# *Post mortem* evaluation of liver iron overload in howler monkeys (*Alouatta clamitans*)

F. S. KUSZKOWSKI<fnref n="1" />*, J. LUCIOLI<fnref n="1" />, J. CÉSAR DE SOUZA JUNIOR<fnref n="2" />, Z. HIRANO<fnref n="2" />

Corresponding author: fsilvak@gmail.com

## SUMMARY

Hemosiderosis is an iron accumulation syndrome that can advance to hemochromatosis, which is the accumulation associated with disorders in organ function. Although howler monkeys have a specific iron metabolism related to their fur colour, iron accumulation in this specie has never been investigated. Hemochromatosis has been reported in other non-human primates. The main goal of the present study is to investigate *post mortem* iron accumulation on 105 livers of *Alouata clamitans*, from the biological material collection of the Centro de Pesquisas Biológicas de Indaial. This paper compares *post mortem* iron accumulation in the liver of free-ranging and captive Howler monkeys through Prussian Blue staining. For microscopic study, Hematoxilin-eosin stain exposed the cellular morphology and Prussian blue stain quantified the amount of iron accumulation. Chi-square test of independence and Prevalence Test were used for statistical evaluation. From 105 animals, 60 (57%) were negative for iron (by Prussian blue staining) and 45 (42%) were positive. There was no significant association between hepatic hemosiderosis and habitat, gender, or age. This is the first study addressing hepatic hemosiderosis in howler monkeys. Iron accumulation was present, however, liver damage (hemochromatosis) was not detected.

**Keywords:** Primates, hemosiderosis, hemochromatosis, histopathology, chi-square.

## RESUME

### Évaluation *post mortem* de la surcharge en fer hépatique chez le singe hurleur (*Alouatta clamitans*)

L'hémosidérose est un syndrome d'accumulation de fer pouvant évoluer vers l'hémochromatose, l'accumulation associée à des troubles de la fonction des organes. Bien que les singes hurleurs aient un métabolisme spécifique du fer lié à la couleur de leur fourrure, l'accumulation de fer chez cette espèce n'a jamais été étudiée. Une hémosidomatose a été rapportée chez d'autres primates non humains. L'objectif principal de la présente étude est d'enquêter sur l'accumulation post mortem de fer sur 105 foies de clamitans Allouata, provenant de la collection de matériel biologique du Centro de Pesquisas Biológicas de Indaial. Cet article compare l'accumulation post mortem de fer dans le foie de singes Howler en liberté et captifs par coloration au bleu de Prusse. Pour une étude microscopique, la coloration à l'hématoxilin-éosine a révélé la morphologie cellulaire et la coloration au bleu de Prusse a quantifié la quantité d'accumulation de fer. Le test d'indépendance du khi-carré et le test de prévalence ont été utilisés pour l'évaluation statistique. Sur 105 animaux, 60 (57%) étaient négatifs pour le fer (par coloration au bleu de Prusse) et 45 (42%) étaient positifs. Il n'y avait pas d'association significative entre l'hémosidérose hépatique et l'habitat, le sexe ou l'âge. Il s'agit de la première étude sur l'hémosidérose hépatique chez des singes hurleurs. L'accumulation de fer était présente, cependant, des dommages au foie (hémochromatose) n'ont pas été détectés.

**Mots-clés :** Primates, hémosidérose, hémochromatose, histopathologie, chi-carré.

## Introduction

Howler monkeys (Allouata clamitans) have a large diversity, living in regions of Atlantic Forests [2]. Prehensile tail and complex increased larynx/hyoid are two important phenotypic characteristics of the Allouata gender. The sexual dimorphism of the specie presents itself in weight and color coat; the male heavier with golden/read coat and the female lighter with a brown coat [20]. The red coat of the male is due to a red epidermal secretion that is rich in iron [14]. The Red List of Threatened Species of the International Union for Conservation of Nature and Natural Resources (IUCN) classifies the howler monkey as last concern (LC) [19]. However, the classification by Brazil and of the state of Santa Catarina classifies the specie as vulnerable (VU) [4; 23].

