Histological liver chances in Swiss mice caused by ... - Springer Link

Environ Sci Pollut Res https://doi.org/10.1007/s11356-017-0647-1

RESEARCH ARTICLE

Histological liver chances in Swiss mice caused by tannery effluent Letícia Martins Rabelo 1 & Abraão Tiago Batista Guimarães 2 & Joyce Moreira de Souza 2 & Wellington Alves Mizael da Silva 2 & Bruna de Oliveira Mendes 1 & Raíssa de Oliveira Ferreira 1 & Aline Sueli de Lima Rodrigues 2 & Guilherme Malafaia 2,3

Received: 4 November 2016 / Accepted: 31 October 2017 # Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract Although tannery effluents are known for being highly toxic to organisms, reports about the effects of the intake of these xenobiotics on experimental mammal models are recent. Studies about the damages the chronic intake of these effluents can cause in the liver of outbred mice remain an unexplored field. Thus, the aim of the present study is to assess (histological) the hepatic condition of Swiss mice (outbred strain) chronically exposed to the intake of different raw tannery effluent concentrations diluted in water for 150 days. Accordingly, the mice (males and females) were divided in the following groups: control group—animals treated with drinking water, only; and groups 5 and 10%—treated with raw tannery effluent diluted in water. After exposure, the animals were subjected to euthanasia for liver fragment sample collection and histological analysis, respectively. Moderate hydropic degeneration was observed in the centrilobular regions of the

liver of mice exposed to 5 and 10% tannery effluent, as well as greater amounts of hepatocytes presenting karyomegaly and necrotic hepatocytes, and a smaller amount of Kuffer cells in the liver of mice exposed to the xenobiotic. Finally, animals exposed to 10% tannery effluent showed mild hyperplasia of the bile ducts in the portal areas and fibroblast proliferation around the bile ducts, thus suggesting a fibrous process. Except for the frequency of hepatocytes presenting karyomegaly (lower in females), the herein observed hepatic changes were similar in male and female Swiss mice. Accordingly, the present data support the hypothesis that the chronic intake of tannery effluent by outbred mice (Swiss) causes damages in the liver, a fact that broadens the knowledge about the toxic potential of this pollutant, which goes beyond that of C57Bl/6J male mice (inbred strain).

Responsible editor: Philippe Garrigues

Introduction

* Guilherme Malafaia [email protected]

Countries such as India, China, Pakistan, and Brazil consider the tannery industry an important socio-economic development driver in regions it is located in. This industry participates in different production chains, because it purchases raw material from slaughterhouses (skin), generates jobs (directly and indirectly), and exports fur and leather (for furniture, footwear, clothing, automotive spare parts industries). Besides, it is one of the main raw material suppliers to different industries (China and Ndaro, 2015; Sabumon, 2016). Although the tannery activity is of great socio-economic importance, it is also of great concern, since tannery activities over pollute the environment (Manivasagam, 1987). The large amount of waste

Aline Sueli de Lima Rodrigues [email protected] 1

Laboratório de Pesquisas Biológicas, Instituto Federal Goiano– Campus Urutaí, Urutaí, GO, Brazil

2

Programa de Pós-Graduação em Conservação de Recursos Naturais do Cerrado, Instituto Federal Goiano–Campus Urutaí, Rodovia Geraldo Silva Nascimento, 2,5 km, Zona Rural, Urutaí, GO CEP: 75790-000, Brazil

3

Programa de Pós-Graduação em Biodiversidade Animal, Universidade Federal de Goiás–Campus Samambaia, Goiânia, GO, Brazil

Keywords Tannery wastewater . Animal model . Histopathology . Xenobiotics . Toxicity

