Abstract
Anurans are exposed to several pollutants. One of these is benzo[α]pyrene (BaP). This compound is produced by incomplete combustion and is toxic to the liver and intestine, where it is metabolized. Here, we tested how different concentrations of BaP affect the thickness of small intestine and liver melanomacrophages (MMCs) of Hypsiboas albopunctatus during short- and long-term exposures. We conducted an experiment with a 3 × 2 factorial design to answer these two questions. Male specimens were separated into groups injected with either 3 or 7 mg/kg of BaP and euthanized after either 72 or 168 h. Then, we measured the thickness of the intestinal epithelium and the area occupied by MMCs. The thickness of intestinal epithelium decreased in both high and low concentration for short-term exposure compared to control, and increased in the long-term group in both low and high concentrations. The short-term decrease in thickness is due to the damage caused by BaP on the absorptive capacity of the epithelium, whereas the epithelium increased its thickness and recovered normal activity in the long-term. High BaP concentration decreased the area of MMCs in the short-term group. The increase in MMCs is associated with the detoxifying role of these cells, while the decrease was triggered by cellular stress due to high BaP concentration. The concentrations of BaP we used are close to those found in polluted environments. Therefore, water contaminated with BaP can potentially affect the morphology of internal organs of anurans.
Introduction
Because anurans have a biphasic life cycle, they can be exposed to pollutants in both aquatic and terrestrial ecosystems. Benzo[α]pyrene (BaP) is a mutagenic, carcinogenic, hepatotoxic, and neurotoxic compound. It also acts as an endocrine disruptor (IARC, 2012; Madureira et al., 2014; Pastore et al., 2014; Gao et al., 2015; Regnault et al., 2016). BaPs belong to the class of Polycyclic Aromatic Hydrocarbons (PAHs) and are formed from incomplete combustion (Page et al., 1999), frequently as a result of forest fires and oil spills (Hartl, 2002). PAHs accumulate in sediments and soils and are also easily adsorbed and bioaccumulated in organisms (Neff, 1984). BaP has been detected in the soil, water, air, and even food (Caruso and Alaburda, 2008), potentially affecting both terrestrial and aquatic ecosystems (Hylland, 2006). BaP can enter an organism via dermal, oral, or respiratory ways (Netto et al., 2000). After intake, BaP reaches organs of the digestive system and their glands, which metabolize it and generate toxic by-products (James et al., 1997; Cavret and Feidt, 2005; Harris et al., 2009; Reynaud et al., 2012; Madureira et al., 2014).
The small intestine is one of the components of the digestive system of anurans responsible for the chemical digestion of food and absorption of nutrients (Moore, 1964; Duellman and Trueb, 1994). Intestines of fish (Van Veld, Patton and Lee, 1988; Lemaire et al., 1992; James et al., 1997) and mammals (Artrup et al., 1977; Stohs et al., 1977; Artrup et al., 1982; Cavret and Feidt, 2005) can metabolize BaP. This metabolic activity is performed by epithelial cells that constitute the mucous layer (Stohs et al., 1977; Cavret and Feidt, 2005).
The liver is one of the accessory glands to the digestive tract. Its main components are hepatocytes, which play key roles in several metabolic processes, such as protein synthesis, bile secretion, and metabolite storage. This organ is also responsible for processing and storing absorbed nutrients (Akiyoshi and Inoue, 2012). The liver of amphibians has a special cell type called melanomacrophages (Oliveira and Franco-Belussi, 2012).
Melanomacrophages are pigment-containing cells that occur in the liver, spleen, and kidney of fish, squamates, and amphibians (Wolke, 1992; Loumbourdis and Vogiatzis, 2002; Fishelson, 2006). Their number, size, and pigment amount vary according to species, age, and stress (Agius, 1981). Melanomacrophages produce and store pigments, such as: melanin, hemosiderin, and lipofuscin and can aggregate to form Melanomacrophage Centres (MMCs) (Oliveira and Franco-Belussi, 2012). These cells have bactericide functions (Franco-Belussi, Castrucci and Oliveira, 2013), besides degrading and recycling toxic substances (Agius and Roberts, 2003; Passantino et al., 2014). MMCs are often used as biomarkers of environmental contamination, since their melanin increase with hepatic stress, temperature (Santos et al., 2014), exposure to toxic substances, and infectious agents (Roberts, 1975; Wolke, 1992; Regnault et al., 2014). Usually, melanomacrophages deal with pollutants that are metabolized in the liver by removing the by-products that have undergone phagocytosis by macrophages (Fournie et al., 2001).