There are two iron oxidation forms found in tissues: ferrous ($Fe^{2+}$) and ferric ($Fe^{3+}$) states. The body has a low excretion rate of this mineral and most excretion occurs on the duodenum and jejunum [21]. Free (unbound) iron

is toxic unless bound to one of several specific proteins including transferrin (for transport), haemoglobin, or ferritin (storage) [3].

Hemosiderin is a brown pigment composed of ferric iron ($Fe^{3+}$) associated with ferritin, and is formed when there is excessive iron in tissues. Hemosiderosis is an excessive presence of hemosiderin in macrophages within the spleen, lymph nodes, and bone marrow or macrophages and/or hepatocytes of the liver. Hemochromatosis, on the other hand, is an iron overload in organs and tissues inducing morphological and functional disorders [3; 28]. Genetic alteration that causes a defective control on iron absorption [3; 11; 17], diets rich in iron, haemolytic and dyserythropoietic anemia, and frequent blood transfusions [3] are potential aetiologies. Sequelae of hepatic hemochromatosis include infections [10; 15; 22], liver failure and hepatic cirrhosis [3].

Information regarding iron overload in primates are scarce, with reports in species such as muriquis [24],

<footnote n="1">¹Veterinary Pathology Laboratory, Universidade Regional de Blumenau, 768 Samuel Morse St., Blumenau SC 89058-010, Brazil.</footnote>

<footnote n="2">²Centro de Pesquisas Biológicas de Indaial (CEPESBI) – Projeto Bugio, 401 Rio de Janeiro St., Indaial SC 89130-000, Brazil.</footnote>
Callitrichidae family [6; 25], lemurs [9; 12; 27; 29; 30], and baboons [5] and, in muriquis, iron overload can be accompanied by hepatic alterations [24]. This paper has as goal to investigate post mortem iron accumulation in howler monkeys, from wild and from captivity, and correlate it with epidemiological, nutritional, and pathological data.

# Materials and methods

## INDIVIDUALS

105 specimens of Allouata clamitans, both wild (54 individuals and captive (18 individuals), were studied. 33 animals did not have any information regarding habitat. Animals were classified as adults (60 individuals) and non-adult (24 individuals). 21 individuals did not have information about age. It was not used the age classification proposed by Mendes (1985) because some categories would have a low number of animals making statistical analysis difficult. Also, 50 animals were males and 39 were female. 16 animals did not have information about gender.

Animal tissues came from the collection of biological materials of the Centro de Pesquisas Biológicas de Indaial – Projeto Bugio, Santa Catarina, Brazil, under registration on Conselho de Gestão do Patrimônio Genético-CEGEN process nº 02000.003226/2006-91, not requiring authorization from the Ethics Committee of Animals Use of the Universidade Regional de Blumenau (CEUA/FURB). Activity for research purposes registered and authorized by the Sistema de Autorização e Informação em Biodiversidade of the Instituto Chico Mendes (ICMBio\SISBIO), respecting the Normative Instruction ICMBio nº 03/2014 that establishes and regulates the SISBIO, under nº 54094-1.

The collection of biological material is composed of formalin fixed tissue from animals that died at the Centro de Pesquisas Biológicas de Indaial – Projeto Bugio. Animals considered as “captive animals” lived at least six months on captivity. On the other hand, “wild animals” lived less than six months on captivity or on wild their whole life.

## HISTOPATHOLOGY

Formalized fragments of liver were dried in alcohol, clarified in xylene and embedded in paraffin, cut in sections of three micrometres (µm) of thickness, and stained by the methods Hematoxylin-Eosin (HE) and Prussian Blue. Two observers carried out the microscopic and morphological evaluation simultaneously.

