Howell-Jolly Bodies in Red Blood Cells of Snow Vole Chionomys Nivalis

Oecologia Montana 2018, Howell-Jolly bodies in red blood cells of snow vole 27, 27-31 Chionomys nivalis

M. BIELESCH and M. JANIGA al. (2012), hematological data are important indica- tors out of the condition of wild animal populations Institute of High Mountain Biology, Žilina University, affected by pollutants. One of the main hematologi- Tatranská Javorina 7, SK-059 56, Slovak Republic; e- cal parameters used to determine the effects of heavy mail: [email protected]; [email protected] metals in small mammals is the presence of Howell- Jolly bodies in erythrocytes. Erythrocytes are blood cells, which transport oxygen to the body’s tissues through the circulatory system. Howell-Jolly bodies were described as micronuclei or “fragment of nuclear Abstract. This study focused on counting Howell- material” in the cytoplasm of erythrocytes by Howell Jolly bodies in peripheral blood of Chionomys nivalis and Jolly in the late 1800s and early 1900s. The first and Myodes glareolus within the study area (High innovators, Evans et al. (1959) discovered the ap- Tatras - Biele plesá). The number of Howell-Jolly plicability of micronuclei as markers for cytogenetic bodies was similar in both species and did not dif- damage. Several researchers have recommended the fer between males and females or adults and non- use of micronuclei as a biomarker in testing on count- adults. Only five samples contained relatively more ing micronuclei in the erythrocytes of small mammals. Howell-Jolly bodies (4 samples contained four and Significant correlation between heavy metal contami- one sample contained five Howell-Jolly bodies per nation and presence of Howell-Jolly bodies in eryth- 1000 erythrocytes). We also compared the number of rocytes has been detected in rodents living near pol- Howell-Jolly bodies to the amount of heavy metals luted areas (Tapisso et al. 2009). found in the vertebrae of snow voles. The quantity In some studies, Snow voles caught in the spring of Howell-Jolly bodies in peripheral blood of animals exhibited significantly higher micronuclei frequen- in relation to the amount of lead in bones was found cies in peripheral blood than individuals trapped in to be statistically insignificant but we found an in- summer or winter. This result correlates with other creased number of Howell-Jolly bodies in relation to studies in small mammals, reporting an association on the concentration of molybdenum. of micronuclei frequencies and metal concentrations (Ieradi et al. 1996, Topashka-Ancheva et al. Key words: Chionomys nivalis, Myodes glareolus, hema- 2003, Metcheva et al. 2008). tology, Howell-Jolly bodies, High Tatras, pollution The main goal of this study was to observe the Howell-Jolly bodies in the peripheral blood of Chionomys nivalis in the Tatra mountains. The Introduction number of Howell-Jolly bodies in Chiononyms nivalis was compared to the number of Howell-Jolly Pollution represents a critical problem, particularly bodies in the blood of Myodes glareolus to exam- in heterogeneous landscapes, such as mountainous ine the relationship lead and molybdenum con- regions, where we observe a higher impact than in centrations in the tail vertebrae and the number of low lying areas. Recent studies demonstrable clear- Howell-Jolly bodies in the blood. ly that high alpine environments are negatively influenced by environmental pollution. Atmospheric inputs at high elevations are greater than those Material and Methods oflow elevation regions, because of orographic effects, cloud deposition and wind speed (Lovett and Local populations of snow voles in the Dolina Kinsman 1990). Although adverse health effects Bielych plies valley region of the High Tatras were of heavy metals have been known for some time, sampled as part of this study. Chionomys nivalis exposure to heavy metals continues, and has even and Myodes glareolus were regularly sampled from increased in some parts of the world, particularly in June to November 2016. The study area included less developed countries (Jarup 2003). The presence alpine meadows, with typical geological, plant, and of metals in the environment always represents a moss features for this type of ecosystem. risk for living organisms, and the effects of heavy Live traps were distributed in the field approxi- metals may lead to morphological, physiological, mately 5 to 10 m apart from one another. Approxi- pathological and genetic changes. The most com- mately 80 traps were checked per day. The field- mon pathological changes affect the blood and work took place over 18 days. Coordinates were organs(Jančová et al. 2004). According to Rostal et recorded with GPS to mark the location of trapped