Previous studies found that a PAH, the 7,12-dimethylbenz(a)anthracene (Abdelmeguid et al., 1999; Al-Attar, 2004) decreases the hepatic enzymatic function in anurans. The sensitivity of anuran larvae to aquatic pollutants is well documented (Camargo and Alonso, 2006; Marquis et al., 2009; Erismis, Ciğerci and Konut, 2013). Conversely, the effects of pollutants on adult anurans are usually poorly known (but see Colombo et al., 2003), specially on their internal morphology. There are a few studies with BaP, and most of them tested its toxic effects on the liver of fish (Padrós, Pelletier and Ribeiro, 2003; Malmström et al., 2004). BaP increased the activity of Glutathione S-Transferase (GST) in fish in the short (8 days), but not in the long term (32 days; Padrós, Pelletier and Ribeiro, 2003). BaP also increased the levels of another hepatic enzyme, Ethoxyresorufin-O-deethylase (EROD) in two fish species (Malmström et al., 2004). Most studies testing the effects of BaP on anurans focused on larval stages (Reynaud et al., 2012; Regnault et al., 2014, 2016). Previous studies (e.g., Padrós, Pelletier and Ribeiro, 2003) have experimentally manipulated the time of exposure to BaP. However, no study has tested both the concentration and time of exposure to BaP on internal morphology of anurans in a crossed factorial experiment.
Hypsiboas albopunctatus is a hylid species widely distributed in Brazil, Argentina, Uruguay, Paraguay, and Bolivia (Frost, 2016), where it calls perched on shrubs near permanent or temporary ponds and streams (Guimarães et al., 2011). Laboratory experiments with wild species are essential to test possible effects of contaminants on their life history. This is the first study to test the effects of BaP on internal morphology of adult anurans at concentrations found in the environment (see table 1). We hypothesize that the time of exposure to BaP will increase both MMCs area and thickness of intestinal epithelium.
Effects of benzo[α]pyrene on different taxa.
Methodology
Specimen sampling
We collected 36 adults of Hypsiboas albopunctatus in the northwestern region of São Paulo state, Brazil (RAN/IBAMA/MMA 18573-1) during the reproductive period (December 2012 to February 2013). Animals remained in acclimatization at room temperature (27 ± 0.5°C) and natural photoperiod for one week prior to experiments. During the experiment, animals were kept in boxes (28 × 21 × 15 cm) containing 2 cm of moist soil and fed on termites.
Experimental design
Our experiment had a 3 × 2 fully-crossed factorial design in which we varied the time of exposure to BaP (72 and 168 h) and the concentration of the compound (control, 3, and 7 mg/kg). Each experimental group had six animals (replicates). We injected animals with benzo[α]pyrene diluted in 0.02 ml of Nujol® mineral oil (Mantecorp Indústria Química e Farmacêutica Ltda., Brazil) subcutaneously every 48 h, following Lemaire et al. (1990), and euthanized them either 72 or 168 h. For example, one group of six animals received 3 mg/kg of BaP twice (every 48 h) and were euthanized after 72 h of the first injection (see Fanali et al., 2017). The control group received only mineral oil. The half-life of BaP in the liver of fish is 48 h (Lemaire et al., 1990). Thus, here we tested for both short- and long-term effects of BaP.
We applied subcutaneous injections on the dorsum, instead of intraperitoneal in the ventral region, as in fish (Padrós, Pelletier and Ribeiro, 2003; Malmström et al., 2004), due to the smaller size of anurans. This was necessary to avoid damage to the organs, since the skin musculature of H. albopunctatus is very thin. The concentrations of BaP used in the experiment were also adapted from fish.
Histological processing
Fragments of the liver and of the posterior portion of the small intestine (ileum) were fixed in Karnovsky solution (0.1 M Sørensen phosphate buffer, phosphate buffer pH 7.2 containing 5% paraformaldehyde and 2.5% glutaraldehyde) for 24 h at 4°C. Thereafter, samples were washed in water, dehydrated in ethanol, and embedded in historesin (Leica Historesin embedding kit, Switzerland). Sections of 2 μm were obtained in microtome (RM 2265, Leica, Switzerland), stained with Hematoxylin-eosin, and observed under a microscope (Leica DM4000 B) equipped with an image capture system (Leica DFC 280).
Anatomy and histopathology
Specimens were euthanized in benzocaine solution (0.5 g/100 ml water). All experimental procedures were approved by the Committee in Research Ethics (#072/2013) of our university, and were are also in accordance with the Guide for Care and Use of Laboratory Animals (US National Research Council Committee, 2011).
