According to the literature, exposure to pesticides may cause alterations in human hearing.1–6 Auditory injury is often due to several factors7 and, when it occurs occupationally in the primary sector of the economy, it may be related primarily to two factors: exposure to ototoxic chemicals, such as solvents and pesticides, as well as noise from agricultural machinery. The hearing loss due to ototoxic drugs’ exposure is similar to the one that occurs at high sound pressure levels, being sensory-neural, bilateral, symmetrical, irreversible, at high frequencies and damaging to the hair cells of the cochlea.8

Agrochemicals belonging to the class of Organophosphates (OPs) are widely used in agriculture as insecticides. Among them is dichlorvos (2,2 Dichlorovinyl Dimethyl Phosphate – DDVP), used in public health campaigns to control vectors, as a resource in veterinary medicine – in the fight against ectoparasites9 in the primary sector of the Brazilian economy (agriculture). OPs act as cholinesterase inhibitors and they are responsible for many intentional or accidental poisonings.10

OPs are important to control vectors considering that Brazil has a high number of reported cases of dengue, chikungunya and Zika, and nowadays the country could be considered as the most suffering one from Aedes aegypti.11 Therefore, insecticides, such as organophosphates, are widely used in public health for vector control. Dichlorvos, an OP, is also used in domestic disinfestations. Furthermore, there is a concern about its indoor use that may be related to critical phases of human development. Children can be exposed to pesticides, considering that they tend to play and explore their environment, touching surfaces as the floor12 and have a hand-to-mouth behavior, absorbing more pesticides than adults.13

Due to the existence of several etiologic agents that cause occupational hearing loss, such as machinery noise and exposure to chemical agents, it is difficult to correlate this auditory pathology. Furthermore, even those who do not work directly with these substances are exposed to them and hearing loss may be multifactorial.7

The study in animals exposed only to pesticides, free from the bias such as noise and vibration, allows a characterization of the hearing loss and the study of its mechanisms. In the literature, there are few studies evaluating ototoxicity due to exposure to agrochemicals in animal models, and these often use routes not correlated with occupational exposure, such as intraperitoneal injection.14–16 Therefore, the present study has aimed to evaluate the ototoxic effects based in cochlear function of the subchronic inhalation exposure of DBOI in rats through distortion product otoacoustic emissions.

Exposure to the DBOI significantly reduced post-exposure DPOAE (p<0.05, Mann–Whitney) compared to pre-exposure measurements (Fig. 1) at frequencies 8 and 10kHz in both ears (p=0.034 and 0.045 on the right and p=0.015 and 0.013 on the left ear, respectively). As well, the cisplatin treatment significantly reduced DPOAE at (Fig. 1) frequencies 12kHz (p=0.006) in the right ear and 4kHz in the left ear (p=0.032). The control group showed no significant difference (p>0.05; Mann–Whitney) in post-exposure assessments in relation to pre-exposure (Fig. 1), confirming the standardization of the experimental model.

Figure 1.

Median and interquartile ranges of DPOAE amplitudes pre- and post-exposure to DBOI, cisplatin (positive control por hearing damage) and water (control).

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There was a statistically significant difference between the groups (p<0.05; Kruskal–Wallis) in relation to the variation of the medians post- and pre-exposure (Fig. 2).

Figure 2.

Variation of the amplitudes (medians and interquartile ranges) between the pre and post-exposure period to the control (water), positive control and DBOI.

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According to the p-value presented in Table 1, the DBOI group showed a significant difference (p<0.05) from the control at frequency 8kHz on the right and at frequencies 8kHz and 10kHz in the left ear. Although at the frequency of 10kHz on the right ear the analysis showed a difference between the groups (p=0.026, Kruskal–Wallis), the comparison of means did not identify which groups differ from others. The cisplatin group showed a significant difference in relation to the control group at frequency 8kHz on the left and in relation to DBOI group at frequency 12kHz on the right ear (Table 1).

