From the liver tissues of brown hare individuals that lived in two various habitats, i.e., the agricultural region with the pre- dominant farms and the industrial area near a metallurgical plant, histones H1 were analyzed to compare their within and between population variability. Furthermore, because agricultural production emits mainly organic pollutants and metal- lurgical industry is a primarily source of inorganic contaminations, we wanted to check how the brown hare individuals are sensitive for both agents. Among brown hare H1 histones, the histone H1.2 was determined as heterogeneous due to its varied mobility in two-dimensional SDS–polyacrylamide gel. The obtained electrophoretic patterns contained differently moving single spots of histone H1.2 and also its double spots have a similar rate of electrophoretic mobility. Based on this, two homozygous phenotypes (slowly migrating 2a and faster moving 2b) and a heterozygous phenotype (2a2b) was distin- guished. The relatively low variable (CV < 0.25) and comparably abundant (p > 0.05) histone H1.2 homozygous phenotypes form a heterozygous phenotype in a similar proportion, at a ratio approximating 0.5. Although the brown hare population originating from agricultural area displayed a slight excess of heterozygous individuals 2a2b (F = − 0.04), it was conformed to the Hardy–Weinberg assumption (χ = 0.035, p = 0.853). Compared with this population, a sevenfold reduced frequency of the phenotype 2b and above tenfold increase of a heterozygosity (F = − 0.53) was observed in the brown hare population inhabiting the vicinity of metallurgical plant. Therefore, this population did not fit to the Hardy–Weinberg law ( χ = 5.65, p = 0.017). Despite the negligible genetic differentiation ( F = 0.026) between brown hare populations inhabiting areas ST with different anthropogenic pressure, a statistically significant difference in the distribution of their phenotypes ( χ = 6.01, p = 0.049) and alleles (χ = 6.50, p = 0.013) was noted. The collected data confirm that the brown hare species is sensitive for environmental quality and may serve as a good indicator of habitat conditions related to both organic pollution emitted by agricultural activities (PIC = 0.48) and inorganic contamination originating from metallurgical processes (PIC = 0.49). These difference in the environmental quality might be assessed by estimation of genetic variability among the brown hare populations, based on the phenotypes distribution of histone H1 variant H1.2, the protein that was not so far employed as a molecular marker of anthropogenic stress. The European brown hare, Lepus europaeus, originat- of hunting bags allowed scientists to follow the long-term ing from the steppes of Eurasia was spread out in the trends in abundance and natural fluctuations of local brown crop regions of Europe (Averianov et al. 2003) becoming hare populations. This enabled a decline of the brown hare in an important mammalian small game species (Pielowski the early 1960s and also a systematic decrease of its number 1976; Chapman and Flux 1990). The regular monitoring in recent decades throughout Europe (Tapper and Parsons 1984; Mary and Trouvilliez 1995; Pielowski 1976). As a result, the brown hare species has been included in the Inter- * Andrzej Kowalski national Union for Conservation of Nature (IUNC) Red List email@example.com and considered to be a species of low-risk extinction under Appendix III of the Berne Convention on the Conservation Department of Biochemistry and Genetics, Institute of Biology, Jan Kochanowski University, Świętokrzyska 15, of European Wildlife and Natural Habitats (Mitchell-Jones 25-406 Kielce, Poland et al. 1999). Department of Biodiversity Studies, Didactics A significant reduction of the whole brown hare popu - and Bioeducation, University of Lodz, Banacha 1/3, lation caused tremendous research interest, and therefore, 90-237 Lodz, Poland Vol:.