Variation of Metallothionein I and II Gene Expression in the Bank Vole (Clethrionomys glareolus) Under Environmental Zinc and Cadmium Exposure

Variation of Metallothionein I and II Gene Expression in the Bank Vole (Clethrionomys glareolus)... The main idea of the study was to assess how environmental metal pollution activates defence responses at transcription levels in the tissues of bank voles (Clethrionomys glareolus). For this purpose, the metallothioneine (MT) genes expression (a well known biomarker of exposure and response to various metals) was measured. The real-time PCR method was used for relative quantification of metallothionein I and metallothionein II expressions in the livers, kidneys and testes of bank voles from six populations exposed to different contaminants, mainly zinc, cadmium and iron. The assessment of Zn, Cu and Fe concentrations in the tissues allowed to study the MTs gene expression responses to these metals. ANOVA analysis showed differences between populations in terms of metal concentration in tissues, livers and kidneys. Student T test showed significant differences in metal concentration between unpolluted and polluted sites only for the liver tissue: significantly lower Zn levels and significantly higher Fe levels in the unpolluted sites. Kruskal–Wallis test performed on C data shows differences in the gene expressions between populations for both MT genes for liver and testes. In the liver metallothionein I gene expression was upregulated in populations considered as more polluted (up to 7.5 higher expression in Miasteczko Śląskie comparing to Mikołajki). Expression of metallothionein II revealed a similar pattern. In kidneys, differences in expression of both MT genes were not that evident. In testes, MT upregulation in polluted sites was noted for metallothionein II. For metallothionein however, we found downregulation in populations from more contaminated sites. The expressions of both MTs were positively influenced by cadmium in kidney (concentration data from the previous study) and zinc and copper in liver, while cadmium had effects only on the liver MT II gene expression. Positive relationship was obtained for lead and metallothionein II expression in the liver. Metal pollution continues to pose a serious problem, espe- responses at different biological organisation levels are use - cially for organisms that inhabit areas near sources of metal ful—starting from the cellular level (e.g., markers such as contamination. Although many actions have been under- glutathione concentration) (Srikanth et al. 2013) to popula- taken to decrease metal emission and environmental con- tion level (e.g., biomarkers of genetic diversity) (Guban et al. tamination, wildlife is still at risk (Bickham et al. 2000). 2015). Due to this, contemporary biology has developed sev- To better describe animal functioning in contaminated eral tools to study different types of biomarkers (Shugart habitats, metal tissue assessment approaches themselves are 2000; Walker et al. 2001; Handy et al. 2003). not enough. Data concerning other biomarkers that show When facing a contaminated environment, organisms activate a number of defence mechanisms, such as reduc- tion of absorption, effective excretion, immobilisation in * Magdalena Mikowska cell granules, or production of binding molecules. The first magdalena.szczyrek@uj.edu.pl molecules that respond in the defence are metallothioneins Barbara Dziublińska (MTs), proteins taking part in metal scavenging (Sigel b.maglysz@gmail.com et al. 2009). Four groups of MTs have been described for Renata Świergosz-Kowalewska mammals, among which MT I and MT II are commonly renata.swiergosz-kowalewska@uj.edu.pl found in various tissues (Andrews 2000), whereas MT III 1 and MT IV are more tissue-specific. Metallothioneins have Institute of Environmental Sciences, Jagiellonian University, been widely described among terrestrial vertebrates (Day Gronostajowa 7, 30-387 Kraków, Poland Vol:.(1234567890) 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 67 et  al. 1981), invertebrates (Roesijadi 1996), and plants Materials and Methods (Cobbett and Goldsbrough 2002). MTs in higher eukary- otes regulate zinc and copper levels and its distribution Study Sites and Animal Trapping within the cell and organism (Bremner 1979; Nordberg 1998). These low-molecular weight proteins are rich in The animals collected for this study originated from cysteines, which increase their capacity for scavenging six study sites located in the northern and southern of metal ions, such as Zn, Cd, Cu, Ag, and Hg (Miles part of Poland. Three populations (Mikołajki, Teleśnica et al. 2000; Wang and Fowler 2008). Their protective role Oszwarowa, and Niepołomice) inhabited unpolluted sites; against cadmium toxicity and other oxidative stress is well the other three (Miasteczko Śląskie, Katowice and Olkusz) known (Andrews 2000). Previous studies have shown that were located in the vicinity of zinc/lead smelters in South- metal stress is a good MT inducer (Świergosz-Kowalewska ern Poland in the heavily metal-polluted Silesia region. et al. 2007). The strength of this defence depends on sev- Based on the information concerning the industrial opera- eral factors connected with the metal characteristics and tions, the sites were classified as unpolluted and polluted. organism attributes. The literature also shows that metal- Detailed information on the location of the sites was pre- lothioneins may be induced by nonmetallic toxic agents sented by Mikowska et al. (2014). (glucocorticoids) and physiological conditions, such as The animals were trapped during early autumn using a nutritional status or pregnancy (Gil and Pla 2001). standard live-trapping method (Southwood and Hender- Metallothionein has been used as a biomarker of metal son 2000) and were transported to the laboratory, where exposure in several investigations (Amiard et al. 2006; Atli they were killed by decapitation and dissected according and Canli 2008; Espinoza et al. 2012); however, the Cd- to standard procedure. The livers, kidneys, and testes were saturation method (Onosaka and Cherian 1982) was imple- collected and homogenised. Part of each tissue was frozen mented more frequently; it is less costly and laborious than at – 70 °C and kept for chemical (metal tissue contents) the gene expression method (real-time polymerase chain analyses. The other part of each tissue, approximately reaction [RT-PCR]) used in our studies. Research shows that 30 mg, was stored in RNAlater buffer (Sigma Aldrich, Cd–MT, Cu–MT, and Zn–MT fractions in the kidneys and St. Louis, MO) in − 70 °C for gene expression analysis. liver increase with increasing cytosolic metal concentrations (Rogival et al. 2007). Our approach focused on this specific detoxification process, because it may help to decrease metal Metal Analyses in the Tissues toxicity through binding them in stable molecules and then facilitating effective excretion. It is not well documented Tissue samples (kidney and liver) were dried at 70  °C how metallothionein gene expression varies in natural pop- until they reached constant weight. Next, they were wet- ulations of rodents. Additionally, valuable data that show digested in nitric acid (Suprapur; Merck, Darmstadt, Ger- variation of this parameter in wild rodent populations would many). The metal tissue contents (Cu in the liver; Zn and serve in future studies on bank vole populations. Fe in the liver and kidneys) were determined with atomic As a result, we assessed metallothioneins gene expres- absorption spectrometry (AAnalyst 800; Perkin-Elmer, sions (MT I and