicate that cypermethrin may be transplacentally genotoxic. The authors propose more detailed investigations for validating the current findings.

Keywords

cypermethrin transplacental genotoxicity alkaline comet assay

Introduction

Pesticides are well known to have the potential to affect human health and reproduction through direct and indirect mechanisms.¹ Fetuses and infants are extremely susceptible to the health effects caused by pesticides. The National Research Council recorded spontaneous abortions, growth retardation, structural birth defects and functional deficits as some of the developmental toxic effects of pesticides.² Further studies have acknowledged that prolonged exposure to pesticides and other pollutants may lead to various cancers, chronic neurologic syndrome, immunosuppression, malignancy, teratogenicity, abortions and reproductive failure.³ Such exposures, especially during embryogenesis, pose higher risk than similar exposure during adult life. In view of the growing concern over potential adverse health effects through transplacental exposure, various international agencies like World Health Organization (WHO) and Environmental protection Agency (EPA) have emphasized investigations on such chemicals since heritable damages in developing fetus can provoke neoplastic lesion and other diseases in postnatal life.⁴

Cypermethrin, a pyrethroid insecticide, has been extensively used in the last two decades in many of the developing countries for combating many pests and insects of agricultural, animal and human concern. Toxicity of this chemical insecticide to mammals has received attention owing to its effects on the physiological activities of the exposed population.⁵ Literature search revealed that this chemical has controversial reports on genotoxicity; however, the availability of a number of positive reports support the genotoxic potential of cypermethrin. Neonatal rats are reported to be relatively more sensitive to cypermethrin. Developmental delays and other adverse effects were recorded in pups transplacentally or perinatally exposed to cypermethrin,^6–9 suggesting transfer of cypermethrin and/or its metabolites from the mother to the fetus. From an extensive literature survey, it is seen that there is no human data on the potential of cypermethrin to permeate the placental barrier, although there is limited animal data showing this possibility.¹⁰ This led us to speculate whether genotoxic changes are evident in the fetus exposed to cypermethrin in utero.

Transplacental genotoxicity has been demonstrated for some chemicals using various cytogenetic end points such as chromosomal aberrations, micronuclei and sister chromatid exchange.^11–19 Recently, the alkaline comet assay has been applied successfully for the detection of transplacental genotoxicants in mice.²⁰ It has been recommended for investigating the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. In the present study, the alkaline comet assay was employed to evaluate the transplacental genotoxic potential of cypermethrin. This assay was used owing to its relative ease of application and versatility to detect diverse classes of DNA damage.

Materials and methods

Animals

The present study was approved by the Institutional Animal Ethics Committee of International Institute of Biotechnology and Toxicology (IIBAT). Experiments were performed on female Crl: Wistar rats procured from the animal house facility of the IIBAT. The animals were maintained in standard animal house conditions (temperature 22 ± 2°C, Humidity 30–70%, 12-h:12-h dark/light cycle). The γ-irradiated rodent pellet feed was supplied by M/s. Tetragon Chemie Pvt. Ltd., Bangalore, India, and reverse osmosis water were provided ad libitum. Animals were acclimatized in test rooms for 5 days before mating. Pregnancy was confirmed by the presence of a copulatory plug and/or sperm in vaginal lavage and designated as Day 0 of pregnancy.

Chemicals

Technical grade cypermethrin (95%) was received as a gift from M/s. Tagros chemicals Ltd., Chennai, India. Cyclophosphamide, normal melting agarose (NMA) and low melting agarose (LMA) were obtained from Sigma & Co., USA. All other chemicals were of laboratory grade and obtained from HiMedia Laboratories, India.

Experimental design

Animals were divided into five groups of three pregnant females each. Three doses (25, 50 and 75 mg/kg body weight [b.w.] cypermethrin) were suspended in corn oil and administered to animals by oral gavage during organogenesis period, another group of females administered with corn oil alone served as the control. Cyclophosphamide (10 mg/kg b.w.) was administered to the positive control group of animals. Mortality, morbidity and signs of toxicity were observed daily before and after dosing. Animals were killed on Day 20 of pregnancy. Cord blood and fetal liver were collected during necropsy. In addition, external, skeletal (Alizarin red-stained preparations) and visceral examinations of the fetuses were performed in substantial number of fetuses from each group for evidences of toxic effects and malformations, if any.