Using Hematoxylin-Eosin (HE) staining it was possible to analyse the histological findings: (1) congestion; (2) haemorrhage; (3) necrosis; (4) vacuolation of hepatocytes; (5) lymphocytic plasmacytic infiltrate; and (6) presence of brown granular pigments compatible with hemosiderin or ferritin deposition within hepatocytes and macrophages, exposing possible abnormalities in cells morphologies.

On the other hand, Prussian Blue's method highlighted and differentiated iron from bilirubin, melanin and lipofuscin [28]. By this method, it was possible to classify the degree of deposition of blue granular pigments in: ‘1’ mild, when 25% of the focal area was affected; ‘2’ moderate, when 50% of the focal area had blue granular pigments; ‘3’ severe, when there was involvement of 75% or more of the focal area. An animal was considered positive to iron accumulation when it had any degree of histochemical marking in Prussian Blue staining and negative when it had no histochemical marking in Prussian Blue staining.

# Statistical analysis

Statistical analysis were conducted in the Epi Info software, using Chi-Square Test of Independence (significance level when P<0,05) to analyse the variables habitat, gender, age, and hepatic lesions. The Prevalence Test made possible to do descriptive analysis.

# Results

Of the 105 liver samples analysed, 57% (60/15) did not contain pigment, while 42% (45/105) had pigment deposits consistent with hemosiderin or ferritin (ref). Among the positive animals 18% (19/45) had iron accumulation classified as ‘1’ mild, 15% (16/45) as ‘2’ moderate, and 9% (10/45) as ‘3’ severe (Figure 1).

<figure><img src="image_1.png" /><figcaption>FIGURE I: Comparative analysis of the prevalence of hepatic iron according to the degree of iron deposition in (A) normal, (B) mild intensity (1), (C) moderate (2) and (D) severe (3). Liver, howler monkey. Blue of Prussia (Perls), obj. 40x.
Source: Research data (2016).</figcaption></figure>

Samples of animals from captivity and wild were analysed. Among animals that did not have information regarding habitat (33/105), 15 (15/33) were positive for hepatic hemosiderosis and 18 (18/33) were negative. 72 (72/105) animals had information about habitat, being 41%
(30/72) positive for hepatic hemosiderosis and 58% (42/72) negative. 63% (19/30) of positive animals were from wild and 36% (11/30) from captivity. Among the positive animals from wild, had 57% (11/19) of the animals classified as ‘1’ mild; 26% (5/19) as ‘2’ moderate; and 15% (3/19) as ‘3’ severe (Figure 1). The negative animals, on the other hand, had 27% (3/11) of the animals classified as ‘1’ mild; 36% (4/11) as ‘2’ moderate; and 36% (4/11) as ‘3’ severe.

It was not possible to identify significant correlations between iron accumulation and habitat (P=0,053) (Table I), age (P=0,3272) (Table II), or gender (P=0,0678) (Table II).

The hepatic lesions also did not significantly correlate with iron accumulation (P=0,22 – 0,81) (Table III). However, histopathological evaluation identified that iron deposits were accompanied by: necrosis in 10% (24,4%); congestion in 46% (21/45); haemorrhage in 24% (11/45); vacuolation of hepatocytes in 35% (16/45); and inflammatory infiltrate in 33% (15/45) of the cases. These finding can be correlated with different diseases that were not the focus of the study.

Discussion

Iron accumulation in non-human primates has been associated with unbalanced diet in captivity [1; 27; 30]; accumulations have been associated with: (1) a high percentage of elements that increase iron toxicity, such as vitamin C; (2) elements with a high percentage of iron in its composition [7; 8; 12; 30]; (3) and/or a lack of secondary components

that bind to iron and make it unavailable to absorption [16]. Animals from Centro de Pesquisas Biológicas de Indaiatuba (CEPESBI) – Projeto Bugio receive six meals a day, whose schedule and foods were determined according to study of Hirano et al. (1997) which documented by observation of the natural diet of wild howler monkeys. Captivity diet consists in various fruits, leaves of embaúba and chuchu, and 12 pellets of laboratory animals ration. In addition, in winter animals receive vitamin C supplementation, at a dose of 100 mg per day for adults and 50 mg per day for non-adults. Natural diet of howler monkeys is composed in most part of leaves (57%), followed by fruits (41%), and flowers (1,7%).