After dissecting the organs, we visually checked for any abnormalities in the following parameters: size, position, colour, necrotic areas, superficial vascularization, edema, and haemorrhage. After histological processing, cuts were analysed to detect possible histopathological changes of vacuolization, mononuclear cell infiltration, and hypervascularization.
Morphometric analysis
The pigmented area in the liver was quantified based on differential staining intensity (Santos et al., 2014). We randomly took 25 pictures (200× magnification) from 25 histological sections per animal to quantify melanomacrophage area. All measurements were performed using Image Pro-Plus v. 6.0 (Media Cybernetics Inc.).
We randomly captured 25 pictures (100× magnification) per animal of histological sections of the intestinal epithelium. In each picture, we took 30 measurements of the epithelium thickness, i.e., from the basal lamina to the luminal surface. For further analysis, we averaged these 30 measurements, so as to have only one measurement per picture and 25 measurements per animal.
Statistical analysis
Our experiment had a 3 × 2 factorial design with three treatments (two concentrations of BaP, plus a control), crossed with two exposure times (72 h and 168 h). We only could use five out of six animals per treatment to measure intestinal epithelium thickness. The sampling units (Pictures), in which we estimated our response variables, were nested within each Animal (true replicate).
Results of the linear mixed-effects models for intestinal epithelium thickness and area of Melanomacrophage centres (MMC). The levels of the treatments with 3 mg and 72 h were taken as reference levels to test significance.
To model intestinal epithelium thickness (continuous response variable), we fitted a Linear Mixed-Effects Model (Zuur et al., 2009) with restricted maximum likelihood (REML; Bolker et al., 2009) including treatment (categorical predictor with three levels) and time of exposure (categorical predictor with two levels) as fixed factors, along with their interaction. To control for dependency among the 25 pictures from the same animal (Crawley, 2012: 703), we included a random intercept for animal (categorical with 5 levels; see Moen et al., 2016). To model melanomacrophage area, we used the same model with the response variable log-transformed. Analysis was conducted using the R v. 3.3.2 (R Core Team, 2016) package lme4 (Bates et al., 2015). We tested model assumptions using diagnostic plots with the R package sjPlot (Lüdecke, 2016). Residuals of both models had homogeneity of variance and normal distribution (see Fanali et al., 2017). We used least-squares means to test for differences among levels of treatment and time of exposure with the R package lsmeans (Lenth, 2016). Models were summarized and the P-values estimated based on conditional F-tests with Kenward and Roger (1997) approximation for the degrees of freedom using sjPlot.
The relationship between the thickness of intestinal epithelium and MMCs area was tested using a Pearson’s product-moment correlation. The thickness of intestinal epithelium and MMCs area were not correlated (Pearson’s ; ) in the experiment. Therefore, the responses of these organs to BaP were independent. All data, a chart explaining the experimental design, and an R Markdown dynamic document with the description of analyses are available at FigShare (Fanali et al., 2017).
Results
Anatomy and histopathology
The gross anatomical characteristics of both organs were very similar between control and treated groups regarding size, position, colour, and superficial vascularization. There was no vacuolization, mononuclear cell infiltration, or hypervascularization. However, both intestinal epithelium thickness and melanomacrophage morphology changed with BaP treatment.
Morphological effects of BaP
The interaction between time of exposure and concentration of BaP was significant for both organs (table 2), showing that the response of the intestine and liver MMCs to BaP depended on these two factors manipulated in the experiment. Changes in the intestinal epithelium (fig. 1) occurred in both short- and long-term exposures (72 and 168 h) and low and high concentrations (3 and 7 mg/kg). However, the response of MMCs (fig. 2) occurred in both low and higher concentration, but only in the short-time exposure group.
Representative images of the variation in the thickness of intestinal epithelium of Hypsiboas albopunctatus exposed for short- and long-term to different concentrations of benzo[α]pyrene. Coloration: Hematoxilin-eosin. Bars: 5 μm.
Citation: Amphibia-Reptilia 38, 2 (2017) ; 10.1163/15685381-00003101
Representative images of the variation in the thickness of intestinal epithelium of Hypsiboas albopunctatus exposed for short- and long-term to different concentrations of benzo[α]pyrene. Coloration: Hematoxilin-eosin. Bars: 5 μm.
Citation: Amphibia-Reptilia 38, 2 (2017) ; 10.1163/15685381-00003101
Representative images of the variation in the thickness of intestinal epithelium of Hypsiboas albopunctatus exposed for short- and long-term to different concentrations of benzo[α]pyrene. Coloration: Hematoxilin-eosin. Bars: 5 μm.