Alkaline comet assay

Pooled (dam-wise) samples of umbilical cord blood were collected in heparinized tubes. The blood samples were taken directly for comet assay. Samples of fetal liver were homogenized in Hank’s balanced salt solution (HBSS) containing 20 mM EDTA and 10% dimethyl sulfoxide (DMSO). From this, 10 μL was used for the alkaline comet assay. The comet assay was performed under alkaline conditions as suggested by Singh et al.²¹ with minor modifications. Electrophoresis was performed for 25 minutes by applying an electric field of 25 V (0.8 V/cm). Slides were neutralized with Tris buffer. For staining, the slides were immersed in double distilled water for 30 minutes then stained with 200 μl of ethidium bromide and examined at ×400 using a fluorescence microscope (Axiophot, Carl Zeiss, Germany). The slides belonging to various groups (including positive and negative controls) were independently coded and blind scoring was performed. Comets near the edges were not scored.²² Images were analysed according to the method described by Collins et al.²³ A total of 100 comets on each slide were scored visually as belonging to 1 of 5 classes according to tail intensity and given a value of 0, 1, 2, 3, or 4 (from 0 [undamaged] to 4 [maximally damaged]). Thus, the total score for 100 comets could range from 0 (all undamaged) to 400 (all maximally damaged). The percentage of damaged cells was calculated. The ‘arbitrary units’ (AU) were used to express the extent of DNA damage and were calculated as follows:

F value = (auDNA - ssDNA) / (dsDNA - ssDNA)

Where N i = number of cells in i degree; i = degree of damage (0, 1, 2, 3, 4).

Student’s ‘t’ test was employed to assess the impact of cypermethrin on the fetal blood and liver after pre-natal exposure. The p value was set at 0.05.

Results and discussion

There was no mortality in any of the treated group. Females from all three treated groups showed various signs of toxicity (ataxia, tremors and convulsions). No malformations (external, visceral and skeletal) were observed in any of the cypermethrin-treated groups. However, few observations like bent or kinked tail, subcutaneous hemorrhages and small fetus were recorded during external examination. Visceral examination revealed dilated renal pelvis, while in skeletal examination variants like incomplete ossification of sternabre and/or ribs, rudimentary rib and dumbbell centrum were recorded. All these observations were found to be scattered among all groups including control and/or they are commonly occurring variants, hence considered to be of spontaneous, incidental and not attributed to treatment with cypermethrin.

Table 1 represents the distribution of damaged and undamaged nucleoids among the groups. Under the conditions employed, cyclophosphamide, a well-known positive chemical, induced statistically significant percentage of DNA strand breaks in both the fetal blood and liver samples, which demonstrates the technical sensitivity of the experiment.

Table 1.

