genotoxicity assessment of fipronil (frontline plus®) in canis familiaris1

frontline plus
(frontline plus®) in Canis familiaris1

genotoxicity assessment of fipronil (frontline plus®) in canis familiaris1

Evaluation of Fipronil genotoxicity (frontline plus®) in dogs.] Fipronil is an insecticide/ widely used herbicide to control fleas and ticks on pets. Your short exposure has had deleterious effects on animals. However, the possible genotoxic effect of this compound is still hasn't been investigated in target animals. Based on the hypothesis that Fipronil can induce genotoxicity, this study evaluated the deleterious effect of Fipronil on the genetic material of peripheral blood cells. To this end, ten healthy dogs of both sexes were employed in this test. The product (6.7 mg/kg) was applied to the dorsal region of the neck of every animal. The blood samples were collected immediately before the application of the product (control) and after three, eight and 24 hours of application. The samples were immediately processed to conduct the test of the comet in order to assess the basal damage to the DNA. No there was a significant difference between the four moments of the collection in relation to damage to genetic material. The study suggests, for the first time, that a unique exposure to this... Insecticides do not activate the toxic gene effect in dogs.
TERMS OF INDEXATION: Fipronil, frontline plus®, pesticide, DNA damage, comet rehearsal, dogs.

Frontline plus® is a pesticide formed by the compounds fipronil (active principle) and methoprene. It is a highly efficient second generation-pesticide and widely used in veterinary products for controlling agricultural pests and ectoparasites in domestic animals, including those resistant to pyrethroid, organophosphate, and carbamate insecticides (Kidd & James 1991) Fipronil is applied specifically in dogs and cats to control fleas and ticks (Tingle et al. 2003).

The control of ectoparasites is of fundamental importance because they are vectors of various protozoa, bacteria, and viruses that cause diseases in animals and humans.

Once used, the product spreads throughout the body, and It is stored in the fat layer of skin and hair follicles Continue to release the skin and hair cover, Faisal to long-term activity Cells are likely to remain in large quantities a week after the application of the product (Hugnet et al. 1999).

Short-term exposure to fipronil can lead to serious
effects on fetal and postnatal development, such as learning disability, reflex reduction, sterility, and also increased susceptibility to many diseases, including cancer (Lyons 2000). Rats exposed to fipronil demonstrated a significant increase in thyroid cells, leading to tumor formation. In addition, this compound was capable of changing the values of thyroxine hormones (Hurley et al. 1998). Fipronil has been classified by the U.S.
Environmental Protection Agency (EPA), as a possible human carcinogen (group C) based on an increase in thyroid follicular cell tumors in rats of both sexes (EPA 1996).
Since the advent of Genetic Toxicology, it has become possible to assess the toxicity of pesticides in the genetic material. Thus, it is relevant to evaluate whether these compounds have genotoxic/mutagenic effects and their possible mechanisms. Taking into account that the genotoxic effect of fipronil remains unknown and that there is a scarcity of data about the genotoxicity of pesticides evaluated in exposed mammals, the current study was designed to evaluate the genotoxic potential of fipronil by detecting systemic DNA damage in dogs.

MATERIALS AND METHODS:
Ethics statement. The Animal Experimentation Ethics Committee of the local institution approved the study, under the protocol number 154/2014 in adherence to the Principles of the National Council for the Control of Animal Experimentation, from the Ministry of Science and Technology.
Animal preparation. Ten healthy adult crossbred dogs of both sexes (5 male and 5 female), weighing between 15kg and 20kg were used in the study. The animals were considered healthy after clinical, hematological, biochemical, serological, urinalysis and fecal routine examination. Before the beginning of the study, the animals had been vaccinated and vermifuge on a regular and conventional basis, and were housed in pairs in each kennel, where they were given commercial food and drinking water ad libitum. The animals were acclimated for a period of 30 days before the start of the experiment.

Study design. The fipronil (Frontline plus®) was applied on the dorsal neck region of each animal at a dose of 6.7mg/kg, according to the manufacturer’s recommendation for the control of
ectoparasites in dogs and cats in the routine.
Blood samples were collected by venipuncture (cephalic vein) in heparinized syringes immediately prior to the application of the product (T0 - control), then at 3 hours (T1), 8 hours (T2) and 24 hours (T3) after the application. The samples were coded and blindly analyzed.

Genotoxicity assay. The comet assay was performed in duplicate to evaluate DNA damage in peripheral blood cells by following the guidelines (Singh et al. 1988) with some modifications (Tice et al. 1991, Braz & Salvadori 2007). Every step was carried out under indirect light. A volume of 5µl of fresh blood was added to 100µl of 0.5% low-melting-point agarose at 37°C, layered onto a pre-coated slide with 1.5% normal agarose, covered with Keep the lid, for 5 minutes at 4 ° C to solidify the agarose. Afterward, the coverslip was carefully removed and slides were immersed overnight into a cold lysis solution. 

Slides were washed In phosphate store the salts (PBS) for 5 minutes, and immersed in freshly prepared alkaline buffer (pH>13) in a horizontal electrophoresis tank. After 20 min of DNA unwinding period, electrophoresis was conducted at 25 V and 300mA for 30 min. Following 15 min of neutralization, slides were fixed with absolute ethanol for 5 min and stored at 4°C. Prior to analysis, the slides were stained with 50µl of ethidium bromide and scored in a fluorescent microscope at 400 x magnification. Images from 50 nucleoids (25 from each replicate slide) were analyzed using the Comet Assay II image system (Perceptive Instruments, UK). Two parameters (tail intensity and tail moment) were used to estimate DNA damage. As the tail moment gave similar results, only tail intensity (percentage of tail DNA) values were presented.
Statistics. Since the data presented a normal distribution, the four-time points were compared by repeated measures analysis of variance; the p-value less than 0.05 was considered statistically significant.

