Effects of low-dose subchronic exposure to the phenylpyrazole insecticide fipronil in juvenile mice

Abstract

Fipronil (FPN) inhibits gamma-aminobutyric acid type A receptors and exhibits strong insecticidal effects. Although FPN’s adverse effects on mammals have been reported recently, few studies have examined subchronic exposure to low doses. We orally administered FPN (0.43 mg/kg) to 4-week-old male mice for 6 wk and assessed their behavior and blood characteristics. Compared with the control group, the FPN-treated group presented reduced weight gain but upward trends in locomotor activity and blood histamine levels. Fipronil sulfone (FPNS) was detected in all individuals, whereas FPN was not detected in any individual. The present study shows for the first time that subchronic exposure to low-dose FPN in mice led to FPNS conversion in the body, affecting weight gain and behavior.

Fipronil (FPN), a phenylpyrazole insecticide developed in the late 1980s and brought to market in 1993, is a widely applied systemic insecticide with high efficacy against a broad range of insects. The binding of FPN to gamma-aminobutyric acid type A receptors (GABA_AR) inhibits their binding to gamma-aminobutyric acid (GABA) and blocks GABAergic chloride ion channels, causing neural hyperexcitability in a wide range of insects, leading to death. Owing to its strong action, FPN is effective at low doses against insects resistant to conventional insecticides. Because FPN is slow-acting, insects that ingest it die after returning to the nest. As the nestmates eat the carcasses, the effect spreads throughout the colony. On the other hand, FPN also acts on GABA receptors in nontarget organisms, and systemic insecticides containing FPN are considered causative agents of colony collapse disorder (CCD) in European honeybees [11, 30]. Because of FPN’s high toxicity to honey bees, the European Food Safety Authority banned its use throughout the EU beginning in 2013. In the USA, FPN-treated rice seeds have caused mass mortality of crayfish [33], and in Japan, FPN has been found to be highly toxic to dragonfly larvae, resulting in a drastic decline in dragonfly populations [17].

The binding affinity of FPN for insect GABA_AR is thought to be stronger than that for mammalian GABA_AR [7]. For this reason, it is widely used as an insecticide and to control fleas and ticks on companion animals. In mice and rats, however, FPN has been reported to be toxic to reproductive function [31], thyroid function [27], and liver cells [9]. In mice and rats, a single administration of FPN has been observed to induce increases in rearing, freezing, grooming, exploratory behaviors [35], and locomotor activity [29] as well as reductions in anxiety-like behaviors [34]. These reports suggest that FPN administration affects emotion and behavior. In the mature mammalian central nervous system, GABA is the primary inhibitory neurotransmitter that binds to GABA receptors and modulates various signaling processes. When GABA binds to GABA_AR on neurons, neuronal excitation is suppressed [18], and mental stability is achieved [14]. Decreased GABA levels have been implicated in psychiatric disorders such as anxiety, sleep disturbance, depression, and schizophrenia [10, 28]. Mood and emotion are modulated by monoamines, which are neurotransmitters acting in the brain. It has also been reported that cognitive functions and behavior are modulated not only by monoamines but also by steroid hormones [13, 23, 26]. Steroid hormone levels in rats have been reportedly reduced by the administration of commercial preparations containing FPN [31]. FPN is metabolized in the body and converted primarily to the sulfone form, fipronil sulfone (FPNS), which, like FPN, inhibits GABA_AR and exerts its effects. FPNS is more lipophilic than FPN and accumulates well in lipid-rich adipose and brain tissues [8]. FPNS has been suggested also to have a high affinity for GABA_AR in the brain [16]. Therefore, it was speculated that FPN and FPNS may disrupt the secretion of monoamines and steroid hormones.

As described above, FPN is thought to exert GABA inhibitory effects on cognitive and emotional behaviors in mammals. Its effects are thought to be concentration-dependent, with several reports showing little effect after single doses at low concentrations [15, 29]. However, most of the studies conducted thus far have been based on exposure experiments with high doses or single doses, so toxicity after chronic administration remains unclear. It is also unknown whether subchronic FPN administration affects the concentrations of monoamines and steroid hormones. In companion animals, FPN must be administered regularly to maintain its anthelmintic effect, which may result in long-term exposure. Chronic FPN exposure is also a concern for humans, as it has been found in surface water, drinking water, and dust [5]. In addition, children are presumed to be more vulnerable than adults to insecticide toxicity [6]. In the present study, we investigated the effects of subchronic administration of low-dose FPN on the cognitive and emotional systems as well as blood characteristics of mice during the growth period.