(1234567890) 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:576–584 577 various reasons explaining this phenomenon have been by anthropogenic activity. The histone H1.2 presented in suggested. A probable cause of the individual decrease this work seems to be informative in monitoring the regions in the brown hare population pointed to the agricultural contaminated by chemical agents of different origins. intensification and reduction of habitat heterogeneity, the Among them are gas and heavy metals pollution emitted fields enlargement, and decreased crop diversity, as well as especially from works of heavy industries and contamination the landscape fragmentation by roads and climate changes by macro elements, which are predominantly accumulate in (Smith et al. 2005). Likewise, an increase in the preda- the plants and soil as a consequence of agricultural produc- tor numbers and infectious diseases, including the Euro- tion (Malinowska 2004). pean Brown Hare Syndrome (EBHS) occurring in Poland Histone H1, a ubiquitous protein that determines chro- (Kałuzinski and Pielowski 1976; Panek and Kamieniarz matin structure and modulates its activity (Parseghian 2015; 1999; Panek et al. 2006) and other European countries Kowalski and Pałyga 2016), is highly heterogeneous (Kow- (Edwards et al. 2000; Schmidt et al. 2004), were consid- alski and Pałyga 2012a). By interaction with DNA (Izzo and ered as the factors that favor reduction of the brown hare Schneider 2015) and partnering proteins (McBryant et al. abundance. Since the 1980s, the European brown hare 2010), the individual histone H1 variants regulate various has become a target of genetic studies. Research focused cell activities, including gene expression and cell cycle mainly on testing whether such a decline of brown hares progression as well as biogenesis and metabolism of the might be associated with diminished overall fitness linked RNA (Kalashnikova et al. 2016). The nonallelic histone H1 to the reduction of genetic variability. As the first molecu- variants, which on average comprise ten forms in mammals lar markers, the allozymes have been used extensively to and birds (Kowalski and Pałyga 2017), are polymorphic. In describe the genetic structure of brown hare populations birds, histone H1 allelic variants are widespread. Among the (Hartl et al. 1990, 1992, 1994; Suchentrunk et al. 2000; Ben nine histone H1 nonallelic forms, only one variant (H1.c’) Slimen et al. 2008). These studies revealed that brown hare has not been recognized to date as polymorphic (Kowalski populations are locally subjected to genetic drift and also on and Pałyga 2012a). Whereas the functions of avian histone a low exchange of genes between the populations (Wincentz H1 allelic variants are not clearly determined, their poten- 2009). In addition, the brown hare allozymes can have differ - tial link with the specific phenotypic effects (Kowalski and ent metabolic functions (Mitton and Lewis 1989); therefore, Pałyga 2014) and some physiological traits of the organisms a selection can act on the allozyme frequencies (Mitton and (Kowalski et al. 2015) has been reported. The rarely detected Koehn 1975; Powers and Place 1978; Powers et al. 1986; histone H1 polymorphic subtypes in mammals include the Mitton 1993). As shown by Markowski et al. (1990), the rabbit histone H1.4 (Pałyga 1990) and mouse histone H1.S haptoglobin can be used as an informative marker of the (Zweidler 1984) as well as the human histone H1.2 and H1.4 brown hare health status. An influence of contamination on (Sarg et al. 2005). While a functional importance of mam- brown hare population genetic structure was documented malian histone H1 allelic variants remains unknown, the last previously as linked to the physiological processes (Pav and findings of Flanagan et al. (2016) revealed that the mouse Zahradnikova 1987; Paukert 1988). Thus, investigations of histone H1 polymorphic variants, H1.1 and H1.5, differ in the factors that may affect brown hare genetic variability their interaction with chromatin. Thus, histone H1 allelic evoked by the man-induced environmental changes are still variants may have a specialized role related to their indi- needed. The current study was undertaken to determine if vidual impact on the modulation of chromatin structure and brown hare populations are diverse in the agricultural and function. Likewise, the brown hare histone H1.2 presented industrial regions contaminated by organic and inorganic in this