Effects of Oral Exposure to Captan on Growth Plate in Sus scrofa domesticus

Abstract

The expansion of agricultural activities led to the need for the intensive use of pesticides, which allowed large-scale food production. Worldwide reports of intoxication and deleterious effects on humans and livestock animals have raised the need to better understand the effects of pesticides. The species Sus scrofa domesticus, in addition to being a livestock species, is also considered an excellent translational animal model for research. We analyzed the effects of the fungicide Captan on the cartilage of the growth plate after oral subchronic exposure. Captan was added to the animals' food at a concentration of 500 mg/kg of food, and this was administered for a period of 7 weeks. Our results showed that even without macroscopic damage, several foci of fibrosis, chondrocyte necrosis, and tissue disorganization were observed. These types of damage highlight the risk arising from exposure to this fungicide for the normal development of long bones, suggesting that the indiscriminate use of Captan represents real risks for humans and animals.

Keywords: Fungicide; cartilage; pigs; fibrosis; chondrocyte necrosis; tissue disorganization

HIGHLIGHTS

All swine exposed to Captan suffered chondrocyte disorganization.

In exposed pigs, the cartilaginous tissue areas completely lost their characteristics.

Cartilage thickness did not differ between swine exposed and non-exposed to Captan.

Swine exposed to Captan showed significantly larger collagen/aggrecan/proteoglycan areas.

INTRODUCTION

Agriculture has been present in our lives since the neolithic revolution, precipitating community stability and uplifting food availability, thus, enhancing population growth. Plant and animal domestication associated with the implementation of high-tech farming systems drastically changed natural landscapes [¹, ²]. Brazil became the largest consumer of pesticides globally partly due to its political commitment to maximizing productivity per acre of land. According to the Brazilian Institute of Environment and Renewable Natural Resources (in Portuguese, Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis - IBAMA), 800.652,17 tons of active ingredients of pesticides were sold in the Brazilian market throughout 2022 of which 697,62 tons are of the active ingredient Captan [³]. The largest Captan consumers were the states of Santa Catarina (159,38 tons), Rio Grande do Sul (135,07 tons), São Paulo (113,62 tons) [³].

The selectivity and mechanism of action for several pesticides, such as Captan, are not fully known. There are concerns regarding the risks associated with environmental exposure and its impacts on population health, especially of agricultural workers, people living near contaminated fields, and free-roaming wild and domesticated animals [⁴]. For example, during the agricultural application of pesticides based on Captan, the concentrations of the active ingredient itself in the air of the working environment can reach between 0.2 and 0.75 mg/m³. An 8-hour workday can result in an absorbed inhaled dose of Captan between 2.4 and 9.0 mg or 0.034 - 0.128 mg/kg of body weight [⁵]. There are also risks associated to indirect exposure which may occur via the consumption of contaminated food and water [⁴]. Captan is a non-systemic broad-spectrum fungicide belonging to the phthalimide family. Its mechanism of action hinges on inhibiting fungal respiration and metabolic processes through an irreversible non-enzymatic reaction with thiols. The reaction produces small quantities of thiophosgene, a potent and unstable compound that acts at the cellular level by interacting with several enzymes (e.g. sulfhydryl -, amino-, or hydroxyl-), impacting fungal metabolism lethally [⁶, ⁷]. Controversial findings have resulted in Captan being classified as a carcinogen in the European Union, but not in the United States [⁸]. However, Captan exposure has been associated to statistically significant higher risk of multiple myeloma in humans [⁹], inducing the development of intestinal tumors in mice [¹⁰], inducing changes in the shape and size of liver cells in swine [¹¹], and inducing lesions in the gills, liver, spleen, and kidneys of fish which can lead to higher mortality [¹²].

Data on the occurrence of Captan in foods is scarce. In grapes, a concentration of 0.02 mg/kg was reported [¹³]. In the 2017-2022 Multiannual Plan report of the Pesticide Residue Analysis Program in Food (in Portuguese, Programa de Análise de Resíduos de Agrotóxicos em Alimentos - PARA), Captan was found in three types of food analyzed at concentrations below the Brazilian Maximum Residue Limit (MRL). In the 3.53% of apple samples in which Captan was quantified, its concentrations were below the MRL of 25 mg/kg, in 53.28% of the pear samples the concentrations found were below the MRL of 25 mg/kg, and in the 3.21% of the orange samples the concentrations found were below the MRL of 15 mg/kg. Captam was also found in 0.68% of Passion Fruit samples, a crop where its use is prohibited [¹⁴]. The relationship between pesticides and livestock is of grave cause for concern. Specially for animals whose diet consists mainly of grain-based feed and may contain small concentrations of Captan. Effects due to daily or long-term exposure are largely unknown for these. For example, in soybeans the concentrations of Captan were found to be 0.076 ± 0.003 mg/kg [¹⁵].