The relative body mass gain (mean±standard error) in the period was similar (p=0.328; Student's t test) between the control (145.3±5.93%) and DBOI (154.1±5.91%). However, the animals in both groups showed transient clinical signs such as piloerection and dyspnea. The clinical signs (Fig. 3) were more evident at the beginning of the exposure (between 30 and 240min) and in the first two weeks, but only the animals exposed to DBOI had transient depression (evidenced by drowsiness and decreased response to stimuli) that lasted until the 4th week.

Figure 3.

Clinical signs.

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Analyzing the behaviors in the open field test, the animals treated with DBOI were not different from the control in any of the analyzed characteristics (latency to move from the first quadrant (p=0.765; Test T of independent samples), external crossing (p=0.299; Test T of independent samples), internal crossings (p=0.569; Test T of independent samples), rearing (p=0.370; Test T of independent samples), grooming (p=0.145; Test T of independent samples). These results explain that the spontaneous locomotor activity was normal and that the treatment caused no harm to the animals.

The plus maze test (Fig. 4) shows that animals treated with positive control (Diazepam) spent more time in the open arm (p<0.001 one-way ANOVA, post hoc Bonferroni). The animals treated with positive control stayed less time in the closed arm, but the animals treated with DBOI were not different from positive control (p<0.01 oneway ANOVA, post hoc Bonferroni). The time spent in the center of the apparatus shows no difference between the groups (p=0.112 one-way ANOVA, post hoc Bonferroni). The head dipping demonstrates again the anxiolytic action of positive control (p<0.05 one-way ANOVA, post hoc Bonferroni) and no difference between DBOI and positive control (p>0.05 one-way ANOVA, post hoc Bonferroni).

Figure 4.

Plus maze test.

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Plasma cholinesterase values were similar (mean±standard error: control=5958.0±145.0U/L; DBOI=5886.0±112.9U/L, respectively, p=0.735; Student's t test). No macroscopic alterations were found in the organs of the animals used in the experimental protocol.

The present study has demonstrated ototoxicity induced by subchronic inhalation exposure of OP DBOI in rats, which was comparable to cisplatin-induced cochlear damage. The ototoxicity assessment protocol used in this study was consistent, considering all control groups (positive and negative) demonstrated expected behaviors. The choice of the rats as the experimental model for the DPOAE test to protocol is consistent with previous literature data, in which the authors concluded that DPOAE was the most frequent auditory evaluation used in the studies analyzed utilizing animal models, such as rats.24

The use of an animal model in the present study has allowed the identification of the outcome investigated (ototoxicity resulting from inhalation exposure to pesticide), controlling for possible bias (concomitant exposure to noise). Moreover, the inhalation route in an acoustically treated chamber, with an alternating exposure frequency that mimics the working day (5:2 days) has allowed us to consider that the effects observed here in rats could be comparable to those reported in the previous literature involving farmers.3,4,6

In addition, frequently previous studies evaluating cochlear function by otoacoustic emissions have used anesthetics, which have been reported as likely to cause changes in DPOAE responses.25–27 In the present research, it was possible to carry out the evaluations without anesthesia, due to the previous acclimatization of the animals to the instruments used in the auditory evaluations. Furthermore, the finding of ototoxicity secondary after the exposure to DBOI of rats corroborates previous studies that evaluated the hearing of individuals who had contact with pesticides.6,28 Although studies in humans may present some bias, due to the difficulty to completely isolating the noise and other confounding factors inherent in the clinical history, such as exposure to vibration, occurrence of congenital pathologies such as toxoplasmosis, herpes and rubella, or even presbycusis, since damage to hearing may be multifactorial.7

Post-exposure results have indicated that even though dichlorvos has been an irreversible cholinesterase inhibitor, the concentration of the enzyme in the experimental group were similar to those in the control group. This fact suggests that levels of exposure to DBOI that do not cause significant enzymatic inhibition have ototoxic effect. Although studies have suggested the association between auditory damage and exposure to pesticides,1,2,5,6,15,16 the current regulatory norm in Brazil29 (NR7) do not specify reference and sequential auditory exams for workers exposed to chemical agents, and the exposition to pesticides is evaluated by tests such as cholinesterase level. However, our results indicate that – even without alteration in cholinesterase levels – there was ototoxicity due to exposure to DBOI.