Environ Sci Pollut Res

(solid or liquid) that in many circumstances discarded in watercourses without any or with inadequate/ineffective treatment has negative impact on ecosystems and on the biota (Roy et al., 2015). With regard to studies related to mammals’ exposure to tannery effluent, recent results point out the potential toxicological effects of it on this group of animals. Siqueira et al. (2011), Rabelo et al. (2016), Souza et al. (2016a), Guimarães et al. (2016a, b), Almeida et al. (2016), and Silva et al. (2016) have considered the potential of many metals or toxic organic compounds found in effluents to damage the central nervous system of animals causing anxiolytic/anxiogenic and depression/antidepressant effects on rodents, as well as memory deficits. Despite the complexity of these xenobiotics, a fact that would explain these different results, there is consensus that the exposure to tannery effluent causes a range of relatively complex effects. Aspects such as sex, age, effluent type (from different bovine skin processing stages), species, and the strain of the studied rodent, as well as effluent concentration and exposure route, are factors directly influencing research results. On the other hand, so far, only the study by Souza et al. (2016b) has focused on the histological evaluation of mammals exposed to drinking water added with tannery effluent. They histologically assessed the organs of C57Bl/6J male mice chronically treated with different concentrations of lime-type tannery effluent—resulting from the unhairing and liming processes—diluted in water at ratio 0.1, 1, and 5%. Liver changes such as mild hydropic degeneration, necrotic hepatocytes, and a larger number of hepatocytes presenting karyomegaly were identified in animals exposed to higher effluent concentrations (1 and 5%). Souza et al. (2016b) have studied the effects of tannery effluent on C57Bl/6J male mice and pointed out many toxic effects never reported before. In addition, they did not study the females and it was a limitation in their study. However, it is important to notice that the authors assessed an inbred mice strain also called key isogenic, which holds homozygous animals of phenotypic uniformity and long-term stability. Therefore, results of isogenic strains cannot be extrapolated to heterogeneous strains (outbred) resembling natural mammal species able to intake water contaminated with xenobiotics. Such limitation shows the need of further research on the topic. Accordingly, the aim of the present study is to perform a histological evaluation of the liver of male and female Swiss mice (outbred strain) chronically exposed to the intake of different tannery effluent concentrations diluted in water. The herein advocated hypothesis is that these xenobiotics could cause damages in the liver of both male and female Swiss mice.

Materials and methods Animals and experimental design Male and female Swiss mice were obtained and kept in the animal house (vivarium/biotherium) of the Biological Research Laboratory of Goiano Federal Institute-Urutaí Campus (Urutaí, Goiás State, Brazil), under conventional animal house sanitary conditions (without environmental enrichment elements) (from 22C to 24 °C). The animals were kept in collective standard mice polypropylene cages (30 × 20 × 18 cm) covered with antioxidant-treated galvanized wire mesh lids (only five animals per cage). The cages were cleaned three times a week, and the pine shavings were changed (bedding material), as well as the food. Standard rodent diet (Nuvilab CR 1) and fresh water, or water with tannery effluent, were offered ad libitum. All animals were kept under standard 12-h light/dark photoperiod (light was on at 7.00 a.m. and off at 7.00 p.m.). All procedures were approved by the Institutional Committee for Animal Care and Use of Goiano Federal Institute (Goiás State, Brazil) (protocol no. 17/2014) according to the Guide for Care and Use of Laboratory Animals, 8th edition. Animal handling and all the experiments were performed according to the International Guidelines for Animal Welfare. Initially, the 60 animals (40 to 45 days old) were categorized according to the co-variable Bbody mass,^ so that the mean body mass of the experimental groups was statistically equal. Subsequently, mice were distributed in three groups: control—animals treated with drinking water (0% tannery effluent) and groups treated with 5 and 10% raw tannery effluent diluted in drinking water. Animals in groups 5 and 10% were exposed to tannery effluent diluted in water for 150 days. Water and food consumption did not differ between experimental groups (data not shown). Data about effluent generation provided by a local tannery company were based on the company’s operation and on the amount of regulated leather treated on a daily basis at the time the tannery effluent concentrations in the present study were set. Data available about the two watercourses illegally used as effluent receptors were also taken into account to calculate the tannery effluent concentrations. The 5% tannery effluent concentration was set to the largest watercourse and the 10% concentration was set to the smallest one. The name of the company that provided the effluents, as well as that of the receptor watercourses, was kept secret for ethical reasons. Furthermore, the tannery effluent at 5 and 10% concentrations diluted in water corresponded to 1/12 LD50 in mice and 1/6 LD50 in mice, respectively, according to Ferreira et al. (2015).