27 © 2018 Prunella Publishers 28 individuals. Photo and video samples were taken per 1000 erythrocytes M. Bielesch for use in additional evaluation of plants, mosses, - Category 2: 2 Howell-Jolly bodies per 1 000 & M. Janiga and geological features of the study area. erythrocytes Sherman traps were baited with fresh apples, - Category 3: more than 2 Howell-Jolly bodies seeds, cereals and peanut butter. Dry grass was also per 1 000 erythrocytes added to the traps as bedding material, mainly during cold days. Each of the captured individuals was It was found that hematological parameters are not identified. Part of the tail (app. 0.03 cm) was clipped significantly related to the number of Howell-Jolly from each captured animal, after animals were bodies found in the blood. Myodes glareolus lives in anaesthetized with Isoflurane. Peripheral blood was this habitat at the upper limit and can be more sen- collected from the tail vena (vena caudalis). sitive than Chionomys nivalis, which has an upper Two slides were prepared for each individual. A limit at higher altitudes. It appears that habitat is a drop of blood was smeared on each slide to make a thin factor in higher numbers of Howell-Jolly bodies in smear. The blood smears were air-dried and placed in erythrocytes of Myodes glarelous. Ratios of males to a slide box. Following measurement, the blood smears females in both species did not vary over the three were stained according to Doubek et al. (2003). categories based on the number of Howell-Jolly bod- Stained blood smears were numerically ies (Table 1 and 3). Ratios of adults and non-adults evaluated with the use of immersion oil under 1 000 were also unchanged in all three categories based on times magnification using a meandering movement the amount of Howell-Jolly bodies (Table 2 and 4). of the slides. For each individual, micronucleus fre- Chionomys nivalis individuals with higher quency was scored on 1 000 erythrocytes. amounts of lead in the tail vertebrae tended to The sex was identified through location of geni- have more Howell-Jolly bodies, though differences tals. Genitals of female rodents are situated closer among the three Howell-Jolly categories were than male genitals. In sub-adult animals, there are not statistically significantly different (Fig. 1). Ra- distinguishable small nipples in females and ab- tios of males and females did not vary in the three dominal testes in males (Balčiauskiené et al. 2009). categories based on the amount of Howell-Jolly We also recorded the date individuals were bodies (Table 1). Ratios of adults and non-adults trapped, body length, tail length and hind foot were not different in the three categories based on length of individuals. Weight was taken using a amount of Howell-Jolly bodies too (Table 2). spring scale (Pesola 100 g). After these measure- Animals with more Howell-Jolly bodies tended ments animals were immediately released at the to have statistically significant increased levels of point of capture. molybdenum in their tail vertebrae (Fig. 2). Ratios The ED-XRF spectrometer delta was used of males and female were not different between the to determine chemical composition, mainly the three categories, based on the amount of Howell-Jolly amount of heavy metals (Pb, Mo, and etc.) in bones bodies (Table 1). Ratios of adults and non-adults were from the small part off the tail. not different between the three categories based on Animals were categorized into three groups ac- amount of Howell-Jolly bodies too (Table 2). cording to the number of Howell-Jolly bodies present. Individuals from the first group had less than 2 Howell-Jolly bodies per 1 000 erythrocytes (Category Discussion 1), the second group presented with 2 Howell-Jolly bodies per 1 000 erythrocytes (Category 2) and the Chionomys nivalis has been used as a bioindica- third group with more than 2 Howell-Jolly bodies per tor in environmental studies performed in Bulgaria, 1 000 erythrocytes (Category 3). This data was in- Spain, Czech Republic, Italy, Slovakia and others put from Microsoft Excel to STATISTICS 12.1, which (Ieradi et al. 1996, Topashka-Ancheva et al. 2003, was used for statistical analysis. The data was stan- Metcheva et al. 2008, Janiga et al. 2012). dardized and one-way analysis of variance (ANOVA) Çavuoglu et al. (2010) observed the number of was used to determine any significant differences Howell-Jolly bodies in the blood of animals and de- between two or more independent groups of param- scribed inclusion as an important indicator to detect eters. The data are expressed as mean ± standard genetic damage induced by chemicals or radiation. error. P-values less than 0.05 were considered to be The inclusion test showed a significantly higher fre- statistically significant and values more than 0.05 quency of Howell-Jolly bodies found in animals from were considered to be statistically insignificant. polluted areas in comparison to un-polluted localities. In Bulgaria a study compared species in different sites of the Rila mountain with different altitudes, Results where they were collected. They found Howell-Jolly bodies in free-living rodents, as well as in Chionomys In this study we evaluated peripheral blood sam- nivalis and Myodes glareolus. The authors described ples of Chionomys nivalis and Myodes glareolus. A that the presence of Howell-Jolly bodies could be total of 95 samples were taken in the High Tatras resulting in erythrocyte destruction due to the tox- during 2016. The main objective was to count the ic content of cooper found in samples. The results number of Howell-Jolly bodies per 1 000 erythro- showed genetic damage in rodents from the lower cytes in blood smears taken from peripheral blood study area, where it was realistic to assume trans- of Chionomys nivalis. The animals were classified boundary pollution as higher lead concentrations into three groups. They were based on the number were found. Animals trapped in localities with high- of Howell-Jolly bodies found in peripheral blood: er altitudes presented with fewer Howell-Jolly bod- - Category 1: less than 2 Howell-Jolly bodies ies per 1 000 erythrocytes (Metcheva et al. 2003). 29 Males Females Adults Non-adults Howell-Jolly bodies in erythrocytes Category 1 16 10 Category 1 14 12 of snow vole Category 2 13 11 Category 2 15 9 Category 3 9 8 Category 3 9 8

