Abstract
Background/Aim: The pesticide dimethoate (O-dimethyl-S- Nmethylcarbamoylmethyl phosphorodithioate) is able to induce severe acute toxicity in living organisms. The aim of this study was to evaluate the effects of ultraviolet radiation, alone or combined with exposure to dimethoate, on the rat skin. Materials and Methods: A total of 38 Wistar female rats (Rattus norvegicus albinus), were distributed into four groups: A (n=9) control group, B (n=10) exposed to ultraviolet-B radiation (UV-B), C (n=10) exposed to UV-B followed by application of dimethoate (UV-B+AGRO) and group D (n=9) exposed to dimethoate (AGRO). Histological examination of the tissues, as well as immunohistochemistry for cleaved caspase 3, Ki-67 and COX-2 expression were performed to all groups. Results: Animals submitted to UV-B exhibited hyperkeratosis with moderate cell atypia. Regarding exposure to UV-B+AGRO, the animals presented hyperkeratosis and atrophy, whereas in animals exposed to AGRO, only atrophy was noticed. The immunohistochemical results on skin revealed that UVB, AGRO and UVB+AGRO decreased cleaved caspase 3 and Ki-67 expression when compared to the control group (p<0.05). COX-2 expression decreased to UVB or AGRO groups compared to controls (p<0.05). Conclusion: UV-B or AGRO exposure is able to induce histopathological changes and altered expression of cleaved caspase-3 and Ki-67 in rat skin, thus being categorized as a risk condition for skin carcinogenesis.
According to the International Agency for Research on Cancer (IARC), occupational exposure is the main form of human contact with carcinogens, such as pesticides and ultraviolet (UV) radiation (1). Work-related neoplasms are found in some organs, such as skin, lungs, stomach and bladder, among others, being directly absorbed or even eliminated from various carcinogenic agents (2).
The National Cancer Institute in Brazil estimated in 2016 about 596,000 new cases of cancer, of which non-melanoma skin cancer is the most common (180,000). This is the most frequent type of human cancer and shows an increase in its incidence in recent years, becoming a growing public health problem worldwide (3). On the other hand, melanoma skin cancer has a high lethality, but its incidence is low, affecting 5,670 new cases annually (3).
Occupational exposure to UV radiation is a known risk factor for skin cancer (4). The action of UV radiation on the skin is a complex process that is associated with chemical and morphological changes, such as formation of reactive oxygen species, histochemical changes of different severities, thickening of the prickly layer and rectification of the basal layer of the epidermis. It also activates components of the cutaneous immune system and the release of inflammatory mediators by direct activation of keratinocytes and suppression of Langhans cells (5). DNA is one of the major molecules that absorbs UV radiation and undergoes several mutations that can subsequently result in malignant transformations of the eukaryotic cell (6). It has been demonstrated that continuous exposure to arsenic and others pesticides, ionizing radiation and chronic inflammation of the skin contribute to carcinogenesis as well (7). Epidemiological studies suggest a cause-effect relationship between malignant dermal neoplasms and exposure to pesticides in the work environment (8).
Pesticides are considered extremely relevant to the model of agricultural development of Brazil, with the country being the largest consumer of these products in the world up to now (9). The genotoxicity of these compounds is caused by means of adducts, oxidative alterations or even breaking of the DNA molecule, modifying qualitatively and quantitatively the genetic status (9). At high doses, pesticides increase DNA replication and cell proliferation, which can give rise to neoplastic cells; the target organ depends on the route of absorption and metabolization, while environmental factors, and factors inherent to the individual play a pivotal role in genomic instability (10).
Organophosphorus compounds are absorbed from the body through oral, respiratory, and cutaneous routes and can easily cross epithelial, dermal, and respiratory cell membranes due to their lipophilic structures (11). These compounds are distributed throughout the body, particularly in the fatty tissues, and their rapid degradation generally inhibits their accumulation (11).