Pig farming is one of the most profitable livestock-related activities in Brazil and the country occupies the fourth position among the largest swine livestock producers worldwide [¹⁶]. There are records of contaminated production in the state of Mato Grosso as well as cases of sickly livestock that were exposed to pesticides (Captan being cited among these) presenting several yet unexplained clinical-pathological symptoms involving the digestive, neurological, and musculoskeletal systems. The etiopathogenesis of these cases is inconclusive since attempts to elucidate the mechanisms and processes responsible for the symptoms have so far been unsuccessful. The low success rate stems mainly from the complexity of the physiological pathways between pesticide dose and exposure time muddling the determination of cause-and-effect relationships.

Swine have largely been considered good specimens for toxicology studies due to their complex mammal-like metabolism and similarities to humans regarding xenobiotic biotransformation processes. The length of their lifespan also lends itself to monitoring and characterizing the development and progression of pathological symptoms during a period of time that is comparable to those experienced by humans. Therefore, studies investigating swine responses to pesticide exposure may reveal important information about their toxicity in biological systems that are analogous to humans, enabling result extrapolation with higher validity than studies using other model organisms [¹⁷-²⁰]. From a genetic point of view, pigs are three times closer to humans than mice at the nucleotide level, although the latter remains the most used species in experimental studies [²¹]. Swine genes in the cytochromes P450 family are of special interest since they play a direct role in drug metabolism, elimination, and detoxification in a similar manner to the processes happening in humans [²²]. Muscle proportion and distribution across bones in the swine musculoskeletal system is very similar to its human analogue [²³, ²⁴]. The period of time during which growth plate closure occurs in long bones counts as another positive argument towards using swine as experimental model organisms [²⁵]. It occurs between 3 and 3.5 years of age in swine [²⁵], whereas it could take decades in humans (McCrackin e Swindle, 2015). As such, swines are important model organisms for histopathological studies evaluating the effects of pesticide exposure [²⁶].

Our objective was to evaluate the subchronic toxic effects of Captan on the growth of plate (femur and tibia) of swine pelvic limbs in order to contribute to the understanding of reports of damage observed in livestock animals reported by Brazilian farmers.

MATERIAL AND METHODS

All protocols of this study were approved by the Ethics and Animal Use Committee of the Federal University of Mato Grosso - Brazil, protocol Nº 23108.087455/2015-13.

Model organism

3-month-old pigs (Sus scrofa domesticus) of the same genetic lineage and both genders were used. They were fed with a balanced diet in line with the expected life stage growth according to recommendations by National Research Council (NRC) [²⁷]. Individuals were kept in experimental pens in the Swine Sector of the Experimental Farm operated by the Federal University of Mato Grosso (Santo Antônio Leverger/MT, Brazil).

Experimental design

Sixteen pigs of the same age group were selected from a population of 45 swine and randomly allocated into one of two experimental groups: G1 (n = 8) or G2 (n = 8). The first group (G1) was fed with the experimental contrast feed amounting to daily doses of 500 mg of Captan/kg of feed, whereas the second group (G2) was given untreated feed and was kept as a control group. The experiment lasted 7 weeks and the total amount of feed given during this period was 4kg/day/pig, divided equally between two feeding periods, one in the morning (09:00 AM) and one in the afternoon (04:00 PM). The feed amount was measured so that there were no leftovers, ensuring complete ingestion of the experimental contrast.

The Captan concentration in the feed was chosen to reflect a relevant case and realistic scenario. The 500 mg/kg concentration reflects the most commonly commercialized products in the Brazilian territory. It represents a moderate dose, but it is considerably lower than the LD50 (lethal dose for 50% of the population) of 7000 mg/kg previously established for mice [²⁸] or the 12,000 mg/kg dose used in previous studies investigating the potential of Captan to induce intestinal tumors in mice [¹⁰].

Euthanasia procedure

Each pig was put under general anesthesia after a period of 7 weeks by administering an intravenous dose of 10 mg/kg of sodium thiopental, and subsequently euthanized by exsanguination [¹¹].