Table 1. Ratios of males and females in dependence on Table 2. Ratios of adults and non-adults in dependence the number of Howell-Jolly bodies in the peripheral blood on the number of Howell-Jolly bodies in the peripheral of Chionomys nivalis (x2=0.40, p=0.81). For detailed expla- blood of Chionomys nivalis (x2=0.51, p=0.77). For detailed nation of categories see the text. explanation of categories see the text.

Males Females Adults Non-adults Category 1 6 4 Category 1 4 6 Category 2 4 5 Category 2 5 4 Category 3 2 7 Category 3 3 6

Table 3. Ratios of males and females in dependence on Table 4. Ratios of adults and non-adults in dependence the number of Howell-Jolly bodies in the peripheral blood on the number of Howell-Jolly bodies in the peripheral of Myodes glareolus (x2=2.77, p=0.24). For detailed expla- blood of Myodes glareolus (x2=0,95, p=0,61). For detailed nation of categories see the text. explanation of categories see the text.

An insignificant dependence was found between damage to organ systems. In comparison, normal val- the number of Howell-Jolly bodies and mean values ues of leucocytes and normal numbers of Howell-Jolly of leucocytes and erythrocytes. Small differences bodies found in a present study of Chionomys nivalis were also found between species. In Myodes glareo- and Myodes glareolus may indicate that the habitat of lus, a growing tendency of leucocytes with higher free-living rodents in the observed locality is relatively values of Howell-Jolly bodies was found, which may unpolluted and exposure to lead is not high. be attributable to higher sensitivity in this species, In the current study of Chionomys nivalis we whereas habitats of Chionomys nivalis tend to have found that the number of Howell-Jolly bodies was better conditions, due to their higher upper limit. independent of lead concentration in vertebrae of Farkhondeh et al. (2014) observed leucocytes in the Chionomys nivalis. In a study by Chassovnikarova et blood of guinea pigs that were exposed to intraperi- al. (2010) Howell-Jolly bodies were counted in sam- toneal lead. Animals exposed to lead had significantly ples from a polluted locality where high lead con- higher leucocyte counts. Changes to all parameters centrations were recorded. Significant differences exhibited by lead-exposed animals were statistically were recorded between Howell-Jolly frequencies significantly higher than those before exposure. The in animals captured in un-polluted areas compared higher total white blood cells (WBC) count in Farkhon- to individuals sampled from polluted regions. The deh’s results agreed with the results of Kadhim Al-Ali results showed differences in sensitivity among the and Abdula (2007), where they found a large increase collected three species and significant differences in total WBC count after acute and chronic exposure to in Howell-Jolly frequency in A. flavicollis, M. arvalis lead. They demonstrated that acute high lead exposure and M. macedonicus. It is interesting to note the can cause serious effects, including death or long-term study of Sawicka-Kapusta et al. (1987) who investi-