work is rare histone H1 polymorphic variant whose pollutants, respectively. A genetic variation between the phenotypic heterogeneity may be useful for evaluation of the brown hare populations, which reflects a susceptibility of changes that take place in populations living in the regions animals to the environmental changes, was assessed based exposed to different environmental pollutants. on the polymorphic variability of histone H1.2, a representa- tive of histone H1 mammalian somatic subtypes. To monitor the shifts of allele frequency at the brown Materials and Methods hare polymorphic loci, the varied methods using a set of molecular markers are usually adopted. Besides the above- Animal Material mentioned allozymes, the mitochondrial and microsatellite DNA is widely used for estimation of brown hare population The liver tissue samples of brown hares were collected status (Soós and Kusza 2015). However, suitable for this during the hunting seasons, years 1993–1995, from two purpose also may be the other indicators, such as H1 histone areas representing different physiographic regions in proteins, which seems to be effective for the evaluation of Poland, i.e., from area of Wopławki in the Mazurian Lake- brown hare population structure and its variations caused land with predomination of agricultural farms and from 1 3 578 Archives of Environmental Contamination and Toxicology (2018) 75:576–584 industrial region of Małopolska province in the immediate Isolation of Nuclei and Extraction of Histone H1 vicinity of metallurgical plant in the Nowa Huta (Fig. 1). The culled hares were immediately weighed, to the nearest Liver nuclei were prepared by a modified method of Bush 0.1 kg, and dissected for inspection of sex organs to deter- and Daskal (1977), with the use of 0.1% Triton X-100 for mine their gender. The livers and eye balls were extracted lysing the cells and a subsequent sedimentation of nuclei and put into the zip lock plastic bags in which they were through 1.75 M sucrose buffered with a solution contain- transported to the laboratory in ice. The eye balls were left ing 0.25 mM KCl, 0.5 mM MgCl , 0.5 mM Tris–HCl, pH for 3 days in a 10% formalin. Then, the lenses were excised 7.5. and cleaned thoroughly with deionized water. After dried Histones H1 were extracted according to the procedure in an oven at 80 °C for one night and cooled in a desicca- of Neelin et al. (1995), first with 1 M perchloric acid solu- tor, the lenses were weighed to the nearest of 0.1 mg on tion and then with the perchloric acid in the concentration a microbalance in pairs. Determination of the animal age of 0.5 M. The combined supernatants were precipitated based on the lens weight is possible, because the growth of with 20% trichloroacetic acid and washed twice with the lens lasts to grow up to the death (Lord 1959; Augusteyn acetone acidified with HCl (250:1 v/v) and doubly with 2008) and, hence, the dry mass of the eye lens has com- acetone alone. Dried histone H1 preparations (1 mg) were monly been used to estimate the age of mammalian spe- added to a solution (100 mL) containing 8 M urea, 0.9 M cies (Lord 1959; Friend 1967; Dapson 1980; Augusteyn acetic acid, and 10% 2-mercaptoethanol to prepare the his- 2014). The individuals with a dry weight of the lenses tone H1 samples for electrophoresis. less than 275 mg were classified as juveniles (Andersen and Jensen 1972; Cabon-Raczynska and Raczynski 1972; Suchentrunk et al. 1991). The liver samples were perfused Electrophoresis of Histone H1 with a cold solute on containing 0.13 M NaCl, 0.5 mM KCl, and 0.8 mM MgCl supplemented with 0.1 mM phe- Electrophoretic separation of H1 histones was done as nylmethylsulfonyl fluoride (PMSF) and stored at − 20 °C described by Kowalski and Pałyga (2012b). Both first until H1 histones were isolated. dimension (acetic acid-urea) and second dimension (SDS) electrophoretic gels were 24-cm long. First dimen- sion electrophoretic gels were prepared with 15% acryla- mide, 0.5% methylenebisacrylamide, and 8 M urea. The second dimension electrophoretic gels were composed of 13.5% polyacrylamide and 0.1% SDS. Proteins were stained with the Coomassie Blue R-250, in the concen- trations of 0.05 