MT II) in the tissues of wild bank voles Waltham, MA). The copper concentration in the kidneys (Clethrionomys glareolus), originating from metal polluted was not assessed due to technical limitation (a low amount and unpolluted areas. The chosen study sites have a long his- of sample material). Certified reference material was used tory of smelting and mining and are still at risk from metal to check the analytical precision that was achieved (SRM pollution (Łaszczyca et al. 2004; Augustyniak and Migula 1577c Bovine Liver; National Institute of Standards and 2000). The tissue-dependent intensity of MT I and MT II Technology, Gaithersburg, MD). The metal concentration gene expressions and the effects of environmental realistic is presented in units of metal milligrams per kilogram of −1 exposure on this process are estimated. We hypothesised that dry weight tissue (mg kg  dw). Concentrations of Cd and the level of MT I and MT II gene expression would be cor- Pb in the liver and kidney samples were measured in our related positively with increasing metal concentrations in the previous study (Mikowska et al. 2014). tissues, both essential (Cu, Zn, Fe) and toxic (Pb, Cd—data from our previous study, Mikowska et al. 2014), as measured in the kidneys and livers. RNA Isolation and Reverse Transcription We chose the RT-PCR method, because we wanted to check the MTs defence responses at a transcriptional level, RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, more connected with current exposure to pollution, and Germany) from the livers, kidneys, and testes (according show the potential to activate this detoxifying mechanism. 1 3 68 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 to the vendor’s protocol) from tissues stored in RNAlater sample from the Mikołajki population. This population buffer at − 70 °C. was set as a reference base for low concentrations of lead Approximately 30  mg of each tissue was manually and cadmium in the tissues obtained in the previous study homogenised. Isolation steps were performed using micro- (Mikowska et al. 2014), as well as the expected low exposure centrifuge (Centrifuge 5424, Eppendorf, Hamburg, Ger- to other pollutants, which might be conditioned by its loca- many) with the maximum speed of 14,650 rpm. RNA was tion in a region of low urbanisation level. Relative expres- −ΔΔCт eluted with 50 µL of nuclease-free water (Qiagen) and stored sion was calculated as RQ = 2 . The mean RQ for the at − 70 °C. RNA quality (absorbance ratio A260/A280), and Mikołajki population was set as a reference expression = 1 quantity were verified using the NanoDrop ND-1000 spec- to calculate the multiplication ratio for the other populations trophotometer (PEQLAB Biotechnologie GmbH, Erlangen, (Microsoft Excel 2003). Germany). Comparisons between the populations in terms of gene expressions (ΔC ) were done with the Kruskal–Wallis test Real‑Time Polymerase Chain Reaction (Statistica ver. 10). Regression analysis was performed for each tissue between metal concentrations and ΔC values RT-PCR was performed by using the QuantiFast SybrGreen of MT I or MT II. RT PCR Kit (Qiagen) according to the protocol. The reac- tions were conducted with a 7500 Fast RT-PCR instrument (Applied Biosystems, Foster City, CA). The housekeeping Results gene 18S was used as a reference gene. Primer’s sequences for metallothionein I and II and 18S known from previ- Metal Content ous studies by Świergosz-Kowalewska et al. (2007) were synthesised by Genomed (Warsaw, Poland) and then used Mean Zn levels in the liver tissue ranged from 88.8 mg/ for gene expression analyses. To amplify the target genes, kg dw for animals from the T. Oszwarowa site to 128.0 mg/ 20 µg of RNA from each tissue sample was used. At the kg dw for the animals from the Katowice site (Table 1). For end of each RT-PCR reaction, the melting curve procedure this tissue, the average Fe concentration was the highest in was performed to check for possible unspecific products. the bank voles from Mikołajki (924.5 mg/kg dw) and the All the reactions were run in three replicates. On each plate lowest in the Katowice polluted site (494.3  mg/kg  dw). one identical sample (SS—RNA isolated from one of ran- Mean copper concentration in the livers of animals from dom chosen bank vole liver samples) was run to standardise all the studied populations was comparatively low: range readings between plates. The results of MT I, MT II, and 15.3–18.9 mg/kg dw. 18S gene expressions were determined as C (cycle number) The highest average value for zinc concentration in values. the kidneys was noted for animals originating from Kato- wice (101.3 mg/kg dw). Analysis of metal concentration Statistical Analysis showed the lowest Zn levels in the kidneys of animals from Niepołomice (38.2 mg/kg dw). The Fe concentrations in The means and standard error (± SE) were determined for the kidneys of animals ranged from 281.4 (Katowice) to the metal contents in the kidneys (zinc and iron) and livers 447.5 mg/kg dw (Olkusz). (zinc, iron, and copper). The data were log transformed to fit The ANOVA analysis showed a variation between popu- normal distribution. The tissue metal levels between popu- lations in metal concentration for both tissues—the liver and lations were compared by using one-way ANOVA (using kidney (Table 1b). Results of the T test used to calculate the the Statistica ver. 10 software package; StatSoft Inc., Tulsa, difference in metal concentration between the unpolluted OK). The T test was conducted to compare metal levels in and polluted sites was statistically significant only in the the tissues of the bank voles from the two types of popula- case of the liver tissue: significantly lower Zn levels and tions: unpolluted site populations (Mikołajki, T. Oszwarowa, higher Fe levels in bank voles from the unpolluted sites Niepołomice) and polluted site populations (M. Śląskie, (Table 2). The results also were statistically significant for Katowice, Olkusz). lead and cadmium in the kidneys and liver, showing a high The obtained the C value for the SS sample, which was accumulation of both metals in animals originating from the located on each analysed plate, was used to standardise all polluted sites (Mikowska et al. 2014). of the C readings (Microsoft Office Excel 2003; Microsoft Corporation). Then, ΔC for each sample was calculated as Gene Expression a difference between the C value of the target gene (MT I or MT II) and the C value of the endogenous control (18S). The results of the Kruskal–Wallis test performed on ΔC T T The values of ΔΔC were determined based on ΔC of one data show statistical differences in genes expressions T T 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 69 Table 1 a Metal concentrations (in mg/kg dw) in the tissues of bank voles trapped in unpolluted (marked with *) and polluted sites. b Statisti- cally significant differences in metal tissue concentration between sites (for liver: above the diagonal; for kidney: under the diagonal) (a) (b) X arithmetic mean concentration, SE standard error, N number of individuals, F number of females, M number of males p < 0.05 times that of Mikołajki. For both MTs, the lowest values Table 2 Results of T test analyses (p value and F ratio) for differences were obtained in the livers of animals from Mikołajki site. in studied metal concentrations between unpolluted and polluted types of populations Expressions of both MTs in the kidney tissue were not such strongly regulated as in the liver (Fig. 1b). Both Element/tissue p value F ratio metallothioneins in the kidney tissues of animals from T. Zn liver < 0.001 3.09 