Male C57BL/6NCrSlc mice (4 wk old) were purchased from Japan SLC (Hamamatsu, Japan) and maintained as described elsewhere [20]. This study was approved by the Institutional Animal Care and Use Committee (Permission # 30-01-01, 2023-05-01) and carried out according to the Kobe University Animal Experimental Regulations. FPN (5-amino-1-[2,6-dichloro-α,α,α-trifluoro-p-tolyl]-4-trifluoromethylsulfinylpyrazole-3-carbonitrile, CAS No.120068-37-3) was purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). The mice were divided into two groups: a control group (vehicle) and an FPN group (0.43 mg/kg/day). This concentration was based on the nontoxic dose derived from a general pharmacological test in females in an experiment where mice were exposed for 90 days to fipronil desulfinyl, an FPN metabolite [12]. Several studies have shown that FPN metabolites are equally or more toxic to non-target organisms compared to parent compound [16, 33]. Therefore, the FPN dose used in this study is considered low for mice. FPN was dissolved in 600 µL of dimethyl sulfoxide (DMSO, Wako Pure Chemical), injected into 60 g of rehydration gel (MediGel Sucralose: ClearH₂O, Westbrook, ME, USA), and stirred. The amount of FPN to be added was calculated weekly based on the average weight of the mice in the FPN group. The control group received a gel that was stirred with only DMSO. For both groups, the gel was placed in the cage and administered continuously from 4 to 10 wk of age. The mice and their gel intake were weighed weekly. Behavioral tests were conducted at 10 wk of age.

An open field test (OF) and an elevated plus maze test (EPM) were performed under conditions described previously [29]. OF was used to assess locomotor activity and anxiety-like behavior in a novel environment. The total distance traveled and moving speed (total distance traveled [cm]/total moving duration [sec]) were measured as indices of locomotor activity in the novel environment, and the time spent in the center zone (30 × 30 cm) was measured as an index of anxiety-like behavior. The EPM was conducted to evaluate behavior under fear-inducing conditions in a high position without walls. The total distance traveled and the total number of arm entries were measured as indices of motor activity, and both the percentage of open arm entries and the time spent in the open arms were measured as indices of anxiety-like behavior. An increase in anxiety-like behavior was defined as a decrease in the percentage of open arm entries and/or the time spent in the open arm. The novel object recognition test (NOR), which evaluates short-term memory and object recognition, was performed as previously described [24]. In the acclimation trial, the mice were allowed to explore freely for 5 min in an empty field (60 × 60 × 40 cm) and were then moved to their home cages. In the acquisition trial 24 hr later, the mice explored freely for 5 min in a field containing four black bottles (5.7 cm in diameter and 11.3 cm high) and then moved to their home cages. In the test trial that started 30 min later, one of the bottles used in the acquisition trial was replaced by a tower of blocks (Lego bricks; 4.8 × 4.8 × 11.3 cm) as a novel object, which the mice were allowed to explore freely for 5 min in the same field. The time spent in the vicinity of an object was defined as exploration. The percentage of time spent exploring the object before replacement and the novel object (Lego tower) out of the exploration of all objects was calculated. Individuals with trials where the total exploration time was less than 4 sec were excluded. All of their activities were recorded by a video camera.

After completing the behavioral test, the animals were euthanized by opening the chest under deep anesthesia with isoflurane, at which time a total blood sample was taken from the heart. FPN, FPNS, monoamines, and steroid hormones in the blood samples were measured. The target substances were measured by liquid chromatography/electrospray ionization mass spectrometry (LC-ESI/MS/MS) according to a previous report [19]. The results of each behavioral test were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Statistical analyses were performed using BellCurve for Excel (version 4.05; SSRI, Tokyo, Japan). Two-way repeated-measures ANOVA followed by multiple Tukey comparisons was used to analyze body weight changes and NOR behavior. Welch’s t-test or the Mann-Whitney U-test and F-test were used to compare the two groups. The Smirnov–Grubbs two-tailed test was used to identify and exclude outliers, whereas the Kolmogorov–Smirnov test was used to assess normality. The results were considered significant when the P-values were less than 0.05.