Results of comet assay^a

Treatment	Dose (mg/kg b.w.)	Sample	Mean % of cells in different damage categories					% D	% I	AU
Treatment	Dose (mg/kg b.w.)	Sample	0	1	2	3	4	% D	% I	AU
Vehicle control (corn oil)	0	Liver	96.15 ± 0.59	3.27 ± 0.49	0.58 ± 0.18	0.00 ± 0.00	0.00 ± 0.00	3.85	—	4
Vehicle control (corn oil)	0	Blood	97.33 ± 1.53	1.67 ± 2.00	1.00 ± 0.58	0.00 ± 0.00	0.00 ± 0.00	3	—	4
Cyper	25	Liver	91.26 ± 0.66	6.62 ± 0.76	1.91 ± 0.25	0.11 ± 0.03	0.11 ± 0.00	8.75	4.9	11
Cyper	25	Blood	90.00 ± 3.00	2.67 ± 4.58	3.00 ± 2.52	3.00 ± 1.00	1.33 ± 0.58	10	7	23
Cyper	50	Liver	90.35 ± 2.48	5.66 ± 0.62	1.94 ± 0.93	1.26 ± 0.41	0.79 ± 0.00	9.65	5.8	16
Cyper	50	Blood	86.34 ± 2.52	2.33 ± 0. 13	5.00 ± 0.58	4.33 ± 2.08	2.00 ± 1.73	14	11	33
Cyper	75	Liver	84.91 ± 1.55	3.89 ± 2.67	4.60 ± 0.85	4.52 ± 0.67	2.08 ± 0.00	15.09	11.24	34.97
Cyper	75	Blood	92.5 ± 7.07	2.5 ± 0.54	2.3 ± 0.71	2.00 ± 1.41	1.00 ± 0.41	8	5	17
CP	10	Liver	3.00 ± 1.50	3.00 ± 2.60	19.00 ± 6.40	31.00 ± 10.40	45.00 ± 11.60	97.00^b	93.15	312^b
CP	10	Blood	5.67 ± 2.52	3.67 ± 4.72	11.70 ± 5.03	55.33 ± 7.50	23.7 ± 5.86	94.33^b	91.33	288^b

% D: percentage DNA damage, % I: percentage increase in DNA damage over control, CP: cyclophosphamide, Cyper—cypermethrin, AU: arbitrary units; b.w.: body weight.

^aThis table represents the consolidated data on the distribution of undamaged and damaged nucleoids of various degrees in different treatment groups.

^b p < 0.05—students ‘t’ test.

There is an observable increase in treatment group in the number of cells in Categories 1 and 2 in the blood samples of treated groups with respect to the control. Cells with moderate and marked DNA damage (Categories 3 and 4) were found in the treated groups only, proving that cypermethrin interacts with the fetal blood DNA to causes strand breaks. When the percentage of increase over controls is considered, it is pertinent to say that cypermethrin induces low levels of DNA damage in rat fetal blood cells, though it is not statistically significant. In the liver sample, with respect to the control group, the proportion of damaged nucleoids in the 2, 3 and 4 categories was consistently increasing with dose in the treated group. The percentage increase in DNA damage in treated animals with respect to controls also appeared to be consistently increasing with dose, indicating that cypermethrin induces strand breaks in rat liver cells. This increase was also not statistically significant.

Genotoxicity has a very important role in reproductive effects, since it can lead to other toxicological phenomena which are of considerable significance like those including oncogenesis, teratogenesis, sterility, heart diseases and aging.^24–29 DNA damage in the germ cells may lead to the production of abnormal or mutated spermatozoa which results in spontaneous abortion, malformations and/or genetic defects in the offspring.^30,31

Cypermethrin exposure has been reported to cause a transient increase in chromosomal aberrations, micronuclei in bone marrow cells of mice and rat³² and sister chromatid exchanges in the bone marrow of mice.^33–35 It affects the cell cycle causing a decrease in proliferative index in human peripheral blood lymphocytes.³⁶ It also induces systemic genotoxicity in mammals and DNA damage in vital organs and hematopoietic system.³⁷ It has been reported that cypermethrin forms DNA monoadducts and DNA interstrand cross-links in hepatocytes.³⁸ Both in vitro and in vivo experiments with rat peripheral blood lymphocytes showed that cypermethrin severely damages DNA and causes imbalance in the prooxidant and antioxidant status in lymphocytes.^39,40 Recently, genotoxic effects of cypermethrin have been reported in human peripheral lymphocytes.^41,42

There are conflicting reports with regard to the teratogenic potential of cypermethrin. According to the EPA,⁴³ there were no treatment-related developmental toxic effects in pregnant female Sprague Dawley rats administered with cypermethrin at doses 17.5, 35 or 70 mg/kg/day on gestation days 6–15. On the contrary, Khurshid⁴⁴ reported teratogenic effects in chick embryos and suggested that the effect could partially be due to the genotoxic potential of this insecticide, which might have caused some mutations and also may be attributed to some changes in the biochemical components in maternal and fetal compartments. A recent study reported that exposure of pregnant female Wistar rats at 55.1 mg/kg b.w. orally at Days 6–15 of gestation to the insecticide cypermethrin resulted in the development of a lot of external morphological deformities and visceral malformations in their offspring, which signify the potential of such insecticide to induce reproductive toxicity and teratogenesis.¹⁰