Oszwarowa and Niepołomice were downregulated compared Fe liver < 0.001 1.03 to the Mikołajki site. The levels of MTs expressions in the Pb liver* 0.014 10.54 other populations were around the levels obtained for the Cd liver* < 0.001 8.60 Mikołajki site. Pb kidney* < 0.001 1.25 The lowest level of MT I expression in the testes was Cd kidney* < 0.001 3.09 found in the animals from the Olkusz and Katowice sites, whereas the highest was at the Mikołajki site (Fig. 1c). MT *Metal concentration data obtained in previous study (Mikowska II expressions in the testes were more pronounced than in et al. 2014) the case of MT I-up to four times higher in the Olkusz than in Mikołajki site. between populations in the case of both MT genes for the The results of the regression analysis showed a significant liver and testes (Table  3). The highest MT I expressions relationship between cadmium and lead concentrations and (RQ values) in the liver tissue of animals were found in the expressions of both MTs in the kidney (Table 4; Fig. 2). In M. Śląskie polluted site, up to 7.5 time higher than in the the liver, we found major effects of zinc and copper concen - Mikołajki site (Fig. 1a). Similarly, the MT II expressions trations on both MT I and MT II gene expressions, as well were the highest in Katowice and M. Śląskie-up to eight 1 3 70 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 Table 3 Significant between-population differences (p  <  0.05) in expressions of MT I (above diagonal) and MT II (under diagonal) found for studied tissues Liver MT I Liver MT II Table 4 Summary of regression analyses (p value, F ratio, and degrees of freedom) between the tissue metals concentrations and MT (a) I or MT II gene expressions (based on ΔC values) relationship 8 T 6 Pb Cd Zn Cu MT I Liver p = 0.015 p = 0.005 p = 0.025 F = 6.215 F = 8.349 F = 5.284 df = 67 df = 65 df = 66 Mikołajki T.Oszwarowa Niepołomice Katowice M. Śląskie Olkusz (−) (−) (−) Kidney MT I Kidney MT II Kidney p = 0.003 p = 0.004 NA 1,4 F = 9.548 F = 9.136 (b) 1,2 df = 66 df = 65 (−) (−) 1,0 0,8 MT II Liver p < 0.001 p < 0.001 p = 0.003 0,6 F = 18.751 F = 48.905 F = 9.217 0,4 df = 63 df = 64 df = 65 0,2 (−) (−) (−) 0,0 Kidney p = 0.016 p = 0.018 NA Mikołajki T.Oszwarowa Niepołomice KatowiceM. Śląskie Olkusz F = 6.151 F = 5.878 df = 66 df = 65 Testes MT I Testes MT II (c) (−) (−) Only significant p values are reported 3 Directions of relationships are put in brackets (−) NA concentration not measured, df degrees of freedom Discussion MikołajkiT.OszwarowaNiepołomiceKatowice M. Śląskie Olkusz The main goal of this work was to ascertain how animals Fig. 1 Normalised relative MT I/MT II gene expressions in the tis- cope with metal contamination in terms of transcriptional sues of bank voles from unpolluted and polluted sites. Liver (a); kid- regulation of the MT I and MT II gene expressions. Both ney (b); testis (c) the results of metal concentrations and gene expressions (quantitative PCR results) studied in the same tissue sam- as between cadmium and MT II gene expression (Table 4; ples allowed us to correlate exposure directly with the Fig. 2). level of tissue MT defence and assess the potential effects 1 3 Relative gene expression (RQ) Relative gene expression (RQ) Relative gene expression (RQ) Archives of Environmental Contamination and Toxicology (2018) 75:66–74 71 Fig. 2 Relationships between metal concentrations (mg/kg dw) in the liver or kidney of bank voles and MT I or MT II gene expressions (expressed as ΔC values) of environmental contamination to these organisms. The fact rather comparable to Zn levels obtained for the polluted uniqueness of this kind of study lies in an approach of sites (Table 1). assessing this biomarker in naturally exposed wild popula- Interesting findings concerning Fe levels in the livers, tions and also showing its natural variation. Because the which are comparatively low at polluted sites, suggest iron literature shows that photoperiod may change the level of depletion rather than enhancement. The lowest Cu concen- cadmium concentrations in organs (Włostowski et al. 2000) tration found in the livers of animals from T. Oszwarowa and also the level of metallothioneins—higher level of MTs significantly differed compared with the values obtained in winter than in summer (Marques et al. 2008)—all animals for M. Śląskie and Niepołomice, but all of the values were were trapped during one season. In this way, data variance within the range of natural levels (Mikowska et al. 2014). obtained in the study was not due to different photoperiods. The zinc and iron levels identified in the tissues of the stud- By separately grouping the data for populations located near ied animals were comparable to those found in bank voles by zinc–lead smelters (polluted sites) and those occupying rela- Swiergosz-Kowalewska et al. (2007), and they are within a tively unpolluted sites, we could study not only the differ - range of environmental variation typical for both unpolluted ences between single populations, but also the differences and polluted sites. The cadmium and lead concentrations between two types of populations: unpolluted and polluted. in the liver and kidney of the studied animals were already By choosing populations for the study from differently published in a previous paper (Mikowska et al. 2014) and contaminated sites (for more detail, cf. Mikowska et  al. also reflected between-population differences in the levels 2014), we expected to find distinct differences for both of these metals (Fig. 3). These results confirmed our expec- metal levels and MTs expressions between them. Statis- tations about increased cadmium and lead levels in the tis- tical analysis did not fully confirm our expectations and sues of animals inhabiting areas in close proximity to main initially conducted site classification for unpolluted and sources of pollutants (Mikowska et al. 2014). Generally, the polluted. Analyses of the differences between single popu- animal exposure to metals and their uptake in the polluted lations showed that Zn concentrations in the livers of ani- sites were higher than in the unpolluted sites. mals from Niepołomice, classified as unpolluted, differed As a response to lead and cadmium accumulation in the from the other results for the unpolluted sites, and are in tissues of bank voles, the study clearly showed between- population differences in MT I and MT II expression for the 1 3 72 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 concentration and MTs genes expressions, indicated that the metals accumulated in some tissues positively influenced MT I and MT II genes expression (Table 4; Fig. 2). Thus, MT I and II expressions were positively influenced by accu- mulated zinc and copper in the liver and by lead and cad- mium in the kidney. Additionally, MT II expression in the liver was upregulated by cadmium. Research similar to ours was performed by Fritsch et al. (2010) on seven sympatric small mammal species collected near former smelters along a pollution gradient, in an area considered as highly polluted with Cd, Pb, and Zn. The authors found increasing-with-age Cd concentrations in the Fig. 3 Cadmium concentrations (mg/kg dw) in the tissues of bank liver and kidney in the case of all the studied species and voles trapped in unpolluted and polluted sites (modified from Mikowska et al. 2014). Bars indicate standard errors. Different letters some patterns of low tissue metal concentrations and low above bar indicate significant difference between populations sepa- metallothioneins levels. A slight increase of MT with Cd rately for each tissue