Macroscopic evaluation and material collection

The femur and tibia of the pelvic limbs were dissected following euthanasia. The bones were sectioned longitudinally (in relation to its main axis) using a 250 mm hacksaw (Starrett, Model 152) - Figure 1. Photographs were taken of the exposed sections at the proximal and distal ends on the left side bones using a Cannon Sx400ls camera (16 megapixels) for macroscopic analyses. The proximal and distal portions of the right-side femur and tibia bones were collected and fixed in 10% buffered formalin during 48h for microscopic analyses. The shape and size of the growth plate was analyzed using Adobe Photoshop (v22.x) by comparing lines drawn on each plate between samples of different groups.

Figure 1
Longitudinal section of the proximal region on a tibia.

Decalcification procedure

The femur and tibia samples were decalcified immediately after fixation using a 10% EDTA solution at room temperature for 90 days until the samples became malleable.

Paraffin inclusion

The samples were embedded in paraffin and processed into blocks using a Leica TP1020 automatic tissue processor and a Leica EG1150H embedding station in accordance with the following protocol: sample dehydration in 70%, 80%, 90%, and 100% ethanol, followed by 3 passes through xylene, and 3 paraffin baths performed automatically overnight by the available equipment. Each cycle was one hour long.

Microscopic analyses

The slices made for histopathological analyses were 7 µm thick. Hematoxylin and Eosin stain (HE) was used in order to assess cartilage structure and thickness, while Picrosirius Red (PR) was used to evaluate the collagen/ aggrecan/proteoglycan in the cartilage under white polarized light.

Quantitative analyses

Growth plate cartilage thickness was measured according to [²⁹]. Each cartilage was subdivided into 0-10 equidistant points, totaling 11 points which were averaged and compared between groups. Total collagen area was estimated according [²⁹, ³⁰]. All quantitative analyses were performed using ImageJ (v.1.53r). The data was statistically analyzed by employing ANOVAs and Tukey tests in GraphPad Prism 5.

RESULTS

Macroscopic analyses

Some commonly observed alterations were noted in the shape of the growth plate cartilage due to the higher weight of domesticated swine. However, these alterations were not significantly different between the femurs and tibiae of the two experimental groups (Figure 2).

Figure 2
Superposition of the growth lines in sampled bones. Red lines represent the control group, while blue lines represent the measurements from individuals dosed with Captan. A - Proximal portion of the femur; B - Distal portion of the femur; C - Proximal portion of the tibia; D - Distal portion of the tibia.

Microscopic analyses

Results from the proximal and distal regions of both the femurs and tibiae are presented together since they were very similar. Bone morphology for the control group was unremarkable as the reserve, proliferative, and hypertrophic zones maintained their expected morphological characteristics. In the reserve region, chondrocytes with rounded to flatter shapes are observed scattered in the extracellular matrix. The proliferative region has several rows of chondrocytes and between them we observe bundles of extracellular matrix. The hypertrophic region already has larger chondrocytes, some with more rounded nuclei, and these cells have an even more disorganized appearance within the extracellular matrix (Figure 3).

Figure 3
Growth plate cartilage of control group samples. 1 - Reserve zone; 2 - Proliferative zone; 3 - Hypertrophic zone. Black arrows - chondrocytes

Although the Captan absorption route certainly occurs via the perichondrium/periosteum, no changes were observed in its structure and cells in the treated groups, which is why these structures were not addressed in this study.

The bone of individuals exposed with Captan displayed morphological alterations mainly in the proliferative and hypertrophic zones. All swine exposed to the Captan suffered from chondrocytes disorganization and the cartilage tissue zones completely lost their identifying characteristics (Figure 4 A and B).

Specific signs of tissue lesion were found that should be highlighted, that is, the presence of necrotic chondrocytes and increases in the cartilaginous matrix area (Figure 5). Fibrous tissue incursions were noted forming bundles and/or small trabeculae randomly across the cartilaginous matrix (Figure 4C and 5A and B). Additionally, neovascularization with erythrocytes was noted (Figure 5A). These results indicate higher blood flow in that region (Figure 4C and 4F). We confirmed the aforementioned morphological changes with the results from the Picrosirius Red stained samples (Figure 8).

Figure 4
A - Tissue disorganization in the hypertrophic zone of the growth plate cartilage at the proximal end of a femur collected from a specimen dosed with Captan. B - Tissue disorganization in the proliferative zone of the growth plate cartilage at the proximal end of a tibia belonging to the Captan exposed group. C - Growth plate cartilage from the proximal end of a femur collected from a specimen exposed with Captan. Yellow circles - Signs of tissue fibrosis with invading connective tissue trabeculae.