RESULTS:
Results are presented. Despite a slight increase of DNA damage at 3 hours and 8 hours after exposure, no significant differences were found among the four-time points (p-value = 0.52), showing that a single exposure to fipronil (frontline plus®) does not produce short-term genotoxicity in dogs.

DISCUSSION:
There are many reports showing that pesticides can produce toxic effects directly and indirectly exposed non-target organisms, including humans (Dulot et al. 1985). Therefore, the genotoxicity assessment of pesticides is of great importance. Since pesticides are widely used worldwide and information on the possible toxic effect of fipronil is limited, this study was designed to evaluate the ability of fipronil to induce DNA damage in whole blood cells. To the best of our knowledge, our findings suggest for the first time that this pesticide does not induce the genotoxic effect in dogs.

The dose investigated appeared to be the ideal as it is the dose clinically utilized by pet owners according to an indication of the laboratory and veterinarians. In addition, we investigated the genotoxic potential at three-time points based on the guidelines for in vivo genetic toxicology after a single acute treatment (Sasaki et al. 2000, Tice et al. 2000).
The comet assay is a relatively simple, rapid and low-cost test used for detection in DNA of single- and double strand breaks, alkali-labile sites and oxidative lesions (Tice et al. 2000). It is also known as the single-cell gel electrophoresis (SCGE) assay, which consists of immersing the eukaryotic cells in agarose gel to lyse the cell membrane by detergents and alkaline salts, and detecting damaged DNA by differential migration of the nuclear material when subjected to electrophoresis. This essay is an important tool to evaluate DNA damage in environmental monitoring, genetic toxicology, ecotoxicology, and clinical studies. Thus, the use of biomarkers as a measure of biological responses in affected organisms is a very important factor for simplification and cost reduction of biological monitoring.

Fipronil induced in vitro dose-dependent genetic lesion in lymphocytes exposed to 0.1, 0.3 and 0.7µg/ml (Çelik et al. 2014). Male rats orally given fipronil (2.5, 5.0 and 10mg/ kg/day) for 28 days showed increased DNA damage when sperm comet assay was evaluated (Khan et al. 2015). There Are clear manifestations of the carcinogenesis of this factor in mice 300ppm, but not in female mice at doses of 30ppm (Tingle et al. 2003). 

And on the other hand, direct local use
Flea products and ticks containing fipronil do not increase the risk of transitional cell carcinoma (TCC) of the urinary system bladder in Scottish Terriers (Raghavan et al. 2004).

Our findings are in agreement with other reports regarding the genotoxic/mutagenic potential of fipronil. The pesticide was found negative in bacterial mutagenic tests and showed no clastogenic effect when exposed to human lymphocytes at doses of 75, 150 or 300µg/ml with or without metabolic activation (EPA 1997). None of the tested quantities of fipronil (0.05 to 0.23 μg / L) are sufficient to adjust DNA integrity in gill cells assessed by the comet Preview of the new tropical fish . Also, fipronil has not shown the effects of genetic toxicity 24 After hours of exposure or the effect of mutations when tested It started from 24 hours to 72 hours after occurrence at 15 mg/kg or 25 mg/kg in the peripheral blood of mice.
These authors observed that only the highest dose tested (50mg/kg - LD50) induced DNA damage and micronuclei 24 hours after the exposure.

Pesticides are noxious chemicals widely used in agriculture, either in isolation or combined with other substances and may affect long-term health (WHO 1992). The levels The fipronil remains on the gloves after wearing Frontline in dogs after 24 hours of exposure and the result was undetectable after 5 weeks. Repeated use of such effects may pose risks to human health. In fact, appears in patients with self-poisoning fipronil vomiting, stimulation and seizures, usually a favorable outcome. Several insecticides, including fipronil, were shown to be genotoxic to mucosal epithelial cells taken from human tonsil tissue whereas inhalation of these substances can damage epithelial cells of the upper aerodigestive tract.
Fipronil may lead to poor normal functioning of the endocrine system and has adverse positive effects in the Female Rats. This molecule is metabolized rapidly and its residues are dispersed, especially in subcutaneous adipose tissue and hair follicles. The main metabolite of fipronil is fipronil sulfone.
Although less selective, fipronil sulfone lasts longer in relation to GABA receptors and it is found in many organs of mice and other vertebrates after exposure, playing an important role in the adverse effects of the pesticide. The increased power of fipronil sulfone to sensitize GABA receptors intensifies the concern about the toxicity of this pesticide, especially in mammals. The metabolites fipronil sulfone and desulfinyl fipronil are more toxic to aquatic organisms than fipronil. The U.S. EPA states that fipronil is highly toxic to fishes and aquatic invertebrates but relatively less toxic to mammals and birds (EPA 1996).

CONCLUSION:
The result that was reached in this study,
concluded that a single exposure to the clinically recommended dose of fipronil (frontline plus®) does not induce short-term systemic DNA damage when topically applied in dogs. Further studies should be conducted to evaluate the possible genotoxic effects in other tissues and/or after chronic exposure to fipronil.

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