and 0.0035%, mixed with the mixture of acetic acid-propanol-2 (10–25%) and destained with 10% acetic acid solution. After resolution of proteins in the first dimension, the gel fragments containing the stained H1 histone protein bands were cut out and equilibrated (2 × 15 min) for the second dimension in the buffer con- taining 100 mM Tris-base pH 6.8, 10% glycerol, 2.1% SDS, and 2% 2-mercaptoethanol. Gel Images Processing and Measurements of Histone H1.2 Phenotypes Quantity The obtained electrophoretic patterns of H1 histones were recorded with the gel imaging system Doc-Print II (Vil- ber Lourmat) and processed by the software ImageJ 1.44c (www.rsbwe b.nih.gov/ij). A raw integrated density, which Fig. 1 A map of Poland presenting the hunting areas, i.e., Wopławki (WO) in the Mazurian Lakes and Nowa Huta (NH) in the Małopolska indicate a sum of the values of the pixels in the selected province (black points), from which the brown hares were analyzed in gel area, was measured to evaluate an abundance of histone this work and the regions from which the brown hares were caught in H1.2 phenotypes protein spots. The measurements were the same hunting seasons, i.e., Czempiń (CZ) in the Leszno Lakes as repeated for the seven preparations (n = 7) of each histone well as Płock (PŁ) in the Mazovian industrial region and Rogów (RO) in the Mazovian agricultural space (grey points) H1.2 phenotype. 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:576–584 579 Statistical Evaluation Results A significance of difference between the levels of histone Characteristics of Brown Hare Histone H1.2 H1.2 phenotypes was evaluated with the Student’s t test. Heterogeneity The coefficient of variation for histone H1.2 phenotypes was calculated as a ratio of standard deviation and the Histone H1 preparations obtained from brown hare livers in mean, assuming that the value less than 0.25 corresponds the perchloric acid soluble fraction were initially analyzed to the low variability. Testing for fit of brown hare popula- in the first dimension acetic acid urea polyacrylamide gel. tions to the Hardy–Weinberg equilibrium was done with As seen in the Fig. 2, this electrophoretic technique allows the use of Chi square (χ ) test of goodness-of-fit. A differ - to separate the H1 histones into two fractions only, i.e., a ence among the brown hare age and sex as well as between slow migrating bulk of unresolved histone H1 subtypes the levels of histone H1.2 alleles and phenotypes was eval- protein bands and a faster moving individual band of the uated with the use of Chi square (χ ) test of homogeneity. histone H1.0. However, the more precisely separated H1 In all tests, a p value <0.05 was regarded as statistically histones were visible in the second dimension SDS poly- significant for rejecting the null hypothesis. The genetic acrylamide gel (Fig. 3), in which besides the low mobile differentiation of brown hare population was evaluated by histone H1 variants, H1.3, H1.4, and H1.5, a far migrating Wright’s F statistics indices (Excoffier 2007). The poly - histone H1.2 also was present. According to the differential morphic information content (PIC) of the histone H1.2 rate of histone H1.2 electrophoretic migration, the homozy- locus was evaluated through the allelic frequency with the gous phenotypes (2a and 2b) formed by single protein spots use of formula adopted by Anderson et al. (1993). and a heterozygous phenotype (2a2b) composed of double spot was identified. The co-electrophoresed protein spots belonging to the homozygous phenotypes (2a and 2b) were found as possessing similar in-gel location to the protein spots constituting a heterozygous phenotype 2a2b. Such an electrophoretic pattern confirmed the presence of histone H1.2 heterozygotes (Fig. 3). Quantitative evaluation of the levels of histone H1.2 homozygous phenotypes protein spots revealed their similar amount (p = 0.571) and a comparable share in the heterozygous phenotype (Table 1). Also, a simi- lar proteins abundance was characteristic for both heterozy- gotes and their co-electrophoresed counterparts (p = 0.415). Based on the calculated values of the coefficient of vari- ation, not exceeding 0.25, a relative variability