Figure 5
A Growth plate cartilage from the distal end of a femur collected from a specimen dosed with Captan. B and C - Growth plate cartilage from the proximal and distal ends respectively of a tibia belonging to the Captan exposed group. Black arrows - Necrotic chondrocytes. Yellow circles - Signs of tissue fibrosis with invading connective tissue trabeculae. Red Cicles - neovascularization

Quantitative analyses

Cartilage thickness did not differ between groups in a statistically significant manner (p > 0.05) as illustrated in Figure 6.

Figure 6
Average growth plate cartilage thickness on the proximal and distal ends of femurs and tibiae.

Individuals exposed to Captan presented significantly larger collagen/aggrecan/proteoglycan areas with p < 0.05 (Figure 7). This was most pronounced in the reserve and proliferative zones (Figure 8).

Figure 7
Average area occupied by total collagen/ aggrecan/proteoglycan in bones from the control and Captan treatment groups. It should be noted that the area occupied both proximal and distal regions of the bones is higher for samples from the treatment group in comparison to the control group samples. * Significantly differed from the control group measurements with p < 0.05.

Figure 8
A - Total collagen/ aggrecan/proteoglycan in the growth plate cartilage on the proximal end of a femur from the control group stained using Picrosirius Red. B and C - Total collagen/ aggrecan/proteoglycan in the growth plate cartilage on the proximal end of a femur from the Captan exposure group stained using Picrosirius Red.

DISCUSSION

Captan is a source of grave concern both in Brazil and globally due to common practices by farmers to use it in doses well above the recommended limits and its potential side effects for non-target co-occurring species. One such case investigated [³¹] showed a dose 4 times higher than recommended being used in order to control a disease caused by the fungus Neonectria ditissima. The species is commonly found in apple orchards and seemed to have developed a resistance to the pesticide [³¹]. Further scientific studies show that there is a real probability that Captan is being used in concentrations that far exceed the recommended doses. Previous research investigating honey samples found that 75% of the honey in the world contains quantifiable amounts of pesticides. These are being detected in honey even after being absorbed by the plant and undergoing transformation by bees. This is specially concerning if we consider that these compounds could impact not only honey bees which are environmental sentinels and an important biomonitoring species in environmental impact assessment studies, but also humans [³²]. The aforementioned study found Captan concentrations in honey to be approximately 50% of that used on the field. In fact, the concentrations measured for honey samples were significantly higher than the Maximum Residue Limit (MRL) permitted for Captan, representing a percentage of the maximum of 103.04% which is not appropriate for human consumption [³³]. This highlights how Captan has a high residual rate and can be found directly in plants or secondarily in plant-derived products which are consumed by human beings and/or animals.

Captan exposure can result in several toxic effects already described in literature. For example, it may impact the growth of the walking catfish Clarias batrachus by disrupting physiological and biochemical processes [³⁴], induce behavioral alterations on worms of the Branchiura sowerbyi species [³⁵], induce apoptosis in eggs cells from mice [³⁶], and result in morphological changes in the hepatocytes of Sus scrofa swine [¹¹]. Although several studies have been conducted on its toxicity, the present study is the first work to date addressing the effects of Captan exposure on cartilage associated with body growth.

The specimens in the present study were 140 days (4.7 months) old at the moment of euthanasia. Works centers comparing the development of humans and pigs, suggests that the developmental stage of a 6-month-old swine is comparable to that of a 10-year-old human child [³⁷]. Therefore, the results from the present study about the effects of Captan exposure on swine growth plate tissue may be considered analogous to cases involving children between 7.5- and 8-year-old. Evidence of the effects on growth plate tissue from exposure to different pesticides has been previously described by Maghfiroh and coauthors [³⁸] and Huang and coauthors [³⁹]. They reported cell death and decreased vascularization in growth plate tissue of chickens due to exposure to a pesticide named Thiram used in crops employed as feed in the poultry industry. Furthermore, Huang and coauthors [⁴⁰] have reported various impacts from heavy metals and organic contaminants on bone and cartilage development in the zebrafish Danio rerio.

The exposure to propiconazole may impact gene expression in swine regardless of their weight, specifically the expression of genes involved in fibroplasia on different tissues (e.g. liver, kidney, muscle, and ileum) [⁴¹]. The study exposed swine to the propiconazole fungicide for 28 days, resulting in severe morphological alterations. The current study reports on findings using the Picrosirius Red stain that are similar to those of Garg and coauthors [⁴²] with higher amounts of growth plate extracellular matrix being found in tissue samples belonging to the group exposed to Captan rather than the control group. Craig and coauthors [⁴³] showed that exposure to organophosphate, pyrethroid, and chlorinated pesticides may result in thinner cartilage discs in the growth plate due to decreases in cell density. Our current findings partially corroborate those of Shapiro [⁴⁴] since we also observed cell death in the cartilage tissue, albeit without a reduction in disc thickness. This could be explained by a buildup of the connective tissue infiltrating the affected areas.