Tannery effluent The effluent discharged by the tannery industry (Pires do Rio, GO, Brazil) did not contain chromium, because it belonged to the bovine skin removal stage, in other words, the stage prior to leather tanning, when large amounts of chromium salts are often used. The effluent used in the present study was the same used by Souza et al. (2016b). The choice was made to use this effluent type, because many tannery industries discharge it directly into water bodies bordering their properties, and mainly because this effluent does not contain chromium. The physicochemical and chemical analyses of raw tannery effluent and drinking water (Table 1) were carried out according to recommendations by the American Public Health Association (APHA, 2005).

on 10% buffered formalin, embedded in paraffin blocks, and thin-sliced (thickness 5 μm). The hematoxylin and eosin staining technique (HE) was used. The thin-session descriptions were carried out in bright-field Carl Zeiss® optical microscope, Jenaval model, in order to compare the tissue structures of organs removed from animals belonging to different experimental groups. Five liver fragments were collected from each animal (with some adjustments by Souza et al. (2016b)), and the changes in them were classified and quantified through the assessment of ten fields randomly selected from these fragments. The analysis was performed in microscope, by the same observer at magnifications 40×, 100×, and 400×. The images were captured using the image analysis system TSView, version 7.1.

Statistical analysis Histological evaluation The animals were subjected to euthanasia after 150 days of exposure in order to enable the collection of liver samples for histological analysis. These liver fragments were subjected to histological preparation, according to Luna et al. (1968) and Behmer et al. (1976). Briefly, the liver was collected and fixed Table 1 Characteristics of the raw tannery effluent diluted in water at different tannery concentrations and offered to male and female Swiss mice

Initially, the residual normality was assessed through the Shapiro-Wilk test. Bartlett’s test was used to check data homoscedasticity. Subsequently, the quantitative data were subjected to factorial analysis of variance (ANOVA) 3 × 2, using factor 1 Btreatments^ (control (0%), 5, and 10%) and factor 2 Bsex^ (male and female). All analyses were performed in the Assistat software, version 7.7 beta (freely distributed).

Parameters

Tannery effluent (100%)

Drinking water



pH at 25 °C (UpH) Turbidity (NTU) Ammonia nitrogen turbidity (mg.L−1) Total nitrogen (mg.L−1)

8.19 382.00

7.19 < 1.00

7.21 19.10

7.23 38.20

2.10

0.01

0.11

0.22

110.00

1.20

6.64

12.08

Nitrate (mg.L−1)

23.00 72.10

0.30 52.00

1.44 53.01

2.57 54.01

33.61 77.09 9333.33

0.11 0.26 0.50

1.79 4.10 467.14

3.46 7.94 933.78

82,190.00 < 0.01 < 0.10 1.91 < 0.01 5680.00 243.20 2805.00 833.33 122.00 93.32

30.00 0.04 ND 0.09 1.06 5.01 1.21 4.00 1.00 1.60 8.20

4138.00 0.04 0.00 0.18 1.01 288.76 13.31 144.05 42.62 7.62 12.46

8246.00 0.04 0.00 0.27 0.95 572.51 25.41 284.10 84.23 13.64 16.71

Electrical conductivity at 25 ° C (μS.cm−1) Total phosphorus (mg.L−1) Orthophosphate (mg.L−1) Biochemical oxygen demand (BOD) (mg.L−1) Total solids (mg.L−1) Dissolved copper (mg.L−1) Dissolved manganese (mg.L−1) Dissolved iron (mg.L−1) Zinc (mg.L−1) Sodium (mg.L−1) Magnesium (mg.L−1) Calcium (mg.L−1) Sulfur (mg.L−1) Potassium (mg.L−1) Total organic carbon (TOC) (mg.L−1)


Results and discussion The present results evidenced that the exposure to different tannery effluent concentrations diluted in water caused histological changes in the livers of the assessed animals. Moderate hydropic degeneration was observed in the centrilobular region of the liver of animals (male and female) exposed to 5 and 10% tannery effluent (Fig. 1). Such degeneration indicated electrochemical disorders resulting in electrolyte and water retention in the cells (Thoolen et al., 2010). Although the specific action mechanisms of inorganic and organic compounds found in the effluent have not been investigated in the present study, it can be assumed that these agents probably caused such disturbance, because they acted in the appropriate functioning of the ion channels in the membrane (cytoplasmic and organelles). Therefore, one can assume that different organic and inorganic constituents found in the herein used tannery effluent may have caused sodium retention, potassium reduction, and increased intracellular osmotic pressure in the hepatocytes of animals belonging to the 5 and 10% groups, a fact that thereby leads to water accumulation in the cytoplasm and cell isosmotic expansion. Additionally, cellular changes in hepatocytes such as necrosis, karyomegaly, and change in the amount of Kupffer cells were identified in the liver of animals exposed to tannery effluent. The statistical analysis revealed the effect of factor 1 Btreatment^ (F(5;54) = 0.540, p = 0.015) and factor 2 Bsex^ (F (5;54) = 38.681; p < 0.0001) on the results of hepatocytes presenting karyomegaly. As it can be seen in Fig. 2a, animals Fig. 1 Representative photomicrographs of the liver of male and female Swiss mice exposed, or not, to different tannery effluent concentrations diluted in water. a Liver from one animal belonging to the control group (dashed arrows indicate normal hepatocytes). b Liver from one animal belonging to the 5% tannery effluent group (presenting centrilobular ballooning degeneration) and c 10% tannery effluent group. c Detail of hepatocytes presenting hydropic degeneration. The arrows highlight the swollen and granular appearance of cytoplasm hepatocytes. CV—centrilobular vein. Bar = 100 μm. HE stain