of histone H1.2 phenotypes is low (Table 1). Thus, brown hare histone Fig. 2 The electrophoretic pattern of brown hare histones H1 resolved H1.2 is a polymorphic protein determined by the presence in the first dimension, acetic acid-urea polyacrylamide gel. The of three phenotypes (2a, 2b, and 2a2b), which are coded by slowly migrating intense bands of H1 subtypes (H1s) and the faster two codominant alleles (2a and 2b) at a locus. moving minute bands of subtype H1 (H1 ) that belong to different individuals, numbered 162, 172, 176, 180, and 187, are depicted Fig. 3 The second dimension, SDS polyacrylamide gel, electro- of histone H1.2 reflected by the presence of separately migrating phe- phoretic pattern of brown hare histone H1. On the left, three slow notype 2a2b (individual 180), 2a (individual 167), and 2b (individual migrating protein spots of subtype H1.3, H1.4, and H1.5 and the fast 172) and a joint migration of the co-electrophoresed phenotype 2a migrating protein spot of subtype H1.2. On the right, the variability (individual 167) and 2b (individual 172) 1 3 580 Archives of Environmental Contamination and Toxicology (2018) 75:576–584 Table 1 Abundance of histone a b x y Histone H1.2 phenotype 2a 2b 2a2b co-2a2b H1.2 phenotypes detected in the two-dimensional electrophoretic Mean ± standard deviation 1062.3 ± 112.5 1088.4 ± 88.1 1972.6 ± 211.7 1969.4 ± 171,7 gel patterns Coefficient of variation 0.105 0.081 0.107 0.087 The level of histone H1.2 phenotypes protein spots resolved separately (2a and 2b) and co-electrophoresed in common (co-2a2b) were measured as an integrated density, which is a sum of the values of the pixels in the selected gal area, with the ImageJ processing program for separate individuals (n = 10) belonging to a given phenotype a,b p = 0.571 x,y p = 0.415 Table 2 Age and sexes structure of whole collected brown hare indi- of brown hare populations structure originating from the viduals from agricultural, i.e. Czempiń, Rogów and Wopławki, and regions exposed to different environmental pollutants is pre- industrial, i.e. Płock and Nowa Huta, areas sented in Table 3. While the brown hare population origi- Histone H1.2 Number of individuals (observed/expected) nating from agricultural area of Wopławki conformed to phenotype the Hardy–Weinberg proportions (Χ = 0.035, p = 0.853), a Age Sexes population living in the vicinity of metallurgical plant in the a a b b Juvenile Adult Male Female Nowa Huta significantly differ from the Hardy–Weinberg expectation (Χ = 5.65, p = 0.017). A severe shortage of the 2a 5/4.09 10/10.9 11/9.24 8/9.76 homozygous 2b individuals (frequency 0.05) was detected 2b 1/3.81 13/10.18 4/6.82 10/7.18 in the amount of seven times less compared with the same 2a2b 12/10.09 25/26.9 21/19.95 20/21.05 individuals from the area of Wopławki (frequency 0.35). a 2 Χ = 3.54, p = 0.17 Besides, the surplus of heterozygous 2a2b individuals, exem- b 2 Χ = 2.92, p = 0.23 plified by a negative value of within population inbreeding coefficient (F = − 0.53; Table 4), also was characteristic for this population. Thus, a statistically significant difference in Histone H1.2 Genotypes Variability Within the distribution of histone H1.2 alleles (Χ = 6.50, p = 0.013) and Between Brown Hare Population and phenotypes (Χ = 6.01, p = 0.049) frequency was found between populations. To determine differentiation of brown Determination of the brown hare age structure indicates that hare populations, the Wright’s F-statistics was used as a the adult individuals constitute a major part, i.e., 73.6% of measure of their genetic variation. The different values of the population while a share of sexes is almost the same and individuals (H = 0.625) and subpopulations (H = 0.485) amount to 48.6 and 51.4% of males and females, respec- I S heterozygosity confirmed deviation from the Hardy–Wein- tively. Thus, no statistically significant difference between berg assumption, which also was identified in relation to juvenile and adult individuals (Χ = 3.54, p = 0.17) as well the negative value of F (− 0.288) and F (− 0.255) indi- as between both groups of sexes (Χ = 2.92, p = 0.23) has IS IT ces calculated from observed and expected heterozygosity, been