Osteochondrosis (OCD) is one of the main diseases afflicting the long bones of swine. It is characterized by a failure in the endochondral ossification process which may occur either on the physeal portion of the growth plate and/or on the articular epiphyseal cartilage. This disease is multi-factorial in that several factors can interact to result in its manifestation: genetics, physical trauma, nutrition, and growth rates. As such, it may develop into one of three types: (1) Latens - necrosis of ischemic origin focused on the cartilage associated to body growth but not articular cartilage, manifesting without clinical symptoms; (2) Manifesta - retention of necrotic cartilage on the subchondral bone as a result of endochondral ossification failure which may manifest with or without clinical symptoms; (3) Dissecans - necrosis which desiccates (flap) the articular cartilage, forming cracks and clefts in the tissue, and occasionally manifesting with clinical symptoms [⁴⁵].

The primary mechanism responsible for OCD injuries revolves around a lack of blood supply (ischemia) to the cartilage. Articular cartilage is avascular however, physeal cartilage receives nutrients from blood vessels running within cartilage channels. Should any of the contributing factors impact structural and/or functional aspects of this cartilage, the blood vessels within the channels may regress and the channels become filled with more cartilage (chondrification) which may lead to the development of local ischemia and cartilage necrosis [⁴⁵]. These lesions are found more frequently at specific regions of swine anatomy: at the distal portions of the femur and ulna, at the femoral and humerus heads, at the ischial tuberosity, and at the costochondral junctions. Complete rupture of the physis may also occur, separating the epiphysis from the rest of the bone, configuring a condition named epiphysiolysis [⁴⁵]. The morphology of cartilage channels is fairly similar across vertebrates [⁴⁶]. Therefore, the knowledge gained on the mechanisms responsible for OCD injuries in other vertebrates can also be applied to humans [⁴⁷].

Raimann and coauthors [⁴⁷] compared the development of vertebrae and long bones between swine of different age groups - i.e. between 2 and 6 weeks old. They found similar results to ours regarding growth and morphological characteristics. However, the reserve, proliferative, and hypertrophic zones in the vertebrae were considerably smaller than the same zones in long bones. This means that the toxicity effects observed on the growth plate of long bones could also theoretically be found in vertebrae. Therefore, the results of the present study in conjunction with the findings from Raimann and coauthors [⁴⁷] suggest that the degenerative and fibroblastic alterations found in the femur and tibia presently may manifest even more aggressively in flat and curved bones. The appearance of blood vessels in the cartilage, associated with injuries, can lead us to two options. The first would be the evolution of this condition into a tumor condition, with this contamination with the pesticide being a factor in promoting tumors [⁴⁸]. Another relevant point that we can highlight would be the appearance of these vessels linked to cartilage remodeling. This could occur due to the need for restructuring after tissue destruction after exposure to Captan [⁴⁹]. We believe that chronic experiments should be carried out to verify the final fate of these lesions.

Our study used the swine as an experimental model, and its results can be extrapolated to children aged 7.5 to 8.0 years. As seen, the replacement of cartilaginous tissue by fibrous tissue produces catastrophic effects for the development of the growth plate. As the possibility to accelerate the failure in the endochondral ossification process, as well as the loss of resistance, traction, and shear of the cartilaginous tissue. Both situations in the face of risk factors such as trauma and obesity in children can initiate early insidious changes, which in the future will become osteoarthritis, one of the main diseases of old age in humans [⁴⁷, ⁵⁰, ⁵¹]. Together, these results suggest that exposure to Captan for long periods may become a risk factor especially for children before puberty in triggering osteoarthritis.

Acknowledgments

The authors are grateful to Ana Cláudia Tenfen Das Chagas Lima for technical support at the beginning of this study.

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• Funding:
  This research was funded by Fundação de Amparo a Pesquisa do Estado de São Paulo - FAPESP, grant number 2021/03091-3.

Edited by

• Editor-in-Chief:
  Paulo Vitor Farago

• Associate Editor:
  Renata Marino Romano

Publication Dates

• Publication in this collection
  15 Nov 2024
• Date of issue
  2024

History

• Received
  15 Feb 2024
• Accepted
  20 July 2024