Particularly, the insecticide dimethoate (O-dimethyl-S-Nmethylcarbamoylmethyl) phosphorodithioate and molecular formula C5H12NO3PS2) is able to induce severe acute toxicity according to the Hazard Classification System (ABNT-NBR 14725 - Part 2: 2009) on living organisms. In fact, some authors have postulated that dimethoate may cause dermatitis and sensitization in close contact with skin (12, 13). However, there are no reports investigating the harmful effects induced by dimethoate combined with UV on skin carcinogenesis so far.
The aim of this study was to evaluate the effects of UV, alone or combined with exposure to dimethoate, on the skin of Wistar rats.
Materials and Methods
Animals and experimental design. The study was approved by the Animal Ethics Committee at Federal University of Vale do Sao Francisco (UNIVASF) (n° 0015/120215), and Federal University of Sao Paulo (UNIFESP) (n° 6407040516). It was an experimental study, which employs an animal model aligned to a follow-up study. A total of 38 Wistar female rats (Rattus norvegicus albinus), 3 months old, and average weight of 200±30 g was used. The time period of performance and observation of the animals was 15 weeks. This time was determined by considering previous studies published in the literature (14-16). The animals were distributed into four study groups, as follows: A (n=9) control group, B (n=10) exposed to ultraviolet-B radiation (UV-B), C (n=10) exposed to UV-B followed by application of dimethoate (UV-B+AGRO), and group D (n=9) exposed to dimethoate only (AGRO).
Pesticide application. The pesticide under study was dimethoate (Agritoato 400®). The chemical name is O, O-dimethyl-S-(Nmethylcarbamoylmethyl) phosphorodithioate and molecular formula C5H12NO3PS2. For exposure to UVR-B and pesticide, the animals had a 3×3 cm area of fur removed from their backs each three days, according to experiments conducted by Tang et al. (16). A solution containing the organophosphate “Agritoato®” diluted in distilled water containing 0.75 ml of the pesticide for each 500 ml of water, was applied topically to the back of the animals of group C and D three times a week, on non-consecutive days, using a wood stamp with a sponge on its end, measuring 6×4 cm, for a period of 15 weeks.
UV radiation. An UV-B lamp model G15T8E (UV-BRAVO, Bravoluz Lâmpadas Especiais®, Curtiba, Brazil) with a wavelength of 280-360 nm (peak at 305-310 nm) and, power of 9 J/s (W), which does not produce thermal alteration, was used. The rats from group A and B were submitted to exposure for 60 s, 3 times a week, on non-consecutive days, on the same days of the pesticide application. The period of exposure to UV-B was based on the power of the lamp and the minimum time to obtain two times the Minimal Erythema Dose at the animal's skin (16). At the end of the experiment, 24,300 J of total energy for each animal were obtained as described elsewhere (14-16).
Microscopic analysis. For the resection of dermatological lesions (altered skin areas), the animals were euthanized under anesthesia, using a solution of ketamine hydrochloride (60 mg/ml) and xylazine hydrochloride (18 mg/ml). Skin tissues were resected, processed, and stained by the hematoxylin and eosin (H&E) method. The following parameters were examined: hyperkeratosis, parakeratosis, cellular atypia, hypogranulosis, epidermal atrofia, spongiosis, and apoptosis. Apoptosis in skin tissue was evaluated as recommended by Elmore et al. (17).
Immunohistochemistry. To confirm the histopathological changes induced by UV and/or dimethoate, the expression of Ki-67 (proliferation activity), cleaved caspase-3 (apoptosis), and COX-2 (inflammation) was examined by immunohistochemistry in rat skin. The method was manually performed using the EnVision FLEX+, Mouse, High pH (Link) (Dako, Copenhagen, Denmark) and the Novolink (Novocastra Laboratories, Newcastle, UK) polymers according to the manual from the manufacturer. The EnVision polymer conjugate was used for the detection of mouse anti-COX 2 (C0X-2- D12) (Santa Cruz Biotechnology) (1:150) and mouse anti-Ki67 (MIB-1) (Novocastra) (1:30) antibodies, while Novolink for the cleaved rabbit polyclonal anti-caspase3 (Cell Signalling) (1:200).