Citation: Amphibia-Reptilia 38, 2 (2017) ; 10.1163/15685381-00003101
Representative images of variation in the area of melanomacrophages (MMCs) of Hypsiboas albopunctatus exposed for short- and long-terms to different concentrations of benzo[α]pyrene. Arrows indicate MMCs in the liver tissue. Coloration: Hematoxilin-eosin. Bars: 25 μm.
Citation: Amphibia-Reptilia 38, 2 (2017) ; 10.1163/15685381-00003101
Representative images of variation in the area of melanomacrophages (MMCs) of Hypsiboas albopunctatus exposed for short- and long-terms to different concentrations of benzo[α]pyrene. Arrows indicate MMCs in the liver tissue. Coloration: Hematoxilin-eosin. Bars: 25 μm.
Citation: Amphibia-Reptilia 38, 2 (2017) ; 10.1163/15685381-00003101
Representative images of variation in the area of melanomacrophages (MMCs) of Hypsiboas albopunctatus exposed for short- and long-terms to different concentrations of benzo[α]pyrene. Arrows indicate MMCs in the liver tissue. Coloration: Hematoxilin-eosin. Bars: 25 μm.
Citation: Amphibia-Reptilia 38, 2 (2017) ; 10.1163/15685381-00003101
Small intestine
The mean thickness of the intestinal epithelium of the low concentration group decreased about 14% and about 7% in the high concentration one compared to the control in the short-term exposure (table 2; fig. 3A). Conversely, the epithelium thickness of the low concentration group increased approximately 20% after long-term exposure in relation to the control, while it increased only 18% in the high concentration one (fig. 3A).
(A) Thickness (μm) of intestinal epithelium, and (B) Area (μm2) of liver melanomacrophages of H. albopunctatus exposed for short- (72 h) and long-term (168 h) to different concentrations of benzo[α]pyrene. Different letters indicate significant differences between groups based on least-square means (). Data are presented as mean ± SE.
Citation: Amphibia-Reptilia 38, 2 (2017) ; 10.1163/15685381-00003101
(A) Thickness (μm) of intestinal epithelium, and (B) Area (μm2) of liver melanomacrophages of H. albopunctatus exposed for short- (72 h) and long-term (168 h) to different concentrations of benzo[α]pyrene. Different letters indicate significant differences between groups based on least-square means (). Data are presented as mean ± SE.
Citation: Amphibia-Reptilia 38, 2 (2017) ; 10.1163/15685381-00003101
(A) Thickness (μm) of intestinal epithelium, and (B) Area (μm2) of liver melanomacrophages of H. albopunctatus exposed for short- (72 h) and long-term (168 h) to different concentrations of benzo[α]pyrene. Different letters indicate significant differences between groups based on least-square means (). Data are presented as mean ± SE.
Citation: Amphibia-Reptilia 38, 2 (2017) ; 10.1163/15685381-00003101
Liver
The response of MMCs to BaP depended on exposure times (table 2; fig. 3B). The area of MMCs of the treated groups increased 20%, but was not different from the control of the long-term exposure. However, for the short-term exposure it significantly increased 35% in the low concentration, decreasing to levels lower than the control in the high concentration.
Discussion
Intestinal epithelium
The ileum of H. albopunctatus had no histopathological lesions other than the change in epithelial thickness at the concentrations used. However, previous studies have shown exfoliation of cells and foci of necrosis in the intestines of fish exposed to hydrocarbons (Hawkes, Gruger and Olson, 1980). An insecticide (carbaryl) caused the collapse of villi of intestinal cells of lizards, besides disintegration of epithelial cells in small intestine (Çakici and Akat, 2012). Carbaryl also caused necrosis, hemorrhage, vacuolization, and inflammation in intestinal cells of anurans (Çakici, 2014, 2016). However, BaP did not induce histopathological lesions in the intestine in our study. This is probably due to the different action pathways of these two substances on the epithelial cells. The way the substance is administrated to the animal can also be responsible for these differences (see Lemaire et al., 1992). Lemaire et al. (1992) administrated BaP orally to the animals, making it directly in contact with intestinal epithelial cells, whereas we injected it in the dorsum of animals.