detected in the brown hares originating from all respectively (Table 4). However, the value of F amounting regions in which they were caught (Table 2). The analyses ST Table 3 Histone H1.2 phenotypes and alleles frequency of brown hare population living in the agricultural region of Wopławki and around the metallurgic industry in Nowa Huta a b Phenotype 2a 2b 2a2b Allele 2a 2b Wopławki Number of individuals (observed/expected) 3/3.2 7/7.2 10/9.6 Frequency of allele 0.4 0.6 Frequency of phenotype (observed/expected) 0.15/0.16 0.35/0.36 0.5/0.48 HWE Χ = 0.035, p = 0.853 Nowa Huta Number of individuals (observed/expected) 4/6.6 1/3.6 15/9.8 Frequency of allele 0.575 0.425 Frequency of phenotype (observed/expected) 0.2/0.33 0.05/0.18 0.75/0.48 = 5.65, p = 0.017 HWE Χ a 2 The phenotypic (Χ = 6.01, p = 0.049) b 2 Allelic (Χ = 6.50, p = 0.013) diversity between populations 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:576–584 581 Table 4 Genetic variation Population Heterozygosity Local inbreeding Wright’s F-statistics Polymorphic among the brown hare coefficient (F ) indices information content populations (PIC) Observed Expected F F F IS ST IT Wopławki 0.5 0.48 0.04 − 0.028 0.026 − 0.255 0.48 Nowa Huta 0.75 0.49 − 0.53 0.49 0.026 (Table 4) indicate a negligible genetic differentiation population (Kowalski 2016) was observed after a reloca- between populations and shows that only approximately 3% tion of bird individuals from natural habitat to the breeding. of the total genetic variation concerns a population differ - Thus, the fluctuation of histone H1 polymorphic variants is ence and 97% correspond to the difference between individ- related to the differences in animal living places, i.e., natural uals. The relatively low values of F among the populations versus breeding habitats, and also linked to some physiologi- ST may be caused by a lack of equilibrium between migration cal traits of the organisms. It seems, however, that a scope of and drift. It also seems that brown hare histone H1.2 can be the factors that influence on the distribution of histone H1 used as an reasonably informative marker of environmental phenotypes and alleles might be broader and correspond- changes evoked by both agricultural pollution (PIC = 0.48) ing to the environmental heterogeneity. An example is the and petrochemical contamination (PIC = 0.49; Table 4). brown hare histone H1.2, whose polymorphic variants were The most suitable marker of pollution-induced changes identified in the current study as differently arranged in the is probably more sensitive variant H1.2b (phenotype 2b), area polluted by agricultural and metallurgic activity. The frequency of which was strongly reduced, i.e., seven times evidence for a selective value of H1 histone molecular poly- lower frequency, in the population inhabiting the industrial- morphism was shown in a natural population of the wild ized region. leguminous plant Vicia unijuga inhabiting the territory and surroundings of Novosibirsk. The distribution of four alleles belonging to one of the histone H1 subtype revealed a radial Discussion cline of their pattern, which was likely due to some of man- caused factor, presumably insecticide, that has been used As already reported, the histone H1 has multiple subtypes for a period of time not exceeding the 25 years (Berdnikov in various organisms (for a review, see Parseghian 2015; et al. 1992). Kowalski and Pałyga 2016; Fyodorov et al. 2017). In the set The toxicants that are produced by agricultural farms of mammalian H1 histones, there are regular variants present and industrial plants are different (Malinowska 2004). The in the somatic cells (H1.1, H1.2, H1.3, H1.4, H1.5, H1.0, nitrogen derivatives, i.e., nitrous oxide, nitrogen oxides, and and H1.10) and the variants characteristic for specialized ammonia, are primarily released from fertilizers in the agri- cells, such as sperm (H1t) and oocyte (H1oo) (Happel and cultural production (Parris 2011; Savci 2012) and the vari- Doenecke 2009). Histone H1 polymorphic variants fluctu- ous mixtures of heavy metals, i.e., cadmium, nickel, zinc, ate in a population. In the rabbit breeds, a rare histone H1.4 and lead, are the prevalent side