Concerning the effects of inhaled subchronic treatment DBOI in the Open Field and Plus Maze behaviors tests, in this research we demonstrate that DBOI administrated in Wistar rats was not an anxiogenic drug and had no impact on the locomotor activity. However, using another route of administration (oral), Silva et al. (2017) had different results in their study,30 in which Wistar rats were treated with chlorpyrifos (0.01, 0.1, 1 and 10mg/kg/day) on gestational days (14–20). Male offspring behavior was evaluated on post-natal days 21 and 70 by the elevated plus-maze test and open field test. The results demonstrated that exposure to 0.1, 1 or 10mg/kg/day of chlorpyrifos could induce anxiogenic behavior, but this signal was reversed on post-natal day 70.

The few clinical signs manifested by animals have indicated that toxicity to cochlear function, after the exposure to DBOI, was present at a level where rats did not exhibit relevant systemic toxicity – as in central nervous system (based on behavior tests) – only transient toxicity. The clinical signs were more frequent in the first hours of exposure, as well as in the first weeks of exposure, with no appearance in the last two weeks, which suggests a possible progressive tolerance to the same concentration of the substance. In addition, the transient alterations were reinforced by similar results between the groups regarding weight development.

The statistically significant difference between the control group and the positive control for injury was expected, since the ototoxicity of cisplatin has been well established in the literature.19,31–36 Additionally, the reduction of the DPOAE amplitude between the experimental group and the control group has indicated that exposure to OP actually could damage the cochlear function of the animals. The change in cochlear function values before and after DBOI exposure corroborates previous literature, which verified alterations in the cochlear morphology of guinea pigs due to exposure to an OP (0.3mg/kg/day or 3mg/kg/day during seven consecutive days, intraperitoneally), with alteration in the outer hair cells cytoarchitecture.16 This hypothesis is reinforced by the previous literature,37,38 which indicated that exposure to insecticides could interact at the cochlear level.

Although the DPOAE test does not evaluate the hearing loss itself, the outer hair cells activity could be referred here. We highlight that the bilaterality of the 8 and 10kHz damage in the experimental group is one of the characteristics of the sensorineural hearing loss secondary from exposure to ototoxic substances. In addition, the affected frequencies in animals exposed to DBOI are considered high, which also resembles the toxic agent induced hearing loss. On the other hand, the occurrence of loss in the high frequencies of 8 and 10kHz bilaterally, but not in 12kHz, is intriguing, and no similar result was found in the previous literature. This may be explained by the fact that usually DPOAE studies1,39 do not consider 12kHz frequency, and this might occur due to limitation of the equipment used.

The mechanism of hearing loss after exposure to organophosphates has not yet been fully elucidated.2 Ototoxicity secondary to some substances, such as gentamicin and cisplatin, has been related to possible alterations in the antioxidant functions in the sensory cells of the organ of Corti, especially the outer hair cells, due to lipid peroxidation.40

This study has potential limitations. Although Auditory Brainstem Response (ABR) testing would be a better indicator of cochlear function and the presence of hearing impairment than DPOAE, the ABR test was not performed, since the ABR equipment was in a building inaccessible to animals. DPOAE could be performed, since the equipment is portable, and can be taken where the animals were housed. In addition, the institution's equipment did not have the ABR screening function (ABR-A). The second limitation is the lack of previous research studies analyzing frequencies such as 10kHz and 12kHz, which makes it difficult to compare our results with another findings (such as statistical difference at 8kHz and 10kHz in the DBOI group, but not at 12kHz). Another limitation is that the evaluation of DPOAE without the measurement of antioxidant enzymes in the auditory system does not justify the statement that ototoxicity to DBOI could be caused by dysfunction in antioxidant enzymes; however, it suggests that this may be related to it, as the decrease or absence of DPOAE can indicate damage in hair cells function. In order to fully clarify this mechanism, new functional research studies will be necessary, associated with methods that evaluate antioxidant enzymes and the morphology of hair cells, such as scanning electron microscopy or even fluorescence microscopy.

DBOI caused ototoxicity in the animals evaluated, comparable to cisplatin-induced damage in DPOAE, without significant systemic effects, with loss at frequencies of 8 and 10kHz bilaterally.

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.

The authors declare no conflicts of interest.