chronically exposed to tannery effluent (10% group) had larger number of hepatocytes presenting karyomegaly than those in the control group; this variation was significantly lower between female mice. According to Harada et al. (1999), kariocytomegaly can result from polyploidy hepatocyte reflection, when there is nuclear material duplication in the absence of cytokinesis. Accordingly, it is possible in recording increased number of nuclei per diploid hepatocytes or ploidy level increase in a single core hepatocyte. Except for the study by Souza et al. (2016b), there is no information in the literature about tannery effluent exposure (orally) and oversized core hepatocytes (karyomegaly). Souza et al. (2016b) have also observed kariocytomegaly in animals by assessing the chronic exposure of C57Bl/6J male mice to tannery effluent (5%). Other studies have shown the strong association between this anomaly and senescence in murine models (Percy & Barthold, 2001) and chemical carcinogenicity in rodents (rats and/or mice), regardless of the genotoxic potential of compounds animals are exposed to (Kitaura et al., 1999; Inoue et al., 2008; Inoue et al., 2009; Thoolen et al., 2010). Thus, we suggest that different components (inorganic and/or organic) found in tannery effluent may have helped triggering biological processes leading to such hepatocellular abnormality, thus reinforcing the hypothesis that chronic exposure to this pollutant can cause hepatic tumors. On the other hand, it was observed that Swiss female mice, regardless of the treatment (control, 5, or 10%), showed lower

A

VC

C

B

CV


N ú m e ro d e h e p a tó c ito s c o m c a rio m e g a lia

A

F a to r 2

F a to r 1

80

a a ab

60

Hyd Deg Hp

b b

40

Hyd Deg Hp 20

Hyd Deg Hp

0

C o n tro le

5%

10%

M achos

F êm eas

F a to r 1 250

B

N ú m e ro d e c é lu la s d e K u p ffe r

a

K

b

200

b

150

100

CV

50

K

0

C o n tro le

N ú m e ro d e h e p a tó c ito s c o m n e c r o s e

10%

F a to r 1

10

C

5%

a 8

Hyd Deg Hp a

6

4

Necr Hp

2

b 0

C o n tro le

5%

10%

Fig. 2 a Number of hepatocytes presenting karyomegaly. b Frequency of Kupffer cells. c Number of necrotic hepatocyte in Swiss mice (male and female) exposed, or not, to different tannery effluent concentrations diluted in water, for 150 days. Different letters indicate statistically

significant differences between experimental groups. Hyd Deg Hp: hepatocyte presenting hydropic degeneration; k: Kupffer cell; Necr Hp: necrosis in hepatocyte; CV: centrilobular vein. Bar = 100 μm. HE stain

amount of hepatocytes presenting karyomegaly in comparison to male mice. In this case, the present data support the hypothesis that the response of Swiss mice to oral tannery effluent exposure may be different between male and female animals, just as it was found by Lee et al. (2015), who showed that the response of males and females exposed to contaminants can be different. Regarding the results of Kupffer cell amount in the livers of the assessed animals, factor Btreatment^ (F(5.54) = 7.517, p = 0.001) has had effect on them and factor 2 Bsex^ (F(5.54) = 1.169, p = 0.284) did otherwise; moreover, there was no interaction between factors (F(5.54) = 1.877, p = 0.162). Mice exposed to tannery effluent (5 and 10% groups) showed smaller number of Kupffer cells than those in the control group (Fig. 2b).