At the beginning of the experiment (4 wk), the body weights of both groups were almost the same, but from 5 wk onward, the mean body weight of the FPN group (0.43 mg/kg/day) was always lower than that of the control group. In the two-way repeated-measures ANOVA, the main effect of time was significant, and the main effect of FPN administration showed a low P-value (P=0.11); however, there was no significant interaction effect between FPN administration and time. Between-group comparisons showed that the FPN group was significantly lower than the control group at all time points after 5 wk (Fig. 1A). Gel intake was significantly lower in the FPN group than in the control group at 5 wk of age (i.e., 1 wk into the experiment), but did not differ at other time points (Fig. 1B). According to previous reports, a single oral administration of FPN (63.3 mg/kg) to hens decreased their food intake [36], and oral administration of FPN (3 mg/kg) to 9-wk-old female rats for 2 or 4 wk decreased thyroid function as well as plasma T3 and T4 concentrations [27]. It has also been reported that treatment to decrease T3 production induces a decrease in food intake and body weight in rats [2], and food intake and water intake are interrelated [4]. Food intake and thyroid hormone levels were not evaluated in the present study, but effects of FPN on food intake and thyroid function should be considered in future studies. The measurement of FPN and FPNS in blood showed that FPN itself was below the detection limit in all individuals in the FPN group, whereas FPNS, the primary metabolite, was detected in all mice at approximately 400–500 ng/mL (Fig. 1C). No FPN or FPNS was detected in the control group (Fig. 1C). FPN metabolites (fipronil sulfide, fipronil desulfinyl) other than FPNS were below the detection limit in both groups. This indicates that FPN itself is rapidly metabolized and converted to FPNS. In a pharmacokinetic study in rats, high concentrations of FPN residues were detected in adipose tissue, adrenal glands, intestine, liver, and brain after administration, with FPNS accounting for more than 90% of the total [8]. After a single dermal dose of FPN (100 mg/kg) in mice, the C_max, T_max, T_1/2, and MRTinf of FPN were 450 ± 26 ng/mL, 11 ± 1 hr, 60 ± 14 hr, and 56 ± 7 hr, respectively, whereas the C_max, T_max, T_1/2, and MRTinf of FPNS were 2,700 ± 590 ng/mL, 45 ± 5.2 hr, 120 ± 26 hr, and 200 ± 35 hr, respectively, indicating that both substances are remain in the body for a long time [34]. A similar phenomenon was speculated to have occurred in the mice subchronically exposed to low doses of FPN in the present study. Neither FPN itself nor any metabolites other than FPNS were detected in the blood after 6 wk of treatment, suggesting that FPN may have been converted to and accumulated in the more toxic FPNS. However, details of its pharmacokinetics remain unknown and further research is needed.

Fig. 1.

A: Effect of subchronic exposure of fipronil (FPN) on body weight. Mice were weighed weekly from 4 to 10 wk of age. The main effect of time (weeks of age) is significantly different (P<0.001), and there is a possible effect of FPN administration (P=0.11). There is no interaction between time and FPN administration. Between-group comparisons show significantly lower values in the FPN group from 5 wk onward. Two-way repeated-measures ANOVA. Values are mean ± SD (n=12 mice each), **: P<0.01, ***: P<0.001. B: Change in gel intake at each week of age due to subchronic exposure to FPN. Gel intake at 5-6 wk of age is significantly lower in the FPN group compared to the control group. Welch’s t-test or the Mann-Whitney U-test. Values are mean ± SD (n=4–6 mice each), and circles show the values for individual mice. *P<0.05. C: Blood FPN and fipronil sulfone (FPNS) levels in mice after treatment with FPN for 6 wk. FPN is below the detection limit in both the FPN group and the control group, but FPNS is detected in all individuals in the FPN group (P<0.001). Welch’s t-test. Values are mean ± SD (n=12 mice each), and circles show the values for individual mice. ***P<0.01.