products of metallurgic pro- (H1e) phenotype B was found as occurring at a frequency cesses (Dai et al. 2015; Mizerna 2106). Although there are that ranges from 0.10 to 0.28 only (Pałyga 1990). However, no data about contamination released by agricultural activ- the changes in the distribution of histone H1 polymorphic ity in the region of Wopławki, a higher concentration of variants are well known mainly in the avian H1 histones. nitrates has been observed in the water of lakes located in For example, the duck histone H1 variant H1.a2 was shown the same region (Zieliński et al. 2013; Mioduszewski 2015). as missing in the conservative flocks, in contrast to the vari- Likewise, a determination of pollutions in the close vicin- ant H1.a1 commonly present in all strains tested (Górnicka- ity of Wopławki indicated a higher content of magnesium, Michalska et al. 2014). Likewise, the phenotype b2 and z2 nitrogen, and phosphorus compounds in the places lying of histone H1.b and H1.z, respectively, was not detected near the agricultural areas (Domska et al. 2010). However, in general in the production and conservative groups of as shown by Sobolewski (2016), a predominant number of the duck (Kowalski and Pałyga 2014). A disproportion of Masurian lakes possess transparent water with low level allele frequency in the histone H1.b, H1.d, and H1.z was of the nitrogen compounds. A much higher content of the revealed between quail control line and the line selected for heavy metals, i.e., zinc, cadmium, and lead, was detected in a high yolk cholesterol content (Kowalski et al. 2015). Fur- the soil samples around steel plant in the Nowa Huta (Lenart thermore, a complete lack of histone H1 phenotype b1b2 and Wolny-Koladka 2013). Similarly, the same agents were and c1c2 in the guinea fowl strains (Kowalski et al. 2011) highly concentrated in the tissues of animals, i.e., goat and and an absence of histone H5 phenotype ab in the pheasant sheep (Mundała et al. 2016), bank vole (Myodes glareolus) 1 3 582 Archives of Environmental Contamination and Toxicology (2018) 75:576–584 Acknowledgements This study is part of a project supported by (Damek-Poprawa 2002; Topolska et al. 2004), and yellow- the “Ministry of Science and Higher Education” (granted to J. necked mice (Apodemus flavicollis) (Damek-Poprawa 2002), Markowski in 1992–1995). The authors thank the local hunters for which originated from this area. Thus, the brown hare indi- their cooperation. viduals from populations living in the contaminated areas might be variously sensitive for such agents, serving simul- Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat iveco taneously as an indicators of their incidence. These environ- mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- mental changes might be monitored with the help of histone tion, and reproduction in any medium, provided you give appropriate H1 variant H1.2, which could be considered as a reasonably credit to the original author(s) and the source, provide a link to the informative marker (Botstein et al. 1980) of anthropogenic Creative Commons license, and indicate if changes were made. activity related to the pollutions. It seems that especially vul- nerable for potential toxicants released by metallurgic pro- duction is a phenotype 2b, very poorly represented in popu- lation living in the vicinity of steel plant, in contrast to the References phenotype 2a that is similarly abundant in both agricultural Andersen J, Jensen B (1972) The weight of the eye lens in the European and industrial space. Such a change in the histone H1.2 phe- hares of known age. Acta Theriol 17:87–92 notypes arrangement implies that histone H1 polymorphic Anderson JA, Churchill GA, Autrique JE, Tanksley SD, Sorrells ME variants might not be functionally equivalent and, hence, (1993) Optimizing parental selection for genetic linkage maps. could act individually to regulate the chromatin-dependent Genome 36:181–186 Augusteyn RC (2008) Growth of the lens: in vitro observations. Clin physiological processes. Exp Optom 91:226–239 The environmental stress associated with habitat pol- Augusteyn RC (2014) Growth of the eye lens. I. 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