Our data presented no significant correlation between iron accumulation and habitat. This finding corroborates with studies of Williams et al. (2006) and Santos et al. (2011), thus, showing that captivity animals do not appear to be more susceptible to hemosiderosis.

In regard to captive animals, iron accumulation was observed in 61,11% (11/18) of animals, percentage lower than the results of study of similar methodology of Santos et al. (2011), which analysed hepatic hemosiderosis in muriquis (Brachyteles spp.) and obtained 100% (15/15) of positive animals. Hence, howler monkeys appear to be less susceptible to hepatic hemosiderosis than muriquis, which may be due to the excretion of iron in their perspiration [14], mobilizing part of ingested and stored iron, and, therefore, decreasing its accumulation.

<table><thead><tr><th rowspan="2">Habitat</th><th colspan="2">Prussian Blue</th><th rowspan="2">Total</th><th rowspan="2">P</th></tr><tr><th>YES</th><th>NO</th></tr></thead><tbody><tr><td>Free life</td><td>19 (63.3%)</td><td>35 (83.3%)</td><td>54 (75%)</td><td rowspan="2">0,053</td></tr><tr><td>Captivity</td><td>11 (36.7%)</td><td>7 (16.7%)</td><td>18 (25%)</td></tr><tr><td><strong>Total</strong></td><td>30 (100%)</td><td>42 (100%)</td><td>72 (100%)</td><td></td></tr></tbody></table>

P: *P-value* of the Chi-square test of independence. If *P* <0.05 then significant differences. Individuals without identification were excluded from the test.

Source: Research data (2016).

<table><caption>TABLE I: Analysis of the frequency of iron accumulation in red howler monkeys (*Alouatta clamitans*), evaluated by the Prussian Blue technique (Perls) in ‘free life’ and ‘captivity’ variables (n = 72).</caption><thead><tr><th rowspan="2">Variables</th><th colspan="2">Prussian Blue</th><th rowspan="2">Total</th><th rowspan="2">P</th></tr><tr><th>YES</th><th>NO</th></tr></thead><tbody><tr><td><strong>SEX</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Male</td><td>25 (67.6%)</td><td>25 (48.1%)</td><td>50 (56.2%)</td><td rowspan="2">0.0678</td></tr><tr><td>Female</td><td>12 (32.4%)</td><td>27 (51.9%)</td><td>39 (43.8%)</td></tr><tr><td><strong>Total</strong></td><td>37 (100%)</td><td>52 (100%)</td><td>89 (100%)</td><td></td></tr><tr><td><strong>AGE</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Adult</td><td>23 (65.7%)</td><td>37 (75.5%)</td><td>60 (71.4%)</td><td rowspan="2">0.3272</td></tr><tr><td>Non-adult</td><td>12 (34.3%)</td><td>12 (24.5%)</td><td>24 (28.6%)</td></tr><tr><td><strong>Total</strong></td><td>35 (100%)</td><td>49 (100%)</td><td>84 (100%)</td><td></td></tr></tbody></table>

P: *P-value* of the Chi-square test of independence. If *P* <0.05 then significant differences. Individuals without identification were excluded from the test.

Source: Research data (2016).