In the three behavioral tests, the OF and EPM were administered to assess changes in locomotor activity and anxiety-like behavior due to FPN exposure. In the OF, no significant differences were found in total distance traveled or moving speed, both of which are indices of locomotor activity (Fig. 2A, 2B). Moreover, no significant differences were found in time spent in the center zone, an index of anxiety-like behavior (Fig. 2C). In the EPM, the total distance traveled, an index of locomotor activity, tended to be higher in the FPN group than in the control group (P=0.09) (Fig. 2D). There was no significant difference between the groups in the number of arm entries (Fig. 2E). No significant differences were found in the time spent in the open arm or in the number of open arm entries, both of which are indices of anxiety-like behavior (Fig. 2F, 2G). These results suggest that exposure to subchronic administration of FPN for 6 wk may increase locomotor activity. In our previous study, a single oral dose of 0.5 mg/kg FPN had no significant effect on locomotor activity in 7-wk-old male mice, whereas a dose of 5 mg/kg increased locomotor activity in the OF [29]. In the present study, we comprehensively evaluated locomotor activity and anxiety-like behavior in mice under two different conditions: OF (in a novel environment) and EPM (in fear-inducing conditions). The difference between the two experimental conditions may explain why OF did not increase locomotor activity. Behavioral studies with larger numbers of mice or higher doses of FPN are needed to clarify the effects of FPN on locomotor activity. Like FPN, FPNS is a GABA_AR inhibitor, but it has greater affinity for brain receptors than FPN does, suggesting that it accumulates in brain tissue [8, 16]. It has not been reported whether FPNS has a direct effect on the brain in vivo, but it has been shown to be toxic to the neuroblastoma cell line SH-SY5Y [32]. In the present study, the upward trend in locomotor activity in the EPM, despite the dose concentration being less than 0.5 mg/kg, is thought to the accumulation of FPNS due to subchronic exposure.

Fig. 2.

Results of behavioral changes in the open field test (OF) and the elevated plus maze test (EPM) due to subchronic exposure to fipronil. A–C: Total distance traveled (A), moving speed (B), and time in the center zone (C) in OF do not significantly differ between the control and fipronil groups. D–G: Total distance traveled (D), number of arm entries (E), time spent in open arms (F), and number of open arm entries (G) in the EPM are shown. For total distance traveled (D), an increasing trend is observed in the treatment group compared to the control group (P=0.09). Welch’s t-test or the Mann-Whitney U-test. Values are mean ± SD (OF: n=6, EPM: n=4–5 mice each), and circles show the values for individual mice.

Next, the effects of FPN exposure on short-term memory and object recognition performance were evaluated via the NOR. In terms of total exploration time, no significant effects were observed regarding training, FPN administration, or their interaction (Fig. 3A). Analysis of the frequency of object exploration revealed a significant main effect of FPN administration, and there was also a trend toward a main effect of training (P=0.08), but no interaction effect was observed. During the training trials, the FPN group stayed in the same location significantly more frequently than the control group did (Fig. 3B). Taken together, these results show that FPN administration did not affect total exploration time, but the training effect observed in the control group was not evident in the FPN group. According to a previous report, 10 mg/kg/day FPN administration to rats for 15 days did not significantly decrease short-term memory, but 30 mg/kg/day administration significantly decreased short-term memory. [15]. As the present study is the first in which a behavioral test has been conducted with low-dose subchronic FPN administration, it is unclear whether FPN administration affects short-term memory and/or the training trial process.

Fig. 3.

Results of behavioral changes in the novel object recognition test (NOR) due to subchronic exposure to fipronil (FPN). A (total exploration time): There are no significant main effects (FPN administration, training) or interaction between the control and FPN groups. B (object exploration frequency): A significant main effect is observed for FPN administration, and a trend for a main effect of training (P=0.08) is also observed, but no interaction is observed. Two-way repeated-measures ANOVA. Values are mean ± SD (n=5–7 mice each), and circles show the values for individual mice. *P<0.05.