The thickness of the intestinal epithelium decreased in the short term and increased in the long term. In the short term, BaP increased the number of lysosomes in enterocytes of fish (Lemaire et al., 1992). Accordingly, the increase in lysosome may be involved in epithelial cell degeneration of anurans during metamorphosis (Bonneville, 1963). Therefore, it is reasonable to suppose that BaP may have decreased the thickness of epithelium in the short term by increasing the number of lysosomes in the intestinal epithelial cells. With continuing exposure, there is hypertrophy of both the endoplasmic reticulum and Golgi apparatus in fish (Lemaire et al., 1992), increasing cell size. Both organelles are associated with adaptive responses to hydrocarbon intoxication (Lemaire et al., 1992). Thus, the thickness of the epithelium may have increased as a result of changes in these organelles caused by long-term exposure to BaP.
Other substances also cause atrophy of epithelial cells, like lambda-cyhalothrin in fish (Velmurugan et al., 2007) and mycotoxins in birds (Girish and Smith, 2008). These alterations compromise the digestive and absorptive activity of the intestine, causing feeding dysfunctions. Consequently, the reduction of thickness of epithelium reported here may well compromise the absorption of nutrients in the intestine of H. albopunctatus.
Liver and MMCs
We have found neither macroscopic nor histological lesions in the liver. However, fish exposed to BaP up to 17 days have developed degenerative changes in the liver (Lemaire et al., 1992). Similarly, previous studies have found necrosis and basophilic foci in fish (Ribeiro et al., 2007; Oliveira et al., 2015) exposed to BaP up to 56 days. We probably did not detect any changes in the treated groups due to the short term exposure, compared to those previous studies.
Melanomacrophage area increased in the low concentration, with a fall in the high concentration in the short-term group, whereas there was a slight increase in the high concentration in the long-term treatment. BaP increases lipid content in the liver of amphibians, which in turn induce oxidative stress (Regnault et al., 2014, 2016). Melanin has an anti-oxidant function. Therefore, the melanin in melanomacrophages may have increased as a physiological response to oxidative stress caused by BaP. The pigmented area in the liver of Xenopus tropicalis exposed to 10 μg/l of BaP for 12 h decreased. This may be related to hepatic stress and hepatocytes apoptosis (Regnault et al., 2014). At low concentration of PAHs, melanomacrophages can still remove cellular debris by increasing phagocytosis, while phagocytosis is impaired at high PAH concentration (see Weeks and Warinner, 1984), leading to a decrease in the area of MMCs (Kranz, 1986 apud Payne and Fancey, 1989). Therefore, our results showing an increase followed by a decrease in MMCs area in the short term may be related to the dysfunction of their phagocytic activity caused by BaP.
The area of melanomacrophages in the long-term was lower than the short-term treatment, but it did not change in the treated groups compared to the control. The herbicide glyphosate alters cell pH at a low concentration (6 g/l), inhibiting intracellular transport by degrading the cytoskeleton (Hedberg and Wallin, 2010). This alteration prevents transport of melanosomes, changing both cell morphology and functions. BaP may have caused a similar dysfunction in MMCs in the long term, making melanin less abundant inside the cell.
Conclusion
Benzo[α]pyrene decreased the thickness of the intestinal epithelium and increased the area of MMCs of H. albopunctatus in the short term. These responses are probably related to the primary function of each organ. Although both organs are part of the digestive system, they had different morphological and functional responses to BaP. Morphological changes in organs that metabolize xenobiotics may have systemic effects, since they can release toxic by-products in the circulation and consequently damage animal health.
Conflicts of interest
None declared.
Acknowledgements
FAPERP provided financial support for translating (233/2014) and revising (040/2016) this manuscript. FAPESP provided financial support to this project (2013/02067-5), a master’s fellowship to LZF (2013/05907-4), a scientific initiation fellowship to BSLV (2013/18469-5), and post-doctoral fellowship to LFB (2014/00946-4) and to DBP (2016/13494-7). LFB was also supported by a CAPES-PNPD postdoc fellowship during the final preparation of this manuscript. CO received a grant (305081/2015-2) and a fellowship from CNPq.
The authors thank Rodolfo M. Pelinson for helping with field work, Ruth V. Kakogiannos for helping with the translation, Ivã G. Lopes for revising the translation, and Luiz R. Falleiros Jr. for helping with the staining. Author contribution statement: LZF designed the experiment and collected specimens. LZF and BSLV conducted the experiment and collected the data, assisted by LFB. LZF, BSLV, LFB and CO prepared the first draft of the manuscript, to which all authors contributed subsequently. DBP revised the text, conducted data analysis, curation, and presentation. CO contributed chemicals and equipment.
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Footnotes
Associate Editor: Caitlin Gabor.