Pb means +-St.Errors One way ANOVA: F(2, 20)=1,3687, p=,27725 18

14 Pb (ug/g) 13

10 Less than 2 2 More than 2 Number of H-J bodies

Fig. 1. Amount of lead in bones of Chionomys nivalis and the number of Howell-Jolly bodies in their peripheral blood. 30 Mo means+-St.Errors M. Bielesch One way ANOVA: F(2, 20)=3,7406, p=,04168 & M. Janiga 9,0

8,5

8,0

7,5

Mo in vertebrae Mo (ug/g) in vertebrae 7,0

6,5

6,0 Less than 2 2 More than 2 Number of H-J bodies in the blood of Chionomys nivalis

Fig. 2. Amount of molybdenum in bones of Chionomys nivalis and the number of Howell-Jolly bodies in their peripheral blood.

gated heavy metal content in rodents living in pol- (Chan et al. 1998). Higher concentration of molyb- luted forests in Poland, recording lead concentra- denum in inhaled air was found to significantly tions in M. glareolus at significantly higher levels increase degeneration of the respiratory system than A. flavicollis. In the present study, indepen- in all exposed males and females compared with dence between the number of Howell-Jolly bodies controls. The incidence of alveolar/bronchiolar car- and lead concentration in Chionomys nivalis may cinoma was significantly greater in exposed groups by the result of low concentrations of lead in the of males and females than in the control groups. observed locality as well as the relatively high resis- Rosoff and Spencer (1964) demonstrated that tance to heavy metals in the alpine zone. Mathieu concentrations of molybdenum in the tissue, bones, (1996) came to a similar conclusion in her study, and blood rise rapidly after administration of mo- where she described that different species may re- lybdenum compounds. In their study, the effect of spond differently to the same exposure level of lead. water-soluble molybdenum compounds, molybde- In the present study, molybdenum was found to num trioxide and calcium molybdate was observed be the most important factor significantly influencing in animals (guinea pigs, rabbits, rats, sheep) after the number of Howell-Jolly bodies in erythrocytes of exposure through the intestinal tract. Chionomys nivalis. Molybdenum is a heavy metal, Asadi et al. (2017) investigated effects of molyb- similar to lead, which exhibits similar effects on or- denum injected intraperitoneally into Sprague-Daw- ganisms and impacts on the environment. Molybde- ley rats at different doses of Mo-nanoparticles over num is an essential element for both plants and ani- a period of 28 days. Hematological and biochemi- mals but high dietary levels can result in molybdenum cal parameters as well as sexual hormones and his- toxicity in some mammals (Mathieu 1996). Molybde- topathological examinations of the liver and testes num compounds are water soluble and are well ab- were assessed and compared with a control group. sorbed through inhalation and oral exposure. The rate The results showed that serum levels of testoster- of absorption of molybdenum is influenced both by its one, aspartate aminotransferase (AST), and lactate chemical form and the animal species (WHO 2011). dehydrogenase (LDH) all decreased significantly Molybdenum appears the most rapidly in the blood with higher concentration of Mo. The histopatho- and most organs after gastrointestinal absorption. logical examination of testes showed a decrease in The effects of dietary molybdenum (1.7 g/day) the number of Leydig cells while chronic inflamma- were tested in four Holstein cows (Huber et al. tory cells increased in portal triad and parenchyma 1971). After the molybdenum intake was increased in liver tissue of rats exposed to Mo nanoparticles. to 7 mg/kg of body weight per day, one cow de- In British Columbia, Mathieu (1996) examined veloped severe diarrhoea and exhibited signs of the potential toxic effects of