Blood histamine concentrations tended to be higher in the FPN group than in the control group (P=0.07) (Fig. 4A). The concentrations of 3-methoxytyramine (3-MT), a dopamine metabolite, as well as serotonin and tryptamine did not differ significantly between the control and FPN groups (Fig. 4B–D). Histamine is normally stored in mast cells and neutrophils in vivo but is released from these cells upon exposure to external stimuli such as antigen binding to IgE antibodies on the cell surface. In a previous report, repeated administration of FPN (9.7 mg/kg/day) to 6-wk-old rats for 30 days resulted in increased malondialdehyde (MDA), an indicator of in vivo oxidative stress in blood; decreased total antioxidant capacity (TAC) content, an indicator of resistance to oxidative stress; and increased IL4, IL12, IgE, and histamine [1]. In addition, the oral administration of 5 mg/kg FPN to mice for two weeks affected immune responses and GABAergic gene expression [25]. These results suggest that FPN is immunotoxic due to its GABAergic gene expression, endocrine-disrupting, allergenic, and proinflammatory effects. The upward trend in histamine levels in the present study suggests that subchronic exposure to low-dose FPN may cause immunotoxicity, but further investigation is needed. Furthermore, histaminergic neurons in the brain project to a large portion of the central nervous system, and histamine is known to act as a neurotransmitter. Histamine release in the brain is involved in the regulation of behaviors such as learning, cognition, memory, arousal, and attention [3]. Administration of a neonicotinoid, an insecticide with a different mechanism of action than FPN, reduced histamine levels in the plasma and brain of mice [19]. Changes in blood histamine and 3-MT levels, along with the modulation of behavioral effects, were observed in treated mice [22]. On the other hand, since FPN administration had no effect on the blood concentrations of 3-MT, serotonin, or tryptamine, these factors cannot explain the results of the behavioral tests. This study did not quantify brain histamine levels. Therefore, the behavioral effects remain unknown and need to be considered in future studies.

Fig. 4.

Results of changes in blood monoamine levels with fipronil (FPN) subchronic exposure. A trend toward an increase in histamine (A) is observed in the FPN group compared to the control group (P=0.07). There are no significant differences between the groups in 3-methoxytyramine (3-MT) (B), serotonin (C), or tryptamine (D). Welch’s t-test, the Mann-Whitney U-test and F-test. values are mean ± SD (n=10–12 mice each), and circles show the values for individual mice.

There were no significant differences in the blood levels of progesterone, 17-hydroxyprogesterone, corticosterone, 11-deoxycortisol, aldosterone, or testosterone between the control and FPN groups (Fig. 5). The levels of 11-deoxycorticosterone, 21-deoxycortisol, androstenedione, cortisol, and cortisone were below the detection limits in all individuals. It has been reported that a single topical administration (70 mg/kg) of a commercial preparation containing FPN to the cervix of female rats decreased plasma progesterone and estradiol concentrations [31]. Oral administration of FPN (2.26 mg/kg) to male Japanese quail for 15 days also increased the serum estradiol level and decreased the testosterone level [21]. Steroid hormones are also involved in the regulation of cognitive function and behavior [13, 23, 26]. In the present study, no changes were observed in the blood concentrations of steroid hormones, suggesting that steroid hormones did not influence the behavioral changes induced by FPN administration. The difference in steroid hormone concentrations after FPN administration compared with previous reports may be due to differences in animal species or sex, but the most significant factor is thought to be the low concentration of FPN administered.

Fig. 5.

Changes in blood steroid hormone concentrations with subchronic exposure to fipronil (FPN). There are no significant differences between the control and FPN groups. Welch’s t-test, the Mann-Whitney U-test and F-test. Values are mean ± SD (n=11–12 mice each), and circles show the values for individual mice.

This study showed that subchronic administration of low doses of FPN to mice during the growth period resulted in the accumulation of the metabolite FPNS in the blood and a tendency toward increased blood histamine levels. Furthermore, the increasing trend of locomotor activity in the EPM and the behavioral changes in the NOR suggest, for the first time, that FPN administration may affect emotional behavior. These results offer an opportunity to reevaluate the safety of the FPN.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.