<caption>TABLE II: Correlation between iron accumulation analyzed by Prussian Blue (Perls) technique, sex (n = 89) and age (n = 84).</caption>
<table><thead><tr><th rowspan="2">Variables</th><th colspan="2">Iron accumulation</th><th rowspan="2">Total</th><th rowspan="2">P</th></tr><tr><th>YES</th><th>NO</th></tr></thead><tbody><tr><td><strong>Necrosis</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>YES</td><td>11 (24.4%)</td><td>9 (15%)</td><td>20 (19%)</td><td rowspan="2">0.2226</td></tr><tr><td>NO</td><td>34 (75.6%)</td><td>51 (85%)</td><td>85 (81%)</td></tr><tr><td><strong>Total</strong></td><td>45 (100%)</td><td>60 (100%)</td><td>105 (100%)</td><td></td></tr><tr><td><strong>Congestion</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>YES</td><td>21 (46.7%)</td><td>24 (40%)</td><td>45 (42.9%)</td><td rowspan="2">0.4945</td></tr><tr><td>NO</td><td>24 (53.3%)</td><td>36 (60%)</td><td>60 (57.1%)</td></tr><tr><td><strong>Total</strong></td><td>45 (100%)</td><td>60 (100%)</td><td>105 (100%)</td><td></td></tr><tr><td><strong>Hemorragy</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>YES</td><td>11 (24.4%)</td><td>16 (26.7%)</td><td>27 (25.7%)</td><td rowspan="2">0.7965</td></tr><tr><td>NO</td><td>34 (75.6%)</td><td>44 (73.3%)</td><td>78 (74.3%)</td></tr><tr><td><strong>Total</strong></td><td>45 (100%)</td><td>60 (100%)</td><td>105 (100%)</td><td></td></tr><tr><td><strong>Vacuolization hepatocytes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>YES</td><td>16 (35.6%)</td><td>20 (33.3%)</td><td>36 (34.3%)</td><td rowspan="2">0.8123</td></tr><tr><td>NO</td><td>29 (64.4%)</td><td>40 (66.7%)</td><td>69 (65.7%)</td></tr><tr><td><strong>Total</strong></td><td>45 (100%)</td><td>60 (100%)</td><td>105 (100%)</td><td></td></tr><tr><td><strong>Inflammatory infiltrate</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>YES</td><td>15 (33.3%)</td><td>22 (36.7%)</td><td>37 (35.2%)</td><td rowspan="2">0.7235</td></tr><tr><td>NO</td><td>30 (66.7%)</td><td>38 (63.3%)</td><td>68 (64.8%)</td></tr><tr><td><strong>Total</strong></td><td>45 (100%)</td><td>60 (100%)</td><td>105 (100%)</td><td></td></tr></tbody></table>

Chi-square test. P: P-value of the Chi-square test of independence. If P <0.05 then significant differences.

Source: Research data (2016).

<caption>TABLE III: Frequency of hepatic alterations analyzed by Hematoxilin-Eosin technique (HE) (n=105).</caption>

It could be expected that female howler monkeys were more affected by hepatic hemosiderosis, since this gender has a lower release of iron in their perspiration than males [14]. However, our results showed no significant correlation between hepatic hemosiderosis and gender in howler monkeys, corroborating with findings of Crawford et al. (2005), Santos et al. (2011), Smith et al. (2008), and Wood et al. (2003).

Equally, there were no significant correlation between hemosiderosis and age, as in previous studies [24; 30]. In contrast, studies of Crawford et al. (2005) and Glenn et al. (2006) found a significantly higher level of iron accumulation in older ages of captive lemurs.

In humans hereditary hemochromatosis iron accumulation can lead to progressive tissue damage, especially in the liver, due to lipid peroxidation and production of free radicals [26]. Even though hepatic lesions were present, there were no significant correlation of these with hepatic hemosiderosis.

Results of this study showed presence of hemosiderosis in howler monkeys, however hemochromatosis was not detected. In addiction, iron accumulation could not be

correlated with age, gender or habitat. As this is a retrospective study and it was not possible to have access to clinical data of all animals, further investigations in the field are needed to understand if iron accumulation represents a risk to the specie (such as quantitative chemical determination of iron). Considering the conservation status of the specie Allouata clamitans in Brazil and Santa Catarina [4; 23] studies related to these animals are extremely important.

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