In the present study, technical grade cypermethrin was administered to pregnant rats at the doses 25, 50 and 75 mg/kg at 6–15 days of gestation. Although we did not record any obvious teratogenic effect, which is in line with the observations of the EPA,⁴³ we did record a marginal increase in the percentage of DNA damage in the blood and liver cells of the fetuses. This increase was not statistically significant, however, taking into account the positive effect of this chemical in other studies; it is pertinent to say that cypermethrin could be transplacentally genotoxic. Precise molecular mechanisms of the genotoxicity of cypermethrin are not yet elucidated and require further studies. It has been reported that due to the hydrophobic nature and small molecular size, cypermethrin passes through the cell membrane and reaches the nucleus. It is suggested that within the nucleus, cypermethrin binds to DNA through the reactive groups of its acid moiety, leading to destabilization as well as unwinding of the DNA, which could be a possible mechanism for its genotoxicity.²⁵ Intoxication with cypermethrin is reported to cause free radical-mediated tissue damage and oxidize total reduced gilutaihione (GISH) in rats,^45,46 and DNA damage caused by reactive oxygen species such as hydroxyl radicals, hydrogen peroxides and singlet oxygen has been implicated in mutagenesis, oncogenesis and aging.⁴⁷ Hence, the present observations indicate that the marginal increase in the percentage of DNA damage could be possibly due to the free radicals generated during the metabolism of this chemical or alternatively because of its hydrophobic nature. The marginal increase can be probably due to increased absorption of the administered cypermethrin by the maternal tissues and a very less concentration must have reached the fetus. These are only assumptions and need to be exemplified by further experimentation in this regard.

Efforts were made in the recent past to review newer techniques used in the detection of transplacental genotoxins. DNA strand breakage is a sensitive marker of genotoxicity, since they are potential pre-mutagenic lesions.⁴⁸ Comet assay is a well-established direct method to detect a broad spectrum of DNA damage and has been applied in the evaluation of the possible genotoxic action of various pesticides.^49–55 Current results using this assay suggest that, cypermethrin does induce strand breaks in blood and liver cells of transplacentally exposed rat pups. Relative to other genotoxicity tests, such as chromosomal aberrations, sister chromatid exchanges, alkaline elution and micronucleus assay, the advantages of the comet assay include its demonstrated sensitivity for detecting low levels of DNA damage.⁵⁶

Tripathi et al.²⁰ suggested the use of alkaline comet assay in mice for the detection of transplacental genotoxins. The present study demonstrates that rats can also be a useful model for studying transplacental genotoxicity using alkaline comet assay. Multiple organs of mouse or rat, including brain, blood, kidney, lungs liver and bone marrow, have been utilized for the comprehensive understanding of the systemic genotoxicity of chemicals.^6,57,58 Cypermethrin has been reported to cause systemic genotoxicity in mammals and DNA damage in vital organs as revealed by the comet assay.³⁷

Conclusion

In the present study, the ability of cypermethrin to be a transplacental genotoxin was evaluated using alkaline comet assay. There was no statistically significant increase in the percentage of DNA damage assessed; however, a marginal increase was observed, which cannot be neglected owing to the positive effects of this chemical in other studies mentioned previously. Hence, it is concluded that cypermethrin could be a transplacental genotoxin, nevertheless further investigations are warranted with additional genotoxicity endpoints to acknowledge this effect, since the results are preliminary with respect to the number of animals. Furthermore, if a comparison is made between the effect on maternal and fetal tissues and if a difference exists, then the precise mechanism can be delineated. We are currently investigating this subject.

Footnotes

The authors wish to thank the Management of IIBAT for financial support and encouragement.

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