molybdenum in the en- lethargy, cessation of milk synthesis and general vironment surrounding an active molybdenum mine emaciation. When the molybdenum dose was in- in small rodents, including the red-backed vole creased to 10 mg/kg of body weight per day, two of (Clethrionomys gapperi), the deer mouse (Peromys- three cows exhibited these symptoms. cus maniculatus), and the meadow vole (Microtus In a study by Miller et al. (1956) in which pennsylvanicus). Results from this study indicated Holtzman rats were fed diets containing hydrogen that molybdenum concentrations in small mammals molybdate at 75 or 300 mg/kg, molybdenum signifi- were not higher in individuals captured in treat- cantly inhibited growth and increased molybdenum ment areas than in those which were captured in concentrations in the liver. control areas. She thought that this could be the Inhalation studies of molybdenum trioxide were result of observed toxic effects as the different conducted by Chan et al. (1998). They found signifi- species of rodents respond differently to the same cant exposure-dependent increases in blood mo- exposure level of molybdenum and it is also pos- lybdenum concentration in exposed rats and mice sible that the populations of small mammals found 31 around the mine area have developed a resistance diation biology, 1: 216-229. Howell-Jolly bo- to the toxic levels of molybdenum over time. Farkhondeh, T., Boskabady, M.H., Kohi, M.K., Sadeghi- dies in erythrocytes In the present study, increasing numbers of Hashjin, G. and Moin, M. 2014: Lead exposure affects of snow vole Howell-Jolly bodies were found in erythrocytes as inflammatory mediators, total and differential white blood cells in sensitized guinea pigs during and after molybdenum concentrations in the tail vertebrae of sensitization. Drug and Chemical Toxicology 37:329-35. Chionomys nivalis increased. The amount of mo- Huber, J.T., Price, N.O. and Engel, R.W. 1971: Response lybdenum is expected to influence distribution and of lactating dairy cows to high levels of dietary molyb- concentration of molybdenum in the bodies of small denum. Journal of Animal Science, 32:364–367. mammals and their physiology which may result in Ieradi, L.A., Cristaldi, M., Mascanzoni, D., Cardarelli, E., changes to homeostasis. Results from the present Grossi, R. and Campanella L. 1996: Genetic damage in study showed that higher amounts of molybdenum urban mice exposed to traffic pollution.Environmental may change morphology of blood cells and influence Pollution, 92: 323-328. the number of Howell-Jolly bodies in peripheral Jančová, A., Massányi, P., Naď, P., Skalická, M., Koré- neková, B. and Drábeková, J. 2004: Obsah ťažkých ko- blood, when compared to other studies. This may vov v orgánoch drobných zemných cicavcov z emisne support the hypothesis that compounds of molybde- zaťaženej oblasti. In: Rizikové faktory potravového num are equally toxic to organisms as other heavy reťazca IV. pp. 90-94. Slovenská poľnohospodárska metals, especially for micromammals living in alpine univerzita, Nitra. zones, which have a high sensitivity to heavy metal Janiga M., Hrehová Z., Kostková-Zelinová, V. 2012: Sea- pollution. However, as Mathieu (1996) described, sonal effects of lead uptake by snow vole Chionomys it is possible that these populations of small mam- nivalis (Martins, 1842) in West Tatra Mts: Bone metal mals have developed a resistance to toxic levels of concentrations and hematological indices. Polish Jour- , : 611-619. molybdenum and other heavy metals and thus may nal of Ecology 60 Jarup, L. 2003: Hazards of heavy metal contamination. respond differently to the same exposure level of mo- British Medical Bulletin, 68: 167-182. lybdenum when compared to other species. 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