accumulation was noted by Fritsch et al. (2010) for bank vole species. The literature provides more examples of MT livers and testes of the animals but not for the kidney tis- induction (e.g., the findings of Chater et al. 2008) than inhi- sue (ANOVA test). Between-population differences in MTs bition after metal exposure. Chater et al. (2008) studied the gene expressions are more visible in the case of liver tissue, effects of sub-acute cadmium treatment (3 mg of cadmium where the expression is up to eight times higher (MT II) in chloride per kg of bw) on pregnant female rats and noted polluted sites compared with the unpolluted reference—the MT increase (measured with Cd method) in the liver tissues. Mikołajki site. In contrast, the analysis of MT I expression in In such a treatment type, MTs induction is supposed to be the animal testes gave a surprising result of downregulation more evident than after exposure to mostly low environmen- in animals from all the sites when compared to the Mikołajki tal contamination, as we hypothesised in our study. site. Contrary to MT I expression, analysis of the testes of Metallothionein I and II expression in the kidney of animals inhabiting polluted sites and also the Niepołomice Sprague–Dawley rats after intoxication with lead acetate site showed a clear pattern of upregulation of the MT II (300 mg/L) and/or cadmium dichloride (50 mg/L), sepa- gene (up to 4 times higher than those from the Mikołajki rately and in combination, was studied by Wang and Fowler reference site). Taking into account the results of MTs in the (2008). As they revealed, there were no effects of lead on liver and MT II in the testes, the results for animals from the both MTs. Animals from the Cd and Cd–Pb treated groups Niepołomice site were close to the results for animals from had significantly higher expression in the kidney than from sites initially classified as polluted. These expression data the control and, additionally, animals from the Cd–Pb group also reflect the results concerning zinc and cadmium levels had significantly higher MTs expressions than the Cd and Pb in these studied tissues. separately treated groups, which suggests a synergistic effect As mentioned earlier, the statistical results for kidney of these metals. Despite the fact that our studied populations tissues show a similar level of expression for both the stud- were exposed to both cadmium and lead, we may only con- ied MT genes (lower than for Mikołajki) among the studied clude that the main MTs inducer was cadmium because the populations. However, there are some signs of slight upregu- tissue lead concentration was very low. lation in the tissue of bank voles from polluted sites. In the According to some literature data rodent testes are study by Dondero et al. (2005), the authors assessed gene more sensitive to cadmium toxicity than the liver tissue expression of MT10 and MT20, representing the metal- (Mckenna et al. 1996; Ren 2003). Available figures are lothionein gene family. They found that heavy metals influ- inconclusive about the levels of metallothioneins gene enced the expression of the MT10 and MT20 genes and the expressions in the testes samples under environmental level of expression depended on the metal and gene. In case conditions. In some studies, the authors noted no induction of cadmium and MT20 expression reached up to 2200-fold of MT genes expression after Cd exposure (Shiraishi et al. induction. However, in most cases MT expression varied 1995), whereas others confirmed upregulation in rodents between 2- and 30-fold. These results suggest that, in our intoxicated with Cd and Cd/Zn mixture (Messaoudi et al. case, the impact of metal exposure was rather moderate. 2010). In the studied bank vole testes, the MTs expres- Our results concerning MT regulations in the animals sions were not high. What more, MT I expression in from the unpolluted and polluted sites were confirmed the testes of animals from the reference site (Mikołajki) by the results of the regression analysis. The regression was the highest. Downregulation was distinguished for analysis, which takes into account individual tissue metal the remaining sites—up to five times lower than at the 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 73 Open Access This article is distributed under the terms of the Creative Mikołajki site. Bonda et al. (2004) showed that testicular Commons Attribution 4.0 International License (http://creativecom- MT levels after cadmium intake are lower among adult mons.org/licenses/by/4.0/), which permits unrestricted use, distribu- individuals when compared to young animals. Similar to tion, and reproduction in any medium, provided you give appropriate our research, cadmium contamination seemed to decrease credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. the levels of metallothioneins gene expression. However, in our study, regression analysis did not demonstrate any relationship between metal concentration in the testes and References gene expression of MT I, suggesting that this low expres- sion was not due to the studied pollutants but rather that Amiard J-C, Amiard-Triquet C, Barka S, Pellerin J, Rainbow PS the basal expression was low in all populations due to (2006) Metallothioneins in aquatic invertebrates: their role in other potential factors. metal detoxification and their use as biomarkers. Aquat Toxicol 76:160–202 Andrews GK (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol 59(1):95–104 Atli G, Canli M (2008) Responses of metallothionein and reduced Conclusions glutathione in a freshwater fish Oreochromis niloticus following metal exposures. 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Aquat Toxicol PL0419, from the Polish Ministry of Science and Education, the EEA 110–111:37–44. https://doi.org/10.1016/j.aquatox.2011.12.012 Financial Mechanism, the Norwegian Financial Mechanism. Fritsch C, Cosson RP, Cœurdassier M, Raoul F, Giraudoux P et al (2010) Responses of wild small mammals to a pollution gradient: host factors influence metal and metallothionein levels. Environ Compliance with Ethical Standards Pollut 158:827–840 Gil F, Pla A (2001) Biomarkers as biological indicators of xenobiotic Ethical Standards All research complied with ethical standards con- exposure. J Appl Toxicol 21(4):245–255 cerning the treatment of animals. The authors obtained the agreement Guban P, Wennerström L, Elfwing T, Sundelin B, Laikre L (2015) of the I Local Ethic Committee (Kraków, Poland, Decision No. 48/2007 Genetic diversity in Monoporeia affinis at polluted and reference from 10.05.2007). sites of the Baltic Bothnian Bay. 