Immunohistochemical data analysis. For the analysis, the Nikon optical microscope model Eclipse E600 was used. The percent expression of each marker was evaluated in 10 fields of 400× magnification and the semi-quantitative scoring system was used. For this purpose, a numerical scoring system with two categories was used. First, the number of immunoreactive cells was classified as 0 or negative (no immunoreactive cells or <10% immunoreactivity), 1 (10 to 25%), 2 (25 to <50%) and 3 (>50% immunoreactivity). These values correspond to category A. In category B, the intensity of immunostaining was categorized as 0 or negative, 1 (no or weak immunostaining), 2 (moderate) and 3 (strong). After that, the numerical data from categories A and B were multiplied, resulting in an immunoreactive score ranging from 0 to 9, with 0-3 considered negative and 4-9 positive (18). All results were expressed to percentage (%).
Statistical analysis. All data were evaluated by Fischer's exact test. Statistical analyses were performed using the statistical program R version 3.0.2. (www.r-project.org). All p-values <0.0 were considered statistically significant.
Results
Microscopic findings. In the control group, none of the animals presented microscopic changes within the scope of this investigation. In Group B (UV-B exposed rats), we observed hyperkeratosis and parakeratosis in all the animals; and 8 animals exhibited cellular atypia and hypogranulosis. Spongiosis and apoptosis were also observed in 5 animals. In regard to UV-B+AGRO exposure, a total of 8 of the animals presented hyperkeratosis, and 2 specimens exhibited parakeratosis. Four animals in this group presented spongiosis and 6 rats presented apoptosis. It was also observed that atrophy was detected to three animals only. In the group D, atrophy of the skin was evident in all animals evaluated in this setting. Hyperkeratosis, parakeratosis, cellular atypia, hypogranulosis or spongiosis were not detected in this group.
Distribution of variables analyzed in microscopic slides from animals exposed to dimethoate (AGRO) and ultraviolet radiation (UV-B).
Hyperkeratosis (p<0.001), parakeratosis (p<0.001), cellular atypia, (p<0.001), hypogranulosis (p<0.001), and spongiosis (p=0.005) were more frequent in the UV-B and UV-B+AGRO groups, when compared to the AGRO and control groups. Epidermal atrophy was more frequent in the AGRO group when compared to the control, UV-B or UV-B+AGRO groups (p<0.001). Regarding apoptosis, it was more frequent in the UV-B and RUV-B+AGRO groups when compared to the AGRO group (p=0.022). These results are summarized in Table I.
Expression of cleaved caspase-3, Ki-67, and COX-2 in rats exposed to diamethoate (AGRO) and ultraviolet radiation (UV-B), alone or in combination.
Immunohistochemistry. None of the control group animals expressed COX-2, while one animal demonstrated immunopositivity for Ki-67 and cleaved caspase-3 only. The animals in the UV-B group were positive for all markers investigated in this study. More specifically, cleaved caspase-3 (p=0.011), Ki-67 (p<0.001), and COX-2 (p<0.001) were significantly increased in the UV-B group, compared to the control group. Regarding the UV-B+AGRO group, all animals were positive for cleaved caspase-3 (p=0.011, compared to the control group), and 9 animals for Ki-67 (p<0.001, compared to the control group), whereas only one was positive for COX-2 (p>0.05, compared to the control group).
In the group exposed to dimethoate only, all animals expressed positivity for the cleaved caspase-3 (p=0.011) when compared to negative control. Regarding Ki-67 expression, a total of six animals expressed the proliferative marker (p<0.001), and two animals expressed COX-2 (p<0.001). The results demonstrated that groups B, C, and D expressed more cleaved caspase-3 (p=0.011) and Ki-67 (p<0.001) compared to negative control. These findings are summarized in Table II.