These results are new and were not yet reported in any other study; thus, these data show that mice exposed to xenobiotics face Kupffer cell population reduction in the liver. It is possible saying that the different chemical compounds found in the effluent may (together or separately) have negatively interfered in MCSF production (necessary for the differentiation of these cells), as suggested by Mosher et al. (2001). With regard to the parameter Bhepatocyte presenting necrosis,^ it was also observed that factor 1 (F(5.54) = 9.097, p = 0.0003) only had one effect. There was no effect from factor 2 Bsex^ (F(5.54) = 2.963, p = 0.090) nor interaction between factors (F(5,54) = 0.624, p = 0.539). Animals exposed to tannery effluent showed higher number of necrotic hepatocytes than those in the control group (Fig. 2c). They showed nuclear


pyknosis and cytoplasm with acidophilus, granular and gelled aspect compatible with hepatocellular necrosis (Fig. 2c). Similar results were reported by Souza et al. (2016b) after they exposed inbred male mice (C57Bl/6) to chronic amounts of tannery effluent diluted in 5% water. Thus, the present data support the findings in their study and reinforce the potential these xenobiotics have to damage liver cells. Although the mechanisms causing these changes remain unknown in mice exposed to tannery effluents, there is strong evidence that these xenobiotics can damage cells and lead them to death. Studies involving microalgae (Ajayan & Selvaraju, 2012), bacteria (Mishra & Mishra, 2015), fish (Prabakaran et al., 2007), and even humans (Ambreen et al., 2014) reinforce the hypothesis that tannery effluent can significantly affect the production of reactive oxygen species and, consequently, free radicals production, a fact that would impair the structure and function of vital organs. Finally, mice exposed to 10% tannery effluent (male or female) showed slight bile duct hyperplasia in portal areas (Fig. 3a) and fibroblast proliferation around bile ducts, thus indicating a fibrous process (Fig. 3b), unlike the control group (Fig. 3c). So far, no study has reported these changes in experimental mammal models exposed to water containing tannery effluent. However, it is known that biliary hyperplasia is a characteristic reaction of the Fig. 3 Representative photomicrographs of the liver of male and female Swiss mice exposed, or not, to tannery effluent diluted in water. a Liver from male and b female Swiss mice belonging to the 10% tannery effluent group; c liver from male Swiss mice belonging to the control group. SP: space port. Bar = 100 μm. HE stain

liver to specific insult types, but much is yet to be learned about the proliferative potential of these structures and the metabolic stimuli that provoke them. The appearance of this type of injury in young animals may be associated with the aggression from toxic agents and with the consequent obstruction of the bile ducts, which irritate the hepatic triads (portal vein, hepatic artery, and bile duct) and/or damage the ducts’ epithelium (Stalker & Hayes, 2007). Given the chemical complexity of the xenobiotic used in the present study, it is likely that its constituents may have synergistically, and/or antagonistically, acted in order to cause the herein observed changes.

Conclusion Based on the results, and according to the experimental conditions, the present data support the hypothesis that the chronic intake of tannery effluent at 5 and 10% by outbred mice (Swiss strain) causes liver damage. It broadens the knowledge about the toxic potential of this xenobiotic in C57Bl/6J male mice (inbred strain). Moreover, male and female Swiss mice presented similar hepatic changes after the chronic intake of xenobiotics; however, female mice have higher frequency of

Bile duct

Bile duct

SP

SP Bile duct

A

B Hepatic artery

SP

C

Bile duct


hepatocytes presenting karyomegaly. Therefore, further research must be conducted in order to help better understand the mechanisms related to the effects observed in the present study, as well as to find out other possible impacts on different organs or tissues of mammals exposed to the chronic intake of water contaminated with the herein studied agro-industrial waste. Funding information The authors are grateful to Brazilian National Council for Research (CNPq) (Brazilian research agency) (Proc. No. 467801/2014-2) and to Instituto Federal Goiano for the financial support. Moreover, the authors are grateful to CNPq for granting a scholarship to the student who developed the current study.s Compliance with ethical standards All procedures were approved by the Institutional Committee for Animal Care and Use of Goiano Federal Institute (Goiás State, Brazil) (protocol no. 17/2014) according to the Guide for Care and Use of Laboratory Animals, 8th edition. Animal handling and all the experiments were performed according to the International Guidelines for Animal Welfare.

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