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Royal Society of Chemistry, Cambridge Messaoudi I, Banni M, Saïd L, Saïd K, Kerkeni A (2010) Evaluation Southwood TRE, Henderson PA (2000) Ecological methods, 3rd edn. of involvement of testicular metallothionein gene expression in Blackwell Publishing Ltd., Oxford, Malden, MA the protective effect of zinc against cadmium-induced testicular Srikanth K, Pereira E, Duarte AC, Ahmad I (2013) Glutathione pathophysiology in rat. Reprod Toxicol 29(3):339–345. https:// and its dependent enzymes’ modulatory responses to toxic doi.org/10.1016/j.reprotox.2010.01.004 metals and metalloids in fish—a review. Environ Sci Pollut R Mikowska M, Gaura A, Sadowska E, Koteja P, Świergosz-Kowalewska 20(4):2133–2149 R (2014) Genetic variation in bank vole populations in natural Świergosz-Kowalewska R, Bednarska A, Callaghan A (2007) Expres- and metal-contaminated areas. Arch Environ Contam Toxicol sion of metallothionein genes I and II in bank vole Clethrionomys 67:535–546 glareolus populations chronically exposed in situ to heavy metals. Miles AT, Hawksworth GM, Beattie JH, Rodilla V (2000) Induction, Environ Sci Technol 41(3):1032–1037 regulation, degradation, and biological significance of mammalian Walker CH, Hopkin SP, Sibly RM, Peakall DB (2001) Principles of metallothioneins. Crit Rev Biochem Mol 35:35–70 ecotoxicology, 2nd edn. Taylor & Francis, London Nordberg M (1998) Metallothioneins: historical review and state of Wang G, Fowler B (2008) Roles of biomarkers in evaluating inter- knowledge. Talanta 46:243–254 actions among mixtures of lead, cadmium and arsenic. Toxi- Onosaka S, Cherian GM (1982) Comparison of metallothionein deter- col Appl Pharmacol 233(1):92–99. https://doi.org/10.1016/j. mination by polarographic and cadmium-saturation methods. taap.2008.01.01 Toxicol Appl Pharmacol 63(2):270–274 Włostowski T, Krasowska A, Laszkiewicz-Tiszczenko B (2000) Ren X (2003) Metallothionein gene expression under different time Dietary cadmium induces histopathological changes despite a in testicular Sertoli and spermatogenic cells of rats treated with sufficient metallothionein level in the liver and kidneys of the cadmium. Reprod Toxicol 17(2):219–227. https://doi.org/10.1016/ bank vole (Clethrionomys glareolus). Comp Biochem Physiol C S0890-6238(02)00151-X 126:21–28 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Environmental Contamination and Toxicology Springer Journals

Variation of Metallothionein I and II Gene Expression in the Bank Vole (Clethrionomys glareolus) Under Environmental Zinc and Cadmium Exposure

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Environment; Ecotoxicology; Pollution, general; Environmental Health; Environmental Chemistry; Soil Science & Conservation; Monitoring/Environmental Analysis
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0090-4341
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10.1007/s00244-017-0485-7
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Abstract

The main idea of the study was to assess how environmental metal pollution activates defence responses at transcription levels in the tissues of bank voles (Clethrionomys glareolus). For this purpose, the metallothioneine (MT) genes expression (a well known biomarker of exposure and response to various metals) was measured. The real-time PCR method was used for relative quantification of metallothionein I and metallothionein II expressions in the livers, kidneys and testes of bank voles from six populations exposed to different contaminants, mainly zinc, cadmium and iron. The assessment of Zn, Cu and Fe concentrations in the tissues allowed to study the MTs gene expression responses to these metals. ANOVA analysis showed differences between populations in terms of metal concentration in tissues, livers and kidneys. Student T test showed significant differences in metal concentration between unpolluted and polluted sites only for the liver tissue: significantly lower Zn levels and significantly higher Fe levels in the unpolluted sites. Kruskal–Wallis test performed on C data shows differences in the gene expressions between populations for both MT genes for liver and testes. In the liver metallothionein I gene expression was upregulated in populations considered as more polluted (up to 7.5 higher expression in Miasteczko Śląskie comparing to Mikołajki). Expression of metallothionein II revealed a similar pattern. In kidneys, differences in expression of both MT genes were not that evident. In testes, MT upregulation in polluted sites was noted for metallothionein II. For metallothionein however, we found downregulation in populations from more contaminated sites. The expressions of both MTs were positively influenced by cadmium in kidney (concentration data from the previous study) and zinc and copper in liver, while cadmium had effects only on the liver MT II gene expression. Positive relationship was obtained for lead and metallothionein II expression in the liver. Metal pollution continues to pose a serious problem, espe- responses at different biological organisation levels are use - cially for organisms that inhabit areas near sources of metal ful—starting from the cellular level (e.g., markers such as contamination. Although many actions have been under- glutathione concentration) (Srikanth et al. 2013) to popula- taken to decrease metal emission and environmental con- tion level (e.g., biomarkers of genetic diversity) (Guban et al. tamination, wildlife is still at risk (Bickham et al. 2000). 2015). Due to this, contemporary biology has developed sev- To better describe animal functioning in contaminated eral tools to study different types of biomarkers (Shugart habitats, metal tissue assessment approaches themselves are 2000; Walker et al. 2001; Handy et al. 2003). not enough. Data concerning other biomarkers that show When facing a contaminated environment, organisms activate a number of defence mechanisms, such as reduc- tion of absorption, effective excretion, immobilisation in * Magdalena Mikowska cell granules, or production of binding molecules. The first magdalena.szczyrek@uj.edu.pl molecules that respond in the defence are metallothioneins Barbara Dziublińska (MTs), proteins taking part in metal scavenging (Sigel b.maglysz@gmail.com et al. 2009). Four groups of MTs have been described for Renata Świergosz-Kowalewska mammals, among which MT I and MT II are commonly renata.swiergosz-kowalewska@uj.edu.pl found in various tissues (Andrews 2000), whereas MT III 1 and MT IV are more tissue-specific. Metallothioneins have Institute of Environmental Sciences, Jagiellonian University, been widely described among terrestrial vertebrates (Day Gronostajowa 7, 30-387 Kraków, Poland Vol:.(1234567890) 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 67 et  al. 1981), invertebrates (Roesijadi 1996), and plants Materials and Methods (Cobbett and Goldsbrough 2002). MTs in higher eukary- otes regulate zinc and copper levels and its distribution Study Sites and Animal Trapping within the cell and organism (Bremner 1979; Nordberg 1998). These low-molecular weight proteins are rich in The animals collected for this study originated from cysteines, which increase their capacity for scavenging six study sites located in the northern and southern of metal ions, such as Zn, Cd, Cu, Ag, and Hg (Miles part of Poland. Three populations (Mikołajki, Teleśnica et al. 2000; Wang and Fowler 2008). Their protective role Oszwarowa, and Niepołomice) inhabited unpolluted sites; against cadmium toxicity and other oxidative stress is well the other three (Miasteczko Śląskie, Katowice and Olkusz) known (Andrews 2000). Previous studies have shown that were located in the vicinity of zinc/lead smelters in South- metal stress is a good MT inducer (Świergosz-Kowalewska ern Poland in the heavily metal-polluted Silesia region. et al. 2007). The strength of this defence depends on sev- Based on the information concerning the industrial opera- eral factors connected with the metal characteristics and tions, the sites were classified as unpolluted and polluted. organism attributes. The literature also shows that metal- Detailed information on the location of the sites was pre- lothioneins may be induced by nonmetallic toxic agents sented by Mikowska et al. (2014). (glucocorticoids) and physiological conditions, such as The animals were trapped during early autumn using a nutritional status or pregnancy (Gil and Pla 2001). standard live-trapping method (Southwood and Hender- Metallothionein has been used as a biomarker of metal son 2000) and were transported to the