Discussion
In response to UV-B, all animals presented moderate focal or multifocal parakeratosis-like hyperkeratosis, which, when associated with cellular atypia and apoptosis, may be a marker for skin carcinogenesis (19). Skin cancer may be produced by UV action on epidermal keratinocytes due to the production of genotoxicity and DNA damage, directly or through reactive oxygen species (ROS), activation of oncogenes and inactivation of tumor suppressor genes (19).
Following the multi-step of skin carcinogenesis, the epidermis may present atrophic, of normal or acanthotic thickness and, in most cases, there is a decrease of the granular layer in the areas underlying the parakeratosis, as described by Smoller et al. (20). This is consistent with our findings animals submitted to UV-B and UV-B+AGRO. Some dermatoses, including contact dermatitis, urticaria, erythema multiforme, gray dermatosis, porphyria cutanea tarda, carcinoid syndrome, Bowen's disease (squamous cell carcinoma in situ), multiple basal and squamous cell carcinomas, and melanoma, are described as caused by pesticides (21). In this study, we also found histopathological evidence (spongiosis) characterized as contact dermatitis in animals submitted to UV-B and UV-B+AGRO (40%).
All groups showed positivity for cleaved caspase-3, except for the control group. In light of these results, we have shown that both exposure to UV-B and dimethoate, alone or in combination, induced intense apoptosis in rat skin. It has been established that UV radiation induces genetic damage by means of generation of reactive oxygen species. By eliminating these damaged cells, apoptosis, induced via caspase-3 activation, plays a crucial role for preventing skin carcinogenesis (22, 23).
Regarding Ki-67, UV-B exposure to rat skin was able to increase ki-67 immunoexpression when compared to control. This is agreement with others (24). Dimethoate also increased cell proliferation in rat kin. The combination of dimethoate and UV-B showed similar values when compared to UV-B only. These results suggest that skin exposure to pesticides may constitute an important factor that induces cell proliferation and neoplasias.
The catalytic activity of COX-2 results in the production and potentiation of DNA damage by free radicals. The combination of these effects contributes to permanent damage to genomic DNA and cell differentiation. Studies that investigated the presence of this enzyme in normal skin and in non-melanoma cutaneous neoplasms, revealed that it was not expressed in healthy skin, but it was strongly expressed in actinic keratosis, and Bowen's disease (25). Jiao et al., similarly to our experimental model, used UV lamps in the 280-400 nm range to irradiate SKH-1 mice 3 times a week and found strong expression of COX-2 in epithelial cells of these animals after a single irradiation with UV-B (26). Another study demonstrated COX-2 overexpression in human keratinocytes irradiated by UV-B, confirming the correlation of this exposure with the increase in COX-2 positivity (27). The causal relationship between pesticide and cutaneous neoplasms has been the subject of many studies, such as those of Gallagher et al. (28), Robinson et al. (29), Krishnatreya et al. (30), Brouwer et al. (31), demonstrating the impact of pesticide on skin cancer. In line with literature evidence, our results revealed that dimethoate increased COX-2 expression in rat skin. However, in the literature, there is an absolute scarcity of experimental studies, using animal model, associating the UV and pesticide variables (32). Interestingly, our results demonstrated that the combination of UV-B and dimethoate decreased COX-2 immunoexpression. Such findings are very new and so, difficult to explain. Further studies are welcomed to elucidate the issue.
In summary, our results suggest that exposure to UV-B and dimethoate is able to induce histopathological changes in rat skin and altered expression of cleaved caspase-3 and Ki-67. Such results are considered as a risk condition for carcinogenesis, in Wistar rats. The causal association between exposure to both pesticide and UV radiation and the onset of skin cancer, despite being strongly emphasized, requires further studies to be fully understood.
Acknowledgements
DAR is a researcher on Productivity at CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico; grant number #001).
Footnotes
Authors' Contributions
Research project conception: TMROF, JPK and HP. Study design: TMROF, JPK, CTFO, LK and MSS. Data analysis: TMROF, CTFO, DAR and HP. Writing of the manuscript: all Authors.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received July 10, 2019.
- Revision received August 7, 2019.
- Accepted August 8, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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