laboratory, where exposure in several investigations (Amiard et al. 2006; Atli they were killed by decapitation and dissected according and Canli 2008; Espinoza et al. 2012); however, the Cd- to standard procedure. The livers, kidneys, and testes were saturation method (Onosaka and Cherian 1982) was imple- collected and homogenised. Part of each tissue was frozen mented more frequently; it is less costly and laborious than at – 70 °C and kept for chemical (metal tissue contents) the gene expression method (real-time polymerase chain analyses. The other part of each tissue, approximately reaction [RT-PCR]) used in our studies. Research shows that 30 mg, was stored in RNAlater buffer (Sigma Aldrich, Cd–MT, Cu–MT, and Zn–MT fractions in the kidneys and St. Louis, MO) in − 70 °C for gene expression analysis. liver increase with increasing cytosolic metal concentrations (Rogival et al. 2007). Our approach focused on this specific detoxification process, because it may help to decrease metal Metal Analyses in the Tissues toxicity through binding them in stable molecules and then facilitating effective excretion. It is not well documented Tissue samples (kidney and liver) were dried at 70  °C how metallothionein gene expression varies in natural pop- until they reached constant weight. Next, they were wet- ulations of rodents. Additionally, valuable data that show digested in nitric acid (Suprapur; Merck, Darmstadt, Ger- variation of this parameter in wild rodent populations would many). The metal tissue contents (Cu in the liver; Zn and serve in future studies on bank vole populations. Fe in the liver and kidneys) were determined with atomic As a result, we assessed metallothioneins gene expres- absorption spectrometry (AAnalyst 800; Perkin-Elmer, sions (MT I and MT II) in the tissues of wild bank voles Waltham, MA). The copper concentration in the kidneys (Clethrionomys glareolus), originating from metal polluted was not assessed due to technical limitation (a low amount and unpolluted areas. The chosen study sites have a long his- of sample material). Certified reference material was used tory of smelting and mining and are still at risk from metal to check the analytical precision that was achieved (SRM pollution (Łaszczyca et al. 2004; Augustyniak and Migula 1577c Bovine Liver; National Institute of Standards and 2000). The tissue-dependent intensity of MT I and MT II Technology, Gaithersburg, MD). The metal concentration gene expressions and the effects of environmental realistic is presented in units of metal milligrams per kilogram of −1 exposure on this process are estimated. We hypothesised that dry weight tissue (mg kg  dw). Concentrations of Cd and the level of MT I and MT II gene expression would be cor- Pb in the liver and kidney samples were measured in our related positively with increasing metal concentrations in the previous study (Mikowska et al. 2014). tissues, both essential (Cu, Zn, Fe) and toxic (Pb, Cd—data from our previous study, Mikowska et al. 2014), as measured in the kidneys and livers. RNA Isolation and Reverse Transcription We chose the RT-PCR method, because we wanted to check the MTs defence responses at a transcriptional level, RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, more connected with current exposure to pollution, and Germany) from the livers, kidneys, and testes (according show the potential to activate this detoxifying mechanism. 1 3 68 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 to the vendor’s protocol) from tissues stored in RNAlater sample from the Mikołajki population. This population buffer at − 70 °C. was set as a reference base for low concentrations of lead Approximately 30  mg of each tissue was manually and cadmium in the tissues obtained in the previous study homogenised. Isolation steps were performed using micro- (Mikowska et al. 2014), as well as the expected low exposure centrifuge (Centrifuge 5424, Eppendorf, Hamburg, Ger- to other pollutants, which might be conditioned by its loca- many) with the maximum speed of 14,650 rpm. RNA was tion in a region of low urbanisation level. Relative expres- −ΔΔCт eluted with 50 µL of nuclease-free water (Qiagen) and stored sion was calculated as RQ = 2 . The mean RQ for the at − 70 °C. RNA quality (absorbance ratio A260/A280), and Mikołajki population was set as a reference expression = 1 quantity were verified using the NanoDrop ND-1000 spec- to calculate the multiplication ratio for the other populations trophotometer (PEQLAB Biotechnologie GmbH, Erlangen, (Microsoft Excel 2003). Germany). Comparisons between the populations in terms of gene expressions (ΔC ) were done with the Kruskal–Wallis test Real‑Time Polymerase Chain Reaction (Statistica ver. 10). Regression analysis was performed for each tissue between metal concentrations and ΔC values RT-PCR was performed by using the QuantiFast SybrGreen of MT I or MT II. RT PCR Kit (Qiagen) according to the protocol. The reac- tions were conducted with a 7500 Fast RT-PCR instrument (Applied Biosystems, Foster City, CA). The housekeeping Results gene 18S was used as a reference gene. Primer’s sequences for metallothionein I and II and 18S known from previ- Metal Content ous studies by Świergosz-Kowalewska et al. (2007) were synthesised by Genomed (Warsaw, Poland) and then used Mean Zn levels in the liver tissue ranged from 88.8 mg/ for gene expression analyses. To amplify the target genes, kg dw for animals from the T. Oszwarowa site to 128.0 mg/ 20 µg of RNA from each tissue sample was used. At the kg dw for the animals from the Katowice site (Table 1). For end of each RT-PCR reaction, the melting curve procedure this tissue, the average Fe concentration was the highest in was performed to check for possible unspecific products. the bank voles from Mikołajki (924.5 mg/kg dw) and the All the reactions were run in three replicates. On each plate lowest in the Katowice polluted site (494.3  mg/kg  dw). one identical sample (SS—RNA isolated from one of ran- Mean copper concentration in the livers of animals from dom chosen bank vole liver samples) was run to standardise all the studied populations was comparatively low: range readings between plates. The results of MT I, MT II, and 15.3–18.9 mg/kg dw. 18S gene expressions were determined as C (cycle number) The highest average value for zinc concentration in values. the kidneys was noted for animals originating from Kato- wice (101.3 mg/kg dw). Analysis of metal concentration Statistical Analysis showed the lowest Zn levels in the kidneys of animals from Niepołomice (38.2 mg/kg dw). The Fe concentrations in The means and standard error (± SE) were determined for the kidneys of animals ranged from 281.4 (Katowice) to the metal contents in the kidneys (zinc and iron) and livers 447.5 mg/kg dw (Olkusz). (zinc, iron, and copper). The data were log transformed to fit The ANOVA analysis showed a variation between popu- normal distribution. The tissue metal levels between popu- lations in metal concentration for both tissues—the liver and lations were compared by using one-way ANOVA (using kidney (Table 1b). Results of the T test used to calculate the the Statistica ver. 10 software package; StatSoft Inc., Tulsa, difference in metal concentration between the unpolluted OK). The T test was conducted to compare metal levels in and polluted sites was statistically significant only in the the tissues of the bank voles from the two types of popula- case of the liver tissue: significantly lower Zn levels and tions: unpolluted site populations (Mikołajki, T. Oszwarowa, higher Fe levels in bank voles from the unpolluted sites Niepołomice) and polluted site populations (M. Śląskie, (Table 2). The results also were statistically significant for Katowice, Olkusz). lead and cadmium in the kidneys and liver, showing a high The obtained the C value for the SS sample, which was accumulation of both metals in animals originating from the located on each analysed plate, was used to standardise all polluted sites (Mikowska et al. 2014). of the C readings (Microsoft Office Excel 2003; Microsoft Corporation). Then, ΔC for each sample was calculated as Gene Expression a difference between the C value of the target gene (MT I or MT II) and the C value of the endogenous control (18S). The results of the Kruskal–Wallis test performed on ΔC T T The values of ΔΔC were determined based on ΔC of one data show statistical differences in genes expressions T T 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 69 Table 1 a Metal concentrations (in mg/kg dw) in the tissues of bank voles trapped in unpolluted (marked with *) and polluted sites. b Statisti- cally significant differences in metal tissue concentration between sites (for liver: above the diagonal; for kidney: under the diagonal) (a) (b) X arithmetic mean concentration, SE standard error, N number of individuals, F number of females, M number of males p < 0.05 times that of Mikołajki. For both MTs, the lowest values Table 2 Results of T test analyses (p value and F ratio) for differences were obtained in the livers of animals from Mikołajki site. in studied metal concentrations between unpolluted and polluted types of populations Expressions of both MTs in the kidney tissue were not such strongly regulated as in the liver (Fig. 1b). Both Element/tissue p value F ratio metallothioneins in the kidney tissues of animals from T. Zn liver < 0.001 3.09 Oszwarowa and Niepołomice were downregulated compared Fe liver < 0.001 1.03 to the Mikołajki site. The levels of MTs expressions in the Pb liver* 0.014 10.54 other populations were around the levels obtained for the Cd liver* < 0.001 8.60 Mikołajki site. Pb kidney* < 0.001 1.25 The lowest level of MT I expression in the testes was Cd kidney* < 0.001 3.09 found in the animals from the Olkusz and Katowice sites, whereas the highest was at the Mikołajki site (Fig. 1c). MT *Metal concentration data obtained in previous study (Mikowska II expressions in the testes were more pronounced than in et al. 2014) the case of MT I-up to four times higher in the Olkusz than in Mikołajki site. between populations in the case of both MT genes for the The results of the regression analysis showed a significant liver and testes (Table  3). The highest MT I expressions relationship between cadmium and lead concentrations and (RQ values) in the liver tissue of animals were found in the expressions of both MTs in the kidney (Table 4; Fig. 2). In M. Śląskie polluted site, up to 7.5 time higher than in the the liver, we found major effects of zinc and copper concen - Mikołajki site (Fig. 1a). Similarly, the MT II expressions trations on both MT I and MT II gene expressions, as well were the highest in Katowice and M. Śląskie-up to eight 1 3 70 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 Table 3 Significant between-population differences (p  <  0.05) in expressions of MT I (above diagonal) and MT II (under diagonal) found for studied tissues Liver MT I Liver MT II Table 4 Summary of regression analyses (p value, F ratio, and degrees of freedom) between the tissue metals concentrations and MT (a) I or MT II gene expressions (based on ΔC values) relationship 8 T 6 Pb Cd Zn Cu MT I Liver p = 0.015 p = 0.005 p = 0.025 F = 6.215 F = 8.349 F = 5.284 df = 67 df = 65 df = 66 Mikołajki T.Oszwarowa Niepołomice Katowice M. Śląskie Olkusz (−) (−) (−) Kidney MT I Kidney MT II Kidney p = 0.003 p = 0.004 NA 1,4 F = 9.548 F = 9.136 (b) 1,2 df = 66 df = 65 (−) (−) 1,0 0,8 MT II Liver p < 0.001 p < 0.001 p = 0.003 0,6 F = 18.751 F = 48.905 F = 9.217 0,4 df = 63 df = 64 df = 65 0,2 (−) (−) (−) 0,0 Kidney p = 0.016 p = 0.018 NA Mikołajki T.Oszwarowa Niepołomice KatowiceM. Śląskie Olkusz F = 6.151 F = 5.878 df = 66 df = 65 Testes MT I Testes MT II (c) (−) (−) Only significant p values are reported 3 Directions of relationships are put in brackets (−) NA concentration not measured, df degrees of freedom Discussion MikołajkiT.OszwarowaNiepołomiceKatowice M. Śląskie Olkusz The main goal of this work was to ascertain how animals Fig. 1 Normalised relative MT I/MT II gene expressions in the tis- cope with metal contamination in terms of transcriptional sues of bank voles from unpolluted and polluted sites. Liver (a); kid- regulation of the MT I and MT II gene expressions. Both ney (b); testis (c) the results of metal concentrations and gene expressions (quantitative PCR results) studied in the same tissue sam- as between cadmium and MT II gene expression (Table 4; ples allowed us to correlate exposure directly with the Fig. 2). level of tissue MT defence and assess the potential effects 1 3 Relative gene expression (RQ) Relative gene expression (RQ) Relative gene expression (RQ) Archives of Environmental Contamination and Toxicology (2018) 75:66–74 71 Fig. 2 Relationships between metal concentrations (mg/kg dw) in the liver or kidney of bank voles and MT I or MT II gene expressions (expressed as ΔC values) of environmental contamination to these organisms. The fact rather comparable to Zn levels obtained for the polluted uniqueness of this kind of study lies in an approach of sites (Table 1). assessing this biomarker in naturally exposed wild popula- Interesting findings concerning Fe levels in the livers, tions and also showing its natural variation. Because the which are comparatively low at polluted sites, suggest iron literature shows that photoperiod may change the level of depletion rather than enhancement. The lowest Cu concen- cadmium concentrations in organs (Włostowski et al. 2000) tration found in the livers of animals from T. Oszwarowa and also the level of metallothioneins—higher level of MTs significantly differed compared with the values obtained in winter than in summer (Marques et al. 2008)—all animals for M. Śląskie and Niepołomice, but all of the values were were trapped during one season. In this way, data variance within the range of natural levels (Mikowska et al. 2014). obtained in the study was not due to different photoperiods. The zinc and iron levels identified in the tissues of the stud- By separately grouping the data for populations located near ied animals were comparable to those found in bank voles by zinc–lead smelters (polluted sites) and those occupying rela- Swiergosz-Kowalewska et al. (2007), and they are within a tively unpolluted sites, we could study not only the differ - range of environmental variation typical for both unpolluted ences between single populations, but also the differences and polluted sites. The cadmium and lead concentrations between two types of populations: unpolluted and polluted. in the liver and kidney of the studied animals were already By choosing populations for the study from differently published in a previous paper (Mikowska et al. 2014) and contaminated sites (for more detail, cf. Mikowska et  al. also reflected between-population differences in the levels 2014), we expected to find distinct differences for both of these metals (Fig. 3). These results confirmed our expec- metal levels and MTs expressions between them. Statis- tations about increased cadmium and lead levels in the tis- tical analysis did not fully confirm our expectations and sues of animals inhabiting areas in close proximity to main initially conducted site classification for unpolluted and sources of pollutants (Mikowska et al. 2014). Generally, the polluted. Analyses of the differences between single popu- animal exposure to metals and their uptake in the polluted lations showed that Zn concentrations in the livers of ani- sites were higher than in the unpolluted sites. mals from Niepołomice, classified as unpolluted, differed As a response to lead and cadmium accumulation in the from the other results for the unpolluted sites, and are in tissues of bank voles, the study clearly showed between- population differences in MT I and MT II expression for the 1 3 72 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 concentration and MTs genes expressions, indicated that the metals accumulated in some tissues positively influenced MT I and MT II genes expression (Table 4; Fig. 2). Thus, MT I and II expressions were positively influenced by accu- mulated zinc and copper in the liver and by lead and cad- mium in the kidney. Additionally, MT II expression in the liver was upregulated by cadmium. Research similar to ours was performed by Fritsch et al. (2010) on seven sympatric small mammal species collected near former smelters along a pollution gradient, in an area considered as highly polluted with Cd, Pb, and Zn. The authors found increasing-with-age Cd concentrations in the Fig. 3 Cadmium concentrations (mg/kg dw) in the tissues of bank liver and kidney in the case of all the studied species and voles trapped in unpolluted and polluted sites (modified from Mikowska et al. 2014). Bars indicate standard errors. Different letters some patterns of low tissue metal concentrations and low above bar indicate significant difference between populations sepa- metallothioneins levels. A slight increase of MT with Cd rately for each tissue accumulation was noted by Fritsch et al. (2010) for bank vole species. The literature provides more examples of MT livers and testes of the animals but not for the kidney tis- induction (e.g., the findings of Chater et al. 2008) than inhi- sue (ANOVA test). Between-population differences in MTs bition after metal exposure. Chater et al. (2008) studied the gene expressions are more visible in the case of liver tissue, effects of sub-acute cadmium treatment (3 mg of cadmium where the expression is up to eight times higher (MT II) in chloride per kg of bw) on pregnant female rats and noted polluted sites compared with the unpolluted reference—the MT increase (measured with Cd method) in the liver tissues. Mikołajki site. In contrast, the analysis of MT I expression in In such a treatment type, MTs induction is supposed to be the animal testes gave a surprising result of downregulation more evident than after exposure to mostly low environmen- in animals from all the sites when compared to the Mikołajki tal contamination, as we hypothesised in our study. site. Contrary to MT I expression, analysis of the testes of Metallothionein I and II expression in the kidney of animals inhabiting polluted sites and also the Niepołomice Sprague–Dawley rats after intoxication with lead acetate site showed a clear pattern of upregulation of the MT II (300 mg/L) and/or cadmium dichloride (50 mg/L), sepa- gene (up to 4 times higher than those from the Mikołajki rately and in combination, was studied by Wang and Fowler reference site). Taking into account the results of MTs in the (2008). As they revealed, there were no effects of lead on liver and MT II in the testes, the results for animals from the both MTs. Animals from the Cd and Cd–Pb treated groups Niepołomice site were close to the results for animals from had significantly higher expression in the kidney than from sites initially classified as polluted. These expression data the control and, additionally, animals from the Cd–Pb group also reflect the results concerning zinc and cadmium levels had significantly higher MTs expressions than the Cd and Pb in these studied tissues. separately treated groups, which suggests a synergistic effect As mentioned earlier, the statistical results for kidney of these metals. Despite the fact that our studied populations tissues show a similar level of expression for both the stud- were exposed to both cadmium and lead, we may only con- ied MT genes (lower than for Mikołajki) among the studied clude that the main MTs inducer was cadmium because the populations. However, there are some signs of slight upregu- tissue lead concentration was very low. lation in the tissue of bank voles from polluted sites. In the According to some literature data rodent testes are study by Dondero et al. (2005), the authors assessed gene more sensitive to cadmium toxicity than the liver tissue expression of MT10 and MT20, representing the metal- (Mckenna et al. 1996; Ren 2003). Available figures are lothionein gene family. They found that heavy metals influ- inconclusive about the levels of metallothioneins gene enced the expression of the MT10 and MT20 genes and the expressions in the testes samples under environmental level of expression depended on the metal and gene. In case conditions. In some studies, the authors noted no induction of cadmium and MT20 expression reached up to 2200-fold of MT genes expression after Cd exposure (Shiraishi et al. induction. However, in most cases MT expression varied 1995), whereas others confirmed upregulation in rodents between 2- and 30-fold. These results suggest that, in our intoxicated with Cd and Cd/Zn mixture (Messaoudi et al. case, the impact of metal exposure was rather moderate. 2010). In the studied bank vole testes, the MTs expres- Our results concerning MT regulations in the animals sions were not high. What more, MT I expression in from the unpolluted and polluted sites were confirmed the testes of animals from the reference site (Mikołajki) by the results of the regression analysis. The regression was the highest. Downregulation was distinguished for analysis, which takes into account individual tissue metal the remaining sites—up to five times lower than at the 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:66–74 73 Open Access This article is distributed under the terms of the Creative Mikołajki site. Bonda et al. (2004) showed that testicular Commons Attribution 4.0 International License (http://creativecom- MT levels after cadmium intake are lower among adult mons.org/licenses/by/4.0/), which permits unrestricted use, distribu- individuals when compared to young animals. Similar to tion, and reproduction in any medium, provided you give appropriate our research, cadmium contamination seemed to decrease credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. the levels of metallothioneins gene expression. However, in our study, regression analysis did not demonstrate any relationship between metal concentration in the testes and References gene expression of MT I, suggesting that this low expres- sion was not due to the studied pollutants but rather that Amiard J-C, Amiard-Triquet C, Barka S, Pellerin J, Rainbow PS the basal expression was low in all populations due to (2006) Metallothioneins in aquatic invertebrates: their role in other potential factors. metal detoxification and their use as biomarkers. Aquat Toxicol 76:160–202 Andrews GK (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol 59(1):95–104 Atli G, Canli M (2008) Responses of metallothionein and reduced Conclusions glutathione in a freshwater fish Oreochromis niloticus following metal exposures. 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Archives of Environmental Contamination and ToxicologySpringer Journals

Published: Dec 16, 2017

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